COMPOSITIONS TARGETING PROSTATE-SPECIFIC MEMBRANE ANTIGEN AND METHODS FOR MAKING AND USING THE SAME

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted herewith and is hereby incorporated by reference in its entirety. Said .xml copy, created on Jun. 10, 2024, is named 747707_SA9-741_ST26.xml, and is 3,320,448 bytes in size.

BACKGROUND

Prostate cancer is the second most common cancer in men and is expected to affect one in nine men in the United States over the course of their lifetimes. Treatment of localized disease (as measured by a Gleason score <6, PSA<10 ng/ml) by radiation, radical prostatectomy, or active surveillance is successful at controlling early-stage disease; however, relapse occurs in 20 to 50% of men. Patients who continue to progress on first- and second-line androgen deprivation therapies (ADT) develop castration-resistant prostate cancer (CRPC), which often metastasizes (mCRPC) to the bone, brain, liver, and lungs. Chemotherapies such as docetaxel and cabazitaxel have demonstrated improved survival in this population, but there is no cure for mCRPC.

Prostate-Specific Membrane Antigen (PSMA; also known as folate hydrolase 1 and FOLH1) is an integral cell surface membrane protein that is frequently overexpressed in prostate cancer and is often associated with androgen-independent prostate cancer and secondary metastatic lesions. PSMA is also expressed within the neovasculature of bladder, renal, gastric, and colorectal carcinomas.

Immunotherapies have demonstrated mixed success, including with respect to prostate cancer. While the first cell-based immunotherapy sipuleucel T (PROVENGE®) was approved in 2010 for mCRPC, checkpoint blockade targeting immunoinhibitory receptors PD-1 and CTLA-4 have shown very little in the way of lowering response rates in prostate cancer compared with other solid tumor malignancies. It has been suggested that this may be due to the immunologically “cold” tumor microenvironment of primary prostate cancer tumors, characterized by low immune cell infiltration, and a weak neoantigen burden shown to be required for response to checkpoint blockade inhibitors.

There is a long-felt and yet unmet need for therapeutic intervention of tumors that express PSMA, including immunologically cold tumors.

BRIEF DESCRIPTION

The present disclosure provides, among other things, antigen-binding molecules with binding specificity to PSMA, antigen-binding molecules with binding specificity to CD3, as well as bispecific antigen-binding molecules that bind both PSMA and CD3 for use in therapeutic settings in which specific targeting and T cell-mediated killing of PSMA-expressing cells is desired. Aspects disclosed herein address a long-felt unmet need for PSMA-targeting cancer therapeutics, including T cell engagers (TCEs) that have an increased therapeutic index. Aspects of the present disclosure also address the long-felt and yet unmet need for the therapeutic intervention of immunologically cold tumors, e.g., solid tumors, that express PSMA. Also included are, e.g., protease cleavable linkers, barcode fragments, antibody domain linkers, and activatable TCEs (including those that do not bind PSMA). Included herein are also fusion proteins, such as non-TCE fusion proteins that target PSMA, CD3, and/or that comprise linkers and other components provided herein.

Certain aspects of the present disclosure include compounds, compositions, and methods for increasing a subject's therapeutic response to a checkpoint inhibitor (e.g., a PD-1 or CTLA-4 inhibitor such as an anti-PD1 antibody or an anti-CTLA4 antibody). In some embodiments, a compound provided herein recruits and activates effector T cells in a major histocompatibility complex-independent manner via engagement of CD3 on T cells. In some embodiments, the compound is bispecific TCE that is administered in an inactive form and that is activated at and/or within tumor site. In some embodiments, a bispecific TCE is administered before a checkpoint inhibitor therapy begins, concurrently with checkpoint inhibitor therapy, or after checkpoint inhibitor has ended.

Certain aspects of the present disclosure are directed to a chimeric polypeptide comprising a bispecific antibody domain, wherein the bispecific antibody domain comprises a first antigen binding domain that specifically binds to prostate-specific membrane antigen (PSMA) and a second antigen binding domain that binds to cluster of differentiation 3 T cell receptor (CD3), wherein the chimeric polypeptide further comprises a mask polypeptide joined to the bispecific antibody domain via a linker comprising a protease-cleavable release segment positioned between the mask polypeptide and the bispecific antibody domain such that the mask polypeptide is capable of reducing the binding of the bispecific antibody domain to CD3 or PSMA, wherein the protease-cleavable release segment is not capable of being cleaved by legumain in human plasma, or wherein legumain cleaves the protease-cleavable release segment in human plasma at a rate that is less than about 25% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain.

In some embodiments, the chimeric polypeptide comprises a structural arrangement from the N-terminal side to the C-terminal side defined as: (first antigen binding domain)-(second antigen binding domain)-(linker)-(mask polypeptide), (second antigen binding domain)-(first antigen binding domain)-(linker)-(mask polypeptide), (mask polypeptide)-(linker)-(first antigen binding domain)-(second antigen binding domain), or (mask polypeptide)-(linker)-(second antigen binding domain)-(first antigen binding domain), wherein each - is a covalent connection or a polypeptide linker.

In some embodiments, the mask polypeptide is an ELNN.

In some embodiments, the linker further comprises a spacer.

In some embodiments, the protease-cleavable release segment is fused to the bispecific antibody domain via the spacer.

In some embodiments, the spacer is characterized in that: (i) at least 90% of its amino acids are glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), proline (P), or any combination thereof, and (ii) it comprises at least 3 types of amino acids selected from the group consisting of G, A, S, T, E, and P.

In some embodiments, the spacer is from 9 to 14 amino acids in length.

In some embodiments, the spacer comprises at least 4 types of amino acids selected from the group consisting of G, A, S, T, E, and P. In some embodiments, the amino acids of the spacer consist of A, E, G, S, P, and/or T.

In some embodiments, the spacer is cleavable by a non-mammalian protease. In some embodiments, the non-mammalian protease is Glu-C.

In some embodiments, the spacer comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to a sequence listed in Table C. In some embodiments, the spacer comprises an amino acid sequence having at least 85% identity to a sequence listed in Table C. In some embodiments, the spacer comprises an amino acid sequence having at least 90% identity to a sequence listed in Table C. In some embodiments, the spacer comprises an amino acid sequence having at least 91% identity to a sequence listed in Table C. In some embodiments, the spacer comprises an amino acid sequence having at least 92% identity to a sequence listed in Table C. In some embodiments, the spacer comprises an amino acid sequence having at least 93% identity to a sequence listed in Table C. In some embodiments, the spacer comprises an amino acid sequence having at least 94% identity to a sequence listed in Table C. In some embodiments, the spacer comprises an amino acid sequence having at least 95% identity to a sequence listed in Table C. In some embodiments, the spacer comprises an amino acid sequence having at least 96% identity to a sequence listed in Table C. In some embodiments, the spacer comprises an amino acid sequence having at least 97% identity to a sequence listed in Table C. In some embodiments, the spacer comprises an amino acid sequence having at least 98% identity to a sequence listed in Table C. In some embodiments, the spacer comprises an amino acid sequence having at least 99% identity to a sequence listed in Table C. In some embodiments, the spacer comprises an amino acid sequence having 100% identity to a sequence listed in Table C.

In some embodiments, the spacer comprises an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GTSESATPES (SEQ ID NO:96) or GTATPESGPG(SEQ ID NO:97). In some embodiments, the spacer comprises an amino acid sequence that has at least 85% identity to GTSESATPES(SEQ ID NO:96) or GTATPESGPG(SEQ ID NO:97). In some embodiments, the spacer comprises an amino acid sequence that has at least 90% identity to GTSESATPES(SEQ ID NO:96) or GTATPESGPG(SEQ ID NO:97). In some embodiments, the spacer comprises an amino acid sequence that has at least 91% identity to GTSESATPES(SEQ ID NO:96) orGTATPESGPG(SEQ ID NO:97). In some embodiments, the spacer comprises an amino acid sequence that has at least 92% identity to GTSESATPES(SEQ ID NO:96) or GTATPESGPG(SEQ ID NO:97). In some embodiments, the spacer comprises an amino acid sequence that has at least 93% identity to GTSESATPES(SEQ ID NO:96) or GTATPESGPG(SEQ ID NO:97). In some embodiments, the spacer comprises an amino acid sequence that has at least 94% identity to GTSESATPES(SEQ ID NO:96) or GTATPESGPG(SEQ ID NO:97). In some embodiments, the spacer comprises an amino acid sequence that has at least 94% identity to GTSESATPES(SEQ ID NO:96) or GTATPESGPG(SEQ ID NO:97). In some embodiments, the spacer comprises an amino acid sequence that has at least 95% identity to GTSESATPES(SEQ ID NO:96) or GTATPESGPG(SEQ ID NO:97). In some embodiments, the spacer comprises an amino acid sequence that has at least 96% identity to GTSESATPES(SEQ ID NO:96) or GTATPESGPG(SEQ ID NO:97). In some embodiments, the spacer comprises an amino acid sequence that has at least 97% identity to GTSESATPES(SEQ ID NO:96) orGTATPESGPG(SEQ ID NO:97). In some embodiments, the spacer comprises an amino acid sequence that has at least 98% identity to GTSESATPES(SEQ ID NO:96) or GTATPESGPG(SEQ ID NO:97). In some embodiments, the spacer comprises an amino acid sequence that has at least 99% identity to GTSESATPES(SEQ ID NO:96) or GTATPESGPG(SEQ ID NO:97). In some embodiments, the spacer comprises an amino acid sequence that has 100% identity to GTSESATPES(SEQ ID NO:96) or GTATPESGPG(SEQ ID NO:97).

In some embodiments, the protease-cleavable release segment comprises an amino acid sequence comprising the sequence: EAGRSAXHTPAGLTGP (SEQ ID NO: 7627), wherein X is any amino acid other than N. In some embodiments, X is S.

Certain aspects of the present disclosure are directed to a chimeric polypeptide comprising a bispecific antibody domain, wherein the bispecific antibody domain comprises a first antigen binding domain that has binding specificity to a cancer cell antigen, and a second antigen binding domain that has binding specificity to an effector cell antigen expressed on an effector cell, wherein the chimeric polypeptide further comprises a first ELNN joined to the first antigen binding domain via a first linker comprising a first protease-cleavable release segment (RS1) positioned between the first ELNN and the first antigen binding domain such that the first ELNN is capable of reducing the binding of the first antigen binding domain to the cancer cell antigen, wherein the RS1 is cleavable by at least one protease that is present in a tumor, wherein the chimeric polypeptide further comprises a second ELNN joined to the second antigen binding domain via a second linker comprising second protease-cleavable release segment (RS2) positioned between the second ELNN and the second antigen binding domain such that the second ELNN is capable of reducing the binding of the first antigen binding domain to the effector cell antigen, wherein the RS2 is cleavable by at least one protease that is present in a tumor, wherein the first ELNN has a shorter amino acid sequence than the second ELNN, and wherein the cancer cell antigen is not HER2.

In some embodiments, the chimeric polypeptide comprises a structural arrangement from the N-terminal side to the C-terminal side defined as: (ELNN1)-(Linker1)-(first antigen binding domain)-(second antigen binding domain)-(Linker2)-(ELNN2), (ELNN1)-(Linker1)-(second antigen binding domain)-(first antigen binding domain)-(Linker2)-(ELNN2), (ELNN2)-(Linker2)-(first antigen binding domain)-(second antigen binding domain)-(Linker1)-(ELNN1), or (ELNN2)-(Linker2)-(second antigen binding domain)-(first antigen binding domain)-(Linker1)-(ELNN1), wherein each - is, individually, a covalent bond or a polypeptide linker.

In some embodiments, each - is a covalent bond. In some embodiments, each - is a peptide bond.

In some embodiments, Linker1 further comprises a first spacer (Spacer1). In some embodiments, Linker2 further comprises a second spacer (Spacer2).

In some embodiments, RS1 is fused to the bispecific antibody domain via Spacer1 and/or RS2 is fused to the bispecific antibody domain via Spacer2.

In some embodiments, the chimeric polypeptide comprises a structural arrangement from the N-terminal side to the C-terminal side defined as: (ELNN1)-(RS1)-(Spacer1)-(first antigen binding domain)-(second antigen binding domain)-(Spacer2)-(RS2)-(ELNN2), (ELNN1)-(RS1)-(Spacer1)-(second antigen binding domain)-(first antigen binding domain)-(Spacer2)-(RS2)-(ELNN2), (ELNN2)-(RS2)-(Spacer2)-(first antigen binding domain)-(second antigen binding domain)-(Spacer1)-(RS1)-(ELNN1), or (ELNN2)-(RS2)-(Spacer2)-(second antigen binding domain)-(first antigen binding domain)-(Spacer1)-(RS1)-(ELNN1), wherein each - is a, individually, covalent bond or a polypeptide linker.

In some embodiments, each - is a covalent bond. In some embodiments, each - is a peptide bond.

In some embodiments, the chimeric polypeptide further comprises an antibody domain linker between the first antigen binding domain and the second antigen binding domain.

Certain aspects of the present disclosure are directed to a chimeric polypeptide comprising a bispecific antibody domain, comprising the formulas that comprises from the N-terminal side to the C-terminal side: Formula 1: (Mask1)-(RS1)-(Spacer1)-(first antigen binding domain)-[antibody domain linker]-(second antigen binding domain); Formula 2: (first antigen binding domain)-[antibody domain linker]-(second antigen binding domain)-(Spacer2)-(RS2)-(Mask2); or Formula 3: (Mask1)-(RS1)-(Spacer1)-(first antigen binding domain)-[antibody domain linker]-(second antigen binding domain)-(Spacer2)-(RS2)-(Mask2), wherein, the first antigen binding domain has binding specificity to a cancer cell antigen; the second antigen binding domain has binding specificity to an effector cell antigen expressed on an effector cell; each - comprises, individually, a covalent connection or a polypeptide linker; the Mask1 is a polypeptide that is capable of reducing binding of the first antigen binding domain to its target; the Mask2 is a polypeptide that is capable of reducing binding of the second antigen binding domain to its target; if the chimeric polypeptide comprises Formula 1 then the Spacer1 consists of A, E, G, S, P, and/or T residues, if the chimeric polypeptide comprises Formula 2 then the Spacer2 consists of A, E, G, S, P, and/or T residues, and if the chimeric polypeptide comprises Formula 3 then the Spacer1 and/or the Spacer2 consists of A, E, G, S, P, and/or T residues; and wherein the cancer cell antigen is not HER2.

In some embodiments, each - is, individually, a covalent connection. In some embodiments, each - is, individually, a covalent bond. In some embodiments, each - is a peptide bond. In some embodiments, each - is, individually, a polypeptide linker of no more than 5 amino acids.

In some embodiments, the cancer cell antigen is human alpha 4 integrin, Ang2, B7-H3, B7-H6, CEACAM5, cMET, CTLA4, FOLR1, EpCAM, CCR5, CD19, HER3, HER4, PD-L1, prostate-specific membrane antigen (PSMA), CEA, MUC1 (mucin), MUC-2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, MUC16 PhCG, Lewis-Y, CD20, CD33, CD38, CD30, CD56 (NCAM), CD133, ganglioside GD3; 9-O-Acetyl-GD3, GM2, Globo H, fucosyl GM1, GD2, carbonicanhydrase IX, CD44v6, Sonic Hedgehog (Shh), Wue-1, plasma cell antigen 1, melanoma chondroitin sulfate proteoglycan (MCSP), CCR8, 6-transmembrane epithelial antigen of prostate (STEAP), mesothelin, A33 antigen, prostate stem cell antigen (PSCA), Ly-6, desmoglein 4, fetal acetylcholine receptor (fnAChR), CD25, cancer antigen 19-9 (CA19-9), cancer antigen 125 (CA-125), Muellerian inhibitory substance receptor type II (MISIIR), sialylated Tn antigen (sTN), fibroblast activation antigen (FAP), endosialin (CD248), tumor-associated antigen L6 (TAL6), SAS, CD63, TAG72, Thomsen-Friedenreich antigen (TF-antigen), insulin-like growth factor I receptor (IGF-IR), Cora antigen, CD7, CD22, CD70, CD79a, CD79b, G250, MT-MMPs, F19 antigen, CA19-9, CA-125, alpha-fetoprotein (AFP), VEGFR1, VEGFR2, DLK1, SP17, ROR1, or EphA2. In some embodiments, the cancer cell antigen is PSMA.

In some embodiments, the effector cell antigen is cluster of differentiation 3 T cell receptor (CD3).

In some embodiments, the second antigen binding domain has binding specificity to human CD3 and cynomolgus monkey CD3.

In some embodiments, the second antigen binding domain has binding specificity to human CD3.

In some embodiments, the effector cell antigen is CD3 epsilon, CD3 delta, CD3 gamma, or CD3 zeta.

In some embodiments, the effector cell antigen is CD3 epsilon.

In some embodiments, Mask1 is a first ELNN and the Mask2 is a second ELNN.

In some embodiments, the Spacer1 and/or the Spacer2 is characterized in that: (i) at least 90% of its amino acids are glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), proline (P), or any combination thereof, and (ii) it comprises at least 3 types of amino acids selected from the group consisting of G, A, S, T, E, and P.

In some embodiments, the Spacer1 and/or the Spacer2 is from 9 to 14 amino acids in length.

In some embodiments, the Spacer1 and/or the Spacer2 comprises at least 4 types of amino acids selected from the group consisting of G, A, S, T, E, and P.

In some embodiments, the amino acids of the Spacer1 and/or the Spacer2 consists of A, E, G, S, P, and/or T.

In some embodiments, the Spacer1 and/or the Spacer2 is cleavable by a non-mammalian protease. In some embodiments, the non-mammalian protease is Glu-C.

The chimeric polypeptide of any one of claims 42 to 47, wherein the Spacer1 and/or the Spacer 2 comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to a sequence listed in Table C. In some embodiments, the Spacer1 and/or the Spacer 2 comprises an amino acid sequence having at least 85% identity to a sequence listed in Table C. In some embodiments, the Spacer1 and/or the Spacer 2 comprises an amino acid sequence having at least 90% identity to a sequence listed in Table C. In some embodiments, the Spacer1 and/or the Spacer 2 comprises an amino acid sequence having at least 91% identity to a sequence listed in Table C. In some embodiments, the Spacer1 and/or the Spacer 2 comprises an amino acid sequence having at least 92% identity to a sequence listed in Table C. In some embodiments, the Spacer1 and/or the Spacer 2 comprises an amino acid sequence having at least 93% identity to a sequence listed in Table C. In some embodiments, the Spacer1 and/or the Spacer 2 comprises an amino acid sequence having at least 94% identity to a sequence listed in Table C. In some embodiments, the Spacer1 and/or the Spacer 2 comprises an amino acid sequence having at least 95% identity to a sequence listed in Table C. In some embodiments, the Spacer1 and/or the Spacer 2 comprises an amino acid sequence having at least 96% identity to a sequence listed in Table C. In some embodiments, the Spacer1 and/or the Spacer 2 comprises an amino acid sequence having at least 97% identity to a sequence listed in Table C. In some embodiments, the Spacer1 and/or the Spacer 2 comprises an amino acid sequence having at least 98% identity to a sequence listed in Table C. In some embodiments, the Spacer1 and/or the Spacer 2 comprises an amino acid sequence having at least 99% identity to a sequence listed in Table C. In some embodiments, the Spacer1 and/or the Spacer 2 comprises an amino acid sequence having 100% identity to a sequence listed in Table C.

In some embodiments, wherein the Spacer1 and/or the Spacer 2 comprises an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GTSESATPES(SEQ ID NO:96) or GTATPESGPG(SEQ ID NO:97). In some embodiments, the Spacer1 and/or the Spacer 2 comprises an amino acid sequence that has at least 85% identity to GTSESATPES(SEQ ID NO:96) orGTATPESGPG(SEQ ID NO:97). In some embodiments, the Spacer1 and/or the Spacer 2 comprises an amino acid sequence that has at least 90% identity to GTSESATPES(SEQ ID NO:96) or GTATPESGPG(SEQ ID NO:97). In some embodiments, the Spacer1 and/or the Spacer 2 comprises an amino acid sequence that has at least 91% identity to GTSESATPES(SEQ ID NO:96) orGTATPESGPG(SEQ ID NO:97). In some embodiments, the Spacer1 and/or the Spacer 2 comprises an amino acid sequence that has at least 92% identity to GTSESATPES(SEQ ID NO:96) or GTATPESGPG(SEQ ID NO:97). In some embodiments, the Spacer1 and/or the Spacer 2 comprises an amino acid sequence that has at least 93% identity to GTSESATPES(SEQ ID NO:96) orGTATPESGPG(SEQ ID NO:97). In some embodiments, the Spacer1 and/or the Spacer 2 comprises an amino acid sequence that has at least 94% identity to GTSESATPES(SEQ ID NO:96) or GTATPESGPG(SEQ ID NO:97). In some embodiments, the Spacer1 and/or the Spacer 2 comprises an amino acid sequence that has at least 94% identity to GTSESATPES(SEQ ID NO:96) orGTATPESGPG(SEQ ID NO:97). In some embodiments, the Spacer1 and/or the Spacer 2 comprises an amino acid sequence that has at least 95% identity to GTSESATPES(SEQ ID NO:96) or GTATPESGPG(SEQ ID NO:97). In some embodiments, the Spacer1 and/or the Spacer 2 comprises an amino acid sequence that has at least 96% identity to GTSESATPES(SEQ ID NO:96) orGTATPESGPG(SEQ ID NO:97). In some embodiments, the Spacer1 and/or the Spacer 2 comprises an amino acid sequence that has at least 97% identity to GTSESATPES(SEQ ID NO:96) or GTATPESGPG(SEQ ID NO:97). In some embodiments, the Spacer1 and/or the Spacer 2 comprises an amino acid sequence that has at least 98% identity to GTSESATPES(SEQ ID NO:96) orGTATPESGPG(SEQ ID NO:97). In some embodiments, the Spacer1 and/or the Spacer 2 comprises an amino acid sequence that has at least 99% identity to GTSESATPES(SEQ ID NO:96) or GTATPESGPG(SEQ ID NO:97). In some embodiments, the Spacer1 and/or the Spacer 2 comprises an amino acid sequence that has 100% identity to GTSESATPES(SEQ ID NO:96) orGTATPESGPG(SEQ ID NO:97).

In some embodiments, the amino acid sequence of the first ELNN is at least 100 amino acids shorter than the amino acid sequence of the second ELNN. In some embodiments, the amino acid sequence of the first ELNN is at least 200 amino acids shorter than the amino acid sequence of the second ELNN. In some embodiments, the amino acid sequence of the first ELNN is at least 250 amino acids shorter than the amino acid sequence of the second ELNN. In some embodiments, the amino acid sequence of the first ELNN is about 294 amino acids in length, and wherein the amino acid sequence of the second ELNN is about 582 amino acids in length.

In some embodiments, the first antigen binding domain comprises a first antibody or an antigen-binding fragment thereof, and wherein the second antigen binding domain is a second antibody or an antigen-binding fragment thereof.

In some embodiments, the first antigen binding domain is a Fab, an scFV, or an ISVD. In some embodiments, the ISVD is a VHH domain. In some embodiments, the second antigen binding domain is a Fab, an scFV, or an ISVD. In some embodiments, the ISVD is a VHH domain. In some embodiments, the first antigen binding domain is a VHH domain. In some embodiments, the second antigen binding domain is an scFV.

In some embodiments, there is an antibody domain linker between the first antigen binding domain and the second antigen binding domain.

In some embodiments, the antibody domain linker comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to a sequence listed in Table A or B. In some embodiments, the antibody domain linker comprises an amino acid sequence that has at least 85% identity to a sequence listed in Table A or B. In some embodiments, the antibody domain linker comprises an amino acid sequence that has at least 90% identity to a sequence listed in Table A or B. In some embodiments, the antibody domain linker comprises an amino acid sequence that has at least 91% identity to a sequence listed in Table A or B. In some embodiments, the antibody domain linker comprises an amino acid sequence that has at least 92% identity to a sequence listed in Table A or B. In some embodiments, the antibody domain linker comprises an amino acid sequence that has at least 93% identity to a sequence listed in Table A or B. In some embodiments, the antibody domain linker comprises an amino acid sequence that has at least 94% identity to a sequence listed in Table A or B. In some embodiments, the antibody domain linker comprises an amino acid sequence that has at least 94% identity to a sequence listed in Table A or B. In some embodiments, the antibody domain linker comprises an amino acid sequence that has at least 95% identity to a sequence listed in Table A or B. In some embodiments, the antibody domain linker comprises an amino acid sequence that has at least 96% identity to a sequence listed in Table A or B. In some embodiments, the antibody domain linker comprises an amino acid sequence that has at least 97% identity to a sequence listed in Table A or B. In some embodiments, the antibody domain linker comprises an amino acid sequence that has at least 98% identity to a sequence listed in Table A or B. In some embodiments, the antibody domain linker comprises an amino acid sequence that has at least 99% identity to a sequence listed in Table A or B. In some embodiments, the antibody domain linker comprises an amino acid sequence that has 100% identity to a sequence listed in Table A or B.

In some embodiments, the antibody domain linker consists of G and S amino residues. In some embodiments, the antibody domain linker is about 9 residues in length. In some embodiments, the antibody domain linker comprises the amino acid sequence GGGGSGGGS(SEQ ID NO:125).

In some embodiments, the scFv comprises a VL domain, a VH domain, and a linker between the VL domain and the VH domain, wherein the linker consists of A, E, G, S, P, and/or T residues.

In some embodiments, the linker is characterized in that: (i) at least 90% of its amino acids are glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), proline (P), or any combination thereof, and (ii) it comprises at least 3 types of amino acids selected from the group consisting of G, A, S, T, E, and P.

In some embodiments, the linker between the VL domain and the VH domain is from 25 to 35 amino acids in length.

In some embodiments, the linker between the VL domain and the VH domain comprises at least 4 types of amino acids selected from the group consisting of G, A, S, T, E, and P.

In some embodiments, the amino acids of the linker between the VL domain and the VH domain consists of A, E, G, S, P, and/or T.

In some embodiments, the linker between the VL domain and the VH domain is cleavable by a non-mammalian protease. In some embodiments, the non-mammalian protease is Glu-C.

In some embodiments, the linker between the VL domain and the VH domain comprises an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81).

In some embodiments, the linker between the VL domain and the VH domain comprises an amino acid sequence that has at least 85% identity to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). In some embodiments, the linker between the VL domain and the VH domain comprises an amino acid sequence that has at least 90% identity to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). In some embodiments, the linker between the VL domain and the VH domain comprises an amino acid sequence that has at least 91% identity to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). In some embodiments, the linker between the VL domain and the VH domain comprises an amino acid sequence that has at least 92% identity to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). In some embodiments, the linker between the VL domain and the VH domain comprises an amino acid sequence that has at least 93% identity to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). In some embodiments, the linker between the VL domain and the VH domain comprises an amino acid sequence that has at least 94% identity to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). In some embodiments, the linker between the VL domain and the VH domain comprises an amino acid sequence that has at least 95% identity to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). In some embodiments, the linker between the VL domain and the VH domain comprises an amino acid sequence that has at least 96% identity to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). In some embodiments, the linker between the VL domain and the VH domain comprises an amino acid sequence that has at least 97% identity to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). In some embodiments, the linker between the VL domain and the VH domain comprises an amino acid sequence that has at least 98% identity to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). In some embodiments, the linker between the VL domain and the VH domain comprises an amino acid sequence that has at least 99% identity to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). In some embodiments, the linker between the VL domain and the VH domain comprises an amino acid sequence that has 100% identity to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81).

In some embodiments, the first antigen binding domain comprises a VHH domain comprising three VHH complementarity determining regions (CDRs), wherein the three VHH CDRs comprise the CDR1, CDR2, and CDR3 of a VHH domain comprising the following amino acid sequence:

(SEQ ID NO: 549)

QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVG

AMSWSGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNKEYGRTWYDFNESDYWGQGTQVTVSS.

In some embodiments, the second antigen binding domain comprises a VL domain comprising three the VL CDRs, wherein the three VL CDRs comprise the CDR1, CDR2, and CDR3 of a VL domain comprising the following amino acid sequence: ELVVTQEPSLTVSPGGTVTLTCRSSX₁GAVTX₂SNYANWVQQKPGQAPRGLIGGTN KRAPGTPARFSGSLLGGKAALTLSGVQPEDEAX₃YYCALWYX₄NLWVFGGGTKLTV L (SEQ ID NO:9001), wherein X₁corresponds to T or N, X₂corresponds to T or S, X₃corresponds to E or V, and X₄corresponds to S or P.

(SEQ ID NO: 361)

ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGL

IGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLW

VFGGGTKLTVL.

In some embodiments, the second antigen binding domain comprises a VH domain comprising three the VH CDRs, wherein the three VH CDRs comprise the CDR1, CDR2, and CDR3 of a VH domain comprising the following amino acid sequence: EVQLX₅ESGGGX₆VQPGGSLX₇LSCAASGFTFX₈TYAMNWVRQAPGKGLEWVX₉RI RX₁₀KX₁₁NNYATYYADSVKX₁₂RFTISRDDSKNTX₁₃YLQMNX₁₄LKTEDTAVYYCVRH X₁₅NFGNSYVSWFAX₁₆WGQGTLVTVSS(SEQ ID NO:9002), wherein X₅corresponds to V or L, X₆corresponds to I or L, X₇corresponds to R or K, X₈corresponds to S or N, X₉corresponds to G or A, X₁₀corresponds to T or S, X₁₁corresponds to R or Y, X₁₂corresponds to G or D, X₁₃corresponds to V or A, X₁₄corresponds to S or N, X₁₅corresponds to E or G, and X₁₆corresponds to H or Y.

(SEQ ID NO: 311)

EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVG

RIRTKRNNYATYYADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYC

VRHENFGNSYVSWFAHWGQGTLVTVSS.

In some embodiments, the second antigen binding domain comprises a VL domain amino acid sequence SEQ ID NO/VH domain amino acid sequence SEQ ID NO pair selected from the group consisting of: 896/897; 902/903; 700/701; 702/703; 716/717; 718/719; 728/729; 736/737; 738/739; 740/741; 742/743; 744/745; 746/747; 748/749; 750/751; 752/753; 754/755; 756/757; 758/759; 760/761; 762/763; 764/765; 766/767; 774/775; 776/777; 790/791; 792/793; 798/799; 800/801; 806/807; 808/809; 814/815; 816/817; 822/823; 824/825; or 826/867.

In some embodiments, (i) the first antigen binding domain is a VHH comprising the following CDRs: a VHH CDR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GRTFGIYVWG(SEQ ID NO:9003); a VHH CDR2 with an amino acid sequence that that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to AMSWSGSNRKVSDSVKG(SEQ ID NO:9004); and a VHH CDR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to AASNKEYGRTWYDFNESDY(SEQ ID NO:9005), and (ii) wherein the second antigen binding domain comprises the following CDRs: a VL domain CDR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to RSSXIGAVTX₂SNYAN (SEQ ID NO:9006), wherein X₁corresponds to T or N, and X₂corresponds to T or S; a VL domain CDR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GTNKRAP(SEQ ID NO:4); a VL domain CDR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to ALWYX₄NLWV(SEQ ID NO:9007), wherein X₄corresponds to S or P; a VH domain CDR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GFTFX₈TYAMN(SEQ ID NO:9008), wherein X₈corresponds to S or N; a VH domain CDR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to RIRX₁₀KX₁₁NNYATYYADSVKX₁₂(SEQ ID NO:9009), wherein X₁₀corresponds to T or S, X₁₁corresponds to R or Y, and X₁₂corresponds to G or D; a VH domain CDR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to HX₁₅NFGNSYVSWFAX₁₆(SEQ ID NO:9010), wherein X₁₅corresponds to E or G, and X₁₆corresponds to H or Y.

In some embodiments, (i) the first antigen binding domain is a VHH comprising the following CDRs: a VHH CDR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GRTFGIYVWG(SEQ ID NO:9003); a VHH CDR2 with an amino acid sequence that that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to AMSWSGSNRKVSDSVKG(SEQ ID NO:9004); and a VHH CDR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to AASNKEYGRTWYDFNESDY(SEQ ID NO:9005), and (ii) wherein the second antigen binding domain comprises the following CDRs: a VL domain CDR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to RSSNGAVTSSNYAN(SEQ ID NO:1); a VL domain CDR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GTNKRAP(SEQ ID NO:4); a VL domain CDR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to ALWYPNLWV(SEQ ID NO:6); a VH domain CDR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GFTFSTYAMN(SEQ ID NO:12); a VH domain CDR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to RIRTKRNNYATYYADSVKG(SEQ ID NO:13); and a VH domain CDR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to HENFGNSYVSWFAH(SEQ ID NO:10).

In some embodiments, the VHH comprises the following framework regions (FRs): a VHH FR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to QVQLVESGGGVVQPGRSLRLSCAAS(SEQ ID NO:9011); a VHH FR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to WFRQAPGKEREFVG(SEQ ID NO:9012); a VHH FR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to RFTISRDNSKNTLYLQMNSLRAEDTAVYYC(SEQ ID NO:9013); and a VHH FR4 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to WGQGTQVTVSS(SEQ ID NO:9014).

In some embodiments, the second antigen binding domain comprises the following FRs: a VL domain FR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to ELVVTQEPSLTVSPGGTVTLTC(SEQ ID NO:51); a VL domain FR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to WVQQKPGQAPRGLIG(SEQ ID NO:52); a VL domain FR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GTPARFSGSLLGGKAALTLSGVQPEDEAVYYC(SEQ ID NO:53); a VL domain FR4 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to FGGGTKLTVL(SEQ ID NO:59); a VH domain FR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to EVQLVESGGGIVQPGGSLRLSCAAS(SEQ ID NO:400); a VH domain FR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to WVRQAPGKGLEWVG(SEQ ID NO:401); a VH domain FR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to RFTISRDDSKNTVYLQMNSLKTEDTAVYYCVR(SEQ ID NO:402); and a VH domain FR4 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to WGQGTLVTVSS (SEQ ID NO:67).

In some embodiments, (i) the first antigen binding domain is a VHH comprising the following CDRs: a VHH CDR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GRTFGIYVWG(SEQ ID NO:9003); a VHH CDR2 with an amino acid sequence that that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to AMSWSGSNRK(SEQ ID NO:9015); and a VHH CDR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to AASNKEYGRTWYDFNESDY(SEQ ID NO:9005), and (ii) wherein the second antigen binding domain comprises the following CDRs: a VL domain CDR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to RSSX₁GAVTX₂SNYAN(SEQ ID NO:9006), wherein X₁corresponds to T or N, and X₂corresponds to T or S; a VL domain CDR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GTNKRAP(SEQ ID NO:4); a VL domain CDR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to ALWYX₄NLWV(SEQ ID NO:9007), wherein X₄corresponds to S or P; a VH domain CDR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GFTFX₈TYAMN(SEQ ID NO:9008), wherein X₈corresponds to S or N; a VH domain CDR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to RIRX₁₀KX₁₁NNYATYYADSVKX₁₂(SEQ ID NO:9009), wherein X₁₀corresponds to T or S, X₁₁corresponds to R or Y, and X₁₂corresponds to G or D; a VH domain CDR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to HX₁₅NFGNSYVSWFAX₁₆(SEQ ID NO:9010), wherein X₁₅corresponds to E or G, and X₁₆corresponds to H or Y.

In some embodiments, (i) the first antigen binding domain is a VHH comprising the following CDRs: a VHH CDR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GRTFGIYVWG(SEQ ID NO:9003); a VHH CDR2 with an amino acid sequence that that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to AMSWSGSNRK(SEQ ID NO:9015); and a VHH CDR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to AASNKEYGRTWYDFNESDY(SEQ ID NO:9005), and (ii) wherein the second antigen binding domain comprises the following CDRs: a VL domain CDR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to RSSNGAVTSSNYAN(SEQ ID NO:1); a VL domain CDR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GTNKRAP(SEQ ID NO:4); a VL domain CDR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to ALWYPNLWV(SEQ ID NO:6); a VH domain CDR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GFTFSTYAMN(SEQ ID NO:12); a VH domain CDR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to RIRTKRNNYATYYADSVKG(SEQ ID NO:13); and a VH domain CDR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to HENFGNSYVSWFAH(SEQ ID NO:10).

In some embodiments, the VHH comprises the following framework regions (FRs): a VHH FR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to QVQLVESGGGVVQPGRSLRLSCAAS(SEQ ID NO:9011); a VHH FR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to WFRQAPGKEREFVG(SEQ ID NO:9012); a VHH FR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to VSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC(SEQ ID NO:9016); and a VHH FR4 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to WGQGTQVTVSS(SEQ ID NO:9014).

In some embodiments, the second antigen binding domain comprises a VL domain comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to: ELVVTQEPSLTVSPGGTVTLTCRSSX₁GAVTX₂SNYANWVQQKPGQAPRGLIGGTN KRAPGTPARFSGSLLGGKAALTLSGVQPEDEAX₃YYCALWYX₄NLWVFGGGTKLTV L(SEQ ID NO:9001), wherein X₁corresponds to T or N, X₂corresponds to T or S, X₃corresponds to E or V, and X₄corresponds to S or P.

(SEQ ID NO: 361)

ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGL

IGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLW

VFGGGTKLTVL.

In some embodiments, the second antigen binding domain comprises a VH domain comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to: EVQLX₅ESGGGX₆VQPGGSLX₇LSCAASGFTFX₈TYAMNWVRQAPGKGLEWVXsRI RX₁₀KX₁₁NNYATYYADSVKX₁₂RFTISRDDSKNTX₁₃YLQMNX₁₄LKTEDTAVYYCVRH X₁₅NFGNSYVSWFAX₁₆WGQGTLVTVSS(SEQ ID NO:9002), wherein X₅corresponds to V or L, X₆corresponds to I or L, X₇corresponds to R or K, X₈corresponds to S or N, X₉corresponds to G or A, X₁₀corresponds to T or S, X₁₁corresponds to R or Y, X₁₂corresponds to G or D, X₁₃corresponds to V or A, X₁₄corresponds to S or N, X₁₅corresponds to E or G, and X₁₆corresponds to H or Y.

(SEQ ID NO: 311)

EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVG

RIRTKRNNYATYYADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYC

VRHENFGNSYVSWFAHWGQGTLVTVSS.

In some embodiments, the VL domain is N-terminal to the VH domain. In some embodiments, the VL domain is C-terminal to the VH domain.

In some embodiments, the second antigen binding domain comprises a scFV comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to:

(SEQ ID NO: 215)

ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGL

IGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLW

VFGGGTKLTVLSESATPESGPGTSPGATPESGPGTSESATPEVQLVESG

GGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKRNN

YATYYADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCVRHENFGN

SYVSWFAHWGQGTLVTVSS.

In some embodiments, the first antigen binding domain comprises a VHH comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to: QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVX₁₇GWFRQAPGKEREFVGAX₁₈S WSGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYX₁₉CX₂₀X₂₁SNKX₂₂Y GRTWYDFNESDYWGQGTQVTVSS(SEQ ID NO:9017), wherein X₁₇, X₁₈, X₁₉, X₂₀, X₂₁, and X₆each, individually, correspond to any naturally occurring amino acid. In some embodiments, X₁₇corresponds to M or W, X₁₈corresponds to M or I, X₁₉corresponds to F or Y, X₂₀corresponds to A or G, X₂₁corresponds to A or G, and/or X₂₂corresponds to L, W, R, D, E, or G.

(SEQ ID NO: 549)

QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVG

AMSWSGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNKEYGRTWYDFNESDYWGQGTQVTVSS.

In some embodiments, the RS comprises a protease cleavage site is cleavable by at least one protease listed in Table 7.

In some embodiments, the RS comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to a sequence listed in Table 8a. In some embodiments, the RS comprises an amino acid sequence having at least 85% identity to a sequence listed in Table 8a. In some embodiments, the RS comprises an amino acid sequence having at least 90% identity to a sequence listed in Table 8a. In some embodiments, the RS comprises an amino acid sequence having at least 91% identity to a sequence listed in Table 8a. In some embodiments, the RS comprises an amino acid sequence having at least 92% identity to a sequence listed in Table 8a. In some embodiments, the RS comprises an amino acid sequence having at least 93% identity to a sequence listed in Table 8a. In some embodiments, the RS comprises an amino acid sequence having at least 94% identity to a sequence listed in Table 8a. In some embodiments, the RS comprises an amino acid sequence having at least 95% identity to a sequence listed in Table 8a. In some embodiments, the RS comprises an amino acid sequence having at least 96% identity to a sequence listed in Table 8a. In some embodiments, the RS comprises an amino acid sequence having at least 97% identity to a sequence listed in Table 8a. In some embodiments, the RS comprises an amino acid sequence having at least 98% identity to a sequence listed in Table 8a. In some embodiments, the RS comprises an amino acid sequence having at least 99% identity to a sequence listed in Table 8a. In some embodiments, the RS comprises an amino acid sequence having 100% identity to a sequence listed in Table 8a.

In some embodiments, the RS is cleavable by uPA, ST14, MMP2, MMP7, MMP9, and MMP14.

In some embodiments, the RS is not cleavable by legumain. In some embodiments, the RS is not cleavable by legumain in human blood, plasma, or serum. In some embodiments, the RS is not cleavable upon incubation with about 1 nM or less legumain for about 20 hours. In some embodiments, the RS is not cleavable upon incubation with about 1 nM or less legumain for about 20 hours in human blood, plasma, or serum.

In some embodiments, legumain cleaves the RS in human plasma at a rate that is less than about 50% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain. In some embodiments, legumain cleaves the RS in human plasma at a rate that is less than about 25% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain. In some embodiments, legumain cleaves the RS in human plasma at a rate that is less than about 10% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain. In some embodiments, legumain cleaves the RS in human plasma at a rate that is less than about 5% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain. In some embodiments, legumain cleaves the RS in human plasma at a rate that is less than about 2.5% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain.

In some embodiments, the RS1 and/or RS2 comprises protease cleavage is cleavable by at least one protease listed in Table 7.

In some embodiments, the RS1 and/or RS2 comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to a sequence listed in Table 8a. In some embodiments, the RS1 and/or RS2 comprises an amino acid sequence having at least 85% identity to a sequence listed in Table 8a. In some embodiments, the RS1 and/or RS2 comprises an amino acid sequence having at least 90% identity to a sequence listed in Table 8a. In some embodiments, the RS1 and/or RS2 comprises an amino acid sequence having at least 91% identity to a sequence listed in Table 8a. In some embodiments, the RS1 and/or RS2 comprises an amino acid sequence having at least 92% identity to a sequence listed in Table 8a. In some embodiments, the RS1 and/or RS2 comprises an amino acid sequence having at least 93% identity to a sequence listed in Table 8a. In some embodiments, the RS1 and/or RS2 comprises an amino acid sequence having at least 94% identity to a sequence listed in Table 8a. In some embodiments, the RS1 and/or RS2 comprises an amino acid sequence having at least 95% identity to a sequence listed in Table 8a. In some embodiments, the RS1 and/or RS2 comprises an amino acid sequence having at least 96% identity to a sequence listed in Table 8a. In some embodiments, the RS1 and/or RS2 comprises an amino acid sequence having at least 97% identity to a sequence listed in Table 8a. In some embodiments, the RS1 and/or RS2 comprises an amino acid sequence having at least 98% identity to a sequence listed in Table 8a. In some embodiments, the RS1 and/or RS2 comprises an amino acid sequence having at least 99% identity to a sequence listed in Table 8a. In some embodiments, the RS1 and/or RS2 comprises an amino acid sequence having 100% identity to a sequence listed in Table 8a.

In some embodiments, the RS1 and/or RS2 is cleavable by uPA, ST14, MMP2, MMP7, MMP9, and MMP14.

In some embodiments, the RS1 and/or RS2 is not cleavable by legumain. In some embodiments, the RS1 and/or RS2 is not cleavable by legumain in human blood, plasma, or serum. In some embodiments, the RS1 and/or RS2 is not cleavable upon incubation with about 1 nM or less legumain for about 20 hours. In some embodiments, the RS1 and/or RS2 is not cleavable upon incubation with about 1 nM or less legumain for about 20 hours in human blood, plasma, or serum.

In some embodiments, legumain cleaves the RS1 and/or RS2 in human plasma at a rate that is less than about 50% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain. In some embodiments, legumain cleaves the RS1 and/or RS2 in human plasma at a rate that is less than about 25% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain. In some embodiments, legumain cleaves the RS1 and/or RS2 in human plasma at a rate that is less than about 10% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain. In some embodiments, legumain cleaves the RS1 and/or RS2 in human plasma at a rate that is less than about 5% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain. In some embodiments, legumain cleaves the RS1 and/or RS2 in human plasma at a rate that is less than about 2.5% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain.

In some embodiments, the RS1 comprises a protease-cleavable amino acid sequence comprising the sequence: EAGRSAXHTPAGLTGP (SEQ ID NO: 7627), wherein X is any amino acid other than N.

In some embodiments, the RS2 comprises a protease-cleavable amino acid sequence comprising the sequence: EAGRSAXHTPAGLTGP (SEQ ID NO: 7627), wherein X is any amino acid other than N.

In some embodiments, RS1 and/or RS2 comprises a protease-cleavable amino acid sequence comprising the sequence: EAGRSASHTPAGLTGP (SEQ ID NO: 7628).

In some embodiments, the RS1 and the RS2 are the same. In some embodiments, the RS1 and the RS2 are different.

In some embodiments, the mask polypeptide is a first mask polypeptide and the protease-cleavable release segment is a first protease-cleavable release segment (RS1), and wherein the chimeric polypeptide further comprises a second mask polypeptide and a second protease-cleavable release segment (RS2), wherein the second mask polypeptide is joined to the second antigen binding domain via a second protease-cleavable release segment (RS2) positioned between the second mask polypeptide and the second antigen binding domain such that the second mask polypeptide reduces the binding of the first antigen binding domain to CD3, wherein the RS2 is cleavable by at least one protease that is present in a tumor.

In some embodiments, the first mask polypeptide is attached to the first antigen binding domain and wherein the second mask polypeptide is attached to the second antigen binding domain.

In some embodiments, the first mask polypeptide is a first ELNN and the second mask polypeptide is a second ELNN.

In some embodiments, the first ELNN and the second ELNN are each individually characterized in that: (i) at least 90% of each of the first ELNN's and the second ELNN's amino acids are glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), proline (P), or any combination thereof; and (ii) each comprises at least 3 types of amino acids selected from the group consisting of G, A, S, T, E, and P.

In some embodiments, the first ELNN and the second ELNN are each individually further characterized in that: (i) each comprises at least 100 amino acid residues; and (ii) each comprises a plurality of non-overlapping sequence motifs that are each from 9 to 14 amino acids in length, wherein the plurality of non-overlapping sequence motifs comprise a set of non-overlapping sequence motives, wherein each non-overlapping sequence motive of the set of non-overlapping sequence motifs is repeated at least two times in the ELNN.

In some embodiments, the plurality of non-overlapping sequence motifs comprises at least one non-overlapping sequence motif that occurs only once within the ELNN. In some embodiments, the non-overlapping sequence motifs comprise one of or any combination of the sequence motifs listed in Table 1. In some embodiments, the non-overlapping sequence motifs comprise at least 2, 3, or 4 of the sequence motifs listed in Table 1. In some embodiments, the non-overlapping sequence motifs comprise any one of or any combination of GTSTEPSEGSAP(SEQ ID NO:189), GTSESATPESGP(SEQ ID NO:188), GSGPGTSESATP (SEQ ID NO:9018), GSEPATSGSETP (SEQ ID NO:187), GSPAGSPTSTEE(SEQ ID NO:186), and GTSPSATPESGP (SEQ ID NO:9019).

In some embodiments, each of the first ELNN and the second ELNN comprises at least 4 types of amino acids selected from the group consisting of G, A, S, T, E, and P. In some embodiments, the amino acids of each of the first ELNN and the second ELNN consists of A, E, G, S, P, and/or T.

In some embodiments, the first ELNN and/or the second ELNN comprises an amino acid sequence that is at least 85% identical to an amino acid sequence listed in Table 3a or 3b.

In some embodiments, the first ELNN comprises an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to: ASSATPESGPGTSTEPSEGSAPGTSESATPESGPGSGPGTSESATPGTSESATPE SGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSE SATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTE EGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPAT SGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPG SEPATSGSETPGTSESATP (SEQ ID NO: 8021). In some embodiments, the first ELNN comprises an amino acid sequence that has at least 85%, identity to SEQ ID NO: 8021. In some embodiments, the first ELNN comprises an amino acid sequence that has at least 90%, identity to SEQ ID NO: 8021. In some embodiments, the first ELNN comprises an amino acid sequence that has at least 91%, identity to SEQ ID NO: 8021. In some embodiments, the first ELNN comprises an amino acid sequence that has at least 92%, identity to SEQ ID NO: 8021. In some embodiments, the first ELNN comprises an amino acid sequence that has at least 93%, identity to SEQ ID NO: 8021. In some embodiments, the first ELNN comprises an amino acid sequence that has at least 94%, identity to SEQ ID NO: 8021. In some embodiments, the first ELNN comprises an amino acid sequence that has at least 95%, identity to SEQ ID NO: 8021. In some embodiments, the first ELNN comprises an amino acid sequence that has at least 96%, identity to SEQ ID NO: 8021. In some embodiments, the first ELNN comprises an amino acid sequence that has at least 97%, identity to SEQ ID NO: 8021. In some embodiments, the first ELNN comprises an amino acid sequence that has at least 98%, identity to SEQ ID NO: 8021. In some embodiments, the first ELNN comprises an amino acid sequence that has at least 99%, identity to SEQ ID NO: 8021. In some embodiments, the first ELNN comprises an amino acid sequence that has 100%, identity to SEQ ID NO: 8021.

In some embodiments, the second ELNN comprises an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to: ATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETP GTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPS EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGT SESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPE SGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPA GSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSET PGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGS PTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEG SPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSPSATP ESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTS TEPSEGSAPGSEPATSGSETPGTSESAGEPEA (SEQ ID NO: 8022). In some embodiments, the first ELNN comprises an amino acid sequence that has at least 85%, identity to SEQ ID NO: 8022. In some embodiments, the first ELNN comprises an amino acid sequence that has at least 90%, identity to SEQ ID NO: 8022. In some embodiments, the first ELNN comprises an amino acid sequence that has at least 91%, identity to SEQ ID NO: 8022. In some embodiments, the first ELNN comprises an amino acid sequence that has at least 92%, identity to SEQ ID NO: 8022. In some embodiments, the first ELNN comprises an amino acid sequence that has at least 93%, identity to SEQ ID NO: 8022. In some embodiments, the first ELNN comprises an amino acid sequence that has at least 94%, identity to SEQ ID NO: 8022. In some embodiments, the first ELNN comprises an amino acid sequence that has at least 95%, identity to SEQ ID NO: 8022. In some embodiments, the first ELNN comprises an amino acid sequence that has at least 96%, identity to SEQ ID NO: 8022. In some embodiments, the first ELNN comprises an amino acid sequence that has at least 97%, identity to SEQ ID NO: 8022. In some embodiments, the first ELNN comprises an amino acid sequence that has at least 98%, identity to SEQ ID NO: 8022. In some embodiments, the first ELNN comprises an amino acid sequence that has at least 99%, identity to SEQ ID NO: 8022. In some embodiments, the first ELNN comprises an amino acid sequence that has 100%, identity to SEQ ID NO: 8022.

In some embodiments, the chimeric polypeptide comprises one or more barcode fragments. In some embodiments, the chimeric polypeptide comprises two or more barcode fragments. In some embodiments, each barcode fragment is different from every other barcode fragment.

In some embodiments, each barcode fragment differs in both sequence and molecular weight from all other peptide fragments that are releasable from the chimeric polypeptide upon complete digestion the chimeric polypeptide by a non-mammalian protease.

In some embodiments, the non-mammalian protease is Glu-C. In some embodiments, the chimeric polypeptide comprises a Glu-C cleavage site comprising one of the following amino acid sequences: ATPESGPG(SEQ ID NO:9020), SGSETPGT(SEQ ID NO:9021), and GTSESATP(SEQ ID NO:9022).

In some embodiments, the chimeric polypeptide comprises at least one of the following amino acid sequences: SGPE.SGPGX_nSGPE.SGPG(SEQ ID NO:9023), SGPE.SGPGX_nATPE.SGPG(SEQ ID NO:9024), SGPE.SGPGX_nGTSE.SATP(SEQ ID NO:9025), SGPE.SGPGX_nTTPE.SGPG(SEQ ID NO:9026), SGPE.SGPGX_nSTPE.SGPG(SEQ ID NO:9027), SGPE.SGPGX_nGTPE.SGPG(SEQ ID NO:9028), SGPE.SGPGX_nGTPE.TPGS (SEQ ID NO:9029), SGPE.SGPGX_nGTPE.TPGS(SEQ ID NO:9029), SGPE.SGPGX_nSGSE.TGTP(SEQ ID NO:9030), SGPE.SGPGX_nGTPE.GSAP(SEQ ID NO:9031), SGPE.SGPGX_nEPSE.SATP(SEQ ID NO:9032), ATPE.SGPGX_nSGPE.SGPG(SEQ ID NO:9033), ATPE.SGPGX_nATPE.SGPG(SEQ ID NO:9034), ATPE.SGPGX_nGTSE.SATP(SEQ ID NO:9035), ATPE.SGPGX_nTTPE.SGPG(SEQ ID NO:9036), ATPE.SGPGX_nSTPE.SGPG(SEQ ID NO:9037), ATPE.SGPGX_nGTPE.SGPG(SEQ ID NO:9043)ATPE.SGPGX_nGTPE.TPGS(SEQ ID NO:9045), ATPE.SGPGX_nSGSE.TGTP(SEQ ID NO:9046), ATPE.SGPGX_nGTPE.GSAP(SEQ ID NO:9047), ATPE.SGPGX_nEPSE.SATP(SEQ ID NO:9048), GTSE.SATPX_nSGPE.SGPG(SEQ ID NO:9049), GTSE.SATPX_nATPE.SGPG(SEQ ID NO:9050), GTSE.SATPX_nGTSE.SATP(SEQ ID NO:9051), GTSE.SATPX_nTTPE.SGPG(SEQ ID NO:9052), GTSE.SATPX_nSTPE.SGPG(SEQ ID NO:9053), GTSE.SATPX_nGTPE.SGPG(SEQ ID NO:9054), GTSE.SATPX_nGTPE.TPGS(SEQ ID NO:9055), GTSE.SATPX_nSGSE.TGTP(SEQ ID NO:9056), GTSE.SATPX_nGTPE.GSAP(SEQ ID NO:9057), GTSE.SATPX_nEPSE.SATP(SEQ ID NO:9058), TTPE.SGPGX_nSGPE.SGPG(SEQ ID NO:9059), TTPE.SGPGX_nATPE.SGPG(SEQ ID NO:9060), TTPE.SGPGX_nGTSE.SATP(SEQ ID NO:9061), TTPE.SGPGX_nTTPE.SGPG(SEQ ID NO:9062), TTPE.SGPGX_nSTPE.SGPG(SEQ ID NO:9064), TTPE.SGPGX_nGTPE.SGPG(SEQ ID NO:9065), TTPE.SGPGX_nGTPE.TPGS(SEQ ID NO:9066), TTPE.SGPGX_nSGSE.TGTP(SEQ ID NO:9067), TTPE.SGPGX_nGTPE.GSAP(SEQ ID NO:9068), TTPE.SGPGX_nEPSE.SATP(SEQ ID NO:9069), STPE.SGPGX_nSGPE.SGPG(SEQ ID NO:9070), STPE.SGPGX_nATPE.SGPG(SEQ ID NO:9071), STPE.SGPGX_nGTSE.SATP(SEQ ID NO:9072), STPE.SGPGX_nTTPE.SGPG (SEQ ID NO:9073), STPE.SGPGX_nSTPE.SGPG(SEQ ID NO:9074), STPE.SGPGX_nGTPE.SGPG(SEQ ID NO:9076), STPE.SGPGX_nGTPE.TPGS(SEQ ID NO:9077), STPE.SGPGX_nSGSE.TGTP(SEQ ID NO:9078), STPE.SGPGX_nGTPE.GSAP(SEQ ID NO:9079), STPE.SGPGX_nEPSE.SATP(SEQ ID NO:9080), GTPE.SGPGX_nSGPE.SGPG(SEQ ID NO:9081), GTPE.SGPGX_nATPE.SGPG(SEQ ID NO:9082), GTPE.SGPGX_nGTSE.SATP(SEQ ID NO:9083), GTPE.SGPGX_nTTPE.SGPG(SEQ ID NO:9084), GTPE.SGPGX_nSTPE.SGPG(SEQ ID NO:9086), GTPE.SGPGX_nGTPE.SGPG(SEQ ID NO:9088), GTPE.SGPGX_nGTPE.TPGS(SEQ ID NO:9090), GTPE.SGPGX_nSGSE.TGTP(SEQ ID NO:9091), GTPE.SGPGX_nGTPE.GSAP(SEQ ID NO:9092), GTPE.SGPGX_nEPSE.SATP(SEQ ID NO:9093), GTPE.TPGSX_nSGPE.SGPG(SEQ ID NO:9094), GTPE.TPGSX_nATPE.SGPG(SEQ ID NO:9095), GTPE.TPGSX_nGTSE.SATP(SEQ ID NO:9096), GTPE.TPGSX_nTTPE.SGPG (SEQ ID NO:9097), GTPE.TPGSX_nSTPE.SGPG(SEQ ID NO:9098), GTPE.TPGSX_nGTPE.SGPG(SEQ ID NO:9099), GTPE.TPGSX_nGTPE.TPGS(SEQ ID NO:9100), GTPE.TPGSX_nSGSE.TGTP(SEQ ID NO:9101), GTPE.TPGSX_nGTPE.GSAP(SEQ ID NO:9103), GTPE.TPGSX_nEPSE.SATP(SEQ ID NO:9104), SGSE.TGTPX_nSGPE.SGPG(SEQ ID NO:9105), SGSE.TGTPX_nATPE.SGPG (SEQ ID NO:9106), SGSE.TGTPX_nGTSE.SATP(SEQ ID NO:9107), SGSE.TGTPX_nTTPE.SGPG (SEQ ID NO:9108), SGSE.TGTPX_nSTPE.SGPG(SEQ ID NO:9109), SGSE.TGTPX_nGTPE.SGPG(SEQ ID NO:9110), SGSE.TGTPX_nGTPE.TPGS(SEQ ID NO:9111), SGSE.TGTPX_nSGSE.TGTP(SEQ ID NO:9112), SGSE.TGTPX_nGTPE.GSAP(SEQ ID NO:9113), SGSE.TGTPX_nEPSE.SATP(SEQ ID NO:9114), GTPE.GSAPX_nSGPE.SGPG(SEQ ID NO:9115), GTPE.GSAPX_nATPE.SGPG(SEQ ID NO:9116), GTPE.GSAPX_nGTSE.SATP(SEQ ID NO:9117), GTPE.GSAPX_nTTPE.SGPG(SEQ ID NO:9118), GTPE.GSAPX_nSTPE.SGPG(SEQ ID NO:9119), GTPE.GSAPX_nGTPE.SGPG(SEQ ID NO:9120), GTPE.GSAPX_nGTPE.TPGS(SEQ ID NO:9121), GTPE.GSAPX_nSGSE.TGTP(SEQ ID NO:9122), GTPE.GSAPX_nGTPE.GSAP(SEQ ID NO:9123), GTPE.GSAPX_nEPSE.SATP(SEQ ID NO:9124), EPSE.SATPX_nSGPE.SGPG(SEQ ID NO:9126), EPSE.SATPX_nATPE.SGPG(SEQ ID NO:9127), EPSE.SATPX_nGTSE.SATP(SEQ ID NO:9128), EPSE.SATPX_nTTPE.SGPG(SEQ ID NO:9129), EPSE.SATPX_nSTPE.SGPG(SEQ ID NO:9130), EPSE.SATPX_nGTPE.SGPG(SEQ ID NO:9131), EPSE.SATPX_nGTPE.TPGS(SEQ ID NO:9132), EPSE.SATPX_nSGSE.TGTP(SEQ ID NO:9133), EPSE.SATPX_nGTPE.GSAP(SEQ ID NO:9134), or EPSE.SATPX_nEPSE.SATP(SEQ ID NO:9135), [111] wherein each “.” is a Glu-C cleavage site and n is any integer from 0 to 50. In some embodiments, the chimeric polypeptide comprises at least one of the following amino acid sequences:

(SEQ ID NO: 9038)

SGPE.SGPGXnATPE.SGPG,

(SEQ ID NO: 9040)

ATPE.SGPGXnGTSE.SATP,

(SEQ ID NO: 9041)

ATPE.SGPGXnTTPE.SGPG,

(SEQ ID NO: 9042)

ATPE.SGPGXnSTPE.SGPG,

(SEQ ID NO: 9039)

ATPE.SGPGXnATPE.SGPG,

(SEQ ID NO: 9044)

ATPE.SGPGXnGTPE.SGPG,

(SEQ ID NO: 9044)

ATPE.SGPGXnGTPE.SGPG,

(SEQ ID NO: 9039)

ATPE.SGPGXnATPE.SGPG,

(SEQ ID NO: 9089)

GTPE.SGPGXnGTPE.SGPG,

(SEQ ID NO: 9087)

GTPE.SGPGXnSTPE.SGPG,

(SEQ ID NO: 9085)

GTPE.SGPGXnTTPE.SGPG,

(SEQ ID NO: 9087)

GTPE.SGPGXnSTPE.SGPG,

(SEQ ID NO: 9102)

GTPE.TPGSXnSGSE.TGTP,

(SEQ ID NO: 9125)

GTPE.GSAPXnEPSE.SATP,

(SEQ ID NO: 9044)

ATPE.SGPGXnGTPE.SGPG,

(SEQ ID NO: 9044)

ATPE.SGPGXnGTPE.SGPG,

(SEQ ID NO: 9039)

ATPE.SGPGXnATPE.SGPG,

(SEQ ID NO: 9044)

ATPE.SGPGXnGTPE.SGPG,

(SEQ ID NO: 9063)

TTPE.SGPGXnTTPE.SGPG,

or

(SEQ ID NO: 9075)

STPE.SGPGXnSTPE.SGPG,

- wherein each “.” is a Glu-C cleavage site and n is any integer from 0 to 30.

In some embodiments, n is any integer from 1 to 20. In some embodiments, n is any integer from 5 to 15. In some embodiments, n is any integer from 3 to 7. In some embodiments, n is any integer from 5 to 10. In some embodiments, n is 9. In some embodiments, n is 4.

In some embodiments, X_n is

(SEQ ID NO: 9136)

PGTGTSAT,

(SEQ ID NO: 9137)

PGSGPGT,

(SEQ ID NO: 9138)

PGTTPGTT,

(SEQ ID NO: 9139)

PGTPPTST,

(SEQ ID NO: 9140)

PGTSPSAT,

(SEQ ID NO: 9141)

PGTGSAGT,

(SEQ ID NO: 9142)

PGTGGAGT,

(SEQ ID NO: 9143)

PGTSPGAT,

(SEQ ID NO: 9144)

PGTSGSGT,

(SEQ ID NO: 9145)

PGTSSAST,

(SEQ ID NO: 9146)

PGTGAGTT,

(SEQ ID NO: 9147)

PGTGSTST,

(SEQ ID NO: 9148)

GSEPATSG,

(SEQ ID NO: 9149)

APGTSTEP,

(SEQ ID NO: 9150)

PGTAGSGT,

(SEQ ID NO: 9151)

PGTSSGGT,

(SEQ ID NO: 9152)

PGTAGPAT,

(SEQ ID NO: 9153)

PGTPGTGT,

(SEQ ID NO: 9154)

PGTGGPTT,

or

(SEQ ID NO: 9155)

PGTGSGST.

In some embodiments, X_n is

(SEQ ID NO: 9156)

TGTS,

SGP,

(SEQ ID NO: 9157)

TTPG,

(SEQ ID NO: 9158)

TPPT,

(SEQ ID NO: 9159)

TSPS,

(SEQ ID NO: 9160)

TGSA,

(SEQ ID NO: 9161)

TGGA,

(SEQ ID NO: 9162)

TSPG,

(SEQ ID NO: 9163)

TSGS,

(SEQ ID NO: 9164)

TSSA,

(SEQ ID NO: 9165)

TGAG,

(SEQ ID NO: 9166)

TGST,

(SEQ ID NO: 9167)

EPAT,

(SEQ ID NO: 9168)

GTST,

(SEQ ID NO: 9169)

TAGS,

(SEQ ID NO: 9170)

TSSG,

(SEQ ID NO: 9171)

TAGP,

(SEQ ID NO: 9172)

TPGT,

(SEQ ID NO: 9173)

TGGP,

or

(SEQ ID NO: 9174)

TGSG.

In some embodiments, neither the N-terminal amino acid nor the C-terminal acid of the chimeric polypeptide is included in a barcode fragment.

In some embodiments, the chimeric polypeptide comprises an ELNN with a non-overlapping sequence motif that occurs only once within the ELNN, wherein the ELNN further comprises a barcode fragment that includes at least part of the non-overlapping sequence motif that occurs only once within the ELNN.

In some embodiments, the chimeric polypeptide comprises a first ELNN with a first barcode fragment and a second ELNN with a second barcode fragment, wherein neither the first barcode fragment nor the second barcode fragment includes a glutamate that is immediately adjacent to another glutamate, if present, in the ELNN that contains the barcode fragment.

In some embodiments, at least one of the barcode fragments comprises a glutamate at the C-terminus thereof.

In some embodiments, at least one of the barcode fragments has an N-terminal amino acid that is immediately preceded by a glutamate in the chimeric polypeptide.

In some embodiments, the glutamate that precedes the N-terminal amino acid of the barcode fragment is not immediately adjacent to another glutamate.

In some embodiments, at least one of the barcode fragments does not include a second glutamate at a position other than the C-terminus of the barcode fragment unless the second glutamate is immediately followed by a proline.

In some embodiments, the chimeric polypeptide comprises a single polypeptide chain, wherein the chimeric polypeptide comprises a barcode fragment that is at a position within the polypeptide chain that is from 10 to 200 amino acids or from 10 to 125 amino acids from the N-terminus or the C-terminus of the chimeric polypeptide. In some embodiments, the first ELNN is at the N-terminal side of the bispecific antibody domain, and wherein the first barcode fragment is positioned within 200, 150, 100, or 50 amino acids of the N-terminus of the chimeric polypeptide. In some embodiments, the second ELNN is at the C-terminal side of the bispecific antibody domain, and wherein the second barcode fragment is positioned within 200, 150, 100, or 50 amino acids of the C-terminus of the chimeric polypeptide.

In some embodiments, at least one of the barcode fragments is at least 4 amino acids in length.

In some embodiments, at least one of the barcode fragments is from 4 to 20, from 5 to 15, from 6 to 12, or from 7 to 10 amino acids in length.

In some embodiments, each mask polypeptide comprises one barcode fragment that is listed in Table 2 or disclosed in Table 3a.

In some embodiments, the chimeric polypeptide comprises a barcode fragment comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to SGPGSGPGTSE(SEQ ID NO:78) or SGPGTSPSATPE(SEQ ID NO:79).

In some embodiments, the chimeric polypeptide comprises one barcode fragment comprising an amino acid sequence that is at least 95% identical to SGPGSGPGTSE(SEQ ID NO:78) and one barcode fragment comprising an amino acid sequence that is at least 95% identical to SGPGTSPSATPE(SEQ ID NO:79).

In some embodiments, the barcode fragment consists of A, E, G, S, P, and/or T residues.

In some embodiments, the barcode fragment is part of a mask peptide.

In some embodiments, the mask peptide is the first ELNN or the second ELNN.

In some embodiments, the chimeric polypeptide comprises an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to a sequence listed in Table D (SEQ ID NOs: 1000-1009). In some embodiments, the chimeric polypeptide comprises an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to SEQ ID NO: 1000. In some embodiments, the chimeric polypeptide comprises an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to SEQ ID NO: 1001. In some embodiments, the chimeric polypeptide comprises an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to SEQ ID NO: 1002. In some embodiments, the chimeric polypeptide comprises an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to SEQ ID NO: 1003. In some embodiments, the chimeric polypeptide comprises an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to SEQ ID NO: 1004. In some embodiments, the chimeric polypeptide comprises an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to SEQ ID NO: 1005. In some embodiments, the chimeric polypeptide comprises an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to SEQ ID NO: 1006. In some embodiments, the chimeric polypeptide comprises an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to SEQ ID NO: 1007. In some embodiments, the chimeric polypeptide comprises an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to SEQ ID NO: 1008. In some embodiments, the chimeric polypeptide comprises an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to SEQ ID NO: 1009.

In some embodiments, the chimeric polypeptide comprises an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to: ASSATPESGPGTSTEPSEGSAPGTSESATPESGPGSGPGTSESATPGTSESATPE SGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSE SATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTE EGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPAT SGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPG SEPATSGSETPGTSESATPEAGRSASHTPAGLTGPGTSESATPESQVQLVESGGG VVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVGAMSWSGSNRKVSDS VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAASNKEYGRTWYDFNESDYWG QGTQVTVSSGGGGSGGGSELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYAN WVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCA LWYPNLWVFGGGTKLTVLSESATPESGPGTSPGATPESGPGTSESATPEVQLVES GGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKRNNYATY YADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWG QGTLVTVSSGTATPESGPGEAGRSASHTPAGLTGPATPESGPGTSESATPESGPG SPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSE GSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTS TEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSE TPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSES ATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGP GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATS GSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGS EPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEG SAPGTSESATPESGPGTSESATPESGPGTSPSATPESGPGSEPATSGSETPGSEP ATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSET PGTSESAGEPEA (SEQ ID NO: 1000). In some embodiments, the chimeric polypeptide comprises an amino acid sequence that has at least 85% identity to SEQ ID NO: 1000. In some embodiments, the chimeric polypeptide comprises an amino acid sequence that has at least 90% identity to SEQ ID NO: 1000. In some embodiments, the chimeric polypeptide comprises an amino acid sequence that has at least 91% identity to SEQ ID NO: 1000. In some embodiments, the chimeric polypeptide comprises an amino acid sequence that has at least 92% identity to SEQ ID NO: 1000. In some embodiments, the chimeric polypeptide comprises an amino acid sequence that has at least 93% identity to SEQ ID NO: 1000. In some embodiments, the chimeric polypeptide comprises an amino acid sequence that has at least 94% identity to SEQ ID NO: 1000. In some embodiments, the chimeric polypeptide comprises an amino acid sequence that has at least 95% identity to SEQ ID NO: 1000. In some embodiments, the chimeric polypeptide comprises an amino acid sequence that has at least 96% identity to SEQ ID NO: 1000. In some embodiments, the chimeric polypeptide comprises an amino acid sequence that has at least 97% identity to SEQ ID NO: 1000. In some embodiments, the chimeric polypeptide comprises an amino acid sequence that has at least 98% identity to SEQ ID NO: 1000. In some embodiments, the chimeric polypeptide comprises an amino acid sequence that has at least 99% identity to SEQ ID NO: 1000. In some embodiments, the chimeric polypeptide comprises an amino acid sequence that has 100% identity to SEQ ID NO: 1000.

In some embodiments, the chimeric polypeptide comprises the following amino acid sequence:

(SEQ ID NO: 1000)

ASSATPESGPGTSTEPSEGSAPGTSESATPESGPGSGPGTSESATPGTS

ESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPA

GSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAG

SPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESA

TPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSP

TSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATP

EAGRSASHTPAGLTGPGTSESATPESQVQLVESGGGWVQPGRSLRLSCA

ASGRTFGIYVWGWFRQAPGKEREFVGAMSWSGSNRKVSDSVKGRFTISR

DNSKNTLYLQMNSLRAEDTAVYYCAASNKEYGRTWYDFNESDYWGQGTQ

VTVSSGGGGSGGGSELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYA

NWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPED

EAVYYCALWYPNLWVFGGGTKLTVLSESATPESGPGTSPGATPESGPGT

SESATPEVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGK

GLEWVGRIRTKRNNYATYYADSVKGRFTISRDDSKNTVYLQMNSLKTED

TAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSSGTATPESGPGEAGRSA

SHTPAGLTGPATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATP

ESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEG

SAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS

APGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSET

PGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP

GTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEG

TSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGT

SESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSP

AGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPA

GSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSES

ATPESGPGTSPSATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGS

PTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAG

EPEA.

Certain aspects of the present disclosure are directed to a pharmaceutical composition comprising the chimeric polypeptide described herein and at least one pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is in a liquid form or is frozen. In some embodiments, the pharmaceutical composition is formulated as a lyophilized powder or cake to be reconstituted prior to administration.

Certain aspects of the present disclosure are directed to an injection device comprising the pharmaceutical composition described herein. In some embodiments, injection device comprises a syringe.

Certain aspects of the present disclosure are directed to a polynucleotide sequence encoding the chimeric polypeptide described herein.

Certain aspects of the present disclosure are directed to an expression vector comprising the polynucleotide described herein.

Certain aspects of the present disclosure are directed to a host cell comprising the expression vector described herein.

Certain aspects of the present disclosure are directed to a method of producing the chimeric polypeptide described herein. In some embodiments, the method further comprises isolating the chimeric polypeptide from a host cell.

Certain aspects of the present disclosure are directed to a method of treating cancer in a subject in need thereof, the method comprising administering an effective amount of the chimeric polypeptide described herein to the subject.

In some embodiments, the cancer comprises a solid tumor. In some embodiments, the cancer is a carcinoma. In some embodiments, the cancer is prostate cancer. In some embodiments, the prostate cancer is metastatic prostate cancer. In some embodiments, the prostate cancer is androgen-independent. In some embodiments, the prostate cancer is non-metastatic castration-resistant prostate cancer (nmCRPC). In some embodiments, the prostate cancer is metastatic castration-resistant prostate cancer (mCRPC).

In some embodiments, the method further comprises administering docetaxel to the subject.

In some embodiments, the method further comprises administering a checkpoint inhibitor to the subject. In some embodiments, the checkpoint inhibitor is a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor. In some embodiments, the checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody. In some embodiments, the checkpoint inhibitor is pembrolizumab or cemiplimab.

Certain aspects of the present disclosure are directed to a linker polypeptide comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). Certain aspects of the present disclosure are directed to a linker polypeptide comprising an amino acid sequence that has at least 85% identity to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). Certain aspects of the present disclosure are directed to a linker polypeptide comprising an amino acid sequence that has at least 90% identity to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). Certain aspects of the present disclosure are directed to a linker polypeptide comprising an amino acid sequence that has at least 91% identity to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). Certain aspects of the present disclosure are directed to a linker polypeptide comprising an amino acid sequence that has at least 92% identity to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). Certain aspects of the present disclosure are directed to a linker polypeptide comprising an amino acid sequence that has at least 93% identity to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). Certain aspects of the present disclosure are directed to a linker polypeptide comprising an amino acid sequence that has at least 94% identity to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). Certain aspects of the present disclosure are directed to a linker polypeptide comprising an amino acid sequence that has at least 95% identity to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). Certain aspects of the present disclosure are directed to a linker polypeptide comprising an amino acid sequence that has at least 96% identity to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). Certain aspects of the present disclosure are directed to a linker polypeptide comprising an amino acid sequence that has at least 97% identity to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). Certain aspects of the present disclosure are directed to a linker polypeptide comprising an amino acid sequence that has at least 98% identity to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). Certain aspects of the present disclosure are directed to a linker polypeptide comprising an amino acid sequence that has at least 99% identity to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). Certain aspects of the present disclosure are directed to a linker polypeptide comprising an amino acid sequence that has 100% identity to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81).

In some embodiments, the linker polypeptide is cleavable by a non-mammalian protease. In some embodiments, the non-mammalian protease is Glu-C.

In some embodiments, the linker polypeptide connects a first polypeptide moiety to a second polypeptide moiety. In some embodiments, the first polypeptide moiety is a VL domain and the second polypeptide moiety is a VH domain.

Certain aspects of the present disclosure are directed to an antigen binding polypeptide comprising a VL domain and a VH domain, wherein the VL domain is linked to the VH domain by a linker polypeptide comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81).

In some embodiments, the linker polypeptide is cleavable by a non-mammalian protease. In some embodiments, the non-mammalian protease is Glu-C.

In some embodiments, the antigen binding polypeptide is an scFv.

In some embodiments, the antigen is CD3. In some embodiments, the antigen is CD3 epsilon.

In some embodiments, the VL domain is N-terminal to the VH domain. In some embodiments, the VH domain is N-terminal to the VL domain.

Certain aspects of the present disclosure are directed to a pharmaceutical composition comprising the linker polypeptide described herein or the antigen binding polypeptide described herein, and at least one pharmaceutically acceptable excipient.

In some embodiments, the pharmaceutical composition is in a liquid form or is frozen. In some embodiments, the pharmaceutical composition is formulated as a lyophilized powder or cake to be reconstituted prior to administration.

Certain aspects of the present disclosure are directed to an injection device comprising the pharmaceutical composition described herein. In some embodiments, the injection device comprises a syringe.

Certain aspects of the present disclosure are directed to a polynucleotide sequence encoding the linker described herein or the antigen binding polypeptide described herein.

Certain aspects of the present disclosure are directed to an expression vector comprising the polynucleotide sequence described herein.

Certain aspects of the present disclosure are directed to a host cell comprising the expression vector described herein.

Certain aspects of the present disclosure are directed to a method of producing the linker described herein or the antigen binding polypeptide described herein. In some embodiments, the method further comprises isolating the linker or antigen binding polypeptide from a host cell.

Certain aspects of the present disclosure are directed to an isolated polypeptide comprising a protease-cleavable amino acid sequence comprising the sequence: EAGRSAXHTPAGLTGP (SEQ ID NO: 7627), wherein X is any amino acid other than N. In some embodiments, X is S.

In some embodiments, the isolated polypeptide is not cleavable by legumain. In some embodiments, the isolated polypeptide is not cleavable by legumain in human blood, plasma, or serum. In some embodiments, the isolated polypeptide is not cleavable upon incubation with about 1 nM or less legumain for about 20 hours. In some embodiments, the isolated polypeptide is not cleavable upon incubation with about 1 nM or less legumain for about 20 hours in human blood, plasma, or serum.

In some embodiments, legumain cleaves the isolated polypeptide in human plasma at a rate that is less than about 50% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain. In some embodiments, legumain cleaves the isolated polypeptide in human plasma at a rate that is less than about 25% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain. In some embodiments, legumain cleaves the isolated polypeptide in human plasma at a rate that is less than about 10% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain. In some embodiments, legumain cleaves the isolated polypeptide in human plasma at a rate that is less than about 5% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain. In some embodiments, legumain cleaves the isolated polypeptide in human plasma at a rate that is less than about 2.5% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain.

Certain aspects of the present disclosure are directed to a pharmaceutical composition comprising the isolated polypeptide described herein, and at least one pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is in a liquid form or is frozen. In some embodiments, the pharmaceutical composition is formulated as a lyophilized powder or cake to be reconstituted prior to administration.

Certain aspects of the present disclosure are directed to a polynucleotide sequence encoding the isolated polypeptide described herein.

Certain aspects of the present disclosure are directed to an expression vector comprising the polynucleotide sequence described herein.

Certain aspects of the present disclosure are directed to a host cell comprising the expression vector described herein.

Certain aspects of the present disclosure are directed to a method of producing the isolated polypeptide described herein. In some embodiments, the method further comprises isolating the isolated polypeptide from a host cell.

Certain aspects of the present disclosure are directed to a fusion protein comprising a protease-cleavable amino acid sequence comprising the sequence: EAGRSAXHTPAGLTGP (SEQ ID NO: 7627), wherein X is any amino acid other than N, wherein the protease-cleavable amino acid sequence links a first polypeptide moiety to a second polypeptide moiety. In some embodiments, X is S.

In some embodiments, the fusion protein is not cleavable by legumain. In some embodiments, the fusion protein is not cleavable by legumain in human blood, plasma, or serum. In some embodiments, the fusion protein is not cleavable upon incubation with about 1 nM or less legumain for about 20 hours. In some embodiments, the fusion protein is not cleavable upon incubation with about 1 nM or less legumain for about 20 hours in human blood, plasma, or serum.

In some embodiments, legumain cleaves the protease-cleavable amino acid sequence in human plasma at a rate that is less than about 50% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain. In some embodiments, legumain cleaves the protease-cleavable amino acid sequence in human plasma at a rate that is less than about 25% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain. In some embodiments, legumain cleaves the protease-cleavable amino acid sequence in human plasma at a rate that is less than about 10% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain. In some embodiments, legumain cleaves the protease-cleavable amino acid sequence in human plasma at a rate that is less than about 5% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain. In some embodiments, legumain cleaves the protease-cleavable amino acid sequence in human plasma at a rate that is less than about 2.5% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain.

In some embodiments, the first polypeptide moiety comprises an antigen-binding domain and the second polypeptide moiety comprises a masking polypeptide.

In some embodiments, the first polypeptide moiety comprises an antigen-binding domain and the second polypeptide moiety is a cytokine, an enzyme, a hormone, a growth factor, a chemotherapeutic polypeptide, an antiviral polypeptide, or a toxin.

In some embodiments, the first polypeptide moiety is a cytokine, an enzyme, a hormone, a growth factor, a chemotherapeutic polypeptide, an antiviral polypeptide, or a toxin and the second polypeptide moiety is a masking polypeptide.

In some embodiments, the masking polypeptide comprises an ELNN.

In some embodiments, the fusion protein comprises a single polypeptide chain, which comprises, in the N terminal to C terminal direction, the first polypeptide then the protease-cleavable amino acid sequence then the second polypeptide moiety. In some embodiments, the fusion protein comprises a single polypeptide chain, which comprises, in the N terminal to C terminal direction, the second polypeptide then the protease-cleavable amino acid sequence then the first polypeptide moiety.

Certain aspects of the present disclosure are directed to a pharmaceutical composition comprising the fusion protein described herein, and at least one pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is in a liquid form or is frozen. In some embodiments, the pharmaceutical composition is formulated as a lyophilized powder or cake to be reconstituted prior to administration.

Certain aspects of the present disclosure are directed to a polynucleotide sequence encoding the fusion protein described herein.

Certain aspects of the present disclosure are directed to an expression vector comprising the polynucleotide sequence described herein.

Certain aspects of the present disclosure are directed to a host cell comprising the expression vector described herein.

Certain aspects of the present disclosure are directed to a method of producing the fusion protein described herein. In some embodiments, the method further comprises isolating the fusion protein from a host cell.

Certain aspects of the present disclosure are directed to an ELNN polypeptide comprising the following amino acid sequence:

(SEQ ID NO: 8021)

ASSATPESGPGTSTEPSEGSAPGTSESATPESGPGSGPGTSESATPGTS

ESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPA

GSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAG

SPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESA

TPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSP

TSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAT

P.

Certain aspects of the present disclosure are directed to an ELNN polypeptide comprising the following amino acid sequence:

(SEQ ID NO: 8022)

ATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPAT

SGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPS

EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPT

STEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPE

SGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPES

GPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESG

PGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGP

GSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEG

TSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGS

PAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTS

PSATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTST

EPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAGEPEA.

Certain aspects of the present disclosure are directed to a fusion protein comprising the ELNN polypeptide described herein.

Certain aspects of the present disclosure are directed to a pharmaceutical composition comprising the ELNN polypeptide described herein, or the fusion protein described herein, and at least one pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is in a liquid form or is frozen. In some embodiments, the pharmaceutical composition is formulated as a lyophilized powder or cake to be reconstituted prior to administration.

Certain aspects of the present disclosure are directed to a polynucleotide sequence encoding the ELNN polypeptide described herein, or the fusion protein described herein.

Certain aspects of the present disclosure are directed to an expression vector comprising the polynucleotide sequence described herein.

Certain aspects of the present disclosure are directed to a host cell comprising the expression vector described herein.

Certain aspects of the present disclosure are directed to a method of producing the ELNN polypeptide described herein, or the fusion protein described herein.

In some embodiments, the method of claim 288, further comprising isolating the ELNN polypeptide or the fusion protein, from a host cell.

Certain aspects of the present disclosure are directed to a barcode fragment comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to

(SEQ ID NO: 1010)

SGPGTGTSATPE,

(SEQ ID NO: 78)

SGPGSGPGTSE,

(SEQ ID NO: 1011)

SGPGTTPGTTPE,

(SEQ ID NO: 1012)

SGPGTPPTSTPE,

(SEQ ID NO: 79)

SGPGTSPSATPE,

(SEQ ID NO: 1013)

SGPGTGSAGTPE,

(SEQ ID NO: 1014)

SGPGTGGAGTPE,

(SEQ ID NO: 1015)

SGPGTSPGATPE,

(SEQ ID NO: 1016)

SGPGTSGSGTPE,

(SEQ ID NO: 1017)

SGPGTSSASTPE,

(SEQ ID NO: 1018)

SGPGTGAGTTPE,

(SEQ ID NO: 1019)

SGPGTGSTSTPE,

(SEQ ID NO: 1020)

TPGSEPATSGSE,

(SEQ ID NO: 1021)

GSAPGTSTEPSE,

(SEQ ID NO: 1022)

SGPGTAGSGTPE,

(SEQ ID NO: 1023)

SGPGTSSGGTPE,

(SEQ ID NO: 1024)

SGPGTAGPATPE,

(SEQ ID NO: 1025)

SGPGTPGTGTPE,

(SEQ ID NO: 1026)

SGPGTGGPTTPE,

or

(SEQ ID NO: 1027)

SGPGTGSGSTPE.

In some embodiments, the barcode fragment comprises the amino acid sequence: SGPGSGPGTSE(SEQ ID NO:78). In some embodiments, the barcode fragment comprises the amino acid sequence: SGPGTSPSATPE(SEQ ID NO:79).

Certain aspects of the present disclosure are directed to a fusion protein comprising the barcode fragment described herein.

Certain aspects of the present disclosure are directed to a fusion protein comprising a Glu-C cleavage site comprising one of the following amino acid sequences: ATPESGPG(SEQ ID NO:9020), SGSETPGT(SEQ ID NO:9021), and GTSESATP(SEQ ID NO:9022).

Certain aspects of the present disclosure are directed to a fusion protein comprising at least one of the following amino acid sequences: SGPE.SGPGX_nSGPE.SGPG(SEQ ID NO:9023), SGPE.SGPGX_nATPE.SGPG(SEQ ID NO:9024), SGPE.SGPGX_nGTSE.SATP(SEQ ID NO:9025), SGPE.SGPGX_nTTPE.SGPG(SEQ ID NO:9026), SGPE.SGPGX_nSTPE.SGPG(SEQ ID NO:9027), SGPE.SGPGX_nGTPE.SGPG(SEQ ID NO:9028), SGPE.SGPGX_nGTPE.TPGS(SEQ ID NO:9029), SGPE.SGPGX_nGTPE.TPGS(SEQ ID NO:9029), SGPE.SGPGX_nSGSE.TGTP(SEQ ID NO:9030), SGPE.SGPGX_nGTPE.GSAP(SEQ ID NO:9031), SGPE.SGPGX_nEPSE.SATP(SEQ ID NO:9032), ATPE.SGPGX_nSGPE.SGPG(SEQ ID NO:9033), ATPE.SGPGX_nATPE.SGPG(SEQ ID NO:9034), ATPE.SGPGX_nGTSE.SATP(SEQ ID NO:9035), ATPE.SGPGX_nTTPE.SGPG(SEQ ID NO:9036), ATPE.SGPGX_nSTPE.SGPG(SEQ ID NO:9037), ATPE.SGPGX_nGTPE.SGPG(SEQ ID NO:9043), ATPE.SGPGX_nGTPE.TPGS(SEQ ID NO:9045), ATPE.SGPGX_nSGSE.TGTP(SEQ ID NO:9046), ATPE.SGPGX_nGTPE.GSAP(SEQ ID NO:9047), ATPE.SGPGX_nEPSE.SATP(SEQ ID NO:9048), GTSE.SATPX_nSGPE.SGPG(SEQ ID NO:9049), GTSE.SATPX_nATPE.SGPG(SEQ ID NO:9050), GTSE.SATPX_nGTSE.SATP(SEQ ID NO:9051), GTSE.SATPX_nTTPE.SGPG(SEQ ID NO:9052), GTSE.SATPX_nSTPE.SGPG(SEQ ID NO:9053), GTSE.SATPX_nGTPE.SGPG(SEQ ID NO:9054), GTSE.SATPX_nGTPE.TPGS(SEQ ID NO:9055), GTSE.SATPX_nSGSE.TGTP(SEQ ID NO:9056), GTSE.SATPX_nGTPE.GSAP(SEQ ID NO:9057), GTSE.SATPX_nEPSE.SATP(SEQ ID NO:9058), TTPE.SGPGX_nSGPE.SGPG(SEQ ID NO:9059), TTPE.SGPGX_nATPE.SGPG(SEQ ID NO:9060), TTPE.SGPGX_nGTSE.SATP(SEQ ID NO:9061), TTPE.SGPGX_nTTPE.SGPG(SEQ ID NO:9062), TTPE.SGPGX_nSTPE.SGPG(SEQ ID NO:9064), TTPE.SGPGX_nGTPE.SGPG(SEQ ID NO:9065), TTPE.SGPGX_nGTPE.TPGS(SEQ ID NO:9066), TTPE.SGPGX_nSGSE.TGTP(SEQ ID NO:9067), TTPE.SGPGX_nGTPE.GSAP(SEQ ID NO:9068), TTPE.SGPGX_nEPSE.SATP(SEQ ID NO:9069), STPE.SGPGX_nSGPE.SGPG(SEQ ID NO:9070), STPE.SGPGX_nATPE.SGPG(SEQ ID NO:9071), STPE.SGPGX_nGTSE.SATP(SEQ ID NO:9072), STPE.SGPGX_nTTPE.SGPG(SEQ ID NO:9073), STPE.SGPGX_nSTPE.SGPG(SEQ ID NO:9074), STPE.SGPGX_nGTPE.SGPG(SEQ ID NO:9076), STPE.SGPGX_nGTPE.TPGS(SEQ ID NO:9077), STPE.SGPGX_nSGSE.TGTP(SEQ ID NO:9078), STPE.SGPGX_nGTPE.GSAP(SEQ ID NO:9079), STPE.SGPGX_nEPSE.SATP(SEQ ID NO:9175), GTPE.SGPGX_nSGPE.SGPG(SEQ ID NO:9081), GTPE.SGPGX_nATPE.SGPG(SEQ ID NO:9082), GTPE.SGPGX_nGTSE.SATP(SEQ ID NO:9083), GTPE.SGPGX_nTTPE.SGPG(SEQ ID NO:9084), GTPE.SGPGX_nSTPE.SGPG(SEQ ID NO:9086), GTPE.SGPGX_nGTPE.SGPG(SEQ ID NO:9088), GTPE.SGPGX_nGTPE.TPGS(SEQ ID NO:9090), GTPE.SGPGX_nSGSE.TGTP(SEQ ID NO:9091), GTPE.SGPGX_nGTPE.GSAP(SEQ ID NO:9092), GTPE.SGPGX_nEPSE.SATP(SEQ ID NO:9093), GTPE.TPGSX_nSGPE.SGPG(SEQ ID NO:9094), GTPE.TPGSX_nATPE.SGPG(SEQ ID NO:9095), GTPE.TPGSX_nGTSE.SATP(SEQ ID NO:9096), GTPE.TPGSX_nTTPE.SGPG(SEQ ID NO:9097), GTPE.TPGSX_nSTPE.SGPG(SEQ ID NO:9098), GTPE.TPGSX_nGTPE.SGPG(SEQ ID NO:9099), GTPE.TPGSX_nGTPE.TPGS(SEQ ID NO:9100), GTPE.TPGSX_nSGSE.TGTP(SEQ ID NO:9101), GTPE.TPGSX_nGTPE.GSAP(SEQ ID NO:9103), GTPE.TPGSX_nEPSE.SATP(SEQ ID NO:9104), SGSE.TGTPX_nSGPE.SGPG(SEQ ID NO:9105), SGSE.TGTPX_nATPE.SGPG(SEQ ID NO:9106), SGSE.TGTPX_nGTSE.SATP(SEQ ID NO:9107), SGSE.TGTPX_nTTPE.SGPG(SEQ ID NO:9108), SGSE.TGTPX_nSTPE.SGPG(SEQ ID NO:9109), SGSE.TGTPX_nGTPE.SGPG(SEQ ID NO:9110), SGSE.TGTPX_nGTPE.TPGS(SEQ ID NO:9111), SGSE.TGTPX_nSGSE.TGTP(SEQ ID NO:9112), SGSE.TGTPX_nGTPE.GSAP(SEQ ID NO:9113), SGSE.TGTPX_nEPSE.SATP(SEQ ID NO:9114), GTPE.GSAPX_nSGPE.SGPG(SEQ ID NO:9115), GTPE.GSAPX_nATPE.SGPG(SEQ ID NO:9116), GTPE.GSAPX_nGTSE.SATP(SEQ ID NO:9117), GTPE.GSAPX_nTTPE.SGPG(SEQ ID NO:9118), GTPE.GSAPX_nSTPE.SGPG(SEQ ID NO:9119), GTPE.GSAPX_nGTPE.SGPG(SEQ ID NO:9120), GTPE.GSAPX_nGTPE.TPGS(SEQ ID NO:9121), GTPE.GSAPX_nSGSE.TGTP(SEQ ID NO:9122), GTPE.GSAPX_nGTPE.GSAP(SEQ ID NO:9123), GTPE.GSAPX_nEPSE.SATP(SEQ ID NO:9124), EPSE.SATPX_nSGPE.SGPG(SEQ ID NO:9126), EPSE.SATPX_nATPE.SGPG(SEQ ID NO:9127), EPSE.SATPX_nGTSE.SATP(SEQ ID NO:9128), EPSE.SATPX_nTTPE.SGPG(SEQ ID NO:9129), EPSE.SATPX_nSTPE.SGPG(SEQ ID NO:9130), EPSE.SATPX_nGTPE.SGPG(SEQ ID NO:9131), EPSE.SATPX_nGTPE.TPGS(SEQ ID NO:9132), EPSE.SATPX_nSGSE.TGTP(SEQ ID NO:9133), EPSE.SATPX_nGTPE.GSAP(SEQ ID NO:9134), or EPSE.SATPX_nEPSE.SATP(SEQ ID NO:9135), wherein each “.” is a Glu-C cleavage site and n is any integer from 0 to 50.

In some embodiments, the fusion protein comprises at least one of the following amino acid sequences: SGPE.SGPGX_nATPE.SGPG(SEQ ID NO:9038), ATPE.SGPGX_nGTSE.SATP(SEQ ID NO:9040), ATPE.SGPGX_nTTPE.SGPG(SEQ ID NO:9041), ATPE.SGPGX_nSTPE.SGPG(SEQ ID NO:9042), ATPE.SGPGX_nATPE.SGPG(SEQ ID NO:9039), ATPE.SGPGX_nGTPE.SGPG(SEQ ID NO:9044), ATPE.SGPGX_nGTPE.SGPG(SEQ ID NO:9044), ATPE.SGPGX_nATPE.SGPG(SEQ ID NO:9039), GTPE.SGPGX_nGTPE.SGPG(SEQ ID NO:9089), GTPE.SGPGX_nSTPE.SGPG(SEQ ID NO:9087), GTPE.SGPGX_nTTPE.SGPG(SEQ ID NO:9085), GTPE.SGPGX_nSTPE.SGPG(SEQ ID NO:9087), GTPE.TPGSX_nSGSE.TGTP(SEQ ID NO:9102), GTPE.GSAPX_nEPSE.SATP(SEQ ID NO:9125), ATPE.SGPGX_nGTPE.SGPG(SEQ ID NO:9044), ATPE.SGPGX_nGTPE.SGPG(SEQ ID NO:9044), ATPE.SGPGX_nATPE.SGPG(SEQ ID NO:9039), ATPE.SGPGX_nGTPE.SGPG(SEQ ID NO:9044), TTPE.SGPGX_nTTPE.SGPG(SEQ ID NO:9063), or STPE.SGPGX_nSTPE.SGPG(SEQ ID NO:9075), wherein each “.” is a Glu-C cleavage site and n is any integer from 0 to 30.

Certain aspects of the present disclosure are directed to a pharmaceutical composition comprising the barcode fragment described herein, or the fusion protein described herein, and at least one pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is in a liquid form or is frozen. In some embodiments, the pharmaceutical composition is formulated as a lyophilized powder or cake to be reconstituted prior to administration.

Certain aspects of the present disclosure are directed to a polynucleotide sequence encoding the barcode fragment described herein, or the fusion protein described herein.

Certain aspects of the present disclosure are directed to an expression vector comprising the polynucleotide sequence described herein.

Certain aspects of the present disclosure are directed to a host cell comprising the expression vector described herein.

Certain aspects of the present disclosure are directed to a method of producing the barcode fragment described herein, or the fusion protein described herein. In some embodiments, the method further comprises isolating the barcode fragment or the fusion protein from a host cell.

Certain aspects of the present disclosure are directed to an antibody or an antigen-binding fragment thereof that specifically binds PSMA, comprising a VHH domain or a fragment thereof comprising three VHH CDRs, wherein the three VHH CDRs comprise the CDR1, CDR2, and CDR3 from the following amino acid sequence.

(SEQ ID NO: 549)

QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVG

AMSWSGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNKEYGRTWYDFNESDYWGQGTQVTVSS.

Certain aspects of the present disclosure are directed to an antibody or an antigen-binding fragment thereof that specifically binds PSMA, comprising the following CDRs: a VHH CDR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GRTFGIYVWG(SEQ ID NO:9003); a VHH CDR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to AMSWSGSNRKVSDSVKG(SEQ ID NO:9004); and a VHH CDR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to AASNKEYGRTWYDFNESDY(SEQ ID NO:9005).

In some embodiments, the antibody or fragment comprises one or more of the following FRs: a VHH FR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to QVQLVESGGGVVQPGRSLRLSCAAS(SEQ ID NO:9011); a VHH FR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to WFRQAPGKEREFVG(SEQ ID NO:9012); a VHH FR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to RFTISRDNSKNTLYLQMNSLRAEDTAVYYC(SEQ ID NO:9013); and a VHH FR4 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to WGQGTQVTVSS(SEQ ID NO:9014).

Certain aspects of the present disclosure are directed to an antibody or an antigen-binding fragment thereof that specifically binds PSMA, comprising the following CDRs: a VHH CDR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GRTFGIYVWG(SEQ ID NO:9003); a VHH CDR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to AMSWSGSNRK(SEQ ID NO:9015); and a VHH CDR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to AASNKEYGRTWYDFNESDY(SEQ ID NO:9005).

In some embodiments, the antibody or fragment comprises one or more of the following FRs: a VHH FR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to QVQLVESGGGVVQPGRSLRLSCAAS(SEQ ID NO:9011); a VHH FR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to WFRQAPGKEREFVG(SEQ ID NO:9012); a VHH FR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to VSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC(SEQ ID NO:9016); and a VHH FR4 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to WGQGTQVTVSS(SEQ ID NO:9014).

(SEQ ID NO: 549)

QVQLVESGGGWVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVG

AMSWSGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNKEYGRTWYDFNESDYWGQGTQVTVSS.

In some embodiments, the antibody or fragment is an isolated antibody or fragment thereof.

Certain aspects of the present disclosure are directed to an antibody or an antigen-binding fragment thereof that specifically binds PSMA, comprising a VHH comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to: QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVX₁₇GWFRQAPGKEREFVGAX₁₈S WSGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYX₁₉CX₂₀X₂₁SNKX₂₂Y GRTWYDFNESDYWGQGTQVTVSS(SEQ ID NO:9017), wherein X₁₇, X₁₈, X₁₉, X₂₀, X₂₁, and X₆each, individually, correspond to any naturally occurring amino acid. In some embodiments, X₁₇corresponds to M or W, X₁₈corresponds to M or I, X₁₉corresponds to F or Y, X₂₀corresponds to A or G, X₂₁corresponds to A or G, and/or X₂₂corresponds to L, W, R, D, E, or G.

In some embodiments, the PSMA comprises the following amino acid sequence:

(SEQ ID NO: 1044)

KSSNEATNITPKHNMKAFLDELKAENIKKFLYNFTQIPHLAGTEQNFQL

AKQIQSQWKEFGLDSVELAHYDVLLSYPNKTHPNYISIINEDGNEIFNT

SLFEPPPPGYENVSDIVPPFSAFSPQGMPEGDLVYVNYARTEDFFKLER

DMKINCSGKIVIARYGKVFRGNKVKNAQLAGAKGVILYSDPADYFAPGV

KSYPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVG

LPSIPVHPIGYYDAQKLLEKMGGSAPPDSSWRGSLKVPYNVGPGFTGNF

STQKVKMHIHSTNEVTRIYNVIGTLRGAVEPDRYVILGGHRDSWVFGGI

DPQSGAAVVHEIVRSFGTLKKEGWRPRRTILFASWDAEEFGLLGSTEWA

EENSRLLQERGVAYINADSSIEGNYTLRVDCTPLMYSLVHNLTKELKSP

DEGFEGKSLYESWTKKSPSPEFSGMPRISKLGSGNDFEVFFQRLGIASG

RARYTKNWETNKFSGYPLYHSVYETYELVEKFYDPMFKYHLTVAQVRGG

MVFELANSIVLPFDCRDYAVVLRKYADKIYSISMKHPQEMKTYSVSFDS

LFSAVKNFTEIASKFSERLQDFDKSNPIVLRMMNDQLMFLERAFIDPLG

LPDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDIESKVDPSKAWGEVK

RQIYVAAFTVQAAAETLSEVA.

