EGFR ANTIGEN BINDING FRAGMENTS AND COMPOSITIONS COMPRISING SAME

Abstract
The present disclosure related to antigen-binding units that specifically bind to EGFR or epitopes thereof. Some embodiments include bispecific anti-EGFR/anti-CD3 constructs with improved expression and/or stability. Related methods are also disclosed.
Description
SEQUENCE LISTING

[0001.1] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jun. 25, 2020, is named 32808-779_601_SL.txt and is 2,131,102 bytes in size.


BACKGROUND OF THE INVENTION

Many approved cancer therapeutics are cytotoxic drugs that kill normal cells as well as tumor cells. The therapeutic benefit of these cytotoxic drugs depends on tumor cells being more sensitive than normal cells, thereby allowing clinical responses to be achieved using doses that do not result in unacceptable side effects. However, essentially all of these non-specific drugs result in some if not severe damage to normal tissues, which often limits treatment suitability.


Bispecific antibodies can offer a different approach to cytotoxic drugs by directing immune effector cells to kill cancer cells. Bispecific antibodies combine the benefits of different binding specificities derived from two monoclonal antibodies into a single composition, enabling approaches or combinations of coverages that are not possible with monospecific antibodies. In one embodiment, this approach relies on binding of one arm of the bispecific antibody to a tumor-associated antigen or marker, while the other arm, upon binding the CD3 molecule on T cells, triggers their cytotoxic activity by the release of effector molecules such as such as TNF-α, IFN-γ, interleukins 2, 4 and 10, perforin, and granzymes. Advances in antibody engineering have led to the development of a number of bispecific antibody formats and compositions for redirecting effector cells to tumor targets, including bispecifics that function by recruiting and activating polyclonal populations of T cells at tumor sites, and do so without the need for co-stimulation or conventional MHC recognition. There remains, however, the dual problems of certain patients experiencing serious side effects referred to as “cytokine storm” or “cytokine release syndrome” (Lee DW et al. Current concepts in the diagnosis and management of cytokine release syndrome. Blood. 2014 124(2):188-195) mediated by the release of TNF-α and IFN-γ, amongst other cytokines, in addition to the fact that some bispecific compositions have a very short half-life, necessitating continuous infusions of four to eight weeks in order to maintain circulating concentrations within the therapeutic window for sufficient time to achieve a therapeutic effect, or have a variable effect. Thus, there is an unmet need in the field for the development of effective bispecific antibodies for use in cancer treatment.


SUMMARY OF THE INVENTION

The present invention relates to anti-epidermal growth factor receptor (EGFR) antigen binding fragments incorporated into chimeric fusion proteins and methods of using or making the same. In one aspect, disclosed herein is a polypeptide comprising an antibody binding fragment (AF1), wherein the AF1 comprises light chain complementarity-determining regions (CDR-L), heavy chain complementarity-determining regions (CDR-H), light chain framework regions (FR-L), and heavy chain framework regions (FR-H), and wherein the AF1: a. specifically binds to epidermal growth factor receptor (EGFR); and b. comprises FR-H1, FR-H2, FR-H3, and FR-H4, wherein FR-H1 has an amino acid sequence of any one of SEQ ID NOS: 14-16, FR-H2 has an amino acid sequence of SEQ ID NO:18 or SEQ ID NO: 19, FR-H3 has an amino acid sequence of SEQ ID NO: 20 or SEQ ID NO:21, and FR-H4 has an amino acid sequence of any one of SEQ ID NOS: 22-24. In some embodiments, the AF1 comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% sequence identity or is identical to an amino acid sequence of any one of SEQ ID NOS: 37-51. In certain embodiments, the AF1 is a chimeric or a humanized antigen binding fragment. In one embodiment, the AF1 is selected from the group consisting of Fv, Fab, Fab′, Fab′-SH, linear antibody, and single-chain variable fragment (scFv).


In another embodiment, the AF1 comprises a variable heavy (VH) amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to an amino acid sequence of SEQ ID NO: 28-32. In certain embodiments, the AF1 comprises a variable light (VL) amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to an amino acid sequence of SEQ ID NO: 25-27.


In some embodiments, the AF1 further comprises CDR-H3, wherein the CDRH3 has an amino acid sequence of SEQ ID NO: 6. In certain embodiments, the AF1 further comprises CDR-H1, CDR-H2, and CDR-H3, having amino acid sequences of SEQ ID NOS: 4, 5, and 6, respectively. In certain embodiments, the AF1 CDR-L comprises CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences of SEQ ID NOS: 1, 2, and 3, respectively.


In other embodiments, the AF1 further comprises FR-L comprising FR-L1, FR-L2, FR-L3, and FR-L4, wherein a. FR-L1 exhibits at least 90%, or at least 95% sequence identity or is identical to amino acid sequence of SEQ ID NO: 7; b. FR-L2 exhibits at least 90%, or at least 95% sequence identity or is identical to amino acid sequence of SEQ ID NO:8; c. FR-L3 exhibits at least 90%, or at least 95% sequence identity or is identical to amino acid sequences of SEQ ID NOS: 9-11; and d. FR-L4 exhibits at least 90%, or at least 95% sequence identity or is identical to amino acid sequence of SEQ ID NO: 13. In certain embodiments, the FR-L comprise: a. a FR-L1 having an amino acid sequence of SEQ ID NO: 7, b. a FR-L2 having an amino acid sequence of SEQ ID NO: 8, c. a FR-L3 having an amino acid sequence of SEQ ID NO: 9, d. a FR-L4 having an amino acid sequence of SEQ ID NO: 13. In another embodiment, the FR-L comprises: a. a FR-L1 having an amino acid sequence of SEQ ID NO: 7, b. a FR-L2 having an amino acid sequence of SEQ ID NO: 8, c. a FR-L3 having an amino acid sequence of SEQ ID NO: 10, d. a FR-L4 having an amino acid sequence of SEQ ID NO: 13. In yet another embodiment, the FR-L comprises: a. a FR-L1 having an amino acid sequence of SEQ ID NO: 7, b. a FR-L2 having an amino acid sequence of SEQ ID NO: 8, c. a FR-L3 having an amino acid sequence of SEQ ID NO: 11, and d. a FR-L4 having an amino acid sequence of SEQ ID NO: 13.


In one embodiment, the FR-H comprises: a. a FR-H1 having an amino acid sequence of SEQ ID NO: 14, b. a FR-H2 having an amino acid sequence of SEQ ID NO: 18, b. a FR-H3 having an amino acid sequence of SEQ ID NO: 20, and c. a FR-H4 having an amino acid sequence of SEQ ID NO: 22 or 23. In other embodiments, the FR-H comprises: a. a FR-H1 having an amino acid sequence of SEQ ID NO: 15, b. a FR-H2 having an amino acid sequence of SEQ ID NO: 19, c. a FR-H3 having an amino acid sequence of SEQ ID NO: 21, and d. a FR-H4 having an amino acid sequence of SEQ ID NO: 24. In certain embodiments, FR-H comprises: a. a FR-H1 having an amino acid sequence of SEQ ID NO: 16, b. a FR-H2 having an amino acid sequence of SEQ ID NO: 19, c. a FR-H3 having an amino acid sequence of SEQ ID NO: 20, and d. a FR-H4 having an amino acid sequence of SEQ ID NO: 22 or 23. In some embodiments, the AF1 has at least one or at least two amino acid substitutions, relative to the amino acid sequence of SEQ ID NO: 52, of a hydrophobic amino acid in a framework region wherein the hydrophobic amino acid is selected from isoleucine, leucine or methionine and the substituted amino acid is selected from arginine, threonine, or glutamine.


In one embodiment, the polypeptide further comprises a first release segment peptide (RS1) and/or a first extended recombinant polypeptide (XTEN1), wherein the RS1 is a substrate for cleavage by a mammalian protease. In some embodiments, the fusion protein, in an uncleaved state, has a structural arrangement from N-terminus to C-terminus of AF1-RS1-XTEN1 or XTEN1-RS1-AF1.


In some embodiments, the RS1 is a substrate for a protease selected from the group consisting of legumain, MMP-2, MMP-7, MMP-9, MMP-11, MMP-14, uPA, and matriptase. In other embodiments, the RS1 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from any one of SEQ ID NOS: 53-671. In certain embodiments, the RS1 comprises an amino acid sequence selected from the sequences of RSR-2089, RSR-2295, RSR-2298, RSR-2488, RSR-2599, RSR-2485, RSR-2486, RSR-2728, RSN-2089, RSN-2295, RSN-2298, RSN-2488, RSN-2599, RSN-2485, RSN-2486, RSN-2728, RSC-2089, RSC-2295, RSC-2298, RSC-2488, RSC-2599, RSC-2485, RSC-2486, and RSC-2728, each of which being set forth in Table 5.