Certain aspects of the present disclosure are directed to an antibody or an antigen-binding fragment thereof that specifically binds CD3, comprising a VL domain and a VH domain, wherein: (i) the VL domain comprises the VL CDRs of the amino acid sequence of ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNK RAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL (SEQ ID NO: 361); or (ii) the VH domain comprises the VH CDRs of the amino acid sequence of

(SEQ ID NO: 311)

EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVG

RIRTKRNNYATYYADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYC

VRHENFGNSYVSWFAHWGQGTLVTVSS

Certain aspects of the present disclosure are directed to an anti-CD3 antibody or an antigen-binding fragment thereof, comprising one or more of the following CDRs: a VL domain CDR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to RSSNGAVTSSNYAN(SEQ ID NO:1); a VL domain CDR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GTNKRAP(SEQ ID NO:4); a VL domain CDR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to ALWYPNLWV(SEQ ID NO:6); a VH domain CDR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GFTFSTYAMN(SEQ ID NO:12); a VH domain CDR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to RIRTKRNNYATYYADSVKG(SEQ ID NO:13); and/or a VH domain CDR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to HENFGNSYVSWFAH(SEQ ID NO:10).

In some embodiments, the antibody or fragment comprises one or more of the following FRs: a VL domain FR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to ELVVTQEPSLTVSPGGTVTLTC(SEQ ID NO:51); a VL domain FR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to WVQQKPGQAPRGLIG(SEQ ID NO:52); a VL domain FR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GTPARFSGSLLGGKAALTLSGVQPEDEAVYYC(SEQ ID NO:53); a VL domain FR4 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to FGGGTKLTVL(SEQ ID NO:59); a VH domain FR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to EVQLVESGGGIVQPGGSLRLSCAAS(SEQ ID NO:400); a VH domain FR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to WVRQAPGKGLEWVG(SEQ ID NO:401); a VH domain FR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to RFTISRDDSKNTVYLQMNSLKTEDTAVYYCVR (SEQ ID NO:402); and/or a VH domain FR4 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to WGQGTLVTVSS(SEQ ID NO:67).

In some embodiments, the antibody or fragment comprises a VL domain.

In some embodiments, the VL domain comprises an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to:

(SEQ ID NO: 361)

ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGL

IGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLW

VFGGGTKLTVL.

In some embodiments, the antibody or fragment comprises a VH domain.

In some embodiments, the VH domain comprises an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to:

(SEQ ID NO: 311)

EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVG

RIRTKRNNYATYYADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYC

VRHENFGNSYVSWFAHWGQGTLVTVSS.

Certain aspects of the present disclosure are directed to an antibody or an antigen-binding fragment thereof that specifically binds CD3, comprising a VL domain and a VH domain, wherein the VL domain amino acid sequence SEQ ID NO/VH domain amino acid sequence SEQ ID NO pair is selected from the group consisting of: 896/897; 902/903; 700/701; 702/703; 716/717; 718/719; 728/729; 736/737; 738/739; 740/741; 742/743; 744/745; 746/747; 748/749; 750/751; 752/753; 754/755; 756/757; 758/759; 760/761; 762/763; 764/765; 766/767; 774/775; 776/777; 790/791; 792/793; 798/799; 800/801; 806/807; 808/809; 814/815; 816/817; 822/823; 824/825; or 826/867.

In some embodiments, the antibody or fragment thereof is an isolated antibody or fragment thereof.

In some embodiments, the antibody or fragment thereof is an antibody.

In some embodiments, the antibody or fragment thereof is a Fab, an scFv, or a monoclonal antibody.

In some embodiments, the antibody or fragment thereof is an scFv.

In some embodiments, the VL domain is N-terminal to the VH domain in the scFv

In some embodiments, the VL domain is C-terminal to the VH domain in the scFv.

In some embodiments, the scFv comprises a linker between the VL domain and the VH domain, wherein the linker consists of A, E, G, S, P, and/or T residues.

In some embodiments, the linker is an ELNN.

In some embodiments, the ELNN is cleavable by a non-mammalian protease. In some embodiments, the non-mammalian protease is Glu-C.

In some embodiments, ELNN comprises an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81).

In some embodiments, the scFv comprises an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to:

In some embodiments, the CD3 is CD3 epsilon.

In some embodiments, the CD3 epsilon comprises the following amino acid sequence:

(SEQ ID NO: 1043)

DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDED

DKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCE

NCMEMD

Certain aspects of the present disclosure are directed to a pharmaceutical composition comprising the antibody or an antigen-binding fragment thereof described herein, and at least one pharmaceutically acceptable excipient.

Certain aspects of the present disclosure are directed to a polynucleotide sequence encoding the antibody or an antigen-binding fragment thereof described herein.

Certain aspects of the present disclosure are directed to an expression vector comprising the polynucleotide sequence described herein.

Certain aspects of the present disclosure are directed to a host cell comprising the expression vector described herein.

Certain aspects of the present disclosure are directed to a method of producing the antibody or an antigen-binding fragment thereof described herein. In some embodiments, the method further comprises isolating the antibody or an antigen-binding fragment thereof of from a host cell.

Certain aspects of the present disclosure are directed to a multispecific antibody comprising an anti-PSMA antibody domain comprising an antibody or antibody fragment described herein and/or an anti-CD3 antibody domain comprising an antibody or antibody fragment described herein.

Certain aspects of the present disclosure are directed to a multispecific antibody comprising an anti-PSMA antibody domain comprising an antibody or antibody fragment described herein and an anti-CD3 antibody domain comprising an antibody or antibody fragment described herein.

In some embodiments, the affinity of the anti-PSMA antibody domain to PSMA is higher than the affinity of the anti-CD3 antibody domain to CD3. In some embodiments, the multispecific antibody is a bispecific antibody. In some embodiments, the bispecific antibody is a T cell engager.

Certain aspects of the present disclosure are directed to a pharmaceutical composition comprising the multispecific antibody described herein, and at least one pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is in a liquid form or is frozen. In some embodiments, the pharmaceutical composition is formulated as a lyophilized powder or cake to be reconstituted prior to administration.

Certain aspects of the present disclosure are directed to a polynucleotide sequence encoding the multispecific antibody described herein.

Certain aspects of the present disclosure are directed to an expression vector comprising the polynucleotide sequence described herein.

Certain aspects of the present disclosure are directed to a host cell comprising the expression vector described herein.

Certain aspects of the present disclosure are directed to a method of producing the multispecific antibody described herein. In some embodiments, the method further comprises isolating the multispecific antibody from a host cell.

Certain aspects of the present disclosure are directed to a T cell engager comprising a first antigen binding domain that binds to prostate-specific membrane antigen (PSMA) and a second antigen binding domain that binds to cluster of differentiation 3 T cell receptor (CD3), wherein the first antigen binding domain comprises a VHH comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVGAMSW SGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAASNKEYGRTWY DFNESDYWGQGTQVTVSS (SEQ ID NO: 549); and the second antigen binding domain comprises a VL domain comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNK RAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL (SEQ ID NO: 361) and a VH domain comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to

(SEQ ID NO: 311)

EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVG

RIRTKRNNYATYYADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYC

VRHENFGNSYVSWFAHWGQGTLVTVSS.

Certain aspects of the present disclosure are directed to a pharmaceutical composition comprising the T cell engager described herein, and at least one pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is in a liquid form or is frozen. In some embodiments, the pharmaceutical composition is formulated as a lyophilized powder or cake to be reconstituted prior to administration.

Certain aspects of the present disclosure are directed to a polynucleotide sequence encoding the T cell engager described herein.

Certain aspects of the present disclosure are directed to an expression vector comprising the polynucleotide sequence described herein.

Certain aspects of the present disclosure are directed to a host cell comprising the expression vector described herein.

Certain aspects of the present disclosure are directed to a method of producing the T cell engager described herein. In some embodiments, the method further comprises isolating the T cell engager from a host cell.

Certain aspects of the present disclosure are directed to a protease-activatable T cell engager (paTCE) comprising a T cell engager (TCE) described herein, in the form of a single polypeptide chain, wherein the N-terminus of the TCE is fused to a first masking polypeptide by a first protease-cleavable linker and the C-terminus of the TCE is fused to a second masking polypeptide by a second protease-cleavable linker.

In some embodiments, the first masking polypeptide is a first ELNN. In some embodiments, the second masking polypeptide is a second ELNN.

In some embodiments, the TCE comprises an anti-PSMA VHH comprising the following amino acid sequence:

(SEQ ID NO: 549)

QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVG

AMSWSGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNKEYGRTWYDFNESDYWGQGTQVTVSS.

In some embodiments, the TCE comprises an anti-CD3 scFv comprising a VH domain having the following amino acid sequence: EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKR NNYATYYADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCVRHENFGNSYVS WFAHWGQGTLVTVSS (SEQ ID NO: 311) and a VL domain having the following amino acid sequence:

(SEQ ID NO: 361)

ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGL

IGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLW

VFGGGTKLTVL.

Certain aspects of the present disclosure are directed to a pharmaceutical composition comprising the paTCE described herein, and at least one pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is in a liquid form or is frozen. In some embodiments, the pharmaceutical composition is formulated as a lyophilized powder or cake to be reconstituted prior to administration.

Certain aspects of the present disclosure are directed to a polynucleotide sequence encoding the paTCE described herein.

Certain aspects of the present disclosure are directed to a n expression vector comprising the polynucleotide sequence described herein.

Certain aspects of the present disclosure are directed to a host cell comprising the expression vector described herein.

Certain aspects of the present disclosure are directed to a method of producing the paTCE described herein. In some embodiments, the method further comprises isolating the paTCE from a host cell.

Certain aspects of the present disclosure are directed to a chimeric polypeptide, isolated polypeptide, fusion protein, antigen binding polypeptide, antibody or an antigen-binding fragment thereof that specifically binds PSMA, antibody or an antigen-binding fragment thereof that specifically binds CD3, multispecific antibody, T cell engager, or paTCE, produced by the method described herein.

Certain aspects of the present disclosure are directed to a polynucleotide sequence encoding the amino acid sequence: EAGRSAXHTPAGLTGP (SEQ ID NO: 7627), wherein X is any amino acid other than N.

In some embodiments, the polynucleotide is a vector.

In some embodiments, the polynucleotide is an isolated polynucleotide.

Certain aspects of the present disclosure are directed to a cell line that expresses an exogenous polypeptide comprising the amino acid sequence: EAGRSAXHTPAGLTGP (SEQ ID NO: 7627), wherein X is any amino acid other than N.

In some embodiments, the exogenous polypeptide is a fusion protein described herein.

In some embodiments, the cell line is in culture or is frozen in a glass or plastic container.

In some embodiments, the cell line is in a bioreactor.

In some embodiments, the cell is a stable cell line.

In some embodiments, the cell line is a mammalian cell.

In some embodiments, the cell line is a CHO cell or a HEK293 cell.

In some embodiments, the cell line is a prokaryotic cell.

In some embodiments, the cell line is an Escherichia coli cell.

Certain aspects of the present disclosure are directed to a non-human animal that comprises an exogenous polypeptide comprising the amino acid sequence: EAGRSAXHTPAGLTGP (SEQ ID NO: 7627), wherein X is any amino acid other than N. In some embodiments, X is D, E, or Q. In some embodiments, X is G, A, V, L, I. In some embodiments, X is P. In some embodiments, X is F, Y, or W. In some embodiments, X is H, K, or R. In some embodiments, X is S, C, U, T, or M. In some embodiments, X is S.

Certain aspects of the present disclosure are directed to a fusion protein comprising an anti-PSMA antibody or fragment described herein and a biologically active protein.

Certain aspects of the present disclosure are directed to a fusion protein comprising an anti-CD3 antibody or fragment described herein and a biologically active protein.

In some embodiments, the biologically active protein comprises a cytokine, an enzyme, a hormone, a growth factor, a chemotherapeutic polypeptide, an antiviral polypeptide, or a toxin.

Certain aspects of the present disclosure are directed to an immunoconjugate comprising an anti-PSMA antibody or fragment described herein and a compound.

Certain aspects of the present disclosure are directed to an immunoconjugate comprising an anti-CD3 antibody or fragment described herein and a compound.

In some embodiments, the compound comprises chemotherapeutic agent.

In some embodiments, the compound comprises a diagnostic agent.

In some embodiments, the compound comprises a toxin, a radioactive molecule, a contrast agent, or a drug.

The present disclosure provides an isolated antibody or antigen-binding fragment thereof, which specifically binds CD3, comprising a heavy chain variable region (VH) comprising three heavy-chain CDRs, and a light chain variable region (VL) comprising three light-chain CDRs, wherein the three heavy-chain CDRs comprise the CDR1, CDR2, and CDR3 from EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKR NNYATYYADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCVRHENFGNSYVS WFAHWGQGTLVTVSS (SEQ ID NO: 311) and the three light-chain CDRs comprise the CDR1, CDR2, and CDR3 from ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNK RAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL (SEQ ID NO: 361), and wherein the CDRs are identified by the Kabat definition, the Chothia definition, the AbM definition, the IMGT definition, or the contact definition. In some embodiments, the antibody is an scFv.

Included herein is an antigen binding protein comprising: (i) a light chain variable domain comprising an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to, or 100% identical to, a light chain variable domain sequence comprising a CDR sequence selected from the group consisting of RSSNGAVTSSNYAN(SEQ ID NO:1), GTNKRAP(SEQ ID NO:4), and ALWYPNLWV(SEQ ID NO:6), and (ii) a heavy chain variable domain comprising an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to, or 100% identical to, a heavy chain variable domain sequence comprising a CDR sequence selected from the group consisting of SEQ ID NOs: GFTFSTYAMN(SEQ ID NO:12), RIRTKRNNYATYYADSVKG(SEQ ID NO:13), and HENFGNSYVSWFAH(SEQ ID NO:10), wherein the antigen binding protein specifically binds to CD3. In some embodiments, the antibody is an scFv.

Disclosed herein is an antibody or antigen-binding fragment thereof, which specifically binds CD3, wherein the antibody or antigen-binding fragment thereof comprises three light chain complementarity determining region (CDR) sequences of SEQ ID NOs: RSSNGAVTSSNYAN(SEQ ID NO:1), GTNKRAP(SEQ ID NO:4), and ALWYPNLWV(SEQ ID NO:6), and three heavy chain complementarity determining region (CDR) sequences of GFTFSTYAMN(SEQ ID NO:12), RIRTKRNNYATYYADSVKG(SEQ ID NO:13), and HENFGNSYVSWFAH(SEQ ID NO:10). In some embodiments, the antibody is an scFv.

Included herein is an isolated antibody or antigen-binding fragment thereof, that specifically binds CD3 comprising the amino acid sequence of DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDE DHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMD (SEQ ID NO: 1043), comprising a heavy chain variable region comprising three heavy-chain CDRs, and a light chain variable region comprising three light-chain CDRs, wherein the three heavy-chain CDRs comprise the CDR1, CDR2 and CDR3 from EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKR NNYATYYADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCVRHENFGNSYVS WFAHWGQGTLVTVSS (SEQ ID NO: 311), and the three light-chain CDRs comprise the CDR1, CDR2 and CDR3 from ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNK RAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL (SEQ ID NO: 361). In some embodiments, the heavy chain CDR1 comprises GFTFSTYAMN(SEQ ID NO:12), the heavy chain CDR2 comprises RIRTKRNNYATYYADSVKG(SEQ ID NO:13), the heavy chain CDR3 comprises HENFGNSYVSWFAH(SEQ ID NO:10), the light chain CDR1 comprises RSSNGAVTSSNYAN(SEQ ID NO:1), the light chain CDR2 comprises GTNKRAP(SEQ ID NO:4), and the light chain CDR3 comprises ALWYPNLWV(SEQ ID NO:6). In some embodiments, the heavy chain variable region comprises EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKR NNYATYYADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCVRHENFGNSYVS WFAHWGQGTLVTVSS (SEQ ID NO: 311). In some embodiments, the light chain variable region comprises ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNK RAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL (SEQ ID NO: 361). In some embodiments, the heavy chain variable region comprises EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKR NNYATYYADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCVRHENFGNSYVS WFAHWGQGTLVTVSS (SEQ ID NO: 311) and the light chain variable region comprises ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNK RAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL (SEQ ID NO: 361).

In some embodiments, included herein is an antibody or antigen-binding fragment thereof, that specifically binds CD3 (e.g., a protein having a heavy chain variable region amino acid sequence of SEQ ID NO: 311 and a light chain variable region amino acid sequence of SEQ ID NO: 361) with a Koof about 300 nM or less, e.g., as measured by surface plasmon resonance. In some embodiments, the antibody or antigen-binding portion thereof exhibits a K_Dof about 200 nM or less, about 150 or less, about 100 nM or less, or about 75 nM or less.

Provided herein is an antibody or antigen-binding fragment thereof, that specifically binds CD3 (e.g., a protein having a heavy chain variable region amino acid sequence of SEQ ID NO: 311 and a light chain variable region amino acid sequence of SEQ ID NO: 361), comprising a heavy chain variable region and a light chain variable region, wherein the antibody or antigen-binding fragment comprises: (a) a heavy chain variable region having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKR NNYATYYADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCVRHENFGNSYVS WFAHWGQGTLVTVSS (SEQ ID NO: 311) and a light chain variable region having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNK RAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL (SEQ ID NO: 361), characterized by an affinity for CD3 (K_D) of about 100 nM or less; (b) a heavy chain variable region having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKR NNYATYYADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCVRHENFGNSYVS WFAHWGQGTLVTVSS (SEQ ID NO: 311) and a light chain variable region having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNK RAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL (SEQ ID NO: 361), characterized by an affinity for CD3 (K_D) of about 300 nM or less; or (c) a heavy chain variable region having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKR NNYATYYADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCVRHENFGNSYVS WFAHWGQGTLVTVSS (SEQ ID NO: 311) and a light chain variable region having an amino acid sequence that is at least 90% identical to the amino acid sequence shown in ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNK RAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL (SEQ ID NO: 361), characterized by an affinity for CD3 (K_D) of about 75 nM or less.

The present disclosure provides an isolated antibody or antigen-binding fragment thereof, which specifically binds PSMA, comprising a VHH domain comprising three VHH CDRs, wherein the three VHH CDRs comprise the CDR1, CDR2 and CDR3 from QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVGAMSW SGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAASNKEYGRTWY DFNESDYWGQGTQVTVSS (SEQ ID NO: 549), wherein the CDRs are identified by the Kabat definition, the Chothia definition, the AbM definition, the IMGT definition, or the contact definition.

Included herein is an antigen binding protein comprising: (i) a VHH domain comprising an amino acid sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to, or 100% identical to, a VHH CDR sequence selected from the group consisting of GRTFGIYVWG(SEQ ID NO:9003), AMSWSGSNRK(SEQ ID NO:9015), and AASNKEYGRTWYDFNESDY(SEQ ID NO:9005), wherein the antigen binding protein specifically binds to PSMA.

Included herein is an isolated antibody or antigen-binding fragment thereof, which specifically binds PSMA comprising the amino acid sequence of KSSNEATNITPKHNMKAFLDELKAENIKKFLYNFTQIPHLAGTEQNFQLAKQIQSQW KEFGLDSVELAHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPPGYENVSDIVP PFSAFSPQGMPEGDLVYVNYARTEDFFKLERDMKINCSGKIVIARYGKVFRGNKVK NAQLAGAKGVILYSDPADYFAPGVKSYPDGWNLPGGGVQRGNILNLNGAGDPLTP GYPANEYAYRRGIAEAVGLPSIPVHPIGYYDAQKLLEKMGGSAPPDSSWRGSLKVP YNVGPGFTGNFSTQKVKMHIHSTNEVTRIYNVIGTLRGAVEPDRYVILGGHRDSWV FGGIDPQSGAAVVHEIVRSFGTLKKEGWRPRRTILFASWDAEEFGLLGSTEWAEEN SRLLQERGVAYINADSSIEGNYTLRVDCTPLMYSLVHNLTKELKSPDEGFEGKSLYE SWTKKSPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRARYTKNWETNKFSGYP LYHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVFELANSIVLPFDCRDYAVVLRK YADKIYSISMKHPQEMKTYSVSFDSLFSAVKNFTEIASKFSERLQDFDKSNPIVLRM MNDQLMFLERAFIDPLGLPDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDIESKVD PSKAWGEVKRQIYVAAFTVQAAAETLSEVA (SEQ ID NO: 1044), comprising a VHH region comprising three VHH CDRs from QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVGAMSW SGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAASNKEYGRTWY DFNESDYWGQGTQVTVSS (SEQ ID NO: 549). In some embodiments, the VHH CDR1 comprises GRTFGIYVWG(SEQ ID NO:9003), the VHH CDR2 comprises AMSWSGSNRK(SEQ ID NO:9015), and the VHH CDR3 comprises AASNKEYGRTWYDFNESDY(SEQ ID NO:9005). In some embodiments, the VHH region comprises

(SEQ ID NO: 549)

QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVG

AMSWSGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNKEYGRTWYDFNESDYWGQGTQVTVSS.

In some embodiments, included herein is an antibody or antigen-binding fragment thereof, that specifically binds PSMA (e.g., a protein having the amino acid sequence of SEQ ID NO: 549) with a K_Dof about 300 nM or less, e.g., as measured by surface plasmon resonance. In some embodiments, the antibody or antigen-binding portion thereof exhibits a K_Dof about 200 nM or less, about 150 or less, about 100 nM or less, or about 50 nM or less.

Provided herein is an antibody or antigen-binding fragment thereof, that specifically binds PSMA (e.g., a protein comprising the amino acid sequence of SEQ ID NO: 549), comprising (a) a VHH region having an amino acid sequence that is at least 95% identical to the amino acid sequence shown in QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVGAMSW SGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAASNKEYGRTWY DFNESDYWGQGTQVTVSS (SEQ ID NO: 549), characterized by an affinity for PSMA (K_D) of about 100 nM or less; (b) a VHH region having an amino acid sequence that is at least 95% identical to the amino acid sequence shown in QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVGAMSW SGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAASNKEYGRTWY DFNESDYWGQGTQVTVSS (SEQ ID NO: 549), characterized by an affinity for PSMA (K_D) of about 300 nM or less; or (c) a VHH region having an amino acid sequence that is at least 95% identical to the amino acid sequence shown in QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVGAMSW SGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAASNKEYGRTWY DFNESDYWGQGTQVTVSS (SEQ ID NO: 549) characterized by an affinity for PSMA (K_D) of about 50 nM or less.

The present disclosure includes a bispecific T cell engager comprising (i) an antibody or antigen-binding fragment thereof, that specifically binds human CD3 (a protein having a heavy chain variable region amino acid sequence of SEQ ID NO: 311 and a light chain variable region amino acid sequence of SEQ ID NO: 361) provided herein; and (ii) an antibody or antigen-binding fragment thereof, that specifically binds human PSMA (SEQ ID NO: 549) provided herein.

Various features of this disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts a non-limiting schematic representation of an exemplary paTCE. FIG. 1B depicts a schematic representation of fully unmasked paTCE (a uTCE) and singly masked metabolites paTCE(1x-N) and paTCE(1x-C) from an exemplary paTCE as shown in FIG. 1A.

FIG. 2A -FIG. 2D depict biophysical characterization data of PMSA.2 variant antibodies. FIG. 2A depicts the concentrations of PMSA.2 variant antibodies. FIG. 2B depicts relative binding to PSMA of PMSA.2 variant antibodies. FIG. 2C depicts the thermal stability of PMSA.2 variant antibodies as measured by monomer concentration (pM) at 62° C. FIG. 2D depicts the thermal stability of PMSA.2 variant antibodies as measured by monomer concentration (pM) at 65° C. The AC clone numbers of the tested uTCEs are shown in the figures. uTCEs rather than paTCEs were used in these experiments.

FIG. 3A -FIG. 3C depict biophysical characterization data of PMSA.3 variant antibodies. FIG. 3A depicts relative binding to PSMA of PMSA.3 variant antibodies. FIG. 3B and FIG. 3C depict the thermal stability of PMSA.3 variant antibodies as measured by monomer concentration (FIG. 3B) or aggregate concentration (FIG. 3C) (pM) at 63.5° C. The AC clone numbers of the tested uTCEs are shown in the figures. uTCEs rather than paTCEs were used in these experiments.

FIG. 4 depicts PTE scores of representative PSMA variants and CD3 variants. The graph shows molecules with known Antidrug Antibody (ADA) and their corresponding PTE score. The higher score indicates greater chance of having putative T cell epitopes.

FIG. 5 depicts T cell proliferation in an EpiScreen™ DC: T cell immunogenicity assay. PSMA.350 and the positive control KLH were tested. For each donor date point, each bar from left to right represents Day 9, Day 10, Day 11, and Day 12.

FIG. 6A depicts PTE score evaluations using internal PTE algorithm v22 for anti-CD3 pool2 antibodies. FIG. 6B depicts a percent of remaining antibodies following a thermal stability assay.

FIG. 7A depicts an alignment of the RSR-2295 and RSR-3213 amino acid sequences and proteases capable of cleaving them. FIG. 7B depicts in vitro protease digestion of paTCEs employing RSR-2295 or RSR-3213. The RSR-3213 sequence is modified to substantially reduce cleavage by legumain.

FIG. 8A and FIG. 8B depict relative plasma stability of paTCEs employing RSR-2295 or RSR-3213, measured at Day 0 and Day 7. In FIG. 8A, RSR-2295 employed the SCy5.5 fluorophore and RSR-3213 employed the SCy7.5 fluorophore. In FIG. 8B, the RSR-2295 employed the SCy7.5 fluorophore and RSR-3213 employed the SCy5.5 fluorophore. FIG. 8C depicts the observed cleavability in vivo from tumor homogenates from 3 different mouse tumor models. For each set of bar graphs (i.e., % 1x-C, % 1x-N, % uTCE), each barfrom left to right represents B1, B2, B3, B4, A1, A2, A3, A4, 43-1, 43-2, 43-3, and 43-4. B1-B4 represent 4 different mice from a first tumor model (NCI-N87). A1-A4 represent 4 different mice from a second tumor model (HT-29). 43-1-43-4 represent 4 different mice from a third tumor model (HT-55). FIG. 8D depicts the % of total for the 3 metabolites plus the paTCE (paTCE, 1x-N, 1x-C, and uTCE) when employing RSR-2295 or RSR-3213.

FIG. 9 depicts relative tumor uptake of paTCEs employing RSR-2295 or RSR-3213. The plasma: tumor ratio was calculated in 3 different mouse tumor models (4 mice per tumor model). There is a “Mouse 1” for each of the 3 different tumor models, a “Mouse 2” for each of the 3 different tumor models, a “Mouse 3” for each of the different tumor models, and a “Mouse 4” for each of the 3 different tumor models.

FIG. 10 depicts graphs of PSMA-transfected CHO cell binding activity against human PSMA or cyno PSMA between AC3092 (AMX-500-P1) and AC3896 (AMX-500-P4; also referred to here as simply AMX-500). Surface binding was detected with a labeled secondary antibody specific for the anti-CD3 scFv.

FIG. 11A and FIG. 11B depict dose response curves of relative in vitro cytotoxicity of LNCaP PSMA^highcells (FIG. 11A) and 22Rv1 PSMA^lowcells (FIG. 11B).

FIG. 12A -FIG. 12C depict dose response curves of relative in vitro cytotoxicity of LNCaP PSMA^highcells (FIG. 12A and FIG. 12B) and 22Rv1 PSMA^lowcells (FIG. 12C) with 3 different donor human PBMC samples.

FIG. 13A and FIG. 13B depicts a graph of relative in vitro cytotoxicity of LNCaP PSMA^highcells from donor 1 incubated with various concentrations of AMX-500(uTCE), AMX-500, AMX-500(1x-N), AMX-500(1x-C), and AMX-500(NoClvSite). FIG. 13A depicts the assay results from Donor 1. FIG. 13B depicts similar results from Donors 2-5.

FIG. 14A and FIG. 14B depicts a graph of relative in vitro cytotoxicity of 22Rv1 PSMA^lowcells from donor 1 incubated with various concentrations of AMX-500(uTCE), AMX-500, AMX-500(1x-N), AMX-500(1x-C), and AMX-500(NoClvSite).

FIG. 14A depicts the assay results from Donor 1. FIG. 14B depicts similar results from Donors 2 and 3.

FIG. 15 depicts graphs of in vitro cytokine release from LNCaP PSMA^highcells incubated with various concentrations of AMX-500(uTCE), AMX-500, AMX-500(1x-N), AMX-500(1x-C), and AMX-500(NoClvSite). The cells were co-incubated with PBMCs at a ratio of 10:1 PBMCs to LNCap cells. Levels of cytokines INF-γ, TNF-α, IL-6, IL-10, GM-CSF, IL-β, IL-2, IL-4, and MCP-1 are shown.

FIG. 16 depicts graphs of CD69, CD25, and PD-1 expression on CD4+ T cells from an LNCaP PSMA^high/PBMC co-culture that was incubated with various concentrations of AMX-500(uTCE), AMX-500, AMX-500(1x-N), AMX-500(1x-C), and AMX-500(NoClvSite). The cells were co-incubated with PBMCs at a ratio of 10:1 PBMCs to LNCap cells. PBMCs were taken from Donor 1.

FIG. 17 depicts graphs of CD69, CD25, and PD-1 expression on CD8+ T cells from an LNCaP PSMA^high/PBMC co-culture that was incubated with various concentrations of AMX-500(uTCE), AMX-500, AMX-500(1x-N), AMX-500(1x-C), and AMX-500(NoClvSite). The cells were co-incubated with PBMCs at a ratio of 10:1 PBMCs to LNCap cells. PBMCs were taken from Donor 1.

FIG. 18 depicts graphs of CD69, CD25, and PD-1 expression on CD4+ and CD8+ T cells from an LNCaP PSMA^high/PBMC co-culture that was incubated with various concentrations of AMX-500(uTCE), AMX-500, AMX-500(1x-N), AMX-500(1x-C), and AMX-500(NoClvSite). The cells were co-incubated with PBMCs at a ratio of 10:1 PBMCs to LNCap cells. PBMCs were taken from Donor 2.

FIG. 19 depicts graphs of CD69, CD25, and PD-1 expression on CD4+ and CD8+ T cells from an LNCaP PSMA^high/PBMC co-culture that was incubated with various concentrations of AMX-500(uTCE), AMX-500, AMX-500(1x-N), AMX-500(1x-C), and AMX-500(NoClvSite). The cells were co-incubated with PBMCs at a ratio of 10:1 PBMCs to LNCap cells. PBMCs were taken from Donor 3.

FIG. 20A and FIG. 20B depict relative target binding of AMX-500 (FIG. 20A) and AMX-500-P7 (AC3934, FIG. 20B) to about 6,000 different HEK293T membrane proteins.

FIG. 21 depicts graphs of tumor volume from human prostate tumor mouse models. Tumor mouse models were generated with 22Rv1 PSMA^lowcells, LNCaP PSMA^highcells, or C4-2 PSMA^highcells. For the 22Rv1 model, AMX-500 paTCE was dosed at 2 mg/kg, 16 nmol/kg and AMX-500 unmasked TCE (uTCE) was dosed at 0.35 mg/kg, 7.6 nmol/kg. For the LNCaP model, AMX-500 paTCE was dosed at 3 mg/kg, 24 nmol/kg and AMX-500 uTCE was dosed at 0.35 mg/kg, 7.6 nmol/kg. For the C4-2 model, dose A was 7.5 mg/kg, 59 nmol/kg, BIW and dose B was 3.5 mg/kg, 27 nmol/kg, BIW.

FIG. 22 depicts graphs of tumor volume from human prostate tumor mouse models of LNCaP PSMA^highcells.

FIG. 23 depicts graphs of tumor volume from human prostate tumor mouse models of 22Rv1 PSMA^lowcells.

FIG. 24 depicts a graph of tumor volume from a human prostate tumor mouse model administered AMX-500, the anti-PD-1 antibody pembrolizumab, or the combination of AMX-500 and pembrolizumab.

FIG. 25 depicts tissue distribution of AMX-500 in a mouse tumor model.

DETAILED DESCRIPTION

There is a significant unmet need in cancer therapeutics for a PSMA-targeted bispecific treatment modality that is efficacious against solid tumors, particularly solid tumors that are present in an immunologically cold microenvironment. While TCEs have been shown to be effective in inducing remission in certain cancers, they have not led to the development of widespread therapeutics due to their extreme potency and on target, off tumor toxicities in healthy tissues.

Without being bound by any scientific theory, TCEs form a bridge between T cells and tumor cells and activate T cell-mediated killing of the tumor cells and further initiating a cytokine amplification cascade. The cytokine amplification cascade can promote further killing of tumor cells and potentially provide long term immunity. T cells activated by TCEs release cytolytic perforin/granzymes in a manner that is independent of antigen-MHC recognition. This creates a two-fold response: direct tumor cell death and amplification of tumor killing through initiation of a powerful cytokine response from the tumor cells. The direct tumor cell death results in release of tumor antigens. The cytokine response may include, among others, increased interferon-g which stimulates CD8 T cell activity and stimulates antigen presentation by APCs; increased IL2 which causes increased proliferation of activated T-cells, and increased CXCL9 and 10 response which increases T cell recruitment. Together the release of tumor antigens and the initiation of the cytokine response results in activation of the endogenous T cell response which potentially causes epitope spreading to induce long term immunity.

One toxicity challenge with TCEs arises out the fact that many tumor targets are, to some extent, also expressed in healthy tissue, and normal cells also can produce the cytokines response resulting in cytokine release syndrome (CRS). These two powerful responses of health tissue to T cell activation by TCEs often results in an overall lack of acceptable therapeutic index for these agents.

The present disclosure provides protease-activatable TCEs (paTCEs) that address an unmet need and are superior in one or more aspects including enhanced terminal half-life, targeted delivery, and/or improved therapeutic ratio with reduced toxicity to healthy tissues compared to conventional antibody therapeutics or bispecific antibody therapeutics that are active upon injection.

Included herein are compounds, compositions and methods that overcome the drawbacks in the existing TCEs by providing paTCEs that target PSMA (referred to herein as PSMA-paTCEs and exemplified as AMX-500).

AMX-500 comprises the amino acid sequence set forth as SEQ ID NO: 1000. Without being bound by any scientific theory, the paTCEs described herein are understood to exploit the dysregulated protease activity present in tumors vs. healthy tissues, enabling expansion of the therapeutic index. The paTCE core comprises antigen binding domains; one targets CD3 and the other targets PSMA. The two antigen binding domains may, in exemplary embodiments, be in two different antibody formats (such as, e.g., a single chain antibody fragment (scFv) and a VHH), or the same antibody format (such as, e.g., scFvs). Many different antibody fragments or formats may be used.

In some embodiments, a PSMA-targeting paTCE comprises a first portion that is a VHH that binds to PSMA and a second portion that is an scFv that binds to CD3. One or more (e.g., two) unstructured polypeptide masks are attached to the core. In some embodiments, these unstructured polypeptide masks sterically reduce target engagement of either the tumor target and/or CD3, and also extend protein half-life. In some embodiments, the unstructured polypeptide masks are extended length non-natural polypeptides (ELNNs).

In some embodiments, the properties of ELNNs also minimize the potential for immunogenicity, as their lack of stable tertiary structures disfavors antibody binding, and the absence of hydrophobic, aromatic, and positively charged residues that serve as anchor residues for peptide MHC II binding reduces the potential for T cell epitopes.

In some embodiments, protease cleavage sites at the base of the ELNN or ELNNs enable proteolytic activation of paTCEs in the tumor microenvironment, unleashing a smaller, highly potent TCEs that are capable redirecting cytotoxic T cells to kill target-expressing tumor cells. In some embodiments, in healthy tissues, where protease activity is tightly regulated, paTCEs remain predominantly inactive, thus expanding the therapeutic index compared to unmasked TCEs.

In some embodiments, in addition to localized activation, the short half-life of the unmasked TCE form further widens the therapeutic index while providing the potency of T-cell immunity to improve the eradication of solid tumors. In some embodiments, the release sites used in the paTCEs can be cleaved across a broad array of tumors by proteases that are collectively involved in every cancer hallmark (growth; survival and death; angiogenesis; invasion and metastasis; inflammation; and immune evasion). Thus, TCE activity of the paTCEs is localized to tumors by exploiting the enhanced protease activity that is upregulated in all stages of cancer and tumor development but is tightly regulated in healthy tissues.

Terminology

As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

As used in the specification and claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes a plurality of cells, including mixtures thereof, unless the context clearly dictates otherwise.

Furthermore, “and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following aspects: “A, B, and C”; “A, B, or C”; “A or C”; “A or B”; “B or C”; “A and C”; “A and B”; “B and C”; “A” (alone); “B” (alone); and “C” (alone).

It is understood that wherever aspects are described herein with the language “comprising,” otherwise analogous aspects described in terms of “consisting of” and/or “consisting essentially of” are also provided.

Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not limitations of the various aspects of the disclosure. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

The term “about” is used herein to mean approximately, roughly, around, or in the regions of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” can modify a numerical value above and below the stated value by a variance of, e.g., 10 percent, up or down (higher or lower). In some embodiments, the term indicates deviation from the indicated numerical value by ±10%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, ±0.05%, or ±0.01%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±10%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±5%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±4%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±3%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±2%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±1%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±0.9%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±0.8%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±0.7%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±0.6%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±0.5%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±0.4%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±0.3%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±0.1%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±0.05%. In some embodiments, “about” indicates deviation from the indicated numerical value by ±0.01%.

With respect to naturally occurring compounds, the term “isolated” refers to a compound (i.e., a polypeptide or polynucleotide) that is not in its native state (e.g., free to varying degrees from components that naturally accompany the compound in nature). No particular level of purification is required. For example, an isolated polypeptide can simply be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for the purpose of the disclosure, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique. “Isolate” and “isolated” may also denote a degree of separation from an original source or surrounding, depending on context.

The term “polypeptide” refers to any polymer of two or more amino acids. Thus, the terms peptide, dipeptide, tripeptide, oligopeptide, protein, amino acid chain, or any other term used to refer to a chain of two or more amino acids, is included within the definition of “polypeptide.” The term “polypeptide” also encompasses an amino acid polymer that has been modified (e.g., by post-translational modification), for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component. Depending on context, the term “polypeptide” may also be used to refer to a protein comprising two or more polymers of two or more amino acids.

A “host cell” includes an individual cell (e.g., in culture) which that comprises an exogenous polynucleotide. Host cells may include progeny of a single host cell. The progeny may not necessarily be completely identical (in morphology or in genomic of total DNA complement) to the original parent cell due to naturally occurring or genetically engineered variation.

A “fusion” or “chimeric” polypeptide or protein comprises a first polypeptide portion linked to a second polypeptide portion with which it is not naturally linked in nature. In some embodiments, the portions may normally exist in separate proteins and are brought together in the fusion polypeptide; they may normally exist in the same protein but are placed in a new arrangement in the fusion polypeptide; or the portions may be brought together from different sources. In some embodiments, a fusion or chimeric protein comprises two or more moieties that do not occur in nature (e.g., are created, designed, or otherwise generated by humans, such as binding domains, masks, linkers, barcodes, and other polypeptides provided herein). A chimeric protein may be created, for example, by chemical synthesis, or by recombinant expression (e.g., comprising creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship).

“Conjugated”, “linked,” “fused,” and “fusion” may be used interchangeably herein, depending on context. These terms may refer to the covalent joining together of two more chemical (e.g., polypeptide) elements or components, by whatever means including chemical conjugation or recombinant means.

As known in the art, “sequence identity” between two polypeptides is determined by comparing the amino acid sequence of one polypeptide to the sequence of a second polypeptide. Similarly, “sequence identity” between two polynucleotides is determined by comparing the nucleotide sequence of one polynucleotide to the sequence of a second polynucleotide. The terms “% identical”, “% identity” or similar terms are intended to refer, in particular, to the percentage of nucleotides or amino acids (as applicable) which are identical in an optimal alignment between the sequences to be compared. Said percentage may be purely statistical, and the differences between the two sequences may be but are not necessarily randomly distributed over the entire length of the sequences to be compared. Comparisons of two sequences are usually carried out by comparing the sequences, after optimal alignment, with respect to a segment or “window of comparison”, in order to identify local regions of corresponding sequences. For example, the optimal alignment for a comparison may be carried out manually or with the aid of the local homology algorithm by Smith and Waterman, 1981, Ads App. Math. 2, 482, with the aid of the local homology algorithm by Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, with the aid of the similarity search algorithm by Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or with the aid of computer programs using the algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). In some embodiments, percent identity of two sequences is determined using the BLASTN or BLASTP algorithm, as available on the United States National Center for Biotechnology Information (NCBI) website (e.g., at blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2se q&LINK_LOC=align2seq). In some embodiments, the algorithm parameters used for BLASTN algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 28; (iii) Max matches in a query range set to 0; (iv) Match/Mismatch Scores set to 1, −2; (v) Gap Costs set to Linear; and (vi) the filter for low complexity regions being used. In some embodiments, the algorithm parameters used for BLASTP algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 3; (iii) Max matches in a query range set to 0; (iv) Matrix set to BLOSUM62; (v) Gap Costs set to Existence: 11 Extension: 1; and (vi) conditional compositional score matrix adjustment. When discussed herein, whether any particular polypeptide is at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to another polypeptide can be determined using methods and computer programs/software known in the art such as, but not limited to, the BESTFIT program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711). BESTFIT uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981), to find the best segment of homology between two sequences. When using BESTFIT or any other sequence alignment program to determine whether a particular sequence is, for example, 95% identical to a reference sequence according to the present disclosure, the parameters are set, of course, such that the percentage of identity is calculated over the full-length of the reference polypeptide sequence and that gaps in homology of up to 5% of the total number of amino acids in the reference sequence are allowed.

As used herein, the terms “mask polypeptide”, “mask”, and “masking moiety” refer to a polypeptide that is capable of reducing the binding of an antigen binding domain (e.g., an antibody) to the target antigen in the context of a fusion protein (such as a chimeric polypeptide) provided herein. Exemplary mask polypeptides include, but are not limited to, the ELNN polypeptides described herein. Additional mask polypeptides include albumin, polypeptides consisting of proline, serine and alanine, coiled-coil domains, albumin binding domains, Fc domains, and binding domains with specificity to conserved regions of an antibody variable domain. Mask polypeptides are described in further detail in Lucchi et al. (ACS Cent Sci. 2021 May 26; 7(5): 724-738).

As used herein, the terms “ELNN polypeptides” and “ELNNs” are synonymous and refer to extended length polypeptides comprising non-naturally occurring, substantially non-repetitive sequences (e.g., polypeptide motifs) that are composed mainly of small hydrophilic amino acids, with the sequence having a low degree or no secondary or tertiary structure under physiologic conditions. ELNN polypeptides include unstructured hydrophilic polypeptides comprising repeating motifs of 6 natural amino acids (G, A, P, E, S, and/or T). In some embodiments, an ELNN polypeptide comprises multiple motifs of 6 natural amino acids (G, A, P, E, S, T), wherein the motifs are the same or comprise a combination of different motifs. In some embodiments, ELNN polypeptides can confer certain desirable pharmacokinetic, physicochemical, and pharmaceutical properties when linked to proteins, including T-cell engagers as disclosed herein. Such desirable properties may include but are not limited to enhanced pharmacokinetic parameters and solubility characteristics, as well as improved therapeutic index. ELNN polypeptides are known in the art, and non-limiting descriptions relating to and examples of ELNN polypeptides known as XTEN® polypeptides are available in Schellenberger et al., (2009) Nat Biotechnol 27(12):1186-90; Brandl et al., (2020) Journal of Controlled Release 327:186-197; and Radon et al., (2021) Advanced Functional Materials 31, 2101633 (pages 1-33), the entire contents of each of which are incorporated herein by reference.

In some embodiments, the repetitiveness of an ELNN sequence refers to the 3-mer repetitiveness and can be measured by computer programs or algorithms or by other means known in the art. In some embodiments, the 3-mer repetitiveness of an ELNN may be assessed by determining the number of occurrences of the overlapping 3-mer sequences within the polypeptide. For example, a polypeptide of 200 amino acid residues has 198 overlapping 3-amino acid sequences (3-mers), but the number of unique 3-mer sequences will depend on the amount of repetitiveness within the sequence. In some embodiments, the score can be generated (hereinafter “subsequence score”) that is reflective of the degree of repetitiveness of the 3-mers in the overall polypeptide sequence. In this context, “subsequence score” means the sum of occurrences of each unique 3-mer frame across a 200 consecutive amino acid sequence of the polypeptide divided by the absolute number of unique 3-mer subsequences within the 200 amino acid sequence. Examples of such subsequence scores derived from the first 200 amino acids of repetitive and non-repetitive polypeptides are presented in Example 73 of International Patent Application Publication No. WO 2010/091122 A1, which is incorporated by reference in its entirety.

In some embodiments, and in the context of ELNNs, a “substantially non-repetitive sequence,” refers to an ELNN sequence, wherein (1) there are few or no instances of four identical amino acids in a row in the ELNN sequence and wherein (2) the ELNN has a subsequence score (defined in the preceding paragraph herein) of 12, or 10 or less or that there is not a pattern in the order, from N- to C-terminus, of the sequence motifs that constitute the polypeptide sequence.

A “vector” is a nucleic acid molecule that transfers an inserted nucleic acid molecule into and/or between host cells. In some embodiments, a vector self-replicates in an appropriate host. The term includes vectors that function primarily for insertion of DNA or RNA into a cell, replication of vectors that function primarily for the replication of DNA or RNA, and expression vectors that function for transcription and/or translation of the DNA or RNA. Also included are vectors that provide more than one of the above functions. An “expression vector” is a polynucleotide which, when introduced into an appropriate host cell, can be used for the transcription of mRNA that is translated into a polypeptide(s). In some embodiments, an “expression system” is a suitable host cell comprising an expression vector that can function to yield a desired expression product. The terms “treatment” or “treating,” and “ameliorating” may be used interchangeably herein. These terms refer to an approach for obtaining beneficial or desired results including but not limited to a therapeutic benefit. By “therapeutic benefit” is meant eradication or amelioration of the underlying disorder being treated. In some embodiments, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disease condition such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder. In some embodiments, a therapeutic benefit comprises slowing or halting the growth of one or more tumors. In some embodiments, a therapeutic benefit comprises reducing the size of one or more tumors. In some embodiments, a therapeutic benefit comprises eradicating one or more tumors from a subject. In some embodiments, a therapeutic benefit comprises effecting the death of cancer cells.

As used herein, the term “therapeutically effective amount” refers to an amount of a biologically active agent (such as a fusion protein provided herein, e.g., as part of a pharmaceutical composition), that is capable of having any detectable, beneficial effect on any symptom, aspect, measured parameter or characteristics of a disease state or condition when administered in one or repeated doses to a subject. Such effect need not be absolute to be beneficial. The disease condition can refer to a disorder or a disease, e.g., cancer or a symptom of cancer.

Antigen Binding Domains, Cleavage Sequences, Barcode Fragments, and Fusion Polypeptides

The present disclosure provides, inter alia, new and useful anti-PSMA antibodies, new and useful anti-CD3 antibodies, cleavage sequences, barcode fragments, and fusion proteins comprising the same. Included herein are fusion polypeptides comprising (i) one or more mask polypeptides (such as ELNNs), (ii) a bispecific antibody (BsAb, e.g., a TCE) linked to the mask polypeptide(s), and (iii) one or more protease-cleavable release segments (RS), wherein an RS is positioned between the mask polypeptide(s) and the BsAb.

In some embodiments, anti-PSMA antibodies provided herein include a VHH domain comprising the CDRs of a VHH domain comprising the sequence:

(SEQ ID NO: 549)

QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVG

AMSWSGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNKEYGRTWYDFNESDYWGQGTQVTVSS.

In some embodiments, anti-CD3 antibodies provided herein comprise a VH domain comprising the CDRs of a VH domain comprising the sequence:

(SEQ ID NO: 311)

EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVG

RIRTKRNNYATYYADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYC

VRHENFGNSYVSWFAHWGQGTLVTVSS.

and/or a VL domain comprising the CDRs of a VL domain comprising the sequence:

(SEQ ID NO: 361)

ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGL

IGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLW

VFGGGTKLTVL.

Also provided are BsAbs comprising, e.g., anti-PSMA antibodies and/or anti-CD3 antibodies disclosed herein. In some embodiments, the bispecific antibodies comprise an anti-PSMA VHH region disclosed herein. In some embodiments, the BsAbs comprise the VH and VL regions of an anti-CD3 antibody disclosed herein. In some embodiments, the BsAbs comprise an anti-PSMA VHH region herein and an anti-CD3 scFV comprising a VH and VL pair disclosed herein. In some embodiments, the BsAbs are TCEs.

In some embodiments, the fusion polypeptide comprises a first ELNN (such as an ELNN described herein). In some embodiments, the polypeptide further comprises a second ELNN (such as an ELNN described herein). In some embodiments, the polypeptide comprises an ELNN at or near its N-terminus (an “N-terminal ELNN”). In some embodiments, the polypeptide comprises an ELNN at or near its C-terminus (a “C-terminal ELNN”). In some embodiments, the polypeptide comprises both an N-terminal ELNN and a C-terminal ELNN.

In some embodiments, a fusion polypeptide comprises a BsAb and a first ELNN is attached to the N-terminus of the BsAb by a first RS and a second ELNN is attached to the C-terminus of the BsAb by a second RS. In some embodiments, each RS is cleavable by a protease mentioned herein. In some embodiments, each RS comprises an RS sequence disclosed herein. In some embodiments, the fusion polypeptide is a paTCE.

Included herein are polypeptide sequences that may be used, e.g., to link one polypeptide moiety to another within a fusion protein. For example, useful linkers are provided that are cleaved by multiple proteases but not legumain. In some embodiments, such linkers may be used outside the context of antibodies such as those described herein.

In some embodiments, a fusion polypeptide (e.g., one or more ELNNs of a paTCE and/or another portion of a fusion polypeptide such as a linker or spacer sequence) can comprise one or more barcode fragments (e.g., as described herein) releasable (e.g., configured to be released) the fusion polypeptide upon cleavage or digestion of the fusion polypeptide (e.g., a paTCE) by a protease. In some embodiments, the protease is a non-mammalian protease. In some embodiments, each barcode fragment differs in sequence and molecular weight from all other peptide fragments (including all other barcode fragments if present) that are releasable from the polypeptide upon complete digestion of the polypeptide by the protease, thereby making it unique and making its presence detectable through techniques such as mass spectrometry.

Extended Recombinant Polypeptides (ELNNS)
Chain Length and Amino Acid Composition

In some embodiments, an ELNN comprises at least 100, or at least 150 amino acids. In some embodiments, an ELNN is from 100 to 3,000, or from 150 to 3,000 amino acids in length. In some embodiments, an ELNN is from 100 to 1,000, or from 150 to 1,000 amino acids in length. In some embodiments, an ELNN is at least (about) 100, at least (about) 150, at least (about) 200, at least (about) 250, at least (about) 300, at least (about) 350, at least (about) 400, at least (about) 450, at least (about) 500, at least (about) 550, at least (about) 600, at least (about) 650, at least (about) 700, at least (about) 750, at least (about) 800, at least (about) 850, at least (about) 900, at least (about) 950, at least (about) 1,000, at least (about) 1,100, at least (about) 1,200, at least (about) 1,300, at least (about) 1,400, at least (about) 1,500, at least (about) 1,600, at least (about) 1,700, at least (about) 1,800, at least (about) 1,900, or at least (about) 2,000 amino acids in length. In some embodiments, an ELNN is at most (about) 100, at most (about) 150, at most (about) 200, at most (about) 250, at most (about) 300, at most (about) 350, at most (about) 400, at most (about) 450, at most (about) 500, at most (about) 550, at most (about) 600, at most (about) 650, at most (about) 700, at most (about) 750, at most (about) 800, at most (about) 850, at most (about) 900, at most (about) 950, at most (about) 1,000, at most (about) 1,100, at most (about) 1,200, at most (about) 1,300, at most (about) 1,400, at most (about) 1,500, at most (about) 1,600, at most (about) 1,700, at most (about) 1,800, at most (about) 1,900, or at most (about) 2,000 amino acids in length. In some embodiments, an ELNN has (about) 100, (about) 150, (about) 200, (about) 250, (about) 300, (about) 350, (about) 400, (about) 450, (about) 500, (about) 550, (about) 600, (about) 650, (about) 700, (about) 750, (about) 800, (about) 850, (about) 900, (about) 950, (about) 1,000, (about)1,100, (about) 1,200, (about) 1,300, (about) 1,400, (about) 1,500, (about) 1,600, (about) 1,700, (about) 1,800, (about) 1,900, or (about) 2,000 amino acids in length, or of a range between any two of the foregoing. In some embodiments, at least 90% of the amino acid residues of the ELNN are glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) or proline (P). In some embodiments, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the amino acid residues of the ELNN are glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) or proline (P). In some embodiments, an ELNN comprises at least 3 different types of amino acids selected from the group consisting of G, A, S, T, E, and P. In some embodiments, an ELNN comprises at least 4 different types of amino acids selected from the group consisting of G, A, S, T, E, and P. In some embodiments, an ELNN comprises at least 5 different types of amino acids selected from the group consisting of G, A, S, T, E, and P. In some embodiments, an ELNN consists of amino acids selected from the group consisting of G, A, S, T, E, and P. In some embodiments, an ELNN comprises G, A, S, T, E, or P amino acids. In some embodiments, an ELNN (e.g., ELNN1, ELNN2, etc.) is characterized in that: (i) it comprises at least 100, or at least 150 amino acids; (ii) at least 90% of the amino acid residues of the ELNN are glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) or proline (P); and (iii) it comprises at least 4 different types of the amino acids from G, A, S, T, E, or P. As used herein, the term “glutamate” is a synonym for “glutamic acid,” and refers to the glutamic acid residue whether or not the side-chain carboxyl is deprotonated. In some embodiments, the ELNN-containing fusion polypeptide comprises a first ELNN and a second ELNN. In some embodiments, the sum of the total number of amino acids in the first ELNN and the total number of amino acids in the second ELNN is at least 300, at least 350, at least 400, at least 500, at least 600, at least 700, or at least 800 amino acids.

Non-Overlapping Sequence Motif

In some embodiments, the ELNN comprises, or is formed from, a plurality of non-overlapping sequence motifs. In some embodiments, at least one of the non-overlapping sequence motifs is recurring (or repeated at least two times in the ELNN). In some embodiments, the ELNN comprises at least one other non-overlapping sequence motif that is non-recurring (or found only once within the ELNN). In some embodiments, the plurality of non-overlapping sequence motifs comprises (a) a set of (recurring) non-overlapping sequence motifs, wherein each non-overlapping sequence motif of the set of non-overlapping sequence motifs is repeated at least two times in the ELNN; and (b) a non-overlapping (non-recurring) sequence motif that occurs (or is found) only once within the ELNN. In some embodiments, each non-overlapping sequence motif is from 9 to 14 (or 10 to 14, or 11 to 13) amino acids in length. In some embodiments, each non-overlapping sequence motif is 12 amino acids in length. In some embodiments, the plurality of non-overlapping sequence motifs comprises a set of non-overlapping (recurring) sequence motifs, wherein each non-overlapping sequence motif of the set of non-overlapping sequence motifs is (1) repeated at least two times in the ELNN; and (2) is between 9 and 14 amino acids in length. In some embodiments, the set of (recurring) non-overlapping sequence motifs comprises 12-mer sequence motifs identified herein by SEQ ID NOs: 179-200 and 1715-1722 in Table 1. In some embodiments, the set of (recurring) non-overlapping sequence motifs comprise 12-mer sequence motifs identified herein by SEQ ID NOs: 186-189 in Table 1. In some embodiments, the set of (recurring) non-overlapping sequence motifs comprise at least two, at least three, or all four of 12-mer sequence motifs of SEQ ID NOs: 186-189 in Table 1. In some embodiments, an ELNN further comprises a sequence other than a 12-mer sequence motif shown in Table 1. In some embodiments, an ELNN comprises a sequence that is not in Table 1 such as ASSATPESGP(SEQ ID NO:9176), GSGPGTSESATP(SEQ ID NO:9018), or GTSESATP(SEQ ID NO:9022). In some embodiments, an ELNN comprises a sequence that is not in Table 1 such as ATPESGP(SEQ ID NO:9177), GTSPSATPESGP(SEQ ID NO:9019), or GTSESAGEPEA. In some embodiments, an ELNN comprises a barcode sequence.

TABLE 1

Exemplary 12-Mer Sequence Motifs for

Construction of ELNNs

Amino Acid
SEQ ID

Motif Family*
Sequence
NO.

AD
GESPGGSSGSES
182

AD
GSEGSSGPGESS
183

AD
GSSESGSSEGGP
184

AD
GSGGEPSESGSS
185

AE, AM
GSPAGSPTSTEE
186

AE, AM, AQ
GSEPATSGSETP
187

AE, AM, AQ
GTSESATPESGP
188

AE, AM, AQ
GTSTEPSEGSAP
189

AF, AM
GSTSESPSGTAP
190

AF, AM
GTSTPESGSASP
191

AF, AM
GTSPSGESSTAP
192

AF, AM
GSTSSTAESPGP
193

AG, AM
GTPGSGTASSSP
194

AG, AM
GSSTPSGATGSP
195

AG, AM
GSSPSASTGTGP
196

AG, AM
GASPGTSSTGSP
197

AQ
GEPAGSPTSTSE
198

AQ
GTGEPSSTPASE
199

AQ
GSGPSTESAPTE
200

AQ
GSETPSGPSETA
179

AQ
GPSETSTSEPGA
180

AQ
GSPSEPTEGTSA
181

BC
GSGASEPTSTEP
1715

BC
GSEPATSGTEPS
1716

BC
GTSEPSTSEPGA
1717

BC
GTSTEPSEPGSA
1718

BD
GSTAGSETSTEA
1719

BD
GSETATSGSETA
1720

BD
GTSESATSESGA
1721

BD
GTSTEASEGSAS
1722

*Denotes individual motif sequences that, when used together in various permutations, results in a ″family sequence″

Unstructured Polypeptide Confirmation

In various embodiments, an ELNN component (or the ELNN components) of a fusion protein has an unstructured conformation under physiological conditions, regardless of the length (e.g., extended length) of the polymer. For example, the ELNN is characterized by a large conformational freedom of the peptide backbone. In some embodiments, the ELNN is characterized by a lack of long-range interactions as determined by NMR. In some embodiments, the present disclosure provides ELNNs that, under physiologic conditions, resemble the structure of denatured sequences largely devoid in secondary structure. In some embodiments, the ELNNs can be substantially devoid of secondary structure under physiologic conditions. “Largely devoid,” as used in this context, means that less than 50% of the ELNN amino acid residues of the ELNN contribute to secondary structure as measured or determined by the means described herein. “Substantially devoid,” as used in this context, means that at least about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or at least about 99% of the ELNN amino acid residues of the ELNN sequence do not contribute to secondary structure, as measured or determined by the means described herein.

A variety of methods have been established in the art to discern the presence or absence of secondary and tertiary structures in a given polypeptide. In some embodiments, ELNN secondary structure can be measured spectrophotometrically, e.g., by circular dichroism spectroscopy in the “far-UV” spectral region (190-250 nm). Secondary structure elements, such as alpha-helix and beta-sheet, each give rise to a characteristic shape and magnitude of CD spectra. Secondary structure can also be predicted for a polypeptide sequence via certain computer programs or algorithms, such as the well-known Chou-Fasman algorithm (Chou, P. Y., et al. (1974) Biochemistry, 13: 222-45) and the Garnier-Osguthorpe-Robson (“GOR”) algorithm (Garnier J, Gibrat J F, Robson B. (1996), GOR method for predicting protein secondary structure from amino acid sequence. Methods Enzymol 266:540-553), as described in US Patent Application Publication No. 20030228309A1 (the entire contents of which are incorporated herein by reference). For a given sequence, the algorithms can predict whether there exists some or no secondary structure at all, expressed as the total and/or percentage of residues of the sequence that form, for example, alpha-helices or beta-sheets or the percentage of residues of the sequence predicted to result in random coil formation (which lacks secondary structure).

In some embodiments, the ELNNs used in a fusion protein composition can have an alpha-helix percentage ranging from 0% to less than about 5% as determined by a Chou-Fasman algorithm. In some embodiments, the ELNNs of the fusion protein compositions can have a beta-sheet percentage ranging from 0% to less than about 5% as determined by a Chou-Fasman algorithm. In some embodiments, the ELNNs of the fusion protein compositions can have an alpha-helix percentage ranging from 0% to less than about 5% and a beta-sheet percentage ranging from 0% to less than about 5% as determined by a Chou-Fasman algorithm. In some embodiments, the ELNNs of the fusion protein compositions will have an alpha-helix percentage less than about 2% and a beta-sheet percentage less than about 2%. In some embodiments, the ELNNs of the fusion protein compositions can have a high degree of random coil percentage, as determined by a GOR algorithm. In some embodiments, an ELNN can have at least about 80%, more preferably at least about 90%, more preferably at least about 91%, more preferably at least about 92%, more preferably at least about 93%, more preferably at least about 94%, more preferably at least about 95%, more preferably at least about 96%, more preferably at least about 97%, more preferably at least about 98%, and most preferably at least about 99% random coil, as determined by a GOR algorithm.

Net Charge

In some embodiments, the ELNN polypeptides can have an unstructured characteristic imparted by incorporation of amino acid residues with a net charge and/or reducing the proportion of hydrophobic amino acids in the ELNN sequence. The overall net charge and net charge density may be controlled, e.g., by modifying the content of charged amino acids in the ELNNs. In some embodiments, the net charge density of the ELNN of the compositions may be above +0.1 or below −0.1 charges/residue. In some embodiments, the net charge of a ELNN can be about 0%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10% about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20% or more.

Since most tissues and surfaces in a human or animal have a net negative charge, the ELNNs can optionally be designed to have a net negative charge to minimize non-specific interactions between the ELNN containing compositions and various surfaces such as blood vessels, healthy tissues, or various receptors. Not to be bound by a particular theory, an ELNN may adopt open conformations due to electrostatic repulsion between individual amino acids of the ELNN polypeptide that individually carry a high net negative charge and that are distributed across the sequence of the ELNN polypeptide. Such a distribution of net negative charge in the extended sequence lengths of ELNN can lead to an unstructured conformation that, in turn, can result in an effective increase in hydrodynamic radius. Accordingly, in some embodiments the ELNNs contain glutamic acid such that the glutamic acid is at about 8, 10, 15, 20, 25, or even about 30% of the amino acids in the sequences. The ELNN of the compositions of the present disclosure generally have no or a low content of positively charged amino acids. In some embodiments the ELNN may have less than about 10% amino acid residues with a positive charge, or less than about 7%, or less than about 5%, or less than about 2% amino acid residues with a positive charge. However, the present disclosure contemplates polypeptides where a limited number of amino acids with a positive charge, such as lysine, may be incorporated into an ELNN, e.g., to permit conjugation between the epsilon amine of the lysine and a reactive group on a peptide, a linker bridge, or a reactive group on a drug or small molecule to be conjugated to the ELNN backbone.

In some embodiments, an ELNN may comprise charged residues separated by other residues such as serine or glycine, which may lead to better expression or purification behavior. Based on the net charge, ELNNs of the subject compositions may have an isoelectric point (pl) of 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, or even 6.5. In some embodiments, the ELNN will have an isoelectric point between 1.5 and 4.5. In some embodiments, an ELNN incorporated into an paTCE fusion protein carries a net negative charge under physiologic conditions contributes to the unstructured conformation and reduced binding of the ELNN component to mammalian proteins and tissues.

As hydrophobic amino acids can impart structure to a polypeptide, in some embodiments the content of hydrophobic amino acids in the ELNN is less than 5%, or less than 2%, or less than 1% hydrophobic amino acid content. In some embodiments, an ELNN has no hydrophobic amino acids. In some embodiments, the amino acid content of methionine and tryptophan in the ELNN component of a paTCE fusion protein is less than 5%, or less than 2%, and most preferably less than 1%. In some embodiments, the ELNN has a sequence that has less than 10% amino acid residues with a positive charge, or less than about 7%, or less that about 5%, or less than about 2% amino acid residues with a positive charge, the sum of methionine and tryptophan residues will be less than 2%, and the sum of asparagine and glutamine residues will be less than 10% of the total ELNN sequence. In some embodiments, the ELNN has no methionine or tryptophan residues.

Increased Hydrodynamic Radius

In some embodiments, the ELNN can have a high hydrodynamic radius, conferring a corresponding increased Apparent Molecular Weight to the paTCE fusion protein which incorporates the ELNN. The linking of ELNNs to BsAb (e.g., TCE) sequences can result in paTCE compositions that can have increased hydrodynamic radii, increased Apparent Molecular Weight, and increased Apparent Molecular Weight Factor compared to BsAbs (e.g., TCEs) not linked to an ELNN. For example, in some therapeutic applications in which prolonged half-life is desired, one or more ELNNs with a high hydrodynamic radius are incorporated into a fusion protein comprising a BsAb (e.g., a TCE) to effectively enlarge the hydrodynamic radius of the fusion protein beyond the glomerular pore size of approximately 3-5 nm (corresponding to an apparent molecular weight of about 70 kDa) (Caliceti. 2003. Pharmacokinetic and biodistribution properties of poly(ethylene glycol)-protein conjugates. Adv. Drug Deliv. Rev. 55:1261-1277), resulting in reduced renal clearance of circulating proteins. In some embodiments, the hydrodynamic radius of a protein is determined by its molecular weight as well as by its structure, including shape and compactness. Not to be bound by a particular theory, the ELNN may adopt open conformations due to electrostatic repulsion between individual charges of the peptide or the inherent flexibility imparted by the particular amino acids in the sequence that lack potential to confer secondary structure. In some embodiments, the open, extended and unstructured conformation of the ELNN polypeptide has a greater proportional hydrodynamic radius compared to polypeptides of a comparable sequence length and/or molecular weight that have secondary and/or tertiary structure, such as typical globular proteins. Methods for determining the hydrodynamic radius are well known in the art, such as by the use of size exclusion chromatography (SEC), as described in U.S. Pat. Nos. 6,406,632 and 7,294,513. In some embodiments, the addition of increasing lengths of ELNN results in proportional increases in the parameters of hydrodynamic radius, Apparent Molecular Weight, and Apparent Molecular Weight Factor, permitting the tailoring of paTCE to desired characteristic cut-off Apparent Molecular Weights or hydrodynamic radii. Accordingly, in some embodiments, the paTCE fusion protein can be configured with an ELNN such that the fusion protein can have a hydrodynamic radius of at least about 5 nm, or at least about 8 nm, or at least about 10 nm, or 12 nm, or at least about 15 nm. In some embodiments, the large hydrodynamic radius conferred by the ELNN in an paTCE fusion protein can lead to reduced renal clearance of the resulting fusion protein, leading to a corresponding increase in terminal half-life, an increase in mean residence time, and/or a decrease in renal clearance rate.

In some embodiments, an ELNN (or multiple ELNNs, such as two ELNNs) of a chosen length and sequence can be selectively incorporated into a paTCE to create a fusion protein that will have, under physiologic conditions, an Apparent Molecular Weight of at least about 150 kDa, or at least about 300 kDa, or at least about 400 kDa, or at least about 500 kDa, or at least about 600 kDa, or at least about 700 kDa, or at least about 800 kDa, or at least about 900 kDa, or at least about 1000 kDa, or at least about 1200 kDa, or at least about 1500 kDa, or at least about 1800 kDa, or at least about 2000 kDa, or at least about 2300 kDa or more. In some embodiments, an ELNN (or multiple ELNNs, such as two ELNNs) of a chosen length and sequence can be selectively linked to a BsAb (e.g., a TCE) to result in a paTCE fusion protein that has, under physiologic conditions, an Apparent Molecular Weight Factor of at least 3, alternatively of at least 4, alternatively of at least 5, alternatively of at least 6, alternatively of at least 7, alternatively of at least 8, alternatively of at least 9, alternatively of at least 10, alternatively of at least 15, or an Apparent Molecular Weight Factor of at least 20 or greater. In some embodiments, the paTCE fusion protein has, under physiologic conditions, an Apparent Molecular Weight Factor that is about 4 to about 20, or is about 6 to about 15, or is about 8 to about 12, or is about 9 to about 10 relative to the actual molecular weight of the fusion protein. In some embodiments, the fusion polypeptide exhibits an apparent molecular weight factor under physiological conditions that is greater than about 6.

Increased Terminal Half-Life

In some embodiments, a fusion polypeptide comprising an ELNN (such as a paTCE) has a terminal half-life that is at least two-fold longer, or at least three-fold longer, or at least four-fold longer, or at least five-fold longer, compared to a corresponding biologically active polypeptide that is not linked to the ELNN. In some embodiments, the (fusion) polypeptide has a terminal half-life that is at least two-fold longer compared to the biologically active polypeptide not linked to the ELNN.

In some embodiments, administration of a therapeutically effective amount of a paTCE fusion protein to a subject in need thereof results in a gain in time of at least two-fold, or at least three-fold, or at least four-fold, or at least five-fold or more spent within a therapeutic window for the fusion protein compared to the corresponding BsAb (e.g., TCE) not linked to the ELNN(s) when administered at a comparable dose to a subject.

In some embodiments, a TCE released from a paTCE upon protease cleavage comprises one or more short polypeptides (e.g., about 30, 25, 20, 15, 14, 13, 12, 11, 10, or less amino acids in length) that has no amino acids other than G, A, P, E, S, and/or T. For example, a short polypeptide that has no amino acids other than G, A, P, E, S, and/or T might be incorporated into one or more spacer or linker sequences of the TCE, and/or a portion of one or more spacers or linkers that remain part of the TCE after cleavage. In some embodiments, a TCE that is released from a paTCE comprises a GTSESATPES(SEQ ID NO:96) on the N-terminal side (e.g., the closest amino acid of the sequence is within 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid positions of the N-terminal amino acid or the sequence includes the N-terminus) of the TCE. In some embodiments, a TCE that is released from a paTCE comprises a GTATPESGPG(SEQ ID NO:97) on the C-terminal side (e.g., the closest amino acid of the sequence is within 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid positions of the N-terminal amino acid or the sequence includes the N-terminus) of the TCE. In some embodiments, a TCE comprises an internal linker (e.g., between a VL region and a VH region of a scFV) that comprises a polypeptide sequence with no amino acids other than G, A, P, E, S, and/or T, such as

(SEQ ID NO: 81)

SESATPESGPGTSPGATPESGPGTSESATP.

Low Immunogenicity

In some embodiments, the present disclosure provides compositions in which the ELNNs have a low degree of immunogenicity or are substantially non-immunogenic. Several factors can contribute to the low immunogenicity of an ELNN, e.g., the substantially non-repetitive sequence, the unstructured conformation, the high degree of solubility, the low degree or lack of self-aggregation, the low degree or lack of proteolytic sites within the sequence, and the low degree or lack of epitopes in the ELNN.

One of ordinary skill in the art will understand that, in general, polypeptides having highly repetitive short amino acid sequences (e.g., wherein a 200 amino acid-long sequence contain on average 20 repeats or more of a limited set of 3- or 4-mers) and/or having contiguous repetitive amino acid residues (e.g., wherein 5- or 6-mer sequences have identical amino acid residues) have a tendency to aggregate or form higher order structures or form contacts resulting in crystalline or pseudo-crystalline structures.

In some embodiments, a ELNN sequence is substantially non-repetitive, wherein (1) the ELNN sequence has no three contiguous amino acids that are identical amino acid types, unless the amino acid is serine, in which case no more than three contiguous amino acids can be serine residues, and wherein (2) the ELNN contains no 3-amino acid sequences (3-mers) that occur more than 16, more than 14, more than 12, or more than 10 times within an at least 200 amino acid-long sequence of the ELNN (e.g., the entire span of an ELNN that is at least amino acids long). Without being bound by any scientific theory, such substantially non-repetitive sequences have less tendency to aggregate and, thus, enable the design of long-sequence ELNNs with a relatively low frequency of charged amino acids that would be likely to aggregate if the sequences or amino acid residues were otherwise more repetitive.

Conformational epitopes can be formed by regions of protein surfaces that are composed of multiple discontinuous amino acid sequences of a protein antigen. Without being bound by any scientific theory, the precise folding of the protein may bring these sequences into well-defined, stable spatial configurations or epitopes that can be recognized as “foreign” by the host humoral immune system, resulting in the production of antibodies to the protein and/or triggering a cell-mediated immune response. In the latter case, the immune response to a protein in an individual is heavily influenced by T-cell epitope recognition that is a function of the peptide binding specificity of that individual's HLA-DR allotype. Engagement of an MHC Class II peptide complex by a cognate T-cell receptor on the surface of the T-cell, together with the cross-binding of certain other co-receptors such as the CD4 molecule, can induce an activated state within the T-cell. Activation may lead to the release of cytokines further activating other lymphocytes such as B cells to produce antibodies or activating T killer cells as a full cellular immune response.

Without being bound by any scientific theory, the ability of a peptide to bind a given MHC Class II molecule for presentation on the surface of an APC (antigen presenting cell) may depend on a number of factors; most notably its primary sequence. In some embodiments, a lower degree of immunogenicity may be achieved by designing ELNNs that resist antigen processing in antigen presenting cells, and/or choosing sequences that do not bind MHC receptors well. In some embodiments, ELNN-containing fusion proteins have substantially non-repetitive ELNN polypeptides designed to reduce binding with MHC II receptors, as well as to avoid formation of epitopes for T-cell receptor or antibody binding, resulting in a low degree of immunogenicity. Without being bound by any scientific theory, avoidance of immunogenicity is, in part, a direct result of the conformational flexibility of ELNNs; i.e., the lack of secondary structure due to the selection and order of amino acid residues. For example, of particular interest are sequences having a low tendency to adapt compactly folded conformations in aqueous solution or under physiologic conditions that could result in conformational epitopes. The administration of fusion proteins comprising ELNNs, using conventional therapeutic practices and dosing, would generally not result in the formation of neutralizing antibodies to the ELNNs, and may also reduce the immunogenicity of BsAb (e.g., TCE) fusion partners in paTCE compositions.

In some embodiments, the ELNNs utilized in the subject fusion proteins can be substantially free of epitopes recognized by human T cells. The elimination of such epitopes for the purpose of generating less immunogenic proteins has been disclosed previously, see for example WO 98/52976, WO 02/079232, and WO 00/3317 which are incorporated by reference herein. Assays for human T cell epitopes have been described (Stickler, M., et al. (2003) J Immunol Methods, 281: 95-108). Of particular interest are peptide sequences that can be oligomerized without generating T cell epitopes or non-human sequences. This can be achieved by testing direct repeats of these sequences for the presence of T-cell epitopes and for the occurrence of 6 to 15-mer and, in particular, 9-mer sequences that are not human, and then altering the design of the ELNN sequence to eliminate or disrupt the epitope sequence. In some embodiments, the ELNNs are substantially non-immunogenic by the restriction of the numbers of epitopes of the ELNN predicted to bind MHC receptors. With a reduction in the numbers of epitopes capable of binding to MHC receptors, there is a concomitant reduction in the potential for T cell activation as well as T cell helper function, reduced B cell activation or upregulation and reduced antibody production. The low degree of predicted T-cell epitopes can be determined by epitope prediction algorithms such as, e.g., TEPITOPE (Sturniolo, T., et al. (1999) Nat Biotechnol, 17: 555-61), as shown in Example 74 of International Patent Application Publication No. WO 2010/144502 A2, which is incorporated by reference in its entirety. Aspects of the TEPITOPE score of a given peptide frame within a protein are disclosed in Sturniolo, T. et al. (1999) Nature Biotechnology 17:555). The score ranges over at least 20 logs, from about 10 to about −10 (corresponding to binding constraints of 10e¹⁰K_Dto 10e⁻¹⁰K_D), and can be reduced by avoiding hydrophobic amino acids that can serve as anchor residues during peptide display on MHC, such as M, I, L, V, or F. In some embodiments, an ELNN component incorporated into a paTCE does not have a predicted T-cell epitope at a TEPITOPE score of about −5 or greater, or −6 or greater, or −7 or greater, or −8 or greater, or at a TEPITOPE score of −9 or greater. As used herein, a score of “−9 or greater” would encompass TEPITOPE scores of 10 to −9, inclusive, but would not encompass a score of −10, as −10 is less than −9.

In some embodiments, the ELNNs, including those incorporated into the subject paTCE fusion proteins, can be rendered substantially non-immunogenic by the restriction of known proteolytic sites from the sequence of the ELNN, reducing the processing of ELNN into small peptides that can bind to MHC II receptors. In some embodiments, the ELNN sequence can be rendered substantially non-immunogenic by the use a sequence that is substantially devoid of secondary structure, conferring resistance to many proteases due to the high entropy of the structure. Accordingly, the reduced TEPITOPE score and elimination of known proteolytic sites from the ELNN may render the ELNN compositions, including the ELNN of the paTCE fusion protein compositions, substantially unable to be bound by mammalian receptors, including those of the immune system. In some embodiments, an ELNN of a paTCE fusion protein can have >100 nM K_Dbinding to a mammalian receptor, or greater than 500 nM K_D, or greater than 1 μM K_Dtowards a mammalian cell surface or circulating polypeptide receptor.

Additionally, the substantially non-repetitive sequence and corresponding lack of epitopes of such embodiments of ELNNs can limit the ability of B cells to bind to or be activated by the ELNNs. In some embodiments, while an ELNN can make contacts with many different B cells over its extended sequence, each individual B cell may only make one or a small number of contacts with an individual ELNN. As a result, ELNNs typically may have a much lower tendency to stimulate proliferation of B cells and thus an immune response. In some embodiments, the paTCE may have reduced immunogenicity as compared to the corresponding BsAb (e.g., TCE) that is not fused to a mask polypeptide such as an ELNN. In some embodiments, the administration of up to three parenteral doses of a paTCE to a mammal may result in detectable anti-paTCE IgG at a serum dilution of 1:100 but not at a dilution of 1:1000. In some embodiments, the administration of up to three parenteral doses of an paTCE to a mammal may result in detectable anti-BsAb (e.g., TCE) IgG at a serum dilution of 1:100 but not at a dilution of 1:1000. In some embodiments, the administration of up to three parenteral doses of an paTCE to a mammal may result in detectable anti-ELNN IgG at a serum dilution of 1:100 but not at a dilution of 1:1000. In some embodiments, the mammal can be, e.g., a mouse, a rat, a rabbit, cynomolgus monkey, or human. In some embodiments, the mammal is a human.

An additional feature of certain ELNNs with substantially non-repetitive sequences relative to those less non-repetitive sequences (such as one having three contiguous amino acids that are identical) can be that non-repetitive ELNNs form weaker contacts with antibodies (e.g., monovalent interactions), thereby resulting in less likelihood of immune clearance such that the paTCE compositions can remain in circulation for an increased period of time.

In some embodiments, a biologically active polypeptide (such as a BsAb, e.g., a TCE) comprising an ELNN is less immunogenic compared to the fusion polypeptide not linked to any ELNN, wherein immunogenicity is ascertained by measuring production of IgG antibodies that selectively bind to the biologically active polypeptide after administration of comparable doses to a subject.

Barcode Fragment

In some embodiments, a polypeptide (e.g., a fusion polypeptide or a portion thereof such as an ELNN) comprises one or more barcode fragments (e.g., a first, second, or third barcode fragment) releasable from the polypeptide upon digestion by a protease. In some embodiments, the protease is a non-mammalian protease. In some embodiments, the protease is a prokaryotic protease. As used herein, the term “barcode fragment” (or “barcode,” or “barcode sequence”) can refer to either the portion of the polypeptide cleavably fused within the polypeptide, or the resulting peptide fragment released from the polypeptide.

In some embodiments, a barcode fragment (1) is a portion of an ELNN that includes at least part of the (non-recurring, non-overlapping) sequence motif that occurs (or is found) only once within the ELNN; and (2) differs in sequence and molecular weight from all other peptide fragments that are releasable from the polypeptide upon cleavage or complete digestion of the polypeptide by the protease.

In some embodiments, a barcode fragment does not include the N-terminal amino acid or the C-terminal amino acid of the fusion polypeptide. As described herein, in some embodiments, a barcode fragment is releasable (e.g., configured to be released) upon Glu-C digestion of the fusion polypeptide. In some embodiments, a barcode fragment is in an ELNN and does not include a glutamic acid that is immediately adjacent to another glutamic acid, if present, in the ELNN. In some embodiments, a barcode fragment has a glutamic acid at its C-terminus. One of ordinary skill in the art will understand that the C-terminus of a barcode fragment can refer to the “last” (or the most C-terminal) amino acid residue within the barcode fragment, when cleavably fused within a polypeptide (such as an ELNN), even if other non-barcode amino acid residues are positioned C-terminal to the barcode fragment within the polypeptide (e.g., ELNN). In some embodiments, a barcode fragment has an N-terminal amino acid that is immediately preceded by a glutamic acid residue. In some embodiments, the glutamic acid residue that precedes the N-terminal amino acid is not immediately adjacent to another glutamic acid residue. In some embodiments, a barcode fragment does not include a (second) glutamic acid residue at a position other than the C-terminus of the barcode fragment unless the glutamic acid is immediately followed by a proline. In some embodiments, a barcode fragment is positioned a distance from either the N-terminus of the polypeptide or the C-terminus of the polypeptide, wherein the distance is from 10 to 150, or 10 to 125 amino acids. In some embodiments, a barcode fragment is positioned within, or at a location of, 300, 280, 260, 250, 240, 220, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 48, 40, 36, 30, 24, 20, 12, or 10 amino acids from the N-terminus of the polypeptide, or at a location in a range between any of the foregoing. In some embodiments, a barcode fragment is positioned within 200, within 150, within 100, or within 50 amino acids of the N-terminus of the polypeptide. In some embodiments, a barcode fragment is positioned at a location that is between 10 and 200, between 30 and 200, between 40 and 150, or between 50 and 100 amino acids from the N-terminus of the polypeptide. In some embodiments, a barcode fragment is positioned within, or at a location of, 300, 280, 260, 250, 240, 220, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 48, 40, 36, 30, 24, 20, 12, or 10 amino acids from the C-terminus of the polypeptide, or at a location in a range between any of the foregoing. In some embodiments, a barcode fragment is positioned within 200, within 150, within 100, or within 50 amino acids of the C-terminus of the polypeptide. In some embodiments, a barcode fragment is positioned at a location that is between 10 and 200, between 30 and 200, between 40 and 150, or between 50 and 100 amino acids from the C-terminus of the polypeptide. In some embodiments, a barcode fragment (BAR) is characterized in that: (i) it does not include a glutamic acid that is immediately adjacent to another glutamic acid, if present, in the ELNN; (ii) it has a glutamic acid at its C-terminus; (iii) it has an N-terminal amino acid that is immediately preceded by a glutamic acid residue; and (iv) it is positioned a distance from either the N-terminus of the polypeptide or the C-terminus of the polypeptide, wherein the distance is from 10 to 150 amino acids, or from 10 to 125 amino acids in length. In some embodiments, a barcode fragment is in an ELNN and (i) does not include the N-terminal amino acid or the C-terminal amino acid of the polypeptide; (ii) does not include a glutamic acid that is immediately adjacent to another glutamic acid in the ELNN; (iii) has a glutamic acid at its C-terminus; (iv) has an N-terminal amino acid that is immediately preceded by a glutamic acid residue; and (v) is positioned a distance from either the N-terminus of the polypeptide or the C-terminus of the polypeptide, wherein the distance is from 10 to 150, or 10 to 125 amino acids in length. In some embodiments, the glutamic acid residue that precedes the N-terminal amino acid is not immediately adjacent to another glutamic acid residue. In some embodiments, a barcode fragment does not include a glutamic acid residue at a position other than the C-terminus of the barcode fragment unless the glutamic acid is immediately followed by a proline. Depending on context herein and when referring to placement within a polypeptide sequence, the term “distance” can refer to the number of amino acid residues from the N-terminus of the polypeptide to the most N-terminal amino acid residue of the barcode fragment, or from the C-terminus of the polypeptide to the most C-terminal amino acid residue of the barcode fragment. In some embodiments, for a barcoded ELNN fused to a biologically active polypeptide, at least one barcode fragment (or at least two barcode fragments, or three barcode fragments) contained in the barcoded ELNN is positioned at least 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300 amino acids from the biologically active polypeptide. In some embodiments, a barcode fragment is at least 4, at least 5, at least 6, at least 7, or at least 8 amino acids in length. In some embodiments, a barcode fragment is at least 4 amino acids in length. In some embodiments, a barcode fragment is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 amino acids in length, or in a range between any of the foregoing values. In some embodiments, a barcode fragment is between 4 and 20, between 5 and 15, between 6 and 12, or between 7 and 10 amino acids in length. In some embodiments, a barcode fragment comprises an amino acid sequence identified herein by SEQ ID NOs: 68-79 and SEQ ID NOs: 1010-1027 in Table 2.

TABLE 2

Exemplary Barcode Fragments Releasable

Upon Glu-C Digest

Amino Acid

Sequence
SEQ ID NO:

SPATSGSTPE
68

GSAPATSE
69

GSAPGTATE
70

GSAPGTE
71

PATSGPTE
72

SASPE
73

PATSGSTE
74

GSAPGTSAE
75

SATSGSE
76

SGPGSTPAE
77

SGPGSGPGTSE
78

SGPGTSPSATPE
79

SGPGTGTSATPE
1010

SGPGTTPGTTPE
1011

SGPGTPPTSTPE
1012

SGPGTGSAGTPE
1013

SGPGTGGAGTPE
1014

SGPGTSPGATPE
1015

SGPGTSGSGTPE
1016

SGPGTSSASTPE
1017

SGPGTGAGTTPE
1018

SGPGTGSTSTPE
1019

TPGSEPATSGSE
1020

GSAPGTSTEPSE
1021

SGPGTAGSGTPE
1022

SGPGTSSGGTPE
1023

SGPGTAGPATPE
1024

SGPGTPGTGTPE
1025

SGPGTGGPTTPE
1026

SGPGTGSGSTPE
1027

In some embodiments, each barcode fragment differs in both sequence and molecular weight from all other peptide fragments that are releasable from the chimeric polypeptides described herein upon complete digestion the chimeric polypeptide by a non-mammalian protease. In some embodiments, the non-mammalian protease is Glu-C.

In some embodiments, the chimeric polypeptides disclosed herein comprises a Glu-C cleavage site comprising one of the following amino acid sequences: ATPESGPG(SEQ ID NO:9020), SGSETPGT(SEQ ID NO:9021), and GTSESATP(SEQ ID NO:9022).

In some embodiments, the chimeric polypeptides disclosed herein comprises at least one of the following amino acid sequences: PE.GSX_nPE.SG(SEQ ID NO:9392), PE.GSX_nSE.GG(SEQ ID NO:9393), PE.GSX_nSE.TG(SEQ ID NO:9395), PE.GSX_nSE.SA(SEQ ID NO:9396), PE.SGX_nPE.SG(SEQ ID NO:9397), PE.SGX_nSE.GG(SEQ ID NO:9399), PE.SGX_nSE.TG(SEQ ID NO:9400), PE.SGX_nSE.SA(SEQ ID NO:9401), and PE.TPX_nPE.SG(SEQ ID NO:9403), PE.TPX_nSE.GG(SEQ ID NO:9404), PE.TPX_nSE.TG(SEQ ID NO:9405), PE.TPX_nSE.SA(SEQ ID NO:9407), wherein each “.” is a Glu-C cleavage site and n is any integer from 0 to 50. In some embodiments, the chimeric polypeptides disclosed herein comprises at least one of the following amino acid sequences: PE.SGX_nPE.SG(SEQ ID NO:9398), PE.GSX_nSE.GG(SEQ ID NO:9394), PE.TPX_nSE.TG(SEQ ID NO:9406), PE.SGX_nSE.SA(SEQ ID NO:9402). In some embodiments, n is any integer from 1 to 20. In some embodiments, n is any integer from 5 to 15. In some embodiments, n is any integer from 5 to 10. In some embodiments, n is 9. In some embodiments, n is any integer from 5 to 15. In some embodiments, X_nis SGPGTGTSATPE(SEQ ID NO:1010), SGPGSGPGTSE(SEQ ID NO:78), SGPGTTPGTTPE(SEQ ID NO:1011), SGPGTPPTSTPE(SEQ ID NO:1012), SGPGTSPSATPE(SEQ ID NO:79), SGPGTGSAGTPE(SEQ ID NO:1013), SGPGTGGAGTPE(SEQ ID NO:1014), SGPGTSPGATPE(SEQ ID NO:1015), SGPGTSGSGTPE(SEQ ID NO:1016), SGPGTSSASTPE(SEQ ID NO:1017), SGPGTGAGTTPE(SEQ ID NO:1018), SGPGTGSTSTPE(SEQ ID NO:1019), TPGSEPATSGSE(SEQ ID NO:1020), GSAPGTSTEPSE(SEQ ID NO:1021), SGPGTAGSGTPE(SEQ ID NO:1022), SGPGTSSGGTPE(SEQ ID NO:1023), SGPGTAGPATPE(SEQ ID NO:1024), SGPGTPGTGTPE(SEQ ID NO:1025), SGPGTGGPTTPE(SEQ ID NO:1026), or SGPGTGSGSTPE(SEQ ID NO:1027).

In some embodiments, a chimeric polypeptide comprises at least one of the following amino acid sequences: SGPE.SGPGX_nSGPE.SGPG(SEQ ID NO:9023), SGPE.SGPGX_nATPE.SGPG(SEQ ID NO:9024), SGPE.SGPGX_nGTSE.SATP(SEQ ID NO:9025), SGPE.SGPGX_nTTPE.SGPG(SEQ ID NO:9026), SGPE.SGPGX_nSTPE.SGPG(SEQ ID NO:9027), SGPE.SGPGX_nGTPE.SGPG(SEQ ID NO:9028), SGPE.SGPGX_nGTPE.TPGS(SEQ ID NO:9029), SGPE.SGPGX_nGTPE.TPGS(SEQ ID NO:9029), SGPE.SGPGX_nSGSE.TGTP(SEQ ID NO:9030), SGPE.SGPGX_nGTPE.GSAP(SEQ ID NO:9031), SGPE.SGPGX_nEPSE.SATP(SEQ ID NO:9032), ATPE.SGPGX_nSGPE.SGPG(SEQ ID NO:9033), ATPE.SGPGX_nATPE.SGPG(SEQ ID NO:9034), ATPE.SGPGX_nGTSE.SATP(SEQ ID NO:9035), ATPE.SGPGX_nTTPE.SGPG(SEQ ID NO:9036), ATPE.SGPGX_nSTPE.SGPG(SEQ ID NO:9037), ATPE.SGPGX_nGTPE.SGPG(SEQ ID NO:9043), ATPE.SGPGX_nGTPE.TPGS(SEQ ID NO:9045), ATPE.SGPGX_nSGSE.TGTP(SEQ ID NO:9046), ATPE.SGPGX_nGTPE.GSAP(SEQ ID NO:9047), ATPE.SGPGX_nEPSE.SATP(SEQ ID NO:9048), GTSE.SATPX_nSGPE.SGPG(SEQ ID NO:9049), GTSE.SATPX_nATPE.SGPG(SEQ ID NO:9050), GTSE.SATPX_nGTSE.SATP(SEQ ID NO:9051), GTSE.SATPX_nTTPE.SGPG(SEQ ID NO:9052), GTSE.SATPX_nSTPE.SGPG(SEQ ID NO:9053), GTSE.SATPX_nGTPE.SGPG(SEQ ID NO:9054), GTSE.SATPX_nGTPE.TPGS(SEQ ID NO:9055), GTSE.SATPX_nSGSE.TGTP(SEQ ID NO:9056), GTSE.SATPX_nGTPE.GSAP(SEQ ID NO:9057), GTSE.SATPX_nEPSE.SATP(SEQ ID NO:9058), TTPE.SGPGX_nSGPE.SGPG(SEQ ID NO:9059), TTPE.SGPGX_nATPE.SGPG(SEQ ID NO:9060), TTPE.SGPGX_nGTSE.SATP(SEQ ID NO:9061), TTPE.SGPGX_nTTPE.SGPG(SEQ ID NO:9062), TTPE.SGPGX_nSTPE.SGPG(SEQ ID NO:9064), TTPE.SGPGX_nGTPE.SGPG(SEQ ID NO:9065), TTPE.SGPGX_nGTPE.TPGS(SEQ ID NO:9066), TTPE.SGPGX_nSGSE.TGTP(SEQ ID NO:9067), TTPE.SGPGX_nGTPE.GSAP(SEQ ID NO:9068), TTPE.SGPGX_nEPSE.SATP(SEQ ID NO:9069), STPE.SGPGX_nSGPE.SGPG(SEQ ID NO:9070), STPE.SGPGX_nATPE.SGPG(SEQ ID NO:9071), STPE.SGPGX_nGTSE.SATP(SEQ ID NO:9072), STPE.SGPGX_nTTPE.SGPG(SEQ ID NO:9073), STPE.SGPGX_nSTPE.SGPG(SEQ ID NO:9074), STPE.SGPGX_nGTPE.SGPG(SEQ ID NO:9076), STPE.SGPGX_nGTPE.TPGS(SEQ ID NO:9077), STPE.SGPGX_nSGSE.TGTP(SEQ ID NO:9078), STPE.SGPGX_nGTPE.GSAP(SEQ ID NO:9079), STPE.SGPGX_nEPSE.SATP(SEQ ID NO:9080), GTPE.SGPGX_nSGPE.SGPG(SEQ ID NO:9081), GTPE.SGPGX_nATPE.SGPG(SEQ ID NO:9082), GTPE.SGPGX_nGTSE.SATP(SEQ ID NO:9083), GTPE.SGPGX_nTTPE.SGPG(SEQ ID NO:9084), GTPE.SGPGX_nSTPE.SGPG(SEQ ID NO:9086), GTPE.SGPGX_nGTPE.SGPG(SEQ ID NO:9088), GTPE.SGPGX_nGTPE.TPGS(SEQ ID NO:9090), GTPE.SGPGX_nSGSE.TGTP(SEQ ID NO:9091), GTPE.SGPGX_nGTPE.GSAP(SEQ ID NO:9092), GTPE.SGPGX_nEPSE.SATP(SEQ ID NO:9093), GTPE.TPGSX_nSGPE.SGPG(SEQ ID NO:9094), GTPE.TPGSX_nATPE.SGPG(SEQ ID NO:9095), GTPE.TPGSX_nGTSE.SATP(SEQ ID NO:9096), GTPE.TPGSX_nTTPE.SGPG(SEQ ID NO:9097), GTPE.TPGSX_nSTPE.SGPG(SEQ ID NO:9098), GTPE.TPGSX_nGTPE.SGPG(SEQ ID NO:9099), GTPE.TPGSX_nGTPE.TPGS(SEQ ID NO:9100), GTPE.TPGSX_nSGSE.TGTP(SEQ ID NO:9101), GTPE.TPGSX_nGTPE.GSAP(SEQ ID NO:9103), GTPE.TPGSX_nEPSE.SATP(SEQ ID NO:9104), SGSE.TGTPX_nSGPE.SGPG(SEQ ID NO:9105), SGSE.TGTPX_nATPE.SGPG(SEQ ID NO:9106), SGSE.TGTPX_nGTSE.SATP(SEQ ID NO:9107), SGSE.TGTPX_nTTPE.SGPG(SEQ ID NO:9108), SGSE.TGTPX_nSTPE.SGPG(SEQ ID NO:9109), SGSE.TGTPX_nGTPE.SGPG(SEQ ID NO:9110), SGSE.TGTPX_nGTPE.TPGS(SEQ ID NO:9111), SGSE.TGTPX_nSGSE.TGTP(SEQ ID NO:9112), SGSE.TGTPX_nGTPE.GSAP(SEQ ID NO:9113), SGSE.TGTPX_nEPSE.SATP(SEQ ID NO:9114), GTPE.GSAPX_nSGPE.SGPG(SEQ ID NO:9115), GTPE.GSAPX_nATPE.SGPG(SEQ ID NO:9116), GTPE.GSAPX_nGTSE.SATP(SEQ ID NO:9117), GTPE.GSAPX_nTTPE.SGPG(SEQ ID NO:9118), GTPE.GSAPX_nSTPE.SGPG(SEQ ID NO:9119), GTPE.GSAPX_nGTPE.SGPG(SEQ ID NO:9120), GTPE.GSAPX_nGTPE.TPGS(SEQ ID NO:9121), GTPE.GSAPX_nSGSE.TGTP(SEQ ID NO:9122), GTPE.GSAPX_nGTPE.GSAP(SEQ ID NO:9123), GTPE.GSAPX_nEPSE.SATP(SEQ ID NO:9124), EPSE.SATPX_nSGPE.SGPG(SEQ ID NO:9126), EPSE.SATPX_nATPE.SGPG(SEQ ID NO:9127), EPSE.SATPX_nGTSE.SATP(SEQ ID NO:9128), EPSE.SATPX_nTTPE.SGPG(SEQ ID NO:9129), EPSE.SATPX_nSTPE.SGPG(SEQ ID NO:9130), EPSE.SATPX_nGTPE.SGPG(SEQ ID NO:9131), EPSE.SATPX_nGTPE.TPGS(SEQ ID NO:9132), EPSE.SATPX_nSGSE.TGTP(SEQ ID NO:9133), EPSE.SATPX_nGTPE.GSAP(SEQ ID NO:9134), or EPSE.SATPX_nEPSE.SATP(SEQ ID NO:9135), wherein each “.” is a Glu-C cleavage site and n is any integer from 0 to 50. In some embodiments, the chimeric polypeptide comprises at least one of the following amino acid sequences: SGPE.SGPGX_nATPE.SGPG(SEQ ID NO:9038), ATPE.SGPGX_nGTSE.SATP(SEQ ID NO:9040), ATPE.SGPGX_nTTPE.SGPG(SEQ ID NO:9041), ATPE.SGPGX_nSTPE.SGPG(SEQ ID NO:9042), ATPE.SGPGX_nATPE.SGPG(SEQ ID NO:9039), ATPE.SGPGX_nGTPE.SGPG(SEQ ID NO:9044), ATPE.SGPGX_nGTPE.SGPG(SEQ ID NO:9044), ATPE.SGPGX_nATPE.SGPG(SEQ ID NO:9039), GTPE.SGPGX_nGTPE.SGPG(SEQ ID NO:9089), GTPE.SGPGX_nSTPE.SGPG(SEQ ID NO:9087), GTPE.SGPGX_nTTPE.SGPG(SEQ ID NO:9085), GTPE.SGPGX_nSTPE.SGPG(SEQ ID NO:9087), GTPE.TPGSX_nSGSE.TGTP(SEQ ID NO:9102), GTPE.GSAPX_nEPSE.SATP(SEQ ID NO:9125), ATPE.SGPGX_nGTPE.SGPG(SEQ ID NO:9044), ATPE.SGPGX_nGTPE.SGPG(SEQ ID NO:9044), ATPE.SGPGX_nATPE.SGPG(SEQ ID NO:9039), ATPE.SGPGX_nGTPE.SGPG(SEQ ID NO:9044), TTPE.SGPGX_nTTPE.SGPG(SEQ ID NO:9063), or STPE.SGPGX_nSTPE.SGPG(SEQ ID NO:9075), wherein each “.” is a Glu-C cleavage site and n is any integer from 0 to 30. In some embodiments, n is any integer from 1 to 20. In some embodiments, n is any integer from 5 to 15. In some embodiments, n is any integer from 3 to 7. In some embodiments, n is any integer from 5 to 10. In some embodiments, n is 9. In some embodiments, n is 4. In some embodiments, n is any integer from 5 to 15. In some embodiments, wherein X_nis PGTGTSAT(SEQ ID NO:9136), PGSGPGT(SEQ ID NO:9137), PGTTPGTT(SEQ ID NO:9138), PGTPPTST(SEQ ID NO:9139), PGTSPSAT(SEQ ID NO:9140), PGTGSAGT(SEQ ID NO:9141), PGTGGAGT(SEQ ID NO:9142), PGTSPGAT(SEQ ID NO:9143), PGTSGSGT(SEQ ID NO:9144), PGTSSAST(SEQ ID NO:9145), PGTGAGTT(SEQ ID NO:9146), PGTGSTST(SEQ ID NO:9147), GSEPATSG(SEQ ID NO:9148), APGTSTEP(SEQ ID NO:9149), PGTAGSGT(SEQ ID NO:9150), PGTSSGGT(SEQ ID NO:9151), PGTAGPAT(SEQ ID NO:9152), PGTPGTGT(SEQ ID NO:9153), PGTGGPTT(SEQ ID NO:9154), or PGTGSGST(SEQ ID NO:9155). In some embodiments, X_nis TGTS(SEQ ID NO:9156), SGP, TTPG(SEQ ID NO:9157), TPPT(SEQ ID NO:9158), TSPS(SEQ ID NO:9159), TGSA(SEQ ID NO:9160), TGGA(SEQ ID NO:9161), TSPG(SEQ ID NO:9162), TSGS(SEQ ID NO:9163), TSSA(SEQ ID NO:9164), TGAG(SEQ ID NO:9165), TGST(SEQ ID NO:9166), EPAT(SEQ ID NO:9167), GTST(SEQ ID NO:9168), TAGS(SEQ ID NO:9169), TSSG(SEQ ID NO:9170), TAGP(SEQ ID NO:9171), TPGT(SEQ ID NO:9172), TGGP(SEQ ID NO:9173), or TGSG(SEQ ID NO:9174).