In some embodiments, the polypeptide disclosed herein further comprises a first extended recombinant polypeptide (XTEN1), wherein the XTEN1 is characterized in that a. it has at least about 36 amino acids; b. at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the amino acid residues of the XTEN1 sequence are selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P); and c. it has at least 4-6 different amino acids selected from G, A, S, T, E and P. In certain embodiments, the XTEN1 comprises an amino acid sequence that comprises at least three of the amino acid sequences of SEQ ID NOS: 672-675. In another embodiment, the XTEN1 comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from any one of SEQ ID NOS: 676-734. In certain embodiments, XTEN1 comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from the sequences of AE144_1A, AE144_2A, AE1442B, AE144_3A, AE144_3B, AE1444A, AE1444B, AE144_5A, AE144_6B, AE144_7A, AE284, AE288_1, AE2882, AE288_3, AE292, AE293, AE300, AE576, AE584, AE864, AE864_2, AE865, AE866, AE867, and AE868, each of which being set forth in Table 7.


In certain embodiments, the AF1 has a higher isoelectric point (pI) relative to that of an antigen binding fragment consisting of a sequence shown in SEQ ID NO: 52. In one embodiment, the AF1 is incorporated into the polypeptide to form an anti-EGFR bispecific antibody, the polypeptide exhibits a higher pI relative to a control bispecific antibody, wherein said polypeptide comprises said AF1 and a reference antigen binding fragment that binds to a cluster of differentiation 3 T cell receptor (CD3), and wherein said control bispecific antigen binding fragment is identical to the polypeptide except that the AF1 is replaced with SEQ ID NO:52 . In another embodiment, the AF1 exhibits a pI 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 higher than the pI of the antigen binding fragment consisting of a sequence shown in SEQ ID NO:52. In certain embodiments, the AF1 exhibits a pI that is between 5.4 and 6.6, inclusive. In other embodiments, the AF1 exhibits a pI of about 5.4 to about 5.6, or about 5.5 to about 5.7, or about 5.6 to about 5.8, or about 5.7 to about 5.9, or about 5.8 to about 6.0, or about 5.9 to about 6.1, or about 6.0 to about 6.2, or about 6.1 to about 6.3, or about 6.2 to about 6.4, or about 6.3 to about 6.5, or about 6.4 to about 6.6. In another embodiment, AF1 exhibits a pI of about 5.4, or about 5.5, or about 5.6, or about 5.7, or about 5.8, or about 5.9, or about 6.0, or about 6.1, or about 6.2, or about 6.3, or about 6.4, or about 6.5, or about 6.6.


In certain embodiments, the AF1 specifically binds human or cynomolgus monkey (cyno) EGFR. In other embodiments, the AF1 specifically binds human and cynomolgus monkey (cyno) EGFR. In some embodiments, the AF1 specifically binds EGFR with a Kd between about 0.1 nM and about 100 nM, as determined in an in vitro antigen-binding assay comprising EGFR or an epitope thereof.


In another embodiment, the polypeptide further comprises a second antigen binding fragment (AF2) that specifically binds to cluster of differentiation 3 T cell receptor (CD3). In certain embodiments, (1) the AF2 fragment is selected from the group consisting of Fv, Fab, Fab′, Fab′-SH, linear antibody, a single domain antibody, and single-chain variable fragment (scFv), or (2) the AF1 and AF2 are configured as an (Fab′)2 or a single chain diabody.


In some embodiments, the AF2 is fused to the AF1 by a flexible peptide linker. In certain embodiment, the flexible linker comprises 2 or 3 types of amino acids selected from the group consisting of glycine, serine, and proline.


In some embodiment, the AF2 comprises a variable heavy (VH) amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to an amino acid sequence of SEQ ID NO:766 or SEQ ID NO:769. In certain embodiments, the AF2 comprises a variable light (VL) amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to an amino acid sequence of any one of SEQ ID NOS: 765, 767, 768, 770, or 771. In another embodiment, the AF2 comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% sequence identity or is identical to an amino acid sequence of any one of SEQ ID NOS:776-780.


In certain embodiments, the AF2 comprises light chain complementarity-determining regions (CDR-L) and heavy chain complementarity-determining regions (CDR-H), and wherein the antigen binding fragment comprises CDR-H1, CDR-H2, and CDR-H3, having amino acid sequences of SEQ ID NOS: 742, 743, and 744, respectively. In some embodiments, the CDR-L comprises: a. a CDR-L1 having an amino acid sequence of SEQ ID NOS: 735 or 736, b. a CDR-L2 having an amino acid sequence of SEQ ID NOS: 738 or 739, and c. a CDR-L3 having an amino acid sequence of SEQ ID NO:740.


In other embodiments, the AF2 further comprises light chain framework regions (FR-L) and heavy chain framework regions (FR-H) wherein AF2 comprises: a. a FR-L1 having an amino acid sequence of SEQ ID NO: 746; b. a FR-L2 having an amino acid sequence of SEQ ID NO: 747; c. a FR-L3 having an amino acid sequence of any one of SEQ ID NOS:748-751; d. a FR-L4 having an amino acid sequence of SEQ ID NO:754; e. a FR-H1 having an amino acid sequence of SEQ ID NO:755 or SEQ ID NO:756; f. a FR-H2 having an amino acid sequence of SEQ ID NO:759; g. a FR-H3 having an amino acid sequence of SEQ ID NO:760; and h. a FR-H4 having an amino acid sequence of any one of SEQ ID NO:764. In another embodiment, the antigen binding fragment comprises: a. a FR-L1 having an amino acid sequence of SEQ ID NO: 746; b. a FR-L2 having an amino acid sequence of SEQ ID NO: 747; c. a FR-L3 having an amino acid sequence of SEQ ID NO: 748; d. a FR-L4 having an amino acid sequence of SEQ ID NO: 754; e. a FR-H1 having an amino acid sequence of SEQ ID NO: 755; f. a FR-H2 having an amino acid sequence of SEQ ID NO: 759; g. a FR-H3 having an amino acid sequence of SEQ ID NO: 760; and h. a FR-H4 having an amino acid sequence of SEQ ID NO: 764. In yet another embodiment, the antigen binding fragment comprises: a. a FR-L1 having an amino acid sequence of SEQ ID NO:746; b. a FR-L2 having an amino acid sequence of SEQ ID NO:747; c. a FR-L3 having an amino acid sequence of SEQ ID NO:749; d. a FR-L4 having an amino acid sequence of SEQ ID NO:754; e. a FR-H1 having an amino acid sequence of SEQ ID NO:756; f. a FR-H2 having an amino acid sequence of SEQ ID NO:759; g. a FR-H3 having an amino acid sequence of SEQ ID NO:760; and h. a FR-H4 having an amino acid sequence of SEQ ID NO:764. In certain embodiment, the antigen binding fragment comprises: a. a FR-L1 having an amino acid sequence of SEQ ID NO: 746; b. a FR-L2 having an amino acid sequence of SEQ ID NO:747; c. a FR-L3 having an amino acid sequence of SEQ ID NO:750; d. a FR-L4 having an amino acid sequence of SEQ ID NO:754; e. a FR-H1 having an amino acid sequence of SEQ ID NO:756; f. a FR-H2 having an amino acid sequence of SEQ ID NO:759; g. a FR-H3 having an amino acid sequence of SEQ ID NO:760; and h. a FR-H4 having an amino acid sequence of SEQ ID NO:764. In yet another embodiment, the antigen binding fragment comprises: a. a FR-L1 having an amino acid sequence of SEQ ID NO: 746; b. a FR-L2 having an amino acid sequence of SEQ ID NO: 747; c. a FR-L3 having an amino acid sequence of SEQ ID NO: 751; d. a FR-L4 having an amino acid sequence of SEQ ID NO: 754; e. a FR-H1 having an amino acid sequence of SEQ ID NO: 756; f. a FR-H2 having an amino acid sequence of SEQ ID NO: 759; g. a FR-H3 having an amino acid sequence of SEQ ID NO: 760; and h. a FR-H4 having an amino acid sequence of SEQ ID NO: 764.


In some embodiments, the polypeptide further comprises a second release segment (RS2) and/or a second extended recombinant polypeptide (XTEN2), wherein the RS2 is a substrate for cleavage by a mammalian protease. In some embodiments, the sequences of RS1 and RS2 are identical. In another embodiment, the sequences of RS1 and RS2 are not identical.


In some embodiments, the polypeptide has a structural arrangement from N-terminus to C-terminus as follows: XTEN1-RS1-AF1-AF2-RS2-XTEN2, XTEN1-RS1-AF2-AF1-RS2-XTEN2, XTEN2-RS2-AF2-AF1-RS1-XTEN1, XTEN2-RS2-AF1-AF2-RS1-XTEN1, XTEN2-RS2-diabody-RS1-XTENI1, or XTEN1-RS1-diabody-RS2-XTEN2, wherein the diabody comprises VL and VH of the AF1 and AF2, wherein the AF2 specifically binds CD3 and AF1 specifically binds EGFR, and wherein XTEN 1 and XTEN2 are of the same or different amino acid length or sequence.


In certain embodiments, the RS2 is a substrate for a protease selected from legumain, MMP-2, MMP-7, MMP-9, MMP-11, MMP-14, uPA, and matriptase. In other embodiments, the RS2 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to a sequence selected from SEQ ID NOS:53-671. In certain 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 XTEN2 is characterized in that a. it has at least about 36 amino acids; b. at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the amino acid residues of the XTEN1 sequence are selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P); and c. it has at least 4-6 different amino acids selected from G, A, S, T, E and P. In certain embodiments, the XTEN2 comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOS: 676-734. In other embodiments, the XTEN2 comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from the sequences of AE144_1A, AE144_2A, AE144_2B, AE144_3A, AE144_3B, AE144_4A, AE144_4B, AE144_5A, AE144_6B, AE144_7A, AE284, AE288_1, AE288_2, AE288_3, AE292, AE293, AE300, AE576, AE584, AE864, AE864_2, AE865, AE866, AE867, and AE868, each of which being set forth in Table 7. In certain embodiments, the XTEN2 comprises an amino acid sequence that comprises at least three of the amino acid sequences of SEQ ID NOS: 672-675.


In some embodiments, the Tm of the AF2 is at least 2° C. greater, or at least 3° C. greater, or at least 4° C. greater, or at least 5° C. greater, or at least 6° C. greater, or at least 7° C. greater, or at least 8° C. greater, or at least 9° C. greater, or at least 10° C. greater than the Tm of an antigen binding fragment consisting of a sequence of SEQ ID NO:781, as determined by an increase in melting temperature in an in vitro assay.


In some embodiments, the AF2 binds a CD3 complex subunit selected from any one of CD3 epsilon, CD3 delta, CD3 gamma, CD3 zeta, CD3 alpha and CD3 beta epsilon. In one embodiment, the AF2 specifically binds human or cynomolgus monkey (cyno) CD3. In yet another embodiment, the AF2 specifically binds human and cynomolgus monkey (cyno) CD3.


In other embodiments, the AF2 specifically binds human or cyno CD3 with a dissociation constant (Kd) constant between about 10 nM and about 400 nM, as determined in an in vitro antigen-binding assay. In certain embodiments, the AF2 specifically binds human or cyno CD3 with a dissociation constant (Kd) 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. In certain embodiments, the AF2 specifically binds human or cyno CD3 with a dissociation constant (Kd) weaker than about 3 nM, or 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 400 nM, as determined in an in vitro antigen-binding assay. In other embodiments, AF2 specifically binds human or cyno 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 antibody-binding fragment consisting of an amino acid sequence of SEQ ID NO: 781, as determined by the respective dissociation constants (Kd) in an in vitro antigen-binding assays. In yet another embodiment, the binding affinity of the AF1 to EGFR is at least 10-fold greater, or at least 100-fold greater, or at least 1000-fold greater than the binding affinity of the AF2 to CD3, as measured in an in vitro antigen-binding assay.


In certain embodiments, the AF2 exhibits an isoelectric point (pI) that is less than or equal to 6.6. In another embodiment, the AF2 exhibits a pI that is between 5.5 and 6.6, inclusive. In other embodiments, the AF2 exhibits a pI that is between about 5.5 and 6.6, or about 5.6 and about 6.4, or about 5.8 and about 6.2, or about 6.0 and about 6.2. In some embodiments, the AF2 exhibits a pI 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 pI of a reference antigen binding fragment consisting of a sequence shown in SEQ ID NO: 781. In another embodiment, the AF2 exhibits a pI that is within at least about 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 about 1.5 pH units of the pI of the AF 1. In certain embodiments, the AF2 exhibits a pI 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 pI of the AF1.


In another aspect, the present disclosure provides a bispecific antigen-binding unit comprising: a. a first antigen-binding fragment (AF1) wherein the AF1 specifically binds to EGFR; and b. a second antigen-binding fragment (AF2) wherein the AF2 specifically binds to cluster of differentiation 3 T cell receptor (CD3); wherein a difference between an isoelectric point (pI) of the second antigen binding fragment and a pI of the first antigen binding fragment is from 0 to about 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, as determined by an in vitro assays. In some embodiments, the AF1 comprises a variable heavy (VH) amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to an amino acid sequence of SEQ ID NO: 28-32. In other embodiments, the AF1 comprises a variable light (VL) amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to an amino acid sequence of SEQ ID NO: 25-27. In certain embodiments, the AF1 comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% sequence identity or is identical to an amino acid sequence of any one of SEQ ID NOS: 37-51. In other embodiments, (1) each of the AF1 and the AF2 fragment is selected from the group consisting of Fv, Fab, Fab′, Fab′-SH, linear antibody, a single domain antibody, and single-chain variable fragment (scFv), or (2) the AF1 and AF2 are configured as an (Fab′)2 or a single chain diabody.


In some embodiments, the AF1 of the bispecific antigen-binding unit comprises light chain complementarity-determining regions (CDR-L), heavy chain complementarity-determining regions (CDR-H), light chain framework regions (FR-L), and heavy chain framework regions (FR-H), and wherein the AF1 comprises FR-H1, FR-H2, FR-H3, and FR-H4.


In other embodiments, the AF1 further comprises CDR-H3, wherein the CDRH3 has an amino acid sequence of SEQ ID NO: 6. In certain embodiments, the AF1 further comprises CDR-H1, CDR-H2, and CDR-H3, having amino acid sequences of SEQ ID NOS: 4, 5, and 6, respectively. In certain embodiments, the CDR-L comprises CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences of SEQ ID NOS: 1, 2, and 3, respectively.


In certain embodiments, FR-H1 has an amino acid sequence of any one of SEQ ID NOS: 14-16, FR-H2 has an amino acid sequence of SEQ ID NO:18 or SEQ ID NO:19, FR-H3 has an amino acid sequence of SEQ ID NO: 20 or SEQ ID NO:21, and FR-H4 has an amino acid sequence of any one of SEQ ID NOS: 22-24. In some embodiments, the FR-H comprises: a FR-H1 having an amino acid sequence of SEQ ID NO: 14, a FR-H2 having an amino acid sequence of SEQ ID NO: 18, a FR-H3 having an amino acid sequence of SEQ ID NO: 20, and a FR-H4 having an amino acid sequence of SEQ ID NO: 22 or 23. In other embodiments, the FR-H comprise: a FR-H1 having an amino acid sequence of SEQ ID NO: 15, a FR-H2 having an amino acid sequence of SEQ ID NO: 19, a FR-H3 having an amino acid sequence of SEQ ID NO: 21, and a FR-H4 having an amino acid sequence of SEQ ID NO: 24. In yet another embodiment, FR-H comprises: a FR-H1 having an amino acid sequence of SEQ ID NO: 16, a FR-H2 having an amino acid sequence of SEQ ID NO: 19, a FR-H3 having an amino acid sequence of SEQ ID NO: 20, and a FR-H4 having an amino acid sequence of SEQ ID NO: 22 or 23.


In certain embodiments, the FR-L1 exhibits at least 90%, or at least 95% sequence identity or is identical to amino acid sequences of SEQ ID NOS: 7; the FR-L2 exhibits at least 90%, or at least 95% sequence identity or is identical to amino acid sequences of SEQ ID NO: 8; the FR-L3 exhibits at least 90%, or at least 95% sequence identity or is identical to amino acid sequences of SEQ ID NOS: 9-11; and the FR-L4 exhibits at least 90%, or at least 95% sequence identity or is identical to amino acid sequence of SEQ ID NO: 13. In other embodiments, the FR-L comprises: a FR-L1 having an amino acid sequence of SEQ ID NO: 7, a FR-L2 having an amino acid sequence of SEQ ID NO: 8, a FR-L3 having an amino acid sequence of SEQ ID NO: 9, and a FR-L4 having an amino acid sequence of SEQ ID NO: 13. In yet another embodiment, the FR-L comprises: a FR-L1 having an amino acid sequence of SEQ ID NO: 7, a FR-L2 having an amino acid sequence of SEQ ID NO: 8, a FR-L3 having an amino acid sequence of SEQ ID NO: 10, and a FR-L4 having an amino acid sequence of SEQ ID NO: 13. In another embodiment, the FR-L comprises: a FR-L1 having an amino acid sequence of SEQ ID NO: 7, a FR-L2 having an amino acid sequence of SEQ ID NO: 8, a FR-L3 having an amino acid sequence of SEQ ID NO: 11, and a FR-L4 having an amino acid sequence of SEQ ID NO: 13.