In some embodiments, barcodes are designed to have improved analytical properties. In some embodiments, such barcodes can be released with relatively modest concentrations of a non-mammalian protease such as Glu-C. This facilitates better detection, e.g., through LC/MS, and also allows measurement of peptides that are generated from the cleavable linker thereby allowing a measurement of cleavage products using, e.g., LC/MS.

In some embodiments of fusion proteins comprising an ELNN, the fusion protein has a single polypeptide chain, and the polypeptide chain comprises a barcode fragment that is at a position within the polypeptide chain that is from 10 to 200 amino acids or from 10 to 125 amino acids from the N-terminus or the C-terminus of the polypeptide chain. In some embodiments, a fusion protein (such as a paTCE) comprises a first ELNN and a second ELNN, the first ELNN is at the N-terminal side of the bispecific antibody domain, and the first barcode fragment is positioned within 200, 150, 100, or 50 amino acids of the N-terminus of the fusion protein. In some embodiments, the second ELNN is at the C-terminal side of the bispecific antibody domain, and the second barcode fragment is positioned within 200, 150, 100, or 50 amino acids of the C-terminus of the chimeric polypeptide.

In some embodiments, an ELNN further comprises one or more additional barcode fragments, wherein the one or more additional barcode fragments each differs in sequence and molecular weight from all other peptides fragments that are releasable from the polypeptide upon complete digestion of the polypeptide by the protease. In some embodiments, a barcoded ELNN comprises only one barcode fragment. In some embodiments, a barcoded ELNN comprises a set of barcode fragments, comprising a first barcode fragment, such as those described herein. In some embodiments, the set of barcode fragments comprises a second barcode fragment (or a further barcode fragment), such as those described herein. In some embodiments, the set of barcode fragments comprises a third barcode fragment, such as those described herein.

A set of barcode fragments fused within an N-terminal ELNN can be referred to as an N-terminal set of barcodes (an “N-terminal set”). A set of barcode fragments fused within a C-terminal ELNN can be referred to as a C-terminal set of barcodes (a “C-terminal set”). In some embodiments, the N-terminal set comprises a first barcode fragment and a second barcode fragment. In some embodiments, the N-terminal set further comprises a third barcode fragment. In some embodiments, the C-terminal set comprises a first barcode fragment and a second barcode fragment. In some embodiments, the C-terminal set further comprises a third barcode fragment. In some embodiments, the polypeptide comprises a set of barcode fragments that includes a first barcode fragment, a further (second) barcode fragment, and at least one additional barcode fragment, wherein each barcode fragment of the set of barcode fragments (1) is a portion of the second ELNN and (2) differs in sequence and molecular weight from all other peptides fragments that are releasable from the polypeptide upon complete digestion of the polypeptide by the protease.

Included herein is a mixture comprising a plurality of polypeptides of varying length, the mixture comprising a first set of polypeptides and a second set of polypeptides. In some embodiments, each polypeptide of the first set of polypeptides comprises a barcode fragment that (a) is releasable from the polypeptide by digestion with a protease and (b) has a sequence and molecular weight that differs from the sequence and molecular weight of all other fragments that are releasable from the first set of polypeptides. In some embodiments, the second set of polypeptides lack the barcode fragment of the first set of polypeptides (e.g., due to truncation). In some embodiments, both the first set of polypeptides and the second set of polypeptides each comprise a reference fragment that (a) is common to the first set of polypeptides and the second set of polypeptides and (b) releasable by digestion with the protease. In some embodiments, the ratio of the first set of polypeptides to polypeptides comprising the reference fragment is greater than 0.70. In some embodiments, the ratio of the first set of polypeptides to polypeptides comprising the reference fragment is greater than 0.80, 0.90, 0.95, or 0.98. In some embodiments, the reference fragment occurs no more than once in each polypeptide of the first set of polypeptides and the second set of polypeptides. In some embodiments, the protease is a protease that cleaves on the C-terminal side of glutamic acid residues. In some embodiments, the protease is a Glu-C protease. In some embodiments, the protease is not trypsin. In some embodiments, the polypeptides of varying lengths comprise polypeptides comprising at least one ELNN, such as any described herein. In some embodiments, the first set of polypeptides comprises a full-length polypeptide, wherein the barcode fragment is a portion of the full-length polypeptide. In some embodiments, the full-length polypeptide is a (fusion) polypeptide, such as any described hereinabove or described anywhere else herein. In some embodiments, the polypeptides of varying lengths in a mixture differ from one another due to N-terminal truncation, C-terminal truncation, or both N- and C-terminal truncation of a full-length polypeptide. In some embodiments, the first set of polypeptides and the second set of polypeptides may differ in one or more pharmacological properties.

The present disclosure also provides methods for assessing, in a mixture comprising polypeptides of varying length, a relative amount of a first set of polypeptides in the mixture to a second set of polypeptides in the mixture, wherein (1) each polypeptide of the first set of polypeptides shares a barcode fragment that occurs once and only once in the polypeptide and (2) each polypeptide of the second set of polypeptides lacks the barcode fragment that is shared by polypeptides of the first set, wherein individual polypeptides of both the first of polypeptides and the second set of polypeptides each comprises a reference fragment. In some embodiments, the methods comprise contacting the mixture with a protease to produce a plurality of proteolytic fragments that result from cleavage of the first set of polypeptides and the second set of polypeptides, wherein the plurality of proteolytic fragments comprise a plurality of reference fragments, and a plurality of barcode fragments. In some embodiments, the methods can further comprise determining a ratio of the amount of barcode fragments to the amount of reference fragments, thereby assessing the relative amounts of the first set of polypeptides to the second set of polypeptides. In some embodiments, the barcode fragment occurs no more than once in each polypeptide of the first set of polypeptides. In some embodiments, the reference fragment occurs no more than once in each polypeptide of the first set of polypeptides and the second set of polypeptides. In some embodiments, the plurality of proteolytic fragments comprises a plurality of reference fragments, and a plurality of barcode fragments. In some embodiments, the protease cleaves the first and second sets of polypeptides (or the polypeptides of varying length) on the C-terminal side of glutamic acid residues that are not followed by a proline residue. In some embodiments, the protease is a Glu-C protease. In some embodiments, the protease is not trypsin. In some embodiments, the step of determining a ratio of the amount of barcode fragments to the amount of reference fragments comprises identifying barcode fragments and reference fragments from the mixture after it has been contacted with the protease. In some embodiments, the barcode fragments and the reference fragments are identified based on their respective masses. In some embodiments, the barcode fragments and the reference fragments are identified via mass spectrometry.

In some embodiments, the barcode fragments and reference fragments are identified via liquid chromatography-mass spectrometry (LC-MS). In some embodiments, the step of determining a ratio of the barcode fragments to the reference fragments comprises isobaric labeling. In some embodiments, the step of determining a ratio of the barcode fragments to the reference fragments comprises spiking the mixture with one or both of an isotope-labeled reference fragment and an isotope labeled barcode fragment. In some embodiments, the polypeptides of varying lengths comprise polypeptides that comprise at least one ELNN, as described hereinabove or described anywhere else herein. In some embodiments, the ELNN is characterized in that (i) it comprises at least 100, or at least 150 amino acids; (ii) at least 90% of the amino acid residues of the ELNN are glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) or proline (P); and (iii) it comprises at least 4 different types of amino acids that are G, A, S, T, E, or P. In some embodiments, the barcode fragment, when present, is a portion of the ELNN. In some embodiments, the mixture of polypeptides of varying lengths comprises a polypeptide as any described hereinabove or described anywhere else herein. In some embodiments, the polypeptides of varying length comprise a full-length polypeptide and truncated fragments thereof. In some embodiments, the polypeptides of varying length consist essentially of the full-length polypeptide and truncated fragments thereof. In some embodiments, the polypeptides of varying lengths in a mixture differ from one another due to N-terminal truncation, C-terminal truncation, or both N- and C-terminal truncation of a full-length polypeptide. In some embodiments, the full-length polypeptide is a polypeptide as described hereinabove or described anywhere else herein. In some embodiments, the ratio of the amount of barcode fragments to reference fragments is greater than 0.50, 0.60, 0.70, 0.80, 0.90, 0.95, 0.98, or 0.99.

Isobaric Labeling-Based Quantification of Peptides

In some embodiments, isobaric labeling can be used for determining a ratio of the barcode fragments to the reference fragments. Isobaric labeling is a mass spectrometry strategy used in quantitative proteomics, wherein peptides or proteins (or portions thereof) are labeled with various chemical groups that are isobaric (identical in mass) but vary in terms of distribution of heavy isotopes around their structure. In some embodiments, these tags, commonly referred to as tandem mass tags, are designed so that the mass tag is cleaved at a specific linker region upon high-energy collision-induced dissociation (CID) during tandem mass spectrometry, thereby yielding reporter ions of different masses. Some of the most common isobaric tags are amine-reactive tags.

Exemplary Barcoded ELNN Polypeptides

Included herein are ELNNs comprising barcode fragments that are portions of the ELNNs.

Amino acid sequences of exemplary barcoded ELNNs, containing one barcode (e.g., SEQ ID NOs: 8002-8003, 8005-8009, and 8013-8022), or two barcodes (e.g., SEQ ID NOS: 8001, 8004, and 8012), or three barcodes (e.g., SEQ ID NO: 8011), are illustrated in Table 3a, with barcodes being identified in bold. In some embodiments, among these exemplary barcoded ELNNs, 12 (SEQ ID NOs: 8001-8003, 8008-8009, 8011, 8015-8019, and 8022) are to be fused to a biologically-active protein (such as a TCE) at the C-terminal of the biologically-active protein, and 10 (SEQ ID NOS: 8004-8007, 8010, 8012-8014, 8020, and 8021) are to be fused at the N-terminal of the biologically-active protein. In some embodiments, the ELNN has at least 90%, at least 92%, at least 95%, at least 98%, at least 99% or 100% sequence identity to a sequence identified herein by SEQ ID NOs: 8001-8022 in Table 3a.

TABLE 3a

Exemplary Barcoded ELNNs

SEQ

ID
ELNN
# of

Total #

NO.
Type
Barcode(s)
Amino Acid Sequence
of AAs

8001
C-
2
PGSPAGSPTSTEEGTSESATPESGPGTSTEPSE
864

terminal

GSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTE

ELNN

PSEGSAPGTSESATPESGPGSEPATSGSETPG

SEPATSGSETPGSPAGSPTSTEEGTSESATPES

GPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGS

PTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTS

ESATPESGPGTSTEPSEGSAPGTSESATPESGP

GSEPATSGSETPGTSTEPSEGSAPGTSTEPSEG

SAPGTSESATPESGPGTSESATPESGPGSPAG

SPTSTEEGTSESATPESGPGSEPATSGSETPGT

SESATPESGPGTSTEPSEGSAPGTSTEPSEGSA

PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE

GSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE

PSEGSAPGTSESATPESGPGSEPATSGSETPG

TSESATPESGPGSEPATSGSETPGTSESATPES

GPGTSTEPSEGSAPGTSESATPESGPGSPAGS

PTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTS

ESATPESGPGTSTEPSEGSAPGTSESATPESGP

GSEPATSGSETPGTSESATPESGPGSEPATSG

SETPGTSESATPESGPGTSTEPSEGSAPGSPA

GSPTSTEEGTSESATPESGPGSEPATSGSETPG

TSESATPESGPGSPAGSPTSTEEGSPAGSPTST

EEGTSTEPSEGSAPGTSESATPESGPGTSESAT

PESGPGftabTSESATPESGPGSEPATSGPTESG

SEPATSGSETPGSPAGSPTSTEEGTSTEPSEGS

APGTE
STPSEGSAPGSEPATSGSETPGTSESAT

PESGPGTSTEPSEGSAPGEPEA

8002
C-
1
PGSPAGSPTSTEEGTSESATPESGPGTSTEPSE
864

terminal

GSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTE

ELNN

PSEGSAPGTSESATPESGPGSEPATSGSETPG

SEPATSGSETPGSPAGSPTSTEEGTSESATPES

GPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGS

PTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTS

ESATPESGPGTSTEPSEGSAPGTSESATPESGP

GSEPATSGSETPGTSTEPSEGSAPGTSTEPSEG

SAPGTSESATPESGPGTSESATPESGPGSPAG

SPTSTEEGTSESATPESGPGSEPATSGSETPGT

SESATPESGPGTSTEPSEGSAPGTSTEPSEGSA

PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE

GSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE

PSEGSAPGTSESATPESGPGSEPATSGSETPG

TSESATPESGPGSEPATSGSETPGTSESATPES

GPGTSTEPSEGSAPGTSESATPESGPGSPAGS

PTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTS

ESATPESGPGTSTEPSEGSAPGTSESATPESGP

GSEPATSGSETPGTSESATPESGPGSEPATSG

SETPGTSESATPESGPGTSTEPSEGSAPGSPA

GSPTSTEEGTSESATPESGPGSEPATSGSETPG

TSESATPESGPGSPAGSPTSTEEGSPAGSPTST

EEGTSTEPSEGSAPGTSESATPESGPGTSESAT

PESGPGTSESATPESGPGSEPATSGPTESGSE

PATSGSETPGSPAGSPTSTEEGTSTEPSEGSAP

GTSTEPSEGSAPGSEPATSGSETPGTSESATPE

SGPGTSTEPSEGSAPGEPEA

8003
C-
1
PGSPAGSPTSTEEGTSESATPESGPGTSTEPSE
864

terminal

GSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTE

ELNN

PSEGSAPGTSESATPESGPGSEPATSGSETPG

SEPATSGSETPGSPAGSPTSTEEGTSESATPES

GPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGS

PTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTS

ESATPESGPGTSTEPSEGSAPGTSESATPESGP

GSEPATSGSETPGTSTEPSEGSAPGTSTEPSEG

SAPGTSESATPESGPGTSESATPESGPGSPAG

SPTSTEEGTSESATPESGPGSEPATSGSETPGT

SESATPESGPGTSTEPSEGSAPGTSTEPSEGSA

PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE

GSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE

PSEGSAPGTSESATPESGPGSEPATSGSETPG

TSESATPESGPGSEPATSGSETPGTSESATPES

GPGTSTEPSEGSAPGTSESATPESGPGSPAGS

PTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTS

ESATPESGPGTSTEPSEGSAPGTSESATPESGP

GSEPATSGSETPGTSESATPESGPGSEPATSG

SETPGTSESATPESGPGTSTEPSEGSAPGSPA

GSPTSTEEGTSESATPESGPGSEPATSGSETPG

TSESATPESGPGSPAGSPTSTEEGSPAGSPTST

EEGTSTEPSEGSAPGTSESATPESGPGTSESAT

PESGPGTSESATPESGPGSEPATSGSETPGSE

PATSGSETPGSPAGSPTSTEEGTSTEPSEGSAP

GTE
STPSEGSAPGSEPATSGSETPGTSESATPE

SGPGTSTEPSEGSAPGEPEA

8004
N-
2
ASSPAGSPTSTESGTSESATPESGPGTETEPSE
288

terminal

GSAPGTSESATPESGPGSEPATSGSETPGTSE

ELNN

SATPESGPGSTPAESGSETPGTSESATPESGP

GTSTEPSEGSAPGSPAGSPTSTEEGTSESATPE

SGPGESPATSGSTPEGTSESATPESGPGSPAG

SPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGT

SESATPESGPGTSESATPESGPGTSESATPESG

PGSEPATSGSETPGSEPATSGSETPGSPAGSP

TSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSA

P

8005
N-
1
ASSPAGSPTSTESGTSESATPESGPGTSTEPSE
288

terminal

GSAPGTSESATPESGPGSEPATSGSETPGTSE

ELNN

SATPESGPGSEPATSGSETPGTSESATPESGP

GTSTEPSEGSAPGSPAGSPTSTEEGTSESATPE

SGPGESPATSGSTPEGTSESATPESGPGSPAG

SPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGT

SESATPESGPGTSESATPESGPGTSESATPESG

PGSEPATSGSETPGSEPATSGSETPGSPAGSP

TSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSA

P

8006
N-
1
ASSPAGSPTSTESGTSESATPESGPGTSTEPSE
288

terminal

GSAPGTSESATPESGPGSEPATSGSETPGTSE

ELNN

SATPESGPGSTPAESGSETPGTSESATPESGP

GTSTEPSEGSAPGSPAGSPTSTEEGTSESATPE

SGPGEEPATSGSTPEGTSESATPESGPGSPAG

SPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGT

SESATPESGPGTSESATPESGPGTSESATPESG

PGSEPATSGSETPGSEPATSGSETPGSPAGSP

TSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSA

P

8007
N-
1
ASSPAGSPTSTESGTSESATPESGPGTSTEPSE
288

terminal

GSAPGTSESATPESGPGSEPATSGSETPGTSE

ELNN

SATPESGPGSTPAESGSETPGTSESATPESGP

GTSTEPSEGSAPGSPAGSPTSTEEGTSESATPE

SGPGSEPATSGSETPGTSESATPESGPGSPAG

SPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGT

SESATPESGPGTSESATPESGPGTSESATPESG

PGSEPATSGSETPGSEPATSGSETPGSPAGSP

TSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSA

P

8008
C-
1
PGSPAGSPTSTEEGTSESATPESGPGTSTEPSE
864

terminal

GSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTE

ELNN

PSEGSAPGTSESATPESGPGSEPATSGSETPG

SEPATSGSETPGSPAGSPTSTEEGTSESATPES

GPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGS

PTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTS

ESATPESGPGTSTEPSEGSAPGTSESATPESGP

GSEPATSGSETPGTSTEPSEGSAPGTSTEPSEG

SAPGTSESATPESGPGTSESATPESGPGSPAG

SPTSTEEGTSESATPESGPGSEPATSGSETPGT

SESATPESGPGTSTEPSEGSAPGTSTEPSEGSA

PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE

GSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE

PSEGSAPGTSESATPESGPGSEPATSGSETPG

TSESATPESGPGSEPATSGSETPGTSESATPES

GPGTSTEPSEGSAPGTSESATPESGPGSPAGS

PTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTS

ESATPESGPGTSTEPSEGSAPGTSESATPESGP

GSEPATSGSETPGTSESATPESGPGSEPATSG

SETPGTSESATPESGPGTSTEPSEGSAPGSPA

GSPTSTEEGTSESATPESGPGSEPATSGSETPG

TSESATPESGPGSPAGSPTSTEEGSPAGSPTST

EEGTSTEPSEGSAPGTSESATPESGPGTSESAT

PESGPGTSESATPESGPGSEPATSGSETPGSE

PATSGSETPGSPAGSPTSTEEGTSTEPSEGSAP

GTE
STPSEGSAPGSEPATSGSETPGTSESATPE

SGPGTSTEPSEGSAPG

8009
C-
1
PGSPAGSPTSTEEGTSESATPESGPGSEPATS
576

terminal

GSETPGTSESATPESGPGTSTEPSEGSAPGTST

ELNN

EPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAP

GTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPT

STEEGTSTEPSEGSAPGTSESATPESGPGSEPA

TSGSETPGTSESATPESGPGSEPATSGSETPGT

SESATPESGPGTSTEPSEGSAPGTSESATPESG

PGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSP

TSTEEGTSESATPESGPGTSTEPSEGSAPGTSE

SATPESGPGSEPATSGSETPGTSESATPESGP

GSEPATSGSETPGTSESATPESGPGTSTEPSEG

SAPGSPAGSPTSTEEGTSESATPESGPGSEPAT

SGSETPGTSESATPESGPGSPAGSPTSTEEGS

PAGSPTSTEEGTSTEPSEGSAPGTSESATPESG

PGTSESATPESGPGTSESATPESGPGSEPATS

GSETPGSEPATSGSETPGSPAGSPTSTEEGTST

EPSEGSAPGTESTPSEGSAPGSEPATSGSETP

GTSESATPESGPGTSTEPSEGSAPG

8010
N-
2
SAGSPGSPAGSPTSTEEGTSESATPESGPGTST
1152

terminal

EPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAP

ELNN

GTSTEPSEGSAPGTSESATPESGPGSTPAESG

SETPGSEPATSGSETPGSPAGSPTSTEEGTSES

ATPESGPGTSTEPSEGSAPGTSTEPSEGSAPG

SPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGS

APGTSESATPESGPGTSTEPSEGSAPGTSESAT

PESGPGSEPATSGSTETPGTSTEPSEGSAPGT

STEPSEGSAPGTSESATPESGPGTSESATPESG

PGSPAGSPTSTEEGTSESATPESGPGSEPATS

GSETPGTSESATPESGPGTSTEPSEGSAPGTST

EPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAP

GTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPT

STEEGTSTEPSEGSAPGTSESATPESGPGSEPA

TSGSETPGTSESATPESGPGSEPATSGSETPGT

SESATPESGPGTSTEPSEGSAPGTSESATPESG

PGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSP

TSTEEGTSESATPESGPGTSTEPSEGSAPGTSE

SATPESGPGSEPATSGSETPGTSESATPESGP

GSEPATSGSETPGTSESATPESGPGTSTEPSEG

SAPGSPAGSPTSTEEGTSESATPESGPGSEPAT

SGSETPGTSESATPESGPGSPAGSPTSTEEGS

PAGSPTSTEEGTSTEPSEGSAPGTSESATPESG

PGTSESATPESGPGTSESATPESGPGSEPATS

GSETPGSEPATSGSETPGSPAGSPTSTEEGTST

EPSEGSAPGTSTEPSEGSAPGSEPATSGSETP

GTSESATPESGPGTSTEPSEGSAPGSPAGSPT

STEEGTSESATPESGPGTSTEPSEGSAPGSPA

GSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPG

TSESATPESGPGSEPATSGSETPGSEPATSGSE

TPGSPAGSPTSTEEGTSESATPESGPGTSTEPS

EGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTST

EPSEGSAPGTSTEPSEGSAPGTSESATPESGP

GTSTEPSEGSAPGTSESATPESGPGSEPATSG

SETPGTSTEPSEGSAPGTSTEPSEGSAPGTSES

ATPESGPGTESAS

8011
C-
3
SAGSPGSPAGSPTSTEEGTSESATPESGPGTST
1152

terminal

EPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAP

ELNN

GTSTEPSEGSAPGTSESATPESGPGSEPATSG

SETPGSEPATSGSETPGSPAGSPTSTEEGTSES

ATPESGPGTSTEPSEGSAPGTSTEPSEGSAPG

SPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGS

APGTSESATPESGPGTSTEPSEGSAPGTSESAT

PESGPGSEPATSGSETPGTSTEPSEGSAPGTST

EPSEGSAPGTSESATPESGPGTSESATPESGP

GSPAGSPTSTEEGTSESATPESGPGSEPATSG

SETPGTSESATPESGPGTSTEPSEGSAPGTSTE

PSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPG

TSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTST

EEGTSTEPSEGSAPGTSESATPESGPGSEPATS

GSETPGTSESATPESGPGSEPATSGSETPGTSE

SATPESGPGTSTEPSEGSAPGTSESATPESGP

GSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPT

STEEGTSESATPESGPGTSTEPSEGSAPGTSES

ATPESGPGSEPATSGSETPGTSESATPESGPG

SEPATSGSETPGTSESATPESGPGTSTEPSEGS

APGSPAGSPTSTEEGTSESATPESGPGSEPATS

GSETPGTSESATPESGPGSPAGSPTSTEEGSP

AGSPTSTEEGTSTEPSEGSAPGTSESATPESGP

GTSESATPESGPGTSESATPESGPGSEPATSG

SETPGSEPATSGSETPGSPAGSPTSTEEGTSTE

PSEGSAPGTSTEPSEGSAPGSEPATSGSETPG

TSESATPESGPGTSTEPSEGSAPGSPAGSPTST

EEGTSESATPESGPGTSTEPSEGSAPGSPAGS

PTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTS

ESATPESGPGSEPATSGSETPGSEPATSGSTET

PGSPAGSPTSTEEGTSESATPESGPGTSTEPSE

GSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE

PSEGSAPGTATESPEGSAPGTSESATPESGPG

TSTEPSEGSAPGTSAESATPESGPGSEPATSG

SETPGTSTEPSEGSAPGTSTEPSEGSAPGTSES

ATPESGPGTESAS

8012
N-
2
GSPAGSPTSTEEGTSESATPESGPGTSTEPSEG
864

terminal

SAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEP

ELNN

SEGSAPATSESATPESGPGSEPATSGSETPGS

EPATSGSETPGSPAGSPTSTEEGTSESASPESG

PGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSP

TSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSE

SATPESGPGTSTEPSEGSAPGTSESATPESGP

GSEPATSGSETPGTSTEPSEGSAPGTSTEPSEG

SAPGTSESATPESGPGTSESATPESGPGSPAG

SPTSTEEGTSESATPESGPGSEPATSGSETPGT

SESATPESGPGTSTEPSEGSAPGTSTEPSEGSA

PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE

GSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE

PSEGSAPGTSESATPESGPGSEPATSGSETPG

TSESATPESGPGSEPATSGSETPGTSESATPES

GPGTSTEPSEGSAPGTSESATPESGPGSPAGS

PTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTS

ESATPESGPGTSTEPSEGSAPGTSESATPESGP

GSEPATSGSETPGTSESATPESGPGSEPATSG

SETPGTSESATPESGPGTSTEPSEGSAPGSPA

GSPTSTEEGTSESATPESGPGSEPATSGSETPG

TSESATPESGPGSPAGSPTSTEEGSPAGSPTST

EEGTSTEPSEGSAPGTSESATPESGPGTSESAT

PESGPGTSESATPESGPGSEPATSGSETPGSE

PATSGSETPGSPAGSPTSTEEGTSTEPSEGSAP

GTSTEPSEGSAPGSEPATSGSETPGTSESATPE

SGPGTSTEPSEGSAP

8013
N-
1
GSPAGSPTSTEEGTSESATPESGPGTSTEPSEG
864

terminal

SAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEP

ELNN

SEGSAPGTSESATPESGPGSESATSGSETPGS

EPATSGSETPGSPAGSPTSTEEGTSESATPESG

PGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSP

TSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSE

SATPESGPGTSTEPSEGSAPGTSESATPESGP

GSEPATSGSETPGTSTEPSEGSAPGTSTEPSEG

SAPGTSESATPESGPGTSESATPESGPGSPAG

SPTSTEEGTSESATPESGPGSEPATSGSETPGT

SESATPESGPGTSTEPSEGSAPGTSTEPSEGSA

PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE

GSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE

PSEGSAPGTSESATPESGPGSEPATSGSETPG

TSESATPESGPGSEPATSGSETPGTSESATPES

GPGTSTEPSEGSAPGTSESATPESGPGSPAGS

PTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTS

ESATPESGPGTSTEPSEGSAPGTSESATPESGP

GSEPATSGSETPGTSESATPESGPGSEPATSG

SETPGTSESATPESGPGTSTEPSEGSAPGSPA

GSPTSTEEGTSESATPESGPGSEPATSGSETPG

TSESATPESGPGSPAGSPTSTEEGSPAGSPTST

EEGTSTEPSEGSAPGTSESATPESGPGTSESAT

PESGPGTSESATPESGPGSEPATSGSETPGSE

PATSGSETPGSPAGSPTSTEEGTSTEPSEGSAP

GTSTEPSEGSAPGSEPATSGSETPGTSESATPE

SGPGTSTEPSEGSAP

8014
N-
1
SPAGSPTSTESGTSESATPESGPGTSTEPSEGS
292

terminal

APGTSESATPESGPGSEPATSGSETPGTSESAT

ELNN

PESGPGSTPAESGSETPGTSESATPESGPGTS

TEPSEGSAPGSPAGSPTSTEEGTSESATPESGP

GSEPATSGSETPGTSESATPESGPGSPAGSPT

STEEGSPAGSPTSTEEGTSTEPSEGSAPGTSES

ATPESGPGTSESATPESGPGTSESATPESGPG

SEPATSGSETPGSEPATSGSETPGSPAGSPTST

EEGTSTEPSEGSAPGTSTEPSEGSAPGGSAP

8015
C-
1
PGSPAGSPTSTEEGTSESATPESGPGSEPATS
582

terminal

GSETPGTSESATPESGPGTSTEPSEGSAPGTST

ELNN

EPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAP

GTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPT

STEEGTSTEPSEGSAPGTSESATPESGPGSEPA

TSGSETPGTSESATPESGPGSEPATSGSETPGT

SESATPESGPGTSTEPSEGSAPGTSESATPESG

PGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSP

TSTEEGTSESATPESGPGTSTEPSEGSAPGTSE

SATPESGPGSEPATSGSETPGTSESATPESGP

GSEPATSGSETPGTSESATPESGPGTSTEPSEG

SAPGSPAGSPTSTEEGTSESATPESGPGSEPAT

SGSETPGTSESATPESGPGSPAGSPTSTEEGS

PAGSPTSTEEGTSTEPSEGSAPGTSESATPESG

PGTSESATPESGPGTSESATPESGPGSEPATS

GSETPGSEPATSGSETPGSPAGSPTSTEEGTST

EPSEGSAPGTESTPSEGSAPGSEPATSGSETP

GTSESATPESGPGTSTEPSEGSAPGEPEA

8016
C-
1
TPESGPGTSESATPESGPGSPAGSPTSTEEGTS
576

terminal

ESATPESGPGSEPATSGSETPGTSESATPESGP

ELNN

GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEG

SAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEP

SEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGT

SESATPESGPGSEPATSGSETPGTSESATPESG

PGSEPATSGSETPGTSESATPESGPGTSTEPSE

GSAPGTSESATPESGPGSPAGSPTSTEEGSPA

GSPTSTEEGSPAGSPTSTEEGTSESATPESGPG

TSTEPSEGSAPGTSESATPESGPGSEPATSGSE

TPGTSESATPESGPGSEPATSGSETPGTSESAT

PESGPGTSTEPSEGSAPGSPAGSPTSTEEGTS

ESATPESGPGSEPATSGSETPGTSESATPESGP

GSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEG

SAPGTSESATPESGPGTSESATPESGPGTSESA

TPESGPGSEPATSGSETPGSESATSGSETPGS

PAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSA

PGSEPATSGSETPGTSESA

8017
C-
1
GTSTEPSEGSAPGTSESATPESGPGSEPATSG
576

terminal

SETPGSEPATSGSETPGSPAGSPTSTEEGTSES

ELNN

ATPESGPGTSTEPSEGSAPGTSTEPSEGSAPG

SPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGS

APGTSESATPESGPGTSTEPSEGSAPGTSESAT

PESGPGSEPATSGSETPGTSTEPSEGSAPGTST

EPSEGSAPGTSESATPESGPGTSESATPESGP

GSPAGSPTSTEEGTSESATPESGPGSEPATSG

SETPGTSESATPESGPGTSTEPSEGSAPGTSTE

PSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPG

TSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTST

EEGTSTEPSEGSAPGTSESATPESGPGSEPATS

GSETPGTSESATPESGPGSEPATSGSETPGTSE

SATPESGPGTSTEPSEGSAPGTSESASPESGP

GSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPT

STEEGTSESATPESGPGTSTEPSEGSAPGTSES

ATPESGPGSEPATSGSETPGTSESATPESGPG

SEPATSGSETPGTSESATPESGP

8018
C-
1
GSETPGSPAGSPTSTEEGTSESATPESGPGTST
576

terminal

EPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEE

ELNN

GTSTEPSEGSAPGTSTEPSEGSAPGTSESATPE

SGPGTSTEPSEGSAPGTSESATPESGPGSEPA

TSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGT

SESATPESGPGTSESATPESGPGSPAGSPTSTE

EGTSESATPESGPGSEPATSGSETPGTSESATP

ESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTE

PSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPG

TSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGS

APGTSESATPESGPGSEPATSGSETPGTSESAT

PESGPGSEPATSGSETPGTSESATPESGPGTST

EPSEGSAPGTSESATPESGPGSPAGSPTSTEE

GSPAGSPTSTEEGSPAGSPTSTEEGTSESATPE

SGPGTSTEPSEGSAPGTSESATPESGPGSEPA

TSGSTE
TGTSESATPESGPGSEPATSGSETPGT

SESATPESGPGTSTEPSEGSAPGSPAGSPTSTE

EGTSESATPESGPGSEPATS

8019
C-
1
EGSAPGTSTEPSEGSAPGTSESATPESGPGTST
576

terminal

EPSEGSAPGTSESATPESGPGSEPATSGSETP

ELNN

GTSTEPSEGSAPGTSTEPSEGSAPGTSESATPE

SGPGTSESATPESGPGSPAGSPTSTEEGTSES

ATPESGPGSEPATSGSETPGTSESATPESGPGT

STEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA

PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE

GSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSE

SATPESGPGSEPATSGSETPGTSESATPESGP

GSEPATSGSETPGTSESATPESGPGTSTEPSEG

SAPGTSESATPESGPGSPAGSPTSTEEGSPAG

SPTSTEEGSPAGSPTSTEEGTSESATPESGPGT

STEPSEGSAPGTSESATPESGPGSEPATSGSET

PGTSESATPESGPGSEPATSGSETPGTSESASP

E
SGPGTSTEPSEGSAPGSPAGSPTSTEEGTSE

SATPESGPGSEPATSGSETPGTSESATPESGP

GSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEG

SAPGTSESATPESGPGTSESAT

8020
N-
1
ASSPAGSPTSTESGTSESATPESGPGTSTEPSE
294

terminal

GSAPGTSESATPESGPGSEPATSGSETPGTSE

ELNN

SATPESGPGSTPAESGSETPGTSESATPESGP

GTSTEPSEGSAPGSPAGSPTSTEEGTSESATPE

SGPGSEPATSGSETPGTSESATPESGPGSPAG

SPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGT

SESATPESGPGTSESATPESGPGTSESATPESG

PGSEPATSGSETPGSEPATSGSETPGSPAGSP

TSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSA

P

8021
N-
1
ASSATPESGPGTSTEPSEGSAPGTSESATPESG
294

terminal

PGSGPGTSE
SATPGTSESATPESGPGSEPATS

ELNN

GSETPGTSESATPESGPGTSTEPSEGSAPGSP

AGSPTSTEEGTSESATPESGPGSEPATSGSETP

GTSESATPESGPGSPAGSPTSTEEGSPAGSPT

STEEGTSTEPSEGSAPGTSESATPESGPGTSES

ATPESGPGTSESATPESGPGSEPATSGSETPG

SEPATSGSETPGSPAGSPTSTEEGTSTEPSEGS

APGTSTEPSEGSAPGSEPATSGSETPGTSESAT

P

8022
C-
1
ATPESGPGTSESATPESGPGSPAGSPTSTEEGT
582

terminal

SESATPESGPGSEPATSGSETPGTSESATPESG

ELNN

PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE

GSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTE

PSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPG

TSESATPESGPGSEPATSGSETPGTSESATPES

GPGSEPATSGSETPGTSESATPESGPGTSTEPS

EGSAPGTSESATPESGPGSPAGSPTSTEEGSP

AGSPTSTEEGSPAGSPTSTEEGTSESATPESGP

GTSTEPSEGSAPGTSESATPESGPGSEPATSG

SETPGTSESATPESGPGSEPATSGSETPGTSES

ATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGT

SESATPESGPGSEPATSGSETPGTSESATPESG

PGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSE

GSAPGTSESATPESGPGTSESATPESGPGTSP

SATPE
SGPGSEPATSGSETPGSEPATSGSETP

GSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEG

SAPGSEPATSGSETPGTSESAGEPEA

In some embodiments, a barcoded ELNN can be obtained by making one or more mutations to existing ELNN, such as any listed in Table 3b, according to one or more of the following criteria: to minimize the sequence change in the ELNN, to minimize the amino acid composition change in the ELNN, to substantially maintain the net charge of the ELNN, to substantially maintain (or improve) low immunogenicity of the ELNN, and to substantially maintain (or improve) the pharmacokinetic properties of the ELNN. In some embodiments, the ELNN sequence has at least 90%, at least 92%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 601-659 listed in Table 3b. In some embodiments, the ELNN sequence, having at least 90% (e.g., at least 92%, at least 95%, at least 98%, or at least 99%) but less than 100% sequence identity to any of SEQ ID NOs: 601-659 listed in Table 3b, is obtained by one or more mutations (e.g., less than 10, less than 8, less than 6, less than 5, less than 4, less than 3, less than 2 mutations) of the corresponding sequence from Table 3b. In some embodiments, the one or more mutations comprise deletion of a glutamic acid residue, insertion of a glutamic acid residue, substitution of a glutamic acid residue, or substitution for a glutamic acid residue, or any combination thereof. In some embodiments, where the ELNN sequence differs from, but has at least 90% (e.g., at least 92%, at least 95%, at least 98%, or at least 99%) sequence identity to, any one of SEQ ID NOs: 601-659 listed in Table 3b, at least 80%, at least 90%, at least 95%, at least 97%, or about 100% of the difference between the ELNN sequence and the corresponding sequence of Table 3b involve deletion of a glutamic acid residue, insertion of a glutamic acid residue, substitution of a glutamic acid residue, or substitution for a glutamic acid residue, or any combination thereof. In some such embodiments, at least 80%, at least 90%, at least 95%, at least 97%, or about 100% of the difference between the ELNN sequence and the corresponding sequence of Table 3b involve a substitution of a glutamic acid residue, or a substitution for a glutamic acid residue, or both.

The “a substitution of a first amino acid,” as used herein, refers to replacement of the first amino acid residue with a second amino acid residue, resulting in the second amino acid residue taking its place at the substitution position in the obtained sequence. For example, “a substitution of glutamic acid” refers to replacement of the glutamic acid (E) residue for a non-glutamic acid residue (e.g., serine (S)).

TABLE 3b

Exemplary Existing ELNNs for Engineering into Barcoded ELNN(s)

ELNN Name
Amino Acid Sequence
SEQ ID NO

AE144
GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPT
601

STEEGTSTEPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEP

ATSGSETPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETP

GTSTEPSEGSAP

AE144_1A
SPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTS
602

TEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPA

TSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPG

TSTEPSEGSAPG

AE144_2A
TSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEG
603

SAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPA

TSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPG

TSESATPESGPG

AE144_2B
TSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEG
604

SAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPA

TSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPG

TSESATPESGPG

AE144_3A
SPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPE
605

SGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTE

PSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEG

TSTEPSEGSAPG

AE144_3B
SPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPE
606

SGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTE

PSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEG

TSTEPSEGSAPG

AE144_4A
TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGS
607

ETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAG

SPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPG

TSTEPSEGSAPG

AE144_4B
TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGS
608

ETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAG

SPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPG

TSTEPSEGSAPG

AE144_5A
TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGS
609

ETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSES

ATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEG

SPAGSPTSTEEG

AE144_6B
TSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPE
610

SGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTE

PSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPG

TSTEPSEGSAPG

AE288_1
GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSG
611

SETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSE

SATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEE

GSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATP

ESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPA

GSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETP

GTSESATPESGPGTSTEPSEGSAP

AE288_2
GSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATP
612

ESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTST

EPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEE

GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATP

ESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSE

SATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEE

GTSESATPESGPGTSTEPSEGSAP

AE576
GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPT
613

STEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEP

ATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGP

GTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSE

GSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSE

SATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAP

GTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATP

ESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTST

EPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAP

GTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATP

ESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSE

SATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEE

GSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSE

GSAP

AE624
MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTS
614

STGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSP

AGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESG

PGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESAT

PESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTS

TEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSA

PGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPS

EGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTS

ESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSA

PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPS

EGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTS

ESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSET

PGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSP

TSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTS

TEPSEGSAP

AE864
GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPT
615

STEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEP

ATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGP

GTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSE

GSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSE

SATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAP

GTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATP

ESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTST

EPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAP

GTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATP

ESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSE

SATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEE

GSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSE

GSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEP

ATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEE

GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPT

STEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSE

SATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETP

GSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSG

SETPGTSESATPESGPGTSTEPSEGSAP

AE865
GGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSP
616

TSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSE

PATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESG

PGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPS

EGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTS

ESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSA

PGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESAT

PESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTS

TEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA

PGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESAT

PESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTS

ESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTE

EGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPS

EGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSE

PATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTE

EGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSP

TSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTS

ESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSET

PGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATS

GSETPGTSESATPESGPGTSTEPSEGSAP

AE866
PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSP
617

TSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSE

PATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESG

PGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPS

EGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTS

ESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSA

PGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESAT

PESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTS

TEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA

PGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESAT

PESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTS

ESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTE

EGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPS

EGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSE

PATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTE

EGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSP

TSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTS

ESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSET

PGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATS

GSETPGTSESATPESGPGTSTEPSEGSAPG

AE1152
GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPT
618

STEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEP

ATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGP

GTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSE

GSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSE

SATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAP

GTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATP

ESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTST

EPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAP

GTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATP

ESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSE

SATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEE

GSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSE

GSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEP

ATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEE

GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPT

STEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSE

SATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETP

GSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSG

SETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEP

ATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGP

GTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSG

SETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTST

EPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGP

GSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSE

GSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTST

EPSEGSAP

AE144A
STEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPES
619

GPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESA

TPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGS

PAGSPTSTEEGS

AE144B
SEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPE
620

SGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPA

TSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEG

TSTEPSEGSAPG

AE180A
TSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSP
621

AGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESG

PGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATS

GSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSE

PATS

AE216A
PESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSE
622

PATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTE

EGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESAT

PESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTS

TEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAT

AE252A
ESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPA
623

GSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGP

GSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATP

ESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEP

ATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAP

GSEPATSGSETPGTSESATPESGPGTSTEPSE

AE288A
TPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGT
624

SESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGS

APGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESA

TPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGT

SESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSE

TPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEP

SEGSAPGSEPATSGSETPGTSESA

AE324A
PESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTS
625

ESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSET

PGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPS

EGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTS

ESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSA

PGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATS

GSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTS

TEPSEGSAPGSEPATS

AE360A
PESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSP
626

AGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSA

PGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATS

GSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTS

ESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTE

EGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESAT

PESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSP

AGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSET

PGTSESAT

AE396A
PESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTS
627

ESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTE

EGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATS

GSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS

TEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSET

PGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPS

EGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSE

PATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSA

PGTSTEPS

AE432A
EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSE
628

PATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESG

PGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSP

TSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTS

ESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSET

PGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESAT

PESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSP

AGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESG

PGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSP

TSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATS

AE468A
EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTS
629

TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESG

PGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESAT

PESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTS

ESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSET

PGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPS

EGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTS

ESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSA

PGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATS

GSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTS

TEPSEGSAPGSEPATSGSETPGTSESAT

AE504A
EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTS
630

TEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESG

PGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESAT

PESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSP

AGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSA

PGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATS

GSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTS

ESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTE

EGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESAT

PESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSP

AGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSET

PGTSESATPESGPGTSTEPS

AE540A
TPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGT
631

SESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS

APGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGS

PTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGT

SESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGS

APGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGS

PTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGS

EPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPES

GPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPAT

SGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGT

STEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPES

GPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEP

SEGSAPGTSTEP

AE576A
TPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGS
632

EPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGS

APGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEP

SEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGS

EPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPES

GPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGS

PTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGT

SESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSE

TPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESA

TPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGS

PAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPES

GPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGS

PTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGT

SESA

AE612A
GSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTS
633

ESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSET

PGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPS

EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSP

AGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSET

PGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPS

EGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSP

AGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESG

PGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESAT

PESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSE

PATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTE

EGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESAT

PESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTS

TEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAT

AE648A
PESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTS
634

TEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESG

PGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESAT

PESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTS

TEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTE

EGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESAT

PESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTS

ESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTE

EGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATS

GSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS

TEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSET

PGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPS

EGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSE

PATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSA

PGTSTEPSEGSAPGSEPATSGSETPGTSESAT

AE684A
EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTS
635

ESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSET

PGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESAT

PESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTS

ESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA

PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSP

TSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTS

ESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSA

PGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSP

TSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSE

PATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESG

PGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATS

GSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTS

TEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESG

PGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPS

EGSAPGTSTEPSEGSAPGSEPATS

AE720A
TSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPG
636

TSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEG

SAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSES

ATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPG

TSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPE

SGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTE

PSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPG

TSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPE

SGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSES

ATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEG

SPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEG

SAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPA

TSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEG

TSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTS

TEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSES

ATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPG

SPAGSPTSTEEGTSTE

AE756A
TSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPG
637

TSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEG

SAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSES

ATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPG

TSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPE

SGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTE

PSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPG

TSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPE

SGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSES

ATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEG

SPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEG

SAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPA

TSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEG

TSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTS

TEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSES

ATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPG

SPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGS

ETPGTSES

AE792A
EGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSP
638

AGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSA

PGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESAT

PESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTS

TEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESG

PGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESAT

PESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTS

TEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTE

EGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESAT

PESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTS

ESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTE

EGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATS

GSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS

TEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSET

PGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPS

EGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSE

PATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSA

PGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPS

AE828A
PESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTS
639

TEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSET

PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPS

EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTS

ESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSET

PGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESAT

PESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTS

ESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA

PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSP

TSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTS

ESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSA

PGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSP

TSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSE

PATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESG

PGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATS

GSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTS

TEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESG

PGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPS

EGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAT

AE869
GSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAG
640

SPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPG

SEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPE

SGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE

PSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPG

TSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEG

SAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSES

ATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG

TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEG

SAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSES

ATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPG

TSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTS

TEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTE

PSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPG

SEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTS

TEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAG

SPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPG

TSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGS

ETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPA

TSGSETPGTSESATPESGPGTSTEPSEGSAPGR

AE144_R1
SAGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSP
641

AGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESG

PGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESAT

PESGPGTESASR

AE288_R1
SAGSPTGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETP
642

GTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATP

ESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPA

GSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGP

GTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPT

STEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSE

SATPESGPGTSTEPSEGSAPSASR

AE432_R1
SAGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSP
643

AGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESG

PGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESAT

PESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTS

TEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSA

PGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPS

EGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTS

ESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSA

PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPS

EGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTESASR

AE576_R1
SAGSPTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGP
644

GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE

GSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTST

EPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGP

GSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATP

ESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSE

SATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETP

GTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSE

GSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSE

SATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAP

GTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSG

SETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTST

EPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP

SASR

AE864_R1
SAGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSP
645

AGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESG

PGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESAT

PESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTS

TEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSA

PGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPS

EGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTS

ESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSA

PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPS

EGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTS

ESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSET

PGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSP

TSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTS

TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESG

PGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSP

TSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSP

AGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESG

PGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATS

GSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSE

PATSGSETPGTSESATPESGPGTESASR

AE712
PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSP
646

TSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSE

PATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESG

PGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPS

EGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTS

ESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSA

PGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESAT

PESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTS

TEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA

PGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESAT

PESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTS

ESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTE

EGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPS

EGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSE

PATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTE

EGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSP

TSTEAHHH

AE864_R2
GSPGAGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSP
647

AGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESG

PGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESAT

PESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTS

TEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSA

PGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPS

EGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTS

ESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSA

PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPS

EGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTS

ESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSET

PGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSP

TSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTS

TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESG

PGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSP

TSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSP

AGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESG

PGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATS

GSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSE

PATSGSETPGTSESATPESGPGTESASR

AE288_3
SPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPE
648

SGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSES

ATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPG

SEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTS

TEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSES

ATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEG

TSTEPSEGSAPGTSTEPSEGSAPG

AE284
GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSG
649

SETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSE

SATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEE

GSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATP

ESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPA

GSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETP

GTSESATPESGPGTSTEPSE

AE292
SPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPE
650

SGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSES

ATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPG

SEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTS

TEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSES

ATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEG

TSTEPSEGSAPGTSTEPSEGSAPGGSAP

AE864_2
AGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTE
651

EGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATS

GSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTS

TEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSA

PGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESAT

PESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTS

ESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESG

PGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPS

EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTS

TEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESG

PGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESAT

PESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSP

AGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSA

PGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATS

GSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTS

ESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTE

EGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESAT

PESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSP

AGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSET

PGTSESATPESGPGTSTEPSEGAAEPEA

AE867
GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPT
652

STEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEP

ATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGP

GTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSE

GSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSE

SATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAP

GTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATP

ESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTST

EPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAP

GTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATP

ESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSE

SATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEE

GSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSE

GSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEP

ATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEE

GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPT

STEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSE

SATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETP

GSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSG

SETPGTSESATPESGPGTSTEPSEGAAEPEA

AE867_2
SPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGS
653

PTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGS

EPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPES

GPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEP

SEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGT

SESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGS

APGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESA

TPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGT

STEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS

APGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESA

TPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGT

SESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTST

EEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEP

SEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGS

EPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTST

EEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGS

PTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGT

SESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSE

TPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPAT

SGSETPGTSESATPESGPGTSTEPSEGSAPG

AE868
PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSP
654

TSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSE

PATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESG

PGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPS

EGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTS

ESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSA

PGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESAT

PESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTS

TEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA

PGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESAT

PESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTS

ESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTE

EGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPS

EGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSE

PATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTE

EGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSP

TSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTS

ESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSET

PGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATS

GSETPGTSESATPESGPGTSTEPSEGAAEPEA

AE144_7A
GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPT
655

STEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEP

ATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGP

GTSTEPSEGSAP

AE292
SPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPE
656

SGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSES

ATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPG

SEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTS

TEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSES

ATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEG

TSTEPSEGSAPGTSTEPSEGSAPGGSAP

AE293
PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSP
657

TSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSE

PATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESG

PGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPS

EGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTS

ESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSA

PGTSESATPESGPGTSESATPEGAAEPEA

AE300
PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSP
658

TSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSE

PATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESG

PGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPS

EGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTS

ESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSA

PGTSESATPESGPGTSESATPESGPGSPAGAAEPEA

AE584
PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSP
659

TSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSE

PATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESG

PGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPS

EGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTS

ESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSA

PGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESAT

PESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTS

TEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA

PGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESAT

PESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTS

ESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTE

EGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPS

EGSAPGAAEPEA

In some embodiments, for constructing the sequence of a barcoded ELNN, amino-acid mutations are performed on ELNN of intermediate lengths to those of Table 3b, as well as ELNN of longer lengths than those of Table 3b, such as those in which one or more 12-mer motifs of Table 1 are added to the N— or C— terminus of a general-purpose ELNN of Table 3b.

Additional examples of existing ELNNs that can be used according to the present disclosure are disclosed in U.S. Patent Publication Nos. 2010/0239554 A1, 2010/0323956 A1, 2011/0046060 A1, 2011/0046061 A1, 2011/0077199 A1, or 2011/0172146 A1, or International Patent Publication Nos. WO 2010091122 A1, WO 2010144502 A2, WO 2010144508 A1, WO 2011028228 A1, WO 2011028229 A1, WO 2011028344 A2, WO 2014/011819 A2, or WO 2015/023891; each of which is herein incorporated by reference.

In some embodiments, a barcoded ELNN fused within a polypeptide chain adjacent to the N-terminus of the polypeptide chain (“N-terminal ELNN”) can be attached to a His tag of HHHHHH (SEQ ID NO: 48) or HHHHHHHH (SEQ ID NO: 49) at the N-terminus to facilitate the purification of the fusion polypeptide. In some embodiments, a barcoded ELNN fused within a polypeptide chain at the C-terminus of the polypeptide chain (“C-terminal ELNN”) can be comprise or be attached to the sequence EPEA at the C-terminus to facilitate the purification of the fusion polypeptide. In some embodiments, the fusion polypeptide comprises both an N-terminal barcoded ELNN and a C-terminal barcoded ELNN, wherein the N-terminal barcoded ELNN is attached to a His tag of HHHHHH (SEQ ID NO: 48) or HHHHHHHH (SEQ ID NO: 49) at the N-terminus; and wherein the C-terminal barcoded ELNN is attached to the sequence EPEA at the C-terminus, thereby facilitating purification of the fusion polypeptide, for example, to at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% purity by chromatography methods known in the art, including but not limited to IMAC chromatography, C-tagXL affinity matrix, and other such methods.

A barcode fragment, as described herein, can be cleavably fused within the ELNN and releasable (i.e., configured to be released) from the ELNN upon digestion of the polypeptide by a protease. In some embodiments, the protease is a Glu-C protease. In some embodiments, the protease cleaves on the C-terminal side of glutamic acid residues that are not followed by proline. In some embodiments, a barcoded ELNN (an ELNN that contains barcode fragment(s) therewithin) is designed to achieve high efficiency, precision and accuracy of the protease digestion. For example, in some embodiments, adjacent Glu-Glu (EE) residues in an ELNN sequence can result in varying cleavage patterns upon Glu-C digestion. Accordingly, when Glu-C protease is used for barcode release, the barcoded ELNN or the barcode fragment(s) may not contain any Glu-Glu (EE) sequence. Additionally, a di-peptide Glu-Pro (EP) sequence, if present in the fusion polypeptide, may not be cleaved by Glu-C protease during the barcode release process.

Structural Configuration of Activatable TCEs

In some embodiments, a fusion protein comprises a single BsAb in the form of a TCE and a single ELNN. In some embodiments, such a fusion protein can have at least the following permutations of configurations, each listed in an N- to C-terminus orientation: (TCE)-(ELNN); (ELNN)-(TCE); (TCE)-(Linker)-(ELNN); and (ELNN)-(Linker)-(TCE).

In some embodiments, the fusion protein comprises a C-terminal ELNN and, optionally, a linker (such as one described herein, e.g., in Table C) between the ELNN and the TCE. In some embodiments, such a fusion protein can be represented by Formula I (depicted N- to C-terminus):

(TCE)-(Linker)-(ELNN) (I),

wherein the TCE is as described herein; Linker is a linker sequence (such as one described herein, e.g., in Table C) comprising between 1 to about 50 amino acid residues that can optionally include a TCE release segment (e.g., as described herein); and the ELNN can be any ELNN described herein.

In some embodiments, the fusion protein comprises an N-terminal ELNN and, optionally, a linker (such as one described herein, e.g., in Table C) between the ELNN and the TCE. In some embodiments, such a fusion protein can be represented by Formula II (depicted N- to C-terminus):

(ELNN)-(Linker)-(TCE) (II),

wherein TCE is as described herein; Linker is a linker sequence (such as one described herein, e.g., in Table C) comprising between 1 to about 50 amino acid residues that can optionally include a TCE release segment (e.g., as described herein); and ELNN can be any ELNN described herein.

In some embodiments, the fusion protein comprises both an N-terminal ELNN and a C-terminal ELNN. In some embodiments, such a fusion protein can be represented by Formula III:

(ELNN)-(Linker)-(TCE)-(Linker)-(ELNN) (III)

wherein TCE is as described herein; each Linker is, individually, a linker sequence (such as one described herein, e.g., in Table C) having between 1 to about 50 amino acid residues that can optionally include a TCE release segment (e.g., as described herein); and each ELNN can be, individually, any ELNN described herein.

The present disclosure provides BsAbs (e.g., TCEs) comprise one or more sequences disclosed herein in any one of Tables 6a-6g.

Of particular interest are BsAbs (e.g., TCEs) for which an increase in a pharmacokinetic parameter, increased solubility, increased stability, masking of activity, or some other enhanced pharmaceutical property is sought, or those BsAbs (e.g., TCEs) for which increasing the terminal half-life would improve efficacy, and/or safety. Thus, the paTCE fusion protein compositions are prepared with various objectives in mind, including improving the therapeutic efficacy of the TCE by, for example, increasing the in vivo exposure or the length that the TCE remains within the therapeutic window when administered to a subject, compared to a TCE not linked to any ELNNs.

It will be appreciated that various amino acid substitutions (especially conservative amino acid substitutions) can be made in a bispecific sequence to create variants without departing from the spirit of the present disclosure with respect to the biological activity or pharmacologic properties of, e.g., a TCE. Examples of conservative substitutions for amino acids in polypeptide sequences are shown in Table 4. In addition, variants can also include, for instance, polypeptides wherein one or more amino acid residues are added or deleted at the N- or C-terminus of the full-length native amino acid sequence of a TCE that retains at least a portion of the biological activity of the native peptide.

In some embodiments, sequences that retain at least about 40%, or about 50%, or about 55%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95% or more of the activity compared to the corresponding original TCE sequence would be considered suitable for inclusion in the subject paTCE. In some embodiments, a TCE found to retain a suitable level of activity can be linked to one or more ELNN polypeptides, having at least about 80% sequence identity (e.g., at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity) to a sequence from Tables 3a-3b.

TABLE 4

Exemplary conservative amino acid substitutions

Original Residue
Exemplary Substitutions

Ala (A)
val; leu; ile

Arg (R)
lys; gin; asn

Asn (N)
gin; his; lys; arg

Asp (D)
Glu

Cys (C)
Ser

Gln (Q)
Asn

Glu (E)
Asp

Gly (G)
Pro

His (H)
asn: gin: lys: arg

Ile (I)
leu; val; met; ala; phe: norleucine

Leu (L)
norleucine: ile: val; met; ala: phe

Lys (K)
arg: gin: asn

Met (M)
leu; phe; ile

Phe (F)
leu: val: ile; ala

Pro (P)
gly

Ser (S)
thr

Thr (T)
ser

Trp (W)
tyr

Tyr(Y)
trp: phe: thr: ser

Val (V)
ile; leu; met; phe; ala; norleucine

The present disclosure provides ELNNylated TCEs (such as paTCEs) that target PSMA, wherein TCE is a bispecific antibody (e.g., a bispecific TCE) that specifically binds to PSMA with one portion of the bispecific TCE and CD3 with the other portion of the bispecific TCE.

In some embodiments, the ELNNylated TCE comprises (1) a first portion comprising a first binding domain and a second binding domain, and (2) a second portion comprising a release segment, and (3) a third portion comprising an unstructured polypeptide mask (also sometimes referred to herein as a masking moiety).

In some embodiments, the ELNNylated TCE comprises the configuration of Formula Ia (depicted N-terminus to C-terminus):

(first portion)-(second portion)-(third portion) (Ia)

wherein first portion is a bispecific antibody domain comprising two antigen binding domains as noted above wherein the first binding domain has specific binding affinity to PSMA (e.g., as expressed on a cancer cell) and the second binding domain has specific binding affinity to a CD3 (e.g., as expressed on an effector cell); the second portion comprises a release segment (RS) capable of being cleaved by a mammalian protease; and the third portion is a masking moiety that serves to mask the biological properties of the bispecific antibody domain. In some embodiments, the RS is a protease-cleavable release segment that is cleavable by a protease that is present in a tumor microenvironment.

In some embodiments in which the first portion comprises a binding domain comprising a VHH and a binding domain comprising a VL and VH, the first portion binding domains can be in the order (VL-VH)1-(VHH)2, wherein “1” and “2” represent the first and second binding domains, respectively, or (VH-VL)1-(VHH)2, or (VHH)1-(VL-VH)2, or (VHH)1-(VH-VL)2, wherein the paired binding domains are linked by a polypeptide linker (e.g., as described herein). In some embodiments in which the first portion comprises two binding domains that each comprise a VL and VH, the first portion binding domains can be in the order (VL-VH)1-(VL-VH)2, wherein “1” and “2” represent the first and second binding domains, respectively, or (VL-VH)1-(VH-VL)2, or (VH-VL)1-(VL-VH)2, or (VH-VL)1-(VH-VL)2, wherein the paired binding domains are linked by a polypeptide linker (e.g., as described herein).

In some embodiments, the domain that binds PSMA is a VHH.

In some embodiments, the first portion binding domains comprise sequences provided in Tables 6a-6g, wherein Tables 6a-e show sequences that bind CD3 and Tables 6f-h show sequences that bind to PSMA; the RS sequence comprises a sequence provided in Tables 8a-8b (e.g., as described herein); and the masking moiety is an ELNN. In some embodiments, the masking moiety is an ELNN having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence comprising the group of sequences set forth in Tables 3a-3b. In some embodiments, the composition is a recombinant fusion protein. In some embodiments, the portions are linked by chemical conjugation.

In some embodiments, the fusion protein comprises the configuration of Formula IIa (depicted N-terminus to C-terminus):

(third portion)-(second portion)-(first portion) (IIa)

wherein first portion is a bispecific comprising two antigen binding domains wherein the first binding domain has specific binding affinity to a PSMA (e.g., as expressed on a cancer cell) and the second binding domain has specific binding affinity to CD3 (e.g., as expressed on an effector cell); the second portion comprises a release segment (RS) capable of being cleaved by a mammalian protease; and the third portion is a masking moiety that serves to mask the biological properties of the bispecific antibody domain. In some embodiments, the RS is a protease-cleavable release segment that is universally cleavable in a tumor microenvironment.

In some embodiments, the domain that binds PSMA is a VHH.

In some embodiments, the first portion binding domains comprise sequences provided in Tables 6a-6g, wherein Tables 6a-e show sequences that bind CD3 and Tables 6f-h shows sequences that bind to PSMA; the RS sequence comprises a sequence provided in Tables 8a-8b (e.g., as described herein); and the masking moiety is an ELNN. In some embodiments, the masking moiety is an ELNN having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence comprising the group of sequences set forth in Tables 3a-3b. In some embodiments, the composition is a recombinant fusion protein. In some embodiments, the portions are linked by chemical conjugation.

In some embodiments, a paTCE composition comprises the configuration of Formula IIIa (depicted N-terminus to C-terminus):

(fifth portion)-(fourth portion)-(first portion)-(second portion)-(third portion) (IIIa)

wherein first portion is a bispecific comprising two antigen binding domains wherein the first binding domain has specific binding affinity to a PSMA (e.g., as expressed on a cancer cell) and the second binding domain has specific binding affinity to CD3 (e.g., as expressed on an effector cell); the second portion comprises a release segment (RS) capable of being cleaved by a mammalian protease; and the third portion is a masking moiety that serves to mask the biological properties of the bispecific antibody domain; the fourth portion comprises a release segment (RS) capable of being cleaved by a mammalian protease which may be identical or different from the second portion; and the fifth portion is a masking moiety that may be identical or may be different from the third portion.

In some embodiments, the domain that binds PSMA is a VHH.

In some embodiments, the first portion binding domains comprise sequences provided in Tables 6a-6g, wherein Tables 6a-e show sequences that bind CD3 and Tables 6f-h shows sequences that bind to PSMA; each RS sequence comprises, individually, a sequence provided in Tables 8a-8b (e.g., as described herein); and each masking moiety is, individually, an ELNN. In some embodiments, each masking moiety is an ELNN having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence comprising the group of sequences set forth in Tables 3a-3b. In some embodiments, the paTCE is a recombinant fusion protein. In some embodiments, one or more portions of the paTCE are linked by chemical conjugation.

Provided herein are compositions that advantageously provide PSMA-targeted bispecific therapeutics that have more selectivity, greater half-life, and result in less toxicity and fewer side effects once they are cleaved by proteases found in the target tissues or tissues rendered unhealthy by a disease, such that the subject compositions have improved therapeutic index compared to bispecific antibody compositions known in the art. Such compositions are useful in the treatment of cancer. In some embodiments, when a paTCE is in proximity to a target tissue or cell bearing or secreting a protease capable of cleaving the RS, the bispecific binding domains are liberated from the ELNN(s) by the action of protease(s), removing a steric hindrance barrier, and rendering the TCE freer to exert its pharmacologic effect. This property is particularly advantageous in treating immunologically cold tumors that express PSMA. In some embodiments, a paTCE provided herein is activated at in a target tissue, wherein the target tissue is a solid tumor of an organ or system.

Binding Domains

In some embodiments, a binding domain provided herein comprises one or more full-length antibodies or one or more antigen-binding fragments thereof. Antigen-binding fragments of antibodies include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptides comprising a portion or portions of an antibody that specifically bind to an antigen. Antigen-binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques, such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains. The terms binding domain and antibody domain are used interchangeably herein.

In some embodiments, single chain binding domains are used, such as but not limited to Fv, Fab, Fab′, Fab′-SH, F(ab′)2, linear antibodies, single domain antibodies, VHHs, single-chain antibody molecules (scFv), and diabodies capable of binding ligands or receptors associated with effector cells and antigens of diseased tissues or cells that are cancers, tumors, or other malignant tissues.

In some embodiments, the binding domain is a bispecific antibody domain, wherein the bispecific antibody domain comprises a first antigen binding domain that specifically binds to a first target and a second antigen binding domain that specifically binds to a second target. In some embodiments, the first antigen binding domain is a first antigen binding fragment (e.g., an scFv or an ISVD, such as a VHH) and the second antigen binding domain is a second antigen binding fragment (e.g., an scFv or an ISVD, such as a VHH).

In some embodiments, an antigen binding fragment (AF) (e.g., a first antigen binding fragment (AF1), and/or a second antigen binding fragment (AF2)) can (each independently) be a chimeric, a humanized, or a human antigen-binding fragment. The antigen binding fragment (AF) (e.g., a first antigen binding fragment (AF1), and/or a second antigen binding fragment (AF2)) can (each independently) be an Fv, Fab, Fab′, Fab′-SH, linear antibody, VHH, or scFv.

In some embodiments, one or both antigen binding fragments (e.g., the first and/or second antigen binding fragments) can be configured as an (Fab′)2 or a single chain diabody. In some embodiments, the bispecific antibody comprises a first binding domain with binding specificity to a cancer cell marker and a second binding domain with binding specificity to an effector cell antigen. In some embodiments, the binding domain for the tumor cell target is a variable domain of a T cell receptor that has been engineered to bind MHC that is loaded with a peptide fragment of a protein that is overexpressed by tumor cells.

In some embodiments, a paTCE is designed with consideration of the location of the target tissue protease as well as the presence of the same protease in healthy tissues not intended to be targeted, as well as the presence of the target ligand in healthy tissue but a greater presence of the ligand in unhealthy target tissue, in order to provide a wide therapeutic window. A “therapeutic window” refers to the difference between the minimal effective dose and the maximal tolerated dose for a given therapeutic composition. In some embodiments, to help achieve a wide therapeutic window for a TCE, the binding domains of the TCE are shielded by the proximity of a masking (e.g., ELNN) moiety or moieties such that the binding affinity of the intact composition for one, or both, of the ligands is reduced compared to the composition that has been cleaved by a mammalian protease, thereby releasing the first portion from the shielding effects of the masking moiety.

In some embodiments, a complete antigen recognition and binding site comprises a dimer of one heavy chain variable domain (VH) and one light chain variable domain (VL). Within each VH and VL chain are three complementarity determining regions (CDRs) that interact to define an antigen binding site on the surface of the VH-VL dimer; the six CDRs of a binding domain confer antigen binding specificity to the antibody or single chain binding domain. Framework sequences flanking the CDRs have a tertiary structure that is essentially conserved in native immunoglobulins across species, and the framework residues (FR) serve to hold the CDRs in their appropriate orientation. In some embodiments, a constant domain is not required for binding function but may aid in stabilizing VH-VL interaction. In some embodiments, a binding site can be a pair of VH-VL, VH-VH or VL-VL domains either of the same or of different immunoglobulins, however it is generally preferred to make single chain binding domains using the respective VH and VL chains from the parental antibody. In some embodiments, the order of VH and VL domains within the polypeptide chain is not limiting, provided the VH and VL domains are arranged so that the antigen binding site can properly fold. Thus, in some embodiments, a single chain binding domains comprising a VH and a VL (e.g., in an scFv)can have the VH and VL arranged as VL-VH or VL-VH.

In some embodiments, the arrangement of the V chains may be VH(cancer cell surface antigen)-VL(cancer cell surface antigen)-VL(effector cell antigen)-VH(effector cell antigen), VH(cancer cell surface antigen)-VL(cancer cell surface antigen)-VH(effector cell antigen)-VL(effector cell antigen), VL(cancer cell surface antigen)-VH(cancer cell surface antigen)-VL(effector cell antigen)-VH(effector cell antigen), VL(cancer cell surface antigen)-VH(cancer cell surface antigen)-VH(effector cell antigen)-VL(effector cell antigen), VHH(cancer cell surface antigen)-VH(effector cell antigen)-VL(effector cell antigen), VHH(cancer cell surface antigen)-VL(effector cell antigen)-VH(effector cell antigen), VL(cancer cell surface antigen)-VH(cancer cell surface antigen)-VHH(effector cell antigen), or VH(cancer cell surface antigen)-VL(cancer cell surface antigen)-VHH(effector cell antigen).

In some embodiments, the following orders are possible: VH (effector cell antigen)-VL(effector cell antigen)-VL(cancer cell surface antigen)-VH(cancer cell surface antigen), VH(effector cell antigen)-VL(effector cell antigen)-VH(cancer cell surface antigen)-VL(cancer cell surface antigen), VL(effector cell antigen)-VH(effector cell antigen)-VL(cancer cell surface antigen)-VH(cancer cell surface antigen), VL(effector cell antigen)-VH(effector cell antigen)-VH(cancer cell surface antigen)-VL(cancer cell surface antigen), VHH(effector cell antigen)-VH(cancer cell surface antigen)-VL(cancer cell surface antigen), VHH(effector cell antigen)-VL(cancer cell surface antigen)-VH(cancer cell surface antigen), VL(effector cell antigen)-VH(effector cell antigen)-VHH(cancer cell surface antigen), or VH(effector cell antigen)-VL(effector cell antigen)-VHH(cancer cell surface antigen).

As used herein, “N-terminally to” or “C-terminally to” and grammatical variants thereof denote relative location within the primary amino acid sequence rather than placement at the absolute N- or C-terminus of the bispecific single chain antibody. Hence, as a non-limiting example, a first binding domain which is “located C-terminally to” a second binding domain denotes that the first binding is located on the carboxyl side of the second binding domain within a bispecific single chain antibody, and does not exclude the possibility that an additional sequence, for example a linker and/or an ELNN, a His-tag, or another compound such as a radioisotope, is located at the C-terminus of the bispecific single chain antibody.

In some embodiments, a paTCE comprises a first portion comprising a first binding domain and a second binding domain wherein each of the binding domains is an scFv and wherein each scFv comprises one VL and one VH. In some embodiments, the paTCE compositions comprise a first portion comprising a first binding domain and a second binding domain wherein one of the binding domains is an scFV and the other binding domain is a VHH. In some embodiments, the CD3 binding domain may be an scFV (comprising example a sequence shown in any of Tables 6a-e) and the second binding domain is a VHH that binds PSMA. In some embodiments, a paTCE comprises a first portion comprising a first binding domain and a second binding domain wherein the binding domains are in a diabody configuration and wherein one domain comprises one VHH region and the other domain comprises one VL region and one VH region. Exemplary PSMA-binding VHH binding domains are shown in Table 6f. In some embodiments, a paTCE comprises a first portion comprising a first binding domain and a second binding domain wherein the binding domains are in a diabody configuration and wherein each domain comprises one VL region and one VH region. Exemplary PSMA-binding VH and VL regions can be derived from the sequences shown in Table 6g.

In non-limiting examples, a TCE can comprise a sequence that exhibits at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an antibody sequence identified herein. In some embodiments, a TCE comprises a bispecific sequence (e.g., a BsAb) comprising a first binding domain and a second binding domain, wherein the first binding domain has specific binding affinity to a tumor-specific marker or a cancer cell antigen, and exhibits at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to paired to a VHH sequence of an anti-PSMA antibody disclosed herein in Table 6f or paired VL and VH sequences of an anti-PSMA antibody disclosed herein in Table 6g; and wherein the second binding domain has specific binding affinity to an effector cell, and exhibits at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to paired VL and VH sequences of an anti-CD3 antibody disclosed herein in any of Tables 6a-e.

In some embodiments, a TCE can comprise a binding domain (e.g., a VH and/or VL amino acid sequence) of or derived from an anti-CD3 antibody. Non-limiting examples of anti-CD3 antibodies include OKT3 (also called muromonab) and humanized anti-CD3 monoclonal antibody (hOKT31(Ala-Ala))(KC Herold et al., New England Journal of Medicine 346:1692-1698. 2002), as well as fragments and derivatives thereof that selectively bind to CD3. Additional examples are described in U.S. Pat. Nos. 5,885,573; 6,491,916; and US Patent Application Publication No. 2021/0054077-A1, the entire contents of each of which are incorporated herein by reference. Additional non-limiting examples of anti-CD3 antibody sequences include those of pasotuxizumab (also known as AMG-212) and acapatamab (also known as AMG-160).

In some embodiments, a TCE can comprise a binding domain (e.g., a VH and/or VL amino acid sequence) of or derived from an anti-PSMA antibody. Non-limiting examples of anti-PSMA antibody sequences include those of pasotuxizumab and acapatamab.

In some embodiments, the TCE is pasotuxizumab. In some embodiments, the TCE is acapatamab.

The present disclosure provides immunoglobulin single variable domains (ISVDs) that bind PSMA. The present disclosure further provides nucleic acids encoding the ISVDs or polypeptides as well as vectors, hosts and methods to produce these ISVDs or polypeptides. Also provided are multispecific polypeptides comprising an ISVD according to the present disclosure and at least one CD3 binding domain, including paTCEs. Included are methods for treatment making use of the ISVDs or polypeptides according to the present disclosure. In some embodiments, the ISVD is a heavy-chain ISVD. In some embodiments, the ISVD is a VHH, a humanized VHH, or a camelized VH.

In some embodiments, the ISVD is a VHH.

Also provided is a nucleic acid molecule encoding the ISVD or polypeptide of the present disclosure or a vector comprising the nucleic acid.

The present disclosure also relates to a non-human host or host cell transformed or transfected with the nucleic acid or vector that encodes an ISVD or polypeptide disclosed herein.

The present disclosure furthermore relates to compositions comprising an ISVD or polypeptide disclosed herein, such as a pharmaceutical composition.

Included herein is a method for producing an ISVD or polypeptide as disclosed herein, the method comprising the steps of:

- a. expressing, in a host cell or host organism or in another expression system, a nucleic acid sequence encoding the ISVD or polypeptide; optionally followed by:
- b. isolating and/or purifying the ISVD or polypeptide.

Provided herein are compositions and polypeptides comprising an ISVD for use as a medicament. In some embodiments, the polypeptide or composition is for use in the treatment of a proliferative disease. In some embodiments, the proliferative disease is cancer.

The present disclosure also provides a method of treatment comprising the step of administering a composition or polypeptide comprising an ISVD to a subject in need thereof. In some embodiments, the method of treatment is for treating a proliferative disease. In some embodiments, the proliferative disease is cancer.

Included herein are composition and polypeptides comprising an ISVD for use in the preparation of a medicament. In some embodiments, the medicament is used in the treatment of a proliferative disease. In some embodiments, the proliferative disease is cancer.

The term “immunoglobulin single variable domain” (ISVD), defines immunoglobulin molecules wherein the antigen binding site is present on, and formed by, a single immunoglobulin domain. This sets immunoglobulin single variable domains apart from “conventional” immunoglobulins (e.g. monoclonal antibodies) or their fragments (such as Fab, Fab′, F(ab′)2, scFv, di-scFv), wherein two immunoglobulin domains, in particular two variable domains, interact to form an antigen binding site. Typically, in conventional immunoglobulins, a heavy chain variable domain (VH) and a light chain variable domain (VL) interact to form an antigen binding site. In this case, the complementarity determining regions (CDRs) of both VH and VL will contribute to the antigen binding site, i.e. a total of 6 CDRs will be involved in antigen binding site formation, whereas in an ISVD only 3 CDRs from a single domain are contributing to the antigen binding site formation.

In view of the above definition, the antigen-binding domain of a conventional 4-chain antibody (such as an IgG, IgM, IgA, IgD or IgE molecule; known in the art) or of a Fab fragment, a F(ab′)2 fragment, an Fv fragment such as a disulphide linked Fv or a scFv fragment, or a diabody (all known in the art) derived from such conventional 4-chain antibody, would normally not be regarded as an immunoglobulin single variable domain, as, in these cases, binding to the respective epitope of an antigen would normally not occur by one (single) immunoglobulin domain but by a pair of (associating) immunoglobulin domains such as light and heavy chain variable domains, i.e., by a VH-VL pair of immunoglobulin domains, which jointly bind to an epitope of the respective antigen.

In contrast, immunoglobulin single variable domains are capable of specifically binding to an epitope of the antigen without pairing with an additional immunoglobulin variable domain. The binding site of an immunoglobulin single variable domain is formed by a single VH, a single VHH or single VL domain.

As such, the single variable domain may be a light chain variable domain sequence (e.g., a VL-sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a VH-sequence or VHH sequence) or a suitable fragment thereof; as long as it is capable of forming a single antigen binding unit (i.e., a functional antigen binding unit that essentially consists of the single variable domain, such that the single antigen binding domain does not need to interact with another variable domain to form a functional antigen binding unit).

An immunoglobulin single variable domain (ISVD) can for example be a heavy chain ISVD, such as a VH, VHH, including a camelized VH or humanized VHH. In some embodiments, it is a VHH, including a camelized VH or humanized VHH. Heavy chain ISVDs can be derived from a conventional four-chain antibody or from a heavy chain antibody.

For example, the immunoglobulin single variable domain may be a single domain antibody (or an amino acid sequence that is suitable for use as a single domain antibody), a “dAb” or dAb (or an amino acid sequence that is suitable for use as a dAb); other single variable domains, or any suitable fragment of any one thereof.

In some embodiments, the immunoglobulin single variable domain may be a NANOBODY® molecule or a suitable antigen-binding fragment thereof. NANOBODY® is a registered trademark of Ablynx N.V.

“VHH domains”, also known as VHHs, VHH regions, VHH antibody fragments, and VHH antibodies, have originally been described as the antigen binding immunoglobulin variable domain of “heavy chain antibodies” (i.e., of “antibodies devoid of light chains”; Hamers-Casterman et al. Nature 363: 446-448, 1993). The term “VHH domain” has been chosen in order to distinguish these variable domains from the heavy chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as “VH domains”, “VH regions”, and “VHs”) and from the light chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as “VL domains”, “VL regions”, and “VLs”). For a further description of VHHs, reference is made to the review article by Muyldermans (Reviews in Molecular Biotechnology 74: 277-302, 2001).

Typically, the generation of immunoglobulins involves the immunization of experimental animals, fusion of immunoglobulin producing cells to create hybridomas and screening for the desired specificities. Alternatively, immunoglobulins can be generated by screening of naïve or synthetic libraries e.g. by phage display.

The generation of immunoglobulin sequences has been described extensively in various publications, among which WO 94/04678, Hamers-Casterman et al. 1993 and Muyldermans et al. 2001 can be exemplified. In these methods, camelids are immunized with the target antigen in order to induce an immune response against the target antigen. The repertoire of VHHs obtained from the immunization is further screened for VHHs that bind the target antigen.

In these instances, the generation of antibodies requires purified antigen for immunization and/or screening. Antigens can be purified from natural sources, or in the course of recombinant production.

Immunization and/or screening for immunoglobulin sequences can be performed using peptide fragments of such antigens.

The present technology may use immunoglobulin sequences of different origins, comprising mouse, rat, rabbit, donkey, human and camelid immunoglobulin sequences. The technology also includes fully human, humanized, or chimeric sequences. For example, the technology comprises camelid immunoglobulin sequences and humanized camelid immunoglobulin sequences, or camelized domain antibodies, e.g. camelized dAb as described by Ward et al. (see for example WO 94/04678 and Davies and Riechmann (1994 and 1996)). In some embodiments, the technology also uses fused immunoglobulin sequences, e.g. forming a multivalent and/or multispecific construct (for multivalent and multispecific polypeptides containing one or more VHH domains and their preparation, reference is also made to Conrath et al., J. Biol. Chem., Vol. 276, 10. 7346-7350, 2001, as well as to for example WO 96/34103 and WO 99/23221), and immunoglobulin sequences comprising tags or other functional moieties, e.g. toxins, labels, radiochemicals, etc., which are derivable from the immunoglobulin sequences of the present technology.

A “humanized VHH” comprises an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VHH domain, but that has been “humanized”, i.e. by replacing one or more amino acid residues in the amino acid sequence of the naturally occurring VHH sequence (and in particular in the framework sequences) by one or more of the amino acid residues that occur at the corresponding position(s) in a VH domain from a conventional 4-chain antibody from a human being (e.g., indicated above). This can be performed in a manner known per se, which will be clear to the skilled person, for example based on the further description herein and the prior art (e.g., WO 2008/020079). Again, it should be noted that such humanized VHHs can be obtained in any suitable manner known per se and thus are not strictly limited to polypeptides that have been obtained using a polypeptide that comprises a naturally occurring VHH domain as a starting material.

A “camelized VH” comprises an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VH domain, but that has been “camelized”, i.e., by replacing one or more amino acid residues in the amino acid sequence of a naturally occurring VH domain from a conventional 4-chain antibody by one or more of the amino acid residues that occur at the corresponding position(s) in a VHH domain of a heavy chain antibody. This can be performed in a manner known per se, which will be clear to the skilled person, for example based on the further description herein and the prior art (e.g., WO 2008/020079). Such “camelizing” substitutions are preferably inserted at amino acid positions that form and/or are present at the VH-VL interface, and/or at the so-called Camelidae hallmark residues, as defined herein (see for example WO 94/04678 and Davies and Riechmann (1994 and 1996), supra). In some embodiments, the VH sequence that is used as a starting material or starting point for generating or designing the camelized VH is preferably a VH sequence from a mammal, e.g., the VH sequence of a human being, such as a VH3 sequence. However, it should be noted that such camelized VH can be obtained in any suitable manner known per se and thus are not strictly limited to polypeptides that have been obtained using a polypeptide that comprises a naturally occurring VH domain as a starting material.

In some embodiments, the structure of an immunoglobulin single variable domain sequence can be considered to be comprised of four framework regions (“FRs”), which are referred to in the art and herein as “Framework region 1” (“FR1”); as “Framework region 2” (“FR2”); as “Framework region 3” (“FR3”); and as “Framework region 4” (“FR4”), respectively; which framework regions are interrupted by three complementary determining regions (“CDRs”), which are referred to in the art and herein as “Complementarity Determining Region 1” (“CDR1”); as “Complementarity Determining Region 2” (“CDR2”); and as “Complementarity Determining Region 3” (“CDR3”), respectively.

As further described in paragraph q) on pages 58 and 59 of WO 08/020079, the amino acid residues of an immunoglobulin single variable domain can be numbered according to the general numbering for VH domains given by Kabat et al. (“Sequence of proteins of immunological interest”, US Public Health Services, NIH Bethesda, MD, Publication No. 91), as applied to VHH domains from Camelids in the article of Riechmann and Muyldermans, 2000 (J. Immunol. Methods 240 (1-2): 185-195; see for example FIG. 2 of this publication). It should be noted that—as is well known in the art for VH domains and for VHH domains—the total number of amino acid residues in each of the CDRs may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (that is, one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed for by the Kabat numbering). This means that, generally, the numbering according to Kabat may or may not correspond to the actual numbering of the amino acid residues in the actual sequence. In some embodiments, the total number of amino acid residues in a VH domain and a VHH domain is in the range of from 110 to 135. It should however be noted that smaller and longer sequences may also be suitable for the purposes described herein.

Determination of CDR regions may also be done according to different methods.

In some embodiments, VHH CDR sequences were determined according to the AbM definition as described in Martin 2010 (In: Kontermann and Dubel (Eds.) 2010, Antibody Engineering, vol 2, Springer Verlag Heidelberg Berlin, Chapter 3, pp. 33-51). According to this method, FR1 comprises the amino acid residues at positions 1-25, CDR1 comprises the amino acid residues at positions 26-35, FR2 comprises the amino acids at positions 36-49, CDR2 comprises the amino acid residues at positions 50-58, FR3 comprises the amino acid residues at positions 59-94, CDR3 comprises the amino acid residues at positions 95-102, and FR4 comprises the amino acid residues at positions 103-113.

In some embodiments, CDR sequences are determined according to Kabat (Martin 2010, In: Kontermann and Dubel (eds.), Antibody Engineering Vol. 2, Springer Verlag Heidelberg Berlin, Chapter 3, pp. 33-51). According to this method, FR1 of an immunoglobulin single variable domain comprises the amino acid residues at positions 1-30, CDR1 of an immunoglobulin single variable domain comprises the amino acid residues at positions 31-35, FR2 of an immunoglobulin single variable domain comprises the amino acids at positions 36-49, CDR2 of an immunoglobulin single variable domain comprises the amino acid residues at positions 50-65, FR3 of an immunoglobulin single variable domain comprises the amino acid residues at positions 66-94, CDR3 of an immunoglobulin single variable domain comprises the amino acid residues at positions 95-102, and FR4 of an immunoglobulin single variable domain comprises the amino acid residues at positions 103-113.

In some embodiments, FR1 comprises the amino acid residues at positions 1-25, CDR1 comprises the amino acid residues at positions 26-35, FR2 comprises the amino acids at positions 36-49, CDR2 comprises the amino acid residues at positions 50-58, FR3 comprises the amino acid residues at positions 59-94, CDR3 comprises the amino acid residues at positions 93-102, and FR4 comprises the amino acid residues at positions 103-113.

In such an immunoglobulin sequence, the framework sequences may be any suitable framework sequences, and examples of suitable framework sequences will be clear to the skilled person, for example on the basis the standard handbooks and the further disclosure and references mentioned herein.

In some embodiments, the framework sequences are a suitable combination of immunoglobulin framework sequences or framework sequences that have been derived from immunoglobulin framework sequences (for example, by humanization or camelization). For example, the framework sequences may be framework sequences derived from a light chain variable domain (e.g. a VL-sequence) and/or from a heavy chain variable domain (e.g. a VH-sequence or VHH sequence). In some embodiments, the framework sequences are either framework sequences that have been derived from a VHH-sequence (in which the framework sequences may optionally have been partially or fully humanized) or are conventional VH sequences that have been camelized (as defined herein).

In some embodiments, the framework sequences present in the ISVD sequence used in the technology may contain one or more of hallmark residues (as defined herein), such that the ISVD sequence is a VHH, including a humanized VHH or camelized VH. Some non-limiting examples of (suitable combinations of) such framework sequences will become clear from the further disclosure herein.

Again, as generally described herein for the immunoglobulin sequences, it is also possible to use suitable fragments (or combinations of fragments) of any of the foregoing, such as fragments that contain one or more CDR sequences, suitably flanked by and/or linked via one or more framework sequences (for example, in the same order as these CDR's and framework sequences may occur in the full-sized immunoglobulin sequence from which the fragment has been derived).

However, it should be noted that the technology is not limited as to the origin of the ISVD sequence (or of the nucleotide sequence used to express it), nor as to the way that the ISVD sequence or nucleotide sequence is (or has been) generated or obtained. Thus, the ISVD sequences may be naturally occurring sequences (from any suitable species) or synthetic or semi-synthetic sequences. In a specific but non-limiting aspect, the ISVD sequence is a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence, including but not limited to “humanized” (as disclosed herein) immunoglobulin sequences (such as partially or fully humanized mouse or rabbit immunoglobulin sequences, and in particular partially or fully humanized VHH sequences), “camelized” (as disclosed herein) immunoglobulin sequences, as well as immunoglobulin sequences that have been obtained by techniques such as affinity maturation (for example, starting from synthetic, random or naturally occurring immunoglobulin sequences), CDR grafting, veneering, combining fragments derived from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques for engineering immunoglobulin sequences well known to the skilled person, or any suitable combination of any of the foregoing.

Similarly, nucleotide sequences may be naturally occurring nucleotide sequences or synthetic or semi-synthetic sequences, and may for example be sequences that are isolated by PCR from a suitable naturally occurring template (e.g. DNA or RNA isolated from a cell), nucleotide sequences that have been isolated from a library (and in particular, an expression library), nucleotide sequences that have been prepared by introducing mutations into a naturally occurring nucleotide sequence (using any suitable technique known per se, such as mismatch PCR), nucleotide sequence that have been prepared by PCR using overlapping primers, or nucleotide sequences that have been prepared using techniques for DNA synthesis known per se.

As described above, an ISVD may be an ISVD or a suitable fragment thereof. For a general description of ISVDs, reference is made to the further description below, as well as to the references cited herein. In this respect, it should however be noted that this description and the prior art mainly described ISVDs of the so-called “VH3 class” (i.e. ISVDs with a high degree of sequence homology to human germline sequences of the VH3 class such as DP-47, DP-51, or DP-29). It should however be noted that the technology in its broadest sense can generally use any type of ISVD, and for example also uses the ISVDs belonging to the so-called “VH4 class” (i.e. ISVDs with a high degree of sequence homology to human germline sequences of the VH4 class such as DP-78), as for example described in WO 2007/118670.

Generally, ISVDs (in particular VHH sequences, including (partially) humanized VHH sequences and camelized VH sequences) can be characterized by the presence of one or more “Hallmark residues” (as described herein) in one or more of the framework sequences (again as further described herein). Thus, generally, an ISVD can be defined as an immunoglobulin sequence with the (general) structure

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

In some embodiments, an ISVD can be an immunoglobulin sequence with the (general) structure

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which the framework sequences are as further defined herein.

In some embodiments, an ISVD can be an immunoglobulin sequence with the (general) structure

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which one or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering are selected from the Hallmark residues mentioned in Table 5 below.

TABLE 5

Hallmark Residues in ISVDs

Position
Human VH3
Hallmark Residues

11
L, V; predominantly L
L, S, V, M, W, F, T, Q, E, A, R, G, K, Y, N, P,

I; preferably L

37
V, I, F; usually V
F⁽¹⁾, Y, V, L, A, H, S, I, W, C, N, G, D, T, P,

preferably F⁽¹⁾ or Y

44⁽⁸⁾
G
E⁽³⁾, Q⁽³⁾, G⁽²⁾, D, A, K, R, L, P, S, V, H, T, N,

W, M, l;

preferably G⁽²⁾, E⁽³⁾ or Q⁽³⁾; most preferably G⁽²⁾

or Q⁽³⁾

45⁽⁸⁾
L
L⁽²⁾, R⁽³⁾, P, H, F, G, Q, S, E, T, Y, C, I, D, V;

preferably L⁽²⁾ or R⁽³⁾

47⁽⁸⁾
W, Y
F⁽¹⁾, L⁽¹⁾ or W⁽²⁾ G, I, S, A, V, M, R, Y, E, P, T,

C, H, K, Q, N, D; preferably W⁽²⁾ , L⁽¹⁾ or F⁽¹⁾

83
R or K; usually R
R, K⁽⁵⁾, T, E⁽⁵⁾, Q, N, S, I, V, G, M, L, A, D, Y,

H; preferably K or R; most preferably K

84
A, T, D; predominantly
P⁽⁵⁾, S, H, L, A, V, I, T, F, D, R, Y, N, Q, G, E;

A
preferably P

103
W
W⁽⁴⁾, R⁽⁶⁾, G, S, K, A, M, Y, L, F, T, N, V, Q,

P⁽⁶⁾, E, C; preferably W

104
G
G, A, S, T, D, P, N, E, C, L; preferably G

108
L, M or T;
Q, L⁽⁷⁾, R, P, E, K, S, T, M, A, H; preferably Q

predominantly L
or L⁽⁷⁾

Notes:

⁽¹⁾In particular, but not exclusively, in combination with KERE (SEQ ID NO: 9408) or

KQRE(SEQ ID NO:9409) at positions 43-46.

⁽²⁾Usually as GLEW(SEQ ID NO: 9410) at positions 44-47.

⁽³⁾Usually as KERE(SEQ ID NO: 9408) or KQRE(SEQ ID NO: 9409) at positions 43-46, e.g.

as KEREL(SEQ ID NO: 9411), KEREF(SEQ ID NO: 9412), KQREL(SEQ ID NO: 9413), KQREF

(SEQ ID NO: 9414), KEREG(SEQ ID NO: 9415), KQREW(SEQ ID NO: 9416) or KQREG(SEQ ID

NO: 9417) at positions 43-47. Alternatively, also sequences such as TERE(SEQ ID

NO: 9418) (for example TEREL(SEQ ID NO: 9419)), TQRE(SEQ ID NO: 9420) (for example

TQREL(SEQ ID NO: 9421)), KECE(SEQ ID NO: 9422) (for example KECEL(SEQ ID NO: 9423)

or KECER(SEQ ID NO: 9424)), KQCE(SEQ ID NO: 9425) (for example KQCEL(SEQ ID NO: 9426)),

RERE(SEQ ID NO: 9427) (for example REREG(SEQ ID NO: 9428)), RQRE(SEQ ID NO: 9429)

(for example RQREL(SEQ ID NO: 9430), RQREF(SEQ ID NO: 9431) or RQREW(SEQ ID NO: 9432)),

QERE(SEQ ID NO: 9433) (for example QEREG(SEQ ID NO: 9434)), QQRE(SEQ ID NO: 9435),

(for example QQREW(SEQ ID NO: 9436), QQREL(SEQ ID NO: 9437) or QQREF(SEQ ID NO: 9438)),

KGRE(SEQ ID NO: 9439) (for example KGREG(SEQ ID NO: 9440)), KDRE(SE ID NO: 9441)

(for example KDREV(SEQ ID NO: 9442)) are possible. Some other possible, but less

preferred sequences include for example DECKL(SEQ ID NO: 9443) and NVCEL(SEQ ID NO: 9444).

⁽⁴⁾With both GLEW (SEQ ID NO: 9410) at positions 44-47 and KERE(SEQ ID NO: 9408) or

KQRE(SEQ ID NO: 9409) at positions 43-46.

⁽⁵⁾Often as KP or EP at positions 83-84 of naturally occurring VHH domains.

(6)In particular, but not exclusively, in combination with GLEW(SEQ ID NO: 9410) at

positions 44-47.

⁽⁷⁾With the proviso that when positions 44-47 are GLEW(SEQ ID NO: 9410), position

108 is always Q in (non-humanized) VHH sequences that also contain a W at 103.

⁽⁸⁾The GLEW(SEQ ID NO: 9410) group also contains GLEW(SEQ ID NO: 9410) -like sequences

at positions 44-47, such as for example GVEW(SEQ ID NO: 9445), EPEW(SEQ ID NO: 9446),

GLER(SEQ ID NO: 9447), DQEW(SEQ ID NO: 9448), DLEW(SEQ ID NO: 9449), GIEW(SEQ ID

NO: 9450), ELEW(SEQ ID NO: 9451), GPEW(SEQ ID NO: 9452), EWLP(SEQ ID NO: 9453),

GPER(SEQ ID NO: 9454), GLER(SEQ ID NO: 9447) and ELEW(SEQ ID NO: 9451).

In some embodiments, technology provided herein uses ISVDs that can bind to PSMA. In the context of the present technology, “binding to” a certain target molecule has the usual meaning in the art as understood in the context of antibodies and their respective antigens.

In some embodiments, an ISVD (such as a VHH) or multispecific-multivalent polypeptide exhibits reduced binding by pre-existing antibodies in human serum. To this end, in some embodiments, the polypeptide exhibits a valine (V) at amino acid position 11 and a leucine (L) at amino acid position 89 (according to Kabat numbering) in an ISVD. For example, the following sequence:

(SEQ ID NO: 549)

QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVG

AMSWSGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNKEYGRTWYDFNESDYWGQGTQVTVSS

may be modified to be the following sequence:

(SEQ ID NO: 566)

QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVG

AMSWSGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRALDTAVYYCAA

SNKEYGRTWYDFNESDYWGQGTQVTVSS

In some embodiments, the polypeptide exhibits an extension of 1 to 5 (preferably naturally occurring) amino acids, such as a single alanine (A) extension, at the C-terminus of an ISVD (e.g., a C-terminal ISVD of a fusion protein or an ISVD that is not fused to any other polypeptide). The C-terminus of an ISVD is normally VTVSS (SEQ ID NO: 574). For example, the following sequence:

(SEQ ID NO: 549)

QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVG

AMSWSGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNKEYGRTWYDFNESDYWGQGTQVTVSS,

may be modified to be any one of the following

sequences:

(SEQ ID NO: 567)

QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVG

AMSWSGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNKEYGRTWYDFNESDYWGQGTQVTVSSA,

(SEQ ID NO: 568)

QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVG

AMSWSGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNKEYGRTWYDFNESDYWGQGTQVTVSSAA,

(SEQ ID NO: 569)

QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVG

AMSWSGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNKEYGRTWYDFNESDYWGQGTQVTVSSAAA,

(SEQ ID NO: 570)

QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVG

AMSWSGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNKEYGRTWYDFNESDYWGQGTQVTVSSAAAA,

or

(SEQ ID NO: 571)

QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVG

AMSWSGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNKEYGRTWYDFNESDYWGQGTQVTVSSAAAAA.

In some embodiments, the polypeptide exhibits a lysine (K) or glutamine (Q) at position 110 (according to Kabat numbering) in at least one ISVD.

For example, the following sequence:

(SEQ ID NO: 549)

QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVG

AMSWSGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNKEYGRTWYDFNESDYWGQGTQVTVSS

may be modified to be any one of the following

sequences:

(SEQ ID NO: 572)

QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVG

AMSWSGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNKEYGRTWYDKNESDYWGQGTQVTVSS

or

(SEQ ID NO: 573)

QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVGA

MSWSGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAASN

KEYGRTWYDQNESDYWGQGTQVTVSS

In some embodiments, the ISVD exhibits a lysine (K) or glutamine (Q) at position 112 (according to Kabat numbering) in at least on ISVD. In these embodiments, the C-terminus of the ISVD is VKVSS (SEQ ID NO: 575), VQVSS (SEQ ID NO: 576), VTVKS (SEQ ID NO: 577), VTVQS (SEQ ID NO: 578), VKVKS (SEQ ID NO: 579), VKVQS (SEQ ID NO: 580), VQVKS (SEQ ID NO: 581), or VQVQS (SEQ ID NO: 582) such that after addition of a single alanine the C-terminus of the polypeptide for example exhibits the sequence VTVSSA (SEQ ID NO: 583), VKVSSA (SEQ ID NO: 584), VQVSSA (SEQ ID NO: 585), VTVKSA (SEQ ID NO: 586), VTVQSA (SEQ ID NO: 587), VKVKSA (SEQ ID NO: 588), VKVQSA (SEQ ID NO: 589), VQVKSA (SEQ ID NO: 590), or VQVQSA (SEQ ID NO: 591), preferably VTVSSA (SEQ ID NO: 583).

In some embodiments, the polypeptide exhibits a valine (V) at amino acid position 11 and a leucine (L) at amino acid position 89 (according to Kabat numbering) in at least the C-terminal ISVD, optionally a lysine (K) or glutamine (Q) at position 110 (according to Kabat numbering) in at least one ISVD, and exhibits an extension of 1 to 5 (preferably naturally occurring) amino acids, such as a single alanine (A) extension, at the C-terminus of the C-terminal ISVD (such that the C-terminus of the polypeptide for example consists of the sequence VTVSSA (SEQ ID NO: 583), VKVSSA (SEQ ID NO: 584) or VQVSSA (SEQ ID NO: 585), preferably VTVSSA (SEQ ID NO: 583)). See e.g., WO2012/175741 and WO2015/173325 for further information in this regard.

As will be clear from the further description above and herein, the ISVDs of the present technology can be used as “building blocks” to form polypeptides of the present technology, e.g., by suitably combining them with other groups, residues, moieties or binding units, in order to form compounds or fusion proteins as described herein (such as, without limitations, the bi-/tri-/tetra-/multivalent and bi-/tri-/tetra-/multispecific polypeptides of the present technology described herein), which combine within one molecule one or more desired properties or biological functions. A polypeptide with multiple ISVDs is also referred to herein as a “ISVD construct” or “ISVD format”.

The terms “specificity”, “binding specifically” or “specific binding” refer to the number of different target molecules, such as antigens, from the same organism to which a particular binding unit, such as an ISVD (e.g., a VHH) or an scFv, can bind with sufficiently high affinity (see below). “Specificity”, “binding specifically” or “specific binding” are used interchangeably herein with “selectivity”, “binding selectively” or “selective binding”. Binding units, such as VHHs and scFvs, preferably specifically bind to their designated targets.

The specificity/selectivity of a binding unit can be determined based on affinity. The affinity denotes the strength or stability of a molecular interaction. The affinity is commonly given as by the K_Dwhich is expressed in units of mol/liter (or M).

The affinity is a measure for the binding strength between a moiety and a binding site on the target molecule: the lower the value of the K_D, the stronger the binding strength between a target molecule and a targeting moiety.

Typically, binding units used in the present technology (such as ISVDs or scFvs) will bind to their targets with a K_Dof 10⁻⁵to 10⁻¹²moles/liter or less, and preferably 10⁻⁷to 10⁻¹²moles/liter or less and more preferably 10⁻⁸to 10⁻¹²moles/liter.

In some embodiments, a K_Dvalue greater than 10⁻⁴mol/liter is considered nonspecific. In some embodiments, a K_Dvalue less than 10⁻⁴mol/liter is considered specific.

The K_Dfor biological interactions, such as the binding of antibody sequences to an antigen, which are considered specific are typically in the range of 10000 nM or 10 μM to 0.001 nM or 1 μM or less.

Accordingly, specific/selective binding may mean that—using the same measurement method, e.g. SPR—a binding unit (or polypeptide comprising the same) binds to PSMA with a K_Dvalue of 10⁻⁵to 10⁻¹²moles/liter or less and binds to different targets with a K_Dvalue greater than 10⁻⁴moles/liter.

Thus, the ISVD preferably exhibits at least half the binding affinity, e.g., at least the same binding affinity, to human PSMA as compared to an ISVD consisting of the amino acid sequence of QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVGAMSW SGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAASNKEYGRTWY DFNESDYWGQGTQVTVSS (SEQ ID NO: 549), wherein the binding affinity is measured using the same method, such as SPR.

Specific binding to a certain target from a certain species does not exclude that the binding unit can also specifically bind to the analogous target from a different species. For example, specific binding to human PSMA does not exclude that the binding unit (or a polypeptide comprising the same) can also specifically bind to PSMA from cynomolgus monkeys.