In certain embodiments, the AF2 of the bispecific antigen-binding unit comprises light chain complementarity-determining regions (CDR-L) and heavy chain complementarity-determining regions (CDR-H), and wherein the antigen binding unit comprises CDR-H1, CDR-H2, and CDR-H3, having amino acid sequences of SEQ ID NOS: 742, 743, and 744, respectively. In some embodiments, the AF2 comprises a variable heavy (VH) amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to an amino acid sequence of SEQ ID NO:766 or SEQ ID NO:769. In other embodiments, the AF2 comprises a variable light (VL) amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to an amino acid sequence of any one of SEQ ID NOS: 765, 767, 768, 770, or 771. In certain embodiments, the AF2 comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% sequence identity or is identical to an amino acid sequence of any one of SEQ ID NOS:776-780.


In other embodiments, the CDR-L of AF2 comprises: a CDR-L1 having an amino acid sequence of SEQ ID NOS: 735 or 736, a CDR-L2 having an amino acid sequence of SEQ ID NOS: 738 or 739, and a CDR-L3 having an amino acid sequence of SEQ ID NO:740.


In other embodiments, the AF2 further comprises light chain framework regions (FR-L) and heavy chain framework regions (FR-H) wherein AF2 comprises: a. a FR-L1 having an amino acid sequence of SEQ ID NO:746; b. a FR-L2 having an amino acid sequence of SEQ ID NO:747; c. a FR-L3 having an amino acid sequence of any one of SEQ ID NOS:748-751; d. a FR-L4 having an amino acid sequence of SEQ ID NO:754; e. a FR-H1 having an amino acid sequence of SEQ ID NO:755 or SEQ ID NO:756; f. a FR-H2 having an amino acid sequence of SEQ ID NO:759; g. a FR-H3 having an amino acid sequence of SEQ ID NO:760; and h. a FR-H4 having an amino acid sequence of any one of SEQ ID NO:764. In certain embodiments, the AF2 further comprises a light chain framework region (FR-L) and a heavy chain framework region (FR-H), and wherein the antigen binding unit comprises: a. a FR-L1 having an amino acid sequence of SEQ ID NO: 746; b. a FR-L2 having an amino acid sequence of SEQ ID NO: 747; c. a FR-L3 having an amino acid sequence of SEQ ID NO: 748; d. a FR-L4 having an amino acid sequence of SEQ ID NO:754; e. a FR-H1 having an amino acid sequence of SEQ ID NO: 755; f. a FR-H2 having an amino acid sequence of SEQ ID NO: 759; g. a FR-H3 having an amino acid sequence of SEQ ID NO: 760; and h. a FR-H4 having an amino acid sequence of SEQ ID NO: 764. In other embodiments, the AF2 further comprises a light chain framework region (FR-L) and a heavy chain framework region (FR-H), and wherein the antigen binding unit comprises: a. a FR-L1 having an amino acid sequence of SEQ ID NO:746; b. a FR-L2 having an amino acid sequence of SEQ ID NO:747; c. a FR-L3 having an amino acid sequence of SEQ ID NO:749; d. a FR-L4 having an amino acid sequence of SEQ ID NO:754; e. a FR-H1 having an amino acid sequence of SEQ ID NO:756; f. a FR-H2 having an amino acid sequence of SEQ ID NO:759; g. a FR-H3 having an amino acid sequence of SEQ ID NO:760; and h. a FR-H4 having an amino acid sequence of SEQ ID NO:764. In another embodiment, the AF2 further comprises a light chain framework region (FR-L) and a heavy chain framework region (FR-H), and wherein the antigen binding unit comprises: a. a FR-L1 having an amino acid sequence of SEQ ID NO:746; b. a FR-L2 having an amino acid sequence of SEQ ID NO:747; c. a FR-L3 having an amino acid sequence of SEQ ID NO:750; d. a FR-L4 having an amino acid sequence of SEQ ID NO:754; e. a FR-H1 having an amino acid sequence of SEQ ID NO:756; f. a FR-H2 having an amino acid sequence of SEQ ID NO:759; g. a FR-H3 having an amino acid sequence of SEQ ID NO:760; and h. a FR-H4 having an amino acid sequence of SEQ ID NO:764. In certain embodiments, the AF2 further comprises a light chain framework region (FR-L) and a heavy chain framework region (FR-H), and wherein the antigen binding unit comprises: a. a FR-L1 having an amino acid sequence of SEQ ID NO:746; b. a FR-L2 having an amino acid sequence of SEQ ID NO:747; c. a FR-L3 having an amino acid sequence of SEQ ID NO:751; d. a FR-L4 having an amino acid sequence of SEQ ID NO:754; e. a FR-H1 having an amino acid sequence of SEQ ID NO:756; f. a FR-H2 having an amino acid sequence of SEQ ID NO:759; g. a FR-H3 having an amino acid sequence of SEQ ID NO:760; and h. a FR-H4 having an amino acid sequence of SEQ ID NO:764.


In some embodiments, the AF2 is fused to the AF1 by a flexible peptide linker. In certain embodiments, the flexible linker comprises 2 or 3 types of amino acids selected from the group consisting of glycine, serine, and proline.


In certain embodiments, the bispecific antigen binding unit further comprises a first release segment peptide (RS1) and a second release segment peptide (RS2), wherein each of the RS1 and RS2 is a substrate for cleavage by a mammalian protease. In one embodiment, he RS1 and RS2 are identical. In another embodiment, the RS1 and RS2 are different. In some embodiments, each of the RS1 and RS2 is a substrate for a protease selected from the group consisting of legumain, MMP-2, MMP-7, MMP-9, MMP-11, MMP-14, uPA, and matriptase. In other embodiments, each of the RS1 and RS2 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from any one of SEQ ID NOS:53-671. In another embodiment, each of the RS1 and RS2 comprises an amino acid sequence selected from the sequences of RSR-2089, RSR-2295, RSR-2298, RSR-2488, RSR-2599, RSR-2485, RSR-2486, RSR-2728, RSN-2089, RSN-2295, RSN-2298, RSN-2488, RSN-2599, RSN-2485, RSN-2486, RSN-2728, RSC-2089, RSC-2295, RSC-2298, RSC-2488, RSC-2599, RSC-2485, RSC-2486, and RSC-2728, each of which being set forth in Table 5.


In some embodiments, the bispecific antigen binding unit further comprises a first extended recombinant polypeptide (XTEN1) and a second extended recombinant polypeptide, wherein each of the XTEN1 and XTEN2 is characterized in that it has a. at least about 36 amino acids; b. at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the amino acid residues of the XTEN1 sequence are selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P); and c. at least 4-6 different amino acids selected from G, A, S, T, E and P. In one embodiment, the XTEN1 and XTEN2 are identical. In another embodiment, the XTEN1 and XTEN2 are different.


In certain embodiments, each of the XTEN1 and XTEN2 comprises an amino acid sequence that comprises at least three of the amino acid sequences of SEQ ID NOS: 672-675. In yet another embodiment, each of the XTEN1 and XTEN2 comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from any one of SEQ ID NOS: 676-734. In other embodiments, each of the XTEN1 and XTEN2 comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from the sequences of AE144_1A, AE144_2A, AE1442B, AE144_3A, AE144_3B, AE144_4A, AE1444B, AE144_5A, AE144_6B, AE144_7A, AE284, AE288_1, AE288_2, AE288 3, AE292, AE293, AE300, AE576, AE584, AE864, AE864_2, AE865, AE866, AE867, and AE868, each of which being set forth in Table 7.


In some embodiments, the bispecific antigen binding unit has a structural arrangement from N-terminus to C-terminus as follows: XTEN1-RS1-AF1-AF2-RS2-XTEN2, XTEN1-RS1-AF2-AF1-RS2-XTEN2, XTEN2-RS2-AF2-AF1-RS1-XTEN1, XTEN2-RS2-AF1-AF2-RS1-XTEN1, XTEN2-RS2-diabody-RS1-XTEN1, or XTEN1-RS1-diabody-RS2-XTEN2, wherein the diabody comprises VL and VH of the AF1 and AF2.


In some embodiments, the AF1 specifically binds human or cynomolgus monkey (cyno) EGFR. In other embodiments, the AF1 specifically binds human and cynomolgus monkey (cyno) EGFR. In certain embodiments, the AF2 binds a CD3 complex subunit selected from any one of CD3 epsilon, CD3 delta, CD3 gamma, CD3 zeta, CD3 alpha and CD3 beta epsilon. In another embodiment, the AF2 specifically binds human or cynomolgus monkey (cyno) CD3. In yet another embodiment, the AF2 specifically binds human and cynomolgus monkey (cyno) CD3.