Specific binding of a binding unit to its designated target can be determined in any suitable manner known per se, including, for example, Scatchard analysis and/or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, and the different variants thereof known per se in the art, as well as the other techniques mentioned herein.

The dissociation constant may be, e.g., the actual or apparent dissociation constant, as will be clear to the skilled person. Methods for determining the dissociation constant will be clear to the skilled person, and for example include the techniques mentioned below.

The affinity of a molecular interaction between two molecules can be measured via different techniques known per se, such as the well-known surface plasmon resonance (SPR) biosensor technique (see for example Ober et al. 2001, Intern. Immunology 13: 1551-1559). The term “surface plasmon resonance”, as used herein, refers to an optical phenomenon that allows for the analysis of real-time biospecific interactions by detection of alterations in protein concentrations within a biosensor matrix, where one molecule is immobilized on the biosensor chip and the other molecule is passed over the immobilized molecule under flow conditions yielding k_on, k_offmeasurements and hence K_Dvalues. This can for example be performed using the well-known BIAcore® system (BIAcore International AB, a GE Healthcare company, Uppsala, Sweden and Piscataway, NJ). For further descriptions, see Jonsson et al. (1993, Ann. Biol. Clin. 51: 19-26), Jonsson et al. Biotechniques 11: 620-627), Johnsson et al. (1995, J. Mol. Recognit. 8: 125-131), and Johnnson et al. (1991, Anal. Biochem. 198: 268-277).

Another well-known biosensor technique to determine affinities of biomolecular interactions is bio-layer interferometry (BLI) (see for example Abdiche et al. 2008, Anal. Biochem. 377: 209-217). The term “bio-layer Interferometry” or “BLI”, as used herein, refers to a label-free optical technique that analyzes the interference pattern of light reflected from two surfaces: an internal reference layer (reference beam) and a layer of immobilized protein on the biosensor tip (signal beam). A change in the number of molecules bound to the tip of the biosensor causes a shift in the interference pattern, reported as a wavelength shift (nm), the magnitude of which is a direct measure of the number of molecules bound to the biosensor tip surface. Since the interactions can be measured in real-time, association and dissociation rates and affinities can be determined. BLI can for example be performed using the well-known Octet® Systems (ForteBio, a division of Pall Life Sciences, Menlo Park, USA).

Alternatively, affinities can be measured in Kinetic Exclusion Assay (KinExA) (see for example Drake et al. 2004, Anal. Biochem., 328: 35-43), using the KinExA® platform (Sapidyne Instruments Inc, Boise, USA). The term “KinExA”, as used herein, refers to a solution-based method to measure true equilibrium binding affinity and kinetics of unmodified molecules. Equilibrated solutions of an antibody/antigen complex are passed over a column with beads precoated with antigen (or antibody), allowing the free antibody (or antigen) to bind to the coated molecule. Detection of the antibody (or antigen) thus captured is accomplished with a fluorescently labeled protein binding the antibody (or antigen).

The GYROLAB® immunoassay system provides a platform for automated bioanalysis and rapid sample turnaround (Fraley et al. 2013, Bioanalysis 5: 1765-74).

In some embodiments, an ISVD provided herein has an on-rate constant (k_on) for binding to the human PSMA selected from the group consisting of at least about 10³M⁻¹s⁻¹, at least about 10⁴M⁻¹s⁻¹, and at least about 10⁵M⁻¹s⁻¹, e.g., as measured by SPR, such as performed on a ProteOn XPR36 instrument, e.g., at 25°.

In some embodiments, an ISVD provided herein has an a k_onfor binding to the non-human primate PSMA selected from the group consisting of at least about 10³m⁻¹s⁻¹, at least about 10⁴m⁻¹s⁻¹, and at least about 10⁵m⁻¹s⁻¹, e.g., as measured by SPR, such as performed on a ProteOn XPR36 instrument, e.g., at 25° C.

In some embodiments, an ISVD provided herein has a k_offfor binding to the human PSMA selected from the group consisting of at most about 10⁻²s⁻¹, at most about 10⁻³s⁻¹, and at most about 10⁻⁴s⁻¹, e.g., as measured by SPR, such as performed on a ProteOn XPR36 instrument, preferably at 25° C.

In some embodiments, an ISVD provided herein has a k_offfor binding to the non-human primate PSMA selected from the group consisting of at most about 10⁻¹s⁻¹, at most about 10⁻²s⁻¹, at most about 10⁻³s⁻¹, and at most about 10⁻⁴s⁻¹, e.g., as measured by SPR, such as performed on a ProteOn XPR36 instrument, e.g., at 25° C.

In some embodiments, an ISVD provided herein has an affinity (K_D) for binding to the human PSMA selected from the group consisting of at most about 10⁻⁶M, at most about 10⁻⁷M, at most about 10⁻⁸M, at most about 10⁻⁸M, and at most about 10⁻⁹M, e.g., as measured by SPR, such as performed on a ProteOn XPR36 instrument, e.g., at 25° C.

In some embodiments, an ISVD provided herein has a K_Dfor binding to the non-human primate PSMA selected from the group consisting of at most about 10⁻⁶M, at most about 10⁻⁷M, and at most about 10⁻⁸M, e.g., as measured by SPR, such as performed on a ProteOn XPR36 instrument, e.g., at 25° C.

In some embodiments, the PSMA binding ISVD of the present technology bind to the human PSMA with the same or lower off rate constant (k_off) compared to QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVGAMSW SGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAASNKEYGRTWY DFNESDYWGQGTQVTVSS (SEQ ID NO: 549). In some embodiments, the ISVD of the present technology binds to non-human primate PSMA with the same or lower k_offcompared to an ISVD of

(SEQ ID NO: 549)

QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVG

AMSWSGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNKEYGRTWYDFNESDYWGQGTQVTVSS.

In some embodiments, a paTCE comprises a binding domain that is an scFv and a binding domain that is a VHH. In some embodiments, the scFv comprises VL and VH domains and specificity binds to an effector cell antigen (such as CD3), and the VHH domain specifically binds a cancer cell antigen (such as PSMA). In some embodiments, the scFv comprises six CDRs. In some embodiments, the scFv that comprises VH and VL regions comprising amino acid sequences that are at least about 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% identical to, or are identical to, paired VL and VH sequences of an anti-CD3 antibody identified in Table 6a. In some embodiments, the scFv comprises a CDR-H1 region, a CDR-H2 region, a CDR-H3 region, a CDR-L1 region, a CDR-L2 region, and a CDR-L3 region of paired VL and VH sequences of an anti-CD3 antibody identified in Table 6a. In some embodiments, the VHH is derived from an anti-PSMA antibody identified as the antibodies set forth in Table 6f. In some embodiments, the VHH comprises an amino acid sequence that is at least about 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% identical to, or is identical to, a VHH sequence disclosed in Table 6f. In some embodiments, the VHH comprises a CDR-1 region, a CDR-2 region, and a CDR-3 region of a VHH sequence in Table 6f. In some embodiments, the scFv that comprises VH and VL regions comprising amino acid sequences that are at least about 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% identical to, or are identical to, paired VL and VH sequences of an anti-PSMA antibody identified in Table 6g. In some embodiments, the scFv comprises a CDR-H1 region, a CDR-H2 region, a CDR-H3 region, a CDR-L1 region, a CDR-L2 region, and a CDR-L3 region of paired VL and VH sequences of an anti-PSMA antibody identified in Table 6g.

In some embodiments, a paTCE comprises a first binding domain that is an scFv and a second binding domain that is also an scFv. In some embodiments, the scFvs comprise VL and VH domains that are derived from monoclonal antibodies with binding specificity to the tumor-specific marker or an antigen of a cancer cell and effector cell antigen, respectively. In some embodiments, the first and second binding domains each comprise six CDRs derived from monoclonal antibodies with binding specificity to a cancer cell marker, such as a tumor-specific marker and effector cell antigens, respectively. In some embodiments, the first and second binding domains of the first portion of the subject compositions can have 3, 4, 5, or 6 CDRs within each binding domain. In some embodiments, a paTCE comprises a first binding domain and a second binding domain wherein each comprises a CDR-H1 region, a CDR-H2 region, a CDR-H3 region, a CDR-L1 region, a CDR-L2 region, and a CDR-L3 region, wherein each of the regions is derived from a monoclonal antibody capable of binding a tumor-specific marker or an antigen of a cancer cell, and an effector cell antigen, respectively.

In some embodiments, the second binding domain comprises VH and VL regions derived from a monoclonal antibody capable of binding human CD3. In some embodiments, the second binding domain comprises a scFv that comprises VH and VL regions wherein each VH and VL regions exhibit at least about 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% identity to or is identical to paired VL and VH sequences of an anti-CD3 antibody identified in Table 6a. In some embodiments, the second domain comprises a CDR-H1 region, a CDR-H2 region, a CDR-H3 region, a CDR-L1 region, a CDR-L2 region, and a CDR-L3 region, wherein each of the regions is derived from a monoclonal antibody identified herein as the antibodies set forth in Table 6a. In some embodiments, the VH and/or VL domains can be configured as scFvs or diabodies.

In some embodiments, a paTCE comprises a first binding domain that is a diabody and a second binding domain that is also a diabody. In some embodiments, the diabodies comprise VL and VH domains that are derived from monoclonal antibodies with binding specificity to the tumor-specific marker or an antigen of a cancer cell and the effector cell antigen, respectively.

In some embodiments, the present disclosure provides a paTCE composition, wherein the diabody second binding domain comprises VH and VL regions wherein each of the VH and VL regions exhibits at least about 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% identity to or is identical to the VL and a VH sequence of the huUCHT1 antibody of Table 6a. In some embodiments, the diabody second domain of the composition is derived from an anti-CD3 antibody described herein. In some embodiments, the anti-CD3 diabody is linked to an anti-PSMA-binding VHH sequence disclosed herein.

Methods to measure binding affinity and/or other biologic activity of an antigen binding domain can be those disclosed herein or methods generally known in the art. For example, the binding affinity of a binding pair (e.g., antibody and antigen), denoted as K_D, can be determined using various suitable assays including, but not limited to, radioactive binding assays, non-radioactive binding assays such as fluorescence resonance energy transfer and surface plasmon resonance (SPR, Biacore), and enzyme-linked immunosorbent assays (ELISA), kinetic exclusion assay (KinExA®) or as described in the Examples. An increase or decrease in binding affinity, for example the increased binding affinity of a TCE that has been cleaved to remove a masking moiety compared to the paTCE with the masking moiety attached, can be determined by measuring the binding affinity of the TCE to its target binding partner with and without the masking moiety.

Measurement of half-life of a subject chimeric assembly can be performed by various suitable methods. For example, the half-life of a substance can be determined by administering the substance to a subject and periodically sampling a biological sample (e.g., biological fluid such as blood or plasma or ascites) to determine the concentration and/or amount of that substance in the sample over time. The concentration of a substance in a biological sample can be determined using various suitable methods, including enzyme-linked immunosorbent assays (ELISA), immunoblots, and chromatography techniques including high-pressure liquid chromatography and fast protein liquid chromatography. In some cases, the substance may be labeled with a detectable tag, such as a radioactive tag or a fluorescence tag, which can be used to determine the concentration of the substance in the sample (e.g., a blood sample or a plasma sample. The various pharmacokinetic parameters are then determined from the results, which can be done using software packages such as SoftMax Pro software, or by manual calculations known in the art.

In addition, the physicochemical properties of the paTCE compositions may be measured to ascertain the degree of solubility, structure, and retention of stability. Assays of the subject compositions are conducted that allow determination of binding characteristics of the binding domains towards a ligand, including affinity and binding constants (K_D, k_onand k_off), the half-life of dissociation of the ligand-receptor complex, as well as the activity of the binding domain to inhibit the biologic activity of the sequestered ligand compared to free ligand (IC₅₀values). The term “EC₅₀” refers to the concentration needed to achieve half of the maximum biological response of the active substance, and is generally determined by ELISA or cell-based assays, including the methods of the Examples described herein.

Anti-CD3 Binding Domains

Also provided are anti-CD3 antibodies, fragments thereof, and fusion proteins comprising such antibodies and/or fragments.

In some embodiments, the present disclosure provides paTCE compositions comprising a binding domain of a first portion with binding affinity to T cells. In some embodiments, the binding domain comprises VL and VH derived from a monoclonal antibody that binds CD3. In some embodiments, the binding domain comprises VL and VH derived from a monoclonal antibody to CD3 epsilon and/or CD3 delta. In some embodiments, the binding domain comprises VL and VH derived from a monoclonal antibody to CD3 epsilon. In some embodiments, the binding domain comprises VL and VH derived from a monoclonal antibody to CD3 delta. Exemplary, non-limiting examples of VL and VH sequences of monoclonal antibodies to CD3 are presented in Table 6a. In some embodiments, the present disclosure provides a paTCE comprising a binding domain with binding affinity to CD3 comprising anti-CD3 VL and VH sequences set forth in Table 6a. In some embodiments, the present disclosure provides a paTCE comprising a binding domain of the first portion with binding affinity to CD3epsilon comprising anti-CD3epsilon VL and VH sequences set forth in Table 6a. In some embodiments, the present disclosure provides a paTCE composition, wherein a binding domain of the first portion comprises an scFv that comprises VH and VL regions wherein each VH and VL regions exhibit at least about 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% identity to or is identical to paired VL and VH sequences of the huUCHT1 anti-CD3 antibody of Table 6a. In some embodiments, the present disclosure provides a paTCE composition comprising a binding domain with binding affinity to CD3 comprising the CDR-L1 region, the CDR-L2 region, the CDR-L3 region, the CDR-H1 region, the CDR-H2 region, and the CDR-H3 region, wherein each is derived from the respective anti-CD3 VL and VH sequences set forth in Table 6a. In some embodiments, the present disclosure provides a paTCE composition comprising a binding domain with binding affinity to CD3 comprising an CDR-L1 region of RSSNGAVTSSNYAN (SEQ ID NO: 1), an CDR-L2 region of GTNKRAP (SEQ ID NO: 4), an CDR-L3 region of ALWYPNLWV (SEQ ID NO: 6), an CDR-H1 region of GFTFSTYAMN (SEQ ID NO: 12), an CDR-H2 region of RIRTKRNNYATYYADSVKG (SEQ ID NO: 13), and an CDR-H3 region of HENFGNSYVSWFAH (SEQ ID NO: 10).

The CD3 complex is a group of cell surface molecules that associates with the T-cell antigen receptor (TCR) and functions in the cell surface expression of TCR and in the signaling transduction cascade that originates when a peptide:MHC ligand binds to the TCR. Without being bound by any scientific theory, typically, when an antigen binds to the T-cell receptor, the CD3 sends signals through the cell membrane to the cytoplasm inside the T cell. This causes activation of the T cell that rapidly divide to produce new T cells sensitized to attack the particular antigen to which the TCR was exposed. The CD3 complex is comprised of the CD3epsilon molecule, along with four other membrane-bound polypeptides (CD3-gamma, -delta, and/or -zeta). In humans, CD3-epsilon is encoded by the CD3E gene on Chromosome 11. The intracellular domains of each of the CD3 chains contain immunoreceptor tyrosine-based activation motifs (ITAMs) that serve as the nucleating point for the intracellular signal transduction machinery upon T cell receptor engagement.

A number of therapeutic strategies modulate T cell immunity by targeting TCR signaling, particularly the anti-human CD3 monoclonal antibodies (mAbs) that are widely used clinically in immunosuppressive regimes. The CD3-specific mouse mAb OKT3 was the first mAb licensed for use in humans (Sgro, C. Side-effects of a monoclonal antibody, muromonab CD3/orthoclone OKT3: bibliographic review. Toxicology 105:23-29, 1995) and is widely used clinically as an immunosuppressive agent in transplantation (Chatenoud, Clin. Transplant 7:422-430, (1993); Chatenoud, Nat. Rev. Immunol. 3:123-132 (2003); Kumar, Transplant. Proc. 30:1351-1352 (1998)), type 1 diabetes, and psoriasis. Importantly, anti-CD3 mAbs can induce partial T cell signaling and clonal anergy (Smith, J A, Nonmitogenic Anti-CD3 Monoclonal Antibodies Deliver a Partial T Cell Receptor Signal and Induce Clonal Anergy J. Exp. Med. 185:1413-1422 (1997)). OKT3 has been described in the literature as a T cell mitogen as well as a potent T cell killer (Wong, JT. The mechanism of anti-CD3 monoclonal antibodies. Mediation of cytolysis by inter-T cell bridging. Transplantation 50:683-689 (1990)). In particular, the studies of Wong demonstrated that by bridging CD3 T cells and target cells, one could achieve killing of the target and that neither FcR-mediated ADCC nor complement fixation was necessary for bivalent anti-CD3 MAB to lyse the target cells.

OKT3 exhibits both a mitogenic and T-cell killing activity in a time-dependent fashion; following early activation of T cells leading to cytokine release, upon further administration OKT3 later blocks all known T-cell functions. It is due to this later blocking of T cell function that OKT3 has found such wide application as an immunosuppressant in therapy regimens for reduction or even abolition of allograft tissue rejection. Other antibodies specific for the CD3 molecule are disclosed in Tunnacliffe, Int. Immunol. 1 (1989), 546-50, WO2005/118635 and WO2007/033230 describe anti-human monoclonal CD3 epsilon antibodies, U.S. Pat. No. 5,821,337 describes the VL and VH sequences of murine anti-CD3 monoclonal Ab UCHT1 (muxCD3, Shalaby et al., J. Exp. Med. 175, 217-225 (1992) and a humanized variant of this antibody (hu UCHT1), and United States Patent Application 20120034228 discloses binding domains capable of binding to an epitope of human and non-chimpanzee primate CD3 epsilon chain.

In some embodiments, an anti-CD3 antibody domain comprises a VH region comprising the sequence EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKR NNYATYYADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCVRHENFGNSYVS WFAHWGQGTLVTVSS (SEQ ID NO: 311), or the CDRs thereof, and a VL region comprising the sequence ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANVQQKPGQAPRGLIGGTNK RAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL (SEQ ID NO: 361), or the CDRs thereof.

TABLE 6a

Anti-CD3 Monoclonal Antibodies and Sequences

Clone
Antibody

Name
Name
Target
VH Sequence
SEQ ID NO.
VL Sequence
SEQ ID NO.

huOKT3

CD3
QVQLVQSGGG
301
DIQMTQSPSSL
351

WVQPGRSLRLS

SASVGDRVTIT

CKASGYTFTRY

CSASSSVSYM

TMH
WVRQAPG

N
WYQQTPGKA

KGLEWIGYINP

PKRWIYDTSKL

SRGYTNYNQK

AS
GVPSRFSGS

VKD
RFTISRDN

GSGTDYTFTIS

SKNTAFLQMDS

SLQPEDIATYY

LRPEDTGVYFC

CQQWSSNPFT

ARYYDDHYCLD

FGQGTKLQITR

Y
WGQGTPVTV

SS

huUCHT1

CD3
EVQLVESGGGL
302
DIQMTQSPSSL
352

VQPGGSLRLSC

SASVGDRVTIT

AASGYSFTGYT

CRASQDIRNYL

MN
WVRQAPGK

N
WYQQKPGKA

GLEWVALINPY

PKLLIYYTSRLE

KGVST
YNQKFK

S
GVPSRFSGS

DRFTISVDKSK

GSGTDYTLTISS

NTAYLQMNSLR

LQPEDFATYYC

AEDTAVYYCAR

QQGNTLPWT
F

SGYYGDSDWY

GQGTKVEIK

FDV
WGQGTLV

TVSS

hu12F6

CD3
QVQLVQSGGG
303
DIQMTQSPSSL
353

WVQPGRSLRLS

SASVGDRVTMT

CKASGYTFTSY

CRASSSVSYM

TMH
WVRQAPG

H
WYQQTPGKA

KGLEWIGYINP

PKPWIYATSNL

SSGYTKYNQKF

AS
GVPSRFSGS

KD
RFTISADKS

GSGTDYTLTISS

KSTAFLQMDSL

LQPEDIATYYC

RPEDTGVYFCA

QQWSSNPPT
F

RWQDYDVYFD

GQGTKLQITR

Y
WGQGTPVTV

SS

mOKT3

CD3
QVQLQQSGAE
304
QIVLTQSPAIMS
354

LARPGASVKMS

ASPGEKVTMTC

CKASGYTFTRY

SASSSVSYMN

TMH
WVKQRPG

WYQQKSGTSP

QGLEWIGYINP

KRWIYDTSKLA

SRGYTNYNQK

S
GVPAHFRGS

FKD
KATLTTDK

GSGTSYSLTIS

SSSTAYMQLSS

GMEAEDAATYY

LTSEDSAVYYC

CQQWSSNPFT

ARYYDDHYCLD

FGSGTKLEINR

Y
WGQGTTLTV

SS

MT103
blinatumomab
CD3
DIKLQQSGAEL
305
DIQLTQSPAIMS
355

ARPGASVKMS

ASPGEKVTMTC

CKTSGYTFTRY

RASSSVSYMN

TMH
WVKQRPG

WYQQKSGTSP

QGLEWIGYINP

KRWIYDTSKVA

SRGYTNYNQK

S
GVPYRFSGS

FKD
KATLTTDK

GSGTSYSLTISS

SSSTAYMQLSS

MEAEDAATYYC

LTSEDSAVYYC

QQWSSNPLT
F

ARYYDDHYCLD

GAGTKLELK

Y
WGQGTTLTV

SS

MT110
solitomab
CD3
DVQLVQSGAEV
306
DIVLTQSPATLS
356

KKPGASVKVSC

LSPGERATLSC

KASGYTFTRYT

RASQSVSYMN

MH
WVRQAPGQ

WYQQKPGKAP

GLEWIGYINPS

KRWIYDTSKVA

RGYTNYADSV

S
GVPARFSGS

KG
RFTITTDKST

GSGTDYSLTIN

STAYMELSSLR

SLEAEDAATYY

SEDTATYYCAR

CQQWSSNPLT

YYDDHYCLDY

FGGGTKVEIK

WGQGTTVTVS

S

CD3.7

CD3
EVQLVESGGGL
307
QTVVTQEPSLT
357

VQPGGSLKLSC

VSPGGTVTLTC

AASGFTFNKYA

GSSTGAVTSGY

MN
WVRQAPGK

YPN
WVQQKPG

GLEWVARIRSK

QAPRGLIGGTK

YNNYATYYADS

FLAPGTPARFS

VKD
RFTISRDD

GSLLGGKAALT

SKNTAYLQMNN

LSGVQPEDEAE

LKTEDTAVYYC

YYCALWYSNR

VRHGNFGNSYI

WV
FGGGTKLT

SYWAY
WGQGT

VL

LVTVSS

CD3.8

CD3
EVQLVESGGGL
308
QAVVTQEPSLT
358

VQPGGSLRLSC

VSPGGTVTLTC

AASGFTFNTYA

GSSTGAVTTSN

MN
WVRQAPGK

YA
NWVQQKPG

GLEWVGRIRSK

QAPRGLIGGTN

YNNYATYYADS

KRAP
GVPARFS

VKG
RFTISRDD

GSLLGGKAALT

SKNTLYLQMNS

LSGAQPEDEAE

LRAEDTAVYYC

YYCALWYSNL

VRHGNFGNSY

WV
FGGGTKLT

VSWFAY
WGQG

VL

TLVTVSS

CD3.9

CD3
EVQLLESGGGL
309
ELVVTQEPSLT
359

VQPGGSLKLSC

VSPGGTVTLTC

AASGFTFNTYA

RSSTGAVTTSN

MN
WVRQAPGK

YAN
WVQQKPG

GLEWVARIRSK

QAPRGLIGGTN

YNNYATYYADS

KRAP
GTPARFS

VKD
RFTISRDD

GSLLGGKAALT

SKNTAYLQMNN

LSGVQPEDEAE

LKTEDTAVYYC

YYCALWYSNL

VRHGNFGNSY

WV
FGGGTKLT

VSWFAY
WGQG

VL

TLVTVSS

CD3.10

CD3
EVKLLESGGGL
310
QAVVTQESALT
360

VQPKGSLKLSC

TSPGETVTLTC

AASGFTFNTYA

RSSTGAVTTSN

MN
WVRQAPGK

YAN
WVQEKPD

GLEWVARIRSK

HLFTGLIGGTN

YNNYATYYADS

KRAP
GVPARFS

VKD
RFTISRDD

GSLIGDKAALTI

SQSILYLQMNN

TGAQTEDEAIY

LKTEDTAMYYC

FCALWYSNLW

VRHGNFGNSY

V
FGGGTKLTVL

VSWFAY
WGQG

TLVTVSS

CD3.228

CD3
EVQLVESGGGI
311
ELVVTQEPSLT
361

VQPGGSLRLSC

VSPGGTVTLTC

AASGFTFSTYA

RSSNGAVTSSN

MN
WVRQAPGK

YAN
WVQQKPG

GLEWVGRIRTK

QAPRGLIGGTN

RNNYATYYADS

KRAP
GTPARFS

VKG
RFTISRDD

GSLLGGKAALT

SKNTVYLQMNS

LSGVQPEDEAV

LKTEDTAVYYC

YYCALWYPNL

VRHENFGNSYV

WV
FGGGTKLT

SWFAH
WGQGT

VL

LVTVSS

CD3.23

CD3
EVQLLESGGGI
102
ELVVTQEPSLT
101

VQPGGSLKLSC

VSPGGTVTLTC

AASGFTFNTYA

RSSNGAVTSSN

MN
WVRQAPGK

YAN
WVQQKPG

GLEWVARIRSK

QAPRGLIGGTN

YNNYATYYADS

KRAP
GTPARFS

VKD
RFTISRDD

GSLLGGKAALT

SKNTVYLQMNN

LSGVQPEDEAV

LKTEDTAVYYC

YYCALWYPNL

VRHENFGNSYV

WV
FGGGTKLT

SWFAH
WGQGT

VL

LVTVSS

CD3.24

CD3
EVQLLESGGGI
102
ELVVTQEPSLT
103

VQPGGSLKLSC

VSPGGTVTLTC

AASGFTFNTYA

RSSNGEVTTSN

MN
WVRQAPGK

YAN
WVQQKPG

GLEWVARIRSK

QAPRGLIGGTIK

YNNYATYYADS

RAP
GTPARFSG

VKD
RFTISRDD

SLLGGKAALTL

SKNTVYLQMNN

SGVQPEDEAVY

LKTEDTAVYYC

YCALWYPNLW

VRHENFGNSYV

V
FGGGTKLTVL

SWFAH
WGQGT

LVTVSS

CD3.30

CD3
EVQLQESGGGI
105
ELVVTQEPSLT
104

VQPGGSLKLSC

VSPGGTVTLTC

AASGFTFNTYA

RSSNGAVTSSN

MN
WVRQAPGK

YAN
WVQQKPG

GLEWVARIRSK

QAPRGLIGGTN

YNNYATYYADS

KRAP
GTPARFS

VKD
RFTISRDD

GSSLGGKAALT

SKNTVYLQMNN

LSGVQPEDEAV

LKTEDTAVYYC

YYCALWYPNL

VRHENFGNSYV

WV
FGGGTKLT

SWFAH
WGQGT

VL

LVTVSS

CD3.31

CD3
EVQLQESGGGI
105
ELVVTQEPSLT
106

VQPGGSLKLSC

VSPGGTVTLTC

AASGFTFNTYA

RSSNGAVTSSN

MN
WVRQAPGK

YAN
WVQQKPG

GLEWVARIRSK

QAPRGLIGGTN

YNNYATYYADS

KRAP
GTPARFS

VKD
RFTISRDD

GSLLGGSAALT

SKNTVYLQMNN

LSGVQPEDEAV

LKTEDTAVYYC

YYCALWYPNL

VRHENFGNSYV

WV
FGGGTKLT

SWFAH
WGQGT

VL

LVTVSS

CD3.32

CD3
EVQLQESGGGI
105
ELVVTQEPSLT
107

VQPGGSLKLSC

VSPGGTVTLTC

AASGFTFNTYA

RSSNGAVTSSN

MN
WVRQAPGK

YAN
WVQQKPG

GLEWVARIRSK

QAPRGLIGGTN

YNNYATYYADS

KRAP
GTPARFS

VKD
RFTISRDD

GSSLGGSAALT

SKNTVYLQMNN

LSGVQPEDEAV

LKTEDTAVYYC

YYCALWYPNL

VRHENFGNSYV

WV
FGGGTKLT

SWFAH
WGQGT

VL

LVTVSS

CD3.33

CD3
EVQLQESGGG
111
ELVVTQEPSLT
110

LVQPGGSLKLS

VSPGGTVTLTC

CAASGFTFNTY

RSSTGAVTTSN

AMN
WVRQAPG

YAN
WVQQKPG

KGLEWVARIRS

QAPRGLIGGTN

KYNNYATYYAD

KRAP
GTPARFS

SVKDRFTISRD

GSSLGGSAALT

DSKNTAYLQMN

LSGVQPEDEAE

NLKTEDTAVYY

YYCALWYSNL

CVRHGNFGNS

WV
FGGGTKLT

YVSWFAY
WGQ

VL

GTLVTVSS

*underlined sequences, if present, are CDRs within the VL and VH

In some embodiments, the disclosure relates to antigen binding fragments (AF) having specific binding affinity for an effector cell antigen.

Various AF that bind effector cell antigens, particularly CD3 on T cells, have particular utility for pairing with an antigen binding fragment with binding affinity to PSMA antigens associated with a diseased cell or tissue in composition formats in order to recruit and effect effector cell-mediated cell killing of the diseased cell or tissue.

Binding specificity to the antigen of interest can be determined by complementarity determining regions, or CDRs, such as light chain CDRs or heavy chain CDRs. In many cases, binding specificity is determined by light chain CDRs and heavy chain CDRs. A given combination of heavy chain CDRs and light chain CDRs provides a given binding pocket that confers greater affinity and/or specificity towards an effector cell antigen as compared to other reference antigens. The resulting bispecific compositions which on the one hand bind to an effector cell antigen and on the other hand bind to an antigen on the diseased cell or tissue, having a first antigen binding fragment to PSMA linked by a short, flexible peptide linker to a second antigen binding fragment with binding specificity to an effector cell antigen are bispecific, with each antigen binding fragment having specific binding affinity to their respective ligands.

It will be understood that in such compositions, an AF directed against PSMA of a disease tissue is used in combination with an AF directed towards an effector cell marker in order to bring an effector cell in close proximity to the cell of a disease tissue in order to effect the cytolysis of the cell of the diseased tissue. Further, the first antigen fragment (AF1) and the second antigen fragment (AF2) are incorporated into the specifically designed polypeptides comprising cleavable release segments and ELNN segments in order to confer inactive characteristics on the compositions that becomes activated by release of the fused AF1 and AF2 upon the cleavage of the release segments when in proximity to the disease tissue having proteases capable of cleaving the release segments in one or more locations in the release segment sequence.

In some embodiments, the AF2 of the subject compositions has binding affinity for an effector cell antigen expressed on the surface of a T cell. In some embodiments, the AF2 of the subject compositions has binding affinity for CD3. In some embodiments, the AF2 of the subject compositions has binding affinity for a member of the CD3 complex, which includes in individual form or independently combined form all known CD3 subunits of the CD3 complex; for example, CD3 epsilon, CD3 delta, CD3 gamma, and CD3 zeta. In some embodiments, the AF2 has binding affinity for CD3 epsilon, CD3 delta, CD3 gamma, or CD3 zeta.

In some embodiments, the disclosure provides an antigen binding domain (e.g., antibody or an antigen-binding fragment thereof) that binds to cluster of differentiation 3 T cell receptor (CD3), comprising the following CDRs: a VL region CDR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to RSSX₁GAVTX₂SNYAN(SEQ ID NO:9006), wherein X₁corresponds to T or N, and X₂corresponds to T or S; a VL region CDR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GTNKRAP(SEQ ID NO:4); a VL region CDR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to ALWYX₄NLWV(SEQ ID NO:9007), wherein X₄corresponds to S or P; a VH region CDR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GFTFX₈TYAMN (SEQ ID NO:9008), wherein X₈corresponds to S or N; a VH region CDR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to RIRX₁₀KX₁₁NNYATYYADSVKX₁₂(SEQ ID NO:9009), wherein X₁₀corresponds to T or S, X₁₁corresponds to R or Y, and X₁₂corresponds to G or D; and a VH region CDR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to HX₁₅NFGNSYVSWFAX₁₆(SEQ ID NO:9010), wherein X₁₅corresponds to E or G, and X₁₆corresponds to H or Y.

In some embodiments, the disclosure provides an antigen binding domain (e.g., antibody or an antigen-binding fragment thereof) that binds to cluster of differentiation 3 T cell receptor (CD3), comprising the following CDRs: a VL region CDR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to RSSNGAVTSSNYAN(SEQ ID NO:1); a VL region CDR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GTNKRAP(SEQ ID NO:4); a VL region CDR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to ALWYPNLWV(SEQ ID NO:6); a VH region CDR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GFTFSTYAMN(SEQ ID NO:12); a VH region CDR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to RIRTKRNNYATYYADSVKG(SEQ ID NO:13); and a VH region CDR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to HENFGNSYVSWFAH(SEQ ID NO:10).

In some embodiments, the antigen binding domain comprises the following FRs: a VL region FR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to ELVVTQEPSLTVSPGGTVTLTC(SEQ ID NO:51); a VL region FR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to WVQQKPGQAPRGLIG(SEQ ID NO:52); a VL region FR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GTPARFSGSLLGGKAALTLSGVQPEDEAVYYC(SEQ ID NO:53); a VL region FR4 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to FGGGTKLTVL(SEQ ID NO:59); a VH region FR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to EVQLVESGGGIVQPGGSLRLSCAAS(SEQ ID NO:400); a VH region FR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to WVRQAPGKGLEWVG(SEQ ID NO:401); a VH region FR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to RFTISRDDSKNTVYLQMNSLKTEDTAVYYCVR(SEQ ID NO:402); and a VH region FR4 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to WGQGTLVTVSS(SEQ ID NO:67).

In some embodiments, the disclosure provides an antigen binding domain (e.g., antibody or an antigen-binding fragment thereof) that binds to CD3, comprising: a VL region comprising three the VL CDRs, wherein the three VL CDRs comprise the CDR1, CDR2, and CDR3 of a VL region comprising the following amino acid sequence: ELVVTQEPSLTVSPGGTVTLTCRSSX₁GAVTX₂SNYANWVQQKPGQAPRGLIGGTN KRAPGTPARFSGSLLGGKAALTLSGVQPEDEAX₃YYCALWYX₄NLWVFGGGTKLTV L, (SEQ ID NO:9001) wherein X₁corresponds to T or N, X₂corresponds to T or S, X₃corresponds to E or V, and X₄corresponds to S or P; and a VH region comprising three VH CDRs, wherein the three VH CDRs comprise the CDR1, CDR2, and CDR3 of a VH region comprising the following amino acid sequence: EVQLX₅ESGGGX₆VQPGGSLX₇LSCAASGFTFX₈TYAMNWVRQAPGKGLEWVXsRI RX₁₀KX₁₁NNYATYYADSVKX₁₂RFTISRDDSKNTX₁₃YLQMNX₁₄LKTEDTAVYYCVRH X₁₅NFGNSYVSWFAX₁₆WGQGTLVTVSS(SEQ ID NO:9002), wherein X₅corresponds to V or L, X₆corresponds to I or L, X₇corresponds to R or K, X₈corresponds to S or N, X₉corresponds to G or A, X₁₀corresponds to T or S, X₁₁corresponds to R or Y, X₁₂corresponds to G or D, X₁₃corresponds to V or A, X₁₄corresponds to S or N, X₁₅corresponds to E or G, and X₁₆corresponds to H or Y.

(SEQ ID NO: 311)

EVLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGR

IRTKRNNYATYYADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCV

RHENFGNSYVSWFAHWGQGTLVTVSS.

In some embodiments, the disclosure provides an antigen binding domain (e.g., antibody or an antigen-binding fragment thereof) that binds to CD3, comprising a VL region amino acid sequence SEQ ID NO/VH region amino acid sequence SEQ ID NO pair selected from the group consisting of: 896/897; 902/903; 700/701; 702/703; 716/717; 718/719; 728/729; 736/737; 738/739; 740/741; 742/743; 744/745; 746/747; 748/749; 750/751; 752/753; 754/755; 756/757; 758/759; 760/761; 762/763; 764/765; 766/767; 774/775; 776/777; 790/791; 792/793; 798/799; 800/801; 806/807; 808/809; 814/815; 816/817; 822/823; 824/825; or 826/867.

In some embodiments, the present disclosure provides an antigen binding fragment (e.g., AF1 or AF2) that binds to the CD3 protein complex that has enhanced stability compared to CD3 binding antibodies or antigen binding fragments known in the art. In some embodiments, a CD3 antigen binding fragment of the disclosure is designed to confer a higher degree of stability on the chimeric bispecific antigen binding fragment compositions into which they are integrated, leading to improved expression and recovery of the fusion protein, increased shelf-life and enhanced stability when administered to a subject. In some embodiments, an anti-CD3 AF of the present disclosure has a higher degree of thermal stability compared to certain CD3-binding antibodies and antigen binding fragments known in the art. In some embodiments, an anti-CD3 AF of the present disclosure has a higher degree of thermal stability compared to SP34 or an antigen binding fragment thereof. In some embodiments, an anti-CD3 AF of the present disclosure has a higher degree of thermal stability compared to CD3.9 and/or CD3.23 as disclosed in PCT International Patent Application Publication No. WO2021263058, the entire content of which is hereby incorporated herein by reference. In some embodiments, the anti-CD3 AF of the present disclosure is less immunogenic in a human compared to certain CD3-binding antibodies and antigen binding fragments known in the art. In some embodiments, an anti-CD3 AF of the present disclosure is less immunogenic than SP34 or an antigen binding fragment thereof. In some embodiments, an anti-CD3 AF of the present disclosure is less immunogenic than CD3.9 and/or CD3.23 as disclosed in PCT International Patent Application Publication No. WO2021263058, the entire content of which is hereby incorporated herein by reference. In some embodiments, the degree to which an AF is immunogenic is determined by an immunogenicity prediction method such as TEPITOPEpan (described in Zhang et al. PLoS One. 2012;7(2):e30483. doi: 10.1371/journal.pone.0030483, PMID: 22383964, the entire content of which is incorporated herein by reference) or NetMHCpan-4.1 and NetMHCllpan-4.0 (each described in Reynisson et al., Nucleic Acids Res 2020;48(W1):W449-W454. doi: 10.1093/nar/gkaa379., PMID: 32406916, the entire content of which is hereby incorporated herein by reference). In some embodiments, the anti-CD3 AF utilized as components of the chimeric bispecific antigen binding fragment compositions into which they are integrated exhibit favorable pharmaceutical properties, including high thermostability and low aggregation propensity, resulting in improved expression and recovery during manufacturing and storage, as well promoting long serum half-life. Biophysical properties such as thermostability are often limited by the antibody variable domains, which differ greatly in their intrinsic properties. High thermal stability is often associated with high expression levels and other desired properties, including being less susceptible to aggregation (Buchanan A, et al. Engineering a therapeutic IgG molecule to address cysteinylation, aggregation and enhance thermal stability and expression. MAbs 2013; 5:255). In some embodiments, thermal stability is determined by measuring the “melting temperature” (T_m), which is defined as the temperature at which half of the molecules are denatured. The melting temperature of each heterodimer is indicative of its thermal stability. In vitro assays to determine T_mare known in the art, including methods described in the Examples, below. The melting point of the heterodimer may be measured using techniques such as differential scanning calorimetry (Chen et al (2003) Pharm Res 20:1952-60; Ghirlando et al (1999) Immunol Lett 68:47-52). Alternatively, the thermal stability of the heterodimer may be measured using circular dichroism (Murray et al. (2002) J. Chromatogr Sci 40:343-9), or as described in the Examples, below.

In some embodiments of the polypeptides of this disclosure, the antigen binding fragment (e.g., AF1 or AF2) can exhibit a higher thermal stability than an anti-CD3 binding fragment consisting of a sequence of SEQ ID NO: 206 (see Table 6e), as evidenced in an in vitro assay by a higher melting temperature (T_m) of the first antigen binding fragment relative to that of the anti-CD3 binding fragment; or upon incorporating the first antigen binding fragment into a test bispecific antigen binding domain, a higher T_mof the test bispecific antigen binding domain relative to that of a control bispecific antigen binding domain, wherein the test bispecific antigen binding domain comprises the first antigen binding fragment and a reference antigen binding fragment that binds to an antigen other than CD3; and wherein the control bispecific antigen binding domain consists of the anti-CD3 binding fragment consisting of the sequence of SEQ ID NO:206 (see Table 6e) and the reference antigen binding fragment. In some embodiments, the melting temperature (T_m) of the first antigen binding fragment can be at least 2° C. greater, or at least 3° C. greater, or at least 4° C. greater, or at least 5° C. greater than the T_mof the anti-CD3 binding fragment consisting of the sequence of SEQ ID NO: 206 (see Table 6e).

In some embodiments, the polypeptides of any of the subject composition embodiments described herein comprise an antigen binding fragment (AF) that specifically bind human CD3. The antigen binding fragment (AF) can specifically bind human CD3. In some embodiments, the antigen binding fragment (AF) can bind a CD3 complex subunit identified herein as CD3 epsilon, CD3 delta, CD3 gamma, or CD3 zeta unit of CD3. The antigen binding fragment (AF) can bind a CD3 epsilon fragment of CD3. In some embodiments, the antigen binding fragment (AF) can specifically bind human CD3 with a binding affinity (K_D) constant between about 10 nM and about 400 nM, or between about 50 nM and about 350 nM, or between about 100 nM and 300 nM, as determined in an in vitro antigen-binding assay comprising a human CD3 antigen. In some embodiments, the polypeptides of any of the subject composition embodiments described herein comprise an antigen binding fragment (AF) that specifically binds human CD3 with a binding affinity (K_D) weaker than about 10 nM, or about 50 nM, or about 100 nM, or about 150 nM, or about 200 nM, or about 250 nM, or about 300 nM, or about 350 nM, or weaker than about 400 nM as determined in an in vitro antigen-binding assay. For clarity, an antigen binding fragment (AF) with a K_Dof 400 binds its ligand more weakly than one with a K_Dof 10 nM. In some embodiments, the polypeptides of any of the subject composition embodiments described herein comprise an antigen binding fragment (AF) that specifically binds human CD3 with at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or at least 10-fold weaker binding affinity than an antigen binding fragment consisting of an amino acid sequence of Table 6f-h, as determined by the respective binding affinities (K_D) in an in vitro antigen-binding assay.

In some embodiments, the present disclosure provides bispecific polypeptides comprising an antigen binding fragment (AF) that exhibits a binding affinity to CD3 (anti-CD3 AF) that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 50-fold, 100-fold, or at least 1000-fold at weaker relative to that of an anti-PSMA AF embodiments described herein that are incorporated into the subject polypeptides, as determined by the respective binding affinities (K_D) in an in vitro antigen-binding assay.

The binding affinity of the subject compositions for the target ligands can be assayed, e.g., using binding or competitive binding assays, such as Biacore assays with chip-bound receptors or binding proteins or ELISA assays, as described in U.S. Pat. No. 5,534,617, assays described in the Examples herein, radio-receptor assays, or other assays known in the art. The binding affinity constant can then be determined using standard methods, such as Scatchard analysis, as described by van Zoelen, et al., Trends Pharmacol Sciences (1998) 19)12):487, or other methods known in the art.

In some embodiments, the present disclosure provides an antigen binding fragment (AF) that binds to CD3 (anti-CD3 AF) and is incorporated into a chimeric, bispecific polypeptide composition that is designed to have an isoelectric point (pl) that confers enhanced stability on the composition compared to corresponding compositions comprising CD3 binding antibodies or antigen binding fragments known in the art. In some embodiments, the polypeptides of any of the subject composition embodiments described herein comprise AF that bind to CD3 (anti-CD3 AF) wherein the anti-CD3 AF exhibits a pl that is between 6.0 and 6.6, inclusive. In some embodiments, the polypeptides of any of the subject composition embodiments described herein comprise AF that bind to CD3 (anti-CD3 AF) wherein the anti-CD3 AF exhibits a pl that is at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 pH unit lower than the pl of a reference antigen binding fragment (e.g., consisting of a sequence shown in SEQ ID NO: 206 (see Table 6e)). In some embodiments, the polypeptides of any of the subject composition embodiments described herein comprise an AF that binds to CD3 (anti-CD3 AF) fused to another AF that binds to a PSMA antigen (anti-PSMA AF) wherein the anti-CD3 AF exhibits a pl that is within at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5 pH units of the pl of the AF that binds PSMA antigen or an epitope thereof. In some embodiments, the polypeptides of any of the subject composition embodiments described herein comprise an AF that binds to CD3 (anti-CD3 AF) fused to an AF that binds to a PSMA antigen (anti-PSMA AF) wherein the AF exhibits a pl that is within at least about 0.1 to about 1.5, or at least about 0.3 to about 1.2, or at least about 0.5 to about 1.0, or at least about 0.7 to about 0.9 pH units of the pl of the anti-CD3 AF. It is specifically intended that by such design wherein the pl of the two antigen binding fragments are within such ranges, the resulting fused antigen binding fragments will confer a higher degree of stability on the chimeric bispecific antigen binding fragment compositions into which they are integrated, leading to improved expression and enhanced recovery of the fusion protein in soluble, non-aggregated form, increased shelf-life of the formulated chimeric bispecific polypeptide compositions, and enhanced stability when the composition is administered to a subject. In some embodiments, having the two AFs (the anti-CD3 AF and the anti-PSMA AF) within a relatively narrow pl range of may allow for the selection of a buffer or other solution in which both the AFs (anti-CD3 AF and anti-PSMA AF) are stable, thereby promoting overall stability of the composition. In some embodiments, the antigen binding fragment (AF) can exhibit an isoelectric point (pl) that is less than or equal to 6.6. In some embodiments, the antigen binding fragment (AF) can exhibit an isoelectric point (pl) that is between 6.0 and 6.6, inclusive. In some embodiments, the antigen binding fragment (AF) can exhibit an isoelectric point (pl) that is at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 pH units lower than the pl of a reference antigen binding fragment consisting of a sequence shown in SEQ ID NO: 206 (see Table 6e). In some embodiments, the antigen binding fragment (AF) can specifically bind human CD3 with a binding affinity (K_D) constant between about between about 10 nM and about 400 nM (such as determined in an in vitro antigen-binding assay comprising a human CD3 antigen). In some embodiments, the antigen binding fragment (AF) can specifically bind human CD3 with a binding affinity (K_D) of less than about 10 nM, or less than about 50 nM, or less than about 100 nM, or less than about 150 nM, or less than about 200 nM, or less than about 250 nM, or less than about 300 nM, or less than about 350 nM, or less than about 400 nM (such as determined in an in vitro antigen-binding assay). In some embodiments, the antigen binding fragment (AF) can exhibit a binding affinity to CD3 that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or at least 10-fold weaker relative to that of an antigen binding fragment consisting of an amino acid sequence of SEQ ID NO: 206 (see Table 6e) (such as determined by the respective binding affinities (K_D) in an in vitro antigen-binding assay).

In some embodiments, the VL and VH of the antigen binding fragments are fused by relatively long linkers, consisting of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 hydrophilic amino acids that, when joined together, have a flexible characteristic. In some embodiments, the VL and VH of any of the scFv embodiments described herein are linked by a relatively long linker having the sequence SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). In some embodiments, the VL and VH of any of the scFv embodiments described herein are linked by relatively long linkers of hydrophilic amino acids having the sequences GSGEGSEGEGGGEGSEGEGSGEGGEGEGSG (SEQ ID NO: 82), TGSGEGSEGEGGGEGSEGEGSGEGGEGEGSGT (SEQ ID NO: 83), GATPPETGAETESPGETTGGSAESEPPGEG (SEQ ID NO: 84), or GSAAPTAGTTPSASPAPPTGGSSAAGSPST (SEQ ID NO: 85). In some embodiments, the AF1 and AF2 are linked together by a short linker of hydrophilic amino acids having 3, 4, 5, 6, or 7 amino acids. In some embodiments, the short linker sequences are identified herein as the sequences SGGGGS (SEQ ID NO: 86), GGGGS (SEQ ID NO: 87), GGSGGS (SEQ ID NO: 88), GGS, or GSP. In some embodiments, the disclosure provides compositions comprising a single chain diabody in which after folding, the first domain (VL or VH) is paired with the last domain (VH or VL) to form one scFv and the two domains in the middle are paired to form the other scFv in which the first and second domains, as well as the third and last domains, are fused together by one of the foregoing short linkers and the second and the third variable domains are fused by one of the foregoing relatively long linkers. In some embodiments, the selection of the short linker and relatively long linker is to prevent the incorrect pairing of adjacent variable domains, thereby facilitating the formation of a single chain configuration comprising the VL and VH of the first antigen binding fragment and the second antigen binding fragment.

TABLE 6b

Exemplary CD3 CDR Sequences

CDR

Antibody Domain
REGION
Amino Acid Sequence
SEQ ID NO:

3.23, 3.30, 3.31, 3.32,
CDR-L1
RSSNGAVTSSNYAN
1

3.228

3.24
CDR-L1
RSSNGEVTTSNYAN
2

3.33, 3.9
CDR-L1
RSSTGAVTTSNYAN
3

3.23, 3.30, 3.31, 3.32, 3.9,
CDR-L2
GTNKRAP
4

3.33, 3.228

3.24
CDR-L2
GTIKRAP
5

3.23, 3.24, 3.30, 3.31, 3.32,
CDR-L3
ALWYPNLWV
6

3.228

3.33, 3.9
CDR-L3
ALWYSNLWV
7

3.23, 3.24, 3.30, 3.31, 3.32,
CDR-H1
GFTFNTYAMN
8

3.9, 3.33

3.228
CDR-H1
GFTFSTYAMN
12

3.23, 3.24, 3.30, 3.31, 3.32,
CDR-H2
RIRSKYNNYATYYADSVKD
9

3.9, 3.33

3.228
CDR-H2
RIRTKRNNYATYYADSVKG
13

3.23. 3.24, 3.30, 3.31, 3.32,
CDR-H3
HENFGNSYVSWFAH
10

3.228

3.9, 3.33
CDR-H3
HGNFGNSYVSWFAY
11

TABLE 6c

Exemplary CD3 FR Sequences

SEQ

Antibody
FR

ID

Domain
REGION
Amino Acid Sequence
NO:

3.23, 3.24, 3.30,
FR-L1
ELVVTQEPSLTVSPGGTVTLTC
51

3.31, 3.32, 3.9,

3.33, 3.228

3.23, 3.24, 3.30,
FR-L2
WVQQKPGQAPRGLIG
52

3.31, 3.32, 3.9,

3.33, 3.228

3.23, 3.24, 3.228
FR-L3
GTPARFSGSLLGGKAALTLSGVQPEDEAVYYC
53

3.30
FR-L3
GTPARFSGSSLGGKAALTLSGVQPEDEAVYYC
54

3.31
FR-L3
GTPARFSGSLLGGSAALTLSGVQPEDEAVYYC
55

3.32
FR-L3
GTPARFSGSSLGGSAALTLSGVQPEDEAVYYC
56

3.9
FR-L3
GTPARFSGSLLGGKAALTLSGVQPEDEAEYYC
57

3.33
FR-L3
GTPARFSGSSLGGSAALTLSGVQPEDEAEYYC
58

3.23, 3.24, 3.30,
FR-L4
FGGGTKLTVL
59

3.31, 3.32, 3.9,

3.33, 3.228

3.228
FR-H1
EVQLVESGGGIVQPGGSLRLSCAAS
400

3.23, 3.24
FR-H1
EVQLLESGGGIVQPGGSLKLSCAAS
60

3.30, 3.31, 3.32
FR-H1
EVQLQESGGGIVQPGGSLKLSCAAS
61

3.33
FR-H1
EVQLQESGGGLVQPGGSLKLSCAAS
62

3.9
FR-H1
EVQLLESGGGLVQPGGSLKLSCAAS
63

3.23, 3.24, 3.30,
FR-H2
WVRQAPGKGLEWVA
64

3.31, 3.32, 3.9,

3.33

3.228
FR-H2
WVRQAPGKGLEWVG
401

3.23, 3.24, 3.30,
FR-H3
RFTISRDDSKNTVYLQMNNLKTEDTAVYYCVR
65

3.31, 3.32

3.9, 3.33
FR-H3
RFTISRDDSKNTAYLQMNNLKTEDTAVYYCVR
66

3.228
FR-H3
RFTISRDDSKNTVYLQMNSLKTEDTAVYYCVR
402

3.23, 3.24, 3.30,
FR-H4
WGQGTLVTVSS
67

3.31, 3.32, 3.9,

3.33, 3.228

TABLE 6d

Exemplary CD3 VL & VH Sequences

SEQ

Antibody

ID

Domain
REGION
Amino Acid Sequence
NO:

3.23
VL
ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYA
101

NWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLL

GGKAALTLSGVQPEDEAVYYCALWYPNLWVFGG

GTKLTVL

3.23,
VH
EVQLLESGGGIVQPGGSLKLSCAASGFTFNTYAMN
102

3.24

WVRQAPGKGLEWVARIRSKYNNYATYYADSVKDR

FTISRDDSKNTVYLQMNNLKTEDTAVYYCVRHENF

GNSYVSWFAHWGQGTLVTVSS

3.24
VL
ELVVTQEPSLTVSPGGTVTLTCRSSNGEVTTSNYA
103

NWVQQKPGQAPRGLIGGTIKRAPGTPARFSGSLL

GGKAALTLSGVQPEDEAVYYCALWYPNLWVFGG

GTKLTVL

3.30
VL
ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYA
104

NWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSSL

GGKAALTLSGVQPEDEAVYYCALWYPNLWVFGG

GTKLTVL

3.30,
VH
EVQLQESGGGIVQPGGSLKLSCAASGFTFNTYAM
105

3.31,

NWVRQAPGKGLEWVARIRSKYNNYATYYADSVKD

3.32

RFTISRDDSKNTVYLQMNNLKTEDTAVYYCVRHEN

FGNSYVSWFAHWGQGTLVTVSS

3.31
VL
ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYA
106

NWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLL

GGSAALTLSGVQPEDEAVYYCALWYPNLWVFGG

GTKLTVL

3.32
VL
ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYA
107

NWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSSL

GGSAALTLSGVQPEDEAVYYCALWYPNLWVFGG

GTKLTVL

3.9
VL
ELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYA
108

NWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLL

GGKAALTLSGVQPEDEAEYYCALWYSNLWVFGG

GTKLTVL

3.9
VH
EVQLLESGGGLVQPGGSLKLSCAASGFTFNTYAM
109

NWVRQAPGKGLEWVARIRSKYNNYATYYADSVKD

RFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGN

FGNSYVSWFAYWGQGTLVTVSS

3.33
VL
ELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYA
110

NWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSSL

GGSAALTLSGVQPEDEAEYYCALWYSNLWVFGG

GTKLTVL

3.33
VH
EVQLQESGGGLVQPGGSLKLSCAASGFTFNTYAM
111

NWVRQAPGKGLEWVARIRSKYNNYATYYADSVKD

RFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGN

FGNSYVSWFAYWGQGTLVTVSS

3.228
VL
ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYA
361

NWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLL

GGKAALTLSGVQPEDEAVYYCALWYPNLWVFGG

GTKLTVL

3.228
VH
EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAM
311

NWVRQAPGKGLEWVGRIRTKRNNYATYYADSVK

GRFTISRDDSKNTVYLQMNSLKTEDTAVYYCVRHE

NFGNSYVSWFAHWGQGTLVTVSS

TABLE 6e

Exemplary CD3 scFv Sequences

SEQ

Antibody

ID

Domain
Amino Acid Sequence
NO:

3.23
ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQ
201

APRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYY

CALWYPNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAES

EPPGEGEVQLLESGGGIVQPGGSLKLSCAASGFTFNTYAMNWVR

QAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTVYL

QMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS

3.24
ELVVTQEPSLTVSPGGTVTLTCRSSNGEVTTSNYANWVQQKPGQ
202

APRGLIGGTIKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYC

ALWYPNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESE

PPGEGEVQLLESGGGIVQPGGSLKLSCAASGFTFNTYAMNWVRQ

APGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTVYLQ

MNNLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS

3.30
ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQ
203

APRGLIGGTNKRAPGTPARFSGSSLGGKAALTLSGVQPEDEAVYY

CALWYPNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAES

EPPGEGEVQLQESGGGIVQPGGSLKLSCAASGFTFNTYAMNWVR

QAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTVYL

QMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS

3.31
ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQ
204

APRGLIGGTNKRAPGTPARFSGSLLGGSAALTLSGVQPEDEAVYY

CALWYPNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAES

EPPGEGEVQLQESGGGIVQPGGSLKLSCAASGFTFNTYAMNWVR

QAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTVYL

QMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS

3.32
ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQ
205

APRGLIGGTNKRAPGTPARFSGSSLGGSAALTLSGVQPEDEAVYY

CALWYPNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAES

EPPGEGEVQLQESGGGIVQPGGSLKLSCAASGFTFNTYAMNWVR

QAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTVYL

QMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSS

3.9
ELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQ
206

APRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYY

CALWYSNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAES

EPPGEGEVQLLESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVR

QAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYL

QMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS

3.33
ELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQ
207

APRGLIGGTNKRAPGTPARFSGSSLGGSAALTLSGVQPEDEAEYY

CALWYSNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAES

EPPGEGEVQLQESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVR

QAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYL

QMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS

4.11
QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAP
208

KLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAA

WDDSLSGLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAES

EPPGEGQVQLQQWGGGLVKPGGSLRLSCAASGFTFSSYSMNWV

RQAPGKGLEWVSRINSDGSSTNYADSVKGRFTISRDNAKNTLYLQ

MNSLRAEDTAVYYCARELRWGNWGQGTLVTVSS

4.12
QAGLTQPPSASGTPGQRVTLSCSGSYSNIGTYYVYWYQQLPGTA
209

PKLLIYSNDQRLSGVPDRFSGSKSGTSASLAISGLQSEDEAAYYCA

AWDDSLNGWAFGGGTKLTVLGATPPETGAETESPGETTGGSAES

EPPGEGQVQLQQWGGGLVKPGGSLRLSCAASGFTFSSYSMNWV

RQAPGKGLEWVSRINSDGSSTNYADSVKGRFTISRDNAKNTLYLQ

MNSLRAEDTAVYYCARELRWGNWGQGTLVTVSS

4.13
QPGLTQPPSASGTPGQRVTLSCSGRSSNIGSYYVYWYQHLPGMA
210

PKLLIYRNSRRPSGVPDRFSGSKSGTSASLVISGLQSDDEADYYCA

AWDDSLKSWVFGGGTKLTVLGATPPETGAETESPGETTGGSAES

EPPGEGQVQLQQWGGGLVKPGGSLRLSCAASGFTFSSYSMNWV

RQAPGKGLEWVSRINSDGSSTNYADSVKGRFTISRDNAKNTLYLQ

MNSLRAEDTAVYYCARELRWGNWGQGTLVTVSS

4.14
QSVLTQPPSASGTPGQRVTISCSGSSSNIGTNYVYWYQQFPGTAP
211

KLLIYSNNQRPSGVPDRFSGSKSGTSGSLAISGLQSEDEADYSCAA

WDDSLNGWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESE

PPGEGQVQLVQWGGGLVKPGGSLRLSCAASGFTFSSYSMNWVR

QAPGKGLEWVSRINSDGSSTNYADSVKGRFTISRDNAKNTLYLQM

NSLRAEDTAVYYCARELRWGNWGQGTLVTVSS

4.15
QPGLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAP
212

KLLIYRNNQRPSGVPDRLSGSKSGTSASLAISGLRSEDEADYYCAA

WDDSLSGWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESE

PPGEGQVQLVQWGGGLVKPGGSLRLSCAASGFTFSSYSMNWVR

QAPGKGLEWVSRINSDGSSTNYADSVKGRFTISRDNAKNTLYLQM

NSLRAEDTAVYYCARELRWGNWGQGTLVTVSS

4.16
QAVLTQPPSASGTPGQRVTISCSGSSSNIGSYYVYWYQQVPGAAP
213

KLLMRLNNQRPSGVPDRFSGAKSGTSASLVISGLRSEDEADYYCA

AWDDSLSGQWVFGGGTKLTVLGATPPETGAETESPGETTGGSAE

SEPPGEGQVQLQQWGGGLVKPGGSLRLSCAASGFTFSSYSMNW

VRQAPGKGLEWVSRINSDGSSTNYADSVKGRFTISRDNAKNTLYL

QMNSLRAEDTAVYYCARELRWGNWGQGTLVTVSS

4.17
QAGLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAP
214

KLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAT

WDASLSGWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESE

PPGEGEVQLVQWGGGLVKPGGSLRLSCAASGFTFSSYSMNWVR

QAPGKGLEWVSRINSDGSSTNYADSVKGRFTISRDNAKNTLYLQM

NSLRAEDTAVYYCARELRWGNWGQGTLVTVSS

3.228
ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQ
215

APRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYY

CALWYPNLWVFGGGTKLTVL

SESATPESGPGTSPGATPESGPGTSESATPEVQLVESGGGIVQPG

GSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKRNNYA

TYYADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCVRHENF

GNSYVSWFAHWGQGTLVTVSS

Anti-PSMA Binding Domains

Also provided are anti-PSMA antibodies, fragments thereof, and fusion proteins comprising such antibodies and/or fragments.

In some embodiments, the present disclosure provides paTCE compositions comprising a first portion binding domain that binds to the tumor-specific marker PSMA and a second binding domain that binds to an effector cell antigen, such as CD3 antigen.

In some embodiments, the first portion binding domain is a VHH domain. Non-limiting examples of VHH domain sequences are provided in Table 6f. In some embodiments, the binding domain with binding affinity for the tumor-specific marker PSMA is a VHH domain, listed in Table 6f. In some embodiments, the binding domain with binding affinity for PSMA is a VHH domain comprising three CDRs from a VHH domain listed in Table 6f.

In some embodiments, the present disclosure provides a paTCE composition comprising a first portion binding domain with binding affinity to the tumor-specific marker PSMA comprising anti-PSMA VHH sequences set forth in Table 6f. In some embodiments, the binding has a K_Dvalue of about 10⁻¹⁰to 10⁻⁷M, as determined in an in vitro binding assay. In some embodiments, the binding has a K_Dvalue of about 44 nM, as determined in an in vitro binding assay. It is specifically contemplated that the paTCE composition can comprise any one of the binding domains disclosed herein or sequence variants thereof so long as the variants exhibit binding specificity for the described antigen.

TABLE 6f

Anti-PSMA VHH Sequences

SEQ

Antibody
AC

ID

Name
Number
VHH Sequence
NO:

PSMA.301
AC3703
QVQLVESGGGVVQPGRSLRLSCAASG
500

RTFGIYVMGWFRQAPGKEREFVGAIS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYFCAASNKLYGR

TWYDFNESDYWGQGTQVTVSS

PSMA.302
AC3704
QVQLVESGGGVVQPGRSLRLSCAASG
501

RTFGIYVWGWFRQAPGKEREFVGAIS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYFCAASNKLYGR

TWYDFNESDYWGQGTQVTVSS

PSMA.303
AC3705
QVQLVESGGGVVQPGRSLRLSCAASG
502

RTFGIYVMGWFRQAPGKEREFVGAMS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYFCAASNKLYGR

TWYDFNESDYWGQGTQVTVSS

PSMA.304
AC3706
QVQLVESGGGVVQPGRSLRLSCAASG
503

RTFGIYVWGWFRQAPGKEREFVGAMS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYFCAASNKLYGR

TWYDFNESDYWGQGTQVTVSS

PSMA.305
AC3707
QVQLVESGGGVVQPGRSLRLSCAASG
504

RTFGIYVWGWFRQAPGKEREFVGAIS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYYCAASNKLYG

RTWYDFNESDYWGQGTQVTVSS

PSMA.306
AC3708
QVQLVESGGGVVQPGRSLRLSCAASG
505

RTFGIYVMGWFRQAPGKEREFVGAMS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYYCAASNKLYG

RTWYDFNESDYWGQGTQVTVSS

PSMA.307
AC3709
QVQLVESGGGVVQPGRSLRLSCAASG
506

RTFGIYVWGWFRQAPGKEREFVGAMS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYYCAASNKLYG

RTWYDFNESDYWGQGTQVTVSS

PSMA.308
AC3710
QVQLVESGGGVVQPGRSLRLSCAASG
507

RTFGIYVWGWFRQAPGKEREFVGAMS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYYCAASNKWYG

RTWYDFNESDYWGQGTQVTVSS

PSMA.309
AC3711
QVQLVESGGGVVQPGRSLRLSCAASG
508

RTFGIYVWGWFRQAPGKEREFVGAMS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYFCAASNKWYG

RTWYDFNESDYWGQGTQVTVSS

PSMA.310
AC3712
QVQLVESGGGVVQPGRSLRLSCAASG
509

RTFGIYVMGWFRQAPGKEREFVGAIS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYYCGGSNKLYG

RTWYDFNESDYWGQGTQVTVSS

PSMA.312
AC3714
QVQLVESGGGVVQPGRSLRLSCAASG
511

RTFGIYVMGWFRQAPGKEREFVGAIS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYYCGASNKLYG

RTWYDFNESDYWGQGTQVTVSS

PSMA.314
AC3716
QVQLVESGGGVVQPGRSLRLSCAASG
513

RTFGIYVMGWFRQAPGKEREFVGAIS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYYCAASNKDYG

RTWYDFNESDYWGQGTQVTVSS

PSMA.315
AC3717
QVQLVESGGGVVQPGRSLRLSCAASG
514

RTFGIYVMGWFRQAPGKEREFVGAIS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYYCAASNKEYG

RTWYDFNESDYWGQGTQVTVSS

PSMA.316
AC3718
QVQLVESGGGVVQPGRSLRLSCAASG
515

RTFGIYVMGWFRQAPGKEREFVGAIS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYYCAASNKGYG

RTWYDFNESDYWGQGTQVTVSS

PSMA.331
AC3733
QVQLVESGGGVVQPGRSLRLSCAASG
530

RTFGIYVMGWFRQAPGKEREFVGAIS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYFCGASNKLYG

RTWYDFNESDYWGQGTQVTVSS

PSMA.332
AC3734
QVQLVESGGGVVQPGRSLRLSCAASG
531

RTFGIYVMGWFRQAPGKEREFVGAIS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYFCAGSNKLYG

RTWYDFNESDYWGQGTQVTVSS

PSMA.334
AC3736
QVQLVESGGGVVQPGRSLRLSCAASG
533

RTFGIYVMGWFRQAPGKEREFVGAIS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYFCAASNKDYG

RTWYDFNESDYWGQGTQVTVSS

PSMA.335
AC3737
QVQLVESGGGVVQPGRSLRLSCAASG
534

RTFGIYVMGWFRQAPGKEREFVGAIS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYFCAASNKEYG

RTWYDFNESDYWGQGTQVTVSS

PSMA.336
AC3738
QVQLVESGGGVVQPGRSLRLSCAASG
535

RTFGIYVMGWFRQAPGKEREFVGAIS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYFCAASNKGYG

RTWYDFNESDYWGQGTQVTVSS

PSMA.344
AC3746
QVQLVESGGGVVQPGRSLRLSCAASG
543

RTFGIYVMGWFRQAPGKEREFVGAIS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYFCGGSNKLYG

RTWYDFNESDYWGQGTQVTVSS

PSMA.345
AC3747
QVQLVESGGGVVQPGRSLRLSCAASG
544

RTFGIYVWGWFRQAPGKEREFVGAMS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYYCGGSNKLYG

RTWYDFNESDYWGQGTQVTVSS

PSMA.347
AC3749
QVQLVESGGGVVQPGRSLRLSCAASG
546

RTFGIYVWGWFRQAPGKEREFVGAMS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYYCGASNKLYG

RTWYDFNESDYWGQGTQVTVSS

PSMA.348
AC3750
QVQLVESGGGVVQPGRSLRLSCAASG
547

RTFGIYVWGWFRQAPGKEREFVGAMS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYYCAASNKRYG

RTWYDFNESDYWGQGTQVTVSS

PSMA.349
AC3751
QVQLVESGGGVVQPGRSLRLSCAASG
548

RTFGIYVWGWFRQAPGKEREFVGAMS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYYCAASNKDYG

RTWYDFNESDYWGQGTQVTVSS

PSMA.350
AC3752
QVQLVESGGGVVQPGRSLRLSCAASG
549

RTFGIYVWGWFRQAPGKEREFVGAMS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYYCAASNKEYG

RTWYDFNESDYWGQGTQVTVSS

PSMA.351
AC3753
QVQLVESGGGVVQPGRSLRLSCAASG
550

RTFGIYVWGWFRQAPGKEREFVGAMS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYYCAASNKGYG

RTWYDFNESDYWGQGTQVTVSS

PSMA.353
AC3755
QVQLVESGGGVVQPGRSLRLSCAASG
552

RTFGIYVWGWFRQAPGKEREFVGAMS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYFCGASNKLYG

RTWYDFNESDYWGQGTQVTVSS

PSMA.354
AC3756
QVQLVESGGGVVQPGRSLRLSCAASG
553

RTFGIYVWGWFRQAPGKEREFVGAMS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYFCAASNKRYG

RTWYDFNESDYWGQGTQVTVSS

PSMA.355
AC3757
QVQLVESGGGVVQPGRSLRLSCAASG
554

RTFGIYVWGWFRQAPGKEREFVGAMS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYFCAASNKDYG

RTWYDFNESDYWGQGTQVTVSS

PSMA.356
AC3758
QVQLVESGGGVVQPGRSLRLSCAASG
555

RTFGIYVWGWFRQAPGKEREFVGAMS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYFCAASNKEYG

RTWYDFNESDYWGQGTQVTVSS

PSMA.357
AC3759
QVQLVESGGGVVQPGRSLRLSCAASG
556

RTFGIYVWGWFRQAPGKEREFVGAMS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYFCAASNKGYG

RTWYDFNESDYWGQGTQVTVSS

PSMA.358
AC3760
QVQLVESGGGVVQPGRSLRLSCAASG
557

RTFGIYVWGWFRQAPGKEREFVGAMS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYFCGGSNKLYG

RTWYDFNESDYWGQGTQVTVSS

In some embodiments, the disclosure provides an anti-PSMA antibody VHH region comprising the following CDRs: a VHH region CDR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GRTFGIYVWG(SEQ ID NO:9003); a VHH region CDR2 with an amino acid sequence that that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to AMSWSGSNRKVSDSVKG(SEQ ID NO:9004); and a VHH region CDR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to AASNKEYGRTWYDFNESDY(SEQ ID NO:9005).

In some embodiments, the anti-PSMA antibody VHH region comprises the following framework regions (FRs): a VHH region FR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to QVQLVESGGGVWQPGRSLRLSCAAS(SEQ ID NO:9011); a VHH region FR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to WFRQAPGKEREFVG(SEQ ID NO:9012); a VHH region FR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to RFTISRDNSKNTLYLQMNSLRAEDTAVYYC(SEQ ID NO:9013); and a VHH region FR4 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to WGQGTQVTVSS(SEQ ID NO:9014).

In some embodiments, the disclosure provides an anti-PSMA antibody VHH region comprising the sequence QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVGAMSW SGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAASNKEYGRTWY DFNESDYWGQGTQVTVSS (SEQ ID NO: 549), or the CDRs thereof.

Though in some embodiments the binding domain with binding affinity for PSMA is a VHH domain, it is contemplated that in other embodiments a binding domain is used that comprises VL and VH regions from or derived from a monoclonal antibody to PSMA. Exemplary, non-limiting examples of VL and VH sequences are presented in Table 6g. In some embodiments, the present disclosure provides a paTCE composition comprising a first portion binding domain with binding affinity to the tumor-specific marker PSMA comprising anti-PSMA VH and VL sequences set forth in Table 6g. In some embodiments, the present disclosure provides a paTCE composition comprising a first portion binding domain with binding affinity to PSMA tumor-specific marker comprising the CDR-L1 region, the CDR-L2 region, the CDR-L3 region, the CDR-H1 region, the CDR-H2 region, and the CDR-H3 region, wherein each is derived from the respective VL and VH sequences set forth in Table 6g. In some embodiments, the binding has a K_Dvalue of about 10¹⁰to 10⁻⁷M, as determined in an in vitro binding assay. It is specifically contemplated that the paTCE composition can comprise any one of the binding domains disclosed herein or sequence variants thereof so long as the variants exhibit binding specificity for the described antigen.

TABLE 6g

Anti-PSMA VH and VL Sequences

SEQ

SEQ

Tar-

ID

ID

get
VH Sequence
NO:
VL Sequence
NO:

PSMA
QVQLVESGGGLVKPGES
560
DIQMTQSPSSLSASVGDRV
561

LRLSCAASGFTFSDYYM

TITCKASQNVDTNVAWYQQ

YWVRQAPGKGLEWVAII

KPGQAPKSLIYSASYRYSD

SDGGYYTYYSDIIKGRF

VPSRFSGSASGTDFTLTIS

TISRDNAKNSLYLQMNS

SVQSEDFATYYCQQYDSYP

LKAEDTAVYYCARGFPL

YTFGGGTKLEIK

LRHGAMDYWGQGTLVTV

SS

PSMA
QVQLVESGGGLVKPGES
562
DIQMTQSPSSLSASVGDRV
563

LRLSCAASGFTFSDYYM

TITCKASQNVDTNVAWYQQ

YWVRQAPGKCLEWVAII

KPGQAPKSLIYSASYVYWD

SDGGYYTYYSDIIKGRF

VPSRFSGSASGTDFTLTIS

TISRDNAKNSLYLQMNS

SVQSEDFATYYCQQYDQQL

LKAEDTAVYYCARGFPL

ITFGCGTKLEIK

LRHGAMDYWGQGTLVTV

SS

In some embodiments, an anti-PSMA antibody domain comprises a VH region comprising the sequence QVQLVESGGGLVKPGESLRLSCAASGFTFSDYYMYWVRQAPGKGLEWVAIISDGG YYTYYSDIIKGRFTISRDNAKNSLYLQMNSLKAEDTAVYYCARGFPLLRHGAMDYW GQGTLVTVSS (SEQ ID NO: 560), or the CDRs thereof, and a VL region comprising the sequence DIQMTQSPSSLSASVGDRVTITCKASQNVDTNVAWYQQKPGQAPKSLIYSASYRYS DVPSRFSGSASGTDFTLTISSVQSEDFATYYCQQYDSYPYTFGGGTKLEIK (SEQ ID NO: 561), or the CDRs thereof. In some embodiments, an anti-PSMA antibody domain comprises the following sequence:

(SEQ ID NO: 564)

QVQLVESGGGLVKPGESLRLSCAASGFTFSDYYMYWVRQAPGKGLEWVAI

ISDGGYYTYYSDIIKGRFTISRDNAKNSLYLQMNSLKAEDTAVYYCARGF

PLLRHGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSAS

VGDRVTITCKASQNVDTNVAWYQQKPGQAPKSLIYSASYRYSDVPSRFSG

SASGTDFTLTISSVQSEDFATYYCQQYDSYPYTFGGGTKLEIK.

In some embodiments, an anti-PSMA antibody domain comprises a VH region comprising the sequence QVQLVESGGGLVKPGESLRLSCAASGFTFSDYYMYWVRQAPGKCLEWVAIISDGG YYTYYSDIIKGRFTISRDNAKNSLYLQMNSLKAEDTAVYYCARGFPLLRHGAMDYW GQGTLVTVSS (SEQ ID NO: 562), or the CDRs thereof, and a VL region comprising the sequence DIQMTQSPSSLSASVGDRVTITCKASQNVDTNVAWYQQKPGQAPKSLIYSASYVY WDVPSRFSGSASGTDFTLTISSVQSEDFATYYCQQYDQQLITFGCGTKLEIK (SEQ ID NO: 563), or the CDRs thereof. In some embodiments, an anti-PSMA antibody domain comprises the following sequence:

(SEQ ID NO: 565)

QVQLVESGGGLVKPGESLRLSCAASGFTFSDYYMYWVRQAPGKCLEWVAI

ISDGGYYTYYSDIIKGRFTISRDNAKNSLYLQMNSLKAEDTAVYYCARGF

PLLRHGAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSAS

VGDRVTITCKASQNVDTNVAWYQQKPGQAPKSLIYSASYVYWDVPSRFSG

SASGTDFTLTISSVQSEDFATYYCQQYDQQLITFGCGTKLEIK.

Linkers and Spacers Between Antibody Regions in Bispecific Antibodies

In some embodiments of the polypeptides of this disclosure, a pair of the light chain variable region (VL) and the heavy chain variable region (VH) of an antigen binding fragment can be linked by a linker, or a long linker (e.g., of hydrophilic amino acids). In some embodiments, a first antigen binding fragment (AF1) (e.g., a VHH domain, such as an anti-PSMA VHH domain) and a second antigen binding fragment (AF2) (e.g., an scFv, such as an anti-CD3 scFv) are linked by a linker, or a long linker (e.g., of hydrophilic amino acids). In some embodiments, a linker linking the light chain variable region (VL) and the heavy chain variable region (VH) of an antigen binding fragment (e.g., a first antigen binding fragment (AF1) and/or a second antigen binding fragment (AF2)), can (each independently) comprise an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence set forth in Table A. In some embodiments, a linker linking the light chain variable region (VL) and the heavy chain variable region (VH) of an antigen binding fragment (e.g., a first antigen binding fragment (AF1) and/or a second antigen binding fragment (AF2)), can (each independently) comprise an amino acid sequence identical to a sequence set forth in Table A. In some embodiments of the polypeptides of this disclosure, two antigen binding fragments (e.g., a first and a second antigen binding fragments) can be fused together by a peptide linker, or a short linker. In some embodiments, the peptide linker linking two antigen binding fragments (e.g., a first and a second antigen binding fragments), can comprise an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence set forth in Table B. In some embodiments, the peptide linker linking two antigen binding fragments (e.g., a first and a second antigen binding fragments), can comprise an amino acid sequence identical to a sequence set forth in Table B. In some cases, the first antigen binding fragment is a single-chain variable fragment (scFv). In some cases, the second antigen binding fragment is a single-chain variable fragment (scFv). The two single-chain variable fragments of the first and second antigen binding fragments can be linked together by the peptide linker. In some embodiments of the polypeptides of this disclosure, the linker used to link the VHH of the first antigen binding fragment (e.g., an anti-PSMA VHH) and the linker used to link the VL and VH of the second antigen binding fragment (e.g., an anti-CD3 scFv) can be GGGGSGGGS (SEQ ID NO: 125) of Table A. In other embodiments, the linker used to link the VL and VH of an antigen binding fragment (e.g., an anti-CD3 scFv) can be SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81). In some embodiments, the disclosure provides polypeptides comprising a single chain diabody in which after folding, the first domain (VL or VH) is paired with the last domain (VH or VL) to form one scFv and the two domains in the middle are paired to form the other scFv in which the first and second domains, as well as the third and last domains, are fused together by a short linker of hydrophilic amino acids identified herein by the sequences set forth in Table B and the second and the third variable domains are fused by a long linker identified in Table A. In some embodiments, the selection of the short linker and long linker is to prevent the incorrect pairing of adjacent variable domains, thereby facilitating the formation of the single chain configuration comprising the VL and VH of the first binding moiety and the second binding moiety.

TABLE A

Intramolecular Long Linkers

Linker

SEQ

#
Name
ID
Amino Acid Sequence

L1
(G4S)3
112
GGGGSGGGGSGGGGS

L2
MT110_18
113
GEGTSTGSGGSGGSGGAD

L3
MT103_18
114
VEGGSGGSGGSGGSGGVD

L4
UCHT1 29
115
RTSGPGDGGKGGPGKGPGGEGTKGTGPGG

L5
Y30
116
GSGEGSEGEGGGEGSEGEGSGEGGEGEGS

G

L6
Y32
117
TGSGEGSEGEGGGEGSEGEGSGEGGEGEG

SGT

L7
G1_30_3
118
GATPPETGAETESPGETTGGSAESEPPGEG

L8
G9_30_1
119
GSAAPTAGTTPSASPAPPTGGSSAAGSPST

L9
Y30_
120
GEGGESGGSEGEGSGEGEGGSGGEGESEG

modified

G

L10
G1_30_1
121
STETSPSTPTESPEAGSGSGSPESPSGTEA

L11
G1_30_2
122
PTGTTGEPSGEGSEPEGSAPTSSTSEATPS

L12
G1_30_4
123
SESESEGEAPTGPGASTTPEPSESPTPETS

L13
UCHT1_
124
PEGGESGEGTGPGTGGEPEGEGGPGGEGG

modified

T

TABLE B

Intermolecular Short Linkers

Name
Amino Acid Sequence

S-1
GGGGSGGGS (SEQ ID

NO: 125)

S-2
SGGGGS (SEQ ID NO: 86)

S-3
GGGGS (SEQ ID NO: 87)

S-4
GGS

S-5
GSP

Spacers & TCE Release Segments

Included herein are fusion proteins comprising TCE components that either becomes biologically active or have an increase in biological activity upon release from an ELNN by cleavage of an optional cleavage sequence incorporated within optional spacer sequences into the fusion protein, e.g., as described herein.

In some embodiments, the spacer may be provided to enhance expression of the fusion protein from a host cell and/or to decrease steric hindrance such that the TCE component may assume its desired tertiary structure and/or interact appropriately with its target molecule. For spacers and methods of identifying desirable spacers, see, for example, George, et al. (2003) Protein Engineering 15:871-879, specifically incorporated by reference herein. In some embodiments, the spacer comprises one or more peptide sequences that are between 1 to 50 amino acid residues in length, or about 1 to 25 residues, or about 1 to 10 residues in length. Spacer sequences, exclusive of cleavage sites, can comprise any of the 20 natural L amino acids, and will preferably comprise hydrophilic amino acids that are sterically unhindered that can include, but not be limited to, glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) or proline (P). In some embodiments, the spacer can be a polyglycine or polyalanine, or predominately a mixture of combinations of glycine and alanine residues. In some embodiments, the spacer polypeptide exclusive of a cleavage sequence is substantially devoid of secondary structure. In some embodiments, one or both spacer sequences in a paTCE fusion protein composition may each further contain a cleavage sequence, which may be identical or may be different, wherein the cleavage sequence may be acted on by a protease to release the TCE from the fusion protein.

TABLE C

Exemplary Spacers between a Release Segment and a

Bispecific Antibody Domain

Amino Acid

Sequence
SEQ ID NO:

STEPS
89

SATPESGPGT
90

ATSGSETPGT
91

GTAEAASASG
92

STEPSEGSAPGTS
93

SGPGTS
94

GTSTEPS
95

GTSESATPES
96

GTATPESGPG
97

In some embodiments of the polypeptides of this disclosure, a release segment (RS) (e.g., a first release segment (RS1), a second release segment (RS2), etc.) can be fused to a bispecific antibody domain (BsAb) by a spacer. In some embodiments, a spacer can (each independently) comprise at least 4 types of amino acids that are glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) or proline (P). In some embodiments, the peptides of this disclosure can comprise a first release segment fused to the bispecific antibody domain via a first spacer and a second release segment fused to the bispecific antibody domain via a second spacer. In some embodiments, a spacer (e.g., a first spacer, a second spacer, etc.) can (each independently) comprise an amino acid sequence having at least (about) 80%, at least (about) 90%, or 100% sequence identity to a sequence set forth in Table C. In some embodiments, the spacer (e.g., the first spacer, the second spacer, etc.) can (each independently) comprise an amino acid sequence identical to a sequence set forth in Table C.

In some embodiments, the incorporation of the cleavage sequence into a fusion protein is designed to permit release of a TCE that becomes active or more active upon its release from one or more ELNNs. In some embodiments, the cleavage sequences are located sufficiently close to the TCE sequences, generally within 18, or within 12, or within 6, or within 2 amino acids of the TCE sequence terminus, such that any remaining residues attached to the TCE after cleavage do not appreciably interfere with the activity (e.g., such as binding to a receptor) of the TCE yet provide sufficient access to the protease to be able to effect cleavage of the cleavage sequence. In some embodiments, the cleavage site is a sequence that can be cleaved by a protease endogenous to the mammalian subject such that a paTCE can be cleaved after administration to a subject. In such cases, the paTCE can serve as a circulating depot for the TCE. Examples of cleavage sites contemplated herein include, but are not limited to, a polypeptide sequence cleavable by a mammalian endogenous protease listed in Table 7.

In some embodiments, a paTCE fusion protein comprises spacer sequences that comprise one or more cleavage sequences configured to release the TCE from the fusion protein when acted on by a protease. In some embodiments, a spacer sequence does not comprise a cleavage sequence. In some embodiments, the one or more cleavage sequences can be a sequence having at least about 80% (e.g., at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%) sequence identify to a sequence from Table 8a or b.

In some embodiments, the disclosure provides TCE release segment polypeptides (or release segments (RSs)) that are substrates for one or more mammalian proteases associated with or produced by disease tissues or cells found in proximity to disease tissues. Such proteases can include, but not be limited to the classes of proteases such as metalloproteinases, cysteine proteases, aspartate proteases, and serine proteases, including, but not limited to, the proteases of Table 7. The RSs are useful for, amongst other things, incorporation into the subject recombinant polypeptides, conferring an inactive format that can be activated by the cleavage of the RSs by mammalian proteases. As described herein, the RSs are incorporated into the subject recombinant polypeptide compositions, linking the incorporated binding moieties to the ELNN (exemplary configurations of which are described herein) such that upon cleavage of the RSs by action of the one or more proteases for which the RSs are substrates, the binding moieties and ELNN are released from the composition and the binding moieties, no longer shielded by the ELNN, regain their full potential to bind their ligands.

TABLE 7

Proteases of Target Tissues

Class of Proteases
Protease

Metalloproteinases
Meprin

Neprilysin (CD10)

PSMA

BMP-1

A disintegrin and metalloproteinases

(ADAMs)

ADAM8

ADAM9

ADAM10

ADAM12

ADAM15

ADAM17 (TACE)

ADAM19

ADAM28 (MDC-L)

ADAM with thrombospondin motifs

(ADAMTS)

ADAMTS1

ADAMTS4

ADAMTS5

Matrix Metalloproteinases (MMPs)

MMP-1 (Collagenase 1)

MMP-2 (Gelatinase A)

MMP-3 (m1)

MMP-7 (Matrilysin 1)

MMP-8 (Collagenase 2)

MMP-9 (Gelatinase B)

MMP-10 (Stromelysin 2)

MMP-11(Stromelysin 3)

MMP-12 (Macrophage elastase)

MMP-13 (Collagenase 3)

MMP-14 (MT1-MMP)

MMP-15 (MT2-MMP)

MMP-19

MMP-23 (CA-MMP)

MMP-24 (MT5-MMP)

MMP-26 (Matrilysin 2)

MMP-27 (CMMP)

Cysteine Proteases
Legumain

Cysteine cathepsins

Cathepsin B

Cathepsin C

Cathepsin K

Cathepsin L

Cathepsin S

Cathepsin X

Aspartate Proteases
Cathepsin D

Cathepsin E

Secretase

Serine Proteases
Urokinase (uPA)

Tissue-type plasminogen activator (tPA)

Plasmin

Thrombin

Prostate-specific antigen (PSA, KLK3)

Human neutrophil elastase (HNE)

Elastase

Tryptase

Type II transmembrane serine proteases

(TTSPs)

DESC1

Hepsin (HPN)

Matriptase

Matriptase-2

TMPRSS2

TMPRSS3

TMPRSS4 (CAP2)

Fibroblast Activation Protein (FAP)

kallikrein-related peptidase (KLK family)

KLK4

KLK5

KLK6

KLK7

KLK8

KLK10

KLK11

KLK13

KLK14

In some embodiments, the disclosure provides activatable recombinant polypeptides comprising a first release segment (RS1) sequence having at least 88%, or at least 94%, or 100% sequence identity, when optimally aligned, to a sequence identified in Table 8a, wherein the RS1 is a substrate for one or more mammalian proteases. In some embodiments, the RS is further engineered to remove a legumain cleavage site. In some embodiments, the disclosure provides activatable recombinant polypeptides comprising a RS1 and a second release segment (RS2) sequence, each having at least 88%, or at least 94%, or 100% sequence identity, when optimally aligned, to a sequence identified herein by the sequences set forth in Table 8a, wherein the RS1 and the RS2 each are a substrate for one or more mammalian proteases. In some embodiments, the RS1 and RS2 each do not serve as substrates for legumain.

In some embodiments, disclosure provides activatable recombinant polypeptides comprising a first RS (RS1) sequence having at least 90%, at least 93%, at least 97%, or 100% identity, when optimally aligned, to a sequence identified in Table 8b, wherein the RS1 is a substrate for one or more mammalian proteases. In some embodiments, the disclosure provides activatable recombinant polypeptides comprising a RS1 and a second release segment (RS2) sequence, each having at least 88%, or at least 94%, or 100% sequence identity, when optimally aligned, to a sequence identified herein by the sequences set forth in Table 8b, wherein the RS1 and the RS2 are each a substrate for one or more mammalian proteases (e.g., at one, two, or three cleavage sites within each release segment sequence). In some embodiments of activatable recombinant polypeptides comprising RS1 and RS2, the two release segments can be identical. In some embodiments of activatable recombinant polypeptides comprising RS1 and RS2, the two release segments can be different.

The present disclosure contemplates release segments that are substrates for one, two or three different classes of proteases that are metalloproteinases, cysteine proteases, aspartate proteases, or serine proteases, including the proteases of Table 7. In some embodiments, a paTCE comprises RSs (e.g., RS1 and RS2) that serve as substrates for one or more proteases found in close association with or are co-localized with tumors or cancer cells, and upon cleavage of the RSs, the binding moieties that are otherwise shielded by ELNNs of the paTCE (and thus have a lower binding affinity for their respective ligands) are released from the ELNNs and regain their full potential to bind target and effector cell ligands. In some embodiments, a paTCE comprises RSs (e.g., RS1 and RS2), that each comprise an amino acid sequence that is a substrate for one or more cellular proteases located within a targeted cell, including but not limited to a protease of Table 7. In some embodiments, RSs are substrates for two or three classes of proteases that cleave different portions of each RS. In some embodiments, each RS that is a substrate for two, three, or more classes of proteases has two, three, or more distinct cleavage sites, but cleavage by a single protease nevertheless results in the release of the binding moieties from an ELNN.

In some embodiments, an RS of the disclosure for incorporation into a fusion protein (such as a paTCE) is a substrate for one or more proteases including but not limited to meprin, neprilysin (CD10), PSMA, BMP-1, A disintegrin and metalloproteinases (ADAMs), ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 (TACE), ADAM19, ADAM28 (MDC-L), ADAM with thrombospondin motifs (ADAMTS), ADAMTS1, ADAMTS4, ADAMTS5, MMP-1 (collagenase 1), matrix metalloproteinase-1 (MMP-1), matrix metalloproteinase-2 (MMP-2, gelatinase A), matrix metalloproteinase-3 (MMP-3, stromelysin 1), matrix metalloproteinase-7 (MMP-7, Matrilysin 1), matrix metalloproteinase-8 (MMP-8, collagenase 2), matrix metalloproteinase-9 (MMP-9, gelatinase B), matrix metalloproteinase-10 (MMP-10, stromelysin 2), matrix metalloproteinase-11 (MMP-11, stromelysin 3), matrix metalloproteinase-12 (MMP-12, macrophage elastase), matrix metalloproteinase-13 (MMP-13, collagenase 3), matrix metalloproteinase-14 (MMP-14, MT1-MMP), matrix metalloproteinase-15 (MMP-15, MT2-MMP), matrix metalloproteinase-19 (MMP-19), matrix metalloproteinase-23 (MMP-23, CA-MMP), matrix metalloproteinase-24 (MMP-24, MT5-MMP), matrix metalloproteinase-26 (MMP-26, matrilysin 2), matrix metalloproteinase-27 (MMP-27, CMMP), legumain, cathepsin B, cathepsin C, cathepsin K, cathepsin L, cathepsin S, cathepsin X, cathepsin D, cathepsin E, secretase, urokinase (uPA), tissue-type plasminogen activator (tPA), plasmin, thrombin, prostate-specific antigen (PSA, KLK3), human neutrophil elastase (HNE), elastase, tryptase, Type II transmembrane serine proteases (TTSPs), DESC1, hepsin (HPN), matriptase, matriptase-2, TMPRSS2, TMPRSS3, TMPRSS4 (CAP2), fibroblast activation protein (FAP), kallikrein-related peptidase (KLK family), KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, and KLK14. In some embodiments, the RS is a substrate for ADAM17. In some embodiments, the RS is a substrate for BMP-1. In some embodiments, the RS is a substrate for cathepsin. In some embodiments, the RS is a substrate for HtrA1. In some embodiments, the RS is a substrate for legumain. In some embodiments, the RS is a substrate for MMP-1. In some embodiments, the RS is a substrate for MMP-2. In some embodiments, the RS is a substrate for MMP-7. In some embodiments, the RS is a substrate for MMP-9. In some embodiments, the RS is a substrate for MMP-11. In some embodiments, the RS is a substrate for MMP-14. In some embodiments, the RS is a substrate for uPA. In some embodiments, the RS is a substrate for matriptase. In some embodiments, the RS is a substrate for MT-SP1. In some embodiments, the RS is a substrate for neutrophil elastase. In some embodiments, the RS is a substrate for thrombin. In some embodiments RS is a substrate for TMPRSS3. In some embodiments, the RS is a substrate for TMPRSS4. In some embodiments, the RS of the subject recombinant polypeptide compositions is a substrate for at least two proteases including but not limited to legumain, MMP-1, MMP-2, MMP-7, MMP-9, MMP-11, MMP-14, uPA, and matriptase. In some embodiments, the RS of the subject recombinant polypeptide compositions is a substrate for legumain, MMP-1, MMP-2, MMP-7, MMP-9, MMP-11, MMP-14, uPA, and matriptase. In specific embodiments, the RS of the subject recombinant polypeptide compositions is not a substrate for legumain. In some embodiments, the RS of the subject recombinant polypeptide compositions is a substrate for uPA, matriptase (also known as MT-SP1 and ST14), MMP2, MMP7, MMP9, and MMP14. In some embodiments, the RS of the subject recombinant polypeptide compositions is substrate for uPA, matriptase, MMP2, MMP7, MMP9, and MMP14 but not legumain.

TABLE 8a

TCE Release Segment Sequences.

Name
Amino Acid Sequence
SEQ ID NO

RSR-1517
EAGRSANHEPLGLVAT
7001

BSRS-A1-1
ASGRSTNAGPSGLAGP
7002

BSRS-A2-1
ASGRSTNAGPQGLAGQ
7003

BSRS-A3-1
ASGRSTNAGPPGLTGP
7004

VP-1
ASSRGTNAGPAGLTGP
7005

RSR-1752
ASSRTTNTGPSTLTGP
7006

RSR-1512
AAGRSDNGTPLELVAP
7007

RSR-1517
EAGRSANHEPLGLVAT
7008

VP-2
ASGRGTNAGPAGLTGP
7009

RSR-1018
LFGRNDNHEPLELGGG
7010

RSR-1053
TAGRSDNLEPLGLVFG
7011

RSR-1059
LDGRSDNFHPPELVAG
7012

RSR-1065
LEGRSDNEEPENLVAG
7013

RSR-1167
LKGRSDNNAPLALVAG
7014

RSR-1201
VYSRGTNAGPHGLTGR
7015

RSR-1218
ANSRGTNKGFAGLIGP
7016

RSR-1226
ASSRLTNEAPAGLTIP
7017

RSR-1254
DQSRGTNAGPEGLTDP
7018

RSR-1256
ESSRGTNIGQGGLTGP
7019

RSR-1261
SSSRGTNQDPAGLTIP
7020

RSR-1293
ASSRGQNHSPMGLTGP
7021

RSR-1309
AYSRGPNAGPAGLEGR
7022

RSR-1326
ASERGNNAGPANLTGF
7023

RSR-1345
ASHRGTNPKPAILTGP
7024

RSR-1354
MSSRRTNANPAQLTGP
7025

RSR-1426
GAGRTDNHEPLELGAA
7026

RSR-1478
LAGRSENTAPLELTAG
7027

RSR-1479
LEGRPDNHEPLALVAS
7028

RSR-1496
LSGRSDNEEPLALPAG
7029

RSR-1508
EAGRTDNHEPLELSAP
7030

RSR-1513
EGGRSDNHGPLELVSG
7031

RSR-1516
LSGRSDNEAPLELEAG
7032

RSR-1524
LGGRADNHEPPELGAG
7033

RSR-1622
PPSRGTNAEPAGLTGE
7034

RSR-1629
ASTRGENAGPAGLEAP
7035

RSR-1664
ESSRGTNGAPEGLTGP
7036

RSR-1667
ASSRATNESPAGLTGE
7037

RSR-1709
ASSRGENPPPGGLTGP
7038

RSR-1712
AASRGTNTGPAELTGS
7039

RSR-1727
AGSRTTNAGPGGLEGP
7040

RSR-1754
APSRGENAGPATLTGA
7041

RSR-1819
ESGRAANTGPPTLTAP
7042

RSR-1832
NPGRAANEGPPGLPGS
7043

RSR-1855
ESSRAANLTPPELTGP
7044

RSR-1911
ASGRAANETPPGLTGA
7045

RSR-1929
NSGRGENLGAPGLTGT
7046

RSR-1951
TTGRAANLTPAGLTGP
7047

RSR-2295
EAGRSANHTPAGLTGP
7048

RSR-2298
ESGRAANTTPAGLTGP
7049

RSR-2038
TTGRATEAANLTPAGLTGP
7050

RSR-2072
TTGRAEEAANLTPAGLTGP
7051

RSR-2089
TTGRAGEAANLTPAGLTGP
7052

RSR-2302
TTGRATEAANATPAGLTGP
7053

RSR-3047
TTGRAGEAEGATSAGATGP
7054

RSR-3052
TTGEAGEAANATSAGATGP
7055

RSR-3043
TTGEAGEAAGLTPAGLTGP
7056

RSR-3041
TTGAAGEAANATPAGLTGP
7057

RSR-3044
TTGRAGEAAGLTPAGLTGP
7058

RSR-3057
TTGRAGEAANATSAGATGP
7059

RSR-3058
TTGEAGEAAGATSAGATGP
7060

RSR-2485
ESGRAANTEPPELGAG
7061

RSR-2486
ESGRAANTAPEGLTGP
7062

RSR-2488
EPGRAANHEPSGLTEG
7063

RSR-2599
ESGRAANHTGAPPGGLTGP
7064

RSR-2706
TTGRTGEGANATPGGLTGP
7065

RSR-2707
RTGRSGEAANETPEGLEGP
7066

RSR-2708
RTGRTGESANETPAGLGGP
7067

RSR-2709
STGRTGEPANETPAGLSGP
7068

RSR-2710
TTGRAGEPANATPTGLSGP
7069

RSR-2711
RTGRPGEGANATPTGLPGP
7070

RSR-2712
RTGRGGEAANATPSGLGGP
7071

RSR-2713
STGRSGESANATPGGLGGP
7072

RSR-2714
RTGRTGEEANATPAGLPGP
7073

RSR-2715
ATGRPGEPANTTPEGLEGP
7074

RSR-2716
STGRSGEPANATPGGLTGP
7075

RSR-2717
PTGRGGEGANTTPTGLPGP
7076

RSR-2718
PTGRSGEGANATPSGLTGP
7077

RSR-2719
TTGRASEGANSTPAPLTEP
7078

RSR-2720
TYGRAAEAANTTPAGLTAP
7079

RSR-2721
TTGRATEGANATPAELTEP
7080

RSR-2722
TVGRASEEANTTPASLTGP
7081

RSR-2723
TTGRAPEAANATPAPLTGP
7082

RSR-2724
TWGRATEPANATPAPLTSP
7083

RSR-2725
TVGRASESANATPAELTSP
7084

RSR-2726
TVGRAPEGANSTPAGLTGP
7085

RSR-2727
TWGRATEAPNLEPATLTTP
7086

RSR-2728
TTGRATEAPNLTPAPLTEP
7087

RSR-2729
TQGRATEAPNLSPAALTSP
7088

RSR-2730
TQGRAAEAPNLTPATLTAP
7089

RSR-2731
TSGRAPEATNLAPAPLTGP
7090

RSR-2732
TQGRAAEAANLTPAGLTEP
7091

RSR-2733
TTGRAGSAPNLPPTGLTTP
7092

RSR-2734
TTGRAGGAENLPPEGLTAP
7093

RSR-2735
TTSRAGTATNLTPEGLTAP
7094

RSR-2736
TTGRAGTATNLPPSGLTTP
7095

RSR-2737
TTARAGEAENLSPSGLTAP
7096

RSR-2738
TTGRAGGAGNLAPGGLTEP
7097

RSR-2739
TTGRAGTATNLPPEGLTGP
7098

RSR-2740
TTGRAGGAANLAPTGLTEP
7099

RSR-2741
TTGRAGTAENLAPSGLTTP
7100

RSR-2742
TTGRAGSATNLGPGGLTGP
7101

RSR-2743
TTARAGGAENLTPAGLTEP
7102

RSR-2744
TTARAGSAENLSPSGLTGP
7103

RSR-2745
TTARAGGAGNLAPEGLTTP
7104

RSR-2746
TTSRAGAAENLTPTGLTGP
7105

RSR-2747
TYGRTTTPGNEPPASLEAE
7106

RSR-2748
TYSRGESGPNEPPPGLTGP
7107

RSR-2749
AWGRTGASENETPAPLGGE
7108

RSR-2750
RWGRAETTPNTPPEGLETE
7109

RSR-2751
ESGRAANHTGAEPPELGAG
7110

RSR-2754
TTGRAGEAANLTPAGLTES
7111

RSR-2755
TTGRAGEAANLTPAALTES
7112

RSR-2756
TTGRAGEAANLTPAPLTES
7113

RSR-2757
TTGRAGEAANLTPEPLTES
7114

RSR-2758
TTGRAGEAANLTPAGLTGA
7115

RSR-2759
TTGRAGEAANLTPEGLTGA
7116

RSR-2760
TTGRAGEAANLTPEPLTGA
7117

RSR-2761
TTGRAGEAANLTPAGLTEA
7118

RSR-2762
TTGRAGEAANLTPEGLTEA
7119

RSR-2763
TTGRAGEAANLTPAPLTEA
7120

RSR-2764
TTGRAGEAANLTPEPLTEA
7121

RSR-2765
TTGRAGEAANLTPEPLTGP
7122

RSR-2766
TTGRAGEAANLTPAGLTGG
7123

RSR-2767
TTGRAGEAANLTPEGLTGG
7124

RSR-2768
TTGRAGEAANLTPEALTGG
7125

RSR-2769
TTGRAGEAANLTPEPLTGG
7126

RSR-2770
TTGRAGEAANLTPAGLTEG
7127

RSR-2771
TTGRAGEAANLTPEGLTEG
7128

RSR-2772
TTGRAGEAANLTPAPLTEG
7129

RSR-2773
TTGRAGEAANLTPEPLTEG
7130

RSR-3213
EAGRSASHTPAGLTGP
7628

TABLE 8b

Release Segment Sequences

SEQ

SEQ

Amino Acid
ID

Amino Acid
ID

Name
Sequence
NO:
Name
Sequence
NO:

RSN-
GSAPGSAGGYAEL
7131
RSC-
GTAEAASASGGSA
7379

0001
RMGGAIATSGSET

0001
GGYAELRMGGAIP

PGT

GSP

RSN-
GSAPGTGGGYAPL
7132
RSC-
GTAEAASASGGTG
7380

0002
RMGGGAATSGSET

0002
GGYAPLRMGGGA

PGT

PGSP

RSN-
GSAPGAEGGYAAL
7133
RSC-
GTAEAASASGGAE
7381

0003
RMGGEIATSGSET

0003
GGYAALRMGGEIP

PGT

GSP

RSN-
GSAPGGPGGYALL
7134
RSC-
GTAEAASASGGGP
7382

0004
RMGGPAATSGSET

0004
GGYALLRMGGPAP

PGT

GSP

RSN-
GSAPGEAGGYAFL
7135
RSC-
GTAEAASASGGEA
7383

0005
RMGGSIATSGSET

0005
GGYAFLRMGGSIP

PGT

GSP

RSN-
GSAPGPGGGYASL
7136
RSC-
GTAEAASASGGPG
7384

0006
RMGGTAATSGSET

0006
GGYASLRMGGTAP

PGT

GSP

RSN-
GSAPGSEGGYATL
7137
RSC-
GTAEAASASGGSE
7385

0007
RMGGAIATSGSET

0007
GGYATLRMGGAIP

PGT

GSP

RSN-
GSAPGTPGGYANL
7138
RSC-
GTAEAASASGGTP
7386

0008
RMGGGAATSGSET

0008
GGYANLRMGGGA

PGT

PGSP

RSN-
GSAPGASGGYAHL
7139
RSC-
GTAEAASASGGAS
7387

0009
RMGGEIATSGSET

0009
GGYAHLRMGGEIP

PGT

GSP

RSN-
GSAPGGTGGYGEL
7140
RSC-
GTAEAASASGGGT
7388

0010
RMGGPAATSGSET

0010
GGYGELRMGGPA

PGT

PGSP

RSN-
GSAPGEAGGYPEL
7141
RSC-
GTAEAASASGGEA
7389

0011
RMGGSIATSGSET

0011
GGYPELRMGGSIP

PGT

GSP

RSN-
GSAPGPGGGYVEL
7142
RSC-
GTAEAASASGGPG
7390

0012
RMGGTAATSGSET

0012
GGYVELRMGGTAP

PGT

GSP

RSN-
GSAPGSEGGYLEL
7143
RSC-
GTAEAASASGGSE
7391

0013
RMGGAIATSGSET

0013
GGYLELRMGGAIP

PGT

GSP

RSN-
GSAPGTPGGYSEL
7144
RSC-
GTAEAASASGGTP
7392

0014
RMGGGAATSGSET

0014
GGYSELRMGGGA

PGT

PGSP

RSN-
GSAPGASGGYTEL
7145
RSC-
GTAEAASASGGAS
7393

0015
RMGGEIATSGSET

0015
GGYTELRMGGEIP

PGT

GSP

RSN-
GSAPGGTGGYQEL
7146
RSC-
GTAEAASASGGGT
7394

0016
RMGGPAATSGSET

0016
GGYQELRMGGPA

PGT

PGSP

RSN-
GSAPGEAGGYEEL
7147
RSC-
GTAEAASASGGEA
7395

0017
RMGGSIATSGSET

0017
GGYEELRMGGSIP

PGT

GSP

RSN-
GSAPGPGIGPAEL
7148
RSC-
GTAEAASASGGPG
7396

0018
RMGGTAATSGSET

0018
IGPAELRMGGTAP

PGT

GSP

RSN-
GSAPGSEIGAAELR
7149
RSC-
GTAEAASASGGSEI
7397

0019
MGGAIATSGSETP

0019
GAAELRMGGAIPG

GT

SP

RSN-
GSAPGTPIGSAELR
7150
RSC-
GTAEAASASGGTPI
7398

0020
MGGGAATSGSETP

0020
GSAELRMGGGAP

GT

GSP

RSN-
GSAPGASIGTAELR
7151
RSC-
GTAEAASASGGASI
7399

0021
MGGEIATSGSETP

0021
GTAELRMGGEIPG

GT

SP

RSN-
GSAPGGTIGNAEL
7152
RSC-
GTAEAASASGGGTI
7400

0022
RMGGPAATSGSET

0022
GNAELRMGGPAPG

PGT

SP

RSN-
GSAPGEAIGQAEL
7153
RSC-
GTAEAASASGGEAI
7401

0023
RMGGSIATSGSET

0023
GQAELRMGGSIPG

PGT

SP

RSN-
GSAPGPGGPYAEL
7154
RSC-
GTAEAASASGGPG
7402

0024
RMGGTAATSGSET

0024
GPYAELRMGGTAP

PGT

GSP

RSN-
GSAPGSEGAYAEL
7155
RSC-
GTAEAASASGGSE
7403

0025
RMGGAIATSGSET

0025
GAYAELRMGGAIP

PGT

GSP

RSN-
GSAPGTPGVYAEL
7156
RSC-
GTAEAASASGGTP
7404

0026
RMGGGAATSGSET

0026
GVYAELRMGGGAP

PGT

GSP

RSN-
GSAPGASGLYAEL
7157
RSC-
GTAEAASASGGAS
7405

0027
RMGGEIATSGSET

0027
GLYAELRMGGEIP

PGT

GSP

RSN-
GSAPGGTGIYAELR
7158
RSC-
GTAEAASASGGGT
7406

0028
MGGPAATSGSETP

0028
GIYAELRMGGPAP

GT

GSP

RSN-
GSAPGEAGFYAEL
7159
RSC-
GTAEAASASGGEA
7407

0029
RMGGSIATSGSET

0029
GFYAELRMGGSIP

PGT

GSP

RSN-
GSAPGPGGYYAEL
7160
RSC-
GTAEAASASGGPG
7408

0030
RMGGTAATSGSET

0030
GYYAELRMGGTAP

PGT

GSP

RSN-
GSAPGSEGSYAEL
7161
RSC-
GTAEAASASGGSE
7409

0031
RMGGAIATSGSET

0031
GSYAELRMGGAIP

PGT

GSP

RSN-
GSAPGTPGNYAEL
7162
RSC-
GTAEAASASGGTP
7410

0032
RMGGGAATSGSET

0032
GNYAELRMGGGAP

PGT

GSP

RSN-
GSAPGASGEYAEL
7163
RSC-
GTAEAASASGGAS
7411

0033
RMGGEIATSGSET

0033
GEYAELRMGGEIP

PGT

GSP

RSN-
GSAPGGTGHYAEL
7164
RSC-
GTAEAASASGGGT
7412

0034
RMGGPAATSGSET

0034
GHYAELRMGGPAP

PGT

GSP

RSN-
GSAPGEAGGYAEA
7165
RSC-
GTAEAASASGGEA
7413

0035
RMGGSIATSGSET

0035
GGYAEARMGGSIP

PGT

GSP

RSN-
GSAPGPGGGYAEV
7166
RSC-
GTAEAASASGGPG
7414

0036
RMGGTAATSGSET

0036
GGYAEVRMGGTAP

PGT

GSP

RSN-
GSAPGSEGGYAEI
7167
RSC-
GTAEAASASGGSE
7415

0037
RMGGAIATSGSET

0037
GGYAEIRMGGAIP

PGT

GSP

RSN-
GSAPGTPGGYAEF
7168
RSC-
GTAEAASASGGTP
7416

0038
RMGGGAATSGSET

0038
GGYAEFRMGGGA

PGT

PGSP

RSN-
GSAPGASGGYAEY
7169
RSC-
GTAEAASASGGAS
7417

0039
RMGGEIATSGSET

0039
GGYAEYRMGGEIP

PGT

GSP

RSN-
GSAPGGTGGYAES
7170
RSC-
GTAEAASASGGGT
7418

0040
RMGGPAATSGSET

0040
GGYAESRMGGPA

PGT

PGSP

RSN-
GSAPGEAGGYAET
7171
RSC-
GTAEAASASGGEA
7419

0041
RMGGSIATSGSET

0041
GGYAETRMGGSIP

PGT

GSP

RSN-
GSAPGPGGGYAEL
7172
RSC-
GTAEAASASGGPG
7420

0042
AMGGTRATSGSET

0042
GGYAELAMGGTRP

PGT

GSP

RSN-
GSAPGSEGGYAEL
7173
RSC-
GTAEAASASGGSE
7421

0043
VMGGARATSGSET

0043
GGYAELVMGGARP

PGT

GSP

RSN-
GSAPGTPGGYAEL
7174
RSC-
GTAEAASASGGTP
7422

0044
LMGGGRATSGSET

0044
GGYAELLMGGGRP

PGT

GSP

RSN-
GSAPGASGGYAELI
7175
RSC-
GTAEAASASGGAS
7423

0045
MGGERATSGSETP

0045
GGYAELIMGGERP

GT

GSP

RSN-
GSAPGGTGGYAEL
7176
RSC-
GTAEAASASGGGT
7424

0046
WMGGPRATSGSE

0046
GGYAELWMGGPR

TPGT

PGSP

RSN-
GSAPGEAGGYAEL
7177
RSC-
GTAEAASASGGEA
7425

0047
SMGGSRATSGSET

0047
GGYAELSMGGSRP

PGT

GSP

RSN-
GSAPGPGGGYAEL
7178
RSC-
GTAEAASASGGPG
7426

0048
TMGGTRATSGSET

0048
GGYAELTMGGTRP

PGT

GSP

RSN-
GSAPGSEGGYAEL
7179
RSC-
GTAEAASASGGSE
7427

0049
QMGGARATSGSET

0049
GGYAELQMGGAR

PGT

PGSP

RSN-
GSAPGTPGGYAEL
7180
RSC-
GTAEAASASGGTP
7428

0050
NMGGGRATSGSET

0050
GGYAELNMGGGR

PGT

PGSP

RSN-
GSAPGASGGYAEL
7181
RSC-
GTAEAASASGGAS
7429

0051
EMGGERATSGSET

0051
GGYAELEMGGERP

PGT

GSP

RSN-
GSAPGGTGGYAEL
7182
RSC-
GTAEAASASGGGT
7430

0052
RPGGPIATSGSETP

0052
GGYAELRPGGPIP

GT

GSP

RSN-
GSAPGEAGGYAEL
7183
RSC-
GTAEAASASGGEA
7431

0053
RAGGSAATSGSET

0053
GGYAELRAGGSAP

PGT

GSP

RSN-
GSAPGPGGGYAEL
7184
RSC-
GTAEAASASGGPG
7432

0054
RLGGTIATSGSETP

0054
GGYAELRLGGTIPG

GT

SP

RSN-
GSAPGSEGGYAEL
7185
RSC-
GTAEAASASGGSE
7433

0055
RIGGAAATSGSETP

0055
GGYAELRIGGAAP

GT

GSP

RSN-
GSAPGTPGGYAEL
7186
RSC-
GTAEAASASGGTP
7434

0056
RSGGGIATSGSET

0056
GGYAELRSGGGIP

PGT

GSP

RSN-
GSAPGASGGYAEL
7187
RSC-
GTAEAASASGGAS
7435

0057
RNGGEAATSGSET

0057
GGYAELRNGGEAP

PGT

GSP

RSN-
GSAPGGTGGYAEL
7188
RSC-
GTAEAASASGGGT
7436

0058
RQGGPIATSGSET

0058
GGYAELRQGGPIP

PGT

GSP

RSN-
GSAPGEAGGYAEL
7189
RSC-
GTAEAASASGGEA
7437

0059
RDGGSAATSGSET

0059
GGYAELRDGGSAP

PGT

GSP

RSN-
GSAPGPGGGYAEL
7190
RSC-
GTAEAASASGGPG
7438

0060
REGGTIATSGSETP

0060
GGYAELREGGTIP

GT

GSP

RSN-
GSAPGSEGGYAEL
7191
RSC-
GTAEAASASGGSE
7439

0061
RHGGAAATSGSET

0061
GGYAELRHGGAAP

PGT

GSP

RSN-
GSAPGTPGGYAEL
7192
RSC-
GTAEAASASGGTP
7440

0062
RMPGGIATSGSET

0062
GGYAELRMPGGIP

PGT

GSP

RSN-
GSAPGASGGYAEL
7193
RSC-
GTAEAASASGGAS
7441

0063
RMAGEAATSGSET

0063
GGYAELRMAGEAP

PGT

GSP

RSN-
GSAPGGTGGYAEL
7194
RSC-
GTAEAASASGGGT
7442

0064
RMVGPIATSGSETP

0064
GGYAELRMVGPIP

GT

GSP

RSN-
GSAPGEAGGYAEL
7195
RSC-
GTAEAASASGGEA
7443

0065
RMLGSAATSGSET

0065
GGYAELRMLGSAP

PGT

GSP

RSN-
GSAPGPGGGYAEL
7196
RSC-
GTAEAASASGGPG
7444

0066
RMIGTIATSGSETP

0066
GGYAELRMIGTIPG

GT

SP

RSN-
GSAPGSEGGYAEL
7197
RSC-
GTAEAASASGGSE
7445

0067
RMYGAIATSGSETP

0067
GGYAELRMYGAIP

GT

GSP

RSN-
GSAPGTPGGYAEL
7198
RSC-
GTAEAASASGGTP
7446

0068
RMSGGAATSGSET

0068
GGYAELRMSGGAP

PGT

GSP

RSN-
GSAPGASGGYAEL
7199
RSC-
GTAEAASASGGAS
7447

0069
RMNGEIATSGSET

0069
GGYAELRMNGEIP

PGT

GSP

RSN-
GSAPGGTGGYAEL
7200
RSC-
GTAEAASASGGGT
7448

0070
RMQGPAATSGSET

0070
GGYAELRMQGPAP

PGT

GSP

RSN-
GSAPGANHTPAGL
7201
RSC-
GTAEAASASGGAN
7449

0071
TGPGARATSGSET

0071
HTPAGLTGPGARP

PGT

GSP

RSN-
GSAPGANTAPEGL
7202
RSC-
GTAEAASASGGAN
7450

0072
TGPSTRATSGSET

0072
TAPEGLTGPSTRP

PGT

GSP

RSN-
GSAPGTGAPPGGL
7203
RSC-
GTAEAASASGGTG
7451

0073
TGPGTRATSGSET

0073
APPGGLTGPGTRP

PGT

GSP

RSN-
GSAPGANHEPSGL
7204
RSC-
GTAEAASASGGAN
7452

0074
TEGSPRATSGSET

0074
HEPSGLTEGSPRP

PGT

GSP

RSN-
GSAPGANTEPPEL
7205
RSC-
GTAEAASASGGAN
7453

0075
GAGTERATSGSET

0075
TEPPELGAGTERP

PGT

GSP

RSN-
GSAPGASGPPPGL
7206
RSC-
GTAEAASASGGAS
7454

0076
TGPPGRATSGSET

0076
GPPPGLTGPPGRP

PGT

GSP

RSN-
GSAPGASGTPAPL
7207
RSC-
GTAEAASASGGAS
7455

0077
GGEPGRATSGSET

0077
GTPAPLGGEPGRP

PGT

GSP

RSN-
GSAPGPAGPPEGL
7208
RSC-
GTAEAASASGGPA
7456

0078
ETEAGRATSGSET

0078
GPPEGLETEAGRP

PGT

GSP

RSN-
GSAPGPTSGQGGL
7209
RSC-
GTAEAASASGGPT
7457

0079
TGPESRATSGSET

0079
SGQGGLTGPESRP

PGT

GSP

RSN-
GSAPGSAGGAANL
7210
RSC-
GTAEAASASGGSA
7458

0080
VRGGAIATSGSETP

0080
GGAANLVRGGAIP

GT

GSP

RSN-
GSAPGTGGGAAPL
7211
RSC-
GTAEAASASGGTG
7459

0081
VRGGGAATSGSET

0081
GGAAPLVRGGGAP

PGT

GSP

RSN-
GSAPGAEGGAAAL
7212
RSC-
GTAEAASASGGAE
7460

0082
VRGGEIATSGSETP

0082
GGAAALVRGGEIP

GT

GSP

RSN-
GSAPGGPGGAALL
7213
RSC-
GTAEAASASGGGP
7461

0083
VRGGPAATSGSET

0083
GGAALLVRGGPAP

PGT

GSP

RSN-
GSAPGEAGGAAFL
7214
RSC-
GTAEAASASGGEA
7462

0084
VRGGSIATSGSETP

0084
GGAAFLVRGGSIP

GT

GSP

RSN-
GSAPGPGGGAASL
7215
RSC-
GTAEAASASGGPG
7463

0085
VRGGTAATSGSET

0085
GGAASLVRGGTAP

PGT

GSP

RSN-
GSAPGSEGGAATL
7216
RSC-
GTAEAASASGGSE
7464

0086
VRGGAIATSGSETP

0086
GGAATLVRGGAIP

GT

GSP

RSN-
GSAPGTPGGAAGL
7217
RSC-
GTAEAASASGGTP
7465

0087
VRGGGAATSGSET

0087
GGAAGLVRGGGAP

PGT

GSP

RSN-
GSAPGASGGAADL
7218
RSC-
GTAEAASASGGAS
7466

0088
VRGGEIATSGSETP

0088
GGAADLVRGGEIP

GT

GSP

RSN-
GSAPGGTGGAGNL
7219
RSC-
GTAEAASASGGGT
7467

0089
VRGGPAATSGSET

0089
GGAGNLVRGGPAP

PGT

GSP

RSN-
GSAPGEAGGAPNL
7220
RSC-
GTAEAASASGGEA
7468

0090
VRGGSIATSGSETP

0090
GGAPNLVRGGSIP

GT

GSP

RSN-
GSAPGPGGGAVNL
7221
RSC-
GTAEAASASGGPG
7469

0091
VRGGTAATSGSET

0091
GGAVNLVRGGTAP

PGT

GSP

RSN-
GSAPGSEGGALNL
7222
RSC-
GTAEAASASGGSE
7470

0092
VRGGAIATSGSETP

0092
GGALNLVRGGAIP

GT

GSP

RSN-
GSAPGTPGGASNL
7223
RSC-
GTAEAASASGGTP
7471

0093
VRGGGAATSGSET

0093
GGASNLVRGGGAP

PGT

GSP

RSN-
GSAPGASGGATNL
7224
RSC-
GTAEAASASGGAS
7472

0094
VRGGEIATSGSETP

0094
GGATNLVRGGEIP

GT

GSP

RSN-
GSAPGGTGGAQNL
7225
RSC-
GTAEAASASGGGT
7473

0095
VRGGPAATSGSET

0095
GGAQNLVRGGPAP

PGT

GSP

RSN-
GSAPGEAGGAENL
7226
RSC-
GTAEAASASGGEA
7474

0096
VRGGSIATSGSETP

0096
GGAENLVRGGSIP

GT

GSP

RSN-
GSAPEAGRSANHE
7227
RSC-
GTAEAASASGEAG
7475

1517
PLGLVATATSGSET

1517
RSANHEPLGLVAT

PGT

PGSP

BSRS-
GSAPASGRSTNAG
7228
BSRS-
GTAEAASASGASG
7476

A1-2
PSGLAGPATSGSE

A1-3
RSTNAGPSGLAGP

TPGT

PGSP

BSRS-
GSAPASGRSTNAG
7229
BSRS-
GTAEAASASGASG
7477

A2-2
PQGLAGQATSGSE

A2-3
RSTNAGPQGLAGQ

TPGT

PGSP

BSRS-
GSAPASGRSTNAG
7230
BSRS-
GTAEAASASGASG
7478

A3-2
PPGLTGPATSGSE

A3-3
RSTNAGPPGLTGP

TPGT

PGSP

VP-1
GSAPASSRGTNAG
7231
VP-1
GTAEAASASGASS
7479

PAGLTGPATSGSE

RGTNAGPAGLTGP

TPGT

PGSP

RSN-
GSAPASSRTTNTG
7232
RSC-
GTAEAASASGASS
7480

1752
PSTLTGPATSGSET

1752
RTTNTGPSTLTGPP

PGT

GSP

RSN-
GSAPAAGRSDNGT
7233
RSC-
GTAEAASASGAAG
7481

1512
PLELVAPATSGSET

1512
RSDNGTPLELVAP

PGT

PGSP

RSN-
GSAPEAGRSANHE
7234
RSC-
GTAEAASASGEAG
7482

1517
PLGLVATATSGSET

1517
RSANHEPLGLVAT

PGT

PGSP

VP-2
GSAPASGRGTNAG
7235
VP-2
GTAEAASASGASG
7483

PAGLTGPATSGSE

RGTNAGPAGLTGP

TPGT

PGSP

RSN-
GSAPLFGRNDNHE
7236
RSC-
GTAEAASASGLFG
7484

1018
PLELGGGATSGSE

1018
RNDNHEPLELGGG

TPGT

PGSP

RSN-
GSAPTAGRSDNLE
7237
RSC-
GTAEAASASGTAG
7485

1053
PLGLVFGATSGSET

1053
RSDNLEPLGLVFG

PGT

PGSP

RSN-
GSAPLDGRSDNFH
7238
RSC-
GTAEAASASGLDG
7486

1059
PPELVAGATSGSE

1059
RSDNFHPPELVAG

TPGT

PGSP

RSN-
GSAPLEGRSDNEE
7239
RSC-
GTAEAASASGLEG
7487

1065
PENLVAGATSGSE

1065
RSDNEEPENLVAG

TPGT

PGSP

RSN-
GSAPLKGRSDNNA
7240
RSC-
GTAEAASASGLKG
7488

1167
PLALVAGATSGSET

1167
RSDNNAPLALVAG

PGT

PGSP

RSN-
GSAPVYSRGTNAG
7241
RSC-
GTAEAASASGVYS
7489

1201
PHGLTGRATSGSE

1201
RGTNAGPHGLTGR

TPGT

PGSP

RSN-
GSAPANSRGTNKG
7242
RSC-
GTAEAASASGANS
7490

1218
FAGLIGPATSGSET

1218
RGTNKGFAGLIGPP

PGT

GSP

RSN-
GSAPASSRLTNEA
7243
RSC-
GTAEAASASGASS
7491

1226
PAGLTIPATSGSET

1226
RLTNEAPAGLTIPP

PGT

GSP

RSN-
GSAPDQSRGTNAG
7244
RSC-
GTAEAASASGDQS
7492

1254
PEGLTDPATSGSE

1254
RGTNAGPEGLTDP

TPGT

PGSP

RSN-
GSAPESSRGTNIG
7245
RSC-
GTAEAASASGESS
7493

1256
QGGLTGPATSGSE

1256
RGTNIGQGGLTGP

TPGT

PGSP

RSN-
GSAPSSSRGTNQD
7246
RSC-
GTAEAASASGSSS
7494

1261
PAGLTIPATSGSET

1261
RGTNQDPAGLTIPP

PGT

GSP

RSN-
GSAPASSRGQNHS
7247
RSC-
GTAEAASASGASS
7495

1293
PMGLTGPATSGSE

1293
RGQNHSPMGLTGP

TPGT

PGSP

RSN-
GSAPAYSRGPNAG
7248
RSC-
GTAEAASASGAYS
7496

1309
PAGLEGRATSGSE

1309
RGPNAGPAGLEGR

TPGT

PGSP

RSN-
GSAPASERGNNAG
7249
RSC-
GTAEAASASGASE
7497

1326
PANLTGFATSGSET

1326
RGNNAGPANLTGF

PGT

PGSP

RSN-
GSAPASHRGTNPK
7250
RSC-
GTAEAASASGASH
7498

1345
PAILTGPATSGSET

1345
RGTNPKPAILTGPP

PGT

GSP

RSN-
GSAPMSSRRTNAN
7251
RSC-
GTAEAASASGMSS
7499

1354
PAQLTGPATSGSE

1354
RRTNANPAQLTGP

TPGT

PGSP

RSN-
GSAPGAGRTDNHE
7252
RSC-
GTAEAASASGGAG
7500

1426
PLELGAAATSGSET

1426
RTDNHEPLELGAA

PGT

PGSP

RSN-
GSAPLAGRSENTA
7253
RSC-
GTAEAASASGLAG
7501

1478
PLELTAGATSGSET

1478
RSENTAPLELTAGP

PGT

GSP

RSN-
GSAPLEGRPDNHE
7254
RSC-
GTAEAASASGLEG
7502

1479
PLALVASATSGSET

1479
RPDNHEPLALVAS

PGT

PGSP

RSN-
GSAPLSGRSDNEE
7255
RSC-
GTAEAASASGLSG
7503

1496
PLALPAGATSGSET

1496
RSDNEEPLALPAG

PGT

PGSP

RSN-
GSAPEAGRTDNHE
7256
RSC-
GTAEAASASGEAG
7504

1508
PLELSAPATSGSET

1508
RTDNHEPLELSAPP

PGT

GSP

RSN-
GSAPEGGRSDNH
7257
RSC-
GTAEAASASGEGG
7505

1513
GPLELVSGATSGS

1513
RSDNHGPLELVSG

ETPGT

PGSP

RSN-
GSAPLSGRSDNEA
7258
RSC-
GTAEAASASGLSG
7506

1516
PLELEAGATSGSET

1516
RSDNEAPLELEAG

PGT

PGSP

RSN-
GSAPLGGRADNHE
7259
RSC-
GTAEAASASGLGG
7507

1524
PPELGAGATSGSE

1524
RADNHEPPELGAG

TPGT

PGSP

RSN-
GSAPPPSRGTNAE
7260
RSC-
GTAEAASASGPPS
7508

1622
PAGLTGEATSGSE

1622
RGTNAEPAGLTGE

TPGT

PGSP

RSN-
GSAPASTRGENAG
7261
RSC-
GTAEAASASGAST
7509

1629
PAGLEAPATSGSE

1629
RGENAGPAGLEAP

TPGT

PGSP

RSN-
GSAPESSRGTNGA
7262
RSC-
GTAEAASASGESS
7510

1664
PEGLTGPATSGSE

1664
RGTNGAPEGLTGP

TPGT

PGSP

RSN-
GSAPASSRATNES
7263
RSC-
GTAEAASASGASS
7511

1667
PAGLTGEATSGSE

1667
RATNESPAGLTGE

TPGT

PGSP

RSN-
GSAPASSRGENPP
7264
RSC-
GTAEAASASGASS
7512

1709
PGGLTGPATSGSE

1709
RGENPPPGGLTGP

TPGT

PGSP

RSN-
GSAPAASRGTNTG
7265
RSC-
GTAEAASASGAAS
7513

1712
PAELTGSATSGSET

1712
RGTNTGPAELTGS

PGT

PGSP

RSN-
GSAPAGSRTTNAG
7266
RSC-
GTAEAASASGAGS
7514

1727
PGGLEGPATSGSE

1727
RTTNAGPGGLEGP

TPGT

PGSP

RSN-
GSAPAPSRGENAG
7267
RSC-
GTAEAASASGAPS
7515

1754
PATLTGAATSGSET

1754
RGENAGPATLTGA

PGT

PGSP

RSN-
GSAPESGRAANTG
7268
RSC-
GTAEAASASGESG
7516

1819
PPTLTAPATSGSET

1819
RAANTGPPTLTAPP

PGT

GSP

RSN-
GSAPNPGRAANEG
7269
RSC-
GTAEAASASGNPG
7517

1832
PPGLPGSATSGSE

1832
RAANEGPPGLPGS

TPGT

PGSP

RSN-
GSAPESSRAANLT
7270
RSC-
GTAEAASASGESS
7518

1855
PPELTGPATSGSET

1855
RAANLTPPELTGPP

PGT

GSP

RSN-
GSAPASGRAANET
7271
RSC-
GTAEAASASGASG
7519

1911
PPGLTGAATSGSE

1911
RAANETPPGLTGA

TPGT

PGSP

RSN-
GSAPNSGRGENLG
7272
RSC-
GTAEAASASGNSG
7520

1929
APGLTGTATSGSE

1929
RGENLGAPGLTGT

TPGT

PGSP

RSN-
GSAPTTGRAANLT
7273
RSC-
GTAEAASASGTTG
7521

1951
PAGLTGPATSGSE

1951
RAANLTPAGLTGP

TPGT

PGSP

RSN-
GSAPEAGRSANHT
7274
RSC-
GTAEAASASGEAG
7522

2295
PAGLTGPATSGSE

2295
RSANHTPAGLTGP

TPGT

PGSP

RSN-
GSAPESGRAANTT
7275
RSC-
GTAEAASASGESG
7523

2298
PAGLTGPATSGSE

2298
RAANTTPAGLTGP

TPGT

PGSP

RSN-
GSAPTTGRATEAA
7276
RSC-
GTAEAASASGTTG
7524

2038
NLTPAGLTGPATS

2038
RATEAANLTPAGLT

GSETPGT

GPPGSP

RSN-
GSAPTTGRAEEAA
7277
RSC-
GTAEAASASGTTG
7525

2072
NLTPAGLTGPATS

2072
RAEEAANLTPAGLT

GSETPGT

GPPGSP

RSN-
GSAPTTGRAGEAA
7278
RSC-
GTAEAASASGTTG
7526

2089
NLTPAGLTGPATS

2089
RAGEAANLTPAGL

GSETPGT

TGPPGSP

RSN-
GSAPTTGRATEAA
7279
RSC-
GTAEAASASGTTG
7527

2302
NATPAGLTGPATS

2302
RATEAANATPAGLT

GSETPGT

GPPGSP

RSN-
GSAPTTGRAGEAE
7280
RSC-
GTAEAASASGTTG
7528

3047
GATSAGATGPATS

3047
RAGEAEGATSAGA

GSETPGT

TGPPGSP

RSN-
GSAPTTGEAGEAA
7281
RSC-
GTAEAASASGTTG
7529

3052
NATSAGATGPATS

3052
EAGEAANATSAGA

GSETPGT

TGPPGSP

RSN-
GSAPTTGEAGEAA
7282
RSC-
GTAEAASASGTTG
7530

3043
GLTPAGLTGPATS

3043
EAGEAAGLTPAGL

GSETPGT

TGPPGSP

RSN-
GSAPTTGAAGEAA
7283
RSC-
GTAEAASASGTTG
7531

3041
NATPAGLTGPATS

3041
AAGEAANATPAGL

GSETPGT

TGPPGSP

RSN-
GSAPTTGRAGEAA
7284
RSC-
GTAEAASASGTTG
7532

3044
GLTPAGLTGPATS

3044
RAGEAAGLTPAGL

GSETPGT

TGPPGSP

RSN-
GSAPTTGRAGEAA
7285
RSC-
GTAEAASASGTTG
7533

3057
NATSAGATGPATS

3057
RAGEAANATSAGA

GSETPGT

TGPPGSP

RSN-
GSAPTTGEAGEAA
7286
RSC-
GTAEAASASGTTG
7534

3058
GATSAGATGPATS

3058
EAGEAAGATSAGA

GSETPGT

TGPPGSP

RSN-
GSAPESGRAANTE
7287
RSC-
GTAEAASASGESG
7535

2485
PPELGAGATSGSE

2485
RAANTEPPELGAG

TPGT

PGSP

RSN-
GSAPESGRAANTA
7288
RSC-
GTAEAASASGESG
7536

2486
PEGLTGPATSGSE

2486
RAANTAPEGLTGP

TPGT

PGSP

RSN-
GSAPEPGRAANHE
7289
RSC-
GTAEAASASGEPG
7537

2488
PSGLTEGATSGSE

2488
RAANHEPSGLTEG

TPGT

PGSP

RSN-
GSAPESGRAANHT
7290
RSC-
GTAEAASASGESG
7538

2599
GAPPGGLTGPATS

2599
RAANHTGAPPGGL

GSETPGT

TGPPGSP

RSN-
GSAPTTGRTGEGA
7291
RSC-
GTAEAASASGTTG
7539

2706
NATPGGLTGPATS

2706
RTGEGANATPGGL

GSETPGT

TGPPGSP

RSN-
GSAPRTGRSGEAA
7292
RSC-
GTAEAASASGRTG
7540

2707
NETPEGLEGPATS

2707
RSGEAANETPEGL

GSETPGT

EGPPGSP

RSN-
GSAPRTGRTGESA
7293
RSC-
GTAEAASASGRTG
7541

2708
NETPAGLGGPATS

2708
RTGESANETPAGL

GSETPGT

GGPPGSP

RSN-
GSAPSTGRTGEPA
7294
RSC-
GTAEAASASGSTG
7542

2709
NETPAGLSGPATS

2709
RTGEPANETPAGL

GSETPGT

SGPPGSP

RSN-
GSAPTTGRAGEPA
7295
RSC-
GTAEAASASGTTG
7543

2710
NATPTGLSGPATS

2710
RAGEPANATPTGL

GSETPGT

SGPPGSP

RSN-
GSAPRTGRPGEGA
7296
RSC-
GTAEAASASGRTG
7544

2711
NATPTGLPGPATS

2711
RPGEGANATPTGL

GSETPGT

PGPPGSP

RSN-
GSAPRTGRGGEAA
7297
RSC-
GTAEAASASGRTG
7545

2712
NATPSGLGGPATS

2712
RGGEAANATPSGL

GSETPGT

GGPPGSP

RSN-
GSAPSTGRSGESA
7298
RSC-
GTAEAASASGSTG
7546

2713
NATPGGLGGPATS

2713
RSGESANATPGGL

GSETPGT

GGPPGSP

RSN-
GSAPRTGRTGEEA
7299
RSC-
GTAEAASASGRTG
7547

2714
NATPAGLPGPATS

2714
RTGEEANATPAGL

GSETPGT

PGPPGSP

RSN-
GSAPATGRPGEPA
7300
RSC-
GTAEAASASGATG
7548

2715
NTTPEGLEGPATS

2715
RPGEPANTTPEGL

GSETPGT

EGPPGSP

RSN-
GSAPSTGRSGEPA
7301
RSC-
GTAEAASASGSTG
7549

2716
NATPGGLTGPATS

2716
RSGEPANATPGGL

GSETPGT

TGPPGSP

RSN-
GSAPPTGRGGEGA
7302
RSC-
GTAEAASASGPTG
7550

2717
NTTPTGLPGPATS

2717
RGGEGANTTPTGL

GSETPGT

PGPPGSP

RSN-
GSAPPTGRSGEGA
7303
RSC-
GTAEAASASGPTG
7551

2718
NATPSGLTGPATS

2718
RSGEGANATPSGL

GSETPGT

TGPPGSP

RSN-
GSAPTTGRASEGA
7304
RSC-
GTAEAASASGTTG
7552

2719
NSTPAPLTEPATSG

2719
RASEGANSTPAPL

SETPGT

TEPPGSP

RSN-
GSAPTYGRAAEAA
7305
RSC-
GTAEAASASGTYG
7553

2720
NTTPAGLTAPATSG

2720
RAAEAANTTPAGLT

SETPGT

APPGSP

RSN-
GSAPTTGRATEGA
7306
RSC-
GTAEAASASGTTG
7554

2721
NATPAELTEPATSG

2721
RATEGANATPAELT

SETPGT

EPPGSP

RSN-
GSAPTVGRASEEA
7307
RSC-
GTAEAASASGTVG
7555

2722
NTTPASLTGPATSG

2722
RASEEANTTPASLT

SETPGT

GPPGSP

RSN-
GSAPTTGRAPEAA
7308
RSC-
GTAEAASASGTTG
7556

2723
NATPAPLTGPATS

2723
RAPEAANATPAPLT

GSETPGT

GPPGSP

RSN-
GSAPTWGRATEPA
7309
RSC-
GTAEAASASGTWG
7557

2724
NATPAPLTSPATSG

2724
RATEPANATPAPLT

SETPGT

SPPGSP

RSN-
GSAPTVGRASESA
7310
RSC-
GTAEAASASGTVG
7558

2725
NATPAELTSPATSG

2725
RASESANATPAELT

SETPGT

SPPGSP

RSN-
GSAPTVGRAPEGA
7311
RSC-
GTAEAASASGTVG
7559

2726
NSTPAGLTGPATS

2726
RAPEGANSTPAGL

GSETPGT

TGPPGSP

RSN-
GSAPTWGRATEAP
7312
RSC-
GTAEAASASGTWG
7560

2727
NLEPATLTTPATSG

2727
RATEAPNLEPATLT

SETPGT

TPPGSP

RSN-
GSAPTTGRATEAP
7313
RSC-
GTAEAASASGTTG
7561

2728
NLTPAPLTEPATSG

2728
RATEAPNLTPAPLT

SETPGT

EPPGSP

RSN-
GSAPTQGRATEAP
7314
RSC-
GTAEAASASGTQG
7562

2729
NLSPAALTSPATSG

2729
RATEAPNLSPAALT

SETPGT

SPPGSP

RSN-
GSAPTQGRAAEAP
7315
RSC-
GTAEAASASGTQG
7563

2730
NLTPATLTAPATSG

2730
RAAEAPNLTPATLT

SETPGT

APPGSP

RSN-
GSAPTSGRAPEAT
7316
RSC-
GTAEAASASGTSG
7564

2731
NLAPAPLTGPATSG

2731
RAPEATNLAPAPLT

SETPGT

GPPGSP

RSN-
GSAPTQGRAAEAA
7317
RSC-
GTAEAASASGTQG
7565

2732
NLTPAGLTEPATSG

2732
RAAEAANLTPAGLT

SETPGT

EPPGSP

RSN-
GSAPTTGRAGSAP
7318
RSC-
GTAEAASASGTTG
7566

2733
NLPPTGLTTPATSG

2733
RAGSAPNLPPTGL

SETPGT

TTPPGSP

RSN-
GSAPTTGRAGGAE
7319
RSC-
GTAEAASASGTTG
7567

2734
NLPPEGLTAPATSG

2734
RAGGAENLPPEGL

SETPGT

TAPPGSP

RSN-
GSAPTTSRAGTAT
7320
RSC-
GTAEAASASGTTS
7568

2735
NLTPEGLTAPATSG

2735
RAGTATNLTPEGLT

SETPGT

APPGSP

RSN-
GSAPTTGRAGTAT
7321
RSC-
GTAEAASASGTTG
7569

2736
NLPPSGLTTPATSG

2736
RAGTATNLPPSGLT

SETPGT

TPPGSP

RSN-
GSAPTTARAGEAE
7322
RSC-
GTAEAASASGTTA
7570

2737
NLSPSGLTAPATSG

2737
RAGEAENLSPSGL

SETPGT

TAPPGSP

RSN-
GSAPTTGRAGGAG
7323
RSC-
GTAEAASASGTTG
7571

2738
NLAPGGLTEPATS

2738
RAGGAGNLAPGGL

GSETPGT

TEPPGSP

RSN-
GSAPTTGRAGTAT
7324
RSC-
GTAEAASASGTTG
7572

2739
NLPPEGLTGPATS

2739
RAGTATNLPPEGLT

GSETPGT

GPPGSP

RSN-
GSAPTTGRAGGAA
7325
RSC-
GTAEAASASGTTG
7573

2740
NLAPTGLTEPATSG

2740
RAGGAANLAPTGL

SETPGT

TEPPGSP

RSN-
GSAPTTGRAGTAE
7326
RSC-
GTAEAASASGTTG
7574

2741
NLAPSGLTTPATSG

2741
RAGTAENLAPSGL

SETPGT

TTPPGSP

RSN-
GSAPTTGRAGSAT
7327
RSC-
GTAEAASASGTTG
7575

2742
NLGPGGLTGPATS

2742
RAGSATNLGPGGL

GSETPGT

TGPPGSP

RSN-
GSAPTTARAGGAE
7328
RSC-
GTAEAASASGTTA
7576

2743
NLTPAGLTEPATSG

2743
RAGGAENLTPAGL

SETPGT

TEPPGSP

RSN-
GSAPTTARAGSAE
7329
RSC-
GTAEAASASGTTA
7577

2744
NLSPSGLTGPATS

2744
RAGSAENLSPSGL

GSETPGT

TGPPGSP

RSN-
GSAPTTARAGGAG
7330
RSC-
GTAEAASASGTTA
7578

2745
NLAPEGLTTPATSG

2745
RAGGAGNLAPEGL

SETPGT

TTPPGSP

RSN-
GSAPTTSRAGAAE
7331
RSC-
GTAEAASASGTTS
7579

2746
NLTPTGLTGPATSG

2746
RAGAAENLTPTGLT

SETPGT

GPPGSP

RSN-
GSAPTYGRTTTPG
7332
RSC-
GTAEAASASGTYG
7580

2747
NEPPASLEAEATS

2747
RTTTPGNEPPASLE

GSETPGT

AEPGSP

RSN-
GSAPTYSRGESGP
7333
RSC-
GTAEAASASGTYS
7581

2748
NEPPPGLTGPATS

2748
RGESGPNEPPPGL

GSETPGT

TGPPGSP

RSN-
GSAPAWGRTGASE
7334
RSC-
GTAEAASASGAWG
7582

2749
NETPAPLGGEATS

2749
RTGASENETPAPL

GSETPGT

GGEPGSP

RSN-
GSAPRWGRAETTP
7335
RSC-
GTAEAASASGRWG
7583

2750
NTPPEGLETEATS

2750
RAETTPNTPPEGLE

GSETPGT

TEPGSP

RSN-
GSAPESGRAANHT
7336
RSC-
GTAEAASASGESG
7584

2751
GAEPPELGAGATS

2751
RAANHTGAEPPEL

GSETPGT

GAGPGSP

RSN-
GSAPTTGRAGEAA
7337
RSC-
GTAEAASASGTTG
7585

2754
NLTPAGLTESATSG

2754
RAGEAANLTPAGL

SETPGT

TESPGSP

RSN-
GSAPTTGRAGEAA
7338
RSC-
GTAEAASASGTTG
7586

2755
NLTPAALTESATSG

2755
RAGEAANLTPAALT

SETPGT

ESPGSP

RSN-
GSAPTTGRAGEAA
7339
RSC-
GTAEAASASGTTG
7587

2756
NLTPAPLTESATSG

2756
RAGEAANLTPAPLT

SETPGT

ESPGSP

RSN-
GSAPTTGRAGEAA
7340
RSC-
GTAEAASASGTTG
7588

2757
NLTPEPLTESATSG

2757
RAGEAANLTPEPLT

SETPGT

ESPGSP

RSN-
GSAPTTGRAGEAA
7341
RSC-
GTAEAASASGTTG
7589

2758
NLTPAGLTGAATS

2758
RAGEAANLTPAGL

GSETPGT

TGAPGSP

RSN-
GSAPTTGRAGEAA
7342
RSC-
GTAEAASASGTTG
7590

2759
NLTPEGLTGAATS

2759
RAGEAANLTPEGL

GSETPGT

TGAPGSP

RSN-
GSAPTTGRAGEAA
7343
RSC-
GTAEAASASGTTG
7591

2760
NLTPEPLTGAATSG

2760
RAGEAANLTPEPLT

SETPGT

GAPGSP

RSN-
GSAPTTGRAGEAA
7344
RSC-
GTAEAASASGTTG
7592

2761
NLTPAGLTEAATSG

2761
RAGEAANLTPAGL

SETPGT

TEAPGSP

RSN-
GSAPTTGRAGEAA
7345
RSC-
GTAEAASASGTTG
7593

2762
NLTPEGLTEAATSG

2762
RAGEAANLTPEGL

SETPGT

TEAPGSP

RSN-
GSAPTTGRAGEAA
7346
RSC-
GTAEAASASGTTG
7594

2763
NLTPAPLTEAATSG

2763
RAGEAANLTPAPLT

SETPGT

EAPGSP

RSN-
GSAPTTGRAGEAA
7347
RSC-
GTAEAASASGTTG
7595

2764
NLTPEPLTEAATSG

2764
RAGEAANLTPEPLT

SETPGT

EAPGSP

RSN-
GSAPTTGRAGEAA
7348
RSC-
GTAEAASASGTTG
7596

2765
NLTPEPLTGPATSG

2765
RAGEAANLTPEPLT

SETPGT

GPPGSP

RSN-
GSAPTTGRAGEAA
7349
RSC-
GTAEAASASGTTG
7597

2766
NLTPAGLTGGATS

2766
RAGEAANLTPAGL

GSETPGT

TGGPGSP

RSN-
GSAPTTGRAGEAA
7350
RSC-
GTAEAASASGTTG
7598

2767
NLTPEGLTGGATS

2767
RAGEAANLTPEGL

GSETPGT

TGGPGSP

RSN-
GSAPTTGRAGEAA
7351
RSC-
GTAEAASASGTTG
7599

2768
NLTPEALTGGATS

2768
RAGEAANLTPEALT

GSETPGT

GGPGSP

RSN-
GSAPTTGRAGEAA
7352
RSC-
GTAEAASASGTTG
7600

2769
NLTPEPLTGGATS

2769
RAGEAANLTPEPLT

GSETPGT

GGPGSP

RSN-
GSAPTTGRAGEAA
7353
RSC-
GTAEAASASGTTG
7601

2770
NLTPAGLTEGATS

2770
RAGEAANLTPAGL

GSETPGT

TEGPGSP

RSN-
GSAPTTGRAGEAA
7354
RSC-
GTAEAASASGTTG
7602

2771
NLTPEGLTEGATS

2771
RAGEAANLTPEGL

GSETPGT

TEGPGSP

RSN-
GSAPTTGRAGEAA
7355
RSC-
GTAEAASASGTTG
7603

2772
NLTPAPLTEGATSG

2772
RAGEAANLTPAPLT

SETPGT

EGPGSP

RSN-
GSAPTTGRAGEAA
7356
RSC-
GTAEAASASGTTG
7604

2773
NLTPEPLTEGATSG

2773
RAGEAANLTPEPLT

SETPGT

EGPGSP

RSN-
GSAPTTGRAGEAE
7357
RSC-
GTAEAASASGTTG
7605

3047
GATSAGATGPATS

3047
RAGEAEGATSAGA

GSETPGT

TGPPGSP

RSN-
GSAPEAGRSAEAT
7358
RSC-
GTAEAASASGEAG
7606

2783
SAGATGPATSGSE

2783
RSAEATSAGATGP

TPGT

PGSP

RSN-
GSAPSASGTYSRG
7359
RSC-
GTAEAASASGSAS
7607

3107
ESGPGSPATSGSE

3107
GTYSRGESGPGSP

TPGT

PGSP

RSN-
GSAPSASGEAGRT
7360
RSC-
GTAEAASASGSAS
7608

3103
DTHPGSPATSGSE

3103
GEAGRTDTHPGSP

TPGT

PGSP

RSN-
GSAPSASGEPGRA
7361
RSC-
GTAEAASASGSAS
7609

3102
AEHPGSPATSGSE

3102
GEPGRAAEHPGSP

TPGT

PGSP

RSN-
GSAPSPAGESSRG
7362
RSC-
GTAEAASASGSPA
7610

3119
TTIAGSPATSGSET

3119
GESSRGTTIAGSPP

PGT

GSP

RSN-
GSAPTTGEAGEAA
7363
RSC-
GTAEAASASGTTG
7611

3043
GLTPAGLTGPATS

3043
EAGEAAGLTPAGL

GSETPGT

TGPPGSP

RSN-
GSAPEAGESAGAT
7364
RSC-
GTAEAASASGEAG
7612

2789
PAGLTGPATSGSE

2789
ESAGATPAGLTGP

TPGT

PGSP

RSN-
GSAPSASGAPLEL
7365
RSC-
GTAEAASASGSAS
7613

3109
EAGPGSPATSGSE

3109
GAPLELEAGPGSP

TPGT

PGSP

RSN-
GSAPSASGEPPEL
7366
RSC-
GTAEAASASGSAS
7614

3110
GAGPGSPATSGSE

3110
GEPPELGAGPGSP

TPGT

PGSP

RSN-
GSAPSASGEPSGL
7367
RSC-
GTAEAASASGSAS
7615

3111
TEGPGSPATSGSE

3111
GEPSGLTEGPGSP

TPGT

PGSP

RSN-
GSAPSASGTPAPL
7368
RSC-
GTAEAASASGSAS
7616

3112
TEPPGSPATSGSE

3112
GTPAPLTEPPGSP

TPGT

PGSP

RSN-
GSAPSASGTPAEL
7369
RSC-
GTAEAASASGSAS
7617

3113
TEPPGSPATSGSE

3113
GTPAELTEPPGSP

TPGT

PGSP

RSN-
GSAPSASGPPPGL
7370
RSC-
GTAEAASASGSAS
7618

3114
TGPPGSPATSGSE

3114
GPPPGLTGPPGSP

TPGT

PGSP

RSN-
GSAPSASGTPAPL
7371
RSC-
GTAEAASASGSAS
7619

3115
GGEPGSPATSGSE

3115
GTPAPLGGEPGSP

TPGT

PGSP

RSN-
GSAPSPAGAPEGL
7372
RSC-
GTAEAASASGSPA
7620

3125
TGPAGSPATSGSE

3125
GAPEGLTGPAGSP

TPGT

PGSP

RSN-
GSAPSPAGPPEGL
7373
RSC-
GTAEAASASGSPA
7621

3126
ETEAGSPATSGSE

3126
GPPEGLETEAGSP

TPGT

PGSP

RSN-
GSAPSPTSGQGGL
7374
RSC-
GTAEAASASGSPT
7622

3127
TGPGSEPATSGSE

3127
SGQGGLTGPGSEP

TPGT

PGSP

RSN-
GSAPSESAPPEGL
7375
RSC-
GTAEAASASGSES
7623

3131
ETESTEPATSGSET

3131
APPEGLETESTEPP

PGT

GSP

RSN-
GSAPSEGSEPLEL
7376
RSC-
GTAEAASASGSEG
7624

3132
GAASETPATSGSE

3132
SEPLELGAASETPP

TPGT

GSP

RSN-
GSAPSEGSGPAGL
7377
RSC-
GTAEAASASGSEG
7625

3133
EAPSETPATSGSET

3133
SGPAGLEAPSETP

PGT

PGSP

RSN-
GSAPSEPTPPASLE
7378
RSC-
GTAEAASASGSEP
7626

3138
AEPGSPATSGSET

3138
TPPASLEAEPGSPP

PGT

GSP

In some embodiments, a paTCE comprises an RS1 and an RS2 that have different rates of cleavage and different cleavage efficiencies to multiple proteases for which they are substrates. As a given protease may be found in different concentrations in a tumor, compared to healthy tissues or in circulation, the disclosure provides RSs that have a higher or lower cleavage efficiency for a given protease in order to ensure that a paTCE is preferentially converted from the inactive form to the active form (i.e., by the separation and release of the binding moieties and ELNNs from the paTCE after cleavage of the RSs) when in proximity to the cancer cell or tissue and its co-localized proteases compared to the rate of cleavage of the RSs in healthy tissue or the circulation such that the released binding moieties of the TCE have a greater ability to bind to ligands in the tumor compared to the inactive form that remains in circulation. By such selective designs, the therapeutic index of the resulting compositions can be improved, resulting in reduced side effects relative to convention therapeutics that do not incorporate such site-specific activation.

In some embodiments, cleavage efficiency is the log 2 value of the ratio of the percentage of the test substrate comprising the RS cleaved to the percentage of the control substrate AC1611 cleaved when each is subjected to the protease enzyme in biochemical assays in which reaction in conducted wherein the initial substrate concentration is 6 μM, the reactions are incubated at 37° C. for 2 hours before being stopped by adding EDTA, with the amount of digestion products and uncleaved substrate analyzed by non-reducing SDS-PAGE to establish the ratio of the percentage cleaved. The cleavage efficiency may be calculated as follows:

${Log}_{2} (\frac{% Cleaved for substrate of interest}{% cleaved for AC 1611 in the same experiment}) .$

that the amount of test substrate cleaved was 50% compared to that of the control substrate, while a cleavage efficiency of +1 means that the amount of test substrate cleaved was 200% compared to that of the control substrate. A higher rate of cleavage by the test protease relative to the control would result in a higher cleavage efficiency, and a slower rate of cleavage by the test protease relative to the control would result in a lower cleavage efficiency. A control RS sequence AC1611 (RSR-1517), having the amino acid sequence EAGRSANHEPLGLVAT (SEQ ID NO: 7001), was established as having an appropriate baseline cleavage efficiency by the proteases legumain, MMP-2, MMP-7, MMP-9, MMP-14, uPA, and matriptase, when tested in in vitro biochemical assays for rates of cleavage by the individual proteases. By selective substitution of amino acids at individual locations in the RS peptides, libraries of RS were created and evaluated against the panel of the 7 proteases, resulting in profiles that were used to establish guidelines for appropriate amino acid substitutions in order to achieve RS with desired cleavage efficiencies. In some embodiments, in making RSs with desired cleavage efficiencies, substitutions using the hydrophilic amino acids A, E, G, P, S, and T are preferred, however other L-amino acids can be substituted at given positions in order to adjust the cleavage efficiency so long as the RSs retain at least some susceptibility to cleavage by a given protease. Conservative substitutions of amino acids in a peptide to retain or effect activity is well within the knowledge and capabilities of a person within skill in the art. In some embodiments, the disclosure provides an RS in which the RS is cleaved by a protease including but not limited to MMP-2, MMP-7, MMP-9, MMP-14, uPA, or matriptase (also known as MT-SP1) with at least a 0.2 log₂, or 0.4 log₂, or 0.8 log 2, or 1.0 log 2 higher cleavage efficiency in an in vitro biochemical competitive assay compared to the cleavage by the same protease of a control sequence RSR-1517 having the sequence EAGRSANHEPLGLVAT (SEQ ID NO. 7001). In some embodiments, the disclosure provides an RS in which the RS is cleaved by a protease including but not limited to MMP-2, MMP-7, MMP-9, MMP-11, uPA, or matriptase with at least a 0.2 log 2, or 0.4 log 2, or 0.8 log 2, or 1.0 log 2 lower cleavage efficiency in an in vitro biochemical competitive assay compared to the cleavage by the same protease of a control sequence RSR-1517 having the sequence EAGRSANHEPLGLVAT (SEQ ID NO. 7001). In some embodiments, the disclosure provides an RS in which the rate of cleavage of the RS by a protease including but not limited to MMP-2, MMP-7, MMP-9, MMP-14, uPA, or matriptase is at least 2-fold, or at least 4-fold, or at least 8 fold, or at least 16-fold faster compared to the control sequence RSR-1517 having the sequence EAGRSANHEPLGLVAT (SEQ ID NO. 7001). In some embodiments, the disclosure provides an RS in which the rate of cleavage of the RS by a protease including but not limited to MMP-2, MMP-7, MMP-9, MMP-14, uPA, or matriptase is at least 2-fold, or at least 4-fold, or at least 8-fold, or at least 16-fold slower compared to the control sequence RSR-1517 having the sequence EAGRSANHEPLGLVAT (SEQ ID NO. 7001).

In some embodiments, the RS comprises the amino acid sequence EAGRSAXHTPAGLTGP (SEQ ID NO: 7627), wherein X is any amino acid other than N. In some embodiments, X is S. In some embodiments, X is T. In some embodiments, X is Y. In some embodiments, X is Q. In some embodiments, X is G. In some embodiments, X is A. In some embodiments, X is V. In some embodiments, X is C. In some embodiments, X is P. In some embodiments, X is L. In some embodiments, X is I. In some embodiments, X is M. In some embodiments, X is F. In some embodiments, X is K. In some embodiments, X is R. In some embodiments, X is H. In some embodiments, X is D. In some embodiments, X is E. In some embodiments, the RS is not cleaved by legumain. In some embodiments, the RS is not cleavable by legumain in human blood, plasma, or serum.

In some embodiments, the RS is not cleavable upon incubation with about 1 nM or less legumain for about 20 hours. In some embodiments, the RS is cleaved by legumain less quickly or efficiently than RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain. In some embodiments, the RS is cleaved by legumain at a rate that is less than about 50% of the rate that legumain cleaves RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048). In some embodiments, the RS is cleaved by legumain at a rate that is less than about 25% of the rate that legumain cleaves RSR-2295. In some embodiments, the RS is cleaved by legumain at a rate that is less than about 10% of the rate that legumain cleaves RSR-2295. In some embodiments, the RS is cleaved by legumain at a rate that is less than about 5% of the rate that legumain cleaves RSR-2295. In some embodiments, the RS is cleaved by legumain at a rate that is less than about 2.5% of the rate that legumain cleaves RSR-2295.

In some embodiments, the RS is cleaved by legumain at a rate that is less than about 50% of the rate that legumain cleaves RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) in human plasma. In some embodiments, the RS is cleaved by legumain at a rate that is less than about 25% of the rate that legumain cleaves RSR-2295 in human plasma. In some embodiments, the RS is cleaved by legumain at a rate that is less than about 10% of the rate that legumain cleaves RSR-2295 in human plasma. In some embodiments, the RS is cleaved by legumain at a rate that is less than about 5% of the rate that legumain cleaves RSR-2295 in human plasma. In some embodiments, the RS is cleaved by legumain at a rate that is less than about 2.5% of the rate that legumain cleaves RSR-2295 in human plasma.

In some embodiments, the disclosure provides paTCEs comprising multiple RSs wherein each RS sequence is identified herein by the group of sequences set forth in Table 8a and the RSs are linked to each other by 1 to 6 amino acids that are glycine, serine, alanine, and threonine. In some embodiments, a paTCE comprises a first RS and a second RS different from the first RS wherein each RS sequence is identified herein by a sequence set forth in Table 8a and the RSs are linked to each other by 1 to 6 amino acids that are glycine, serine, alanine, and threonine. In some embodiments, the paTCE comprises a first RS, a second RS different from the first RS, and a third RS different from the first and the second RS wherein each sequence is identified herein by s sequence set forth in Table 8a and the first and the second and the third RS are linked to each other by 1 to 6 amino acids that are glycine, serine, alanine, and threonine. In some embodiments, multiple RS of the paTCE can be concatenated to form a sequence that can be cleaved by multiple proteases at different rates or efficiency of cleavage. In some embodiments, the disclosure provides a paTCE comprising an RS1 and an RS2, wherein each has a sequence set forth in Table 8a or 8b and ELNNs (e.g., an ELNN1 and ELNN2), such as those described herein, wherein the RS1 is fused between the ELNN1 and the binding moieties and the RS2 is fused between the ELNN2 and the binding moieties. In some embodiments, a paTCE is more readily cleaved in target tissues that express multiple proteases (e.g., tumor tissues), compared with healthy tissues or when in the normal circulation, with the result that the resulting fragments bearing the binding moieties would more readily penetrate the target tissue, e.g., a tumor, and have an enhanced ability to bind and link the cancer cell and the effector cell.

In some embodiments, a paTCE comprises a first release segment (RS1) positioned between a first ELNN a bispecific antibody. In some embodiments, the polypeptide further comprises a second release segment (RS2) positioned between the bispecific antibody and a second ELNN. In some embodiments, RS1 and RS2 are identical in sequence. In some embodiments, RS1 and RS2 are not identical in sequence. In some embodiments, the RS1 comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence identified herein in Table 8a or 8b or a subset thereof. In some embodiments, the RS2 comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence identified herein in Table 8a or 8b or a subset thereof. In some embodiments, the RS1 and RS2 are each a substrate for cleavage by multiple proteases at one, two, or three cleavage sites within each release segment sequence.

In some embodiments, the paTCE further comprises one or more reference fragments (e.g., barcode fragments) releasable from the paTCE upon digestion by the protease. In some embodiments, the one or more reference fragments is a single reference fragment that differs in sequence and molecular weight from all other peptide fragments that are releasable from the polypeptide upon digestion of the polypeptide by the protease.

Exemplary paTCEs

In some embodiments, a paTCE comprises an amino acid sequence having at least (about) 80% sequence identity to a sequence set forth in Table D (consisting of SEQ ID NOS: 1000-1009) or a subset thereof. In some embodiments, the paTCE comprises an amino acid sequence having at least (about) 81%, at least (about) 82%, at least (about) 83%, at least (about) 84%, at least (about) 85%, at least (about) 86%, at least (about) 87%, at least (about) 88%, at least (about) 89%, at least (about) 90%, at least (about) 91%, at least (about) 92%, at least (about) 93%, at least (about) 94%, at least (about) 95%, at least (about) 96%, at least (about) 97%, at least (about) 98%, at least (about) 99%, or (about) 100% sequence identity to a sequence set forth in Table D (SEQ ID NOS: 1000-1009) or a subset thereof. In some embodiments, the paTCE comprises an amino acid sequence having at least (about) 90%, at least (about) 91%, at least (about) 92%, at least (about) 93%, at least (about) 94%, at least (about) 95%, at least (about) 96%, at least (about) 97%, at least (about) 98%, at least (about) 99%, or (about) 100% sequence identity to a sequence set forth in Table D (SEQ ID NOS: 1000-1009) or a subset thereof. In some embodiments, the paTCE comprises an amino acid sequence identical to a sequence set forth in Table D (SEQ ID NOS: 1000-1009). It is specifically contemplated that the compositions of this disclosure can comprise sequence variants of the amino acid sequences set forth in Table D, such as with linker sequence(s) substituted or inserted or with purification tag sequence(s) attached thereto, so long as the variants exhibit substantially similar or same bioactivity/bioactivities and/or activation mechanism(s).

TABLE D

Exemplary amino acid sequences of polypeptides

SEQ ID

NO
AMINO ACID SEQUENCE

1000
ASSATPESGPGTSTEPSEGSAPGTSESATPESGPGSGPGTSESATPGTSE

(AMX-500)
SATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGS

PTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPT

STEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPES

GPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEE

GTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPEAGRSA

SHTPAGLTGPGTSESATPESQVQLVESGGGVVQPGRSLRLSCAASGRTFG

IYVWGWFRQAPGKEREFVGAMSWSGSNRKVSDSVKGRFTISRDNSKNTL

YLQMNSLRAEDTAVYYCAASNKEYGRTWYDFNESDYWGQGTQVTVSSGG

GGSGGGSELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKP

GQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCAL

WYPNLWVFGGGTKLTVLSESATPESGPGTSPGATPESGPGTSESATPEVQ

LVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRT

KRNNYATYYADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCVRHENF

GNSYVSWFAHWGQGTLVTVSSGTATPESGPGEAGRSASHTPAGLTGPAT

PESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGS

ETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA

PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEG

TSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSE

PATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAG

SPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSE

GSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSE

TPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGP

GSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGT

STEPSEGSAPGTSESATPESGPGTSESATPESGPGTSPSATPESGPGSEP

ATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEP

SEGSAPGSEPATSGSETPGTSESAGEPEA

1001
ASSPAGSPTSTESGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPG

(AC3092)
SEPATSGSETPGTSESATPESGPGSTPAESGSETPGTSESATPESGPGTS

TEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSES

ATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESAT

PESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGS

ETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSAPEAGRSA

NHTPAGLTGPATSGSETPGTQVQLVESGGGVVQPGRSLRLSCAASGRTFG

IYVMGWVRQAPGKEREFVAAISWSGSNRLVSDSVKGRFTISRDNSKNTLYL

QMNSLRAEDTAVYYCAASNRLYGRTWYDFNESDYWGQGTQVTVSSGGG

GSGGGSELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPG

QAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALW

YPNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQL

LESGGGIVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSK

YNNYATYYADSVKDRFTISRDDSKNTVYLQMNNLKTEDTAVYYCVRHENFG

NSYVSWFAHWGQGTLVTVSSGTAEAASASGEAGRSANHTPAGLTGPPGS

PAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTST

EPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEP

SEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATP

ESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPES

GPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEE

GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGS

EPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTST

EPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESA

TPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATP

ESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSE

TPGSPAGSPTSTEEGTSTEPSEGSAPGTESTPSEGSAPGSEPATSGSETP

GTSESATPESGPGTSTEPSEGSAPGEPEA

1002
ASHHHHHHSATPESGPGTSTEPSEGSAPGTSESATPESGPGSGPGTSESA

(AC3445)*
TPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP

GSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGS

PAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSE

SATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGS

PTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPE

AGRSANHTPAGLTGPGTSESATPESQVQLVESGGGVVQPGRSLRLSCAAS

GRTFGIYVMGWVRQAPGKEREFVAAISWSGSNRKVSDSVKGRFTISRDNS

KNTLYLQMNSLRAEDTAVYYCAASNRLYGRTWYDFNESDYWGQGTQVTV

SSGGGGSGGGSELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWV

QQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVY

YCALWYPNLWVFGGGTKLTVLSESATPESGPGTSPGATPESGPGTSESAT

PEVQLLESGGGIVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWV

ARIRSKYNNYATYYADSVKDRFTISRDDSKNTVYLQMNNLKTEDTAVYYCV

RHENFGNSYVSWFAHWGQGTLVTVSSGTATPESGPGEAGRSANHTPAGL

TGPATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEP

ATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEP

SEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPT

STEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPES

GPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGP

GSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGT

STEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEP

ATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESA

TPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPT

STEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSPSATPES

GPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAP

GTSTEPSEGSAPGSEPATSGSETPGTSESAGEPEA

1003
ASSATPESGPGTSTEPSEGSAPGTSESATPESGPGSGPGTSESATPGTSE

(AC3928)
SATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGS

PTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPT

STEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPES

GPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEE

GTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPEAGRSA

SHTPAGLTGPGTSESATPESQVQLVESGGGVVQPGRSLRLSCAASGRTFG

IYVWGWFRQAPGKEREFVGAMSWSGSNRKVSDSVKGRFTISRDNSKNTL

YLQMNSLRAEDTAVYYCAASNKEYGRTWYDFNESDYWGQGTQVTVSSGG

GGSGGGSELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKP

GQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCAL

WYPNLWVFGGGTKLTVLSESATPESGPGTSPGATPESGPGTSESATPEVQ

LLESGGGIVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRS

KYNNYATYYADSVKDRFTISRDDSKNTVYLQMNNLKTEDTAVYYCVRHENF

GNSYVSWFAHWGQGTLVTVSSGTATPESGPGEAGRSASHTPAGLTGPAT

PESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGS

ETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA

PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEG

TSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSE

PATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAG

SPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSE

GSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSE

TPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGP

GSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGT

STEPSEGSAPGTSESATPESGPGTSESATPESGPGTSPSATPESGPGSEP

ATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEP

SEGSAPGSEPATSGSETPGTSESAGEPEA

1004
ASSATPESGPGTSTEPSEGSAPGTSESATPESGPGSGPGTSESATPGTSE

(AC3934)
SATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGS

PTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPT

STEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPES

GPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEE

GTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPEAGRSA

SHTPAGLTGPGTSESATPESQVQLVESGGGVVQPGRSLRLSCAASGRTFG

IYVWGWVRQAPGKEREFVGAISWSGSNRKVSDSVKGRFTISRDNSKNTLY

LQMNSLRAEDTAVYYCAASNKLYGRTWYDFNESDYWGQGTQVTVSSGGG

GSGGGSELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPG

QAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALW

YPNLWVFGGGTKLTVLSESATPESGPGTSPGATPESGPGTSESATPEVQL

VESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTK

RNNYATYYADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCVRHENF

GNSYVSWFAHWGQGTLVTVSSGTATPESGPGEAGRSASHTPAGLTGPAT

PESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGS

ETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA

PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEG

TSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSE

PATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAG

SPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSE

GSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSE

TPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGP

GSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGT

STEPSEGSAPGTSESATPESGPGTSESATPESGPGTSPSATPESGPGSEP

ATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEP

SEGSAPGSEPATSGSETPGTSESAGEPEA

1005
ASHHHHHHSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESAT

(AC2591)*
PESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPE

SGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSET

PGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPG

TSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSE

PATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSAP

EAGRSANHTPAGLTGPATSGSETPGTEVQLVESGGGSVQAGGSLSLSCVA

SGRTFGIYVMGWFRQAPGKEREFVAAISWSGSNRLVSDSVKGRFTISREN

AKNTIYLQMNGLKPEDTANYFCAASNRLYGRTWYDFNESDYWGQGTQVT

VSSGGGGSGGGSELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANW

VQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAV

YYCALWYPNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPG

EGEVQLLESGGGIVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEW

VARIRSKYNNYATYYADSVKDRFTISRDDSKNTVYLQMNNLKTEDTAVYYC

VRHENFGNSYVSWFAHWGQGTLVTVSSGTAEAASASGEAGRSANHTPAG

LTGPPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTS

TEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSET

PGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPG

TSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTS

ESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTE

PSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSP

TSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEG

SAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA

PGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPG

SEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS

TEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAG

SPTSTEEGTSESATPESGPGTSTEPSEGSAPGAAEPEA

1006
ASHHHHHHATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSA

(AC3353)*
PGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPG

SEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGS

APEAGRSANHTPAGLTGPATSGSETPGTQVQLVESGGGVVQPGRSLRLSC

AASGRTFGIYVMGWVRQAPGKEREFVAAISWSGSNRLVSDSVKGRFTISR

DNSKNTLYLQMNSLRAEDTAVYYCAASNRLYGRTWYDFNESDYWGQGTQ

VTVSSGGGGSELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQ

KPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYC

ALWYPNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGE

VQLLESGGGIVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARI

RSKYNNYATYYADSVKDRFTISRDDSKNTVYLQMNNLKTEDTAVYYCVRHE

NFGNSYVSWFAHWGQGTLVTVSSGTAEAASASGEAGRSANHTPAGLTGP

ESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSES

ATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSP

TSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGAAEPEA

1007
ASHHHHHHATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSA

(AC3354)*
PGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPG

SEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGS

APEAGRSANHTPAGLTGPATSGSETPGTQVQLVESGGGVVQPGRSLRLSC

AASGRTFGIYVMGWVRQAPGKEREFVAAISWSGSNRLVSDSVKGRFTISR

DNSKNTLYLQMNSLRAEDTAVYYCAASNRLYGRTWYDFNESDYWGQGTQ

VTVSSGATPPETGAETESPGELVVTQEPSLTVSPGGTVTLTCRSSNGAVTS

SNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQ

PEDEAVYYCALWYPNLWVFGGGTKLTVLGATPPETGAETESPGETTGGSA

ESEPPGEGEVQLLESGGGIVQPGGSLKLSCAASGFTFNTYAMNWVRQAPG

KGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTVYLQMNNLKTEDT

AVYYCVRHENFGNSYVSWFAHWGQGTLVTVSSGTAEAASASGEAGRSAN

HTPAGLTGPESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSG

SETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTST

EEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP

GAAEPEA

1008
ASHHHHHHATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSA

(AC3356)*
PGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPG

SEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGS

APEAGRSANHTPAGLTGPATSGSETPGTQVQLVESGGGVVQPGRSLRLSC

AASGRTFGIYVMGWVRQAPGKEREFVAAISWSGSNRLVSDSVKGRFTISR

DNSKNTLYLQMNSLRAEDTAVYYCAASNRLYGRTWYDFNESDYWGQGTQ

VTVSSGGGGSGGGSEVQLLESGGGIVQPGGSLKLSCAASGFTFNTYAMN

WVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTVYLQM

NNLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSSGATPPETGA

ETESPGETTGGSAESEPPGEGELVVTQEPSLTVSPGGTVTLTCRSSNGAV

TSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSG

VQPEDEAVYYCALWYPNLWVFGGGTKLTVLGTAEAASASGEAGRSANHTP

AGLTGPESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSET

PGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEG

SPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGAA

EPEA

1009
ASHHHHHHATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSA

(AC3329)*
PGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPG

SEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGS

APEAGRSANHTPAGLTGPRAPPEPEFARATSGSETPGTQVQLVESGGGWV

QPGRSLRLSCAASGRTFGIYVMGWVRQAPGKEREFVAAISWSGSNRKVSD

SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAASNRLYGRTWYDFNE

SDYWGQGTQVTVSSGGGGSGGGSELVVTQEPSLTVSPGGTVTLTCRSSN

GAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALT

LSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVLGATPPETGAETESPGET

TGGSAESEPPGEGEVQLLESGGGIVQPGGSLKLSCAASGFTFNTYAMNWV

RQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTVYLQMNN

LKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSSGTAEAASASGE

AGRSANHTPAGLTGPESATPESGPGSEPATSGSETPGTSESATPESGPGS

EPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPA

GSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEP

SEGSAPGAAEPEA

*The HHHHHH (SEQ ID NO: 48) sequence within this amino acid sequence is dispensable and can be removed. A sequence with the HHHHHH (SEQ ID NO: 48) removed is expressly disclosed herein as well.

Recombinant Production

Also provided are polynucleotides that encode any polypeptide disclosed herein and/or the reverse complements of such polynucleotides.

The disclosure herein includes an expression vector that comprises a polynucleotide sequence, such as any described in the preceding paragraph, and a regulatory sequence operably linked to the polynucleotide sequence.

The disclosure herein includes a host cell comprising an expression vector, such as described any in the preceding paragraph. In some embodiments, the host cell is a prokaryote. In some embodiments, the host cell is E. coli. In some embodiments, the host cell is a mammalian cell.

In some embodiments, the disclosure provides methods of manufacturing the subject compositions. In some embodiments, such a method comprises culturing a host cell comprising a nucleic acid construct that encodes a polypeptide (such as a paTCE) described herein under conditions that promote the expression of the polypeptide, followed by recovery of the polypeptide using standard purification methods (e.g., column chromatography, HPLC, and the like) wherein the composition is recovered wherein at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 97%, or at least 99% of the binding fragments of the expressed polypeptide or paTCE fusion polypeptide are correctly folded. In some embodiments of the method of making, the expressed polypeptide is recovered in which at least or at least 90%, or at least 95%, or at least 97%, or at least 99% of the polypeptide is recovered in monomeric, soluble form.

In some embodiments, the disclosure relates to methods of making a polypeptide (such as a paTCE fusion polypeptide) at high fermentation expression levels of functional protein using an E. coli or mammalian host cell, as well as providing expression vectors encoding the polypeptides useful in methods to produce the cytotoxically active polypeptide compositions at high expression levels. In some embodiments, the method comprises the steps of 1) preparing a polynucleotide encoding a polypeptide disclosed herein, 2) cloning the polynucleotide into an expression vector, which can be a plasmid or other vector under the control of appropriate transcription and translation sequences for high level protein expression in a biological system, 3) transforming an appropriate host cell with the expression vector, and 4) culturing the host cell in conventional nutrient media under conditions suitable for the expression of the polypeptide composition. Where desired, the host cell is E. coli. As used herein, the term “correctly folded” means that the antigen binding fragments component of the composition have the ability to specifically bind their target ligands (e.g., upon activation). In some embodiments, the disclosure provides a method for producing a polypeptide, the method comprising culturing in a fermentation reaction a host cell that comprises a vector encoding a polypeptide comprising the polypeptide under conditions effective to express the polypeptide product.

Pharmaceutical Composition

Disclosed herein includes a pharmaceutical composition comprising a polypeptide (such as a paTCE), such as any described herein, and one or more pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical composition is formulated for intradermal, subcutaneous, intravenous, intra-arterial, intraabdominal, intraperitoneal, intravitreal, intrathecal, or intramuscular administration. In some embodiments, the pharmaceutical composition is formulated for intravenous injection. In some embodiments, the pharmaceutical composition is in a liquid form or frozen. In some embodiments, the pharmaceutical composition is formulated as a lyophilized powder to be reconstituted prior to administration.

The pharmaceutical compositions can be administered for therapy by any suitable route. In some embodiments, the dose is administered intradermally, subcutaneously, intravenously, intra-arterially, intra-abdominally, intraperitoneally, intrathecally, or intramuscularly. In some embodiments, the subject is a mouse, rat, monkey, or human. In preferred embodiments, the subject is a human.

In some embodiments, the pharmaceutical composition can be administered subcutaneously, intramuscularly, or intravenously. In some embodiments, the pharmaceutical composition is administered at a therapeutically effective amount. In some embodiments, the therapeutically effective amount results in a gain in time spent within a therapeutic window for the fusion protein compared to the corresponding TCE of the fusion protein not linked to the ELNN and administered at a comparable dose to a subject.

In some embodiments, the pharmaceutical composition is administered subcutaneously. In some embodiments, the pharmaceutical composition is administered intravenously.

In some embodiments, the composition may be supplied as a lyophilized powder or cake to be reconstituted prior to administration. In some embodiments, the composition may also be supplied in a liquid form or frozen, which can be administered directly to a subject.

Pharmaceutical Kits

In some embodiments, the present disclosure provides kits to facilitate the use of paTCEs. In some embodiments, a kit comprises (a) a first container comprising pharmaceutically effective amount of a paTCE in a lyophilized composition; and (b) a second container comprising a diluent for reconstituting the lyophilized formulation. In some embodiments, the kit further comprises instructions for storage of the kit, information regarding a cancer that is treatable with the paTCE, instructions for the reconstitution of the lyophilized formulation, and/or administration instructions.

Methods of Treatment

Disclosed herein are uses of a polypeptide, such as any described herein, in the preparation of a medicament for the treatment of a disease in a subject. In some embodiments, the particular disease to be treated will depend on the choice of the biologically active proteins. In some embodiments, the disease is cancer (including any form thereof). Included herein are paTCE polypeptides for use in the treatment of cancer. In some cases, the cancer or tumor expresses PSMA. In some embodiments, the cancer or tumor is a solid tumor. In some embodiments, the cancer is a carcinoma. In some embodiments, the carcinoma is a gastric carcinoma. In some embodiments, the carcinoma is a colorectal adenocarcinoma. In some embodiment, the carcinoma is a colon carcinoma

The present disclosure includes a method of treating a disease in a subject, the method comprising administering to the subject in need thereof a therapeutically effective amount of the pharmaceutical composition, such as any described herein. In some embodiments, the disease is cancer. In some embodiments, the subject is a mouse, rat, monkey, or human. In some embodiments, the subject is a human.

In some embodiments, the cancer is prostate cancer. In some embodiments, the prostate cancer is metastatic prostate cancer. In some embodiments, the prostate cancer is androgen-independent. In some embodiments, the prostate cancer is non-metastatic castration-resistant prostate cancer (nmCRPC). In some embodiments, the prostate cancer is metastatic castration-resistant prostate cancer (mCRPC).

In some embodiments, a PSMA-targeted bispecific composition of the present disclosure (such as a paTCE) may be combined with a checkpoint inhibitors. In some embodiments of such combination therapy, a paTCE can be combined with an antagonist of the cell surface receptor programmed cell death protein 1, also known as PD-1, and/or an antagonist of PD-L1.

PD-1 plays an important role in down-regulating the immune system and promoting self-tolerance by suppressing T cell inflammatory activity. Binding of the PD-1 ligands, PD-L1 and PD-L2 to the PD-1 receptor found in T cells inhibits T-cell proliferation and cytokine production. Upregulation of PD-1 ligands occurs in some tumors and signaling through this pathway can contribute to inhibition of active T-cell immune surveillance of tumors. Anti-PD-1 antibodies bind to the PD-1 receptor and block its interaction with PD-L1 and PD-L3, releasing PD-1 pathway-mediated inhibition of the immune response, including the anti-tumor immune response.

Those of skill in the art are aware of various anti-PD-1 antibodies that may be used. In some embodiments, an exemplary anti-PD-1 antibody used in combination with the compounds of the present invention is Pembrolizumab (Keytruda®). In some embodiments, the anti-PD-1 antibody used in combination with the compound described above is Nivolumab (Opdivo®). In some embodiments, the anti-PD-1 antibody used in combination with the compound described above is Pidilizumab (Medivation).

Additional PD-1 antibodies known to those of skill in the art, include AGEN-2034 (Agenus), AMP-224 (Medimmune), BCD-100 (Biocad), BGBA-317 (Beigene), BI-754091 (Boehringer Ingelheim), CBT-501 (Genor Biopharma), CC-90006 (Celgene), cemiplimab (Regeneron Pharmaceuticals), durvalumab+MEDI-0680 (Medimmune), GLS-010 (Harbin Gloria Pharmaceuticals), IBI-308 (Eli Lilly), JNJ-3283 (Johnson & Johnson), JS-001 (Shanghai Junshi Bioscience Co.), MEDI-0680 (Medimmune), MGA-012 (MacroGenics), MGD-013 (Marcogenics), pazopanib hydrochloride+pembrolizumab (Novartis), PDR-001 (Novartis), PF-06801591 (Pfizer), SHR-1210 (Jiangsu Hengrui Medicine Co.), TSR-042 (Tesaro Inc.), LZM-009 (Livzon Pharmaceutical Group Inc) and ABBV-181 (AbbVie Inc).

In some embodiments for combination therapy of the present disclosure, the anti-PD-1 antibody is pembrolizumab (Keytruda®).

In some embodiments, the compositions of the present invention are combined with an anti-PD-L1 antibody. Exemplary such anti-PD-L1 antibodies used in the combinations of the present invention may be selected from the group consisting of Durvalumab (MedImmune LLC), Atezolizumab (Hoffmann-La Roche Ltd, Chugai Pharmaceutical Co Ltd), Avelumab (Merck KGaA), CX-072 (CytomX Therapeutics Inc), BMS-936559 (ViiV Healthcare Ltd), SHR-1316 (Jiangsu Hengrui Medicine Co Ltd), M-7824 (Merck KGaA), LY-3300054 (Eli Lilly and Co), FAZ-053 (Novartis AG), KN-035 (AlphaMab Co Ltd), CA-170 (Curis Inc), CK-301 (TG Therapeutics Inc), CS-1001 (CStone Pharmaceuticals Co Ltd), HLX-10 (Shanghai Henlius Biotech Co Ltd), MCLA-145 (Merus NV), MSB-2311 (MabSpace Biosciences (Suzhou) Co Ltd) and MEDI-4736 (Medimmune).

Other immunotherapies and checkpoint inhibitor-based therapies that may be useful in combination with the compositions of the present disclosure include CTLA4, TIGIT, OX40, and TIM3-based therapies.

In some embodiments, the disclosure provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject an amount of the paTCE described herein to the subject, and a checkpoint inhibitor to the subject, wherein the cancer comprises a solid tumor, and treating the cancer comprises reducing the volume of the solid tumor.

EXEMPLARY EMBODIMENTS

Disclosed herein further provides below non-limiting exemplary embodiments:

- 1. A chimeric polypeptide comprising a bispecific antibody domain, wherein the bispecific antibody domain comprises a first antigen binding domain that specifically binds to prostate-specific membrane antigen (PSMA) and a second antigen binding domain that binds to cluster of differentiation 3 T cell receptor (CD3),
  - wherein
  - the first antigen binding domain is a VHH; or
  - the second antigen binding domain is a Fab or an scFV, and
  - wherein the chimeric polypeptide further comprises a mask polypeptide joined to the bispecific antibody domain via a linker comprising a protease-cleavable release segment positioned between the mask polypeptide and the bispecific antibody domain such that the mask polypeptide is capable of reducing the binding of the bispecific antibody domain to CD3 or PSMA, and wherein the protease-cleavable release segment is cleavable by at least one protease that is present in a tumor.
- 2. A chimeric polypeptide comprising a bispecific antibody domain,
  - wherein the bispecific antibody domain comprises a first antigen binding domain that specifically binds to prostate-specific membrane antigen (PSMA) and a second antigen binding domain that binds to cluster of differentiation 3 T cell receptor (CD3),
  - wherein the chimeric polypeptide further comprises a mask polypeptide joined to the bispecific antibody domain via a linker comprising a protease-cleavable release segment positioned between the mask polypeptide and the bispecific antibody domain such that the mask polypeptide is capable of reducing the binding of the bispecific antibody domain to CD3 or PSMA, wherein the protease-cleavable release segment is not capable of being cleaved by legumain in human plasma, or wherein legumain cleaves the protease-cleavable release segment in human plasma at a rate that is less than about 25% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO: 7048) is cleaved by legumain.
- 3. The chimeric polypeptide of embodiment 1 or 2, which comprises a structural arrangement from the N-terminal side to the C-terminal side defined as: (first antigen binding domain)-(second antigen binding domain)-(linker)-(mask polypeptide), (second antigen binding domain)-(first antigen binding domain)-(linker)-(mask polypeptide), (mask polypeptide)-(linker)-(first antigen binding domain)-(second antigen binding domain), or (mask polypeptide)-(linker)-(second antigen binding domain)-(first antigen binding domain), wherein each - is a covalent connection or a polypeptide linker.
- 4. The chimeric polypeptide of any one of the above embodiments, wherein the mask polypeptide is an extended length non-natural polypeptide (ELNN).
- 5. The chimeric polypeptide of any one of the above embodiments, wherein the linker further comprises a spacer.
- 6. The chimeric polypeptide of any one of the above embodiments, wherein the protease-cleavable release segment is fused to the bispecific antibody domain via the spacer.
- 7. The chimeric polypeptide of embodiment 5 or 6 wherein the spacer is characterized in that:
- (i) at least 90% of its amino acids are glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), proline (P), or any combination thereof, and
- (ii) it comprises at least 3 types of amino acids selected from the group consisting of G, A, S, T, E, and P.
- 8. The chimeric polypeptide of any one of embodiments 5 to 7, wherein the spacer is from 9 to 14 amino acids in length.
- 9. The chimeric polypeptide of any one of embodiments 5 to 8, wherein the spacer comprises at least 4 types of amino acids selected from the group consisting of G, A, S, T, E, and P.
- 10. The chimeric polypeptide of any one of embodiments 5 to 9, wherein the amino acids of the spacer consist of A, E, G, S, P, and/or T.
- 11. The chimeric polypeptide of any one of embodiments 5 to 10, wherein the spacer is cleavable by a non-mammalian protease.
- 12. The chimeric polypeptide of embodiment 11, wherein the non-mammalian protease is Glu-C.
- 13. The chimeric polypeptide of any one of embodiments 5 to 12, wherein the spacer comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to a sequence listed in Table C.
- 14. The chimeric polypeptide of any one of embodiments 5 to 13, wherein the spacer comprises an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to

(SEQ ID NO: 96)

GTSESATPES

or

(SEQ ID NO: 97)

GTATPESGPG

15. The chimeric polypeptide of any one of embodiments 1 to 14, wherein the protease-cleavable release segment comprises an amino acid sequence comprising the sequence: EAGRSAXHTPAGLTGP (SEQ ID NO: 7627), wherein X is any amino acid other than N.

- 16. The chimeric polypeptide of embodiment 15, wherein X is S.
- 17. The chimeric polypeptide of embodiment 1 or 2, comprising
  - a first mask polypeptide joined to the first antigen binding domain via a first linker wherein the first linker comprises a first protease cleavable release segment (RS1) cleavable by at least one protease present in a tumor, and
  - a second mask polypeptide joined to the second antigen binding domain via a second linker wherein the second linker comprises a second protease cleavable release segment (RS2) cleavable by at least one protease present in a tumor.
- 18. The chimeric polypeptide of embodiment 17, which comprises a structural arrangement from the N-terminal side to the C-terminal side defined as: (Mask1)-(Linker1)-(first antigen binding domain)-(second antigen binding domain)-(Linker2)-(Mask2), (Mask1)-(Linker1)-(second antigen binding domain)-(first antigen binding domain)-(Linker2)-(Mask2), (Mask2)-(Linker2)-(first antigen binding domain)-(second antigen binding domain)-(Linker1)-(Mask1), or (Mask2)-(Linker2)-(second antigen binding domain)-(first antigen binding domain)-(Linker1)-(Mask1), wherein each - is, individually, a covalent bond or a polypeptide linker.
- 19. The chimeric polypeptide of embodiment 17 or 18, wherein the first mask polypeptide is a first ELNN (ELNN1) and the second mask polypeptide is a second ELNN (ELNN2).
- 20. The chimeric polypeptide of embodiment 19, which comprises a structural arrangement from the N-terminal side to the C-terminal side defined as: (ELNN1)-(Linker1)-(first antigen binding domain)-(second antigen binding domain)-(Linker2)-(ELNN2), (ELNN1)-(Linker1)-(second antigen binding domain)-(first antigen binding domain)-(Linker2)-(ELNN2), (ELNN2)-(Linker2)-(first antigen binding domain)-(second antigen binding domain)-(Linker1)-(ELNN1), or (ELNN2)-(Linker2)-(second antigen binding domain)-(first antigen binding domain)-(Linker1)-(ELNN1), wherein each - is, individually, a covalent bond or a polypeptide linker.
- 21. The chimeric polypeptide of any one of embodiments 17-20, wherein Linker1 further comprises a first spacer (Spacer1).
- 22. The chimeric polypeptide of any one of embodiments 17-21, wherein Linker2 further comprises a second spacer (Spacer2).
- 23. The chimeric polypeptide of embodiment 21 or 22, wherein RS1 is fused to the bispecific antibody domain via Spacer1 and/or RS2 is fused to the bispecific antibody domain via Spacer2.
- 24. The chimeric polypeptide of embodiment 23, which comprises a structural arrangement from the N-terminal side to the C-terminal side defined as: (ELNN1)-(RS1)-(Spacer1)-(first antigen binding domain)-(second antigen binding domain)-(Spacer2)-(RS2)-(ELNN2), (ELNN1)-(RS1)-(Spacer1)-(second antigen binding domain)-(first antigen binding domain)-(Spacer2)-(RS2)-(ELNN2), (ELNN2)-(RS2)-(Spacer2)-(first antigen binding domain)-(second antigen binding domain)-(Spacer1)-(RS1)-(ELNN1), or (ELNN2)-(RS2)-(Spacer2)-(second antigen binding domain)-(first antigen binding domain)-(Spacer1)-(RS1)-(ELNN1), wherein each - is a, individually, covalent bond or a polypeptide linker.
- 25. The chimeric polypeptide of any one of embodiments 21-24 wherein Spacer1 and/or the Spacer2 is characterized in that:
  - (1) at least 90% of its amino acids are glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), proline (P), or any combination thereof, and
  - (ii) it comprises at least 3 types of amino acids selected from the group consisting of G, A, S, T, E, and P.
- 26. The chimeric polypeptide of any one of embodiments 21-25, wherein Spacer1 and/or the Spacer2 is from 9 to 14 amino acids in length.
- 27. The chimeric polypeptide of any one of embodiments 21-26, wherein Spacer1 and/or the Spacer2 comprises at least 4 types of amino acids selected from the group consisting of G, A, S, T, E, and P.
- 28. The chimeric polypeptide of any one of embodiments 21-27, wherein the amino acids of Spacer1 and/or the Spacer2 consists of A, E, G, S, P, and/or T.
- 29. The chimeric polypeptide of any one of embodiments 21-28, wherein Spacer1 and/or the Spacer2 comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to a sequence listed in Table C.
- 30. The chimeric polypeptide of any one of embodiments 21-29, wherein Spacer1 and/or the Spacer2 comprises an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to

(SEQ ID NO: 96)

GTSESATPES

or

(SEQ ID NO: 97)

GTATPESGPG.

- 31. The chimeric polypeptide of any one of embodiments 19-30, wherein the amino acid sequence of the first ELNN is between 250 amino acids and 350 amino acids in length, and wherein the amino acid sequence of the second ELNN is between 500 amino acids and 600 amino acids in length.
- 32. The chimeric polypeptide of any one of embodiments 19-31, wherein the amino acid sequence of the first ELNN is 294 amino acids in length, and wherein the amino acid sequence of the second ELNN is 582 amino acids in length.
- 33. The chimeric polypeptide of any one of embodiments 17-32, wherein RS1 and/or RS2 comprises an amino acid sequence comprising the sequence: EAGRSAXHTPAGLTGP (SEQ ID NO: 7627), wherein X is any amino acid other than N.
- 34. The chimeric polypeptide of embodiment 33, wherein X is S.
- 35. A chimeric polypeptide comprising a bispecific antibody domain,
- wherein the bispecific antibody domain comprises a first antigen binding domain that has binding specificity to a cancer cell antigen, and a second antigen binding domain that has binding specificity to an effector cell antigen expressed on an effector cell, wherein the chimeric polypeptide further comprises a first ELNN joined to the first antigen binding domain via a first linker comprising a first protease-cleavable release segment (RS1) positioned between the first ELNN and the first antigen binding domain such that the first ELNN is capable of reducing the binding of the first antigen binding domain to the cancer cell antigen, wherein the RS1 is cleavable by at least one protease that is present in a tumor,
- wherein the chimeric polypeptide further comprises a second ELNN joined to the second antigen binding domain via a second linker comprising second protease-cleavable release segment (RS2) positioned between the second ELNN and the second antigen binding domain such that the second ELNN is capable of reducing the binding of the first antigen binding domain to the effector cell antigen, wherein the RS2 is cleavable by at least one protease that is present in a tumor,
- wherein the first ELNN has a shorter amino acid sequence than the second ELNN, and
- wherein the cancer cell antigen is not HER2.
- 36. The chimeric polypeptide of embodiment 35, which comprises a structural arrangement from the N-terminal side to the C-terminal side defined as: (ELNN1)-(Linker1)-(first antigen binding domain)-(second antigen binding domain)-(Linker2)-(ELNN2), (ELNN1)-(Linker1)-(second antigen binding domain)-(first antigen binding domain)-(Linker2)-(ELNN2), (ELNN2)-(Linker2)-(first antigen binding domain)-(second antigen binding domain)-(Linker1)-(ELNN1), or (ELNN2)-(Linker2)-(second antigen binding domain)-(first antigen binding domain)-(Linker1)-(ELNN1), wherein each - is, individually, a covalent bond or a polypeptide linker.
- 37. The chimeric polypeptide of any one of embodiments 3-36, wherein each - is a covalent bond.
- 38. The chimeric polypeptide of any one of embodiments 3-37, wherein each - is a peptide bond.
- 39. The chimeric polypeptide of any one of embodiments 36-38, wherein Linker1 further comprises a first spacer (Spacer1).
- 40. The chimeric polypeptide of any one of embodiments 36-38, wherein Linker2 further comprises a second spacer (Spacer2).
- 41. The chimeric polypeptide of embodiment 39 or 40, wherein RS1 is fused to the bispecific antibody domain via Spacer1 and/or RS2 is fused to the bispecific antibody domain via Spacer2.
- 42. The chimeric polypeptide of embodiment 41, which comprises a structural arrangement from the N-terminal side to the C-terminal side defined as: (ELNN1)-(RS1)-(Spacer1)-(first antigen binding domain)-(second antigen binding domain)-(Spacer2)-(RS2)-(ELNN2), (ELNN1)-(RS1)-(Spacer1)-(second antigen binding domain)-(first antigen binding domain)-(Spacer2)-(RS2)-(ELNN2), (ELNN2)-(RS2)-(Spacer2)-(first antigen binding domain)-(second antigen binding domain)-(Spacer1)-(RS1)-(ELNN1), or (ELNN2)-(RS2)-(Spacer2)-(second antigen binding domain)-(first antigen binding domain)-(Spacer1)-(RS1)-(ELNN1), wherein each - is a, individually, covalent bond or a polypeptide linker.
- 43. The chimeric polypeptide of embodiment 42, wherein each - is a covalent bond.
- 44. The chimeric polypeptide of embodiment 42, wherein each - is a peptide bond.
- 45. The chimeric polypeptide of any one of embodiments 1-44, further comprising an antibody domain linker between the first antigen binding domain and the second antigen binding domain.
- 46. A chimeric polypeptide comprising a bispecific antibody domain, comprising the formulas that comprises from the N-terminal side to the C-terminal side:
  - Formula 1: (Mask1)-(RS1)-(Spacer1)-(first antigen binding domain)-[antibody domain linker]-(second antigen binding domain);
  - Formula 2: (first antigen binding domain)-[antibody domain linker]-(second antigen binding domain)-(Spacer2)-(RS2)-(Mask2); or
  - Formula 3: (Mask1)-(RS1)-(Spacer1)-(first antigen binding domain)-[antibody domain linker]-(second antigen binding domain)-(Spacer2)-(RS2)-(Mask2),
  - wherein,
  - the first antigen binding domain has binding specificity to a cancer cell antigen;
  - the second antigen binding domain has binding specificity to an effector cell antigen expressed on an effector cell;
  - each - comprises, individually, a covalent connection or a polypeptide linker;
  - the Mask1 is a polypeptide that is capable of reducing binding of the first antigen binding domain to its target;
  - the Mask2 is a polypeptide that is capable of reducing binding of the second antigen binding domain to its target;
  - if the chimeric polypeptide comprises Formula 1 then the Spacer1 consists of A, E, G, S, P, and/or T residues, if the chimeric polypeptide comprises Formula 2 then the Spacer2 consists of A, E, G, S, P, and/or T residues, and if the chimeric polypeptide comprises Formula 3 then the Spacer1 and/or the Spacer2 consists of A, E, G, S, P, and/or T residues; and
  - wherein the cancer cell antigen is not HER2.
- 47. The chimeric polypeptide of any one of embodiments 3-46, wherein each - is, individually, a covalent connection.
- 48. The chimeric polypeptide of embodiment 47, wherein each - is, individually, a covalent bond.
- 49. The method of embodiment 47, wherein each - is a peptide bond.
- 50. The chimeric polypeptide of embodiment 29, wherein each - is, individually, a polypeptide linker of no more than 5 amino acids.
- 51. The chimeric polypeptide of any one of embodiments 35-50, wherein the cancer cell antigen is human alpha 4 integrin, Ang2, B7-H3, B7-H6, CEACAM5, cMET, CTLA4, FOLR1, EpCAM, CCR5, CD19, HER3, HER4, PD-L1, prostate-specific membrane antigen (PSMA), CEA, MUC1 (mucin), MUC-2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, MUC16 PhCG, Lewis-Y, CD20, CD33, CD38, CD30, CD56 (NCAM), CD133, ganglioside GD3; 9-O-Acetyl-GD3, GM2, Globo H, fucosyl GM1, GD2, carbonicanhydrase IX, CD44v6, Sonic Hedgehog (Shh), Wue-1, plasma cell antigen 1, melanoma chondroitin sulfate proteoglycan (MCSP), CCR8, 6-transmembrane epithelial antigen of prostate (STEAP), mesothelin, A33 antigen, prostate stem cell antigen (PSCA), Ly-6, desmoglein 4, fetal acetylcholine receptor (fnAChR), CD25, cancer antigen 19-9 (CA19-9), cancer antigen 125 (CA-125), Muellerian inhibitory substance receptor type II (MISIIR), sialylated Tn antigen (sTN), fibroblast activation antigen (FAP), endosialin (CD248), tumor-associated antigen L6 (TAL6), SAS, CD63, TAG72, Thomsen-Friedenreich antigen (TF-antigen), insulin-like growth factor I receptor (IGF-IR), Cora antigen, CD7, CD22, CD70, CD79a, CD79b, G250, MT-MMPs, F19 antigen, CA19-9, CA-125, alpha-fetoprotein (AFP), VEGFR1, VEGFR2, DLK1, SP17, ROR1, or EphA2.
- 52. The chimeric polypeptide of any one of embodiments 35-51, wherein the cancer cell antigen is PSMA.
- 53. The chimeric polypeptide of any one of embodiments 35-52, wherein the effector cell antigen is cluster of differentiation 3 T cell receptor (CD3).
- 54. The chimeric polypeptide of any one of embodiments 1-53, wherein the second antigen binding domain has binding specificity to human CD3 and cynomolgus monkey CD3.
- 55. The chimeric polypeptide of any one of embodiments 1-54, wherein the second antigen binding domain has binding specificity to human CD3.
- 56. The chimeric polypeptide of any one of embodiments 53-55, wherein the CD3 is CD3 epsilon, CD3 delta, CD3 gamma, or CD3 zeta.
- 57. The chimeric polypeptide of embodiment 56, wherein the effector cell antigen is CD3 epsilon.
- 58. The chimeric polypeptide of any one of embodiments 46-57, wherein the Mask1 is a first ELNN and the Mask2 is a second ELNN.
- 59. The chimeric polypeptide of any one of embodiments 46-58, wherein the Spacer1 and/or the Spacer2 is characterized in that:
  - (i) at least 90% of its amino acids are glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), proline (P), or any combination thereof, and
  - (ii) it comprises at least 3 types of amino acids selected from the group consisting of G, A, S, T, E, and P.
- 60. The chimeric polypeptide of embodiment 59, wherein the Spacer1 and/or the Spacer2 is from 9 to 14 amino acids in length.
- 61. The chimeric polypeptide of embodiment 59 or 60, wherein the Spacer1 and/or the Spacer2 comprises at least 4 types of amino acids selected from the group consisting of G, A, S, T, E, and P.
- 62. The chimeric polypeptide of any one of embodiments 59-61, wherein the amino acids of the Spacer1 and/or the Spacer2 consists of A, E, G, S, P, and/or T.
- 63. The chimeric polypeptide of any one of embodiments 59-62, wherein the Spacer1 and/or the Spacer2 is cleavable by a non-mammalian protease.
- 64. The chimeric polypeptide of embodiment 63, wherein the non-mammalian protease is Glu-C.
- 65. The chimeric polypeptide of any one of embodiments 59-64, wherein the Spacer1 and/or the Spacer 2 comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to a sequence listed in Table C.
- 66. The chimeric polypeptide of any one of embodiments 59-65, wherein the Spacer1 and/or the Spacer 2 comprises an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GTSESATPES (SEQ ID NO: 96) or GTATPESGPG (SEQ ID NO: 97).
- 67. The chimeric polypeptide of any one of embodiments 35-66, wherein the amino acid sequence of the first ELNN is at least 100 amino acids shorter than the amino acid sequence of the second ELNN.
- 68. The chimeric polypeptide of embodiment 67, wherein the amino acid sequence of the first ELNN is at least 200 amino acids shorter than the amino acid sequence of the second ELNN.
- 69. The chimeric polypeptide of embodiment 67 or 68, wherein the amino acid sequence of the first ELNN is at least 250 amino acids shorter than the amino acid sequence of the second ELNN.
- 70. The chimeric polypeptide of any one of embodiments 35-69, wherein the amino acid sequence of the first ELNN is about 294 amino acids in length, and wherein the amino acid sequence of the second ELNN is about 582 amino acids in length.
- 71. The chimeric polypeptide of any one of embodiments 1-70, wherein the first antigen binding domain comprises a first antibody or an antigen-binding fragment thereof, and wherein the second antigen binding domain is a second antibody or an antigen-binding fragment thereof.
- 72. The chimeric polypeptide of any one of embodiments 1-71, wherein the first antigen binding domain is a Fab, an scFV, or an ISVD.
- 73. The chimeric polypeptide of embodiment 72, wherein the ISVD is a VHH domain.
- 74. The chimeric polypeptide of any one of embodiments 1-73, wherein the second antigen binding domain is a Fab, an scFV, or an ISVD.
- 75. The chimeric polypeptide of embodiment 74, wherein the ISVD is a VHH domain.
- 76. The chimeric polypeptide of any one of embodiments 1-75, wherein the first antigen binding domain is a VHH domain.
- 77. The chimeric polypeptide of any one of embodiments 1-76, wherein the second antigen binding domain is an scFV.
- 78. The chimeric polypeptide of any one of embodiments 1-77, wherein there is an antibody domain linker between the first antigen binding domain and the second antigen binding domain.
- 79. The chimeric polypeptide of embodiment 78, wherein the antibody domain linker comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to a sequence listed in Table A or B.
- 80. The chimeric polypeptide of embodiment 78, wherein the antibody domain linker consists of G and S amino residues.
- 81. The chimeric polypeptide of embodiment 78 or 79, wherein the antibody domain linker is about 9 residues in length.
- 82. The chimeric polypeptide of embodiment 80 or 81, wherein the antibody domain linker comprises the amino acid sequence GGGGSGGGS (SEQ ID NO: 125).
- 83. The chimeric polypeptide of any one of embodiments 1-82, wherein the scFv comprises a VL domain, a VH domain, and a linker between the VL domain and the VH domain, wherein the linker consists of A, E, G, S, P, and/or T residues.
- 84. The chimeric polypeptide of embodiment 83, wherein the linker is characterized in that:
  - (i) at least 90% of its amino acids are glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), proline (P), or any combination thereof, and
  - (ii) it comprises at least 3 types of amino acids selected from the group consisting of G, A, S, T, E, and P.
- 85. The chimeric polypeptide of embodiment 83 or 84, wherein the linker between the VL domain and the VH domain is from 25 to 35 amino acids in length.
- 86. The chimeric polypeptide of any one of embodiments 83-85, wherein the linker between the VL domain and the VH domain comprises at least 4 types of amino acids selected from the group consisting of G, A, S, T, E, and P.
- 87. The chimeric polypeptide of any one of embodiments 83-86, wherein the amino acids of the linker between the VL domain and the VH domain consists of A, E, G, S, P, and/or T.
- 88. The chimeric polypeptide of any one of embodiments 83-87, wherein the linker between the VL domain and the VH domain is cleavable by a non-mammalian protease.
- 89. The chimeric polypeptide of embodiment 88, wherein the non-mammalian protease is Glu-C.
- 90. The chimeric polypeptide of embodiment 89, wherein linker between the VL domain and the VH domain comprises an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81).
- 91. The chimeric polypeptide of any one of embodiments 1-90, wherein the first antigen binding domain comprises a VHH domain comprising three VHH complementarity determining regions (CDRs), wherein the three VHH CDRs comprise the CDR1, CDR2, and CDR3 of a VHH domain comprising the following amino acid sequence:

(SEQ ID NO: 549)

QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVG

AMSWSGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNKEYGRTWYDFNESDYWGQGTQVTVSS.