In one embodiment, the AF1 specifically binds EGFR with a Kd between about 0.1 nM and about 100 nM, as determined in an in vitro antigen-binding assay comprising EGFR or an epitope thereof.


In other embodiments, the AF1 specifically binds EGFR with a dissociation constant (Kd) constant between about 0.1 nM and about 100 nM, or between about 0.5 nM and about 50 nM, or between about 1.0 nM and about 20 nM, or between about 2.0 nM and about 10 nM, as determined in an in vitro antigen-binding assay. In some embodiments, the AF2 specifically binds human or cyno CD3 with a dissociation constant (Kd) 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. In certain embodiments, the AF2 specifically binds human or cyno CD3 with a dissociation constant (Kd) weaker than about 3 nM, or 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 400 nM, as determined in an in vitro antigen-binding assay. In yet another embodiment, the AF2 specifically binds human or cyno 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 antibody-binding fragment consisting of an amino acid sequence of SEQ ID NO: 781, as determined by the respective dissociation constants (Kd) in an in vitro antigen-binding assays. In some embodiments, the AF2 exhibits 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, 10-fold, 20-fold, 50-fold, 100-fold, or at least 1000-fold at weaker relative to that of the AF1, as determined by the respective dissociation constants (Kd) in an in vitro antigen-binding assay.


In another aspect, the present disclosure provides a pharmaceutical composition comprising the polypeptide disclosed herein and one or more pharmaceutically suitable excipients. In some embodiment, the pharmaceutical composition is formulated for intradermal, subcutaneous, intravenous, intra-arterial, intraabdominal, intraperitoneal, intrathecal, or intramuscular administration. In another embodiment, the pharmaceutical composition is in a liquid form or a frozen form. In certain embodiment, the pharmaceutical composition is in a prefilled syringe for a single injection. In another embodiment, the pharmaceutical composition is formulated as a lyophilized powder to be reconstituted prior to administration.


In yet another aspect, the present disclosure provides a polypeptide disclosed herein for use in the preparation of a medicament for treating a disease in a subject in need thereof. In some embodiments, the disease is selected from the group of cancers consisting of anaplastic and medullary thyroid cancers, appendiceal cancer, arrhenoblastoma, biliary tract carcinoma, bladder cancer, breast cancer, cancers of the bile duct, carcinoid tumor, cervical cancer, cholangiocarcinoma, colon cancer, colorectal cancer, craniopharyngioma, endometrial cancer, epithelial intraperitoneal malignancy with malignant ascites, esophageal cancer, Ewing sarcoma, fallopian tube cancer, follicular cancer, gall bladder cancer, gastric cancer, gastrointestinal stromal tumor (GIST), GE-junction cancer, genito-urinary tract cancer, glioma, glioblastoma, head and neck cancer, hepatoblastoma, hepatocarcinoma, HR+ and HER2+ breast cancer, Hurthle cell cancer, Inflammatory breast cancer, Kaposi sarcoma, kidney cancer, laryngeal cancer, liposarcoma, liver cancer, lung cancer, medulloblastoma, melanoma, Merkel cell carcinoma, neuroblastoma, neuroblastoma, neuroendocrine cancer, non-small cell lung cancer, osteosarcoma (bone cancer), ovarian cancer, ovarian cancer with malignant ascites, pancreatic cancer, pancreatic neuroendocrine tumor, papillary cancer, parathyroid cancer, peritoneal carcinomatosis, peritoneal mesothelioma, primitive neuroectodermal tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland carcinoma, sarcoma, skin cancer, small cell lung cancer, small intestine cancer, stomach cancer, testicular cancer, thyroid cancer, triple negative breast cancer, urothelial cancer, uterine cancer, uterine serous carcinoma, vaginal cancer, vulvar cancer, and Wilms tumor.


In a related aspect, the present disclosure provides a method of treating a disease in a subject, comprising administering to the subject in need thereof one or more therapeutically effective doses of the pharmaceutical composition disclosed herein. In some embodiments, the subject is selected from the group consisting of mouse, rat, monkey, and human.


In certain embodiments, the disease is selected from the group of cancers consisting of anaplastic and medullary thyroid cancers, appendiceal cancer, arrhenoblastoma, biliary tract carcinoma, bladder cancer, breast cancer, cancers of the bile duct, carcinoid tumor, cervical cancer, cholangiocarcinoma, colon cancer, colorectal cancer, craniopharyngioma, endometrial cancer, epithelial intraperitoneal malignancy with malignant ascites, esophageal cancer, Ewing sarcoma, fallopian tube cancer, follicular cancer, gall bladder cancer, gastric cancer, gastrointestinal stromal tumor (GIST), GE-junction cancer, genito-urinary tract cancer, glioma, glioblastoma, head and neck cancer, hepatoblastoma, hepatocarcinoma, HR+ and HER2+ breast cancer, Hurthle cell cancer, Inflammatory breast cancer, Kaposi sarcoma, kidney cancer, laryngeal cancer, liposarcoma, liver cancer, lung cancer, medulloblastoma, melanoma, Merkel cell carcinoma, neuroblastoma, neuroblastoma, neuroendocrine cancer, non-small cell lung cancer, osteosarcoma (bone cancer), ovarian cancer, ovarian cancer with malignant ascites, pancreatic cancer, pancreatic neuroendocrine tumor, papillary cancer, parathyroid cancer, peritoneal carcinomatosis, peritoneal mesothelioma, primitive neuroectodermal tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland carcinoma, sarcoma, skin cancer, small cell lung cancer, small intestine cancer, stomach cancer, testicular cancer, thyroid cancer, triple negative breast cancer, urothelial cancer, uterine cancer, uterine serous carcinoma, vaginal cancer, vulvar cancer, and Wilms tumor. In other embodiments, the pharmaceutical composition is administered to the subject as one or more therapeutically effective doses administered twice weekly, once a week, every two weeks, every three weeks, every four weeks, or monthly. In certain embodiments, the pharmaceutical composition is administered to the subject as one or more therapeutically effective doses over a period of at least two weeks, or at least one month, or at least two months, or at least three months, or at least four months, or at least five months, or at least six months. In some embodiments, the dose is administered intradermally, subcutaneously, intravenously, intra-arterially, intra-abdominally, intraperitoneally, intrathecally, or intramuscularly.


In a certain aspect, the present disclosure provides an isolated nucleic acid, the nucleic acid comprising (a) a polynucleotide encoding a polypeptide disclosed herein; or (b) the complement of the polynucleotide of (a).


In a related aspect, the present disclosure provides an expression vector comprising the polynucleotide sequence disclosed herein and a recombinant regulatory sequence operably linked to the polynucleotide sequence.


In yet another aspect, the present disclosure provides an isolated host cell, comprising the expression vector disclosed herein. In some embodiments, the host cell is a prokaryote. In certain embodiments, the host cell is E. coli or a mammalian cell.


INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in this specification are herein incorporated 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.





BRIEF DESCRIPTION OF THE DRAWINGS

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 of which:



FIG. 1 depicts the individual components of a bispecific antigen binding fragment composition. FIG. 1A depicts an antigen binding fragment having affinity to a target cell marker. FIG. 1B depicts an antigen binding fragment having affinity to an effector cell. FIGS. 1C and 1D depict XTEN polypeptides of different length. FIG. 1E depicts a cleavable release segment.



FIG. 2 depicts 2 different forms of the polypeptide compositions described herein. FIG. 2A depicts, on the left side, an antigen binding fragment to an effector cell fused with a release segment and an XTEN, while the arrow depicts the action of a protease to cleave the release segment leading to, on the right hand side, the release of the XTEN from the antigen binding fragment of the polypeptide, such that the antigen-binding fragment regains binding affinity potential (e.g., its full binding affinity potential) as it is no longer shielded by the XTEN. FIG. 2B depicts, on the left side, a bispecific composition having an antigen binding fragment to an effector cell fused to an antigen binding fragment having binding affinity to a target cell marker. A release segment and an XTEN are also fused to the antigen binding fragment having affinity to the effector cell, while the arrow depicts the action of a protease to cleave the release segment leading to, on the right hand side, the release of the XTEN and the fused antigen binding fragments from the polypeptide, which would then regain their full binding affinity potential as they are no longer shielded by the XTEN.



FIG. 3 depicts two different forms of a bispecific antigen binding polypeptide. On the left side, a bispecific composition having an antigen binding fragment to an effector cell is fused to an antigen binding fragment having binding affinity to a target cell marker with the release segment (with the scissors indicating susceptibility to protease cleavage) and the XTEN is fused to the antigen binding fragment having binding affinity to an effector cell, while on the right hand side, a bispecific composition having an antigen binding fragment to an effector cell is fused to an antigen binding fragment having binding affinity to a target cell marker, with the release segment and the XTEN fused to the antigen binding fragment having binding affinity to the target cell marker.



FIG. 4 depicts three different forms of a bispecific antigen-binding polypeptide. FIG. 4A depicts a bispecific composition having an scFv antigen binding fragment to an effector cell fused to an scFv antigen binding fragment having binding affinity to a target cell marker with a release segment (with the scissors indicating susceptibility to protease cleavage) and an XTEN fused to each antigen binding fragment. FIGS. 4B and 4C are variations of 4A in which the antigen binding fragments are in a diabody configuration, with the release segments (with the scissors indicating susceptibility to protease cleavage) and XTENs fused to the antigen binding fragment to an effector cell or the target cell marker, respectively.



FIG. 5 shows schematic representations of a bispecific antigen binding polypeptide in proximity to tumor tissue (on the top) and normal tissue (on the bottom). The bispecific antigen binding polypeptide is preferentially cleaved at the tumor tissue to release one or more XTEN moieties as compared to that in the normal tissue. The cleaved bispecific antigen binding polypeptide is capable of binding to a T cell and a tumor cell expressing a tumor-specific marker.



FIG. 6 depicts the amino acid sequence of the control release segment RSR-1517 (SEQ ID NO: 53), showing the sites of peptide cleavage for the listed proteases.









LENGTHY TABLES




The patent contains a lengthy table section. A copy of the table is available in electronic form from the USPTO web site (). An electronic copy of the table will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR 1.19(b)(3).









DETAILED DESCRIPTION OF THE INVENTION

While preferred 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. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. 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.


Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention.


Definitions

In the context of the present application, the following terms have the meanings ascribed to them unless specified otherwise:


As used throughout the specification and claims, the terms “a,” “an,” and “the” are used in the sense that they mean “at least one,” “at least a first,” “one or more,” or “a plurality” of the referenced components or steps, except in instances wherein an upper limit is thereafter specifically stated. Therefore, a “release segment,” as used herein, means “at least a first release segment,” but includes a plurality of release segments. The operable limits and parameters of combinations, as with the amounts of any single agent, will be known to those of ordinary skill in the art in light of the present disclosure.


The terms “polypeptide”, “peptide”, and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component.


The term “monomeric” as applied to a polypeptide refers to the state of the polypeptide as being a single continuous amino acid sequence substantially unassociated with one or more additional polypeptides of the same or different sequence.


As used herein, the term “amino acid” refers to either natural and/or unnatural or synthetic amino acids, including but not limited to both the D or L optical isomers, and amino acid analogs and peptidomimetics. Standard single or three letter codes may be used to designate amino acids.


The term “natural L-amino acid” or “L-amino acid” means the L optical isomer forms of glycine (G), proline (P), alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M), cysteine (C), phenylalanine (F), tyrosine (Y), tryptophan (W), histidine (H), lysine (K), arginine (R), glutamine (Q), asparagine (N), glutamic acid (E), aspartic acid (D), serine (S), and threonine (T).


The term “antibody” is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), nanobodies, VHH antibodies, and antibody fragments so long as they exhibit the desired antigen-binding activity or immunological activity. The term “immunoglobulin” (Ig) is used interchangeably with antibody herein. The full-length antibodies may be, for example, monoclonal, recombinant, chimeric, deimmunized, humanized and human antibodies. Antibodies represent a large family of molecules that include several types of molecules, such as IgD, IgG, IgA, IgM and IgE. The term “immunoglobulin molecule” includes, for example, hybrid antibodies, or altered antibodies, and fragments thereof. It has been shown that the antigen binding function of an antibody can be performed by fragments of a naturally-occurring or a monoclonal antibody.


A “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human complementarity-determining regions (CDRs) and amino acid residues from human framework regions (FRs). In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDRs correspond to those of a non-human antibody (which may include amino acid substitutions), and all or substantially all of the FRs correspond to those of a human antibody (which may include amino acid substitutions).


The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being known in the art or described herein.


An “antigen-binding fragment” as used herein refers to an immunoglobulin molecule and immunologically active portions of an immunoglobulin molecule, i.e., a molecule that contains an antigen-binding site which specifically binds (“immunoreacts with”) an antigen. Examples include but are not limited to Fv, Fab, Fab′, Fab′-SH, F(ab′)2, diabodies, linear antibodies (see U.S. Pat. No. 5,641,870), a single domain antibody, a single domain camelid antibody, single-chain fragment variable (scFv) antibody molecules, and multispecific antibodies formed from antibody fragments that retain the ability to specifically bind to antigen. Also encompassed within the term “antigen binding fragment” is any polypeptide chain-containing molecular structure that has a specific shape which fits to and recognizes and binds to an epitope, where one or more non-covalent binding interactions stabilize the complex between the molecular structure and the epitope. An antigen binding fragment “specifically binds to” or is “immunoreactive with” an antigen if it binds with greater affinity or avidity than it binds to other reference antigens including polypeptides or other substances.


“scFv” or “single chain fragment variable” are used interchangeably herein to refer to an antibody fragment format comprising variable regions of heavy (“VH”) and light (“VL”) chains or two copies of a VH or VL chain of an antibody, which are joined together by a short flexible peptide linker which enables the scFv to form the desired structure for antigen binding. The scFv is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, and can be easily expressed in functional form in E. coli or other host cells.


“Diabodies” refers to small antibody fragments prepared by constructing scFv fragments with short linkers (about 5-10 residues) between the VH and VL domains such that inter-chain but not intra-chain pairing of the V domains is achieved, resulting in a bivalent fragment, i.e., fragment having two antigen-binding sites. Bispecific diabodies are heterodimers of two “crossover” scFv fragments in which the VH and VL domains of the two antibodies are present on different polypeptide chains. Diabodies are described more fully in, for example, US7635475.


The term “bispecific antigen-binding fragment” is to be understood as an antigen binding fragment that has binding specificities for at least two different antigens.


The terms “antigen”, “target antigen” and “immunogen” are used interchangeably herein to refer to the structure or binding determinant that an antibody, antibody fragment or an antibody fragment-based molecule binds to or has specificity against. The target antigen may be polypeptide, carbohydrate, nucleic acid, lipid, hapten or other naturally occurring or synthetic compound or portions thereof. An antigen is also a ligand for those antibodies or antibody fragments that have binding affinity for the antigen. Non-limiting exemplary antigens described herein included CD3 and EGFR (and portions thereof) from human, non-human primates, murine, and other homologues thereof.


The term “CD3 antigen binding fragment” refers to an antigen binding fragment that is capable of binding cluster of differentiation 3 (CD3) or a member of the CD3 complex with sufficient affinity such that the antigen binding fragment is useful as a diagnostic and/or therapeutic agent in targeting CD3.


An “EGFR antigen binding fragment” refers to an antigen binding fragment that is capable of binding epidermal growth factor receptor. EGFR is a member of the ErbB family of receptors, a subfamily of four closely related receptor tyrosine kinases: EGFR (ErbB-1), HER2/neu (ErbB-2), Her 3 (ErbB-3) and Her 4 (ErbB-4).


A “target tissue” or “target cell” refers to a tissue or cell bearing the EGFR antigen that is the cause of or is part of a disease condition such as, but not limited to cancer or related conditions. Sources of diseased target tissue or cells include a body organ, a tumor, a cancerous cell or population of cancerous cells or cells that form a matrix or are found in association with a population of cancerous cells, bone, skin, cells that produce cytokines or factors contributing to a disease condition.


The term “epitope” refers to the particular site on an antigen molecule to which an antibody, antibody fragment, or binding domain binds. An epitope is a ligand of an antibody or antibody fragment.


“Affinity” refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1: 1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). As used herein “a greater binding affinity” means a lower Kd value; e.g., 1 × 10-9 M is a greater binding affinity than 1 × 10-8 M. An antibody which binds an antigen of interest, e.g., a tumor-associated EGFR antigen, is one that binds the antigen with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting a cell or tissue expressing the antigen, and does not significantly cross-react with other proteins.


“Dissociation constant”, or “Kd”, are used interchangeably and refer to the affinity between a ligand “L” and a protein “P”; i.e. how tightly a ligand binds to a particular protein. It can be calculated using the formula Kd = [L][P]/[LP], where [P], [L] and [LP] represent molar concentrations of the protein, ligand and complex, respectively.