- 92. The chimeric polypeptide of any one of embodiments 1-91, wherein the second antigen binding domain comprises a VL domain comprising three the VL CDRs, wherein the three VL CDRs comprise the CDR1, CDR2, and CDR3 of a VL domain comprising the following amino acid sequence: ELVVTQEPSLTVSPGGTVTLTCRSSX₁GAVTX₂SNYANWVQQKPGQAPRGLIGGTN KRAPGTPARFSGSLLGGKAALTLSGVQPEDEAX₃YYCALWYX₄NLWVFGGGTKLTV L(SEQ ID NO:9001), wherein X₁corresponds to T or N, X₂corresponds to T or S, X₃corresponds to E or V, and X₄corresponds to S or P.
- 93. The chimeric polypeptide of any one of embodiments 1-92, wherein the second antigen binding domain comprises a VL domain comprising three the VL CDRs, wherein the three VL CDRs comprise the CDR1, CDR2, and CDR3 of a VL domain comprising the following amino acid sequence:

(SEQ ID NO: 361)

ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGL

IGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLW

VFGGGTKLTVL.

- 94. The chimeric polypeptide of any one of embodiments 1-91, wherein the second antigen binding domain comprises a VH domain comprising three the VH CDRs, wherein the three VH CDRs comprise the CDR1, CDR2, and CDR3 of a VH domain comprising the following amino acid sequence: EVQLX₅ESGGGX₆VQPGGSLX₇LSCAASGFTFX₈TYAMNWVRQAPGKGLEWVXsRI RX₁₀KX₁₁NNYATYYADSVKX₁₂RFTISRDDSKNTX₁₃YLQMNX₁₄LKTEDTAVYYCVRH X₁₅NFGNSYVSWFAX₁₆WGQGTLVTVSS(SEQ ID NO:9002), wherein X₅corresponds to V or L, X₆corresponds to I or L, X₇corresponds to R or K, X₈corresponds to S or N, X₉corresponds to G or A, X₁₀corresponds to T or S, X₁₁corresponds to R or Y, X₁₂corresponds to G or D, X₁₃corresponds to V or A, X₁₄corresponds to S or N, X₁₅corresponds to E or G, and X₁₆corresponds to H or Y.
- 95. The chimeric polypeptide of any one of embodiments 1-91, wherein the second antigen binding domain comprises a VH domain comprising three the VH CDRs, wherein the three VH CDRs comprise the CDR1, CDR2, and CDR3 of a VH domain comprising the following amino acid sequence:

(SEQ ID NO: 311)

EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVG

RIRTKRNNYATYYADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYC

VRHENFGNSYVSWFAHWGQGTLVTVSS.

- 96. The chimeric polypeptide of any one of embodiments 1-91, wherein the second antigen binding domain comprises a VL domain amino acid sequence SEQ ID NO/VH domain amino acid sequence SEQ ID NO pair selected from the group consisting of: 896/897; 902/903; 700/701; 702/703; 716/717; 718/719; 728/729; 736/737; 738/739; 740/741; 742/743; 744/745; 746/747; 748/749; 750/751; 752/753; 754/755; 756/757; 758/759; 760/761; 762/763; 764/765; 766/767; 774/775; 776/777; 790/791; 792/793; 798/799; 800/801; 806/807; 808/809; 814/815; 816/817; 822/823; 824/825; or 826/867.
- 97. The chimeric polypeptide of any one of embodiments 1-96, wherein
- (i) the first antigen binding domain is a VHH comprising the following CDRs:
  - a VHH CDR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GRTFGIYVWG(SEQ ID NO:9003);
  - a VHH CDR2 with an amino acid sequence that that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to AMSWSGSNRKVSDSVKG(SEQ ID NO:9004); and
  - a VHH CDR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to AASNKEYGRTWYDFNESDY(SEQ ID NO:9005), and
- (ii) and wherein the second antigen binding domain comprises the following CDRs:
  - a VL domain CDR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to RSSX₁GAVTX₂SNYAN(SEQ ID NO:9006), wherein X₁corresponds to T or N, and X₂corresponds to T or S;
  - a VL domain CDR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GTNKRAP(SEQ ID NO:4);
  - a VL domain CDR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to ALWYX₄NLWV(SEQ ID NO:9007), wherein X₄corresponds to S or P;
  - a VH domain CDR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GFTFX₈TYAMN(SEQ ID NO:9008), wherein X₈corresponds to S or N;
  - a VH domain CDR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to RIRX₁₀KX₁₁NNYATYYADSVKX₁₂(SEQ ID NO:9009), wherein X₁₀corresponds to T or S, X₁₁corresponds to R or Y, and X₁₂corresponds to G or D;
  - a VH domain CDR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to HX₁₅NFGNSYVSWFAX₁₆(SEQ ID NO:9010), wherein X₁₅corresponds to E or G, and X₁₆corresponds to H or Y.
- 98. The chimeric polypeptide of any one of embodiments 1-97, wherein (i) the first antigen binding domain is a VHH comprising the following CDRs:
  - a VHH CDR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GRTFGIYVWG(SEQ ID NO:9003);
  - a VHH CDR2 with an amino acid sequence that that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to AMSWSGSNRKVSDSVKG(SEQ ID NO:9004); and
  - a VHH CDR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to AASNKEYGRTWYDFNESDY(SEQ ID NO:9005), and
- (ii) and wherein the second antigen binding domain comprises the following CDRs:
  - a VL domain CDR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to RSSNGAVTSSNYAN(SEQ ID NO:1);
  - a VL domain CDR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GTNKRAP(SEQ ID NO:4);
  - a VL domain CDR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to ALWYPNLWV(SEQ ID NO:6);
  - a VH domain CDR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GFTFSTYAMN(SEQ ID NO:12);
  - a VH domain CDR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to RIRTKRNNYATYYADSVKG(SEQ ID NO:13); and
  - a VH domain CDR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to HENFGNSYVSWFAH(SEQ ID NO:10).
- 99. The chimeric polypeptide of embodiment 97 or 98, wherein the VHH comprises the following framework regions (FRs):
  - a VHH FR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to QVQLVESGGGVVQPGRSLRLSCAAS(SEQ ID NO:9011);
  - a VHH FR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to WFRQAPGKEREFVG(SEQ ID NO:9012);
  - a VHH FR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to RFTISRDNSKNTLYLQMNSLRAEDTAVYYC(SEQ ID NO:9013); and
  - a VHH FR4 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to WGQGTQVTVSS(SEQ ID NO:9014).
- 100. The chimeric polypeptide of any one of embodiments 97-99, wherein the second antigen binding domain comprises the following FRs:
  - a VL domain FR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to ELVVTQEPSLTVSPGGTVTLTC(SEQ ID NO:51);
  - a VL domain FR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to WVQQKPGQAPRGLIG(SEQ ID NO:52);
  - a VL domain FR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GTPARFSGSLLGGKAALTLSGVQPEDEAVYYC(SEQ ID NO:53);
  - a VL domain FR4 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to FGGGTKLTVL(SEQ ID NO:59);
  - a VH domain FR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to EVQLVESGGGIVQPGGSLRLSCAAS(SEQ ID NO:400);
  - a VH domain FR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to WVRQAPGKGLEWVG(SEQ ID NO:401);
  - a VH domain FR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to RFTISRDDSKNTVYLQMNSLKTEDTAVYYCVR(SEQ ID NO:402); and
  - a VH domain FR4 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to WGQGTLVTVSS(SEQ ID NO:67).
- 101. The chimeric polypeptide of any one of embodiments 1-100, wherein
- (i) the first antigen binding domain is a VHH comprising the following CDRs:
  - a VHH CDR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GRTFGIYVWG(SEQ ID NO:9003);
  - a VHH CDR2 with an amino acid sequence that that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to AMSWSGSNRK(SEQ ID NO:9015); and
  - a VHH CDR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to AASNKEYGRTWYDFNESDY(SEQ ID NO:9005), and
- (ii) and wherein the second antigen binding domain comprises the following CDRs:
  - a VL domain CDR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to RSSX₁GAVTX₂SNYAN(SEQ ID NO:9006), wherein X₁corresponds to T or N, and X₂corresponds to T or S;
  - a VL domain CDR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GTNKRAP(SEQ ID NO:4);
  - a VL domain CDR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to ALWYX₄NLWV(SEQ ID NO:9007), wherein X₄corresponds to S or P;
  - a VH domain CDR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GFTFX₈TYAMN(SEQ ID NO:9008), wherein X₈corresponds to S or N;
  - a VH domain CDR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to RIRX₁₀KX₁₁NNYATYYADSVKX₁₂(SEQ ID NO:9009), wherein X₁₀corresponds to T or S, X₁₁corresponds to R or Y, and X₁₂corresponds to G or D;
  - a VH domain CDR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to HX₁₅NFGNSYVSWFAX₁₆(SEQ ID NO:9010), wherein X₁₅corresponds to E or G, and X₁₆corresponds to H or Y.
- 102. The chimeric polypeptide of any one of embodiments 1-101, wherein
- (i) the first antigen binding domain is a VHH comprising the following CDRs:
  - a VHH CDR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GRTFGIYVWG(SEQ ID NO:9003);
  - a VHH CDR2 with an amino acid sequence that that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to AMSWSGSNRK(SEQ ID NO:9015); and
  - a VHH CDR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to AASNKEYGRTWYDFNESDY(SEQ ID NO:9005), and
- (ii) and wherein the second antigen binding domain comprises the following CDRs:
  - a VL domain CDR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to RSSNGAVTSSNYAN(SEQ ID NO:1);
  - a VL domain CDR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GTNKRAP(SEQ ID NO:4);
  - a VL domain CDR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to ALWYPNLWV(SEQ ID NO:6);
  - a VH domain CDR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GFTFSTYAMN(SEQ ID NO:12);
  - a VH domain CDR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to RIRTKRNNYATYYADSVKG(SEQ ID NO:13); and
  - a VH domain CDR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to HENFGNSYVSWFAH(SEQ ID NO:10).
- 103. The chimeric polypeptide of embodiment 101 or 102, wherein the VHH comprises the following framework regions (FRs):
  - a VHH FR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to QVQLVESGGGVVQPGRSLRLSCAAS(SEQ ID NO:9011);
  - a VHH FR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to WFRQAPGKEREFVG(SEQ ID NO:9012);
  - a VHH FR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to VSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC(SEQ ID NO:9016); and
  - a VHH FR4 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to WGQGTQVTVSS(SEQ ID NO:9014).
- 104. The chimeric polypeptide of embodiment 101 or 102, wherein the second antigen binding domain comprises the following FRs:
  - a VL domain FR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to ELVVTQEPSLTVSPGGTVTLTC(SEQ ID NO:51);
  - a VL domain FR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to WVQQKPGQAPRGLIG(SEQ ID NO:52);
  - a VL domain FR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GTPARFSGSLLGGKAALTLSGVQPEDEAVYYC(SEQ ID NO:53);
  - a VL domain FR4 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to FGGGTKLTVL(SEQ ID NO:59);
  - a VH domain FR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to EVQLVESGGGIVQPGGSLRLSCAAS(SEQ ID NO:400);
  - a VH domain FR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to WVRQAPGKGLEWVG(SEQ ID NO:401);
  - a VH domain FR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to RFTISRDDSKNTVYLQMNSLKTEDTAVYYCVR(SEQ ID NO:402); and
  - a VH domain FR4 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to WGQGTLVTVSS(SEQ ID NO:67).
- 105. The chimeric polypeptide of any one of embodiments 1-91, wherein the second antigen binding domain comprises a VL domain comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to: ELVVTQEPSLTVSPGGTVTLTCRSSX₁GAVTX₂SNYANWVQQKPGQAPRGLIGGTN KRAPGTPARFSGSLLGGKAALTLSGVQPEDEAX₃YYCALWYX₄NLWVFGGGTKLTV L(SEQ ID NO:9001), wherein X₁corresponds to T or N, X₂corresponds to T or S, X₃corresponds to E or V, and X₄corresponds to S or P.
- 106. The chimeric polypeptide of any one of embodiments 1-91, wherein the second antigen binding domain comprises a VL domain comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to:

(SEQ ID NO: 361)

ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGL

IGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLW

VFGGGTKLTVL.

- 107. The chimeric polypeptide of any one of embodiments 1-91, wherein the second antigen binding domain comprises a VH domain comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to: EVQLX₅ESGGGX₆VQPGGSLX₇LSCAASGFTFX₈TYAMNWVRQAPGKGLEWVXsRI RX₁₀KX₁₁NNYATYYADSVKX₁₂RFTISRDDSKNTX₁₃YLQMNX₁₄LKTEDTAVYYCVRH X₁₅NFGNSYVSWFAX₁₆WGQGTLVTVSS(SEQ ID NO:9002), wherein X₅corresponds to V or L, X₆corresponds to I or L, X₇corresponds to R or K, X₈corresponds to S or N, X₉corresponds to G or A, X₁₀corresponds to T or S, X₁₁corresponds to R or Y, X₁₂corresponds to G or D, X₁₃corresponds to V or A, X₁₄corresponds to S or N, X₁₅corresponds to E or G, and X₁₆corresponds to H or Y.
- 108. The chimeric polypeptide of any one of embodiments 1-91, wherein the second antigen binding domain comprises a VH domain comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to:

(SEQ ID NO: 311)

EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVG

RIRTKRNNYATYYADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYC

VRHENFGNSYVSWFAHWGQGTLVTVSS.

- 109. The chimeric polypeptide of any one of embodiments 83-108, wherein the VL domain is N-terminal to the VH domain.
- 110. The chimeric polypeptide of any one of embodiments 83-108, wherein the VL domain is C-terminal to the VH domain.
- 111. The chimeric polypeptide of any one of embodiments 1-91, wherein the second antigen binding domain comprises a scFV comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to:

- 112. The chimeric polypeptide of any one of embodiments 1-91, wherein the first antigen binding domain comprises a VHH comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to the amino acid sequence of PSMA.2, PSMA.3, PSMA.5, PSMA.6, PSMA.262, or PSMA.263.
- 113. The chimeric polypeptide of any one of embodiments 1-91, wherein the first antigen binding domain comprises a VHH comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to: QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVX₁₇GWFRQAPGKEREFVGAX₁₈S WSGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYX₁₉CX₂₀X₂₁SNKX₂₂Y GRTWYDFNESDYWGQGTQVTVSS(SEQ ID NO:9017), wherein X₁₇, X₁₈, X₁₉, X₂₀, X₂₁, and X₆each, individually, correspond to any naturally occurring amino acid.
- 114. The chimeric polypeptide of embodiment 113, wherein X₁₇corresponds to M or W, X₁₈corresponds to M or I, X₁₉corresponds to F or Y, X₂₀corresponds to A or G, X₂₁corresponds to A or G, and/or X₂₂corresponds to L, W, R, D, E, or G.
- 115. The chimeric polypeptide of any one of embodiments 1-91, wherein the first antigen binding domain comprises a VHH comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to:

(SEQ ID NO: 549)

QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVG

AMSWSGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNKEYGRTWYDFNESDYWGQGTQVTVSS.

- 116. The chimeric polypeptide of any one of embodiments 1-115, wherein the RS comprises a protease cleavage site is cleavable by at least one protease listed in Table 7.
- 117. The chimeric polypeptide of any one of embodiments 1-115, wherein the RS comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to a sequence listed in Table 8a.
- 118. The chimeric polypeptide of any one of embodiments 1-117, wherein the RS is cleavable by uPA, ST14, MMP2, MMP7, MMP9, and MMP14.
- 119. The chimeric polypeptide of any one of embodiments 1-118, wherein the RS is not cleavable by legumain.
- 120. The chimeric polypeptide of embodiment 119, wherein the RS is not cleavable by legumain in human blood, plasma, or serum.
- 121. The chimeric polypeptide of embodiment 119 or 120, wherein the RS is not cleavable upon incubation with about 1 nM or less legumain for about 20 hours.
- 122. The chimeric polypeptide of any one of embodiments 119-121, wherein the RS is not cleavable upon incubation with about 1 nM or less legumain for about 20 hours in human blood, plasma, or serum.
- 123. The chimeric polypeptide of embodiment 118, wherein legumain cleaves the RS in human plasma at a rate that is less than about 50% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain.
- 124. The chimeric polypeptide of embodiment 118, wherein legumain cleaves the RS in human plasma at a rate that is less than about 25% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain.
- 125. The chimeric polypeptide of embodiment 118, wherein legumain cleaves the RS in human plasma at a rate that is less than about 10% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain.
- 126. The chimeric polypeptide of embodiment 118, wherein legumain cleaves the RS in human plasma at a rate that is less than about 5% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain.
- 127. The chimeric polypeptide of embodiment 118, wherein legumain cleaves the RS in human plasma at a rate that is less than about 2.5% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain.
- 128. The chimeric polypeptide of any one of embodiments 17-115, wherein the RS1 and/or RS2 comprises protease cleavage is cleavable by at least one protease listed in Table 7.
- 129. The chimeric polypeptide of any one of embodiments 17-115, wherein the RS1 and/or RS2 comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to a sequence listed in Table 8a.
- 130. The chimeric polypeptide of any one of embodiments 17-115, wherein the RS1 and/or RS2 is cleavable by uPA, ST14, MMP2, MMP7, MMP9, and MMP14.
- 131. The chimeric polypeptide of any one of embodiments 17-115, wherein the RS1 and/or RS2 is not cleavable by legumain.
- 132. The chimeric polypeptide of embodiment 131, wherein the RS1 and/or RS2 is not cleavable by legumain in human blood, plasma, or serum.
- 133. The chimeric polypeptide of embodiment 131 or 132, wherein the RS1 and/or RS2 is not cleavable upon incubation with about 1 nM or less legumain for about 20 hours.
- 134. The chimeric polypeptide of embodiment 131 or 132, wherein the RS1 and/or RS2 is not cleavable upon incubation with about 1 nM or less legumain for about 20 hours in human blood, plasma, or serum.
- 135. The chimeric polypeptide of embodiment 130, wherein legumain cleaves the RS1 and/or RS2 in human plasma at a rate that is less than about 50% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain.
- 136. The chimeric polypeptide of embodiment 130, wherein legumain cleaves the RS1 and/or RS2 in human plasma at a rate that is less than about 25% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain.
- 137. The chimeric polypeptide of embodiment 130, wherein legumain cleaves the RS1 and/or RS2 in human plasma at a rate that is less than about 10% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain.
- 138. The chimeric polypeptide of embodiment 130, wherein legumain cleaves the RS1 and/or RS2 in human plasma at a rate that is less than about 5% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain.
- 139. The chimeric polypeptide of embodiment 130, wherein legumain cleaves the RS1 and/or RS2 in human plasma at a rate that is less than about 2.5% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain.
- 140. The chimeric polypeptide of any one of embodiments 17-139, wherein the RS1 comprises a protease-cleavable amino acid sequence comprising the sequence: EAGRSAXHTPAGLTGP (SEQ ID NO: 7627), wherein X is any amino acid other than N.
- 141. The chimeric polypeptide of any one of embodiments 17-140, wherein the RS2 comprises a protease-cleavable amino acid sequence comprising the sequence: EAGRSAXHTPAGLTGP (SEQ ID NO: 7627), wherein X is any amino acid other than N.
- 142. The chimeric polypeptide of any one of embodiments 17-141, wherein RS1 and/or RS2 comprises a protease-cleavable amino acid sequence comprising the sequence: EAGRSASHTPAGLTGP (SEQ ID NO: 7628).
- 143. The chimeric polypeptide of any one of embodiments 17-142, wherein the RS1 and the RS2 are the same.
- 144. The chimeric polypeptide of any one of embodiments 17-142, wherein the RS1 and the RS2 are different.
- 145. The chimeric polypeptide of any one of embodiments 1-144, wherein the mask polypeptide is a first mask polypeptide and the protease-cleavable release segment is a first protease-cleavable release segment (RS1), and wherein the chimeric polypeptide further comprises a second mask polypeptide and a second protease-cleavable release segment (RS2), wherein the second mask polypeptide is joined to the second antigen binding domain via a second protease-cleavable release segment (RS2) positioned between the second mask polypeptide and the second antigen binding domain such that the second mask polypeptide reduces the binding of the first antigen binding domain to CD3, wherein the RS2 is cleavable by at least one protease that is present in a tumor.
- 146. The chimeric polypeptide of any one of embodiments 1-145, wherein the first mask polypeptide is attached to the first antigen binding domain and wherein the second mask polypeptide is attached to the second antigen binding domain.
- 147. The chimeric polypeptide of any one of embodiments 1-146, wherein the first mask polypeptide is a first ELNN and the second mask polypeptide is a second ELNN.
- 148. The chimeric polypeptide of any one of embodiments 1-147, wherein the first ELNN and the second ELNN are each individually characterized in that: (i) at least 90% of each of the first ELNN's and the second ELNN's amino acids are glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), proline (P), or any combination thereof; and (ii) each comprises at least 3 types of amino acids selected from the group consisting of G, A, S, T, E, and P.
- 149. The chimeric polypeptide of embodiment 148, wherein the first ELNN and the second ELNN are each individually further characterized in that: (i) each comprises at least 100 amino acid residues; (ii) each comprises a plurality of non-overlapping sequence motifs that are each from 9 to 14 amino acids in length, wherein the plurality of non-overlapping sequence motifs comprise a set of non-overlapping sequence motives, wherein each non-overlapping sequence motive of the set of non-overlapping sequence motifs is repeated at least two times in the ELNN.
- 150. The chimeric polypeptide of embodiment 149, wherein the plurality of non-overlapping sequence motifs comprises at least one non-overlapping sequence motif that occurs only once within the ELNN.
- 151. The chimeric polypeptide of embodiment 149 or 150, wherein the non-overlapping sequence motifs comprise one of or any combination of the sequence motifs listed in Table 1.
- 152. The chimeric polypeptide of embodiment 149 or 150, wherein the non-overlapping sequence motifs comprise at least 2, 3, or 4 of the sequence motifs listed in Table 1.
- 153. The chimeric polypeptide of embodiment 149 or 150, wherein the non-overlapping sequence motifs comprise any one of or any combination of GTSTEPSEGSAP(SEQ ID NO:189), GTSESATPESGP(SEQ ID NO:188), GSGPGTSESATP(SEQ ID NO:9018), GSEPATSGSETP(SEQ ID NO:187), GSPAGSPTSTEE(SEQ ID NO:186), and GTSPSATPESGP(SEQ ID NO:9019).

(SEQ ID NO: 189)

GTSTEPSEGSAP,

(SEQ ID NO: 188)

GTSESATPESGP,

(SEQ ID NO: 9018)

GSGPGTSESATP,

(SEQ ID NO: 187)

GSEPATSGSETP,

(SEQ ID NO: 186)

GSPAGSPTSTEE,

and

(SEQ ID NO: 9019)

GTSPSATPESGP

- 154. The chimeric polypeptide of any one of embodiments 147-153, wherein each of the first ELNN and the second ELNN comprises at least 4 types of amino acids selected from the group consisting of G, A, S, T, E, and P.
- 155. The chimeric polypeptide of any one of embodiments 147-154, wherein the amino acids of each of the first ELNN and the second ELNN consists of A, E, G, S, P, and/or T.
- 156. The chimeric polypeptide of any one of embodiments 147-155, wherein the amino acid sequence of the first ELNN is at least 100 amino acids shorter than the amino acid sequence of the second ELNN.
- 157. The chimeric polypeptide of any one of embodiments 147-155, wherein the amino acid sequence of the first ELNN is at least 200 amino acids shorter than the amino acid sequence of the second ELNN.
- 158. The chimeric polypeptide of any one of embodiments 147-155, wherein the amino acid sequence of the first ELNN is at least 250 amino acids shorter than the amino acid sequence of the second ELNN.
- 159. The chimeric polypeptide of any one of embodiments 147-155, wherein the amino acid sequence of the first ELNN is about 294 amino acids in length, and wherein the amino acid sequence of the second ELNN is about 582 amino acids in length.
- 160. The chimeric polypeptide of any one of embodiments 147-159, wherein the first ELNN and/or the second ELNN comprises an amino acid sequence that is at least 85% identical to an amino acid sequence listed in Table 3a or 3b.
- 161. The chimeric polypeptide of any one of embodiments 147-160, wherein the first ELNN comprises an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to:

- 162. The chimeric polypeptide of any one of embodiments 147-161, wherein the second ELNN comprises an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to:

- 163. The chimeric polypeptide of any one of embodiments 1-162, comprising one or more barcode fragments.
- 164. The chimeric polypeptide of any one of embodiments 1-163, comprising two or more barcode fragments.
- 165. The chimeric polypeptide of embodiment 163 or 164, wherein each barcode fragment is different from every other barcode fragment.
- 166. The chimeric polypeptide of any one of embodiments 163-165, wherein each barcode fragment differs in both sequence and molecular weight from all other peptide fragments that are releasable from the chimeric polypeptide upon complete digestion the chimeric polypeptide by a non-mammalian protease.
- 167. The chimeric polypeptide of embodiment 166, wherein the non-mammalian protease is Glu-C.
- 168. The chimeric polypeptide of any one of embodiments 1-167, comprising a Glu-C cleavage site comprising one of the following amino acid sequences:

(SEQ ID NO: 9020)

ATPESGPG,

(SEQ ID NO: 9021)

SGSETPGT,

and

(SEQ ID NO: 9022)

GTSESATP

- 169. The chimeric polypeptide of any one of embodiments 1-168, comprising at least one of the following amino acid sequences: SGPE.SGPGX_nSGPE.SGPG(SEQ ID NO:9023), SGPE.SGPGX_nATPE.SGPG(SEQ ID NO:9024), SGPE.SGPGX_nGTSE.SATP(SEQ ID NO:9025), SGPE.SGPGX_nTTPE.SGPG(SEQ ID NO:9026), SGPE.SGPGX_nSTPE.SGPG(SEQ ID NO:9027), SGPE.SGPGX_nGTPE.SGPG(SEQ ID NO:9028), SGPE.SGPGX_nGTPE.TPGS(SEQ ID NO:9029), SGPE.SGPGX_nGTPE.TPGS(SEQ ID NO:9029), SGPE.SGPGX_nSGSE.TGTP(SEQ ID NO:9030), SGPE.SGPGX_nGTPE.GSAP(SEQ ID NO:9031), SGPE.SGPGX_nEPSE.SATP(SEQ ID NO:9032), ATPE.SGPGX_nSGPE.SGPG(SEQ ID NO:9033), ATPE.SGPGX_nATPE.SGPG(SEQ ID NO:9034), ATPE.SGPGX_nGTSE.SATP(SEQ ID NO:9035), ATPE.SGPGX_nTTPE.SGPG(SEQ ID NO:9036), ATPE.SGPGX_nSTPE.SGPG(SEQ ID NO:9037), ATPE.SGPGX_nGTPE.SGPG(SEQ ID NO:9043), ATPE.SGPGX_nGTPE.TPGS(SEQ ID NO:9045), ATPE.SGPGX_nSGSE.TGTP(SEQ ID NO:9046), ATPE.SGPGX_nGTPE.GSAP(SEQ ID NO:9047), ATPE.SGPGX_nEPSE.SATP(SEQ ID NO:9048), GTSE.SATPX_nSGPE.SGPG(SEQ ID NO:9049), GTSE.SATPX_nATPE.SGPG(SEQ ID NO:9050), GTSE.SATPX_nGTSE.SATP(SEQ ID NO:9051), GTSE.SATPX_nTTPE.SGPG(SEQ ID NO:9052), GTSE.SATPX_nSTPE.SGPG(SEQ ID NO:9053), GTSE.SATPX_nGTPE.SGPG(SEQ ID NO:9054), GTSE.SATPX_nGTPE.TPGS(SEQ ID NO:9055), GTSE.SATPX_nSGSE.TGTP(SEQ ID NO:9056), GTSE.SATPX_nGTPE.GSAP(SEQ ID NO:9057), GTSE.SATPX_nEPSE.SATP(SEQ ID NO:9058), TTPE.SGPGX_nSGPE.SGPG(SEQ ID NO:9059), TTPE.SGPGX_nATPE.SGPG(SEQ ID NO:9060), TTPE.SGPGX_nGTSE.SATP(SEQ ID NO:9061), TTPE.SGPGX_nTTPE.SGPG(SEQ ID NO:9062), TTPE.SGPGX_nSTPE.SGPG(SEQ ID NO:9064), TTPE.SGPGX_nGTPE.SGPG(SEQ ID NO:9065), TTPE.SGPGX_nGTPE.TPGS(SEQ ID NO:9066), TTPE.SGPGX_nSGSE.TGTP(SEQ ID NO:9067), TTPE.SGPGX_nGTPE.GSAP(SEQ ID NO:9068), TTPE.SGPGX_nEPSE.SATP(SEQ ID NO:9069), STPE.SGPGX_nSGPE.SGPG(SEQ ID NO:9070), STPE.SGPGX_nATPE.SGPG(SEQ ID NO:9071), STPE.SGPGX_nGTSE.SATP(SEQ ID NO:9072), STPE.SGPGX_nTTPE.SGPG(SEQ ID NO:9073), STPE.SGPGX_nSTPE.SGPG(SEQ ID NO:9074), STPE.SGPGX_nGTPE.SGPG(SEQ ID NO:9076), STPE.SGPGX_nGTPE.TPGS(SEQ ID NO:9077), STPE.SGPGX_nSGSE.TGTP(SEQ ID NO:9078), STPE.SGPGX_nGTPE.GSAP(SEQ ID NO:9079), STPE.SGPGX_nEPSE.SATP(SEQ ID NO:9080), GTPE.SGPGX_nSGPE.SGPG(SEQ ID NO:9081), GTPE.SGPGX_nATPE.SGPG(SEQ ID NO:9082), GTPE.SGPGX_nGTSE.SATP(SEQ ID NO:9083), GTPE.SGPGX_nTTPE.SGPG(SEQ ID NO:9084), GTPE.SGPGX_nSTPE.SGPG(SEQ ID NO:9086), GTPE.SGPGX_nGTPE.SGPG(SEQ ID NO:9088), GTPE.SGPGX_nGTPE.TPGS(SEQ ID NO:9090), GTPE.SGPGX_nSGSE.TGTP(SEQ ID NO:9091), GTPE.SGPGX_nGTPE.GSAP(SEQ ID NO:9092), GTPE.SGPGX_nEPSE.SATP(SEQ ID NO:9093), GTPE.TPGSX_nSGPE.SGPG(SEQ ID NO:9094), GTPE.TPGSX_nATPE.SGPG(SEQ ID NO:9095), GTPE.TPGSX_nGTSE.SATP(SEQ ID NO:9096), GTPE.TPGSX_nTTPE.SGPG(SEQ ID NO:9097), GTPE.TPGSX_nSTPE.SGPG(SEQ ID NO:9098), GTPE.TPGSX_nGTPE.SGPG(SEQ ID NO:9099), GTPE.TPGSX_nGTPE.TPGS(SEQ ID NO:9100), GTPE.TPGSX_nSGSE.TGTP(SEQ ID NO:9101), GTPE.TPGSX_nGTPE.GSAP(SEQ ID NO:9103), GTPE.TPGSX_nEPSE.SATP(SEQ ID NO:9104), SGSE.TGTPX_nSGPE.SGPG(SEQ ID NO:9105), SGSE.TGTPX_nATPE.SGPG(SEQ ID NO:9106), SGSE.TGTPX_nGTSE.SATP(SEQ ID NO:9107), SGSE.TGTPX_nTTPE.SGPG(SEQ ID NO:9108), SGSE.TGTPX_nSTPE.SGPG(SEQ ID NO:9109), SGSE.TGTPX_nGTPE.SGPG(SEQ ID NO:9110), SGSE.TGTPX_nGTPE.TPGS(SEQ ID NO:9111), SGSE.TGTPX_nSGSE.TGTP(SEQ ID NO:9112), SGSE.TGTPX_nGTPE.GSAP(SEQ ID NO:9113), SGSE.TGTPX_nEPSE.SATP(SEQ ID NO:9114), GTPE.GSAPX_nSGPE.SGPG(SEQ ID NO:9115), GTPE.GSAPX_nATPE.SGPG(SEQ ID NO:9116), GTPE.GSAPX_nGTSE.SATP(SEQ ID NO:9117), GTPE.GSAPX_nTTPE.SGPG(SEQ ID NO:9118), GTPE.GSAPX_nSTPE.SGPG(SEQ ID NO:9119), GTPE.GSAPX_nGTPE.SGPG(SEQ ID NO:9120), GTPE.GSAPX_nGTPE.TPGS(SEQ ID NO:9121), GTPE.GSAPX_nSGSE.TGTP(SEQ ID NO:9122), GTPE.GSAPX_nGTPE.GSAP(SEQ ID NO:9123), GTPE.GSAPX_nEPSE.SATP(SEQ ID NO:9124), EPSE.SATPX_nSGPE.SGPG(SEQ ID NO:9126), EPSE.SATPX_nATPE.SGPG(SEQ ID NO:9127), EPSE.SATPX_nGTSE.SATP(SEQ ID NO:9128), EPSE.SATPX_nTTPE.SGPG(SEQ ID NO:9129), EPSE.SATPX_nSTPE.SGPG(SEQ ID NO:9130), EPSE.SATPX_nGTPE.SGPG(SEQ ID NO:9131), EPSE.SATPX_nGTPE.TPGS(SEQ ID NO:9132), EPSE.SATPX_nSGSE.TGTP(SEQ ID NO:9133), EPSE.SATPX_nGTPE.GSAP(SEQ ID NO:9134), or EPSE.SATPX_nEPSE.SATP(SEQ ID NO:9135), wherein each “.” is a Glu-C cleavage site and n is any integer from 0 to 50.
- 170. The chimeric polypeptide of embodiment 169, comprising at least one of the following amino acid sequences: SGPE.SGPGX_nATPE.SGPG(SEQ ID NO:9038), ATPE.SGPGX_nGTSE.SATP(SEQ ID NO:9040), ATPE.SGPGX_nTTPE.SGPG(SEQ ID NO:9041), ATPE.SGPGX_nSTPE.SGPG(SEQ ID NO:9042), ATPE.SGPGX_nATPE.SGPG(SEQ ID NO:9039), ATPE.SGPGX_nGTPE.SGPG(SEQ ID NO:9044), ATPE.SGPGX_nGTPE.SGPG(SEQ ID NO:9044), ATPE.SGPGX_nATPE.SGPG(SEQ ID NO:9039), GTPE.SGPGX_nGTPE.SGPG(SEQ ID NO:9089), GTPE.SGPGX_nSTPE.SGPG(SEQ ID NO:9087), GTPE.SGPGX_nTTPE.SGPG(SEQ ID NO:9085), GTPE.SGPGX_nSTPE.SGPG(SEQ ID NO:9087), GTPE.TPGSX_nSGSE.TGTP(SEQ ID NO:9102), GTPE.GSAPX_nEPSE.SATP(SEQ ID NO:9125), ATPE.SGPGX_nGTPE.SGPG(SEQ ID NO:9044), ATPE.SGPGX_nGTPE.SGPG(SEQ ID NO:9044), ATPE.SGPGX_nATPE.SGPG(SEQ ID NO:9039), ATPE.SGPGX_nGTPE.SGPG(SEQ ID NO:9044), TTPE.SGPGX_nTTPE.SGPG(SEQ ID NO:9063), or STPE.SGPGX_nSTPE.SGPG(SEQ ID NO:9075), wherein each “.” is a Glu-C cleavage site and n is any integer from 0 to 30.
- 171. The chimeric polypeptide of any one of embodiments 169 or 170, wherein n is any integer from 1 to 20.
- 172. The chimeric polypeptide of any one of embodiments 169-171, wherein n is any integer from 5 to 15.
- 173. The chimeric polypeptide of any one of embodiments 169-172, wherein n is any integer from 3 to 7.
- 174. The chimeric polypeptide of any one of embodiments 169-172, wherein n is any integer from 5 to 10.
- 175. The chimeric polypeptide of any one of embodiments 169-172, wherein n is 9.
- 176. The chimeric polypeptide of any one of embodiments 169-174, wherein n is 4.
- 177. The chimeric polypeptide of any one of embodiments 169-176, wherein X_nis PGTGTSAT(SEQ ID NO:9136), PGSGPGT(SEQ ID NO:9137), PGTTPGTT(SEQ ID NO:9138), PGTPPTST(SEQ ID NO:9139), PGTSPSAT(SEQ ID NO:9140), PGTGSAGT(SEQ ID NO:9141), PGTGGAGT(SEQ ID NO:9142), PGTSPGAT(SEQ ID NO:9143), PGTSGSGT(SEQ ID NO:9144), PGTSSAST(SEQ ID NO:9145), PGTGAGTT(SEQ ID NO:9146), PGTGSTST(SEQ ID NO:9147), GSEPATSG(SEQ ID NO:9148), APGTSTEP(SEQ ID NO:9149), PGTAGSGT(SEQ ID NO:9150), PGTSSGGT(SEQ ID NO:9151), PGTAGPAT(SEQ ID NO:9152), PGTPGTGT(SEQ ID NO:9153), PGTGGPTT(SEQ ID NO:9154), or PGTGSGST(SEQ ID NO:9155).
- 178. The chimeric polypeptide of any one of embodiments 169-177, wherein X_nis TGTS(SEQ ID NO:9156), SGP, TTPG(SEQ ID NO:9157), TPPT(SEQ ID NO:9158), TSPS(SEQ ID NO:9159), TGSA(SEQ ID NO:9160), TGGA(SEQ ID NO:9161), TSPG(SEQ ID NO:9162), TSGS(SEQ ID NO:9163), TSSA(SEQ ID NO:9164), TGAG(SEQ ID NO:9165), TGST(SEQ ID NO:9166), EPAT(SEQ ID NO:9167), GTST(SEQ ID NO:9168), TAGS(SEQ ID NO:9169), TSSG(SEQ ID NO:9170), TAGP(SEQ ID NO:9171), TPGT(SEQ ID NO:9172), TGGP(SEQ ID NO:9173), or TGSG(SEQ ID NO:9174).
- 179. The chimeric polypeptide of any one of embodiments 1-178, wherein neither the N-terminal amino acid nor the C-terminal acid of the chimeric polypeptide is included in a barcode fragment.
- 180. The chimeric polypeptide of any one of embodiments 1-179, comprising an ELNN with a non-overlapping sequence motif that occurs only once within the ELNN, wherein the ELNN further comprises a barcode fragment that includes at least part of the non-overlapping sequence motif that occurs only once within the ELNN.
- 181. The chimeric polypeptide of any one of embodiments 1-179, comprising a first ELNN with a first barcode fragment and a second ELNN with a second barcode fragment, wherein neither the first barcode fragment nor the second barcode fragment includes a glutamate that is immediately adjacent to another glutamate, if present, in the ELNN that contains the barcode fragment.
- 182. The chimeric polypeptide of embodiment 181, wherein at least one of the barcode fragments comprises a glutamate at the C-terminus thereof.
- 183. The chimeric polypeptide of embodiments 181 or 182, wherein at least one of the barcode fragments has an N-terminal amino acid that is immediately preceded by a glutamate in the chimeric polypeptide.
- 184. The chimeric polypeptide of embodiment 181, wherein the glutamate that precedes the N-terminal amino acid of the barcode fragment is not immediately adjacent to another glutamate.
- 185. The chimeric polypeptide of any one of embodiments 181-184, wherein at least one of the barcode fragments does not include a second glutamate at a position other than the C-terminus of the barcode fragment unless the second glutamate is immediately followed by a proline.
- 186. The chimeric polypeptide of any one of embodiments 1-185, comprising a single polypeptide chain, wherein the chimeric polypeptide comprises a barcode fragment that is at a position within the polypeptide chain that is from 10 to 200 amino acids or from 10 to 125 amino acids from the N-terminus or the C-terminus of the chimeric polypeptide.
- 187. The chimeric polypeptide of any one of embodiments 181-186, wherein the first ELNN is at the N-terminal side of the bispecific antibody domain, and wherein the first barcode fragment is positioned within 200, 150, 100, or 50 amino acids of the N-terminus of the chimeric polypeptide.
- 188. The chimeric polypeptide of any one of embodiments 181-187, wherein the second ELNN is at the C-terminal side of the bispecific antibody domain, and wherein the second barcode fragment is positioned within 200, 150, 100, or 50 amino acids of the C-terminus of the chimeric polypeptide.
- 189. The chimeric polypeptide of any one of embodiments 163-188, wherein at least one of the barcode fragments is at least 4 amino acids in length.
- 190. The chimeric polypeptide of any one of embodiments 163-189, wherein at least one of the barcode fragments is from 4 to 20, from 5 to 15, from 6 to 12, or from 7 to 10 amino acids in length.
- 191. The chimeric polypeptide of embodiment 190, wherein each mask polypeptide comprises one barcode fragment that is listed in Table 2 or disclosed in Table 3a.
- 192. The chimeric polypeptide of any one of embodiments 1-191, comprising a barcode fragment comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to SGPGSGPGTSE(SEQ ID NO:78) or SGPGTSPSATPE(SEQ ID NO:79).
- 193. The chimeric polypeptide of any one of embodiments 1-192, comprising one barcode fragment comprising an amino acid sequence that is at least 95% identical to SGPGSGPGTSE(SEQ ID NO:78) and one barcode fragment comprising an amino acid sequence that is at least 95% identical to SGPGTSPSATPE(SEQ ID NO:79).
- 194. The chimeric polypeptide of any one of embodiments 163-193, wherein the barcode fragment consists of A, E, G, S, P, and/or T residues.
- 195. The chimeric polypeptide of any one of embodiments 163-194, wherein the barcode fragment is part of a mask peptide.
- 196. The chimeric polypeptide of embodiment 195, wherein the mask peptide is the first ELNN or the second ELNN.
- 197. The chimeric polypeptide of any one of embodiments 1-196, comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to a sequence listed in Table D (SEQ ID NOs: 1000-1009).
- 198. The chimeric polypeptide of any one of embodiments 1-197, comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to:

- 199. The chimeric polypeptide of embodiment 198, comprising the following amino acid sequence:

- 200. A pharmaceutical composition comprising the chimeric polypeptide of any one of embodiments 1-199 and at least one pharmaceutically acceptable excipient.
- 201. The pharmaceutical composition of embodiment 200, which is in a liquid form or is frozen.
- 202. The pharmaceutical composition of embodiment 200, which is formulated as a lyophilized powder or cake to be reconstituted prior to administration.
- 203. An injection device comprising the pharmaceutical composition of embodiment 200.
- 204. The injection device of embodiment 203, which comprises a syringe.
- 205. A polynucleotide sequence encoding the chimeric polypeptide of any one of embodiments 1-204.
- 206. An expression vector comprising the polynucleotide sequence of embodiment 205.
- 207. A host cell comprising the expression vector of embodiment 205.
- 208. A method of producing the chimeric polypeptide of any one of embodiments 1-199.
- 209. The method of embodiment 208, further comprising isolating the chimeric polypeptide from a host cell.
- 210. A method of treating cancer in a subject in need thereof, the method comprising administering an effective amount of the chimeric polypeptide of any one of embodiments 1-199 to the subject.
- 211. The method of embodiment 210, wherein the cancer comprises a solid tumor.
- 212. The method of embodiment 210 or 211, wherein the cancer is a carcinoma.
- 213. The method of any one of embodiments 210-212, wherein the cancer is prostate cancer.
- 214. The method of embodiment 213, wherein the prostate cancer is metastatic prostate cancer.
- 215. The method of embodiment 213, wherein the prostate cancer is androgen-independent.
- 216. The method of embodiment 213, wherein the prostate cancer is non-metastatic castration-resistant prostate cancer (nmCRPC).
- 217. The method of embodiment 213, wherein the prostate cancer is metastatic castration-resistant prostate cancer (mCRPC).
- 218. The method of any one of embodiments 210-217, further comprising administering docetaxel to the subject.
- 219. The method of any one of embodiments 210-218, further comprising administering a checkpoint inhibitor to the subject.
- 220. The method of embodiment 219, wherein the checkpoint inhibitor is a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor.
- 221. The method of embodiment 219, wherein the checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody.
- 222. The method of embodiment 219, wherein the checkpoint inhibitor is pembrolizumab or cemiplimab.
- 223. A linker polypeptide comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81).
- 224. The linker polypeptide of embodiment 223, which is cleavable by a non-mammalian protease.
- 225. The linker polypeptide of embodiment 224, wherein the non-mammalian protease is Glu-C.
- 226. The linker polypeptide of any one of embodiments 223-225, wherein the linker polypeptide connects a first polypeptide moiety to a second polypeptide moiety.
- 227. The linker polypeptide of any one of embodiments 223-226, wherein the first polypeptide moiety is a VL domain and the second polypeptide moiety is a VH domain.
- 228. An antigen binding polypeptide comprising a VL domain and a VH domain, wherein the VL domain is linked to the VH domain by a linker polypeptide comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% 97% 98% or 99% identity, or 100% identity, to

(SEQ ID NO: 81)

SESATPESGPGTSPGATPESGPGTSESATP.

- 229. The antigen binding polypeptide of embodiment 228, wherein the linker polypeptide is cleavable by a non-mammalian protease.
- 230. The antigen binding polypeptide of embodiment 229, wherein the non-mammalian protease is Glu-C.
- 231. The antigen binding polypeptide of any one of embodiments 228-230, which is an scFv.
- 232. The antigen binding polypeptide of any one of embodiments 228-231, wherein the antigen is CD3.
- 233. The antigen binding polypeptide of embodiment 232, wherein the antigen is CD3 epsilon.
- 234. The linker polypeptide of any one of embodiments 223-227 or the antigen binding domain of any one of embodiments 228-233, wherein the VL domain is N-terminal to the VH domain.
- 235. The linker polypeptide of any one of embodiments 223-227 or the antigen binding domain of any one of embodiments 228-233, wherein the VH domain is N-terminal to the VL domain.
- 236. A pharmaceutical composition comprising the linker polypeptide of any one of embodiments 223-227 or the antigen binding polypeptide of any one of embodiments 228-233, and at least one pharmaceutically acceptable excipient.
- 237. The pharmaceutical composition of embodiment 236, which is in a liquid form or is frozen.
- 238. The pharmaceutical composition of embodiment 236, which is formulated as a lyophilized powder or cake to be reconstituted prior to administration.
- 239. An injection device comprising the pharmaceutical composition of embodiment 236.
- 240. The injection device of embodiment 239, which comprises a syringe.
- 241. A polynucleotide sequence encoding the linker of any one of embodiments 223-227 or the antigen binding polypeptide of any one of embodiments 228-233.
- 242. An expression vector comprising the polynucleotide sequence of embodiment 241.
- 243. A host cell comprising the expression vector of embodiment 242.
- 244. A method of producing the linker of any one of embodiments 223-227 or the antigen binding polypeptide of any one of embodiments 228-233.
- 245. The method of embodiment 244, further comprising isolating the linker or antigen binding polypeptide from a host cell.
- 246. An isolated polypeptide comprising a protease-cleavable amino acid sequence comprising the sequence: EAGRSAXHTPAGLTGP (SEQ ID NO: 7627), wherein X is any amino acid other than N.
- 247. The isolated polypeptide of embodiment 246, wherein X is S.
- 248. The isolated polypeptide of embodiment 246, which is not cleavable by legumain.
- 249. The isolated polypeptide of embodiment 246, which is not cleavable by legumain in human blood, plasma, or serum.
- 250. The isolated polypeptide of embodiment 246, which is not cleavable upon incubation with about 1 nM or less legumain for about 20 hours.
- 251. The isolated polypeptide of embodiment 246, which is not cleavable upon incubation with about 1 nM or less legumain for about 20 hours in human blood, plasma, or serum.
- 252. The isolated polypeptide of embodiment 246, wherein legumain cleaves the isolated polypeptide in human plasma at a rate that is less than about 50% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain.
- 253. The isolated polypeptide of embodiment 246, wherein legumain cleaves the isolated polypeptide in human plasma at a rate that is less than about 25% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain.
- 254. The isolated polypeptide of embodiment 246, wherein legumain cleaves the isolated polypeptide in human plasma at a rate that is less than about 10% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain.
- 255. The isolated polypeptide of embodiment 246, wherein legumain cleaves the isolated polypeptide in human plasma at a rate that is less than about 5% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain.
- 256. The isolated polypeptide of embodiment 246, wherein legumain cleaves the isolated polypeptide in human plasma at a rate that is less than about 2.5% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain.
- 257. A pharmaceutical composition comprising the isolated polypeptide of any one of embodiments 246-257, and at least one pharmaceutically acceptable excipient.
- 258. The pharmaceutical composition of embodiment 257, which is in a liquid form or is frozen.
- 259. The pharmaceutical composition of embodiment 257, which is formulated as a lyophilized powder or cake to be reconstituted prior to administration.
- 260. An injection device comprising the pharmaceutical composition of embodiment 259.
- 261. The injection device of embodiment 260, which comprises a syringe.
- 262. A polynucleotide sequence encoding the isolated polypeptide of any one of embodiments 246-257.
- 263. An expression vector comprising the polynucleotide sequence of embodiment 262.
- 264. A host cell comprising the expression vector of embodiment 263.
- 265. A method of producing the isolated polypeptide of any one of embodiments 246-257.
- 266. The method of embodiment 265, further comprising isolating the isolated polypeptide from a host cell.
- 267. A fusion protein comprising a protease-cleavable amino acid sequence comprising the sequence: EAGRSAXHTPAGLTGP (SEQ ID NO: 7627), wherein X is any amino acid other than N, wherein the protease-cleavable amino acid sequence links a first polypeptide moiety to a second polypeptide moiety.
- 268. The fusion protein of embodiment 267, wherein X is S.
- 269. The fusion protein of embodiment 267, which is not cleavable by legumain.
- 270. The fusion protein of embodiment 267, which is not cleavable by legumain in human blood, plasma, or serum.
- 271. The fusion protein of embodiment 267, which is not cleavable upon incubation with about 1 nM or less legumain for about 20 hours.
- 272. The fusion protein of embodiment 267, which is not cleavable upon incubation with about 1 nM or less legumain for about 20 hours in human blood, plasma, or serum.
- 273. The fusion protein of embodiment 267, wherein legumain cleaves the protease-cleavable amino acid sequence in human plasma at a rate that is less than about 50% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain.
- 274. The fusion protein of embodiment 267, wherein legumain cleaves the protease-cleavable amino acid sequence in human plasma at a rate that is less than about 25% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain.
- 275. The fusion protein of embodiment 267, wherein legumain cleaves the protease-cleavable amino acid sequence in human plasma at a rate that is less than about 10% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain.
- 276. The fusion protein of embodiment 267, wherein legumain cleaves the protease-cleavable amino acid sequence in human plasma at a rate that is less than about 5% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain.
- 277. The fusion protein of embodiment 267, wherein legumain cleaves the protease-cleavable amino acid sequence in human plasma at a rate that is less than about 2.5% of the rate that RSR-2295 (EAGRSANHTPAGLTGP) (SEQ ID NO:7048) is cleaved by legumain.
- 278. The fusion protein of any one of embodiments 267-278, wherein the first polypeptide moiety comprises an antigen-binding domain and the second polypeptide moiety comprises a masking polypeptide.
- 279. The fusion protein of any one of embodiments 267-278, wherein the first polypeptide moiety comprises an antigen-binding domain and the second polypeptide moiety is a cytokine, an enzyme, a hormone, a growth factor, a chemotherapeutic polypeptide, an antiviral polypeptide, or a toxin.
- 280. The fusion protein of any one of embodiments 267-278, wherein the first polypeptide moiety is a cytokine, an enzyme, a hormone, a growth factor, a chemotherapeutic polypeptide, an antiviral polypeptide, or a toxin and the second polypeptide moiety is a masking polypeptide.
- 281. The fusion protein of embodiment 280, wherein the masking polypeptide comprises an ELNN.
- 282. The fusion protein of any one of embodiments 267-281, comprising a single polypeptide chain, which comprises, in the N terminal to C terminal direction, the first polypeptide then the protease-cleavable amino acid sequence then the second polypeptide moiety.
- 283. The fusion protein of any one of embodiments 267-281, comprising a single polypeptide chain, which comprises, in the N terminal to C terminal direction, the second polypeptide then the protease-cleavable amino acid sequence then the first polypeptide moiety.
- 284. A pharmaceutical composition comprising the fusion protein of any one of embodiments 267-283, and at least one pharmaceutically acceptable excipient.
- 285. The pharmaceutical composition of embodiment 284, which is in a liquid form or is frozen.
- 286. The pharmaceutical composition of embodiment 284, which is formulated as a lyophilized powder or cake to be reconstituted prior to administration.
- 287. An injection device comprising the pharmaceutical composition of embodiment 284.
- 288. The injection device of embodiment 287, which comprises a syringe.
- 289. A polynucleotide sequence encoding the fusion protein of any one of embodiments 267-283.
- 290. An expression vector comprising the polynucleotide sequence of embodiment 289.
- 291. A host cell comprising the expression vector of embodiment 290.
- 292. A method of producing the fusion protein of any one of embodiments 267-283.
- 293. The method of embodiment 275, further comprising isolating the fusion protein from a host cell.
- 294. An ELNN polypeptide comprising the following amino acid sequence:

- 295. An ELNN polypeptide comprising the following amino acid sequence:

- 296. A fusion protein comprising the ELNN polypeptide of embodiment 294 or 295.
- 297. A pharmaceutical composition comprising the ELNN polypeptide of embodiment 294 or 295, or the fusion protein of any one of embodiments 267-283 and 296, and at least one pharmaceutically acceptable excipient.
- 298. The pharmaceutical composition of embodiment 297, which is in a liquid form or is frozen.
- 299. The pharmaceutical composition of embodiment 297, which is formulated as a lyophilized powder or cake to be reconstituted prior to administration.
- 300. An injection device comprising the pharmaceutical composition of embodiment 297.
- 301. The injection device of embodiment 300, which comprises a syringe.
- 302. A polynucleotide sequence encoding the ELNN polypeptide of embodiment 294 or 295, or the fusion protein of any one of embodiments 267-283 and 296.
- 303. An expression vector comprising the polynucleotide sequence of embodiment 302.
- 304. A host cell comprising the expression vector of embodiment 303.
- 305. A method of producing the ELNN polypeptide of embodiment 294 or 295, or the fusion protein of any one of embodiments 267-283 and 296.
- 306. The method of embodiment 305, further comprising isolating the ELNN polypeptide or the fusion protein, from a host cell.
- 307. A barcode fragment comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to SGPGTGTSATPE(SEQ ID NO:1010), SGPGSGPGTSE(SEQ ID NO:78), SGPGTTPGTTPE(SEQ ID NO:1011), SGPGTPPTSTPE(SEQ ID NO:1012), SGPGTSPSATPE(SEQ ID NO:79), SGPGTGSAGTPE(SEQ ID NO:1013), SGPGTGGAGTPE(SEQ ID NO:1014), SGPGTSPGATPE(SEQ ID NO:1015), SGPGTSGSGTPE(SEQ ID NO:1016), SGPGTSSASTPE(SEQ ID NO:1017), SGPGTGAGTTPE(SEQ ID NO:1018), SGPGTGSTSTPE(SEQ ID NO:1019), TPGSEPATSGSE(SEQ ID NO:1020), GSAPGTSTEPSE(SEQ ID NO:1021), SGPGTAGSGTPE(SEQ ID NO:1022), SGPGTSSGGTPE(SEQ ID NO:1023), SGPGTAGPATPE(SEQ ID NO:1024), SGPGTPGTGTPE(SEQ ID NO:1025), SGPGTGGPTTPE(SEQ ID NO:1026), or SGPGTGSGSTPE(SEQ ID NO:1027).
- 308. A barcode fragment comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to SGPGSGPGTSE(SEQ ID NO:78) or SGPGTSPSATPE(SEQ ID NO:79).
- 309. The barcode fragment of embodiment 307 or 308, comprising the amino acid sequence:

(SEQ ID NO: 78)

SGPGSGPGTSE.

- 310. The barcode fragment of embodiment 307 or 308, comprising the amino acid sequence: SGPGTSPSATPE(SEQ ID NO:79).

(SEQ ID NO: 79)

SGPGTSPSATPE.

- 311. A fusion protein comprising the barcode fragment of any one of embodiments 307-310.
- 312. A fusion protein comprising a Glu-C cleavage site comprising one of the following amino acid sequences: ATPESGPG(SEQ ID NO:9020), SGSETPGT(SEQ ID NO:9021), and GTSESATP(SEQ ID NO:9022).
- 313. A fusion protein comprising at least one of the following amino acid sequences: SGPE.SGPGX_nSGPE.SGPG(SEQ ID NO:9023), SGPE.SGPGX_nATPE.SGPG(SEQ ID NO:9024), SGPE.SGPGX_nGTSE.SATP(SEQ ID NO:9025), SGPE.SGPGX_nTTPE.SGPG(SEQ ID NO:9026), SGPE.SGPGX_nSTPE.SGPG(SEQ ID NO:9027), SGPE.SGPGX_nGTPE.SGPG(SEQ ID NO:9028), SGPE.SGPGX_nGTPE.TPGS(SEQ ID NO:9029), SGPE.SGPGX_nGTPE.TPGS(SEQ ID NO:9029), SGPE.SGPGX_nSGSE.TGTP(SEQ ID NO:9030), SGPE.SGPGX_nGTPE.GSAP(SEQ ID NO:9031), SGPE.SGPGX_nEPSE.SATP(SEQ ID NO:9032), ATPE.SGPGX_nSGPE.SGPG(SEQ ID NO:9033), ATPE.SGPGX_nATPE.SGPG(SEQ ID NO:9034), ATPE.SGPGX_nGTSE.SATP(SEQ ID NO:9035), ATPE.SGPGX_nTTPE.SGPG(SEQ ID NO:9036), ATPE.SGPGX_nSTPE.SGPG(SEQ ID NO:9037), ATPE.SGPGX_nGTPE.SGPG(SEQ ID NO:9043), ATPE.SGPGX_nGTPE.TPGS(SEQ ID NO:9045), ATPE.SGPGX_nSGSE.TGTP(SEQ ID NO:9046), ATPE.SGPGX_nGTPE.GSAP(SEQ ID NO:9047), ATPE.SGPGX_nEPSE.SATP(SEQ ID NO:9048), GTSE.SATPX_nSGPE.SGPG(SEQ ID NO:9049), GTSE.SATPX_nATPE.SGPG(SEQ ID NO:9050), GTSE.SATPX_nGTSE.SATP(SEQ ID NO:9051), GTSE.SATPX_nTTPE.SGPG(SEQ ID NO:9052), GTSE.SATPX_nSTPE.SGPG(SEQ ID NO:9053), GTSE.SATPX_nGTPE.SGPG(SEQ ID NO:9054), GTSE.SATPX_nGTPE.TPGS(SEQ ID NO:9055), GTSE.SATPX_nSGSE.TGTP(SEQ ID NO:9056), GTSE.SATPX_nGTPE.GSAP(SEQ ID NO:9057), GTSE.SATPX_nEPSE.SATP(SEQ ID NO:9058), TTPE.SGPGX_nSGPE.SGPG(SEQ ID NO:9059), TTPE.SGPGX_nATPE.SGPG(SEQ ID NO:9060), TTPE.SGPGX_nGTSE.SATP(SEQ ID NO:9061), TTPE.SGPGX_nTTPE.SGPG(SEQ ID NO:9062), TTPE.SGPGX_nSTPE.SGPG(SEQ ID NO:9064), TTPE.SGPGX_nGTPE.SGPG(SEQ ID NO:9065), TTPE.SGPGX_nGTPE.TPGS(SEQ ID NO:9066), TTPE.SGPGX_nSGSE.TGTP(SEQ ID NO:9067), TTPE.SGPGX_nGTPE.GSAP(SEQ ID NO:9068), TTPE.SGPGX_nEPSE.SATP(SEQ ID NO:9069), STPE.SGPGX_nSGPE.SGPG(SEQ ID NO:9070), STPE.SGPGX_nATPE.SGPG(SEQ ID NO:9071), STPE.SGPGX_nGTSE.SATP(SEQ ID NO:9072), STPE.SGPGX_nTTPE.SGPG(SEQ ID NO:9073), STPE.SGPGX_nSTPE.SGPG(SEQ ID NO:9074), STPE.SGPGX_nGTPE.SGPG(SEQ ID NO:9076), STPE.SGPGX_nGTPE.TPGS(SEQ ID NO:9077), STPE.SGPGX_nSGSE.TGTP(SEQ ID NO:9078), STPE.SGPGX_nGTPE.GSAP(SEQ ID NO:9079), STPE.SGPGX_nEPSE.SATP(SEQ ID NO:9175), GTPE.SGPGX_nSGPE.SGPG(SEQ ID NO:9081), GTPE.SGPGX_nATPE.SGPG(SEQ ID NO:9082), GTPE.SGPGX_nGTSE.SATP(SEQ ID NO:9083), GTPE.SGPGX_nTTPE.SGPG(SEQ ID NO:9084), GTPE.SGPGX_nSTPE.SGPG(SEQ ID NO:9086), GTPE.SGPGX_nGTPE.SGPG(SEQ ID NO:9088), GTPE.SGPGX_nGTPE.TPGS(SEQ ID NO:9090), GTPE.SGPGX_nSGSE.TGTP(SEQ ID NO:9091), GTPE.SGPGX_nGTPE.GSAP(SEQ ID NO:9092), GTPE.SGPGX_nEPSE.SATP(SEQ ID NO:9093), GTPE.TPGSX_nSGPE.SGPG(SEQ ID NO:9094), GTPE.TPGSX_nATPE.SGPG(SEQ ID NO:9095), GTPE.TPGSX_nGTSE.SATP(SEQ ID NO:9096), GTPE.TPGSX_nTTPE.SGPG(SEQ ID NO:9097), GTPE.TPGSX_nSTPE.SGPG(SEQ ID NO:9098), GTPE.TPGSX_nGTPE.SGPG(SEQ ID NO:9099), GTPE.TPGSX_nGTPE.TPGS(SEQ ID NO:9100), GTPE.TPGSX_nSGSE.TGTP(SEQ ID NO:9101), GTPE.TPGSX_nGTPE.GSAP(SEQ ID NO:9103), GTPE.TPGSX_nEPSE.SATP(SEQ ID NO:9104), SGSE.TGTPX_nSGPE.SGPG(SEQ ID NO:9105), SGSE.TGTPX_nATPE.SGPG(SEQ ID NO:9106), SGSE.TGTPX_nGTSE.SATP(SEQ ID NO:9107), SGSE.TGTPX_nTTPE.SGPG(SEQ ID NO:9108), SGSE.TGTPX_nSTPE.SGPG(SEQ ID NO:9109), SGSE.TGTPX_nGTPE.SGPG(SEQ ID NO:9110), SGSE.TGTPX_nGTPE.TPGS(SEQ ID NO:9111), SGSE.TGTPX_nSGSE.TGTP(SEQ ID NO:9112), SGSE.TGTPX_nGTPE.GSAP(SEQ ID NO:9113), SGSE.TGTPX_nEPSE.SATP(SEQ ID NO:9114), GTPE.GSAPX_nSGPE.SGPG(SEQ ID NO:9115), GTPE.GSAPX_nATPE.SGPG(SEQ ID NO:9116), GTPE.GSAPX_nGTSE.SATP(SEQ ID NO:9117), GTPE.GSAPX_nTTPE.SGPG(SEQ ID NO:9118), GTPE.GSAPX_nSTPE.SGPG(SEQ ID NO:9119), GTPE.GSAPX_nGTPE.SGPG(SEQ ID NO:9120), GTPE.GSAPX_nGTPE.TPGS(SEQ ID NO:9121), GTPE.GSAPX_nSGSE.TGTP(SEQ ID NO:9122), GTPE.GSAPX_nGTPE.GSAP(SEQ ID NO:9123), GTPE.GSAPX_nEPSE.SATP(SEQ ID NO:9124), EPSE.SATPX_nSGPE.SGPG(SEQ ID NO:9126), EPSE.SATPX_nATPE.SGPG(SEQ ID NO:9127), EPSE.SATPX_nGTSE.SATP(SEQ ID NO:9128), EPSE.SATPX_nTTPE.SGPG(SEQ ID NO:9129), EPSE.SATPX_nSTPE.SGPG(SEQ ID NO:9130), EPSE.SATPX_nGTPE.SGPG(SEQ ID NO:9131), EPSE.SATPX_nGTPE.TPGS(SEQ ID NO:9132), EPSE.SATPX_nSGSE.TGTP(SEQ ID NO:9133), EPSE.SATPX_nGTPE.GSAP(SEQ ID NO:9134), or EPSE.SATPX_nEPSE.SATP(SEQ ID NO:9135), wherein each “.” is a Glu-C cleavage site and n is any integer from 0 to 50.
- 314. The fusion protein of embodiment 313, comprising at least one of the following amino acid sequences: SGPE.SGPGX_nATPE.SGPG(SEQ ID NO:9038), ATPE.SGPGX_nGTSE.SATP(SEQ ID NO:9040), ATPE.SGPGX_nTTPE.SGPG(SEQ ID NO:9041), ATPE.SGPGX_nSTPE.SGPG(SEQ ID NO:9042), ATPE.SGPGX_nATPE.SGPG(SEQ ID NO:9039), ATPE.SGPGX_nGTPE.SGPG(SEQ ID NO:9044), ATPE.SGPGX_nGTPE.SGPG(SEQ ID NO:9044), ATPE.SGPGX_nATPE.SGPG(SEQ ID NO:9039), GTPE.SGPGX_nGTPE.SGPG(SEQ ID NO:9089), GTPE.SGPGX_nSTPE.SGPG(SEQ ID NO:9087), GTPE.SGPGX_nTTPE.SGPG(SEQ ID NO:9085), GTPE.SGPGX_nSTPE.SGPG(SEQ ID NO:9087), GTPE.TPGSX_nSGSE.TGTP(SEQ ID NO:9102), GTPE.GSAPX_nEPSE.SATP(SEQ ID NO:9125), ATPE.SGPGX_nGTPE.SGPG(SEQ ID NO:9044), ATPE.SGPGX_nGTPE.SGPG(SEQ ID NO:9044), ATPE.SGPGX_nATPE.SGPG(SEQ ID NO:9039), ATPE.SGPGX_nGTPE.SGPG(SEQ ID NO:9044), TTPE.SGPGX_nTTPE.SGPG(SEQ ID NO:9063), or STPE.SGPGX_nSTPE.SGPG(SEQ ID NO:9075), wherein each “.” is a Glu-C cleavage site and n is any integer from 0 to 30.
- 315. The fusion protein of embodiment 313 or 314, wherein n is any integer from 1 to 20.
- 316. The fusion protein of embodiment 315, wherein n is any integer from 5 to 15.
- 317. The fusion protein of embodiment 315, wherein n is any integer from 3 to 7.
- 318. The fusion protein of embodiment 315, wherein n is any integer from 5 to 10.
- 319. The fusion protein of embodiment 315, wherein n is 9.
- 320. The fusion protein of embodiment 315, wherein n is 4.
- 321. The fusion protein of any one of embodiments 313-320, wherein X_nis PGTGTSAT(SEQ ID NO:9136), PGSGPGT(SEQ ID NO:9137), PGTTPGTT(SEQ ID NO:9138), PGTPPTST(SEQ ID NO:9139), PGTSPSAT(SEQ ID NO:9140), PGTGSAGT(SEQ ID NO:9141), PGTGGAGT(SEQ ID NO:9142), PGTSPGAT(SEQ ID NO:9143), PGTSGSGT(SEQ ID NO:9144), PGTSSAST(SEQ ID NO:9145), PGTGAGTT(SEQ ID NO:9146), PGTGSTST(SEQ ID NO:9147), GSEPATSG(SEQ ID NO:9148), APGTSTEP(SEQ ID NO:9149), PGTAGSGT(SEQ ID NO:9150), PGTSSGGT(SEQ ID NO:9151), PGTAGPAT(SEQ ID NO:9152), PGTPGTGT(SEQ ID NO:9153), PGTGGPTT(SEQ ID NO:9154), or PGTGSGST(SEQ ID NO:9155).
- 322. The fusion protein of any one of embodiments 313-320, wherein X_nis TGTS(SEQ ID NO:9156), SGP, TTPG(SEQ ID NO:9157), TPPT(SEQ ID NO:9158), TSPS(SEQ ID NO:9159), TGSA(SEQ ID NO:9160), TGGA(SEQ ID NO:9161), TSPG(SEQ ID NO:9162), TSGS(SEQ ID NO:9163), TSSA(SEQ ID NO:9164), TGAG(SEQ ID NO:9165), TGST(SEQ ID NO:9166), EPAT(SEQ ID NO:9167), GTST(SEQ ID NO:9168), TAGS(SEQ ID NO:9169), TSSG(SEQ ID NO:9170), TAGP(SEQ ID NO:9171), TPGT(SEQ ID NO:9172), TGGP(SEQ ID NO:9173), or TGSG(SEQ ID NO:9174).
- 323. A pharmaceutical composition comprising the barcode fragment of any one of embodiments 307-310, or the fusion protein of any one of embodiments 311-322, and at least one pharmaceutically acceptable excipient.
- 324. The pharmaceutical composition of embodiment 323, which is in a liquid form or is frozen.
- 325. The pharmaceutical composition of embodiment 323, which is formulated as a lyophilized powder or cake to be reconstituted prior to administration.
- 326. An injection device comprising the pharmaceutical composition of embodiment 323.
- 327. The injection device of embodiment 326, which comprises a syringe.
- 328. A polynucleotide sequence encoding the barcode fragment of any one of embodiments 307-310, or the fusion protein of any one of embodiments 311-322.
- 329. An expression vector comprising the polynucleotide sequence of embodiment 328.
- 330. A host cell comprising the expression vector of embodiment 329.
- 331. A method of producing the barcode fragment of any one of embodiments 307-310, or the fusion protein of any one of embodiments 311-322.
- 332. The method of embodiment 331, further comprising isolating the barcode fragment or the fusion protein from a host cell.
- 333. An antibody or an antigen-binding fragment thereof that specifically binds PSMA, comprising a VHH domain or a fragment thereof comprising three VHH CDRs, wherein the three VHH CDRs comprise the CDR1, CDR2, and CDR3 from the following amino acid sequence:

(SEQ ID NO: 549)

QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVG

AMSWSGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNKEYGRTWYDFNESDYWGQGTQVTVSS.

- 334. An antibody or an antigen-binding fragment thereof that specifically binds PSMA, comprising the following CDRs:
  - a VHH CDR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GRTFGIYVWG(SEQ ID NO:9003);
  - a VHH CDR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to AMSWSGSNRKVSDSVKG(SEQ ID NO:9004); and
  - a VHH CDR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to AASNKEYGRTWYDFNESDY(SEQ ID NO:9005).
- 335. The antibody or fragment of embodiment 334, comprising one or more of the following FRs:
  - a VHH FR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to QVQLVESGGGVVQPGRSLRLSCAAS(SEQ ID NO:9011);
  - a VHH FR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to WFRQAPGKEREFVG(SEQ ID NO:9012);
  - a VHH FR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to RFTISRDNSKNTLYLQMNSLRAEDTAVYYC(SEQ ID NO:9013); and
  - a VHH FR4 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to WGQGTQVTVSS(SEQ ID NO:9014).
- 336. An antibody or an antigen-binding fragment thereof that specifically binds PSMA, comprising the following CDRs:
  - a VHH CDR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GRTFGIYVWG(SEQ ID NO:9003);
  - a VHH CDR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to AMSWSGSNRK(SEQ ID NO:9015); and
  - a VHH CDR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to AASNKEYGRTWYDFNESDY(SEQ ID NO:9005).
- 337. The antibody or fragment of embodiment 336, comprising one or more of the following FRs:
  - a VHH FR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to QVQLVESGGGVVQPGRSLRLSCAAS(SEQ ID NO:9011);
  - a VHH FR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to WFRQAPGKEREFVG(SEQ ID NO:9012);
  - a VHH FR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to VSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC(SEQ ID NO:9016); and
- a VHH FR4 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to WGQGTQVTVSS(SEQ ID NO:9014).
- 338. An antibody or an antigen-binding fragment thereof that specifically binds PSMA, comprising a VHH comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to

(SEQ ID NO: 549)

QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVG

AMSWSGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNKEYGRTWYDFNESDYWGQGTQVTVSS.

- 339. The antibody or fragment of any one of embodiments 333-338, which is an isolated antibody or fragment thereof.
- 340. An antibody or an antigen-binding fragment thereof that specifically binds PSMA, comprising a VHH comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to the amino acid sequence of PSMA.2, PSMA.3, PSMA.5, PSMA.6, PSMA.262, or PSMA.263.
- 341. An antibody or an antigen-binding fragment thereof that specifically binds PSMA, comprising a VHH comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to: QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVX₁₇GWFRQAPGKEREFVGAX₁₈S WSGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYX₁₉CX₂X₂₁SNKX₂₂Y GRTWYDFNESDYWGQGTQVTVSS(SEQ ID NO:9017), wherein X₁₇, X₁₈, X₁₉, X₂₀, X₂₁, and X₆each, individually, correspond to any naturally occurring amino acid.
- 342. The antibody or fragment of embodiment 341, wherein X₁₇corresponds to M or W, X₁₈corresponds to M or I, X₁₉corresponds to F or Y, X₂₀corresponds to A or G, X₂₁corresponds to A or G, and/or X₂₂corresponds to L, W, R, D, E, or G.
- 343. The antibody or fragment of embodiment 341 or 342, wherein the PSMA comprises the following amino acid sequence:

- 344. An antibody or an antigen-binding fragment thereof that specifically binds CD3, comprising a VL domain and a VH domain, wherein:
- (i) the VL domain comprises the VL CDRs of the amino acid sequence of ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNK RAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL (SEQ ID NO: 361); or
- (ii) the VH domain comprises the VH CDRs of the amino acid sequence of

(SEQ ID NO: 311)

EVLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGR

IRTKRNNYATYYADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCV

RHENFGNSYVSWFAHWGQGTLVTVSS.

- 345. An anti-CD3 antibody or an antigen-binding fragment thereof, comprising one or more of the following CDRs:
  - a VL domain CDR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to RSSNGAVTSSNYAN(SEQ ID NO:1);
  - a VL domain CDR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GTNKRAP(SEQ ID NO:4);
  - a VL domain CDR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to ALWYPNLWV(SEQ ID NO:6);
  - a VH domain CDR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GFTFSTYAMN(SEQ ID NO:12);
  - a VH domain CDR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to RIRTKRNNYATYYADSVKG(SEQ ID NO:13); and/or
  - a VH domain CDR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to HENFGNSYVSWFAH(SEQ ID NO:10).
- 346. The antibody or fragment of embodiment 345, comprising one or more of the following FRs:
  - a VL domain FR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to ELVVTQEPSLTVSPGGTVTLTC(SEQ ID NO:51);
  - a VL domain FR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to WVQQKPGQAPRGLIG(SEQ ID NO:52);
  - a VL domain FR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to GTPARFSGSLLGGKAALTLSGVQPEDEAVYYC(SEQ ID NO:53);
  - a VL domain FR4 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to FGGGTKLTVL(SEQ ID NO:59);
  - a VH domain FR1 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to EVQLVESGGGIVQPGGSLRLSCAAS(SEQ ID NO:400);
  - a VH domain FR2 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to WVRQAPGKGLEWVG(SEQ ID NO:401);
  - a VH domain FR3 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to RFTISRDDSKNTVYLQMNSLKTEDTAVYYCVR(SEQ ID NO:402); and/or
  - a VH domain FR4 with an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to WGQGTLVTVSS(SEQ ID NO:67).
- 347. The antibody or fragment of embodiment 345 or 346, which comprises a VL domain.
- 348. The antibody or fragment of embodiment 347, wherein the VL domain comprises an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to:

(SEQ ID NO: 361)

ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGL

IGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLW

VFGGGTKLTVL.

- 349. The antibody or fragment of any one of embodiments 345-348, which comprises a VH domain.
- 350. The antibody or fragment of embodiment 349, wherein the VH domain comprises an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to:

(SEQ ID NO: 311)

EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVG

RIRTKRNNYATYYADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYC

VRHENFGNSYVSWFAHWGQGTLVTVSS.

- 351. An antibody or an antigen-binding fragment thereof that specifically binds CD3, comprising a VL domain and a VH domain, wherein the VL domain amino acid sequence SEQ ID NO/VH domain amino acid sequence SEQ ID NO pair is selected from the group consisting of: 896/897; 902/903; 700/701; 702/703; 716/717; 718/719; 728/729; 736/737; 738/739; 740/741; 742/743; 744/745; 746/747; 748/749; 750/751; 752/753; 754/755; 756/757; 758/759; 760/761; 762/763; 764/765; 766/767; 774/775; 776/777; 790/791; 792/793; 798/799; 800/801; 806/807; 808/809; 814/815; 816/817; 822/823; 824/825; or 826/867.
- 352. The antibody or fragment thereof of any one of embodiments 340-351, which is an isolated antibody or fragment thereof.
- 353. The antibody or fragment of any one of embodiments 333-352, which is an antibody.
- 354. The antibody of embodiment 353, which is a Fab, an scFV, or a monoclonal antibody.
- 355. The antibody of embodiment 354, which is an scFV.
- 356. The antibody of embodiment 355, wherein the VL domain is N-terminal to the VH domain in the scFV.
- 357. The antibody of embodiment 355, wherein the VL domain is C-terminal to the VH domain in the scFV.
- 358. The antibody of any one of embodiments 353-357, wherein the scFv comprises a linker between the VL domain and the VH domain, wherein the linker consists of A, E, G, S, P, and/or T residues.
- 359. The antibody of embodiment 358, wherein the linker is an ELNN.
- 360. The antibody of embodiment 359, wherein the ELNN is cleavable by a non-mammalian protease.
- 361. The antibody of embodiment 360, wherein the non-mammalian protease is Glu-C.
- 362. The antibody of embodiment 361, wherein ELNN comprises an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to SESATPESGPGTSPGATPESGPGTSESATP (SEQ ID NO: 81).
- 363. The antibody of embodiment 355, wherein the scFV comprises an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to:

- 364. The antibody or fragment of any one of embodiments 344-364, wherein the CD3 is CD3 epsilon.
- 365. The antibody or fragment of embodiment 364, wherein the CD3 epsilon comprises the following amino acid sequence:

(SEQ ID NO: 1043)

DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDED

DKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCE

NCMEMD

- 366. A pharmaceutical composition comprising the antibody or an antigen-binding fragment thereof of any one of embodiments 333-365, and at least one pharmaceutically acceptable excipient.
- 367. The pharmaceutical composition of embodiment 366, which is in a liquid form or is frozen.
- 368. The pharmaceutical composition of embodiment 366, which is formulated as a lyophilized powder or cake to be reconstituted prior to administration.
- 369. An injection device comprising the pharmaceutical composition of embodiment 366.
- 370. The injection device of embodiment 369, which comprises a syringe.
- 371. A polynucleotide sequence encoding the antibody or an antigen-binding fragment thereof of any one of embodiments 333-365.
- 372. An expression vector comprising the polynucleotide sequence of embodiment 371.
- 373. A host cell comprising the expression vector of embodiment 372.
- 374. A method of producing the antibody or an antigen-binding fragment thereof of any one of embodiments 333-365.
- 375. The method of embodiment 374, further comprising isolating the antibody or an antigen-binding fragment thereof of from a host cell.
- 376. A multispecific antibody comprising an anti-PSMA antibody domain comprising an antibody or antibody fragment according to any one of embodiments 333-343 and/or an anti-CD3 antibody domain comprising an antibody or antibody fragment according to any one of embodiments 344-365.
- 377. A multispecific antibody comprising an anti-PSMA antibody domain comprising an antibody or antibody fragment according to any one of embodiments 333-343 and an anti-CD3 antibody domain comprising an antibody or antibody fragment according to any one of embodiments 344-365.
- 378. The multispecific antibody of embodiment 376 or 377, wherein the affinity of the anti-PSMA antibody domain to PSMA is higher than the affinity of the anti-CD3 antibody domain to CD3.
- 379. The multispecific antibody of any one of embodiments 375-378, which is a bispecific antibody.
- 380. The bispecific antibody of embodiment 379, which is a T cell engager.
- 381. A pharmaceutical composition comprising the multispecific antibody of any one of embodiments 375-380, and at least one pharmaceutically acceptable excipient.
- 382. The pharmaceutical composition of embodiment 381, which is in a liquid form or is frozen.
- 383. The pharmaceutical composition of embodiment 381, which is formulated as a lyophilized powder or cake to be reconstituted prior to administration.
- 384. An injection device comprising the pharmaceutical composition of embodiment 381.
- 385. The injection device of embodiment 384, which comprises a syringe.
- 386. A polynucleotide sequence encoding the multispecific antibody of any one of embodiments 375-380.
- 387. An expression vector comprising the polynucleotide sequence of embodiment 386.
- 388. A host cell comprising the expression vector of embodiment 387.
- 389. A method of producing the multispecific antibody of any one of embodiments 375-380.
- 390. The method of embodiment 389, further comprising isolating the multispecific antibody from a host cell.
- 391. A T cell engager comprising a first antigen binding domain that binds to prostate-specific membrane antigen (PSMA) and a second antigen binding domain that binds to cluster of differentiation 3 T cell receptor (CD3), wherein the first antigen binding domain comprises a VHH comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVGAMSW SGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAASNKEYGRTWY DFNESDYWGQGTQVTVSS (SEQ ID NO: 549); and the second antigen binding domain comprises a VL domain comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNK RAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL (SEQ ID NO: 361) and a VH domain comprising an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or 100% identity, to

(SEQ ID NO: 311)

EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVG

RIRTKRNNYATYYADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYC

VRHENFGNSYVSWFAHWGQGTLVTVSS.

- 392. A pharmaceutical composition comprising the T cell engager of embodiment 391, and at least one pharmaceutically acceptable excipient.
- 393. The pharmaceutical composition of embodiment 392, which is in a liquid form or is frozen.
- 394. The pharmaceutical composition of embodiment 392, which is formulated as a lyophilized powder or cake to be reconstituted prior to administration.
- 395. An injection device comprising the pharmaceutical composition of embodiment 392.
- 396. The injection device of embodiment 395, which comprises a syringe.
- 397. A polynucleotide sequence encoding the T cell engager of embodiment 391.
- 398. An expression vector comprising the polynucleotide sequence of embodiment 397.
- 399. A host cell comprising the expression vector of embodiment 398.
- 400. A method of producing the T cell engager of embodiment 391.
- 401. The method of embodiment 400, further comprising isolating the T cell engager from a host cell.
- 402. A protease-activatable T cell engager (paTCE) comprising a T cell engager (TCE) according embodiment 391, in the form of a single polypeptide chain, wherein the N-terminus of the TCE is fused to a first masking polypeptide by a first protease-cleavable linker and the C-terminus of the TCE is fused to a second masking polypeptide by a second protease-cleavable linker.
- 403. The paTCE of embodiment 402, wherein the first masking polypeptide is a first ELNN.
- 404. The paTCE of embodiment 402 or 402, wherein the second masking polypeptide is a second ELNN.
- 405. The paTCE of any one of embodiments 402-404, wherein TCE comprises an anti-PSMA VHH comprising the following amino acid sequence:

(SEQ ID NO: 549)

QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVWGWFRQAPGKEREFVG

AMSWSGSNRKVSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNKEYGRTWYDFNESDYWGQGTQVTVSS.

- 406. The paTCE of any one of embodiments 402-405, wherein TCE comprises an anti-CD3 scFv comprising a VH domain having the following amino acid sequence: EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKR NNYATYYADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCVRHENFGNSYVS WFAHWGQGTLVTVSS (SEQ ID NO: 311) and a VL domain having the following amino acid sequence:

(SEQ ID NO: 361)

ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGL

IGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLW

VFGGGTKLTVL.

- 407. A pharmaceutical composition comprising the paTCE of any one of embodiments 402-406, and at least one pharmaceutically acceptable excipient.
- 408. The pharmaceutical composition of embodiment 407, which is in a liquid form or is frozen.
- 409. The pharmaceutical composition of embodiment 407, which is formulated as a lyophilized powder or cake to be reconstituted prior to administration.
- 410. An injection device comprising the pharmaceutical composition of embodiment 409.
- 411. The injection device of embodiment 410, which comprises a syringe.
- 412. A polynucleotide sequence encoding the paTCE of any one of embodiments 402-406.
- 413. An expression vector comprising the polynucleotide sequence of embodiment 412.
- 414. A host cell comprising the expression vector of embodiment 413.
- 415. A method of producing the paTCE of any one of embodiments 402-406.
- 416. The method of embodiment 415, further comprising isolating the paTCE from a host cell.
- 417. A chimeric polypeptide, isolated polypeptide, fusion protein, antigen binding polypeptide, antibody or an antigen-binding fragment thereof that specifically binds PSMA, antibody or an antigen-binding fragment thereof that specifically binds CD3, multispecific antibody, T cell engager, or paTCE, produced by the method of any one of embodiments 208, 209, 244, 245, 26, 266, 292, 293, 305, 306, 331, 332, 374, 375, 389, 390, 400, 401, 415, or 416.
- 418. A polynucleotide sequence encoding the amino acid sequence: EAGRSAXHTPAGLTGP (SEQ ID NO: 7627), wherein X is any amino acid other than N.
- 419. The polynucleotide of embodiment 418, which is a vector.
- 420. The polynucleotide of embodiment 418, which is an isolated polynucleotide.
- 421. A cell line that expresses an exogenous polypeptide comprising the amino acid sequence: EAGRSAXHTPAGLTGP (SEQ ID NO: 7627), wherein X is any amino acid other than N.
- 422. The cell line of embodiment 421, wherein the exogenous polypeptide is a fusion protein according to any one of embodiments 267-283.
- 423. The cell line of embodiment 421 or 422, which is in culture or is frozen in a glass or plastic container.
- 424. The cell line of any one of embodiments 421-423, which is in a bioreactor.
- 425. The cell line of any one of embodiments 421-424, which is a stable cell line.
- 426. The cell line of any one of embodiments 421-425, which is a mammalian cell.
- 427. The cell line of embodiment 426, which is a CHO cell or a HEK293 cell.
- 428. The cell line of any one of embodiments 421-425, which is a prokaryotic cell.
- 429. The cell line of embodiment 428, which is an Escherichia coli cell.
- 430. A non-human animal that comprises an exogenous polypeptide comprising the amino acid sequence: EAGRSAXHTPAGLTGP (SEQ ID NO: 7627), wherein X is any amino acid other than N.
- 431. The polynucleotide sequence of any one of embodiments 418-420, the cell line of any one of embodiments 421-429, or the non-human animal of embodiment
- 430, wherein X is D, E, or Q.
- 432. The polynucleotide sequence of any one of embodiments 418-420, the cell line of any one of embodiments 421-429, or the non-human animal of embodiment 430, wherein X is G, A, V, L, I.
- 433. The polynucleotide sequence of any one of embodiments 418-420, the cell line of any one of embodiments 421-429, or the non-human animal of embodiment 430, wherein X is P.
- 434. The polynucleotide sequence of any one of embodiments 418-420, the cell line of any one of embodiments 421-429, or the non-human animal of embodiment 430, wherein X is F, Y, or W.
- 435. The polynucleotide sequence of any one of embodiments 418-420, the cell line of any one of embodiments 421-429, or the non-human animal of embodiment 430, wherein X is H, K, or R.
- 436. The polynucleotide sequence of any one of embodiments 418-420, the cell line of any one of embodiments 421-429, or the non-human animal of embodiment 430, wherein X is S, C, U, T, or M.
- 437. The polynucleotide sequence of any one of embodiments 418-420, the cell line of any one of embodiments 421-429, or the non-human animal of embodiment 430, wherein X is S.
- 438. A fusion protein comprising an anti-PSMA antibody or fragment according to any one of embodiments 333-343 and a biologically active protein.
- 439. A fusion protein comprising an anti-CD3 antibody or fragment according to any one of embodiments 344-365 and a biologically active protein.
- 440. The fusion protein of embodiment 438 or 439, wherein the biologically active protein comprises a cytokine, an enzyme, a hormone, a growth factor, a chemotherapeutic polypeptide, an antiviral polypeptide, or a toxin.
- 441. An immunoconjugate comprising an anti-PSMA antibody or fragment according to any one of embodiments 333-343 and a compound.
- 442. An immunoconjugate comprising an anti-CD3 antibody or fragment according to any one of embodiments 344-365 and a compound.
- 443. The immunoconjugate of embodiment 441 or 442, wherein the compound comprises chemotherapeutic agent.
- 444. The immunoconjugate of embodiment 441 or 442, wherein the compound comprises a diagnostic agent.
- 445. The immunoconjugate of embodiment 441 or 442, wherein the compound comprises a toxin, a radioactive molecule, a contrast agent, or a drug.

The following are examples of compositions and evaluations of compositions of the disclosure. It is understood that various some embodiments may be practiced, given the general description provided above.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

EXAMPLES
Example 1. Production of High Affinity Anti-PSMA VHH Domains

High affinity VHH domains were isolated from immunized llamas through in vitro screening. Multiple VHH domains that bind human PSMA (hPSMA) were identified.

The amino acid sequence of the hPSMA antigen (with an added HHHHHH (SEQ ID NO:48)sequence) was as follows:

(SEQ ID NO: 1028)

HHHHHHKSSNEATNITPKHNMKAFLDELKAENIKKFLYNFTQIPHLAGT

EQNFQLAKQIQSQWKEFGLDSVELAHYDVLLSYPNKTHPNYISIINEDG

NEIFNTSLFEPPPPGYENVSDIVPPFSAFSPQGMPEGDLVYVNYARTED

FFKLERDMKINCSGKIVIARYGKVFRGNKVKNAQLAGAKGVILYSDPAD

YFAPGVKSYPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRG

IAEAVGLPSIPVHPIGYYDAQKLLEKMGGSAPPDSSWRGSLKVPYNVGP

GFTGNFSTQKVKMHIHSTNEVTRIYNVIGTLRGAVEPDRYVILGGHRDS

WVFGGIDPQSGAAVVHEIVRSFGTLKKEGWRPRRTILFASWDAEEFGLL

GSTEWAEENSRLLQERGVAYINADSSIEGNYTLRVDCTPLMYSLVHNLT

KELKSPDEGFEGKSLYESWTKKSPSPEFSGMPRISKLGSGNDFEVFFQR

LGIASGRARYTKNWETNKFSGYPLYHSVYETYELVEKFYDPMFKYHLTV

AQVRGGMVFELANSIVLPFDCRDYAVVLRKYADKIYSISMKHPQEMKTY

SVSFDSLFSAVKNFTEIASKFSERLQDFDKSNPIVLRMMNDQLMFLERA

FIDPLGLPDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDIESKVDPSK

AWGEVKRQIYVAAFTVQAAAETLSEVA

Multiple VHH clones were identified, including those having the following amino acid sequences:

>P01C01R3

(SEQ ID NO: 1029)

QVQLVESGGGLVQAGGSLRLSCAASGRTVNSYAMGWFRQAPGKEREFVA

SQSWMGAITYDADYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYR

CAASRQARPGLHVREYDVWGQGTQVTVSSGPGGQHHHHHH

>P01H01R3

(SEQ ID NO: 1030)

EVQLVESGGGLVQPGGSLRLSCVASVSSFSTNDMGWYRQAPGKQRELVA

GITVGGNTFYAGSVKGRFTISRDNGKNTMYLQMNSLKPEDTAVYFCNVG

AKYRKPEWYSGEYWGQGTQVTVSSGPGGQHHHHHH

>P01G03R3

(SEQ ID NO: 1031)

EVLVESGGGLVQAGGSLRLSCVVSGIAFSPYHMAWYRQAPGKQHEWVA

VITTGGTTAYNETVEGRFSISRDNARSTVYLQMNSLKPEDTAVYYCNIY

GLSLKWGQGTQVTVSSGPGGQHHHHHH

>P01E02R3

(SEQ ID NO: 1032)

EVLVESGGGLVQAGGSLKLSCVANGPTFSTYAMAWFRQAPGKEHEFVAA

ITGDGDTTNNADSVKGRFTISRDNAKNRVYLQLNSLKPEDTAAYYCAAG

VHHTYTIPRLWLYWGQGTQVTVSSGPGGQHHHHHH

>CB01A01R3

(SEQ ID NO: 1033)

EVLVESGGGLVQAGGSLRISCTASERSVSTYTKGWFRQAPGKERHLVAA

ISYNGDTTYYSDSVKGRFTISRDNVKNTVNLQMNSLKPEDTAVYFCAAR

GSSWLYGTWDDYHYWGQGTQVTVSSGPGGQHHHHHH

>CB01B02R3

(SEQ ID NO: 1034)

QVQLVESGGGLVQAGDSLRLSCVTSGRTFDVYAMGWFRQAPGKERELVA

AINWSGSNKFHADSVKGRFTISRDNAWKTLSLQMNSLKPEDTAVYFCAA

STRLYGTTWYEFNDSDYWGQGTQVTVSSGPGGQHHHHHH

>CB01H01R3

(SEQ ID NO: 1035)

EVLVESGGGSVQAGGSLSLSCVASGRTFGIYVMGWFRQAPGKEREFVAA

ISWSGSNRLVSDSVKGRFTISRENAKNTIYLQMNGLKPEDTANYFCAAS

NRLYGRTWYDFNESDYWGQGTQVTVSSGPGGQHHHHHH

Example 2. Humanization of VHH Antibodies

Before humanization, a screen was performed by selecting 7 VHH sequences and testing them together with CD3.23 TCEs to screen for binding and function. Based on this screen, two different leads from Example 1 PSMA.2 (also referred to herein as CB01 H01 R3 and used in a uTCE (without the C-terminal GPGGQHHHHHH(SEQ ID NO:9178) portion of the sequence shown above) together with CD3.23) and PSMA.3 (also referred to herein as CB01B02R3 and used in a uTCE (without the C-terminal GPGGQHHHHHH(SEQ ID NO:9178) portion of the sequence shown above) together with CD3.23) were selected for humanization. The humanized sequences described herein are expected to retain canonical structure of the CDR-loops.

SEQ ID

NO
AMINO ACID SEQUENCE

1036
LTGPATSGSETPGTEVQLVESGGGLVQAGGSLRISCTASERSVSTYTKGWF

RQAPGKERHLVAAISYNGDTTYYSDSVKGRFTISRDNVKNTVNLQMNSLKPE

DTAVYFCAARGSSWLYGTWDDYHYWGQGTQVTVSSGGGGSGGGSELVVT

QEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKR

APGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLT

VLGATPPETGAETESPGETTGGSAESEPPGEGEVQLLESGGGIVQPGGSLKL

SCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFT

ISRDDSKNTVYLQMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVT

VSSGTAEAASASGEAGRSANHTPAG

1037
LTGPATSGSETPGTQVQLVESGGGLVQAGGSLRLSCAASGRTVNSYAMGW

FRQAPGKEREFVASQSWMGAITYDADYADSVKGRFTISRDNAKNTLYLQMN

SLKPEDTAVYRCAASRQARPGLHVREYDVWGQGTQVTVSSGGGGSGGGS

ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIG

GTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGG

GTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLLESGGGIVQPG

GSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSV

KDRFTISRDDSKNTVYLQMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWGQ

GTLVTVSSGTAEAASASGEAGRSANHTPAG

1038*
LTGPATSGSETPGTEVQLVESGGGSVQAGGSLSLSCVASGRTFGIYVMGWF

RQAPGKEREFVAAISWSGSNRLVSDSVKGRFTISRENAKNTIYLQMNGLKPE

DTANYFCAASNRLYGRTWYDFNESDYWGQGTQVTVSSGGGGSGGGSELV

VTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTN

KRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTK

LTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLLESGGGIVQPGGSL

KLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDR

FTISRDDSKNTVYLQMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTL

VTVSSGTAEAASASGEAGRSANHTPAG

1039
LTGPATSGSETPGTQVQLVESGGGLVQAGDSLRLSCVTSGRTFDVYAMGWF

RQAPGKERELVAAINWSGSNKFHADSVKGRFTISRDNAWKTLSLQMNSLKP

EDTAVYFCAASTRLYGTTWYEFNDSDYWGQGTQVTVSSGGGGSGGGSELV

VTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTN

KRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTK

LTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLLESGGGIVQPGGSL

KLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDR

FTISRDDSKNTVYLQMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTL

VTVSSGTAEAASASGEAGRSANHTPAG

1040
LTGPATSGSETPGTEVQLVESGGGLVQPGGSLRLSCVASVSSFSTNDMGWY

RQAPGKQRELVAGITVGGNTFYAGSVKGRFTISRDNGKNTMYLQMNSLKPE

DTAVYFCNVGAKYRKPEWYSGEYWGQGTQVTVSSGGGGSGGGSELVVTQ

EPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRA

PGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTV

LGATPPETGAETESPGETTGGSAESEPPGEGEVQLLESGGGIVQPGGSLKLS

CAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI

SRDDSKNTVYLQMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVT

VSSGTAEAASASGEAGRSANHTPAG

1041
LTGPATSGSETPGTEVQLVESGGGLVQAGGSLKLSCVANGPTFSTYAMAWF

RQAPGKEHEFVAAITGDGDTTNNADSVKGRFTISRDNAKNRVYLQLNSLKPE

DTAAYYCAAGVHHTYTIPRLWLYWGQGTQVTVSSGGGGSGGGSELVVTQE

PSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAP

GTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL

GATPPETGAETESPGETTGGSAESEPPGEGEVQLLESGGGIVQPGGSLKLS

CAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI

SRDDSKNTVYLQMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVT

VSSGTAEAASASGEAGRSANHTPAG

1042
LTGPATSGSETPGTEVQLVESGGGLVQAGGSLRLSCVVSGIAFSPYHMAWY

RQAPGKQHEWVAVITTGGTTAYNETVEGRFSISRDNARSTVYLQMNSLKPED

TAVYYCNIYGLSLKWGQGTQVTVSSGGGGSGGGSELVVTQEPSLTVSPGGT

VTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSL

LGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVLGATPPETGAE

TESPGETTGGSAESEPPGEGEVQLLESGGGIVQPGGSLKLSCAASGFTFNTY

AMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTVYL

QMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTVSSGTAEAASA

SGEAGRSANHTPAG

*This is the uTCE sequence from AC2591 (i.e., the unmasked TCE of AC2591).