The term “hypervariable region,” “HVR,” or “CDR”, when used herein, interchangeably refer to the regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops and/or are involved in antigen recognition. Generally, antibodies comprise six hypervariable regions; three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3). A number of CDR delineations are in use and are encompassed herein; e.g., CDR-L1 refers to the first hypervariable CDR region of the light chain, CDR-H2 refers to the second hypervariable CDR region of the heavy chain, etc. The Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).


“Isoelectric point” or “pI” are used interchangeably herein to refer to the pH at which a particular molecule carries no net electrical charge or is electrically neutral in the statistical mean. The standard nomenclature to represent the isoelectric point is pH, such that the units are pH units; e.g., an antigen binding fragment with a pI of 6.3 would have a neutral charge in solution at pH 6.3. The isoelectric point can be determined mathematically, including a number of algorithms for estimating isoelectric points of peptides and proteins; e.g., the Henderson-Hasselbalch equation with different pK values. The isoelectric point can also be determined experimentally by in vitro assays such as capillary electrophoresis focusing.


“Framework” or “FR” residues are those variable domain residues in antigen binding fragments other than the hypervariable region residues as herein defined, and are generally located between or that flank CDR. A number of FR delineations are in use and are encompassed herein; e.g., FR-L1 refers to the first FR region of the light chain, FR-H2 refers to the second FR region of the heavy chain, etc.


The term “release segment” or “RS” refers to a cleavage sequence in the subject compositions that can be recognized and cleaved by one or more proteases, effecting release of the antigen binding fragments and XTEN from the composition. As used herein, “mammalian protease” means a protease that normally exists in the body fluids, cells, tissues, and may be found in higher levels in certain target tissues or cells, e.g., in diseased tissues (e.g., tumor) of a mammal. RS sequences can be engineered to be cleaved by various mammalian proteases or multiple mammalian proteases that are present in or proximal to target tissues in a subject or are introduced in an in vitro assay. Other equivalent proteases (endogenous or exogenous) that are capable of recognizing a defined cleavage site can be utilized. It is specifically contemplated that the RS sequence can be adjusted and tailored to the protease utilized and can incorporate linker amino acids to join to adjacent polypeptides


The term “cleavage site” refers to that location between adjacent amino acids in a peptide or polypeptide that can be broken or cleaved by enzymes such as proteases; the breaking of the peptide bonds between the adjacent amino acids.


The term “within”, when referring to a first polypeptide being linked to a second polypeptide, encompasses linking or fusion of an additional component that connects the N-terminus of the first or second polypeptide to the C-terminus of the second or first polypeptide, respectively, as well as insertion of the first polypeptide into the sequence of the second polypeptide. For example, when an RS component is linked “within” a chimeric polypeptide assembly, the RS may be linked to the N-terminus, the C-terminus, or may be inserted between any two amino acids of an XTEN polypeptide.


“Activity” as applied to form(s) of a composition provided herein, refers to an action or effect, including but not limited to antigen binding, antagonist activity, agonist activity, a cellular or physiologic response, cell lysis, cell death, or an effect generally known in the art for the effector component of the composition, whether measured by an in vitro, ex vivo or in vivo assay or a clinical effect.


“Effector cell”, as used herein, includes any eukaryotic cells capable of conferring an effect on a target cell. For example, an effector cell can induce loss of membrane integrity, pyknosis, karyorrhexis, apoptosis, lysis, and/or death of a target cell. In another example, an effector cell can induce division, growth, differentiation of a target cell or otherwise altering signal transduction of a target cell. Non-limiting examples of effector cells include plasma cell, T cell, CD4 cell, CD8 cell, B cell, cytokine induced killer cell (CIK cell), master cell, dendritic cell, regulatory T cell (RegT cell), helper T cell, myeloid cell, macrophage, and NK cell.


An “effector cell antigen” refers to molecules expressed by an effector cell, including without limitation cell surface molecules such as proteins, glycoproteins or lipoproteins. Exemplary effector cell antigens include proteins of the CD3 complex or the T cell receptor (TCR), CD4, CD8, CD25, CD38, CD69, CD45RO, CD57, CD95, CD107, and CD154, as well as effector molecules such as cytokines in association with, bound to, expressed within, or expressed and released by, an effector cell. An effector cell antigen can serve as the binding counterpart of a binding domain of the subject chimeric polypeptide assembly.


As used herein, “CD3” or “cluster of differentiation 3” means the T cell surface antigen CD3 complex, which includes in individual form or independently combined form all known CD3 subunits, for example CD3 epsilon, CD3 delta, CD3 gamma, CD3 zeta, CD3 alpha and CD3 beta. The extracellular domains of CD3 epsilon, gamma and delta contain an immunoglobulin-like domain, so are therefore considered part of the immunoglobulin superfamily. CD3 includes, for example, human CD3 epsilon protein (NCBI RefSeq No. NP_000724), which is 207 amino acids in length, and human CD3 gamma protein (NCBI RefSeq No. NP_000064), which is 182 amino acids in length.


As used herein, the term “ELISA” refers to an enzyme-linked immunosorbent assay as described herein or as otherwise known in the art.


A “host cell” includes an individual cell or cell culture which can be or has been a recipient for the subject vectors into which exogenous nucleic acid has been introduced, such as those described herein. Host cells 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 natural, accidental, or deliberate mutation. A host cell includes cells transfected in vivo with a vector of this invention.


“Isolated,” when used to describe the various polypeptides disclosed herein, means a polypeptide that has been identified and separated and/or recovered from a component of its natural environment or from a more complex mixture (such as during protein purification). Contaminant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. As is apparent to those of skill in the art, a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, does not require “isolation” to distinguish it from its naturally occurring counterpart. In addition, a “concentrated”, “separated” or “diluted” polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, is distinguishable from its naturally occurring counterpart in that the concentration or number of molecules per volume is generally greater than that of its naturally occurring counterpart. In general, a polypeptide made by recombinant means and expressed in a host cell is considered to be “isolated.”


An “isolated nucleic acid” is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the polypeptide-encoding nucleic acid. For example, an isolated polypeptide-encoding nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated polypeptide-encoding nucleic acid molecules therefore are distinguished from the specific polypeptide-encoding nucleic acid molecule as it exists in natural cells. However, an isolated polypeptide-encoding nucleic acid molecule includes polypeptide-encoding nucleic acid molecules contained in cells that ordinarily express the polypeptide where, for example, the nucleic acid molecule is in a chromosomal or extra-chromosomal location different from that of natural cells.


A “chimeric” protein or polypeptide contains at least one fusion polypeptide comprising at least one region in a different position in the sequence than that which occurs in nature. The regions may normally exist in separate proteins and are brought together in the fusion polypeptide; or they may normally exist in the same protein but are placed in a new arrangement in the fusion polypeptide. A chimeric protein may be created, for example, by chemical synthesis, or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship.


“Fused,” and “fusion” are used interchangeably herein, and refers to the joining together of two or more peptide or polypeptide sequences by recombinant means. A “fusion protein” or “chimeric protein” comprises a first amino acid sequence linked to a second amino acid sequence with which it is not naturally linked in nature.


“XTENylated” is used to denote a peptide or polypeptide that has been modified by the linking or fusion of one or more XTEN polypeptides (described, below) to the peptide or polypeptide, whether by recombinant or chemical cross-linking means.


“Operably linked” means that the DNA sequences being linked are in reading phase or in-frame. An “in-frame fusion” refers to the joining of two or more open reading frames (ORFs) to form a continuous longer ORF, in a manner that maintains the correct reading frame of the original ORFs. For example, a promoter or enhancer is operably linked to a coding sequence for a polypeptide if it affects the transcription of the polypeptide sequence. Thus, the resulting recombinant fusion protein is a single protein containing two or more segments that correspond to polypeptides encoded by the original ORFs (which segments are not normally so joined in nature).


In the context of polypeptides, a “linear sequence” or a “sequence” is an order of amino acids in a polypeptide in an amino to carboxyl terminus (N- to C-terminus) direction in which residues that neighbor each other in the sequence are contiguous in the primary structure of the polypeptide. A “partial sequence” is a linear sequence of part of a polypeptide that is known to comprise additional residues in one or both directions.


“Heterologous” means derived from a genotypically distinct entity from the rest of the entity to which it is being compared. For example, a glycine-rich sequence removed from its native coding sequence and operatively linked to a coding sequence other than the native sequence is a heterologous glycine-rich sequence. The term “heterologous” as applied to a polynucleotide, a polypeptide, means that the polynucleotide or polypeptide is derived from a genotypically distinct entity from that of the rest of the entity to which it is being compared.


The terms “polynucleotides”, “nucleic acids”, “nucleotides,” and “oligonucleotides” are used interchangeably. They refer to nucleotides of any length, encompassing a singular nucleic acid as well as plural nucleic acids, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.