27 humanized variants of PSMA.2 and 31 humanized variants of PSMA.3 were designed. Table 9a lists the variants of PSMA.2 as VHH1 through VHH27. Table 9b lists the variants of PSMA.3 as VHH1 through VHH31.

As shown in FIG. 2A -FIG. 2D, several biophysical properties of the PSMA.2 variants were tested. Several clones exhibited binding similar or equivalent to the parental clone (FIG. 2B; clones with weaker binding are shown with black dots). Several clones exhibited equivalent thermal stability at 60° C. and 62° C. compared to parental clone (FIG. 2C; close with less stability shown with black dots). uTCEs from clones AC2717 and AC2728 (i.e., the unmasked TCEs of AC2717 and AC2728) survived at 65° C. (FIG. 2E). AC2715 had the highest abundance (FIG. 2A). Based on these properties, AC2728, AC2717 and AC2715 were selected as top humanized leads from the PSMA.2 pool.

As shown in FIG. 3A-FIG. 3C, several biophysical properties of the PSMA.3 variants were tested. uTCEs from clones AC2755 and AC2750 exhibited above average binding and were the most thermally stable clones.

Of these humanized variants, two (PSMA.5 (also known as VHH3 and used in the uTCE of clone AC2717 together with CD3.23) and PSMA.6 (also known as VHH14 and used in the uTCE of clone AC2728 together with CD3.23)) showed slightly reduced but comparable binding affinity and comparable stability relative to PSMA.2.

For the study of bispecific antibodies comprising variants of PSMA.2 and PSMA.3, each variant was linked to CD3.23 to form the bispecific antibody.

TABLE 9a

Humanized variants of PSMA.2

uTCE

Clone #

that the

VHH was

used in

together

VHH
with CD3.23
Sequence

VHH1
uTCE of
EVLVESGGGLVQPGGSLRLSCAASGRTFGIYVMGWVR

AC2715
QAPGKEREFVSAISWSGSNRLVSDSVKGRFTISRDNSKN

TLYLQMNSLRAEDTAVYYCAASNRLYGRTWYDFNESDY

WGQGTQVTVSS (SEQ ID NO: 9179)

VHH2
Utce of
EVQLVESGGGLVQPGGSLRLSCAASGRTFGIYVMGWVR

AC2716
QAPGKEREFVSAISWSGSNRLVSDSVKGRFTISRDNAKN

SLYLQMNSLRAEDTAVYYCAASNRLYGRTWYDFNESDY

WGQGTQVTVSS (SEQ ID NO: 9180)

VHH3
uTCE of
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVMGWV

AC2717
RQAPGKEREFVAAISWSGSNRLVSDSVKGRFTISRDNSK

NTLYLQMNSLRAEDTAVYYCAASNRLYGRTWYDFNESD

YWGQGTQVTVSS (SEQ ID NO: 9181)

VHH4
uTCE of
EVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVMGWVR

AC2718
QAPGKEREFVAAISWSGSNRLVSDSVKGRFTISRDNSKN

TLYLQMNSLRAEDTAVYFCAASNRLYGRTWYDFNESDY

WGQGTLVTVSS (SEQ ID NO: 9182)

VHH5
uTCE of
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVMGWV

AC2719
RQAPGKEREFVAAISWSGSNRLVSDSVKGRFTISRDNSK

NTLYLQMNSLRAEGTAVYFCAASNRLYGRTWYDFNESD

YWGQGTMVTVSS (SEQ ID NO: 9183)

VHH6
uTCE of
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVMGWV

AC2720
RQAPGKEREFVAAISWSGSNRLVSDSVKGRFTISRDNSK

NTLYLQMNSLRAEDTAVYFCAASNRLYGRTWYDFNESD

YWGQGTTVTVSS (SEQ ID NO: 9184)

VHH7
uTCE of
EVQLVESGGGLVQPGGSLRLSCAVSGRTFGIYVMGWVR

AC2721
QAPGKEREFVSAISWSGSNRLVSDSVKGRFTISRDNSKN

TLYLQMNSLRAEDTASYFCAASNRLYGRTWYDFNESDY

WGQGTLVTVSS (SEQ ID NO: 9185)

VHH8
Utce of
EVQLVESGGGVVQPGRSLRISCAASGRTFGIYVMAWFR

AC2722
QAPGKEREFVAVISWSGSNKLVTDSVKGRFTISRDNSKN

TVYLQMNSLRPEDTANYFCAASNRLYGRTWYDFNESDY

WGQGTLVTVSS (SEQ ID NO: 9186)

VHH9
uTCE of
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVMAWVR

AC2723
QAPGKEREFVAAISWSGSNRLVSDSVKGRFTISRDNSKN

TLYLQMNSLRPEDTAVYFCAASNRLYGRTWYDFNESDY

WGQGTLVTVSS (SEQ ID NO: 9187)

VHH10
uTCE of
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVMAWVR

AC2724
QAPGKEREFVAAISWSGSNRLVSDSVKGRFTISRDNSKN

TLYLQMNSLRPEDTAVYFCAASNRLYGRTWYDFNESDY

WGQGTLVTVSS (SEQ ID NO: 9187)

VHH11
Utce of
QVQLVESGGGVVQAGGSLSLSCVASGRTFGIYVMAWFR

AC2725
QAPGKEREFLTVISWSGSNKLVTDSVKGRFTISRDNSKN

TLYLQMNSLRAEDTGLYFCAASNRLYGRTWYDFNESDY

WGQGTLLTVSS (SEQ ID NO: 9188)

VHH12
uTCE of
EVKLVESGGGLVQPGRSLRLSCVASGRTFGIYVMGWVR

AC2726
QVPGKSRQFVSAISWSGSNRLVSDSVKGRFTISRDNAK

NSLFLQMNSLRPEDTALYFCAASNRLYGRTWYDFNESD

YWGQGTQVTVSS (SEQ ID NO: 9189)

VHH13
uTCE of
EVQLLESGGGLVQPGGSLRLSCAASGRTFGIYVMGWVR

AC2727
QAPGKEREFVSAISWSGSNRLVSDSVKGRFTISRDNSKN

TLYLQMNSLRAEDTAIYFCAASNRLYGRTWYDFNESDY

WGQGTLVTVSS (SEQ ID NO: 9190)

VHH14
uTCE of
EVQLVESGGGVVQPGRSLRLSCAASGRTFGIYVMGWVR

AC2728
QAPGKEREFVAAISWSGSNRLVSDSVKGRFTISRDNSKN

TLYLQMNSLRAEDTAVYFCAASNRLYGRTWYDFNESDY

WGQGTTVTVSS (SEQ ID NO: 9191)

VHH15
uTCE of
EVQLVESGGGSVQPGGSLRLSCAASGRTFGIYVMAWFR

AC2729
QAPGKEREFVAVISWSGSNRLVTDSVKGRFTISRENSKN

TLYLQMNSLRAEDTANYFCAASNRLYGRTWYDFNESDY

WGQGTQVTVSS (SEQ ID NO: 9192)

VHH16
uTCE of
EVQLVESGGGLVQPGGSLRLSCAASGRTFGIYVMGWFR

AC2730
QAPGKEREFVSVISWSGSNRLVSDSVKGRFTISRENAKN

SLYLQMNSLRAEDTANYFCAASNRLYGRTWYDFNESDY

WGQGTQVTVSS (SEQ ID NO: 9193)

VHH17
uTCE of
EVQLVESGGGLVQPGGSLRLSCAASGRTFGIYVMAWFR

AC2731
QAPGKEREFVSAISWSGSNRLVSDSVKGRFTISRDNSKN

TLYLQMNSLRAEDTAVYFCAASNRLYGRTWYDFNESDY

WGQGTQVTVSS (SEQ ID NO: 9194)

VHH18
uTCE of
EVQLVESGGGLVQPGGSLRLSCAVSGRTFGIYVMGWVR

AC2732
QAPGKEREFVSAITWSGTNRLVSDSVKGRFTISRDNSKN

TLYLQMNSLRAEDTASYFCAASNRLYGRTWYDFNESDY

WGQGTLVTVSS (SEQ ID NO: 9195)

VHH19
uTCE of
EVQLVESGGGLVQPGGSLRLSCAVSGRTFGIYVLGWFR

AC2733
QAPGKEREFVSAISWSGSNRLVSDSVKGRFTISRDNSKN

TLYLQMNSLRAEDTASYFCAGSNRLYGRTWYDFNESDY

WGQGTQVTVSS (SEQ ID NO: 9196)

VHH20
uTCE of
EVQLVESGGGLVQPGGSLRLSCAVSGRTFGIYVLGWFR

AC2734
QAPGKEREFVSAITWSGTNRLVSDSVKGRFTISRDNSKN

TLYLQMNSLRAEDTASYFCAGSNRLYGRTWYDFNESDY

WGQGTQVTVSS (SEQ ID NO: 9197)

VHH21
uTCE of
EVQLVESGGGLVQPGGSLRLSCAVSGRTFGIYVLGWFR

AC2735
QAPGKEREFVSAITWSGTNRLVSDSVKGRFTISRDNSKN

TLYLQMNSLRAEDTASYFCGASNRLYGRTWYDFNESDY

WGQGTQVTVSS (SEQ ID NO: 9198)

VHH22
uTCE of
EVQLLESGGGLVQPGGSLRLSCAASGRTFGIYVLGWFR

AC2736
QAPGKEREFVSAISYSGSNRLVSDSVKGRFTISRDNSKN

TLYLQMNSLRAEDTAIYFCGASNRLYGRTWYDFNESDY

WGQGTQVTVSS (SEQ ID NO: 9199)

VHH23
uTCE of
EVQLLESGGGLVQPGGSLRLSCAASGRTFGIYVMGWFR

AC2737
QAPGKEREFVSAISWSGSDRLVSDSVKGRFTISRDNSKN

TLYLQMNSLRAEDTAIYFCAASNRLYGRTWYDFNESDY

WGQGTLVTVSS (SEQ ID NO: 9200)

VHH24
uTCE of
EVQLLESGGGLVQPGGSLRLSCAASGRTFGIYVMGWVR

AC2738
QAPGKEREFVSAITWSGTNRLVSDSVKGRFTISRDNSKN

TLYLQMNSLRAEDTAIYFCGASNRLYGRTWYDFNESDY

WGQGTLVTVSS (SEQ ID NO: 9201)

VHH25
uTCE of
EVQLVESGGGSVQPGGSLRLSCAASGRTFGIYVMAWFR

AC2739
QAPGKEREFVAVISWSGTNRLVTDSVKGRFTISRENSKN

TLYLQMNSLRAEDTANYFCAGSNRLYGRTWYDFNESDY

WGQGTQVTVSS (SEQ ID NO: 9202)

VHH26
uTCE of
EVQLVESGGGSVQPGGSLRLSCAASGRTFGIYVMAWFR

QAPGKEREFVAVISWSGTNRLVTDSVKGRFTISRENSKN

TLYLQMNSLRAEDTANYFCGASNRLYGRTWYDFNESDY

WGQGTQVTVSS (SEQ ID NO: 9203)

VHH27
uTCE of
EVQLVESGGGSVQPGGSLRLSCAASGRTFGIYVMAWFR

AC2741
QAPGKEREFVAVITWSGTNRLVTDSVKGRFTISRENSKN

TLYLQMNSLRAEDTANYFCGASNRLYGRTWYDFNESDY

WGQGTQVTVSS (SEQ ID NO: 9204)

TABLE 9b

Humanized variants of PSMA.3

uTCE

Clone #

the VHH

was used in

Together

VHH
with CD3.23
Sequence

VHH28
uTCE of
QVQLVESGGGVVQPGRSLRLSCAASGRTFDVYAMGWF

AC2742
RQAPGKERELVAAINWSGSNKFHADSVKGRFTISRDNS

KNTLYLQMNSLRAEDTAVYFCAASTRLYGTTWYEFNDS

DYWGQGTQVTVSS (SEQ ID NO: 9205)

VHH29
uTCE of
EVQLLESGGGLVQPGGSLRLSCAASGRTFDVYAMGWF

AC2743
RQAPGKERELVSAINWSGSNKFHADSVKGRFTISRDNS

KNTLYLQMNSLRAEDTAVYFCAASTRLYGTTWYEFNDS

DYWGQGTQVTVSS (SEQ ID NO: 9206)

VHH30
uTCE of
EVLVESGGGLVQPGGSLRLSCAASGRTFDVYAMSWV

AC2744
RQAPGKERELVAAINWSGSNKFHADSVKGRFTISRDNA

KNSLYLQMNSLRAEDTAVYFCAASTRLYGTTWYEFNDS

DYWGQGTQVTVSS (SEQ ID NO: 9207)

VHH31
uTCE of
QVQLVESGGGLVQPGGSLRLSCSASGRTFDVYAMGWV

AC2745
RQAPGKERELVSAINWSGSNKFHADSVKGRFTISRDNS

KNTLYLQMNSLRAEDTAVYFCAASTRLYGTTWYEFNDS

DYWGQGTQVTVSS (SEQ ID NO: 9208)

VHH32
uTCE of
QVQLEESGGGVVQPGRSLRLSCVVSGRTFDVYAMGW

AC2746
VRQAPGKERELVAAINWSGSNKFHADSVKGRFTISRDN

SKNTLNLQMNSLRPEDTAVYFCAASTRLYGTTWYEFND

SDYWGQGTQVTVSS (SEQ ID NO: 9209)

VHH33
uTCE of
QVQLVESGGGVVQPGRSLRLSCAASGRTFDVYAMAWF

AC2747
RQAPGKERELVAAINWSGSNKFHADSVKGRFTISRDNS

KNTLFLQMNSLRPEDTAVYFCAASTRLYGTTWYEFNDS

DYWGQGTQVTVSS (SEQ ID NO: 9210)

VHH34
uTCE of
QVQLVQSGGGVVQPGRSLRLSCAASGRTFDVYAMGW

AC2748
FRQAPGKERELVAAINWSGSNKFHADSVKGRFTISRDN

SKNTLSLQMNSLRAEDTAVYFCAASTRLYGTTWYEFND

SDYWGQGTQVTVSS (SEQ ID NO: 9211)

VHH35
uTCE of
QVQLVESGGGVVQPGRSLRLSCAASGRTFDVYAMGWF

AC2749
RQAPGKERELVAAINWSGSNKFHADSVKGRFTISRDNS

RNTLYLQMNSLRAEDTAVYFCAASTRLYGTTWYEFNDS

DYWGQGTQVTVSS (SEQ ID NO: 9212)

VHH36
uTCE of
QVQLVESGGGVVQPGRSLRLSCAASGRTFDVYAMGWF

AC2750
RQAPGKGRELVAAINWSGSNKFHADSVKGRFTISRDNS

KNTLYLQMNSLRPEDTAVYFCAASTRLYGTTWYEFNDS

DYWGQGTQVTVSS (SEQ ID NO: 9213)

VHH37
uTCE of
QVQLVESGGGVVQPGRSLRLSCAASGRTFDVYAMGWF

AC2751
RQAPGKERELVAAINWSGSNKFHADSVKGRFTISRDNS

KKTLYLQMNSLRAEDTAVYFCAASTRLYGTTWYEFNDS

DYWGQGTQVTVSS (SEQ ID NO: 9214)

VHH38
uTCE of
EVLVESGGGLVQPGGSLRLSCAASGRTFDVYAMGWF

AC2752
RQAPGKGRELVSAINWSGSNKFHADSVKGRFTISRDNS

KNTLYLQMNSLRAEDTAVYFCAASTRLYGTTWYEFNDS

DYWGQGTQVTVSS (SEQ ID NO: 9215)

VHH39
uTCE of
QVQLVESGGGVVQPGRSLRLSCAASGRTFDVYAMGWF

AC2753
RQAPGKERELVAAINWSGSNKFHADSVKGRFTISRDNS

KNTLFLQMNSLRADDTAVYFCAASTRLYGTTWYEFNDS

DYWGQGTQVTVSS (SEQ ID NO: 9216)

VHH40
uTCE of
QVQVIESGGGVVQSGKSLRLACTTSGRTFDVYAMGWF

AC2754
RQAPGKGRELVAAINWSGSNKFHADSVKGRFTISRDNS

KNTLYLQMNSLRAEDTAVYFCAASTRLYGTTWYEFNDS

DYWGQGTQVTVSS (SEQ ID NO: 9217)

VHH41
uTCE of
QVQLVESGGGVVQPGRSLRLSCAASGRTFDVYAMGW

AC2755
VRQAPGKERELVAAINWSGSNKFHADSVKGRFTISRDN

SKNTLYLQMNSLKTEDTAMYFCAASTRLYGTTWYEFND

SDYWGQGTQVTVSS (SEQ ID NO: 9218)

VHH42
uTCE of
EVQLLESGGGLVQSGDSLRLSCATSGRTFDVYAMSWF

AC2756
RQAPGKERELVSAIDWSGSNKFHADSVKGRFTISRDNS

WKTLYLQMNSLRPEDTAVYFCAASTRLYGTTWYEFNDS

DYWGQGTQVTVSS (SEQ ID NO: 9219)

VHH43
uTCE of
QVQLVESGGGLVQPGRSLRLSCATSGFTFDVYAMGWF

AC2757
RQAPGKERELVSAINWGGSNKFHADSVKGRFTISRDNA

WKTLYLQMNSLRAEDTAVYFCAASTRLYGTTWYEFNDS

DYWGQGTQVTVSS (SEQ ID NO: 9220)

VHH44
uTCE of
QVQLVESGGGVVQPGDSLRLSCATSGRTFDVYAMGWF

AC2758
RQAPGKERELVAAINWAGSNKFHADSVKGRFTISRDNS

KNTLSLQMNSLRAEDTAVYFCAASTRLYGTTWYEFNDS

DYWGQGTQVTVSS (SEQ ID NO: 9221)

VHH45
uTCE of
QVQLVESGGGVVQPGDSLRLSCATSGRTFDVYAMSWF

AC2759
RQAPGKERELVAAINWAGSNKFHADSVKGRFTISRDNS

KNTLSLQMNSLRPEDTAVYFCAASTRLYGTTWYEFNDS

DYWGQGTQVTVSS (SEQ ID NO: 9222)

VHH46
uTCE of
QVQLVESGGGVVQPGDSLRLSCATSGRTFDVYAMAWF

AC2760
RQAPGKERELVAAINWAGSNKFHADSVKGRFTISRDNS

KNTLSLQMNSLRPEDTAVYFCAASTRLYGTTWYEFNDS

DYWGQGTQVTVSS (SEQ ID NO: 9223)

VHH47
uTCE of
QVQLVESGGGVVQPGDSLRLSCATSGRTFDVYAMAWF

AC2761
RQAPGKERELVAAINWGGSNKFHADSVKGRFTISRDNS

KNTLSLQMNSLRPEDTAVYFCAASTRLYGTTWYEFNDS

DYWGQGTQVTVSS (SEQ ID NO: 9224)

VHH48
uTCE of
EVQLLESGGGLVQSGDSLRLSCATSGRTFDVYAMSWF

AC2762
RQAPGKERELVSAINWSGSNKFHADSVKGRFTISRDNS

WKTLYLQMNSLRPEDTAVYFCGATSRLYGTTWYEFND

SDYWGQGTQVTVSS (SEQ ID NO: 9225)

VHH49
uTCE of
QVQLVESGGGLVQPGRSLRLSCATSGFTFDVYAMGWF

AC2763
RQAPGKERELVSAINWSGSNKFHADSVKGRFTISRDNA

WKTLYLQMNSLRAEDTAVYFCAGSTRLYGTTWYEFND

SDYWGQGTQVTVSS (SEQ ID NO: 9226)

VHH50
uTCE of
QVQLVESGGGVVQPGDSLRLSCATSGRTFDVYAMGWF

AC2764
RQAPGKERELVAAINWSGSNKFHADSVKGRFTISRDNS

KNTLSLQMNSLRAEDTAVYFCGASTRLYGTTWYEFNDS

DYWGQGTQVTVSS (SEQ ID NO: 9227)

VHH51
uTCE of
QVQLVESGGGVVQPGDSLRLSCATSGRTFDVYAMSWF

AC2765
RQAPGKERELVAAINWSGSNKFHADSVKGRFTISRDNS

KNTLSLQMNSLRPEDTAVYFCAGTSRLYGTTWYEFNDS

DYWGQGTQVTVSS (SEQ ID NO: 9228)

VHH52
uTCE of
QVQLVESGGGVVQPGDSLRLSCATSGRTFDVYAMAWF

AC2766
RQAPGKERELVAAINWSGSNKFHADSVKGRFTISRDNS

KNTLSLQMNSLRPEDTAVYFCAGTTRLYGTTWYEFNDS

DYWGQGTQVTVSS (SEQ ID NO: 9229)

VHH53
uTCE of
EVQLLESGGGLVQSGDSLRLSCATSGRTFDVYAMSWF

AC2767
RQAPGKERELVSAIDWSGSNKFHADSVKGRFTISRDNS

WKTLYLQMNSLRPEDTAVYFCGATSRLYGTTWYEFND

SDYWGQGTQVTVSS (SEQ ID NO: 9230)

VHH54
uTCE of
QVQLVESGGGLVQPGRSLRLSCATSGFTFDVYAMGWF

AC2768
RQAPGKERELVSAINWGGSNKFHADSVKGRFTISRDNA

WKTLYLQMNSLRAEDTAVYFCAGSTRLYGTTWYEFND

SDYWGQGTQVTVSS (SEQ ID NO: 9231)

VHH55
uTCE of
QVQLVESGGGVVQPGDSLRLSCATSGRTFDVYAMGWF

AC2769
RQAPGKERELVAAINWAGSNKFHADSVKGRFTISRDNS

KNTLSLQMNSLRAEDTAVYFCGASTRLYGTTWYEFNDS

DYWGQGTQVTVSS (SEQ ID NO: 9232)

VHH56
uTCE of
QVQLVESGGGVVQPGDSLRLSCATSGRTFDVYAMSWF

AC2770
RQAPGKERELVAAINWAGSNKFHADSVKGRFTISRDNS

KNTLSLQMNSLRPEDTAVYFCAGTSRLYGTTWYEFNDS

DYWGQGTQVTVSS (SEQ ID NO: 9233)

VHH57
uTCE of
QVQLVESGGGVVQPGDSLRLSCATSGRTFDVYAMAWF

AC2771
RQAPGKERELVAAINWAGSNKFHADSVKGRFTISRDNS

KNTLSLQMNSLRPEDTAVYFCAGTTRLYGTTWYEFNDS

DYWGQGTQVTVSS (SEQ ID NO: 9234)

VHH58
uTCE of
QVQLVESGGGVVQPGDSLRLSCATSGRTFDVYALAWF

AC2772
RQAPGKERELVAAINWGGSNKFHADSVKGRFTISRDNS

KNTLSLQMNSLRPEDTAVYFCAGTSRLYGTTWYEFNDS

DYWGQGTQVTVSS (SEQ ID NO: 9235)

Example 3. Removal of Putative T Cell Epitopes

PSMA.5 was used as the template for removal of PTE based on a proprietary computer prediction program. Three potential hot spots were identified in PSMA.5 that were labeled as Pep19, Pep26, and Pep33. A round of screening was performed by generating 102 point mutations (Pool 1). Mutations from Pool 1 were screened for binding and thermal stability. A pool (Pool 2) of combined mutations was generated from Pool 1 and comprised 40 clones. Each clone in Pool 2 had more than 2 mutations. Pool 2 was screened for binding and thermal stability. PSMA.263 from Pool 2 was selected as a template and combined with additional mutations from Pool 1 to make Pool 3. A total of 60 variants from Pool 3 were tested. PSMA.350 from Pool 3 was selected as the final VHH lead in AMX-500. FIG. 4 depicts PTE scores of representative PSMA variants and CD3 variants. The graph shows molecules with known Antidrug Antibody (ADA) and their corresponding PTE score. The higher score indicates greater chance of having putative T cell epitopes.

PSMA.350 was further tested for T cell immunogenicity in an EpiScreen™ DC: T cell immunogenicity assay. Briefly, PBMCs were extracted from 20 healthy donors. Monocyte-derived dendritic cells and CD4+ T cells were isolated. On Day 4 of culturing, test antigen was added to the culture. The neoantigen Keyhole limpet haemocyanin (KLH) was used as a positive control in a separate culture. CD4+ T cells were isolated on Day 5. T cell proliferation was monitored on Day 9, 10, 11, and 12. As shown in FIG. 5, PSMA.350 induced positive donor responses in only 5% of donor samples, while the KLH positive control induced a position response in 95% of samples.

Tables 10, 10b, and 10c provide the PSMA binding sequences for Pools 1, 2, and 3, respectively. The tables also provide the TCE clone numbers in which the PSMA binding sequences were used together with CD3.23.

TABLE 10a

PSMA VHH sequences from Pool 1

Clone #

the VHH

was used in

PSMA

Together

Mutation
domain
Sequence
with CD3.23

None
PSMA.5
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC2717

FGWVRQAPGKEREFVAAISWSGSNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRLYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9236)

Y32A
PSMA.41
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3251

MGWARQAPGKEREFVAAISWTGSNRYVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRTYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9237)

Y32K
PSMA.42
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIKV
AC3252

MGWVRQAPGKEREFVAAISWSGSNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRLYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9238)

V33A
PSMA.43
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYA
AC3253

MGWVRQAPGKEREFVAAISWSGSNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRLYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9239)

V33H
PSMA.44
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIY
AC3254

HMGWVRQAPGKEREFVAAISWSGSNRLVSDSV

KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA

ASNRLYGRTWYDFNESDYWGQGTQVTVSS

(SEQ ID NO: 9240)

V33P
PSMA.45
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYP
AC3255

MGWVRQAPGKEREFVAAISWSGSNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRLYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9241)

V33S
PSMA.46
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3256

MGWARQAPGKEREFVAAISWTGSNRYVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRTYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9237)

V33W
PSMA.47
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIY
AC3257

WMGWVRQAPGKEREFVAAISWSGSNRLVSDS

VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC

AASNRLYGRTWYDFNESDYWGQGTQVTVSS

(SEQ ID NO: 9242)

V33Y
PSMA.48
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYY
AC3258

MGWVRQAPGKEREFVAAISWSGSNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRLYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9243)

M34F
PSMA.49
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3259

FGWVRQAPGKEREFVAAISWSGSNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRLYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9236)

M34W
PSMA.50
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3260

WGWVRQAPGKEREFVAAISWSGSNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRLYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9244)

V37A
PSMA.51
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3261

MGWARQAPGKEREFVAAISWSGSNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRLYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9245)

V37I
PSMA.52
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3262

MGWIRQAPGKEREFVAAISWSGSNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRLYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9246)

V37F
PSMA.53
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3263

MGWFRQAPGKEREFVAAISWSGSNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRLYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9247)

V37P
PSMA.54
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3264

MGWPRQAPGKEREFVAAISWSGSNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRLYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9248)

V37Y
PSMA.55
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3265

MGWYRQAPGKEREFVAAISWSGSNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRLYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9249)

R38Q
PSMA.56
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3266

MGWVQQAPGKEREFVAAISWSGSNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRLYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9250)

R38K
PSMA.57
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3267

MGWVKQAPGKEREFVAAISWSGSNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRLYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9251)

R38S
PSMA.58
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3268

MGWVSQAPGKEREFVAAISWSGSNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRLYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9252)

F47L
PSMA.59
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3269

MGWVRQAPGKERELVAAISWSGSNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRLYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9253)

F47W
PSMA.60
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3270

MGWVRQAPGKEREWVAAISWSGSNRLVSDSV

KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA

ASNRLYGRTWYDFNESDYWGQGTQVTVSS (SE

Q ID NO: 9254)

F47Y
PSMA.61
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3271

MGWVRQAPGKEREYVAAISWSGSNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRLYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9255)

V48W
PSMA.62
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3272

MGWVRQAPGKEREFWAAISWSGSNRLVSDSV

KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA

ASNRLYGRTWYDFNESDYWGQGTQVTVSS

(SEQ ID NO: 9256)

A49G
PSMA.63
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3273

MGWVRQAPGKEREFVGAISWSGSNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRLYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9257)

I51A
PSMA.64
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3274

MGWVRQAPGKEREFVAAASWSGSNRLVSDSV

KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA

ASNRLYGRTWYDFNESDYWGQGTQVTVSS

(SEQ ID NO: 9258)

I51R
PSMA.65
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3275

MGWVRQAPGKEREFVAARSWSGSNRLVSDSV

KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA

ASNRLYGRTWYDFNESDYWGQGTQVTVSS

(SEQ ID NO: 9259)

I51E
PSMA.66
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3276

MGWVRQAPGKEREFVAAESWSGSNRLVSDSV

KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA

ASNRLYGRTWYDFNESDYWGQGTQVTVSS

(SEQ ID NO: 9260)

I51Q
PSMA.67
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3277

MGWVRQAPGKEREFVAAQSWSGSNRLVSDSV

KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA

ASNRLYGRTWYDFNESDYWGQGTQVTVSS

(SEQ ID NO: 9261)

I51G
PSMA.68
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3278

MGWVRQAPGKEREFVAAGSWSGSNRLVSDSV

KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA

ASNRLYGRTWYDFNESDYWGQGTQVTVSS

(SEQ ID NO: 9262)

I51H
PSMA.69
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3279

MGWVRQAPGKEREFVAAHSWSGSNRLVSDSV

KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA

ASNRLYGRTWYDFNESDYWGQGTQVTVSS

(SEQ ID NO: 9263)

I51M
PSMA.70
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3280

MGWVRQAPGKEREFVAAMSWSGSNRLVSDSV

KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA

ASNRLYGRTWYDFNESDYWGQGTQVTVSS

(SEQ ID NO: 9264)

I51P
PSMA.71
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3281

MGWVRQAPGKEREFVAAPSWSGSNRLVSDSV

KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA

ASNRLYGRTWYDFNESDYWGQGTQVTVSS

(SEQ ID NO: 9265)

I51S
PSMA.72
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3282

MGWVRQAPGKEREFVAASSWSGSNRLVSDSV

KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA

ASNRLYGRTWYDFNESDYWGQGTQVTVSS

(SEQ ID NO: 9266)

I51W
PSMA.73
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3283

MGWVRQAPGKEREFVAAWSWSGSNRLVSDSV

KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA

ASNRLYGRTWYDFNESDYWGQGTQVTVSS

(SEQ ID NO: 9267)

I51V
PSMA.74
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3284

MGWVRQAPGKEREFVAAVSWSGSNRLVSDSV

KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA

ASNRLYGRTWYDFNESDYWGQGTQVTVSS

(SEQ ID NO: 9268)

S52A
PSMA.75
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3285

MGWVRQAPGKEREFVAAIAWSGSNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRLYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9269)

S52D
PSMA.76
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3286

MGWVRQAPGKEREFVAAIDWSGSNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRLYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9270)

S52E
PSMA.77
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3287

MGWVRQAPGKEREFVAAIEWSGSNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRLYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9271)

S52G
PSMA.78
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3288

MGWVRQAPGKEREFVAAWSWSGSNRLVSDSV

KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA

ASNRLYGRTWYDFNESDYWGQGTQVTVSS

(SEQ ID NO: 9267)

S52H
PSMA.79
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3289

MGWVRQAPGKEREFVAAIHWSGSNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRLYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9272)

S52P
PSMA.80
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3290

MGWVRQAPGKEREFVAAIPWSGSNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRLYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9273)

S52T
PSMA.81
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3291

MGWVRQAPGKEREFVAAITWSGSNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRLYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9274)

S52W
PSMA.82
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3292

MGWVRQAPGKEREFVAAIWWSGSNRLVSDSV

KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA

ASNRLYGRTWYDFNESDYWGQGTQVTVSS

(SEQ ID NO: 9275)

S52Y
PSMA.83
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3293

MGWVRQAPGKEREFVAAIYWSGSNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRLYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9276)

W53V
PSMA.84
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3294

MGWVRQAPGKEREFVAAISWSGSNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRYYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9277)

S54A
PSMA.85
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3295

MGWVRQAPGKEREFVAAISWSGSNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRYYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9277)

S54D
PSMA.86
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3296

MGWVRQAPGKEREFVAAISWSGSNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRYYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9277)

S54E
PSMA.87
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3297

MGWVRQAPGKEREFVAAISWSGSNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRYYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9277)

S54Q
PSMA.88
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3298

MGWVRQAPGKEREFVAAISWSGSNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRYYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9277)

S54G
PSMA.89
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3299

MGWVRQAPGKEREFVAAISWSGSNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRYYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9277)

S54K
PSMA.90
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3300

MGWVRQAPGKEREFVAAISWSGSNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRYYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9277)

S54P
PSMA.91
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3301

MGWVRQAPGKEREFVAAISWSGSNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRYYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9277)

S54W
PSMA.92
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3302

MGWVRQAPGKEREFVAAISWWGSNRLVSDSV

KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA

ASNRLYGRTWYDFNESDYWGQGTQVTVSS

(SEQ ID NO: 9278)

G55D
PSMA.93
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3303

MGWVRQAPGKEREFVAAISWSGSNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRYYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9277)

S56A
PSMA.94
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3304

MGWVRQAPGKEREFVAAISWSGSNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRYYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9277)

S56N
PSMA.95
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3305

MGWVRQAPGKEREFVAAISWSGSNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRYYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9277)

S56E
PSMA.96
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3306

MGWVRQAPGKEREFVAAISWSGSNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRYYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9277)

S56Q
PSMA.97
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3307

MGWVRQAPGKEREFVAAISWSGSNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRYYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9277)

S56G
PSMA.98
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3308

MGWVRQAPGKEREFVAAISWSGSNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRYYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9277)

S56H
PSMA.99
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3309

MGWVRQAPGKEREFVAAISWSGSNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRYYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9277)

S56L
PSMA.100
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3310

MGWVRQAPGKEREFVAAISWSGLNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRLYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9279)

S56P
PSMA.101
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3311

MGWVRQAPGKEREFVAAISWSGPNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRLYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9280)

S56T
PSMA.102
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3312

MGWVRQAPGKEREFVAAISWSGTNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRLYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9281)

N57D
PSMA.103
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3313

MGWVRQAPGKEREFVAAISWSGSDRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRLYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9282)

N57Q
PSMA.104
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3314

MGWVRQAPGKEREFVAAISWSGSQRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRLYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9283)

N57G
PSMA.105
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3315

MGWVRQAPGKEREFVAAISWSGSGRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRLYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9284)

N57H
PSMA.106
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3316

MGWVRQAPGKEREFVAAISWSGSHRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRLYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9285)

N57F
PSMA.107
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3317

MGWVRQAPGKEREFVAAISWSGSFRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRLYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9286)

N57P
PSMA.108
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3318

MGWVRQAPGKEREFVAAISWSGSPRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRLYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9287)

R58A
PSMA.109
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3319

MGWVRQAPGKEREFVAAISWSGSNALVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRLYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9288)

R58Q
PSMA.110
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3320

MGWVRQAPGKEREFVAAISWSGSNQLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRLYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9289)

R58I
PSMA.111
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3321

MGWVRQAPGKEREFVAAISWSGSNILVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRLYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9290)

R58T
PSMA.112
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3322

MGWVRQAPGKEREFVAAISWSGSNTLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRLYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9291)

L59A
PSMA.113
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3323

MGWVRQAPGKEREFVAAISWSGSNRAVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRLYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9292)

L59N
PSMA.114
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3324

MGWVRQAPGKEREFVAAISWSGSNRNVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRLYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9293)

L59E
PSMA.115
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3325

MGWVRQAPGKEREFVAAISWSGSNREVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRLYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9294)

L59Q
PSMA.116
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3326

MGWVRQAPGKEREFVAAISWSGSNRQVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRLYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9295)

L59G
PSMA.117
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3327

MGWVRQAPGKEREFVAAISWSGSNRGVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRLYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9296)

L59H
PSMA.118
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3328

MGWVRQAPGKEREFVAAISWSGSNRHVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRLYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9297)

L59K
PSMA.119
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3329

MGWVRQAPGKEREFVAAISWSGSNRKVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRLYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9298)

L59M
PSMA.120
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3330

MGWVRQAPGKEREFVAAISWSGSNRMVSDSV

KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA

ASNRLYGRTWYDFNESDYWGQGTQVTVSS

(SEQ ID NO: 9299)

L59P
PSMA.121
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3331

MGWVRQAPGKEREFVAAISWSGSNRPVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRLYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9300)

L59S
PSMA.122
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3332

MGWVRQAPGKEREFVAAISWSGSNRSVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRLYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9301)

L59T
PSMA.123
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3333

MGWVRQAPGKEREFVAAISWSGSNRTVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRLYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9302)

L59Y
PSMA.124
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3334

MGWVRQAPGKEREFVAAISWSGSNRYVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRLYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9303)

V60Y
PSMA.125
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3335

MGWVRQAPGKEREFVAAISWSGSNRLYSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRLYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9304)

R67Q
PSMA.126
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3336

MGWVRQAPGKEREFVAAISWSGSNRLVSDSVK

GQFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRLYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9305)

S99E
PSMA.127
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3337

MGWVRQAPGKEREFVAAISWSGSNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

ENRLYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9306)

S99Q
PSMA.128
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3338

MGWVRQAPGKEREFVAAISWSGSNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

HNRLYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9307)

S99H
PSMA.129
QVQLVESGGGWVQPGRSLRLSCAASGRTFGIYV
AC3339

MGWVRQAPGKEREFVAAISWSGSNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

YNRLYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9308)

S99Y
PSMA.130
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3340

MGWVRQAPGKEREFVAAISWSGSNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

QNRLYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9309)

N100E
PSMA.131
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3341

MGWVRQAPGKEREFVAAISWSGSNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SERLYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9310)

R101K
PSMA.132
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3342

MGWVRQAPGKEREFVAAISWSGSNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNKLYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9311)

L102A
PSMA.133
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3343

MGWVRQAPGKEREFVAAISWSGSNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRAYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9312)

L102Q
PSMA.134
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3344

MGWVRQAPGKEREFVAAISWSGSNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRQYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9313)

L102H
PSMA.135
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3345

MGWVRQAPGKEREFVAAISWSGSNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRHYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9314)

L102K
PSMA.136
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3346

MGWVRQAPGKEREFVAAISWSGSNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRKYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9315)

L102M
PSMA.137
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3347

MGWVRQAPGKEREFVAAISWSGSNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRMYGRTWYDFNESDYWGQGTQVTVSS

(SEQ ID NO: 9316)

L102F
PSMA.138
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3348

MGWVRQAPGKEREFVAAISWSGSNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRFYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9317)

L102P
PSMA.139
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3349

MGWVRQAPGKEREFVAAISWSGSNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRPYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9318)

L102T
PSMA.140
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3350

MGWVRQAPGKEREFVAAISWSGSNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRTYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9319)

L102W
PSMA.141
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3351

MGWVRQAPGKEREFVAAISWSGSNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRWYGRTWYDFNESDYWGQGTQVTVSS

(SEQ ID NO: 9320)

L102Y
PSMA.142
QVQLVESGGGVVQPGRSLRLSCAASGRTFGIYV
AC3352

MGWVRQAPGKEREFVAAISWSGSNRLVSDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA

SNRYYGRTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9277)

TABLE 10b

PSMA VHH sequences from Pool 2

huPSMA

PSMA
Binding
PTE

AC

domain
K_D (nM)
score
Sequence
Number

PSMA.5
4.8
69
QVQLVESGGGVVQPGRSLRLSCAASG
AC2717

RTFGIYVMGWFRQAPGKEREFVAAMS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYYCAASNRLYG

RTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9321)

PSMA.119
4.1
46
QVQLVESGGGVVQPGRSLRLSCAASG
AC3329

RTFGIYVMGWVRQAPGKEREFVAAIS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYYCAASNRLYG

RTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9298)

PSMA.250
6.2
26
QVQLVESGGGVVQPGRSLRLSCAASG
AC3461

RTFGIYVMGWYRQAPGKEREFVAAMS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYYCAASNKLYGR

TWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9322)

PSMA.251
5.1
27
QVQLVESGGGVVQPGRSLRLSCAASG
AC3462

RTFGIYVMGWYRQAPGKEREFVAAIS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYYCAASNKLYGR

TWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9323)

PSMA.252
15.9
15
QVQLVESGGGVVQPGRSLRLSCAASG
AC3463

RTFGIYVMGWYRQAPGKEREFVAAMS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYYCAASNRWYG

RTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9324)

PSMA.253
8.5
16
QVQLVESGGGVVQPGRSLRLSCAASG
AC3464

RTFGIYVMGWYRQAPGKEREFVAAIS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYYCAASNRWYG

RTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9325)

PSMA.254
41.0
15
QVQLVESGGGVVQPGRSLRLSCAASG
AC3465

RTFGIYVMGWYRQAPGKEREFVAAMS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYYCAASNRYYG

RTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9326)

PSMA.255
18.9
16
QVQLVESGGGVVQPGRSLRLSCAASG
AC3466

RTFGIYVMGWYRQAPGKEREFVAAIS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYYCAASNRYYG

RTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9327)

PSMA.256
3.4
29
QVQLVESGGGVVQPGRSLRLSCAASG
AC3467

RTFGIYVMGWYRQAPGKEREFVAAMS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYYCAASNRLYG

RTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9328)

PSMA.257
2.3
30
QVQLVESGGGVVQPGRSLRLSCAASG
AC3468

RTFGIYVMGWYRQAPGKEREFVAAIS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYYCAASNRLYG

RTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9329)

PSMA.260
11.4
23
QVQLVESGGGVVQPGRSLRLSCAASG
AC3469

RTFGIYAMGWVRQAPGKEREFVGAIS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYYCAASNKLYGR

TWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9330)

PSMA.261
2.1
23
QVQLVESGGGVVQPGRSLRLSCAASG
AC3470

RTFGIYVFGWVRQAPGKEREFVGAIS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYYCAASNKLYGR

TWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9331)

PSMA.262
2.7
23
QVQLVESGGGVVQPGRSLRLSCAASG
AC3471

RTFGIYVWGWVRQAPGKEREFVGAIS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYYCAASNKLYGR

TWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9332)

PSMA.263
Not
23
QVQLVESGGGVVQPGRSLRLSCAASG
AC3472

Determined

RTFGIYVMGWFRQAPGKEREFVGAIS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYYCAASNKLYGR

TWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9333)

PSMA.264
18.6
26
QVQLVESGGGVVQPGRSLRLSCAASG
AC3473

RTFGIYAMGWVRQAPGKEREFVAAMS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYYCAASNKLYGR

TWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9334)

PSMA.265
3.9
26
QVQLVESGGGVVQPGRSLRLSCAASG
AC3474

RTFGIYVFGWVRQAPGKEREFVAAMS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYYCAASNKLYGR

TWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9335)

PSMA.266
4.6
26
QVQLVESGGGVVQPGRSLRLSCAASG
AC3475

RTFGIYVWGWVRQAPGKEREFVAAMS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYYCAASNKLYGR

TWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9336)

PSMA.267
5.6
20
QVQLVESGGGVVQPGRSLRLSCAASG
AC3476

RTFGIYVMGWFRQAPGKEREFVAAMS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYYCAASNKLYGR

TWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9337)

PSMA.268
131.3
20
QVQLVESGGGVVQPGRSLRLSCAASG
AC3477

RTFGIYAMGWVRQAPGKEREFVGAIS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYYCAASNRTYG

RTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9338)

PSMA.269
5.5
20
QVQLVESGGGVVQPGRSLRLSCAASG
AC3478

RTFGIYVFGWVRQAPGKEREFVGAIS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYYCAASNRTYG

RTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9339)

PSMA.270
6.5
20
QVQLVESGGGVVQPGRSLRLSCAASG
AC3479

RTFGIYVWGWVRQAPGKEREFVGAIS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYYCAASNRTYG

RTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9340)

PSMA.271
9.1
23
QVQLVESGGGVVQPGRSLRLSCAASG
AC3480

RTFGIYVMGWFRQAPGKEREFVGAIS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYYCAASNRTYG

RTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9341)

PSMA.272
503.7
23
QVQLVESGGGVVQPGRSLRLSCAASG
AC3481

RTFGIYAMGWVRQAPGKEREFVAAMS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYYCAASNRTYG

RTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9342)

PSMA.273
16.6
23
QVQLVESGGGVVQPGRSLRLSCAASG
AC3482

RTFGIYVFGWVRQAPGKEREFVAAMS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYYCAASNRTYG

RTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9343)

PSMA.274
39.6
23
QVQLVESGGGVVQPGRSLRLSCAASG
AC3483

RTFGIYVWGWVRQAPGKEREFVAAMS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYYCAASNRTYG

RTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9344)

PSMA.275
20.5
24
QVQLVESGGGVVQPGRSLRLSCAASG
AC3484

RTFGIYVMGWFRQAPGKEREFVAAMS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYYCAASNRTYG

RTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9345)

PSMA.276
10.5
24
QVQLVESGGGVVQPGRSLRLSCAASG
AC3485

RTFGIYAMGWVRQAPGKEREFVGAIS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYYCAASNRMYG

RTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9346)

PSMA.277
3.1
24
QVQLVESGGGVVQPGRSLRLSCAASG
AC3486

RTFGIYVFGWVRQAPGKEREFVGAIS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYYCAASNRMYG

RTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9347)

PSMA.278
3.9
24
QVQLVESGGGVVQPGRSLRLSCAASG
AC3487

RTFGIYVWGWVRQAPGKEREFVGAIS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYYCAASNRMYG

RTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9348)

PSMA.279
2.3
27
QVQLVESGGGVVQPGRSLRLSCAASG
AC3488

RTFGIYVMGWFRQAPGKEREFVGAIS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYYCAASNRMYG

RTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9349)

PSMA.280
16.3
27
QVQLVESGGGVVQPGRSLRLSCAASG
AC3489

RTFGIYAMGWVRQAPGKEREFVAAMS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYYCAASNRMYG

RTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9350)

PSMA.281
5.3
27
QVQLVESGGGVVQPGRSLRLSCAASG
AC3490

RTFGIYVFGWVRQAPGKEREFVAAMS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYYCAASNRMYG

RTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9351)

PSMA.282
8.7
27
QVQLVESGGGVVQPGRSLRLSCAASG
AC3491

RTFGIYVWGWVRQAPGKEREFVAAMS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYYCAASNRMYG

RTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9352)

PSMA.283
3.5
26
QVQLVESGGGVVQPGRSLRLSCAASG
AC3492

RTFGIYVMGWFRQAPGKEREFVAAMS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYYCAASNRMYG

RTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9353)

PSMA.284
4.1
26
QVQLVESGGGVVQPGRSLRLSCAASG
AC3493

RTFGIYAMGWVRQAPGKEREFVGAIS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYYCAASNRLYG

RTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9354)

PSMA.285
3.1
26
QVQLVESGGGVVQPGRSLRLSCAASG
AC3494

RTFGIYVFGWVRQAPGKEREFVGAIS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYYCAASNRLYG

RTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9355)

PSMA.286
No
26
QVQLVESGGGVVQPGRSLRLSCAASG
AC3495

Binding

RTFGIYVWGWVRQAPGKEREFVGAIS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYYCAASNRLYG

RTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9356)

PSMA.287
2.3
29
QVQLVESGGGVVQPGRSLRLSCAASG
AC3496

RTFGIYVMGWFRQAPGKEREFVGAIS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYYCAASNRLYG

RTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9357)

PSMA.288
6.0
29
QVQLVESGGGVVQPGRSLRLSCAASG
AC3497

RTFGIYAMGWVRQAPGKEREFVAAMS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYYCAASNRLYG

RTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9358)

PSMA.289
6.2

QVQLVESGGGVVQPGRSLRLSCAASG
AC3498

RTFGIYVFGWVRQAPGKEREFVAAMS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYYCAASNRLYG

RTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9359)

PSMA.290
5.5
29
QVQLVESGGGVVQPGRSLRLSCAASG
AC3499

RTFGIYVWGWVRQAPGKEREFVAAMS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYYCAASNRLYG

RTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9360)

PSMA.291
2.6
29
QVQLVESGGGVVQPGRSLRLSCAASG
AC3500

RTFGIYVMGWFRQAPGKEREFVAAMS

WSGSNRKVSDSVKGRFTISRDNSKNT

LYLQMNSLRAEDTAVYYCAASNRLYG

RTWYDFNESDYWGQGTQVTVSS (SEQ

ID NO: 9321)

TABLE 10c

PSMA VHH sequences from Pool 3

huPSMA

PSMA
Binding
PTE

AC
SEQ ID

domain
K_D (nM)
score
Sequence
Number
NO

PSMA.301
10.0
23
QVQLVESGGGVVQPGRSLRLSCAAS
AC3703
500

GRTFGIYVMGWFRQAPGKEREFVGA

ISWSGSNRKVSDSVKGRFTISRDNSK

NTLYLQMNSLRAEDTAVYFCAASNKL

YGRTWYDFNESDYWGQGTQVTVSS

PSMA.302
2.8
16
QVQLVESGGGVVQPGRSLRLSCAAS
AC3704
501

GRTFGIYVWGWFRQAPGKEREFVGA

ISWSGSNRKVSDSVKGRFTISRDNSK

NTLYLQMNSLRAEDTAVYFCAASNKL

YGRTWYDFNESDYWGQGTQVTVSS

PSMA.303
10.3
18
QVQLVESGGGVVQPGRSLRLSCAAS
AC3705
502

GRTFGIYVMGWFRQAPGKEREFVGA

MSWSGSNRKVSDSVKGRFTISRDNS

KNTLYLQMNSLRAEDTAVYFCAASNK

LYGRTWYDFNESDYWGQGTQVTVS

S

PSMA.304
5.9
11
QVQLVESGGGVVQPGRSLRLSCAAS
AC3706
503

GRTFGIYVWGWFRQAPGKEREFVGA

MSWSGSNRKVSDSVKGRFTISRDNS

KNTLYLQMNSLRAEDTAVYFCAASNK

LYGRTWYDFNESDYWGQGTQVTVS

S

PSMA.305
N/D
16
QVQLVESGGGVVQPGRSLRLSCAAS
AC3707
504

(has

GRTFGIYVWGWFRQAPGKEREFVGA

strong

ISWSGSNRKVSDSVKGRFTISRDNSK

binding)

NTLYLQMNSLRAEDTAVYYCAASNKL

YGRTWYDFNESDYWGQGTQVTVSS

PSMA.306
10.4
18
QVQLVESGGGVVQPGRSLRLSCAAS
AC3708
505

GRTFGIYVMGWFRQAPGKEREFVGA

MSWSGSNRKVSDSVKGRFTISRDNS

KNTLYLQMNSLRAEDTAVYYCAASNK

LYGRTWYDFNESDYWGQGTQVTVS

S

PSMA.307
6.0
11
QVQLVESGGGVVQPGRSLRLSCAAS
AC3709
506

GRTFGIYVWGWFRQAPGKEREFVGA

MSWSGSNRKVSDSVKGRFTISRDNS

KNTLYLQMNSLRAEDTAVYYCAASNK

LYGRTWYDFNESDYWGQGTQVTVS

S

PSMA.308
8.8
0
QVQLVESGGGVVQPGRSLRLSCAAS
AC3710
507

GRTFGIYVWGWFRQAPGKEREFVGA

MSWSGSNRKVSDSVKGRFTISRDNS

KNTLYLQMNSLRAEDTAVYYCAASNK

WYGRTWYDFNESDYWGQGTQVTVS

S

PSMA.309
8.5
0
QVQLVESGGGVVQPGRSLRLSCAAS
AC3711
508

GRTFGIYVWGWFRQAPGKEREFVGA

MSWSGSNRKVSDSVKGRFTISRDNS

KNTLYLQMNSLRAEDTAVYFCAASNK

WYGRTWYDFNESDYWGQGTQVTVS

S

PSMA.310
23.1
12
QVQLVESGGGVVQPGRSLRLSCAAS
AC3712
509

GRTFGIYVMGWFRQAPGKEREFVGA

ISWSGSNRKVSDSVKGRFTISRDNSK

NTLYLQMNSLRAEDTAVYYCGGSNK

LYGRTWYDFNESDYWGQGTQVTVS

S

PSMA.311
No
12
QVQLVESGGGVVQPGRSLRLSCAAS
AC3713
510

Binding

GRTFGIYVMGWFRQAPGKEREFVGA

ISWSGSNRKVSDSVKGRFTISRDNSK

NTLYLQMNSLRAEDTAVDYCAASNKL

YGRTWYDFNESDYWGQGTQVTVSS

PSMA.312
12.0
13
QVQLVESGGGVVQPGRSLRLSCAAS
AC3714
511

GRTFGIYVMGWFRQAPGKEREFVGA

ISWSGSNRKVSDSVKGRFTISRDNSK

NTLYLQMNSLRAEDTAVYYCGASNKL

YGRTWYDFNESDYWGQGTQVTVSS

PSMA.313
No
12
QVQLVESGGGVVQPGRSLRLSCAAS
AC3715
512

Binding

GRTFGIYVMGWFRQAPGKEREFVGA

ISWSGSNRKVSDSVKGRFTISRDNSK

NTLYLQMNSLRAEDTAVYYCAASNK

RYGRTWYDFNESDYWGQGTQVTVS

S

PSMA.314
69.9
12
QVQLVESGGGVVQPGRSLRLSCAAS
AC3716
513

GRTFGIYVMGWFRQAPGKEREFVGA

ISWSGSNRKVSDSVKGRFTISRDNSK

NTLYLQMNSLRAEDTAVYYCAASNK

DYGRTWYDFNESDYWGQGTQVTVS

S

PSMA.315
24.2
12
QVQLVESGGGVVQPGRSLRLSCAAS
AC3717
514

GRTFGIYVMGWFRQAPGKEREFVGA

ISWSGSNRKVSDSVKGRFTISRDNSK

NTLYLQMNSLRAEDTAVYYCAASNKE

YGRTWYDFNESDYWGQGTQVTVSS

PSMA.316
150.0
13
QVQLVESGGGVVQPGRSLRLSCAAS
AC3718
515

GRTFGIYVMGWFRQAPGKEREFVGA

ISWSGSNRKVSDSVKGRFTISRDNSK

NTLYLQMNSLRAEDTAVYYCAASNK

GYGRTWYDFNESDYWGQGTQVTVS

S

PSMA.317
No
17
QVQLVESGGGVVQPGRSLRLSCAAS
AC3719
516

Binding

GRTFGIYVMGWFRQAPGKEREFVGA

ISWSGSNRKVSDSVKGRFTISRDNSK

NTLYLQMNSLRAEDTAVAYCAGSNKL

YGRTWYDFNESDYWGQGTQVTVSS

PSMA.318
No
16
QVQLVESGGGVVQPGRSLRLSCAAS
AC3720
517

Binding

GRTFGIYVMGWFRQAPGKEREFVGA

ISWSGSNRKVSDSVKGRFTISRDNSK

NTLYLQMNSLRAEDTAVRYCAGSNK

LYGRTWYDFNESDYWGQGTQVTVS

S

PSMA.319
No
16
QVQLVESGGGVVQPGRSLRLSCAAS
AC3721
518

Binding

GRTFGIYVMGWFRQAPGKEREFVGA

ISWSGSNRKVSDSVKGRFTISRDNSK

NTLYLQMNSLRAEDTAVNYCAGSNK

LYGRTWYDFNESDYWGQGTQVTVS

S

PSMA.320
No
17
QVQLVESGGGVVQPGRSLRLSCAAS
AC3722
519

Binding

GRTFGIYVMGWFRQAPGKEREFVGA

ISWSGSNRKVSDSVKGRFTISRDNSK

NTLYLQMNSLRAEDTAVGYCAGSNK

LYGRTWYDFNESDYWGQGTQVTVS

S

PSMA.321
No
17
QVQLVESGGGVVQPGRSLRLSCAAS
AC3723
520

Binding

GRTFGIYVMGWFRQAPGKEREFVGA

ISWSGSNRKVSDSVKGRFTISRDNSK

NTLYLQMNSLRAEDTAVKYCAGSNKL

YGRTWYDFNESDYWGQGTQVTVSS

PSMA.322
No
17
QVQLVESGGGVVQPGRSLRLSCAAS
AC3724
521

Binding

GRTFGIYVMGWFRQAPGKEREFVGA

ISWSGSNRKVSDSVKGRFTISRDNSK

NTLYLQMNSLRAEDTAVSYCAGSNKL

YGRTWYDFNESDYWGQGTQVTVSS

PSMA.323
No
17
QVQLVESGGGVVQPGRSLRLSCAAS
AC3725
522

Binding

GRTFGIYVMGWFRQAPGKEREFVGA

ISWSGSNRKVSDSVKGRFTISRDNSK

NTLYLQMNSLRAEDTAVTYCAGSNKL

YGRTWYDFNESDYWGQGTQVTVSS

PSMA.324
No
17
QVQLVESGGGVVQPGRSLRLSCAAS
AC3726
523

Binding

GRTFGIYVMGWFRQAPGKEREFVGA

ISWSGSNRKVSDSVKGRFTISRDNSK

NTLYLQMNSLRAEDTAVAFCAASNKL

YGRTWYDFNESDYWGQGTQVTVSS

PSMA.325
No
17
QVQLVESGGGVVQPGRSLRLSCAAS
AC3727
524

Binding

GRTFGIYVMGWFRQAPGKEREFVGA

ISWSGSNRKVSDSVKGRFTISRDNSK

NTLYLQMNSLRAEDTAVRFCAASNKL

YGRTWYDFNESDYWGQGTQVTVSS

PSMA.326
No
17
QVQLVESGGGVVQPGRSLRLSCAAS
AC3728
525

Binding

GRTFGIYVMGWFRQAPGKEREFVGA

ISWSGSNRKVSDSVKGRFTISRDNSK

NTLYLQMNSLRAEDTAVNFCAASNKL

YGRTWYDFNESDYWGQGTQVTVSS

PSMA.327
No
12
QVQLVESGGGVVQPGRSLRLSCAAS
AC3729
526

Binding

GRTFGIYVMGWFRQAPGKEREFVGA

ISWSGSNRKVSDSVKGRFTISRDNSK

NTLYLQMNSLRAEDTAVDFCAASNKL

YGRTWYDFNESDYWGQGTQVTVSS

PSMA.328
No
17
QVQLVESGGGVVQPGRSLRLSCAAS
AC3730
527

Binding

GRTFGIYVMGWFRQAPGKEREFVGA

ISWSGSNRKVSDSVKGRFTISRDNSK

NTLYLQMNSLRAEDTAVGFCAASNKL

YGRTWYDFNESDYWGQGTQVTVSS

PSMA.329
No
17
QVQLVESGGGVVQPGRSLRLSCAAS
AC3731
528

Binding

GRTFGIYVMGWFRQAPGKEREFVGA

ISWSGSNRKVSDSVKGRFTISRDNSK

NTLYLQMNSLRAEDTAVKFCAASNKL

YGRTWYDFNESDYWGQGTQVTVSS

PSMA.330
No
17
QVQLVESGGGVVQPGRSLRLSCAAS
AC3732
529

Binding

GRTFGIYVMGWFRQAPGKEREFVGA

ISWSGSNRKVSDSVKGRFTISRDNSK

NTLYLQMNSLRAEDTAVSFCAASNKL

YGRTWYDFNESDYWGQGTQVTVSS

PSMA.331
12.1
14
QVQLVESGGGVVQPGRSLRLSCAAS
AC3733
530

GRTFGIYVMGWFRQAPGKEREFVGA

ISWSGSNRKVSDSVKGRFTISRDNSK

NTLYLQMNSLRAEDTAVYFCGASNKL

YGRTWYDFNESDYWGQGTQVTVSS

PSMA.332
24.1
17
QVQLVESGGGVVQPGRSLRLSCAAS
AC3734
531

GRTFGIYVMGWFRQAPGKEREFVGA

ISWSGSNRKVSDSVKGRFTISRDNSK

NTLYLQMNSLRAEDTAVYFCAGSNKL

YGRTWYDFNESDYWGQGTQVTVSS

PSMA.333
No
14
QVQLVESGGGVVQPGRSLRLSCAAS
AC3735
532

Binding

GRTFGIYVMGWFRQAPGKEREFVGA

ISWSGSNRKVSDSVKGRFTISRDNSK

NTLYLQMNSLRAEDTAVYFCAASNKR

YGRTWYDFNESDYWGQGTQVTVSS

PSMA.334
70.6
12
QVQLVESGGGVVQPGRSLRLSCAAS
AC3736
533

GRTFGIYVMGWFRQAPGKEREFVGA

ISWSGSNRKVSDSVKGRFTISRDNSK

NTLYLQMNSLRAEDTAVYFCAASNKD

YGRTWYDFNESDYWGQGTQVTVSS

PSMA.335
24.8
12
QVQLVESGGGVVQPGRSLRLSCAAS
AC3737
534

GRTFGIYVMGWFRQAPGKEREFVGA

ISWSGSNRKVSDSVKGRFTISRDNSK

NTLYLQMNSLRAEDTAVYFCAASNKE

YGRTWYDFNESDYWGQGTQVTVSS

PSMA.336
129.1
14
QVQLVESGGGVVQPGRSLRLSCAAS
AC3738
535

GRTFGIYVMGWFRQAPGKEREFVGA

ISWSGSNRKVSDSVKGRFTISRDNSK

NTLYLQMNSLRAEDTAVYFCAASNK

GYGRTWYDFNESDYWGQGTQVTVS

S

PSMA.337
No
17
QVQLVESGGGVVQPGRSLRLSCAAS
AC3739
536

Binding

GRTFGIYVMGWFRQAPGKEREFVGA

ISWSGSNRKVSDSVKGRFTISRDNSK

NTLYLQMNSLRAEDTAVAFCAGSNKL

YGRTWYDFNESDYWGQGTQVTVSS

PSMA.338
No
16
QVQLVESGGGVVQPGRSLRLSCAAS
AC3740
537

Binding

GRTFGIYVMGWFRQAPGKEREFVGA

ISWSGSNRKVSDSVKGRFTISRDNSK

NTLYLQMNSLRAEDTAVRFCAGSNKL

YGRTWYDFNESDYWGQGTQVTVSS

PSMA.339
No
16
QVQLVESGGGVVQPGRSLRLSCAAS
AC3741
538

Binding

GRTFGIYVMGWFRQAPGKEREFVGA

ISWSGSNRKVSDSVKGRFTISRDNSK

NTLYLQMNSLRAEDTAVNFCAGSNKL

YGRTWYDFNESDYWGQGTQVTVSS

PSMA.340
No
17
QVQLVESGGGVVQPGRSLRLSCAAS
AC3742
539

Binding

GRTFGIYVMGWFRQAPGKEREFVGA

ISWSGSNRKVSDSVKGRFTISRDNSK

NTLYLQMNSLRAEDTAVGFCAGSNK

LYGRTWYDFNESDYWGQGTQVTVS

S

PSMA.341
No
17
QVQLVESGGGVVQPGRSLRLSCAAS
AC3743
540

Binding

GRTFGIYVMGWFRQAPGKEREFVGA

ISWSGSNRKVSDSVKGRFTISRDNSK

NTLYLQMNSLRAEDTAVKFCAGSNKL

YGRTWYDFNESDYWGQGTQVTVSS

PSMA.342
No
17
QVQLVESGGGVVQPGRSLRLSCAAS
AC3744
541

Binding

GRTFGIYVMGWFRQAPGKEREFVGA

ISWSGSNRKVSDSVKGRFTISRDNSK

NTLYLQMNSLRAEDTAVSFCAGSNKL

YGRTWYDFNESDYWGQGTQVTVSS

PSMA.343
No
17
QVQLVESGGGVVQPGRSLRLSCAAS
AC3745
542

Binding

GRTFGIYVMGWFRQAPGKEREFVGA

ISWSGSNRKVSDSVKGRFTISRDNSK

NTLYLQMNSLRAEDTAVTFCAGSNKL

YGRTWYDFNESDYWGQGTQVTVSS

PSMA.344
Not
12
QVQLVESGGGVVQPGRSLRLSCAAS
None
543

Determined

GRTFGIYVMGWFRQAPGKEREFVGA

ISWSGSNRKVSDSVKGRFTISRDNSK

NTLYLQMNSLRAEDTAVYFCGGSNK

LYGRTWYDFNESDYWGQGTQVTVS

S

PSMA.345
6.1
0
QVQLVESGGGVVQPGRSLRLSCAAS
AC3747
544

GRTFGIYVWGWFRQAPGKEREFVGA

MSWSGSNRKVSDSVKGRFTISRDNS

KNTLYLQMNSLRAEDTAVYYCGGSN

KLYGRTWYDFNESDYWGQGTQVTV

SS

PSMA.346
No
0
QVQLVESGGGVVQPGRSLRLSCAAS
AC3748
545

Binding

GRTFGIYVWGWFRQAPGKEREFVGA

MSWSGSNRKVSDSVKGRFTISRDNS

KNTLYLQMNSLRAEDTAVDYCAASN

KLYGRTWYDFNESDYWGQGTQVTV

SS

PSMA.347
7.3
1
QVQLVESGGGVVQPGRSLRLSCAAS
AC3749
546

GRTFGIYVWGWFRQAPGKEREFVGA

MSWSGSNRKVSDSVKGRFTISRDNS

KNTLYLQMNSLRAEDTAVYYCGASN

KLYGRTWYDFNESDYWGQGTQVTV

SS

PSMA.348
1149.0
0
QVQLVESGGGVVQPGRSLRLSCAAS
AC3750
547

GRTFGIYVWGWFRQAPGKEREFVGA

MSWSGSNRKVSDSVKGRFTISRDNS

KNTLYLQMNSLRAEDTAVYYCAASNK

RYGRTWYDFNESDYWGQGTQVTVS

S

PSMA.349
18.0
0
QVQLVESGGGVVQPGRSLRLSCAAS
AC3751
548

GRTFGIYVWGWFRQAPGKEREFVGA

MSWSGSNRKVSDSVKGRFTISRDNS

KNTLYLQMNSLRAEDTAVYYCAASNK

DYGRTWYDFNESDYWGQGTQVTVS

S

PSMA.350
7.4
0
QVQLVESGGGVVQPGRSLRLSCAAS
AC3752
549

GRTFGIYVWGWFRQAPGKEREFVGA

MSWSGSNRKVSDSVKGRFTISRDNS

KNTLYLQMNSLRAEDTAVYYCAASNK

EYGRTWYDFNESDYWGQGTQVTVS

S

PSMA.351
32.6
1
QVQLVESGGGVVQPGRSLRLSCAAS
AC3753
550

GRTFGIYVWGWFRQAPGKEREFVGA

MSWSGSNRKVSDSVKGRFTISRDNS

KNTLYLQMNSLRAEDTAVYYCAASNK

GYGRTWYDFNESDYWGQGTQVTVS

S

PSMA.352
No
0
QVQLVESGGGVVQPGRSLRLSCAAS
AC3754
551

Binding

GRTFGIYVWGWFRQAPGKEREFVGA

MSWSGSNRKVSDSVKGRFTISRDNS

KNTLYLQMNSLRAEDTAVDFCAASNK

LYGRTWYDFNESDYWGQGTQVTVS

S

PSMA.353
5.1
2
QVQLVESGGGVVQPGRSLRLSCAAS
AC3755
552

GRTFGIYVWGWFRQAPGKEREFVGA

MSWSGSNRKVSDSVKGRFTISRDNS

KNTLYLQMNSLRAEDTAVYFCGASN

KLYGRTWYDFNESDYWGQGTQVTV

SS

PSMA.354
1089.0
2
QVQLVESGGGVVQPGRSLRLSCAAS
AC3756
553

GRTFGIYVWGWFRQAPGKEREFVGA

MSWSGSNRKVSDSVKGRFTISRDNS

KNTLYLQMNSLRAEDTAVYFCAASNK

RYGRTWYDFNESDYWGQGTQVTVS

S

PSMA.355
17.8
0
QVQLVESGGGVVQPGRSLRLSCAAS
AC3757
554

GRTFGIYVWGWFRQAPGKEREFVGA

MSWSGSNRKVSDSVKGRFTISRDNS

KNTLYLQMNSLRAEDTAVYFCAASNK

DYGRTWYDFNESDYWGQGTQVTVS

S

PSMA.356
7.8
0
QVQLVESGGGVVQPGRSLRLSCAAS
AC3758
555

GRTFGIYVWGWFRQAPGKEREFVGA

MSWSGSNRKVSDSVKGRFTISRDNS

KNTLYLQMNSLRAEDTAVYFCAASNK

EYGRTWYDFNESDYWGQGTQVTVS

S

PSMA.357
24.6
2
QVQLVESGGGVVQPGRSLRLSCAAS
AC3759
556

GRTFGIYVWGWFRQAPGKEREFVGA

MSWSGSNRKVSDSVKGRFTISRDNS

KNTLYLQMNSLRAEDTAVYFCAASNK

GYGRTWYDFNESDYWGQGTQVTVS

S

PSMA.358
6.5
0
QVQLVESGGGVVQPGRSLRLSCAAS
AC3760
557

GRTFGIYVWGWFRQAPGKEREFVGA

MSWSGSNRKVSDSVKGRFTISRDNS

KNTLYLQMNSLRAEDTAVYFCGGSN

KLYGRTWYDFNESDYWGQGTQVTV

SS

PSMA.359
No
4
QVQLVESGGGVVQPGRSLRLSCAAS
AC3761
558

Binding

GRTFGIYVWGWFRQAPGKEREFVGA

MSWSGSNRKVSDSVKGRFTISRDNS

KNTLYLQMNSLRAEDTAVRYCAGSN

KLYGRTWYDFNESDYWGQGTQVTV

SS

PSMA.360
No
4
QVQLVESGGGVVQPGRSLRLSCAAS
AC3762
559

Binding

GRTFGIYVWGWFRQAPGKEREFVGA

MSWSGSNRKVSDSVKGRFTISRDNS

KNTLYLQMNSLRAEDTAVNYCAGSN

KLYGRTWYDFNESDYWGQGTQVTV

SS

Example 4. PSMA Binding Analyses

PSMA binding kinetics were determined for select clones from the non-humanized antibodies against human PSMA. The binding affinity values are depicted below in Table 11. Each PSMA antibody was paired with the CD3 antibody CD3.23.

TABLE 11

Binding affinity for select PSMA antibodies

SEQ ID

NO
Description
K_D(NM)
K_on(1/Ms)
K_diss(1/s)

1036
PSMA-VHH; CB01A01R3, CD3.23
12.87
9.87E+04
1.27E−03

1037
PSMA-VHH; P01C01R3, CD3.23
Weak

binding

1038
PSMA-VHH; CB01H01R3, CD3.23
0.79
1.10E+05
8.70E−05

1039
PSMA-VHH; CB01B02R3, CD3.23
1.15
1.78E+05
2.05E−04

1040
PSMA-VHH; P01H01R3, CD3.23
No binding

1041
PSMA-VHH; P01E02R3, CD3.23
0.60
2.61E+05
1.56E−04

1042
PSMA-VHH; P01G03R3, CD3.23
55.74
9.67E+03
5.39E−04

uTCEs having the amino acid sequences of SEQ ID NOs 1038 (the unmasked TOE, or uTCE, of A2591), 1039, and 1041 had sub- to single digit nM affinity.

Epitope competition assays were performed next. An Octet AHC biosensor was loaded with human PSMA-Fc, followed by the uTCE of AC2591. The complex of human PSMA-Fc and the uTCE of AC2591 was then dipped into uTCEs having the amino acid sequences of SEQ ID Nos 1036, 1039 and 1041. The results of the competition assay indicated that uTCEs having the amino acid sequences of SEQ ID NOs 1036, 1038 (the uTCE of AC2591), and 1039 have an overlapping epitope, while the uTCE having the amino acid sequence of SEQ ID NO 1042 binds to a different epitope than the uTCE of AC2591.

Binding affinities and melting temperatures were determined for PSMA.5 and other variants. As shown in Table 12 below, the PSMA PTE removal clones possess binding affinities closer to PSMA.2. All PTE removal clones were combined with CD3.23. All clones were screened as unmasked paTCE (uTCE)

TABLE 12

Binding affinities and melting temperatures of select PSMA antibodies

N_ELNN

Linker

C_ELNN

Length

Length

Length
K_D

(number

(number

(number
PSMA,

of

aPSMA
of
aCD3

of
Octet,

uTCE
N-term
residues)
RS
Domain
residues)
Domain
Order
RS
residues)
(nM)
TM

uTCE
ASHHHH
288
2295
PSMA.2
9
CD3.23
VL-
2295
576
1±0.6
64.3

of
HH (SEQ

VH

AC2591
ID NO:

9361)

uTCE

288
2295
PSMA.5
9
CD3.23
VL-
2295
576
3.9±1.8
64.6

of

VH

AC3092

uTCE
ASHHHH
144
2295
PSMA.5
15
CD3.23
VL-
2295
144
3.5±0.07
64.74

of
HH (SEQ

VH

AC3354
ID NO:

9361)

uTCE
ASHHHH
144
2295
PSMA.5
5
CD3.23
VL-
2295
144
2.1±0.2
64.05

of
HH (SEQ

VH

AC3353
ID NO:

9361)

uTCE
ASHHHH
144
2295
PSMA.5
9
CD3.23
VH-
2295
144
2.4±0.1
63.71

of
HH (SEQ

VL

AC3356
ID NO:

9361)

uTCE
ASHHHH
144
2295
PSMA.
9
CD3.23
VL-
2295
144
5.6±1.8
63.51

of
HH (SEQ

132

VH

AC3342
ID NO:

9361)

uTCE
ASHHHH
144
2295
PSMA.
9
CD3.23
VL-
2295
144
3.2±1.8
64.45

of
HH (SEQ

119

VH

AC3329
ID NO:

9361)

uTCE
ASHHHH
144
2295
PSMA.
5
CD3.23
VL-
2295
144
1.1±0.1
62.57

of
HH (SEQ

37

VH

AC3185
ID NO:

9361)

uTCE
ASHHHH
144
2295
PSMA.
9
CD3.23
VL-
2295
144
2.7±0.9
64.45

of
HH (SEQ

55

VH

AC3265
ID NO:

9361)

Binding affinity of select PSMA PTE variants was tested by Octet. Human PSMA-Fc fusions were used and measured with 300 nM, 100 nM, 33 nM, and 3.6 nM of PSMA-uTCE. PSMA.55, PSMA.119 and PSMA.132 are single point mutants of PTE removal variants which show maintained binding and stability. PSMA.2 is a camelid VHH. PSMA.5 and PSMA.6 are humanized VHH from PSMA. The Y to F mutation in PSMA.6 was determined to eliminate PTE in an assay. The binding affinities are shown below in Table 13.

TABLE 13

Binding affinities of select PSMA antibodies

Molecule
K_D(nM)
k_on(1/Ms)
k_off(1/s)

PSMA.55
2.7 ± 0.9
1.04E+05
2.80E−04

PSMA.119
3.2 ± 1.8
8.00E+04
2.48E−04

PSMA.132
5.6 ± 1.8
8.66E+04
4.79E−04

PSMA.2

1 ± 0.6
1.25E+05
1.22E−04

PSMA.5
3.9 ± 1.8
7.25E+04
2.73E−04

PSMA.6
2.3 ± 1
9.63E+04
2.35E−04

The binding affinity of PSMA antibodies was tested in the paTCE format using different linker lengths between the PSMA antibody and CD3 antibody to determine the effect of linker length on binding affinity. A 5 amino acid (5mer) and 15 amino acid (15mer) linker was tested. A domain swapped CD3 was also tested (VH-VL orientation and VL-VH orientation from N-terminus to C-terminus). The results, shown below in Table 14, show that linker length and domain swapping did not impact binding. PSMA.37 also had similar binding affinity to PSMA.2.

TABLE 14

Binding affinities of select PSMA antibodies with

alternative linker lengths and CD3 domain swapping

Molecule
K_D(nM)
k_on(1/Ms)
k_off(1/s)

PSMA.5 (5 mer)
2.1 ± 0.2
1.1E+05
2.2E−04

PSMA.5 (15 mer)
3.5 ± 0.07
7.7E+04
2.7E−04

PSMA.5 (domain
2.4 ± 0.1
7.6E+04
1.8E−04

swap)

PSMA.37 (scFv)
1.1 ± 0.1
1.2E+05
1.4E−04

PSMA.2

1 ± 0.6
1.2E+05
1.2E−04

PSMA.5
3.9 ± 1.8
7.2E+04
2.7E−04

PSMA.6
2.3 ± 1
9.6E+04
2.4E−04

Variants of the PSMA-binding unmasked PSMA-binding paTCE (uTCE) were tested for binding affinity, each variant employing a different PSMA antibody/CD3 antibody combination. The binding affinities to both human and cyno PSMA and human and cyno CD3 epsilon were determined. The values are reported below in Table 15 and Table 16. The melting temperature for several of these variants was also determined and reported below in Table 17. Cell binding data comparing the uTCEs from AC3092 (AMX-500-P1) and AC3896 (AMX-500-P4; also referred to here as simply AMX-500) is shown in FIG. 10. The EC50 values are reported below in Table 18.

TABLE 15

PSMA binding affinities of select PSMA-binding uTCEs

Octet,
Octet,
Biacore,
Biacore,

huPSMA
cyPSMA
huPSMA
cyPSMA

AC
RS
aPSMA
aCD3
RS
K_D(nM)
K_D(nM)
K_D(nM)
K_D(nM)

uTCE
2295
PSMA.5
CD3.23
2295
4.5
41.1
ND
ND

from

AC3092

uTCE
2295
PSMA.119
CD3.23
2295
6.2
47.5
ND
ND

from

AC3445

uTCE
3213
PSMA.350
CD3.228
3213
12.1
201
11.7
230

from

AC3896

uTCE
3213
PSMA.350
CD3.23
3213
13.2
198
19
354

from

AC3928

uTCE
3213
PSMA.262
CD3.228
3213
1.6
13.1
0.5
13

from

AC3934

ND = Not determined.

TABLE 16

CD3 binding affinities of select PSMA-binding uTCEs

Octet,
Octet,
Biacore,
Biacore,

huCD3e
cyCD3e
huCD3e
cyCD3e

AC
RS
aPSMA
aCD3
RS
K_D(nM)
K_D(nM)
K_D(nM)
K_D(nM)

uTCE
2295
PSMA.5
CD3.23
2295
71.9
64.6
ND
ND

from

AC3092

uTCE
2295
PSMA.119
CD3.23
2295
34.8
40
ND
ND

from

AC3445

uTCE
3213
PSMA.350
CD3.228
3213
33.2
38.8
12.8
13.7

from

AC3896

uTCE
3213
PSMA.350
CD3.23
3213
43
44.9
27.3
26.6

from

AC3928

uTCE
3213
PSMA.262
CD3.228
3213
26.6
33.7
9.83
10.39

from

AC3934

ND = Not Determined

TABLE 17

Melting Temperatures

AC Num
Description
Tm (° C.)
SD

AC2330
paTCE control for target
67.71
0.0001

other than PSMA

AC3896
AMX500-P4
70.92
0.0857

AC3896
AMX500-P4
70.53
0.0001

AC3928
AMX500-P6
68.01
0.1483

AC3928
AMX500-P6
67.61
0.0855

AC3934
AMX500-P7
70.97
0.0001

AC3934
AMX500-P7
70.43
0.0855

TABLE 18

CHO cell binding EC50 values (nM)

of select PSMA-binding paTCEs

Cell Line
AMX-500-P1 (nM)
AMX-500-P4 (nM)

Hu PSMA CHO
9.063
17.42

Cyno PSMA CHO
28.77
ND

The molecule designated AC3896 (AMX-500) was chosen for further characterization.

Unmasked PSMA-binding paTCE (uTCE) leads were screened via BLI (Biolayer Interferometry) or SPR (Surface Plasmon Resonance). The uTCE leads were determined to have a K_Din a range of 1 nM to 100 nM against human PSMA and a K_Din a range of 1 nM to 1000 nM against cynomolgus monkey PSMA. The masked PSMA-binding paTCE AMX-500 was determined to have a K_Dof about 546 nM and about 2900 nM against human and cynomolgus monkey PSMA, respectively. The metabolite AMX-500(1x-N) of AMX-500 has a K_Dof about 230 nM and about 2400 nM against human and cynomolgus monkey PSMA, respectively. The AMX-500(1x-C) metabolite AMX-500 has a K_Dof about 353 nM and about 2900 nM against human and cynomolgus monkey PSMA, respectively. Fully unmasked AMX-500(uTCE) has a K_Dof about 44 nM and about 410 nM against human and cynomolgus monkey PSMA, respectively. The values are reported below in Table 19 for PSMA and Table 20 for CD3.

TABLE 19

Binding kinetics of AMX-500 and its metabolites for human and cynomolgus monkey PSMA

Human PSMA
Cyno PSMA

K_D
ka
kd
K_D
ka
kd

Compound
(nM)
(M − 1 s − 1)
(s − 1)
(nM)
(M − 1 s − 1)
(s − 1)

AMX-500
546 ± 29
(2.3 ± 0.09)E4
(1.2 ± 0.03)E−2
2900 ± 700
(1.4 ± 0.5)E4
(4.0 ± 0.5)E−2

AMX-500
230 ± 18
(5.0 ± 0.2)E4
(1.2 ± 0.05)E−2
2400 ± 300
(1.6 ± 0.2)E4
(3.9 ± 0.6)E−2

(1x-N)

AMX-500
353 ± 11
(3.5 ± 0.1)E4
(1.2 ± 0.03)E−2
2900 ± 800
(1.4 ± 0.3)E4
(3.9 ± 0.09)E−2

(1x-C)

AMX-500
44 ± 1
(2.7 ± 0.06)E5
(1.2 ± 0.008)E−2
410.1 ± 0.8
(1.0 ± 0.01)E5
(4.3 ± 0.06)E−2

(uTCE)

TABLE 20

Binding kinetics of AMX-500 and its metabolites for human and cynomolgus monkey CD3

Human CD3
Cyno CD3

K_D
ka
kd
K_D
ka
kd

Compound
(nM)
(M − 1 s − 1)
(s − 1)
(nM)
(M − 1 s − 1)
(s − 1)

AMX-500
90 ± 3
(1.7 ± 0.09)E6
0.2 ± 0.004
70 ± 2
(2.0 ± 0.07)E6
0.1 ± 0.03

AMX-500
46.3 ± 0.3
(2.8 ± 0.07)E6
0.1 ± 0.002
38 ± 1
(2.9 ± 0.08)E6
0.1 ± 0.002

(1x-N)

AMX-500
34.4 ± 0.6
(3.2 ± 0.08)E6
0.1 ± 0.002
26.7 ± 0.7
(3.4 ± 0.09)E6
0.1 ± 0.0006

(1x-C)

AMX-500
28 ± 2
(4.0 ± 0.2)E6
0.1 ± 0.01
26 ± 3
(5 ± 2)E6
0.1 ± 0.05

(uTCE)

The binding kinetic data recited above in Table 19 demonstrate that AMX-500, comprising both masking polypeptides, has a higher K_Dfor PSMA than the unmasked AMX-500(uTCE). Similarly, the AMX-500 metabolites each have a higher K_Dfor PSMA than the unmasked AMX-500. The AMX-500 1x-N metabolite lacks the masking polypeptide linked to the CD3 antigen binding domain and the AMX-500 1x-C metabolite lacks the masking polypeptide linked to the PSMA antigen binding domain. Table 19 further demonstrates that the PSMA antigen binding domain does not cross react to cyno PSMA. Table 20 demonstrates that the CD3 antigen binding domain binds to human and cyno CD3 with a similar K_D.

Example 5. Design of Barcoded ELNNs by Minimal Mutations in ELNNs

ELNN polypeptide sequences can optionally contain a barcode fragment releasable from the polypeptide upon digestion by a protease. A barcode fragment may be, e.g., (1) a portion of the ELNN that includes at least part of a (non-recurring, non-overlapping) sequence motif that occurs only once within the ELNN; and (2) differs in sequence and molecular weight from all other peptide fragments that are releasable from the polypeptide containing them (e.g., a paTCE) upon complete digestion of the polypeptide by a protease. The term “barcode fragment” (“barcode,” or “barcode sequence”) can refer to either the portion of the ELNN cleavably fused within the polypeptide, or the resulting peptide fragment released from the polypeptide. Previous barcode sequences (see, e.g., PCT International Patent Application No. WO2021/263058, the entire content of which is incorporated herein by reference) were designed with the intention of creating unique barcode polypeptide sequences with as minimal mutations in the original ELNN sequence as possible. However, such barcode sequences required 1000 μg/mL of Glu-C and an overnight digest to release them from peptides containing them, such as paTCEs. The barcode polypeptide sequences described in this Example were designed and tested to perform against a second criteria: That the barcode polypeptide is releasable from the ELNN polypeptide rapidly (in approximately two hours vs an overnight digest) by a low concentration of protease (less than 30 μg/mL protease); in addition to the criteria of introducing the fewest mutations to the original ELNN sequence as possible.

In order to determine which peptide sequences were most favorably cleaved by Glu-C protease in a two-hour protease digest, a library of approximately 1000 peptides was constructed with each peptide containing a different cleavage sequence for the protease Glu-C. Equimolar concentrations of these Glu-C site-containing peptides were tested in a 2-hour digest against a range of Glu-C protease concentrations from 0.05 μg/mL to 1000 μg/mL of protease. After digestion the peptides were analyzed by liquid chromatography mass spectrometry. The Glu-C cleavage site sequences that were cleaved by the lowest concentrations of protease were cataloged. From this list of the fastest sequences, a select few were selected that were most compatible with ELNN polypeptides. These sequences were then implemented to flank new “Generation 2” barcode sequences.

A selection of Generation 2 barcode sequences was cloned into ELNN sequences and their performance as barcode peptides was tested by Glu-C digestion and subsequent liquid chromatography mass spectrometry analyses. Successful barcode sequences from this experiment had 3 criteria: 1.) The barcode peptide was fully releasable from the ELNN polypeptide in a 2-hour digest by a concentration of 40 μg/mL of protease. 2.) The barcode peptide was not cleaved or otherwise degraded by much higher concentrations of protease, and 3.) The barcode peptide that met conditions 1 and 2 contained the fewest mutations from the original ELNN polypeptide sequence. Below are examples of successful Generation 2 barcode sequences according to the criteria of the aforementioned selection process:

TABLE 21

Exemplary Generation 2 Barcode Sequences

Gen 2 Barcode
SGPE.SGPGTGTSATPE.SGPG (SEQ

01
ID NO: 9362)

Gen 2 Barcode
ATPE.SGPGSGPGTSE.SATP (SEQ

02
ID NO: 9363)

Gen 2 Barcode
ATPE.SGPGTTPGTTPE.SGPG (SEQ

03
ID NO: 9364)

Gen 2 Barcode
ATPE.SGPGTPPTSTPE.SGPG (SEQ

04
ID NO: 9365)

Gen 2 Barcode
ATPE.SGPGTSPSATPE.SGPG (SEQ

05
ID NO: 9366)

Gen 2 Barcode
ATPE.SGPGTGSAGTPE.SGPG (SEQ

06
ID NO: 9367)

Gen 2 Barcode
ATPE.SGPGTGGAGTPE.SGPG (SEQ

07
ID NO: 9368)

Gen 2 Barcode
ATPE.SGPGTSPGATPE.SGPG (SEQ

08
ID NO: 9369)

Gen 2 Barcode
GTPE.SGPGTSGSGTPE.SGPG (SEQ

09
ID NO: 9370)

Gen 2 Barcode
GTPE.SGPGTSSASTPE.SGPG (SEQ

10
ID NO: 9371)

Gen 2 Barcode
GTPE.SGPGTGAGTTPE.SGPG (SEQ

11
ID NO: 9372)

Gen 2 Barcode
GTPE.SGPGTGSTSTPE.SGPG (SEQ

12
ID NO: 9373)

Gen 2 Barcode
GTPE.TPGSEPATSGSE.TGTP (SEQ

13
ID NO: 9374)

Gen 2 Barcode
GTPE.GSAPGTSTEPSE.SATP (SEQ

14
ID NO: 9375)

Gen 2 Barcode
ATPE.SGPGTAGSGTPE.SGPG (SEQ

15
ID NO: 9376)

Gen 2 Barcode
ATPE.SGPGTSSGGTPE.SGPG (SEQ

16
ID NO: 9377)

Gen 2 Barcode
ATPE.SGPGTAGPATPE.SGPG (SEQ

17
ID NO: 9378)

Gen 2 Barcode
ATPE.SGPGTPGTGTPE.SGPG (SEQ

18
ID NO: 9379)

Gen 2 Barcode
TTPE.SGPGTGGPTTPE.SGPG (SEQ

19
ID NO: 9380)

Gen 2 Barcode
STPE.SGPGTGSGSTPE.SGPG (SEQ

20
ID NO: 9381)

Example 6. Improved Anti-CD3 Binding Sequences

CD3 scFv paTCE arm optimization was conducted to reduce molecule immunogenicity and improve stability, while maintaining binding affinity with CD3 close to the affinity observed for the CD3.23 parental molecule.

To achieve this, Pool 1 was created, which included 74 paTCE molecules, each containing PSMA.5 and one of the 74 CD3.23 mutation variants. The amino acid sequences of each of the 74 CD3.23 mutation variants are provided in Table 23a. Single mutations were chosen based on analyses including CD3.23 PTE score analysis (using internal PTE algorithm v12) and structural analysis. Structural considerations included: possible contact disruption, anticipated steric clashes, side chain charge maintenance and possible pockets filling. Stability and affinity of the individually expressed molecules in the form of crude lysate was evaluated by Octet (ForteBio).

Based on the results of the Pool 1 screening, mutations that did not disrupt paTCE molecule affinity and stability were taken further to evaluate as combinations in Pool 2. Pool 2 consisted of paTCE molecules each containing PSMA.262 and one of 64 CD3.23 mutation combination variants. The amino acid sequences of each of the 64 CD3.23 mutation combination variants are provided in Table 23b. Stability and affinity of the individually expressed molecules in the form of crude lysate was evaluated by Octet. The four most stable paTCE molecules from Pool 2 were additionally expressed in a larger volume (2.5 L) and purified. The binding of these anti-CD3 molecules (CD3.227, CD3.228, CD3.229 and CD3.230) to human and cynomolgus CD3 was measured by Octet and the T_mwas measured by Differential Scanning Fluorimetry. All variants were paired with PSMA.262 except for CD3.23 which was paired with PSMA.5. Values are reported below in Table 22.

TABLE 22

Binding affinities, melting temperatures, and PTE values for select CD3 antibodies

kon
kdiss

kon
kdiss

K_D,
(1/Ms),
(1/s),

(1/Ms),
(1/s),
PTE

CD3
huCD3e-
huCD3e-
huCD3e-
K_D,
cyCD3e-
cyCD3e-
score

Antibody
Fc
Fc
Fc
cyCD3e
Fc
Fc
(v22)
Tm

CD3.227
57 nM
3.42E+05
1.96E−02
80 nM
3.15E+05
2.53E−02
10
63.71

CD3.228
69 nM
3.17E+05
2.17E−02
80 nM
3.34E+05
2.66E−02
10
64.45

CD3.229
162 nM
3.21E+05
5.20E−02
193 nM
3.17E+05
6.11E−02
15
63.46

CD3.230
195 nM
3.25E+05
6.33E−02
216 nM
3.36E+05
7.26E−02
15
63.71

CD3.23
131 nM
2.20E+05
2.89E−02
130 nM
2.40E+05
3.12E−02
73
62.62

Based on these data that included additional PTE score evaluation using internal PTE algorithm v22 (FIG. 6A), and an additional thermal stability evaluation for which the antibodies were heated at 60° C. for 5 min (FIG. 6B), CD3.228 scFv was chosen over the other leads in Table 22.

An alignment of parental CD3.9 and CD3.23 and selected CD3.228 VL and VH molecules with differences highlighted is provided below. CD3.9 is a humanized version of the SP34 monoclonal mouse antibody. CD3.23 has 8 mutations compared to CD3.9, and has an estimated 2-4 fold lower affinity vs CD3.9 based on ELISA, Octet, and cell binding data. CD3.228 has 8 mutations compared to CD3.23 and 16 mutations compared to CD3.9. CD3.228 has increased stability and lower immunogenicity risk compared to CD3.23.

Mutation numbering is according to Kabat database.

>CD3.9_VL

(SEQ ID NO: 359)

ELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRA

PGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL

>CD3.23_VL

(SEQ ID NO: 361)

ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRA

PGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL

>CD3.228_VL

(SEQ ID NO: 361)

ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRA

PGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL

CD3.9_VL

(SEQ ID NO: 9382)

ELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGT

CD3.23_VL

(SEQ ID NO: 9383)

ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGT

CD3.228_VL

(SEQ ID NO: 9383)

ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIGGTNKRAPGT

*************************.****.*****************************

CD3.9_VL

(SEQ ID NO: 9384)

PARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL

CD3.23_VL

(SEQ ID NO: 9385)

PARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL

CD3.228_VL

(SEQ ID NO: 9385)

PARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGGGTKLTVL

**************************.*******.**************

T27N, T29S, E85V, S93P: differences with CD3.9

>CD3.9_VH

(SEQ ID NO: 309)

EVQLLESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNN

YATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAY

WGQGTLVTVSS

>CD3.23_VH

(SEQ ID NO: 102)

EVQLLESGGGIVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNN

YATYYADSVKDRFTISRDDSKNTVYLQMNNLKTEDTAVYYCVRHENFGNSYVSWFAH

WGQGTLVTVSS

>CD3.228_VH

(SEQ ID NO: 311)

EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKRNN

YATYYADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCVRHENFGNSYVSWFAH

WGQGTLVTVSS

CD3.9_VH

(SEQ ID NO: 9386)

EVQLLESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYAT

CD3.23_VH

(SEQ ID NO: 9387)

EVQLLESGGGIVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYAT

CD3.228_VH

(SEQ ID NO: 9388)

EVQLVESGGGIVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRTKRNNYAT

****:*****:*******:**********.******************.***:*.*****

CD3.9_VH

(SEQ ID NO: 9389)

YYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTL

CD3.23_VH

(SEQ ID NO: 9390)

YYADSVKDRFTISRDDSKNTVYLQMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTL

CD3.228_VH

(SEQ ID NO: 9391)

YYADSVKGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTL

*******.************ *****.************** ***********:******

CD3.9_VH

(SEQ ID NO: 574)

VTVSS

CD3.23_VH

(SEQ ID NO: 574)

VTVSS

CD3.228_VH

(SEQ ID NO: 574)

VTVSS

*****

L11I, A78V, G96E, Y102H: differences with CD3.9 shared between CD3.23 and CD3.228

L5V, K19R, N30S, A49G, D65G, N82bS

: PTE removal, not present in CD3.9 or CD3.23

TABLE 23a

Pool 1 CD3.23 Mutation Variants

VL

VH

SEQ

SEQ

AC
VL sequence
ID
VH sequence
ID

AC3364
ELVVTQEPSLTVSPGGTVTL
834
EVQLLESGGGIVQPGGSLKLSC
835

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVARIRSKYNNYATYYA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3366
ELVVTQEPSLTVSPGGTVTL
836
EVQLLESGGGIVQPGGSLKLSC
837

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVARIRSKYNNYATYYA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3367
ELVVTQEPSLTVSPGGTVTL
838
EVQLLESGGGIVQPGGSLKLSC
839

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEDVARIRSKYNNYATYYAD

PGTPARFSGSLLGGKAALTL

SVKDRFTISRDDSKNTVYLQMN

SGVQPEDEAVYYCALWYPN

NLKTEDTAVYYCVRHENFGNS

LWVFGGGTKLTVL

YVSWFAHWGQGTLVTVSS

AC3368
ELVVTQEPSLTVSPGGTVTL
840
EVQLLESGGGIVQPGGSLKLSC
841

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEEVARIRSKYNNYATYYAD

PGTPARFSGSLLGGKAALTL

SVKDRFTISRDDSKNTVYLQMN

SGVQPEDEAVYYCALWYPN

NLKTEDTAVYYCVRHENFGNS

LWVFGGGTKLTVL

YVSWFAHWGQGTLVTVSS

AC3369
ELVVTQEPSLTVSPGGTVTL
842
EVQLLESGGGIVQPGGSLKLSC
843

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWAARIRSKYNNYATYYA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3370
ELVVTQEPSLTVSPGGTVTL
844
EVQLLESGGGIVQPGGSLKLSC
845

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWEARIRSKYNNYATYYA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3371
ELVVTQEPSLTVSPGGTVTL
846
EVQLLESGGGIVQPGGSLKLSC
847

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWGARIRSKYNNYATYYA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3372
ELVVTQEPSLTVSPGGTVTL
848
EVQLLESGGGIVQPGGSLKLSC
849

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWSARIRSKYNNYATYYA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3373
ELVVTQEPSLTVSPGGTVTL
850
EVQLLESGGGIVQPGGSLKLSC
851

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWTARIRSKYNNYATYYA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3374
ELVVTQEPSLTVSPGGTVTL
852
EVQLLESGGGIVQPGGSLKLSC
853

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWWARIRSKYNNYATYYA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3375
ELVVTQEPSLTVSPGGTVTL
854
EVQLLESGGGIVQPGGSLKLSC
855

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVDRIRSKYNNYATYYA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3376
ELVVTQEPSLTVSPGGTVTL
856
EVQLLESGGGIVQPGGSLKLSC
857

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVERIRSKYNNYATYYA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3377
ELVVTQEPSLTVSPGGTVTL
858
EVQLLESGGGIVQPGGSLKLSC
859

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVGRIRSKYNNYATYYA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3378
ELVVTQEPSLTVSPGGTVTL
860
EVQLLESGGGIVQPGGSLKLSC
861

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVAQIRSKYNNYATYYA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3379
ELVVTQEPSLTVSPGGTVTL
862
EVQLLESGGGIVQPGGSLKLSC
863

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVAGIRSKYNNYATYYA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3380
ELVVTQEPSLTVSPGGTVTL
864
EVQLLESGGGIVQPGGSLKLSC
865

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVAHIRSKYNNYATYYA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3381
ELVVTQEPSLTVSPGGTVTL
866
EVQLLESGGGIVQPGGSLKLSC
867

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVAPIRSKYNNYATYYA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3382
ELVVTQEPSLTVSPGGTVTL
868
EVQLLESGGGIVQPGGSLKLSC
869

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVAWIRSKYNNYATYYA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3383
ELVVTQEPSLTVSPGGTVTL
870
EVQLLESGGGIVQPGGSLKLSC
871

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVARARSKYNNYATYYA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3384
ELVVTQEPSLTVSPGGTVTL
872
EVQLLESGGGIVQPGGSLKLSC
873

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVARGRSKYNNYATYYA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3385
ELVVTQEPSLTVSPGGTVTL
874
EVQLLESGGGIVQPGGSLKLSC
875

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVARTRSKYNNYATYYA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3386
ELVVTQEPSLTVSPGGTVTL
876
EVQLLESGGGIVQPGGSLKLSC
877

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVARINSKYNNYATYYA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3387
ELVVTQEPSLTVSPGGTVTL
878
EVQLLESGGGIVQPGGSLKLSC
879

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVARIDSKYNNYATYYA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3388
ELVVTQEPSLTVSPGGTVTL
880
EVQLLESGGGIVQPGGSLKLSC
881

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVARIESKYNNYATYYA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3389
ELVVTQEPSLTVSPGGTVTL
882
EVQLLESGGGIVQPGGSLKLSC
883

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVARIQSKYNNYATYYA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3390
ELVVTQEPSLTVSPGGTVTL
884
EVQLLESGGGIVQPGGSLKLSC
885

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVARIGSKYNNYATYYA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3391
ELVVTQEPSLTVSPGGTVTL
886
EVQLLESGGGIVQPGGSLKLSC
887

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVARIHSKYNNYATYYA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3392
ELVVTQEPSLTVSPGGTVTL
888
EVQLLESGGGIVQPGGSLKLSC
889

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVARIWSKYNNYATYYA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3393
ELVVTQEPSLTVSPGGTVTL
890
EVQLLESGGGIVQPGGSLKLSC
891

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVARIRNKYNNYATYYA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3394
ELVVTQEPSLTVSPGGTVTL
892
EVQLLESGGGIVQPGGSLKLSC
893

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVARIRDKYNNYATYYA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3395
ELVVTQEPSLTVSPGGTVTL
894
EVQLLESGGGIVQPGGSLKLSC
895

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVARIREKYNNYATYYA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3396
ELVVTQEPSLTVSPGGTVTL
896
EVQLLESGGGIVQPGGSLKLSC
897

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVARIRTKYNNYATYYA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3397
ELVVTQEPSLTVSPGGTVTL
898
EVQLLESGGGIVQPGGSLKLSC
899

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVARIRSPYNNYATYYA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3398
ELVVTQEPSLTVSPGGTVTL
900
EVQLLESGGGIVQPGGSLKLSC
901

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVARIRSKANNYATYYA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3399
ELVVTQEPSLTVSPGGTVTL
902
EVQLLESGGGIVQPGGSLKLSC
903

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVARIRSKRNNYATYYA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3400
ELVVTQEPSLTVSPGGTVTL
904
EVQLLESGGGIVQPGGSLKLSC
905

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVARIRSKGNNYATYYA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3401
ELVVTQEPSLTVSPGGTVTL
906
EVQLLESGGGIVQPGGSLKLSC
907

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVARIRSKKNNYATYYA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3402
ELVVTQEPSLTVSPGGTVTL
908
EVQLLESGGGIVQPGGSLKLSC
909

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVARIRSKPNNYATYYA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3403
ELVVTQEPSLTVSPGGTVTL
910
EVQLLESGGGIVQPGGSLKLSC
911

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVARIRSKTNNYATYYA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3404
ELVVTQEPSLTVSPGGTVTL
912
EVQLLESGGGIVQPGGSLKLSC
913

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVARIRSKWNNYATYYA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3405
ELVVTQEPSLTVSPGGTVTL
914
EVQLLESGGGIVQPGGSLKLSC
915

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVARIRSKYDNYATYYA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3406
ELVVTQEPSLTVSPGGTVTL
916
EVQLLESGGGIVQPGGSLKLSC
917

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVARIRSKYENYATYYA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3407
ELVVTQEPSLTVSPGGTVTL
918
EVQLLESGGGIVQPGGSLKLSC
919

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVARIRSKYNDYATYYA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3408
ELVVTQEPSLTVSPGGTVTL
920
EVQLLESGGGIVQPGGSLKLSC
921

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVARIRSKYNEYATYYA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3409
ELVVTQEPSLTVSPGGTVTL
922
EVQLLESGGGIVQPGGSLKLSC
923

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVARIRSKYNNGATYYA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3410
ELVVTQEPSLTVSPGGTVTL
924
EVQLLESGGGIVQPGGSLKLSC
925

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVARIRSKYNNFATYYA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3411
ELVVTQEPSLTVSPGGTVTL
926
EVQLLESGGGIVQPGGSLKLSC
927

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVARIRSKYNNWATYYA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3412
ELVVTQEPSLTVSPGGTVTL
928
EVQLLESGGGIVQPGGSLKLSC
929

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVARIRSKYNNYGTYYA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3413
ELVVTQEPSLTVSPGGTVTL
930
EVQLLESGGGIVQPGGSLKLSC
931

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVARIRSKYNNYTTYYA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3414
ELVVTQEPSLTVSPGGTVTL
932
EVQLLESGGGIVQPGGSLKLSC
933

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVARIRSKYNNYATDYA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3415
ELVVTQEPSLTVSPGGTVTL
934
EVQLLESGGGIVQPGGSLKLSC
935

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVARIRSKYNNYATEYA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3416
ELVVTQEPSLTVSPGGTVTL
936
EVQLLESGGGIVQPGGSLKLSC
937

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVARIRSKYNNYATTYA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3417
ELVVTQEPSLTVSPGGTVTL
938
EVQLLESGGGIVQPGGSLKLSC
939

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVARIRSKYNNYATYDA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3418
ELVVTQEPSLTVSPGGTVTL
940
EVQLLESGGGIVQPGGSLKLSC
941

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVARIRSKYNNYATYEA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3419
ELVVTQEPSLTVSPGGTVTL
942
EVQLLESGGGIVQPGGSLKLSC
943

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVARIRSKYNNYATYQA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3420
ELVVTQEPSLTVSPGGTVTL
944
EVQLLESGGGIVQPGGSLKLSC
945

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVARIRSKYNNYATYGA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3421
ELVVTQEPSLTVSPGGTVTL
946
EVQLLESGGGIVQPGGSLKLSC
947

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVARIRSKYNNYATYWA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3422
ELVVTQEPSLTVSPGGTVTL
948
EVQLLESGGGIVQPGGSLKLSC
949

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVARIRSKYNNYATYYK

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3423
ELVVTQEPSLTVSPGGTVTL
950
EVQLLESGGGIVQPGGSLKLSC
951

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVARIRSKYNNYATYYP

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3424
ELVVTQEPSLTVSPGGTVTL
952
EVQLLESGGGIVQPGGSLKLSC
953

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVARIRSKYNNYATYYA

PGTPARFSGSLLGGKAALTL

DSVKGRFTISRDDSKNTVYLQ

SGVQPEDEAVYYCALWYPN

MNNLKTEDTAVYYCVRHENFG

LWVFGGGTKLTVL

NSYVSWFAHWGQGTLVTVSS

AC3425
ELVVTQEPSLTVSPGGTVTL
954
EVQLLESGGGIVQPGGSLKLSC
955

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVARIRSKYNNYATYYA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVDLQ

SGVQPEDEAVYYCALWYPN

MNNLKTEDTAVYYCVRHENFG

LWVFGGGTKLTVL

NSYVSWFAHWGQGTLVTVSS

AC3426
ELVVTQEPSLTVSPGGTVTL
956
EVQLLESGGGIVQPGGSLKLSC
957

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVARIRSKYNNYATYYA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVGLQ

SGVQPEDEAVYYCALWYPN

MNNLKTEDTAVYYCVRHENFG

LWVFGGGTKLTVL

NSYVSWFAHWGQGTLVTVSS

AC3427
ELVVTQEPSLTVSPGGTVTL
958
EVQLLESGGGIVQPGGSLKLSC
959

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVARIRSKYNNYATYYA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVSLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3428
ELVVTQEPSLTVSPGGTVTL
960
EVQLLESGGGIVQPGGSLKLSC
961

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVARIRSKYNNYATYYA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NELKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3429
ELVVTQEPSLTVSPGGTVTL
962
EVQLLESGGGIVQPGGSLKLSC
963

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVARIRSKYNNYATYYA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NQLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3430
ELVVTQEPSLTVSPGGTVTL
964
EVQLLESGGGIVQPGGSLKLSC
965

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVARIRSKYNNYATYYA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NSLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3431
ELVVTQEPSLTVSPGGTVTL
966
EVQLLESGGGIVQPGGSLKLSC
967

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVARIRSKYNNYATYYA

PGTPARFSGSLLGGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NYLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3432
ELVVTQEPSLTVSPGGTVTL
968
EVQLLESGGGIVQPGGSLKLSC
969

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVGRIRSKYNNGATYYA

PGTPARFSGSLLGGKAALTL

DSVKGRFTISRDDSKNTVYLQ

SGVQPEDEAVYYCALWYPN

MNSLKTEDTAVYYCVRHENFG

LWVFGGGTKLTVL

NSYVSWFAHWGQGTLVTVSS

AC3433
ELVVTQEPSLTVSPGGTVTL
970
EVQLLESGGGIVQPGGSLKLSC
971

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVARIRSKYNNYATYYA

PGTPARFSGSLQGGKAALT

DSVKDRFTISRDDSKNTVYLQM

LSGVQPEDEAVYYCALWYP

NNLKTEDTAVYYCVRHENFGN

NLWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3434
ELVVTQEPSLTVSPGGTVTL
972
EVQLLESGGGIVQPGGSLKLSC
973

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVARIRSKYNNYATYYA

PGTPARFSGSLLEGKAALTL

DSVKDRFTISRDDSKNTVYLQM

SGVQPEDEAVYYCALWYPN

NNLKTEDTAVYYCVRHENFGN

LWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3435
ELVVTQEPSLTVSPGGTVTL
974
EVQLLESGGGIVQPGGSLKLSC
975

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVARIRSKYNNYATYYA

PGTPARFSGSLDGGKAALT

DSVKDRFTISRDDSKNTVYLQM

LSGVQPEDEAVYYCALWYP

NNLKTEDTAVYYCVRHENFGN

NLWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3436
ELVVTQEPSLTVSPGGTVTL
976
EVQLLESGGGIVQPGGSLKLSC
977

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVARIRSKYNNYATYYA

PGTPARFSGSSLGGKAALT

DSVKDRFTISRDDSKNTVYLQM

LSGVQPEDEAVYYCALWYP

NNLKTEDTAVYYCVRHENFGN

NLWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3437
ELVVTQEPSLTVSPGGTVTL
978
EVQLLESGGGIVQPGGSLKLSC
979

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVARIRSKYNNYATYYA

PGTPARFSGSKLGGKAALT

DSVKDRFTISRDDSKNTVYLQM

LSGVQPEDEAVYYCALWYP

NNLKTEDTAVYYCVRHENFGN

NLWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3438
ELVVTQEPSLTVSPGGTVTL
980
EVQLLESGGGIVQPGGSLKLSC
981

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVARIRSKYNNYATYYA

PGTPARFSGSNLGGKAALT

DSVKDRFTISRDDSKNTVYLQM

LSGVQPEDEAVYYCALWYP

NNLKTEDTAVYYCVRHENFGN

NLWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

AC3439
ELVVTQEPSLTVSPGGTVTL
982
EVQLLESGGGIVQPGGSLKLSC
983

TCRSSNGAVTSSNYANWV

AASGFTFNTYAMNWVRQAPG

QQKPGQAPRGLIGGTNKRA

KGLEWVARIRSKYNNYATYYA

PGTPARFSGSTLGGKAALT

DSVKDRFTISRDDSKNTVYLQM

LSGVQPEDEAVYYCALWYP

NNLKTEDTAVYYCVRHENFGN

NLWVFGGGTKLTVL

SYVSWFAHWGQGTLVTVSS

TABLE 23a cont.

Pool 1 CD3.23 Mutation Variants

Relative

Expression

(1 - lowest

expression, 5 -

highest

expression -

%

PTE
evaluated by PEG

Remaining

CD3.23

score
gel
Primary K_D
after

AC
domain
Mutation
v12
electrophoresis)
(nM)
heating

AC3364
CD3.23-L7
WT
50
4
8.5
56.90%

AC3366
CD3.23-D
WT
50
3
14.0
28.73%

AC3367
CD3.38
W47D
41
4
40.2
ND

AC3368
CD3.39
W47E
42
4
18.1
0

AC3369
CD3.40
V48A
38
4
8.3
0

AC3370
CD3.41
V48E
38
4
164.8
ND

AC3371
CD3.42
V48G
38
4
71.5
ND

AC3372
CD3.43
V48S
38
4
11.2
0

AC3373
CD3.44
V48T
38
4
5.7
0

AC3374
CD3.45
V48W
40
4
34270.0
ND

AC3375
CD3.46
A49D
48
4
Weak binding
ND

AC3376
CD3.47
A49E
46
4
No binding
ND

AC3377
CD3.48
A49G
45
4
4.5
79.06%

AC3378
CD3.49
R50Q
39
4
No binding
ND

AC3379
CD3.50
R50G
39
4
No binding
ND

AC3380
CD3.51
R50H
41
4
No binding
ND

AC3381
CD3.52
R50P
39
4
No binding
ND

AC3382
CD3.53
R50W
39
2
No binding
ND

AC3383
CD3.54
151A
45
2
1182.0
ND

AC3384
CD3.55
151G
42
2
Weak binding
ND

AC3385
CD3.56
151T
44
2
424.6
ND

AC3386
CD3.57
R52N
39
2
No binding
ND

AC3387
CD3.58
R52D
35
2
No binding
ND

AC3388
CD3.59
R52E
35
2
No binding
ND

AC3389
CD3.60
R52Q
48
2
434.4
ND

AC3390
CD3.61
R52G
41
2
No binding
ND

AC3391
CD3.62
R52H
50
2
No binding
ND

AC3392
CD3.63
R52W
42
2
No binding
ND

AC3393
CD3.64
S52aN
48
2
Weak binding
ND

AC3394
CD3.65
S52aD
42
4
No binding
ND

AC3395
CD3.66
S52aE
42
4
No binding
ND

AC3396
CD3.67
S52aT
49
4
4.8
60.06%

AC3397
CD3.68
K52bP
45
4
51.0
ND

AC3398
CD3.69
Y52cA
37
4
11.5
14.99%

AC3399
CD3.70
Y52cR
38
4
3.8
56.68%

AC3400
CD3.71
Y52cG
36
4
20.1
0

AC3401
CD3.72
Y52cK
40
4
5.1
60.72%

AC3402
CD3.73
Y52cP
36
4
33.1
ND

AC3403
CD3.74
Y52cT
36
4
11.1
35.59%

AC3404
CD3.75
Y52cW
48
4
10.5
15.25%

AC3405
CD3.76
N53D
34
4
No binding
ND

AC3406
CD3.77
N53E
34
4
574.6
ND

AC3407
CD3.78
N54D
37
4
7.4
61.45%

AC3408
CD3.79
N54E
42
4
8.3
43.27%

AC3409
CD3.80
Y55G
34
4
11.3
0

AC3410
CD3.81
Y55F
44
4
6.1
23.50%

AC3411
CD3.82
Y55W
38
4
7.8
6.79%

AC3412
CD3.83
A56G
49
4
8.2
9.14%

AC3413
CD3.84
A56T
49
4
10.7
26.45%

AC3414
CD3.85
Y58D
35
4
938.6
ND

AC3415
CD3.86
Y58E
35
4
183.4
ND

AC3416
CD3.87
Y58T
35
4
17.9
26.86%

AC3417
CD3.88
Y59D
42
4
63.2
ND

AC3418
CD3.89
Y59E
42
4
9.7
0

AC3419
CD3.90
Y59Q
42
4
7.2
0

AC3420
CD3.91
Y59G
42
4
8.3
0

AC3421
CD3.92
Y59W
42
4
37.2
ND

AC3422
CD3.93
A60K
37
4
8.0
0

AC3423
CD3.94
A60P
35
4
8.2
0

AC3424
CD3.95
D65G
46
4
5.4
47.80%

AC3425
CD3.96
Y79D
31
4
9.8
0

AC3426
CD3.97
Y79G
31
2
121.1
ND

AC3427
CD3.98
Y79S
31
4
9.6
0

AC3428
CD3.99
N82bE
39
4
5.9
39.70%

AC3429
CD3.100
N82bQ
40
4
7.1
18.12%

AC3430
CD3.101
N82bS
32
4
4.8
4.22%

AC3431
CD3.102
N82bY
46
4
5.2
1.66%

AC3432
CD3.103
A49G,
8
4
11.4
0

Y52cG,

D65G,

N82bS

AC3433
CD3.104
L67Q
55
4
4.6
59.68%

AC3434
CD3.105
G68E
54
4
4.9
70.99%

AC3435
CD3.106
L67D
50
4
6.1
43.75%

AC3436
CD3.107
L66S
50
4
7.3
0

AC3437
CD3.108
L66K
50
4
3.2
0

AC3438
CD3.109
L66N
50
4
8.3
0

AC3439
CD3.110
L66T
50
4
8.9
0

TABLE 23b

Pool 2 CD3.23 Mutation Combination Variants

VL

VH

SEQ

SEQ

AC
VL sequence
ID
VH sequence
ID

AC3632
ELVVTQEPSLTVSPGGTVTL
700
EVQLLESGGGIVQPGGSLRL
701

TCRSSNGAVTSSNYANWV

SCAASGFTFNTYAMNWVRQ

QQKPGQAPRGLIGGTNKRA

APGKGLEWVGRIRSKRNNYA

PGTPARFSGSLLGGKAALTL

TYYADSVKGRFTISRDDSKNT

SGVQPEDEAVYYCALWYPN

VYLQMNSLKTEDTAVYYCVR

LWVFGGGTKLTVL

HENFGNSYVSWFAHWGQGT

LVTVSS

AC3633
ELVVTQEPSLTVSPGGTVTL
702
EVQLVESGGGIVQPGGSLRL
703

TCRSSNGAVTSSNYANWV

SCAASGFTFNTYAMNWVRQ

QQKPGQAPRGLIGGTNKRA

APGKGLEWVGRIRSKRNNYA

PGTPARFSGSLLGGKAALTL

TYYADSVKGRFTISRDDSKNT

SGVQPEDEAVYYCALWYPN

VYLQMNSLKTEDTAVYYCVR

LWVFGGGTKLTVL

HENFGNSYVSWFAHWGQGT

LVTVSS

AC3634
ELVVTQEPSLTVSPGGTVTL
704
EVQLLESGGGIVQPGGSLRL
705

TCRSSNGAVTSSNYANWV

SCAASGFTFNTYAMNWVRQ

QQKPGQAPRGLIGGTNKRA

APGKGLEWVGRIRSKTNNYA

PGTPARFSGSLLGGKAALTL

TYYADSVKGRFTISRDDSKNT

SGVQPEDEAVYYCALWYPN

VYLQMNSLKTEDTAVYYCVR

LWVFGGGTKLTVL

HENFGNSYVSWFAHWGQGT

LVTVSS

AC3635
ELVVTQEPSLTVSPGGTVTL
706
EVQLVESGGGIVQPGGSLRL
707

TCRSSNGAVTSSNYANWV

SCAASGFTFNTYAMNWVRQ

QQKPGQAPRGLIGGTNKRA

APGKGLEWVGRIRSKTNNYA

PGTPARFSGSLLGGKAALTL

TYYADSVKGRFTISRDDSKNT

SGVQPEDEAVYYCALWYPN

VYLQMNSLKTEDTAVYYCVR

LWVFGGGTKLTVL

HENFGNSYVSWFAHWGQGT

LVTVSS

AC3636
ELVVTQEPSLTVSPGGTVTL
708
EVQLLESGGGIVQPGGSLRL
709

TCRSSNGAVTSSNYANWV

SCAASGFTFNTYAMNWVRQ

QQKPGQAPRGLIGGTNKRA

APGKGLEWVGRIRSKYNDYA

PGTPARFSGSLLGGKAALTL

TYYADSVKGRFTISRDDSKNT

SGVQPEDEAVYYCALWYPN

VYLQMNSLKTEDTAVYYCVR

LWVFGGGTKLTVL

HENFGNSYVSWFAHWGQGT

LVTVSS

AC3637
ELVVTQEPSLTVSPGGTVTL
710
EVQLVESGGGIVQPGGSLRL
711

TCRSSNGAVTSSNYANWV

SCAASGFTFNTYAMNWVRQ

QQKPGQAPRGLIGGTNKRA

APGKGLEWVGRIRSKYNDYA

PGTPARFSGSLLGGKAALTL

TYYADSVKGRFTISRDDSKNT

SGVQPEDEAVYYCALWYPN

VYLQMNSLKTEDTAVYYCVR

LWVFGGGTKLTVL

HENFGNSYVSWFAHWGQGT

LVTVSS

AC3638
ELVVTQEPSLTVSPGGTVTL
712
EVQLLESGGGIVQPGGSLRL
713

TCRSSNGAVTSSNYANWV

SCAASGFTFNTYAMNWVRQ

QQKPGQAPRGLIGGTNKRA

APGKGLEWVGRIRSKYNNYA

PGTPARFSGSLLGGKAALTL

TTYADSVKGRFTISRDDSKNT

SGVQPEDEAVYYCALWYPN

VYLQMNSLKTEDTAVYYCVR

LWVFGGGTKLTVL

HENFGNSYVSWFAHWGQGT

LVTVSS

AC3639
ELVVTQEPSLTVSPGGTVTL
714
EVQLVESGGGIVQPGGSLRL
715

TCRSSNGAVTSSNYANWV

SCAASGFTFNTYAMNWVRQ

QQKPGQAPRGLIGGTNKRA

APGKGLEWVGRIRSKYNNYA

PGTPARFSGSLLGGKAALTL

TTYADSVKGRFTISRDDSKNT

SGVQPEDEAVYYCALWYPN

VYLQMNSLKTEDTAVYYCVR

LWVFGGGTKLTVL

HENFGNSYVSWFAHWGQGT

LVTVSS

AC3640
ELVVTQEPSLTVSPGGTVTL
716
EVQLVESGGGIVQPGGSLRL
717

TCRSSNGAVTSSNYANWV

SCAASGFTFNTYAMNWVRQ

QQKPGQAPRGLIGGTNKRA

APGKGLEWVGRIRSKRNNYA

PGTPARFSGSLLGGKAALTL

TYYADSVKGRFTISRDDSKNT

SGVQPEDEAVYYCALWYPN

VYLQMNELKTEDTAVYYCVR

LWVFGGGTKLTVL

HENFGNSYVSWFAHWGQGT

LVTVSS

AC3641
ELVVTQEPSLTVSPGGTVTL
718
EVQLVESGGGIVQPGGSLRL
719

TCRSSNGAVTSSNYANWV

SCAASGFTFSTYAMNWVRQA

QQKPGQAPRGLIGGTNKRA

PGKGLEWVGRIRSKRNNYAT

PGTPARFSGSLLGGKAALTL

YYADSVKGRFTISRDDSKNTV

SGVQPEDEAVYYCALWYPN

YLQMNELKTEDTAVYYCVRH

LWVFGGGTKLTVL

ENFGNSYVSWFAHWGQGTL

VTVSS

AC3642
ELVVTQEPSLTVSPGGTVTL
720
EVQLVESGGGIVQPGGSLRL
72

TCRSSNGAVTSSNYANWV

SCAASGFTFNTYAMNWVRQ

QQKPGQAPRGLIGGTNKRA

APGKGLEWVGRIRSKTNNYA

PGTPARFSGSLLGGKAALTL

TYYADSVKGRFTISRDDSKNT

SGVQPEDEAVYYCALWYPN

VYLQMNELKTEDTAVYYCVR

LWVFGGGTKLTVL

HENFGNSYVSWFAHWGQGT

LVTVSS

AC3643
ELVVTQEPSLTVSPGGTVTL
722
EVQLVESGGGIVQPGGSLRL
723

TCRSSNGAVTSSNYANWV

SCAASGFTFSTYAMNWVRQA

QQKPGQAPRGLIGGTNKRA

PGKGLEWVGRIRSKTNNYAT

PGTPARFSGSLLGGKAALTL

YYADSVKGRFTISRDDSKNTV

SGVQPEDEAVYYCALWYPN

YLQMNELKTEDTAVYYCVRH

LWVFGGGTKLTVL

ENFGNSYVSWFAHWGQGTL

VTVSS

AC3644
ELVVTQEPSLTVSPGGTVTL
724
EVQLVESGGGIVQPGGSLRL
725

TCRSSNGAVTSSNYANWV

SCAASGFTFNTYAMNWVRQ

QQKPGQAPRGLIGGTNKRA

APGKGLEWVGRIRSKYNDYA

PGTPARFSGSLLGGKAALTL

TYYADSVKGRFTISRDDSKNT

SGVQPEDEAVYYCALWYPN

VYLQMNELKTEDTAVYYCVR

LWVFGGGTKLTVL

HENFGNSYVSWFAHWGQGT

LVTVSS

AC3645
ELVVTQEPSLTVSPGGTVTL
726
EVQLVESGGGIVQPGGSLRL
727

TCRSSNGAVTSSNYANWV

SCAASGFTFSTYAMNWVRQA

QQKPGQAPRGLIGGTNKRA

PGKGLEWVGRIRSKYNDYAT

PGTPARFSGSLLGGKAALTL

YYADSVKGRFTISRDDSKNTV

SGVQPEDEAVYYCALWYPN

YLQMNELKTEDTAVYYCVRH

LWVFGGGTKLTVL

ENFGNSYVSWFAHWGQGTL

VTVSS

AC3646
ELVVTQEPSLTVSPGGTVTL
728
EVQLVESGGGIVQPGGSLRL
729

TCRSSNGAVTSSNYANWV

SCAASGFTFSTYAMNWVRQA

QQKPGQAPRGLIGGTNKRA

PGKGLEWVGRIRSKRNNYAT

PGTPARFSGSLLGGKAALTL

YYADSVKGRFTISRDDSKNTV

SGVQPEDEAVYYCALWYPN

YLQMNSLKTEDTAVYYCVRH

LWVFGGGTKLTVL

ENFGNSYVSWFAHWGQGTL

VTVSS

AC364
ELVVTQEPSLTVSPGGTVTL
730
EVQLVESGGGIVQPGGSLRL
731

7
TCRSSNGAVTSSNYANWV

SCAASGFTFSTYAMNWVRQA

QQKPGQAPRGLIGGTNKRA

PGKGLEWVGRIRSKTNNYAT

PGTPARFSGSLLGGKAALTL

YYADSVKGRFTISRDDSKNTV

SGVQPEDEAVYYCALWYPN

YLQMNSLKTEDTAVYYCVRH

LWVFGGGTKLTVL

ENFGNSYVSWFAHWGQGTL

VTVSS

AC3648
ELVVTQEPSLTVSPGGTVTL
732
EVQLVESGGGIVQPGGSLRL
733

TCRSSNGAVTSSNYANWV

SCAASGFTFSTYAMNWVRQA

QQKPGQAPRGLIGGTNKRA

PGKGLEWVGRIRSKYNDYAT

PGTPARFSGSLLGGKAALTL

YYADSVKGRFTISRDDSKNTV

SGVQPEDEAVYYCALWYPN

YLQMNSLKTEDTAVYYCVRH

LWVFGGGTKLTVL

ENFGNSYVSWFAHWGQGTL

VTVSS

AC3649
ELVVTQEPSLTVSPGGTVTL
734
EVQLVESGGGIVQPGGSLRL
735

TCRSSNGAVTSSNYANWV

SCAASGFTFSTYAMNWVRQA

QQKPGQAPRGLIGGTNKRA

PGKGLEWVGRIRSKYNNYAT

PGTPARFSGSLLGGKAALTL

TYADSVKGRFTISRDDSKNTV

SGVQPEDEAVYYCALWYPN

YLQMNSLKTEDTAVYYCVRH

LWVFGGGTKLTVL

ENFGNSYVSWFAHWGQGTL

VTVSS

AC3650
ELVVTQEPSLTVSPGGTVTL
736
EVQLVESGGGIVQPGGSLRL
737

TCRSSNGAVTSSNYANWV

SCAASGFTFNTYAMNWVRQ

QQKPGQAPRGLIGGTNKRA

APGKGLEWVGRIRTKRNNYA

PGTPARFSGSLLGGKAALTL

TYYADSVKGRFTISRDDSKNT

SGVQPEDEAVYYCALWYPN

VYLQMNELKTEDTAVYYCVR

LWVFGGGTKLTVL

HENFGNSYVSWFAHWGQGT

LVTVSS

AC3651
ELVVTQEPSLTVSPGGTVTL
738
EVQLVESGGGIVQPGGSLRL
739

TCRSSNGAVTSSNYANWV

SCAASGFTFSTYAMNWVRQA

QQKPGQAPRGLIGGTNKRA

PGKGLEWVGRIRTKRNNYAT

PGTPARFSGSLLGGKAALTL

YYADSVKGRFTISRDDSKNTV

SGVQPEDEAVYYCALWYPN

YLQMNELKTEDTAVYYCVRH

LWVFGGGTKLTVL

ENFGNSYVSWFAHWGQGTL

VTVSS

AC3652
ELVVTQEPSLTVSPGGTVTL
740
EVQLVESGGGIVQPGGSLRL
741

TCRSSNGAVTSSNYANWV

SCAASGFTFNTYAMNWVRQ

QQKPGQAPRGLIGGTNKRA

APGKGLEWVGRIRTKTNNYA

PGTPARFSGSLLGGKAALTL

TYYADSVKGRFTISRDDSKNT

SGVQPEDEAVYYCALWYPN

VYLQMNELKTEDTAVYYCVR

LWVFGGGTKLTVL

HENFGNSYVSWFAHWGQGT

LVTVSS

AC3653
ELVVTQEPSLTVSPGGTVTL
742
EVQLVESGGGIVQPGGSLRL
743

TCRSSNGAVTSSNYANWV

SCAASGFTFSTYAMNWVRQA

QQKPGQAPRGLIGGTNKRA

PGKGLEWVGRIRTKTNNYAT

PGTPARFSGSLLGGKAALTL

YYADSVKGRFTISRDDSKNTV

SGVQPEDEAVYYCALWYPN

YLQMNELKTEDTAVYYCVRH

LWVFGGGTKLTVL

ENFGNSYVSWFAHWGQGTL

VTVSS

AC3654
ELVVTQEPSLTVSPGGTVTL
744
EVQLVESGGGIVQPGGSLRL
745

TCRSSNGAVTSSNYANWV

SCAASGFTFNTYAMNWVRQ

QQKPGQAPRGLIGGTNKRA

APGKGLEWVGRIRTKYNDYA

PGTPARFSGSLLGGKAALTL

TYYADSVKGRFTISRDDSKNT

SGVQPEDEAVYYCALWYPN

VYLQMNELKTEDTAVYYCVR

LWVFGGGTKLTVL

HENFGNSYVSWFAHWGQGT

LVTVSS

AC3655
ELVVTQEPSLTVSPGGTVTL
746
EVQLVESGGGIVQPGGSLRL
747

TCRSSNGAVTSSNYANWV

SCAASGFTFSTYAMNWVRQA

QQKPGQAPRGLIGGTNKRA

PGKGLEWVGRIRTKYNDYAT

PGTPARFSGSLLGGKAALTL

YYADSVKGRFTISRDDSKNTV

SGVQPEDEAVYYCALWYPN

YLQMNELKTEDTAVYYCVRH

LWVFGGGTKLTVL

ENFGNSYVSWFAHWGQGTL

VTVSS

AC3656
ELVVTQEPSLTVSPGGTVTL
748
EVQLVESGGGIVQPGGSLRL
749

TCRSSNGAVTSSNYANWV

SCAASGFTFNTYAMNWVRQ

QQKPGQAPRGLIGGTNKRA

APGKGLEWVGRIRTKYNNYA

PGTPARFSGSLLGGKAALTL

TTYADSVKGRFTISRDDSKNT

SGVQPEDEAVYYCALWYPN

VYLQMNELKTEDTAVYYCVR

LWVFGGGTKLTVL

HENFGNSYVSWFAHWGQGT

LVTVSS

AC3657
ELVVTQEPSLTVSPGGTVTL
750
EVQLVESGGGIVQPGGSLRL
751

TCRSSNGAVTSSNYANWV

SCAASGFTFSTYAMNWVRQA

QQKPGQAPRGLIGGTNKRA

PGKGLEWVGRIRTKYNNYAT

PGTPARFSGSLLGGKAALTL

TYADSVKGRFTISRDDSKNTV

SGVQPEDEAVYYCALWYPN

YLQMNELKTEDTAVYYCVRH

LWVFGGGTKLTVL

ENFGNSYVSWFAHWGQGTL

VTVSS

AC3658
ELVVTQEPSLTVSPGGTVTL
752
EVQLVESGGGIVQPGGSLRL
753

TCRSSNGAVTSSNYANWV

SCAASGFTFNTYAMNWVRQ

QQKPGQAPRGLIGGTNKRA

APGKGLEWVGRIRTKRNNYA

PGTPARFSGSLLGGKAALTL

TYYADSVKGRFTISRDDSKNT

SGVQPEDEAVYYCALWYPN

VYLQMNSLKTEDTAVYYCVR

LWVFGGGTKLTVL

HENFGNSYVSWFAHWGQGT

LVTVSS

AC3659
ELVVTQEPSLTVSPGGTVTL
754
EVQLVESGGGIVQPGGSLRL
755

TCRSSNGAVTSSNYANWV

SCAASGFTFSTYAMNWVRQA

QQKPGQAPRGLIGGTNKRA

PGKGLEWVGRIRTKRNNYAT

PGTPARFSGSLLGGKAALTL

YYADSVKGRFTISRDDSKNTV

SGVQPEDEAVYYCALWYPN

YLQMNSLKTEDTAVYYCVRH

LWVFGGGTKLTVL

ENFGNSYVSWFAHWGQGTL

VTVSS

AC3660
ELVVTQEPSLTVSPGGTVTL
756
EVQLVESGGGIVQPGGSLRL
757

TCRSSNGAVTSSNYANWV

SCAASGFTFNTYAMNWVRQ

QQKPGQAPRGLIGGTNKRA

APGKGLEWVGRIRTKTNNYA

PGTPARFSGSLLGGKAALTL

TYYADSVKGRFTISRDDSKNT

SGVQPEDEAVYYCALWYPN

VYLQMNSLKTEDTAVYYCVR

LWVFGGGTKLTVL

HENFGNSYVSWFAHWGQGT

LVTVSS

AC3661
ELVVTQEPSLTVSPGGTVTL
758
EVQLVESGGGIVQPGGSLRL
759

TCRSSNGAVTSSNYANWV

SCAASGFTFSTYAMNWVRQA

QQKPGQAPRGLIGGTNKRA

PGKGLEWVGRIRTKTNNYAT

PGTPARFSGSLLGGKAALTL

YYADSVKGRFTISRDDSKNTV

SGVQPEDEAVYYCALWYPN

YLQMNSLKTEDTAVYYCVRH

LWVFGGGTKLTVL

ENFGNSYVSWFAHWGQGTL

VTVSS

AC3662
ELVVTQEPSLTVSPGGTVTL
760
EVQLVESGGGIVQPGGSLRL
761

TCRSSNGAVTSSNYANWV

SCAASGFTFNTYAMNWVRQ

QQKPGQAPRGLIGGTNKRA

APGKGLEWVGRIRTKYNDYA

PGTPARFSGSLLGGKAALTL

TYYADSVKGRFTISRDDSKNT

SGVQPEDEAVYYCALWYPN

VYLQMNSLKTEDTAVYYCVR

LWVFGGGTKLTVL

HENFGNSYVSWFAHWGQGT

LVTVSS

AC3663
ELVVTQEPSLTVSPGGTVTL
762
EVQLVESGGGIVQPGGSLRL
763

TCRSSNGAVTSSNYANWV

SCAASGFTFSTYAMNWVRQA

QQKPGQAPRGLIGGTNKRA

PGKGLEWVGRIRTKYNDYAT

PGTPARFSGSLLGGKAALTL

YYADSVKGRFTISRDDSKNTV

SGVQPEDEAVYYCALWYPN

YLQMNSLKTEDTAVYYCVRH

LWVFGGGTKLTVL

ENFGNSYVSWFAHWGQGTL

VTVSS

AC3664
ELVVTQEPSLTVSPGGTVTL
764
EVQLVESGGGIVQPGGSLRL
765

TCRSSNGAVTSSNYANWV

SCAASGFTFNTYAMNWVRQ

QQKPGQAPRGLIGGTNKRA

APGKGLEWVGRIRTKYNNYA

PGTPARFSGSLLGGKAALTL

TTYADSVKGRFTISRDDSKNT

SGVQPEDEAVYYCALWYPN

VYLQMNSLKTEDTAVYYCVR

LWVFGGGTKLTVL

HENFGNSYVSWFAHWGQGT

LVTVSS

AC3665
ELVVTQEPSLTVSPGGTVTL
766
EVQLVESGGGIVQPGGSLRL
767

TCRSSNGAVTSSNYANWV

SCAASGFTFSTYAMNWVRQA

QQKPGQAPRGLIGGTNKRA

PGKGLEWVGRIRTKYNNYAT

PGTPARFSGSLLGGKAALTL

TYADSVKGRFTISRDDSKNTV

SGVQPEDEAVYYCALWYPN

YLQMNSLKTEDTAVYYCVRH

LWVFGGGTKLTVL

ENFGNSYVSWFAHWGQGTL

VTVSS

AC3666
ELVVTQEPSLTVSPGGTVTL
768
EVQLVESGGGIVQPGGSLRL
769

TCRSSNGAVTSSNYANWV

SCAASGFTFNTYAMNWVRQ

QQKPGQAPRGLIGGTNKRA

APGKGLEWVGRIRSKYNEYA

PGTPARFSGSLLGGKAALTL

TYYADSVKGRFTISRDDSKNT

SGVQPEDEAVYYCALWYPN

VYLQMNSLKTEDTAVYYCVR

LWVFGGGTKLTVL

HENFGNSYVSWFAHWGQGT

LVTVSS

AC3667
ELVVTQEPSLTVSPGGTVTL
770
EVQLVESGGGIVQPGGSLRL
771

TCRSSNGAVTSSNYANWV

SCAASGFTFNTYAMNWVRQ

QQKPGQAPRGLIGGTNKRA

APGKGLEWVGRIRSKYNNGA

PGTPARFSGSLLGGKAALTL

TYYADSVKGRFTISRDDSKNT

SGVQPEDEAVYYCALWYPN

VYLQMNSLKTEDTAVYYCVR

LWVFGGGTKLTVL

HENFGNSYVSWFAHWGQGT

LVTVSS

AC3668
ELVVTQEPSLTVSPGGTVTL
772
EVQLVESGGGIVQPGGSLRL
773

TCRSSNGAVTSSNYANWV

SCAASGFTFNTYAMNWVRQ

QQKPGQAPRGLIGGTNKRA

APGKGLEWVGRIRSKYNNGA

PGTPARFSGSLLGGKAALTL

TYYADSVKGRFTISRDDSKNT

SGVQPEDEAVYYCALWYPN

VYLQMNELKTEDTAVYYCVR

LWVFGGGTKLTVL

HENFGNSYVSWFAHWGQGT

LVTVSS

AC3669
ELVVTQEPSLTVSPGGTVTL
774
EVQLVESGGGIVQPGGSLRL
775

TCRSSNGAVTSSNYANWV

SCAASGFTFNTYAMNWVRQ

QQKPGQAPRGLIGGTNKRA

APGKGLEWVARIRTKYNNYA

PGTPARFSGSLLGGKAALTL

TTYADSVKDRFTISRDDSKNT

SGVQPEDEAVYYCALWYPN

VYLQMNSLKTEDTAVYYCVR

LWVFGGGTKLTVL

HENFGNSYVSWFAHWGQGT

LVTVSS

AC3670
ELVVTQEPSLTVSPGGTVTL
776
EVQLVESGGGIVQPGGSLRL
777

TCRSSNGAVTSSNYANWV

SCAASGFTFNTYAMNWVRQ

QQKPGQAPRGLIGGTNKRA

APGKGLEWVARIRSKRNNYA

PGTPARFSGSLLGGKAALTL

TTYADSVKDRFTISRDDSKNT

SGVQPEDEAVYYCALWYPN

VYLQMNSLKTEDTAVYYCVR

LWVFGGGTKLTVL

HENFGNSYVSWFAHWGQGT

LVTVSS

AC3671
ELVVTQEPSLTVSPGGTVTL
778
EVQLVESGGGIVQPGGSLRL
779

TCRSSNGAVTSSNYANWV

SCAASGFTFNTYAMNWVRQ

QQKPGQAPRGLIGGTNKRA

APGKGLEWVARIRSKKNNYA

PGTPARFSGSLLGGKAALTL

TTYADSVKDRFTISRDDSKNT

SGVQPEDEAVYYCALWYPN

VYLQMNSLKTEDTAVYYCVR

LWVFGGGTKLTVL

HENFGNSYVSWFAHWGQGT

LVTVSS

AC3672
ELVVTQEPSLTVSPGGTVTL
780
EVQLVESGGGIVQPGGSLRL
781

TCRSSNGAVTSSNYANWV

SCAASGFTFNTYAMNWVRQ

QQKPGQAPRGLIGGTNKRA

APGKGLEWVARIRSKTNNYA

PGTPARFSGSLLGGKAALTL

TTYADSVKDRFTISRDDSKNT

SGVQPEDEAVYYCALWYPN

VYLQMNSLKTEDTAVYYCVR

LWVFGGGTKLTVL

HENFGNSYVSWFAHWGQGT

LVTVSS

AC3673
ELVVTQEPSLTVSPGGTVTL
782
EVQLVESGGGIVQPGGSLRL
783

TCRSSNGAVTSSNYANWV

SCAASGFTFNTYAMNWVRQ

QQKPGQAPRGLIGGTNKRA

APGKGLEWVGRIRSKYNDYA

PGTPARFSGSLLGGKAALTL

TYYADSVKGRFTISRDDSKNT

SGVQPEDEAVYYCALWYPN

LYLQMNSLKTEDTAVYYCVR

LWVFGGGTKLTVL

HENFGNSYVSWFAHWGQGT

LVTVSS

AC3674
ELVVTQEPSLTVSPGGTVTL
784
EVQLVESGGGIVQPGGSLRL
785

TCRSSNGAVTSSNYANWV

SCAASGFTFNTYAMNWVRQ

QQKPGQAPRGLIGGTNKRA

APGKGLEWVGRIRSKYNEYA

PGTPARFSGSLLGGKAALTL

TYYADSVKGRFTISRDDSKNT

SGVQPEDEAVYYCALWYPN

LYLQMNSLKTEDTAVYYCVR

LWVFGGGTKLTVL

HENFGNSYVSWFAHWGQGT

LVTVSS

AC3675
ELVVTQEPSLTVSPGGTVTL
786
EVQLVESGGGIVQPGGSLRL
787

TCRSSNGAVTSSNYANWV

SCAASGFTFNTYAMNWVRQ

QQKPGQAPRGLIGGTNKRA

APGKGLEWVGRIRSKYNNGA

PGTPARFSGSLLGGKAALTL

TYYADSVKGRFTISRDDSKNT

SGVQPEDEAVYYCALWYPN

LYLQMNSLKTEDTAVYYCVR

LWVFGGGTKLTVL

HENFGNSYVSWFAHWGQGT

LVTVSS

AC3676
ELVVTQEPSLTVSPGGTVTL
788
EVQLVESGGGIVQPGGSLRL
789

TCRSSNGAVTSSNYANWV

SCAASGFTFNTYAMNWVRQ

QQKPGQAPRGLIGGTNKRA

APGKGLEWVGRIRSKYNNGA

PGTPARFSGSLLGGKAALTL

TYYADSVKGRFTISRDDSKNT

SGVQPEDEAVYYCALWYPN

LYLQMNELKTEDTAVYYCVR

LWVFGGGTKLTVL

HENFGNSYVSWFAHWGQGT

LVTVSS

AC3677
ELVVTQEPSLTVSPGGTVTL
790
EVQLVESGGGIVQPGGSLRL
791

TCRSSNGAVTSSNYANWV

SCAASGFTFNTYAMNWVRQ

QQKPGQAPRGLIGGTNKRA

APGKGLEWVARIRTKYNNYA

PGTPARFSGSLLGGKAALTL

TTYADSVKDRFTISRDDSKNT

SGVQPEDEAVYYCALWYPN

LYLQMNSLKTEDTAVYYCVR

LWVFGGGTKLTVL

HENFGNSYVSWFAHWGQGT

LVTVSS

AC3678
ELVVTQEPSLTVSPGGTVTL
792
EVQLVESGGGIVQPGGSLRL
793

TCRSSNGAVTSSNYANWV

SCAASGFTFNTYAMNWVRQ

QQKPGQAPRGLIGGTNKRA

APGKGLEWVARIRSKRNNYA

PGTPARFSGSLLGGKAALTL

TTYADSVKDRFTISRDDSKNT

SGVQPEDEAVYYCALWYPN

LYLQMNSLKTEDTAVYYCVR

LWVFGGGTKLTVL

HENFGNSYVSWFAHWGQGT

LVTVSS

AC3679
ELVVTQEPSLTVSPGGTVTL
794
EVQLVESGGGIVQPGGSLRL
795

TCRSSNGAVTSSNYANWV

SCAASGFTFNTYAMNWVRQ

QQKPGQAPRGLIGGTNKRA

APGKGLEWVARIRSKKNNYA

PGTPARFSGSLLGGKAALTL

TTYADSVKDRFTISRDDSKNT

SGVQPEDEAVYYCALWYPN

LYLQMNSLKTEDTAVYYCVR

LWVFGGGTKLTVL

HENFGNSYVSWFAHWGQGT

LVTVSS

AC3680
ELVVTQEPSLTVSPGGTVTL
796
EVQLVESGGGIVQPGGSLRL
797

TCRSSNGAVTSSNYANWV

SCAASGFTFNTYAMNWVRQ

QQKPGQAPRGLIGGTNKRA

APGKGLEWVARIRSKTNNYA

PGTPARFSGSLLGGKAALTL

TTYADSVKDRFTISRDDSKNT

SGVQPEDEAVYYCALWYPN

LYLQMNSLKTEDTAVYYCVR

LWVFGGGTKLTVL

HENFGNSYVSWFAHWGQGT

LVTVSS

AC3681
ELVVTQEPSLTVSPGGTVTL
798
EVQLVESGGGIVQPGGSLRL
799

TCRSSNGAVTSSNYANWV

SCAASGFTFNTYAMNWVRQ

QQKPGQAPRGLIGGTNKRA

APGKGLEWVGRIRTKYNNYA

PGTPARFSGSLLGGKAALTL

TTYADSVKDRFTISRDDSKNT

SGVQPEDEAVYYCALWYPN

VYLQMNSLKTEDTAVYYCVR

LWVFGGGTKLTVL

HENFGNSYVSWFAHWGQGT

LVTVSS

AC3682
ELVVTQEPSLTVSPGGTVTL
800
EVQLVESGGGIVQPGGSLRL
801

TCRSSNGAVTSSNYANWV

SCAASGFTFNTYAMNWVRQ

QQKPGQAPRGLIGGTNKRA

APGKGLEWVGRIRSKRNNYA

PGTPARFSGSLLGGKAALTL

TTYADSVKDRFTISRDDSKNT

SGVQPEDEAVYYCALWYPN

VYLQMNSLKTEDTAVYYCVR

LWVFGGGTKLTVL

HENFGNSYVSWFAHWGQGT

LVTVSS

AC3683
ELVVTQEPSLTVSPGGTVTL
802
EVQLVESGGGIVQPGGSLRL
803

TCRSSNGAVTSSNYANWV

SCAASGFTFNTYAMNWVRQ

QQKPGQAPRGLIGGTNKRA

APGKGLEWVGRIRSKKNNYA

PGTPARFSGSLLGGKAALTL

TTYADSVKDRFTISRDDSKNT

SGVQPEDEAVYYCALWYPN

VYLQMNSLKTEDTAVYYCVR

LWVFGGGTKLTVL

HENFGNSYVSWFAHWGQGT

LVTVSS

AC3684
ELVVTQEPSLTVSPGGTVTL
804
EVQLVESGGGIVQPGGSLRL
805

TCRSSNGAVTSSNYANWV

SCAASGFTFNTYAMNWVRQ

QQKPGQAPRGLIGGTNKRA

APGKGLEWVGRIRSKTNNYA

PGTPARFSGSLLGGKAALTL

TTYADSVKDRFTISRDDSKNT

SGVQPEDEAVYYCALWYPN

VYLQMNSLKTEDTAVYYCVR

LWVFGGGTKLTVL

HENFGNSYVSWFAHWGQGT

LVTVSS

AC3685
ELVVTQEPSLTVSPGGTVTL
806
EVQLVESGGGIVQPGGSLRL
807

TCRSSNGAVTSSNYANWV

SCAASGFTFNTYAMNWVRQ

QQKPGQAPRGLIGGTNKRA

APGKGLEWVGRIRTKYNNYA

PGTPARFSGSLLGGKAALTL

TTYADSVKDRFTISRDDSKNT

SGVQPEDEAVYYCALWYPN

VYLQMNELKTEDTAVYYCVR

LWVFGGGTKLTVL

HENFGNSYVSWFAHWGQGT

LVTVSS

AC3686
ELVVTQEPSLTVSPGGTVTL
808
EVQLVESGGGIVQPGGSLRL
809

TCRSSNGAVTSSNYANWV

SCAASGFTFNTYAMNWVRQ

QQKPGQAPRGLIGGTNKRA

APGKGLEWVGRIRSKRNNYA

PGTPARFSGSLLGGKAALTL

TTYADSVKDRFTISRDDSKNT

SGVQPEDEAVYYCALWYPN

VYLQMNELKTEDTAVYYCVR

LWVFGGGTKLTVL

HENFGNSYVSWFAHWGQGT

LVTVSS

AC3687
ELVVTQEPSLTVSPGGTVTL
810
EVQLVESGGGIVQPGGSLRL
811

TCRSSNGAVTSSNYANWV

SCAASGFTFNTYAMNWVRQ

QQKPGQAPRGLIGGTNKRA

APGKGLEWVGRIRSKKNNYA

PGTPARFSGSLLGGKAALTL

TTYADSVKDRFTISRDDSKNT

SGVQPEDEAVYYCALWYPN

VYLQMNELKTEDTAVYYCVR

LWVFGGGTKLTVL

HENFGNSYVSWFAHWGQGT

LVTVSS

AC3688
ELVVTQEPSLTVSPGGTVTL
812
EVQLVESGGGIVQPGGSLRL
813

TCRSSNGAVTSSNYANWV

SCAASGFTFNTYAMNWVRQ

QQKPGQAPRGLIGGTNKRA

APGKGLEWVGRIRSKTNNYA

PGTPARFSGSLLGGKAALTL

TTYADSVKDRFTISRDDSKNT

SGVQPEDEAVYYCALWYPN

VYLQMNELKTEDTAVYYCVR

LWVFGGGTKLTVL

HENFGNSYVSWFAHWGQGT

LVTVSS

AC3689
ELVVTQEPSLTVSPGGTVTL
814
EVQLVESGGGIVQPGGSLRL
815

TCRSSNGAVTSSNYANWV

SCAASGFTFNTYAMNWVRQ

QQKPGQAPRGLIGGTNKRA

APGKGLEWVGRIRTKYNNYA

PGTPARFSGSLLGGKAALTL

TTYADSVKDRFTISRDDSKNT

SGVQPEDEAVYYCALWYPN

LYLQMNSLKTEDTAVYYCVR

LWVFGGGTKLTVL

HENFGNSYVSWFAHWGQGT

LVTVSS

AC3690
ELVVTQEPSLTVSPGGTVTL
816
EVQLVESGGGIVQPGGSLRL
817

TCRSSNGAVTSSNYANWV

SCAASGFTFNTYAMNWVRQ

QQKPGQAPRGLIGGTNKRA

APGKGLEWVGRIRSKRNNYA

PGTPARFSGSLLGGKAALTL

TTYADSVKDRFTISRDDSKNT

SGVQPEDEAVYYCALWYPN

LYLQMNSLKTEDTAVYYCVR

LWVFGGGTKLTVL

HENFGNSYVSWFAHWGQGT

LVTVSS

AC3691
ELVVTQEPSLTVSPGGTVTL
818
EVQLVESGGGIVQPGGSLRL
819

TCRSSNGAVTSSNYANWV

SCAASGFTFNTYAMNWVRQ

QQKPGQAPRGLIGGTNKRA

APGKGLEWVGRIRSKKNNYA

PGTPARFSGSLLGGKAALTL

TTYADSVKDRFTISRDDSKNT

SGVQPEDEAVYYCALWYPN

LYLQMNSLKTEDTAVYYCVR

LWVFGGGTKLTVL

HENFGNSYVSWFAHWGQGT

LVTVSS

AC3692
ELVVTQEPSLTVSPGGTVTL
820
EVQLVESGGGIVQPGGSLRL
821

TCRSSNGAVTSSNYANWV

SCAASGFTFNTYAMNWVRQ

QQKPGQAPRGLIGGTNKRA

APGKGLEWVGRIRSKTNNYA

PGTPARFSGSLLGGKAALTL

TTYADSVKDRFTISRDDSKNT

SGVQPEDEAVYYCALWYPN

LYLQMNSLKTEDTAVYYCVR

LWVFGGGTKLTVL

HENFGNSYVSWFAHWGQGT

LVTVSS

AC3693
ELVVTQEPSLTVSPGGTVTL
822
EVQLVESGGGIVQPGGSLRL
823

TCRSSNGAVTSSNYANWV

SCAASGFTFNTYAMNWVRQ

QQKPGQAPRGLIGGTNKRA

APGKGLEWVGRIRSKRNNYA

PGTPARFSGSLLGGKAALTL

TTYADSVKDRFTISRDDSKNT

SGVQPEDEAVYYCALWYPN

LYLQMNELKTEDTAVYYCVR

LWVFGGGTKLTVL

HENFGNSYVSWFAHWGQGT

LVTVSS

AC3694
ELVVTQEPSLTVSPGGTVTL
824
EVQLVESGGGIVQPGGSLKL
825

TCRSSNGAVTSSNYANWV

SCAASGFTFNTYAMNWVRQ

QQKPGQAPRGLIGGTNKRA

APGKGLEWVGRIRTKRNNYA

PGTPARFSGSLLGGKAALTL

TYYADSVKGRFTISRDDSKNT

SGVQPEDEAVYYCALWYPN

VYLQMNSLKTEDTAVYYCVR

LWVFGGGTKLTVL

HENFGNSYVSWFAHWGQGT

LVTVSS

AC3695
ELVVTQEPSLTVSPGGTVTL
826
EVQLVESGGGIVQPGGSLKL
827

TCRSSNGAVTSSNYANWV

SCAASGFTFSTYAMNWVRQA

QQKPGQAPRGLIGGTNKRA

PGKGLEWVGRIRTKRNNYAT

PGTPARFSGSLLGGKAALTL

YYADSVKGRFTISRDDSKNTV

SGVQPEDEAVYYCALWYPN

YLQMNSLKTEDTAVYYCVRH

LWVFGGGTKLTVL

ENFGNSYVSWFAHWGQGTL

VTVSS

AC3471
ELVVTQEPSLTVSPGGTVTL
828
EVQLLESGGGIVQPGGSLKL
829

TCRSSNGAVTSSNYANWV

SCAASGFTFNTYAMNWVRQ

QQKPGQAPRGLIGGTNKRA

APGKGLEWVARIRSKYNNYA

PGTPARFSGSLLGGKAALTL

TYYADSVKDRFTISRDDSKNT

SGVQPEDEAVYYCALWYPN

VYLQMNNLKTEDTAVYYCVR

LWVFGGGTKLTVL

HENFGNSYVSWFAHWGQGT

LVTVSS

AC3432
ELVVTQEPSLTVSPGGTVTL
830
EVQLLESGGGIVQPGGSLKL
831

TCRSSNGAVTSSNYANWV

SCAASGFTFNTYAMNWVRQ

QQKPGQAPRGLIGGTNKRA

APGKGLEWVGRIRSKYNNGA

PGTPARFSGSLLGGKAALTL

TYYADSVKGRFTISRDDSKNT

SGVQPEDEAVYYCALWYPN

VYLQMNSLKTEDTAVYYCVR

LWVFGGGTKLTVL

HENFGNSYVSWFAHWGQGT

LVTVSS

AC2717
ELVVTQEPSLTVSPGGTVTL
832
EVQLLESGGGIVQPGGSLKL
833

TCRSSNGAVTSSNYANWV

SCAASGFTFNTYAMNWVRQ

QQKPGQAPRGLIGGTNKRA

APGKGLEWVARIRSKYNNYA

PGTPARFSGSLLGGKAALTL

TYYADSVKDRFTISRDDSKNT

SGVQPEDEAVYYCALWYPN

VYLQMNNLKTEDTAVYYCVR

LWVFGGGTKLTVL

HENFGNSYVSWFAHWGQGT

LVTVSS

TABLE 23b cont.

Pool 2 CD3.23 Mutation Combination Variants

Primary

PTE
PTE

Screen
% Remaining

CD3.23

score
score
Expression
Expression
huCD3e
of

AC
domain
Mutation
v12
v22
Level**
Ratio***
(nM)
stability

AC3632
CD3.201
K19R,
9
18
3050.0
1.2
8.6
55.24%

A49G,

Y52cR,

D65G,

N82bS

AC3633
CD3.202
L5V,
9
18
2220.0
0.9
8.4
60.79%

K19R,

A49G,

Y52cR,

D65G,

N82bS

AC3634
CD3.203
K19R,
10
19
ND
ND
ND
ND

A49G,

Y52cT

D65G,

N82bS

AC3635
CD3.204
L5V,
10
19
1870.0
0.8
7.2
36.92%

K19R,

A49G,

Y52cT,

D65G,

N82bS

AC3636
CD3.205
K19R,
10
19
3000.0
1.2
7.2
58.50%

A49G,

N54D,

D65G,

N82bS

AC3637
CD3.206
L5V,
10
19
1450.0
0.6
10.3
28.98%

K19R,

A49G,

N54D,

D65G,

N82bS

AC3638
CD3.207
K19R,
12
21
2420.0
1.0
15.6
1.01%

A49G,

Y58T,

D65G,

N82bS

AC3639
CD3.208
L5V,
12
21
2400.0
1.0
16.8
24.61%

K19R,

A49G,

Y58T,

D65G,

N82bS

AC3640
CD3.209
L5V,
16
25
2280.0
0.9
5.6
37.31%

K19R,

A49G,

Y52cR,

D65G,

N82bE

AC3641
CD3.210
L5V,
16
25
2120.0
0.9
5.7
69.25%

K19R,

N30S,

A49G,

Y52cR,

D65G,

N82bE

AC3642
CD3.211
L5V,
17
26
2610.0
1.1
9.5
29.37%

K19R,

A49G,

Y52cT,

D65G,

N82bE

AC3643
CD3.212
L5V,
17
26
890.0
0.4
35.6
9.81%

K19R,

N30S,

A49G,

Y52CT,

D65G,

N82bE

AC3644
CD3.213
L5V,
17
26
3280.0
1.2
6.6
57.91%

K19R,

A49G,

N54D,

D65G,

N82bE

AC3645
CD3.214
L5V,
17
26
3420.0
1.3
6.6
67.42%

K19R,

N30S,

A49G,

N54D,

D65G,

N82bE

AC3646
CD3.215
L5V,
9
18
2020.0
0.8
9.7
54.85%

K19R,

N30S,

A49G,

Y52cR,

D65G,

N82bS

AC3647
CD3.216
L5V,
10
19
2100.0
0.8
11.2
61.45%

K19R,

N30S,

A49G,

Y52cT,

D65G,

N82bS

AC3648
CD3.217
L5V,
10
19
1040.0
0.4
10.7
31.11%

K19R,

N30S,

A49G,

N54D,

D65G,

N82bS

AC3649
CD3.218
L5V,
12
21
800.0
0.3
52.6
3.83%

K19R,

N30S,

A49G,

Y58T,

D65G,

N82bS

AC3650
CD3.219
L5V,
10
17
2010.0
0.8
4.7
57.93%

K19R,

A49G,

S52aT,

Y52cR,

D65G,

N82bE

AC3651
CD3.220
L5V,
10
17
1950.0
0.7
4.8
63.19%

K19R,

N30S,

A49G,

S52aT,

Y52cR,

D65G,

N82bE

AC3652
CD3.221
L5V,
15
22
2600.0
1.0
11.7
5.17%

K19R,

A49G,

S52aT,

Y52cT,

D65G,

N82bE

AC3653
CD3.222
L5V,
15
22
2440.0
0.9
10.8
33.59%

K19R,

N30S,

A49G,

S52aT,

Y52cT,

D65G,

N82bE

AC3654
CD3.223
L5V,
17
24
2890.0
1.1
6.0
68.99%

K19R,

A49G,

S52aT,

N54D,

D65G,

N82bE

AC3655
CD3.224
L5V,
17
24
2530.0
1.0
6.4
52.55%

K19R,

N30S,

A49G,

S52aT,

N54D,

D65G,

N82bE

AC3656
CD3.225
L5V,
19
26
3110.0
1.0
19.5
37.24%

K19R,

A49G,

S52aT,

Y58T,

D65G,

N82bE

AC3657
CD3.226
L5V,
19
26
1320.0
0.4
50.9
8.94%

K19R,

N30S,

A49G,

S52aT,

Y58T,

D65G,

N82bE

AC3658
CD3.227
L5V,
3
10
2850.0
1.0
8.2
62.96%

K19R,

A49G,

S52aT,

Y52cR,

D65G,

N82bS

AC3659
CD3.228
L5V,
3
10
2750.0
0.9
6.2
80.86%

K19R,

N30S,

A49G,

S52aT,

Y52cR,

D65G,

N82bS

AC3660
CD3.229
L5V,
8
15
3310.0
1.1
11.9
51.28%

K19R,

A49G,

S52aT,

Y52cT,

D65G,

N82bS

AC3661
CD3.230
L5V,
8
15
2740.0
0.9
17.0
62.00%

K19R,

N30S,

A49G,

S52aT,

Y52cT,

D65G,

N82bS

AC3662
CD3.231
L5V,
10
17
3590.0
1.2
5.1
54.69%

K19R,

A49G,

S52aT,

N54D,

D65G,

N82bS

AC3663
CD3.232
L5V,
10
17
3890.0
1.3
5.1
74.10%

K19R,

N30S,

A49G,

S52aT,

N54D,

D65G,

15.01%

N82bS

AC3664
CD3.233
L5V,
12
19
3310.0
1.1
24.9

K19R,

A49G,

S52aT,

Y58T,

D65G,

N82bS

AC3665
CD3.234
L5V,
12
19
3310.0
1.1
17.4
13.49%

K19R,

N30S,

A49G,

S52aT,

Y58T,

D65G,

N82bS

AC3666
CD3.235
L5V,
14
23
3620.0
1.2
6.5
63.15%

K19R,

A49G,

N54E,

D65G,

N82bS

AC3667
CD3.236
L5V,
8
17
3180.0
1.1
21.9
0.65%

K19R,

A49G,

D65G,

N82bS

AC3668
CD3.237
L5V,
15
24
690.0
0.3
22.2
0

K19R,

A49G,

D65G,

N82bE

AC3669
CD3.238
L5V,
16
23
1680.0
0.7
23.1
−5.55%

K19R,

S52aT,

Y58T,

N82bS

AC3670
CD3.239
L5V,
14
21
1590.0
0.7
29.9
−12.81%

K19R,

Y52cR,

Y58T,

N82bS

AC3671
CD3.240
L5V,
16
23
1790.0
0.7
16.5
−25.28%

K19R,

Y52cK,

Y58T,

N82bS

AC3672
CD3.241
L5V,
14
21
2280.0
0.9
92.6
−183.07%

K19R,

Y52cT,

Y58T,

N82bS

AC3673
CD3.242
L5V,
9
18
2620.0
1.1
7.7
34.70%

K19R,

A49G,

N54D,

D65G,

V78L,

N82bS

AC3674
CD3.243
L5V,
13
22
ND
ND
ND
ND

K19R,

A49G,

N54E,

D65G,

V78L,

N82bS

AC3675
CD3.244
L5V,
7
16
740.0
0.3
43.6
−43.16%

K19R,

A49G,

D65G,

V78L,

N82bS

AC3676
CD3.245
L5V,
17
26
2490.0
1.0
19.3
−65.26%

K19R,

A49G,

D65G,

V78L,

N82bE

AC3677
CD3.246
L5V,
15
22
1970.0
0.8
35.2
−0.10%

K19R,

S52aT,

Y58T,

V78L,

N82bS

AC3678
CD3.247
L5V,
13
20
1880.0
0.8
28.9
−59.35%

K19R,

Y52cR,

Y58T,

V78L,

N82bS

AC3679
CD3.248
L5V,
15
22
1700.0
0.7
49.2
−25.51%

K19R,

Y52cK,

Y58T,

V78L,

N82bS

AC3680
CD3.249
L5V,
13
20
3180.0
1.0
128.0
−193.59%

K19R,

Y52cT,

Y58T,

V78L,

N82bS

AC3681
CD3.250
L5V,
12
19
2950.0
0.9
31.0
5.62%

K19R,

A49G,

S52aT,

Y58T,

N82bS

AC3682
CD3.251
L5V,
8
17
2730.0
0.9
32.8
11.12%

K19R,

A49G,

Y52cR,

Y58T,

N82bS

AC3683
CD3.252
L5V,
9
19
2280.0
0.7
22.9
−13.59%

K19R,

A49G,

Y52ck,

Y58T,

N82bS

AC3684
CD3.253
L5V,
10
19
2900.0
0.9
79.9
−105.16%

K19R,

A49G,

Y52cT,

Y58T,

N82bS

AC3685
CD3.254
L5V,
19
26
2650.0
0.8
32.3
24.18%

K19R,

A49G,

S52aT,

Y58T,

N82bE

AC3686
CD3.255
L5V,
15
24
2110.0
0.7
22.4
32.74%

K19R,

A49G,

Y52cR,

Y58T,

N82bE

AC3687
CD3.256
L5V,
16
26
ND
ND
ND
ND

K19R,

A49G,

Y52ck,

Y58T,

N82bE

AC3688
CD3.257
L5V,
17
26
2970.0
0.9
159.0
−92.57%

K19R,

A49G,

Y52cT,

Y58T,

N82bE

AC3689
CD3.258
L5V,
11
18
2840.0
0.9
38.0
22.13%

K19R,

A49G,

S52aT,

Y58T,

V78L,

N82bS

AC3690
CD3.259
L5V,
7
16
2540.0
0.8
29.4
14.38%

K19R,

A49G,

Y52cR,

Y58T,

V78L,

N82bS

AC3691
CD3.260
L5V,
8
18
2730.0
0.9
45.6
27.07%

K19R,

A49G,

Y52ck,

Y58T,

V78L,

N82bS

AC3692
CD3.261
L5V,
9
18
2200.0
0.9
97.0
−122.00%

K19R,

A49G,

Y52cT,

Y58T,

V78L,

N82bS

AC3693
CD3.262
L5V,
17
26
2100.0
0.9
25.4
−4.58%

K19R,

A49G,

Y52cR,

Y58T,

V78L,

N82bE

AC3694
CD3.263
L5V,
17
26
2050.0
0.8
7.2
53.06%

A49G,

S52aT,

Y52cR,

D65G,

N82bS

AC3695
CD3.264
L5V,
17
26
2500.0
1.0
4.5
55.56%

N30S,

A49G,

S52aT,

Y52cR,

D65G,

N82bS

AC3471
CD3.23
WT
50
73
2738.0
1.0
13.5
63.73%

AC3432*
CD3.103
A49G,
8
33
3843.3
1.3
13.5
21.44%

Y52cG,

D65G,

N82bS

AC2717*
CD3.23
WT
50
73
3723.3
1.3
12.9
73.42%

*AC3432 and AC2717 paired with PSMA.5.

**These values are arbitrary reads from the Octet data. A higher number means more protein is presented.

***These values are ratios compared to expression level of CD3.23. Higher ration means higher expression level compared to expression of CD3.23.

Example 7. Release Site Engineering

Incubation of a paTCE comprising RSR-2295 in human plasma showed some cleavage that, though not high, was unexpected. Further investigation revealed that the cleavage was surprisingly due to legumain, which has previously believed to be specifically present in tumor tissues. Additionally, it was initially believed that legumain cleavage provided meaningful levels of paTCE activation in tumor tissues.

A new release site was designed to avoid cleavage by legumain, resulting in RSR-3213. Surprisingly, a paTCE containing RSR-3213 release sequences was cleaved less in plasma but at comparable amounts to a corresponding paTCE comprising RSR-2295 release sequences in multiple tumor types (including gastric carcinoma (NCI-N87), colorectal adenocarcinoma (HT-29), colon carcinoma (HT-55) tumors). Thus, paTCEs comprising RSR-3213 have enhanced specificity for tumor tissues without a significant loss of activation in tumor tissues.

In Vitro Digest:

In vitro digest assays were performed to demonstrate that RSR-3213 is cleaved by MMP and ST14/matriptase, but not legumain. Two EpCAM-targeting paTCE (EpCAM-paTCE) molecules (one of having RSR-2295 on both sides of the TCE, and the other having RSR-3213 on both sides of the TCE) flanking the TCE core were digested with 5-fold dilutions of MMP9, legumain, or ST14/matriptase. Similar banding patterns were observed for both MMP9 and matriptase, suggesting the mutation of the legumain cleavage site did not affect cleavability of the MMP and serine protease cleavage sites. uTCE was observed for the paTCE containing RSR-2295 after digestion with legumain, indicating cleavage at the protease cleavable linker by legumain. uTCE was not observed for the paTCE containing RSR-3213 after digestion with legumain, indicating the mutation successfully prevented cleavage at the protease cleavable linker by legumain (FIG. 7A and FIG. 7B).

Plasma Stability—In Vivo Cleavability

Fluorescently labeled variants of an EpCAM-paTCE containing either RSR-2295 or RSR-3213 were labeled with Sulfo-Cy5.5 or Sulfo-Cy7.5. Opposite colors were co-injected into mice containing NCI-N87, HT-29, or HT-55 xenograft tumors. 48 hours after injection, tumors were harvested, homogenized, and protein extracts were analyzed by SDS-PAGE and LI-COR. Relative abundances for paTCE, 1x-C, 1x-N, and uTCE were quantified. No significant differences were observed in uTCE and 1x-C between the two protease cleavable linkers. paTCE containing RSR-2295 showed a small but statistically significant increase (average 2.19% more) in 1x-N than the corresponding paTCE containing RSR-3213. (FIG. 8A and FIG. 8B).

The observed cleavability in vivo from tumor homogenates was also determined from 3 different mouse tumor models. The % abundance for metabolites 1x-C, 1x-N, and uTCE was measured with results depicted in FIG. 8C. Finally, FIG. 8D depicts the % of total for paTCE plus the 3 metabolites (1x-N, 1x-C, and uTCE) when employing RSR-2295 or RSR-3213.

Overall, these data suggest that differences between in vivo cleavability of RSR-2295 and RSR-3213 are minor across 3 different tumor models.

Tumor Uptake:

Tumor uptake between EpCAM-paTCEs containing either RSR-2295 or RSR-3213 were compared using the ratio of calculated concentrations of total drug (paTCE, 1x-C, 1x-N, and uTCE). While differences in tumor uptake were observed across 3 different tumor models, no significant differences were observed between RSR-2295 and RSR-3213 within each model. This indicates that the changes to the protease cleavable linkers between RSR-2295 and RSR-3213 do not affect tumor uptake of paTCE (FIG. 9).

Example 8. AMX-500, an Exemplary PSMA-Targeting Protease-Activated TCE

This example provides data relating to an exemplary paTCE, referred to as AMX-500.

Method of Production of AMX-500

Methods for producing paTCEs proteins are known in the art, e.g., as described in PCT International Patent Publication No. WO2017/040344. For example, paTCE was expressed in E. coli, which was transformed with an expression vector encoding the paTCE and grown in fermentation. Fermentation cultures were grown with animal-free complex medium at 37° C. and temperature shifted to 26° C. before phosphate depletion, which triggered induction (PhoA). Target protein was partitioned into the periplasm via an N-terminal secretory leader sequence, which was cleaved during translocation. During harvest, fermentation whole broth was centrifuged to pellet the product-containing cells, which were retained and frozen at ≤−70° C. The frozen cell pellet was resuspended and, once homogenous, the resuspension was mechanically lysed and refrigerated. The chilled lysate was centrifuged (12,000 RCF, 10° C., 30 min) and the supernatant was decanted and retained, while the pellet was discarded. The following day, centrifugation was performed again (12,000 RCF, 10° C., 30 min) and the supernatant was decanted, submicron filtered and purified via a chromatographic process comprising an Anion Exchange (AEX) chromatography step. paTCE proteins and their derivatives were prepared as aqueous solutions and stored frozen at ≤−70° C. and, after thawing, at temperatures between 2° C. and 8° C.

An exemplary nucleotide sequence for the production of AMX-500 is provided below:

(SEQ ID NO: 9000)

GCATCTTCGGCGACGCCGGAAAGCGGTCCGGGTACGTCCACCGAACCGA

GCGAGGGTAGCGCTCCGGGCACCAGCGAGTCCGCGACCCCGGAAAGCGG

TCCGGGTAGCGGTCCGGGCACCTCCGAGAGCGCGACCCCGGGCACCTCT

GAATCAGCCACCCCGGAGTCTGGCCCAGGTAGCGAGCCGGCAACCTCTG

GCAGCGAAACCCCGGGCACCAGCGAATCCGCGACGCCAGAGAGCGGTCC

GGGCACCTCTACGGAGCCTAGCGAGGGCTCAGCACCAGGTAGCCCTGCA

GGTTCCCCGACGTCAACCGAGGAAGGTACAAGCGAAAGCGCCACCCCTG

AGTCGGGCCCTGGCAGCGAACCGGCAACTAGCGGCAGCGAGACTCCGGG

TACCAGCGAGTCTGCTACGCCAGAGAGCGGCCCAGGTTCGCCAGCGGGT

TCGCCGACTAGCACGGAGGAGGGCAGCCCAGCGGGTAGCCCTACCAGCA

CTGAAGAGGGTACGTCCACCGAACCGAGCGAAGGTAGCGCACCAGGTAC

CTCCGAGTCTGCCACCCCTGAATCCGGTCCAGGTACCAGCGAATCAGCC

ACCCCGGAGTCGGGTCCAGGTACGAGCGAATCTGCTACCCCGGAATCCG

GCCCAGGCAGCGAACCTGCTACTAGCGGCAGCGAAACGCCGGGCAGCGA

ACCTGCCACGTCAGGCAGCGAGACGCCGGGTTCCCCTGCAGGCTCCCCG

ACCAGCACTGAGGAGGGCACCTCCACCGAACCATCAGAAGGTAGCGCGC

CTGGTACGTCAACCGAACCTTCCGAGGGCAGCGCACCGGGTTCAGAACC

AGCTACGTCTGGGAGCGAGACCCCGGGCACCTCCGAGTCGGCGACCCCG

GAGGCAGGTCGTTCTGCTAGCCATACCCCTGCAGGGTTAACTGGCCCCG

GAACTTCAGAAAGTGCTACACCCGAGTCTCAGGTTCAACTGGTGGAGAG

CGGTGGCGGTGTGGTTCAGCCGGGTCGTAGCCTGCGTCTGAGCTGCGCG

GCGAGCGGTCGTACCTTTGGTATCTATGTGTGGGGTTGGTTTCGTCAGG

CGCCGGGCAAGGAGCGTGAATTCGTGGGCGCGATGAGCTGGAGCGGTAG

CAACCGTAAAGTGAGCGACAGCGTTAAGGGCCGTTTTACCATTAGCCGT

GATAACAGCAAAAACACCCTGTACCTGCAAATGAACAGCCTGCGTGCGG

AGGACACCGCGGTTTACTATTGCGCGGCGAGCAACAAAGAATATGGCCG

TACCTGGTATGATTTCAATGAGAGCGACTACTGGGGCCAAGGCACCCAA

GTGACCGTTAGCAGCGGGGGAGGCGGAAGTGGTGGAGGGTCAGAGTTAG

TTGTGACCCAAGAGCCGAGCCTGACCGTTAGCCCGGGTGGTACGGTCAC

CCTGACGTGCCGTAGCAGCAACGGTGCGGTCACGAGCAGCAACTATGCC

AATTGGGTCCAGCAGAAACCGGGTCAAGCACCGCGTGGCCTGATCGGCG

GCACCAATAAACGTGCCCCGGGTACTCCTGCGCGTTTCTCCGGTAGCCT

GCTGGGCGGCAAAGCCGCTCTGACCCTGAGCGGTGTCCAGCCGGAAGAT

GAAGCGGTGTACTACTGCGCGCTGTGGTATCCGAATCTGTGGGTTTTTG

GCGGCGGTACCAAGCTGACCGTATTGAGCGAGAGCGCAACGCCAGAGAG

CGGTCCAGGCACCAGCCCAGGTGCCACCCCTGAGAGCGGCCCAGGTACT

TCTGAGAGCGCCACTCCGGAGGTCCAACTGGTGGAGTCTGGTGGTGGCA

TTGTTCAACCGGGTGGCTCGTTGCGCCTGAGCTGTGCAGCTAGCGGCTT

TACCTTCAGCACCTATGCGATGAATTGGGTTCGTCAGGCACCGGGTAAG

GGCCTGGAATGGGTGGGCCGTATCCGCACCAAGCGCAACAACTACGCGA

CCTACTACGCGGATAGCGTTAAAGGCCGCTTCACGATTAGCCGTGACGA

TTCCAAGAATACGGTGTATCTGCAAATGAACAGCCTGAAAACCGAAGAT

ACCGCGGTGTATTACTGTGTGCGCCACGAAAATTTCGGCAACAGCTACG

TGAGCTGGTTTGCACATTGGGGTCAGGGCACCCTGGTTACGGTGAGCTC

CGGTACAGCTACTCCAGAATCAGGACCCGGGGAAGCTGGAAGAAGCGCC

TCACACACACCAGCTGGACTTACAGGCCCGGCTACTCCCGAAAGTGGGC

CAGGAACATCAGAGTCCGCGACCCCGGAAAGCGGTCCGGGTTCTCCAGC

TGGCAGCCCGACCTCCACTGAAGAAGGCACCTCTGAGTCTGCTACCCCT

GAATCTGGTCCTGGCTCCGAACCTGCTACCTCTGGTTCCGAAACTCCAG

GTACCTCGGAATCTGCGACTCCGGAATCTGGCCCGGGCACGAGCACGGA

GCCGTCTGAGGGTAGCGCACCAGGTACCAGCACTGAGCCTTCTGAGGGC

TCTGCACCGGGTACCTCCACGGAACCTTCGGAAGGTTCTGCGCCGGGTA

CCTCCACTGAGCCATCCGAGGGTTCAGCACCAGGTACTAGCACGGAACC

GTCCGAGGGCTCTGCACCAGGTACGAGCACCGAACCGTCGGAGGGTAGC

GCTCCAGGTAGCCCAGCGGGCTCTCCGACAAGCACCGAAGAAGGCACCA

GCACCGAGCCGTCCGAAGGTTCCGCACCAGGTACAAGCGAGAGCGCGAC

TCCTGAATCTGGTCCGGGTAGCGAGCCTGCAACCAGCGGTTCTGAGACG

CCGGGCACTTCCGAATCTGCGACCCCGGAGTCCGGTCCAGGTTCAGAGC

CGGCGACGAGCGGTTCGGAAACGCCGGGTACGTCTGAATCAGCCACGCC

GGAGTCTGGTCCGGGTACCTCGACCGAACCAAGCGAAGGTTCGGCACCG

GGTACTAGCGAGAGCGCAACCCCTGAAAGCGGTCCGGGCAGCCCGGCAG

GTTCTCCAACCAGCACCGAAGAAGGTTCCCCTGCTGGTAGCCCGACCTC

TACGGAGGAAGGTAGCCCTGCAGGTTCCCCAACTTCTACTGAGGAAGGT

ACTTCTGAGTCCGCTACCCCAGAAAGCGGTCCTGGTACCTCCACTGAAC

CGTCTGAAGGCTCTGCACCAGGCACTTCTGAGTCTGCTACTCCAGAAAG

CGGCCCAGGTTCTGAACCAGCAACTTCTGGCTCTGAGACTCCAGGCACT

TCTGAGTCCGCAACGCCTGAATCCGGTCCTGGTTCTGAACCAGCTACTT

CCGGCAGCGAAACCCCAGGTACCTCTGAGTCTGCGACTCCAGAGTCTGG

TCCTGGTACTTCCACTGAGCCTAGCGAGGGTTCCGCACCAGGTTCTCCG

GCTGGTAGCCCGACCAGCACGGAGGAGGGTACGTCTGAATCTGCAACGC

CGGAATCGGGCCCAGGTTCGGAGCCTGCAACGTCTGGCAGCGAAACCCC

GGGTACCTCCGAATCTGCTACACCGGAAAGCGGTCCTGGCAGCCCTGCT

GGTTCTCCAACCTCTACCGAGGAGGGTTCACCGGCAGGTAGCCCGACTA

GCACTGAAGAAGGTACTAGCACGGAGCCGAGCGAGGGTAGTGCTCCGGG

TACGAGCGAGAGCGCAACGCCAGAGAGCGGTCCAGGCACCAGCGAATCG

GCCACCCCTGAGAGCGGCCCAGGTACTTCACCCTCTGCTACGCCGGAAA

GCGGTCCGGGTTCCGAGCCGGCGACCAGCGGCTCCGAGACTCCGGGTTC

GGAGCCGGCGACCTCCGGCTCGGAAACCCCGGGTAGCCCGGCTGGTTCT

CCGACCAGCACTGAGGAAGGCACCAGCACCGAACCAAGCGAGGGCAGCG

CGCCAGGTACGAGCACCGAACCGAGCGAGGGTTCAGCCCCTGGCTCTGA

GCCGGCGACGTCTGGCTCCGAAACCCCGGGCACCAGCGAGAGCGCTGGT

GAACCGGAAGCG.

In Vitro Data, Including Cytotoxicity, In Vitro CRA and T-Cell Activation

In vitro cytotoxicity of PSMA-paTCE leads were screened using either LNCaP or 22Rv1 cell lines in an Effector to Target (E:T) ratio of 10 to 1. Unmasked PSMA-paTCE (PSMA-uTCE) leads had a EC50 value between 1 μM to 100 μM in LnCaP cell line (see Table 24 below). Unmasked PSMA-paTCE (PSMA-uTCE) leads had a EC50 value between 1 μM to 2000 μM in 22Rv1 cell line (see Table 25 below). Various linker lengths between TAA and CD3 domains have been evaluated in the in vitro cytotoxicity assay using LNCaP cell line which have EC50 ranged from 1 μM to 50 μM. Different orientation of heavy and light CD3 domains were screened which have EC50 between 1 μM to 50 μM. PSMA-paTCE AMX-500 has an EC50 ranged from 35000 μM to 90000 μM in the LnCaP cell line. The AMX-500(1x-N) metabolite of PSMA-paTCE AMX-500 has an EC50 ranged from 5000 μM to 8000 μM in the LnCaP cell line. The AMX-500(1x-C) metabolite of PSMA-paTCE AMX-500 has an EC50 ranged from 6000 μM to 12000 μM in the LnCaP cell line. Fully unmasked AMX-500(uTCE) has an EC50 ranged from 15 μM to 35 μM in the LnCaP cell line. PSMA-paTCE AMX-500, AMX-500(1x-N) and AMX-500(1x-C) did not exhibit any cytotoxic activity up to 1 μM in the 22Rv1 cell line. Fully unmasked AMX-500(uTCE) has an EC50 ranged from 600 μM to 1500 μM in 22Rv1 cell line (see FIG. 11A -FIG. 11B for dose response curves).

TABLE 24

in vitro cytotoxicity assay EC50 values in the LnCaP cell line

uTCE
uTCE
Masked
uTCE
Masked
uTCE
Masked
uTCE
Masked

paTCE

paTCE

paTCE

paTCE

PSMA.5-
PSMA.119-
PSMA.350-
PSMA.350-
PSMA.262-

CD3.23
CD3.23
CD3.228
CD3.23
CD3.228

AC3092
AC3445
AC3445
AC3896
AC3896
AC3928
AC3928
AC3934
AC3934

20 pM
22 pM
7,909 pM
49 pM
46,806 pM
43 pM
188,626 pM
3 pM
ND

TABLE 25

in vitro cytotoxicity assay EC50 values in the 22Rv1 cell line

uTCE
uTCE
Masked
uTCE
Masked
uTCE
Masked
uTCE
Masked

paTCE

paTCE

paTCE

paTCE

PSMA.5-
PSMA.119-
PSMA.350-
PSMA.350-
PSMA.262-

CD3.23
CD3.23
CD3.228
CD3.23
CD3.228

AC3092
AC3445
AC3445
AC3896
AC3896
AC3928
AC3928
AC3934
AC3934

18 pM
160 pM
6896 pM
523 pM
5640 pM
1294 pM
ND
34 pM
3753 PM

Variable amino acid linker lengths between the PSMA antibody and CD3 antibody were tested to determine their effect on in vitro cytotoxicity in the LNCaP and 22Rv1 cell lines. The results are shown below in Table 26. The dose response curves for Donors A-C are shown in FIG. 12A -FIG. 120.

TABLE 26

in vitro cytotoxicity assay IC50 values in the LnCaP cell line and 22Rv1 cell line with alternative linker lengths

IC50
IC50

(pM)
(pM)
IC50

LNCaP-
LNCaP-
(pM)

FGC
FGC
22Rv1

AC

CD3 Domain
N_ELNN
C_ELNN

Donor
Donor
Donor

uTCE
Domains
Linker
order
Length
Length
Description
A
B
C

uTCE
[PSMA.2]-
9
VL-VH
288
576
Control,
5.1
5.2
146

of
[CD3.23]

initial

AC2591

Camelid

αPSMA

uTCE
[PSMA.5]-
9
VL-VH
144
144
Control,
5.9
ND
418

of
[CD3.23]

initial

AC3092

humanized

αPSMA

uTCE
[PSMA.5]-
5
VL-VH
144
144
5mer GS
ND
15
470

of
[CD3.23]

VHH ScFv

AC3353

linker

UTCE
[PSMA.5]-
15
VL-VH
144
144
15mer
ND
31
1,289

of
[CD3.23]

ELNN

AC3354

VHH ScFv

linker

uTCE
[PSMA.5]-
9
VH-VL
144
144
cd3.23
20.1
29
1,350

of
[CD3.23]

VH-VL

AC3356

domain

swap

uTCE
[PSMA.119]-
9
VL-VH
144
144
L59K
4.8
ND
532

of
[CD3.23]

mutation

AC3329

from

PSMA.5,

PTE

removal

variant

The supernatants of LNCaP cells after cytotoxic reactions were harvested and in vitro cytokine assays were performed. In general, IL-6 and IL-10 induction by PSMA-paTCE lead AMX-500 was 100-1000 fold reduced compared to fully unmasked AMX-500(uTCE). Singly masked metabolite AMX-500(1x-N) and AMX-500(1x-C) induced IL-6 and IL-10 similar to fully masked AMX-500. AMX-500(uTCE) induced GM-CSF and IFN-g release at much lower levels than AMX-500. In general, the singly masked metabolites had intermediate response between uTCE and AMX-500. AMX-500 and the AMX-500-NoClvSite induced minimal GM-CSF and IFN-g. AMX-500 or AMX-500(uTCE) have minimal induction of IL-2 and IL-4. The maximum level of MCP-1 produced by AMX-500, and the various metabolites were the same ˜9000 μg/ml.

To evaluate the activity of T cells, AMX-500 or its metabolites were co-cultured with healthy human PBMCs together with LNCaP cells. Human PBMCs from Donor 1 were incubated with titrations of AMX-500 or metabolites in the presence of LNCaP cells at 37° C. (PMBC:LNCaP cells at 10:1). After 72 hours, PBMCs were analyzed by flow cytometric analysis. Specifically, CD4 and CD8 T cells were interrogated for CD69, CD25, and PD-1 expression. Results are depicted in FIG. 16 and FIG. 17.

The T cell activity assay above was repeated across two additional donors (Donor 2 and Donor 3). Results for Donor 2 are shown in FIG. 18. Results for Donor 3 are shown in FIG. 19. The EC50 values (nM) are summarized below in Table 27.

TABLE 27

EC50 values for T cell activation in CD4+ and CD8+

T cells based on CD69, CD25, and PD-1 expression

CD4 T cells
AMX-500 [EC50, nM]
AMX-500(uTCE) [EC50, nM]

Donor ID
CD69
CD25
PD-1
CD69
CD25
PD-1

Donor 1
N.D.
N.D.
N.D.
0.5726
0.8236
2.415

Donor 2
N.D.
N.D.
N.D.
1.888
0.4784
1.794

Donor 3
N.D.
N.D.
N.D.
0.9662
2.081
0.6407

CD8 T cells
AMX-500 [EC50, nM]
AMX-500(uTCE) [EC50, nM]

Donor ID
CD69
CD25
PD-1
CD69
CD25
PD-1

Donor 1
N.D.
N.D.
N.D.
N.D.
0.5807
0.9478

Donor 2
N.D.
N.D.
N.D.
N.D.
0.5592
4.386

Donor 3
N.D.
N.D.
N.D.
2.148
1.22
1.788

In summary, fully masked AMX-500 protected against T cell activation relative to AMX-500(uTCE). AMX-500 intermediates (1X-C, 1X-N) maintained protection relative to AMX-500(uTCE); albeit to a lesser extent (in general) than AMX-500.

TABLE 28

In vitro cytotoxicity in LNCaP PSMA^highcells from

5 different donors. Values reported as IC50 (pM)

AMX-500

AMX-500
AMX-500
AMX-500-

Donor
(uTCE)
AMX-500
(1X-N)
(1X-C)
NoClvSite

1
24
42190
5377
8579
42005

2
15
44651
5709
6176
383235

3
16
37737
7893
6703
15188

4
ND*
38283
5359
12063
14893

5
32
85371
6439
6287
219590

*ND—not determined

As shown above in Table 28 and FIG. 13A and FIG. 13B, AMX-500 provides ˜2500-fold protection in the in vitro cytotoxicity assay compared to AMX-500(uTCE). Moreover, the AMX-500 cleavage intermediates (1x-N and 1x-C) maintain protection and reduce cytotoxicity by ˜200-500-fold relative to AMX-500(uTCE). Finally, AMX-500(NoClvSite) exhibited cytotoxicity similar to AMX-500. AMX-500(NoClvSite) corresponds to a version of AMX-500 with masking polypeptides that are non-cleavable.

An in vitro cytokine release assay was performed using PBMC: LNCAP cells at a 10:1 effector-target ratio. Cytokines INF-γ, TNF-α, IL-6, IL-10, GM-CSF, IL-β, IL-2, IL-4, and MCP-1 were measured. The results are depicted in FIG. 15 for 1 of 5 donors (Donor 5). Similar results were obtained from samples from Donors 1-4.

In general, IL-6 and IL-10 induction by AMX-500 was 100-1000 fold reduced, relative to AMX-500(uTCE), and the singly masked metabolites induced IL-6 and IL-10 similar to AMX-500.

AMX-500(uTCE) induced GM-CSF and IFN-γ release at much higher levels compared to AMX-500, and the singly masked metabolites had intermediate response between uTCE and AMX-500. AMX-500 and the AMX-500-NoClvSite induced minimal GM-CSF and IFN-γ.

AMX-500(uTCE) induced TNF-α and IL-1R response at a much higher level compared to AMX-500. The singly masked metabolites induced TNF-α and IL-13 levels similar to AMX-500. AMX-500 and AMX-500-NoClvSite produced minimal to not measurable levels of TNF-α and IL-1.

There was minimal induction of IL-2 and IL-4 observed with AMX-500 or AMX-500(uTCE).

The maximum level of MCP-1 produced by AMX-500, and the various formats was same at ˜9000 μg/ml.

TABLE 29

In vitro cytotoxicity in 22Rv1 PSMA^highcells from

3 different donors. Values reported as IC50 (pM)

Donor
AMX-500

AMX-500
AMX-500
AMX-500

ID
(uTCE)
AMX-500
(NoClvSite)
(1X-N)
(1X-C)

1
691
ND
ND
ND
ND

2
1343
ND
ND
ND
ND

3
1298
ND
ND
ND
ND

* ND—not determined

As shown above in Table 29 and FIG. 14A and FIG. 14B, AMX-500(uTCE) has reduced cytotoxicity against the 22Rv1 cells, which have a receptor density of about 4,000 copies of PSMA per cell, compared to the LNCaP cells, which have a receptor density of about 209,000 copies of PSMA per cell.

Off-target Binding Assay

Off-target binding of AMX-500 was measured against about 6,000 different membrane proteins in HEK293T cells. Ligand binding detection was done via immunofluorescence FACS. Validation of hits with signal >3 standard deviations above background was considered. As shown in FIG. 20A and FIG. 20B, AMX-500 and AMX-500-P7 do not bind to any of the 6,000 targets above background other than PSMA, CD3 complex, and CD3 epsilon. Binding to FCGR1A was determined to be an artifact of the secondary antibody binding.

In Vivo Efficacy in Mice

PSMA-paTCE AMX-500 was evaluated in three in vivo cell line-derived xenograft (CDX) models which includes C4-2 (PSMA^high, androgen-independent), LNCaP-FGC (PSMA^high) and 22Rv1 (PSMA^low/neg) cell lines. AMX-500 exhibits tumor growth inhibition in 3.5 mg/kg and 7.5 mg/kg twice weekly via IV injection in C4-2 model. AMX-500 exhibits tumor growth inhibition in 3 mg/kg twice or once weekly via IV injection in LNCaP model. AMX-500(uTCE) exhibits tumor growth inhibition in 0.35 mg/kg twice weekly via IV injection in LNCaP model. AMX-500 exhibits tumor growth inhibition in 2 mg/kg twice weekly via IV injection in 22Rv1 model. AMX-500(uTCE) exhibits tumor growth inhibition in 0.35 mg/kg twice weekly via IV injection in 22Rv1 model (FIG. 21).

Additional in vivo efficacy experiments were performed at a range of alternative doses.

The in vivo efficacy of AMX-500, AMX-500-NoClvSite, and AMX-500(uTCE) was evaluated in the human PBMC-engrafted LNCaP-fast-growing colony (FGC) human prostate tumor model in nonobese diabetic (NOD).Cg-Prkdc^scidII2rg^tm1Wjl/SzJ (NSG) mice.

Mice bearing LNCaP-FGC tumors were randomized into 7 groups of 8 mice each and administered vehicle diluent (no PBMC), vehicle diluent (PBMC), 0.5 mg/kg AMX-500, 1.5 mg/kg AMX-500, 3 mg/kg AMX-500, and 3 mg/kg AMX-500-NoClvSite by weekly bolus IV, and 3 mg/kg AMX-500(uTCE) by twice-weekly bolus IV. The AMX-500(uTCE) was administered twice-weekly to account for the expected more rapid elimination of the unmasked TCE. Experimental design and results summary are shown in Table 30. Tumor growth curves between treatment initiation (Day 8) and study termination (Day 28) are shown in FIG. 22.

All test articles were well tolerated by the experimental animals, as evidenced by the similar average body weight loss (BWL) in the range of 3-9% across all experimental groups.

AMX-500 treatment promoted anti-tumor activity at all dose levels evaluated when compared with the applicable PBMC-engrafted control, Group 2. At Day 30, the end of the study, AMX-500 at a dose level of 3 mg/kg QW showed TGI of 75% (p<0.0001), while the intermediate (1.5 mg/kg QW) and lowest (0.5 mg/kg QW) dose levels showed TGIs of 62% (p=0.0129) and 64% (p=0.0149), respectively. At Day 30, AMX-500 treatment at the highest tested dose of 3 mg/kg QW had similar TGI (75% TGI) as the enzymatically cleaved and activated AMX-500(uTCE) (80% TGI) using a 0.35 mg/kg twice weekly (BIW) dose. As the masks on AMX-500 only reduce binding and activity, AMX-500-NoClvSite did exhibit a partial response in this model (52% TGI). However, the protease-activatable AMX-500 exhibited a greater anti-tumor effect at a dose of 0.5 mg/kg (64% TGI) than that observed with AMX-500-NoClvSite at a QW dose of 3 mg/kg (52% TGI), indicating that the ELNN masks of AMX-500 may be removed in the tumor micro-environment, releasing the potent unmasked TCE.

TABLE 30

Study Design and Results Summary

Study Design

Dosing
Day 30 Results

Dose
Dosing

Frequency

Tumor

Level
Volume

and

Regression ^b

Group
N
Treatment
(mg/kg)
(mL/kg)
Route
Duration
BWL
TGI
(#/n)

1
8
Vehicle diluent,
—
10
IV
QW × 3 weeks
7.5%
—
0/8

no PBMC

2
8
Vehicle diluent,
—
10
IV
QW × 3 weeks
8.4%
—
0/8

PBMC

3
8
AMX-500
0.5
10
IV
QW × 3 weeks
6.5%
64%
2/8

4
8
AMX-500
1.5
10
IV
QW × 3 weeks
7.2%
62%
2/7

5
8
AMX-500
3.0
10
IV
QW × 3 weeks
3.7%
75%
4/8

6
8
AMX-500-NoClv
3.0
10
IV
QW × 3 weeks
8.6%
52%
2/8

Site

7
8
AMX-500(uTCE)
0.35
10
IV
BIW × 3 weeks
8.2%
80%
1/8

Abbreviations: BIW, 2 times a week; BWL, body weight loss compared with body weight at the start of treatment; IV, intravenous; NA, not applicable; PBMC, peripheral blood mononuclear cells; QW, one time a week; TGI, tumor growth inhibition.

^aTGI (%) = (Vc-Vt)/(Vc-Vo) × 100, where Vc and Vt are the mean tumor volume of the control and treated groups at the end of the study (respectively) and Vo is the mean tumor volume of the control group at the start of dosing. TGI was calculated versus Group 2 (Vehicle diluent, PBMC).

^bTumor regression was defined as tumor volume at study end (Day 30), which is less

than the starting tumor volume prior to dosing.

The in vivo efficacy of AMX-500(uTCE) and AMX-500 was evaluated in the human ex vivo-activated pan T cells-engrafted 22Rv1 human prostate tumor model in NSG mice.

Mice bearing 22Rv1 tumors were randomized into 1 group of 10 and 5 groups of 9 mice each and administered either vehicle diluent (no pan T), vehicle diluent (with pan T), 0.35 mg/kg AMX-500(uTCE), 0.5 mg/kg AMX-500(uTCE), 2 mg/kg AMX-500, or 5 mg/kg AMX-500 3 times a week for 3 weeks via bolus IV lateral tail vein injection. Experimental design and results summary are shown in Table 31. Tumor growth curves between treatment initiation (Day 8) and study termination (Day 28) are shown in FIG. 23.

All test articles were generally well tolerated by test animals, as shown by the minimal average BWL in the range of 0.6 to 5.6% across groups on Day 28.

AMX-500(uTCE) and AMX-500 promoted antitumor activity at all dose levels evaluated when compared with the applicable pan T-engrafted control, Group 2. At Day 28, the treatment groups had TGI in the range of 47.4 to 63.7%, indicating that AMX-500 has activity in this PSMA-low expressing mouse tumor model.

TABLE 31

Study Design and Results Summary

Study Design

Dosing

Dose
Dosing

Frequency

Level
Volume

and
Day 28 Results

Group
N
Treatment
(mg/kg)
(mL/kg)
Route
Duration
BWL
TGI

1
10
Vehicle (no pan T)
NA
10
IV
TIW × 3 weeks
1.6%
—

2
9
Vehicle + pan T
NA
10
IV
TIW × 3 weeks
1.9%
—

3
9
AMX-500(uTCE)
0.35
10
IV
TIW × 3 weeks
2.0%
63.7%

4
9
AMX-500(uTCE)
0.5
10
IV
TIW × 3 weeks
5.6%
47.4%

5
9
AMX-500
2
10
IV
TIW × 3 weeks
4.1%
56.7%

6
9
AMX-500
5
10
IV
TIW × 3 weeks
0.6%
63.6%

Abbreviations: BWL = body weight loss compared with body weight at the start of treatment; IV = intravenous; TGI = tumor growth inhibition; TIW = 3 times a week.

^aTGI (%) = (Vc-Vt)/(Vc-Vo) × 100, where Vc and Vt are the mean tumor volume of the control and treated groups at the end of the study (respectively) and Vo is the mean tumor volume of the control group at the start of dosing. TGI was calculated versus Group 2 (vehicle diluent + pan T).

In Vivo Efficacy in Mice—Pembrolizumab Combination

In addition, a combination of AMX-500 and pembrolizumab was tested in LNCaP CDX model. A treatment of 1.5 mg/kg of AMX-500 (once weekly IV injection) together with 10 mg/kg of pembrolizumab (twice weekly IV injection) exhibits enhanced anti-tumor activity up to 90% tumor growth inhibition (TGI) (FIG. 24).

In vivo Toxicity Assessment

AMX-500 was administrated to cynomolgus monkeys. Cynomolgus monkeys were followed by 4-week recovery schedule to evaluate reversibility of any findings. In general, AMX-500 is well tolerated with no apparent treatment related findings observed, including no apparent treatment related clinical signs, no apparent treatment related change in clinical pathology, no apparent treatment related findings in ophthalmology exams and no apparent treatment related findings in gross necropsy.

In Vivo Tumor Distribution and Tumor Cleavage

The tumor tissue distribution and masking polypeptide cleavage of AMX-500 was determined. Tumor-bearing mice were administered fluorescently labeled AMX-500. Multiple xenograft tumor models (LNCAP-FGC, 22RV1) were evaluated and select tissues and plasma was collected 48 hours post-administration. A control paTCE was spiked in during homogenization of tissues. Relative abundance of AMX-500 and cleavage products were quantified by SDS-PAGE and LI-COR detection. As shown in FIG. 26, AMX-500 distributed to healthy tissue and xenografted tumor within 48 hours after administration. As shown in Table 32, AMX-500 cleavage intermediates and fully unmasked AMX-500 were detected in the LNCaP-FGC xenograft. Minimal cleavage of AMX-500 observed in plasma or healthy tissue. AMX-500 cleavage not observed in 22Rv1 xenograft model, nor additional PDx models (n=4).

TABLE 32

In vivo cleavage relative abundance

—
AMX-500
1X-N
1X-C
uTCE

Tumor
86.5%
1.4%
3.1%
8.9%

Brain
100%
ND
ND
ND

Heart
100%
ND
ND
ND

Lung
99.4%
ND
<2%
ND

Liver
98.5%
<2%
<2%
ND

Prostate
100%
ND
ND
ND

Plasma
98.3%
0.6%
1.1%
<0.1%

While embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

	Number	Date	Country
	63499031	Apr 2023	US
	63444839	Feb 2023	US

COMPOSITIONS TARGETING PROSTATE-SPECIFIC MEMBRANE ANTIGEN AND METHODS FOR MAKING AND USING THE SAME

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Inventors

Original Assignees

CPC

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Abstract

Description

Claims

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