The term “complement of a polynucleotide” denotes a polynucleotide molecule having a complementary base sequence and reverse orientation as compared to a reference sequence, such that it could hybridize with a reference sequence with complete fidelity.


“Recombinant” as applied to a polynucleotide means that the polynucleotide is the product of various combinations of recombination steps which may include cloning, restriction and/or ligation steps, and other procedures that result in expression of a recombinant protein in a host cell.


The terms “gene” and “gene fragment” are used interchangeably herein. They refer to a polynucleotide containing at least one open reading frame that is capable of encoding a particular protein after being transcribed and translated. A gene or gene fragment may be genomic or cDNA, as long as the polynucleotide contains at least one open reading frame, which may cover the entire coding region or a segment thereof. A “fusion gene” is a gene composed of at least two heterologous polynucleotides that are linked together.


As used herein, a “coding region” or “coding sequence” is a portion of polynucleotide which consists of codons translatable into amino acids. Although a “stop codon” (TAG, TGA, or TAA) is typically not translated into an amino acid, it may be considered to be part of a coding region, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, and the like, are not part of a coding region. The boundaries of a coding region are typically determined by a start codon at the 5′ terminus, encoding the amino terminus of the resultant polypeptide, and a translation stop codon at the 3′ terminus, encoding the carboxyl terminus of the resulting polypeptide. Two or more coding regions of the present invention can be present in a single polynucleotide construct, e.g., on a single vector, or in separate polynucleotide constructs, e.g., on separate (different) vectors. It follows, then, that a single vector can contain just a single coding region, or comprise two or more coding regions, e.g., a single vector can separately encode a binding domain-A and a binding domain-B as described below. In addition, a vector, polynucleotide, or nucleic acid of the invention can encode heterologous coding regions, either fused or unfused to a nucleic acid encoding a binding domain of the invention. Heterologous coding regions include without limitation specialized elements or motifs, such as a secretory signal peptide or a heterologous functional domain.


The term “downstream” refers to a nucleotide sequence that is located 3′ to a reference nucleotide sequence. In certain embodiments, downstream nucleotide sequences relate to sequences that follow the starting point of transcription. For example, the translation initiation codon of a gene is located downstream of the start site of transcription.


The term “upstream” refers to a nucleotide sequence that is located 5′ to a reference nucleotide sequence. In certain embodiments, upstream nucleotide sequences relate to sequences that are located on the 5′ side of a coding region or starting point of transcription. For example, most promoters are located upstream of the start site of transcription.


“Homology” or “homologous” refers to sequence similarity or interchangeability between two or more polynucleotide sequences or between two or more polypeptide sequences. When using a program such as BestFit to determine sequence identity, similarity or homology between two different amino acid sequences, the default settings may be used, or an appropriate scoring matrix, such as blosum45 or blosum80, may be selected to optimize identity, similarity or homology scores. Preferably, polynucleotides that are homologous are those which hybridize under stringent conditions as defined herein and have at least 70%, preferably at least 80%, more preferably at least 90%, more preferably 95%, more preferably 97%, more preferably 98%, and even more preferably 99% sequence identity compared to those sequences. Polypeptides that are homologous preferably have sequence identities that are at least 70%, preferably at least 80%, even more preferably at least 90%, even more preferably at least 95-99% identical when optimally aligned over sequences of comparable length.


“Ligation” as applied to polynucleic acids refers to the process of forming phosphodiester bonds between two nucleic acid fragments or genes, linking them together. To ligate the DNA fragments or genes together, the ends of the DNA must be compatible with each other. In some cases, the ends will be directly compatible after endonuclease digestion. However, it may be necessary to first convert the staggered ends commonly produced after endonuclease digestion to blunt ends to make them compatible for ligation.


The terms “stringent conditions” or “stringent hybridization conditions” include reference to conditions under which a polynucleotide will hybridize to its target sequence, to a detectably greater degree than other sequences (e.g., at least 2-fold over background). Generally, stringency of hybridization is expressed, in part, with reference to the temperature and salt concentration under which the wash step is carried out. Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short polynucleotides (e.g., 10 to 50 nucleotides) and at least about 60° C. for long polynucleotides (e.g., greater than 50 nucleotides)-for example, “stringent conditions” can include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and three washes for 15 min each in 0.1 xSSC/1% SDS at 60° C. to 65° C. Alternatively, temperatures of about 65° C., 60° C., 55° C., or 42° C. may be used. SSC concentration may be varied from about 0.1 to 2x SSC, with SDS being present at about 0.1%. Such wash temperatures are typically selected to be about 5° C. to 20° C. lower than the thermal melting point for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. An equation for calculating Tm and conditions for nucleic acid hybridization are well known and can be found in Sambrook, J. et al., “Molecular Cloning: A Laboratory Manual,” 3rd edition, Cold Spring Harbor Laboratory Press, 2001. Typically, blocking reagents are used to block non-specific hybridization. Such blocking reagents include, for instance, sheared and denatured salmon sperm DNA at about 100-200 µg/ml. Organic solvent, such as formamide at a concentration of about 35-50% v/v, may also be used under particular circumstances, such as for RNA:DNA hybridizations. Useful variations on these wash conditions will be readily apparent to those of ordinary skill in the art.


The terms “percent identity,” percentage of sequence identity,” and “% identity,” as applied to polynucleotide sequences, refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences. Percent identity may be measured over the length of an entire defined polynucleotide sequence, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polynucleotide sequence, for instance, a fragment of at least 45, at least 60, at least 90, at least 120, at least 150, at least 210 or at least 450 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured. The percentage of sequence identity is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of matched positions (at which identical residues occur in both polypeptide sequences), dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. When sequences of different length are to be compared, the shortest sequence defines the length of the window of comparison. Conservative substitutions are not considered when calculating sequence identity.


The terms “percent identity,” percentage of sequence identity,” and “% identity,” with respect to the polypeptide sequences identified herein, is defined as the percentage of amino acid residues in a query sequence that are identical with the amino acid residues of a second, reference polypeptide sequence of comparable length or a portion thereof, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity, thereby resulting in optimal alignment. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve optimal alignment over the full length of the sequences being compared. Percent identity may be measured over the length of an entire defined polypeptide sequence, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.


“Repetitiveness” used in the context of polynucleotide sequences refers to the degree of internal homology in the sequence such as, for example, the frequency of identical nucleotide sequences of a given length. Repetitiveness can, for example, be measured by analyzing the frequency of identical sequences.


The term “expression” as used herein refers to a process by which a polynucleotide produces a gene product, for example, an RNA or a polypeptide. It includes without limitation transcription of the polynucleotide into messenger RNA (mRNA), transfer RNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA) or any other RNA product, and the translation of an mRNA into a polypeptide. Expression produces a “gene product.” As used herein, a gene product can be either a nucleic acid, e.g., a messenger RNA produced by transcription of a gene, or a polypeptide which is translated from a transcript. Gene products described herein further include nucleic acids with post transcriptional modifications, e.g., polyadenylation or splicing, or polypeptides with post translational modifications, e.g., methylation, glycosylation, the addition of lipids, association with other protein subunits, or proteolytic cleavage.


A “vector” or “expression vector” are used interchangeably and refers to a nucleic acid molecule, preferably self-replicating in an appropriate host, which transfers an inserted nucleic acid molecule into and/or between host cells. 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 transcribed and translated into a polypeptide(s). An “expression system” usually connotes a suitable host cell comprised of an expression vector that can function to yield a desired expression product.


“Serum degradation resistance,” as applied to a polypeptide, refers to the ability of the polypeptides to withstand degradation in blood or components thereof, which typically involves proteases in the serum or plasma. The serum degradation resistance can be measured by combining the protein with human (or mouse, rat, dog, monkey, as appropriate) serum or plasma, typically for a range of days (e.g. 0.25, 0.5, 1, 2, 4, 8, 16 days), typically at about 37° C. The samples for these time points can be run on a Western blot assay and the protein is detected with an antibody. The antibody can be to a tag in the protein. If the protein shows a single band on the western, where the protein’s size is identical to that of the injected protein, then no degradation has occurred. In this exemplary method, the time point where 50% of the protein is degraded, as judged by Western blots or equivalent techniques, is the serum degradation half-life or “serum half-life” of the protein.


The terms “t½”, “half-life”, “terminal half-life”, “elimination half-life” and “circulating half-life” are used interchangeably herein and, as used herein means the terminal half-life calculated as 1n(2)/Ke1. Ke1 is the terminal elimination rate constant calculated by linear regression of the terminal linear portion of the log concentration vs. time curve. Half-life typically refers to the time required for half the quantity of an administered substance deposited in a living organism to be metabolized or eliminated by normal biological processes. When a clearance curve of a given polypeptide is constructed as a function of time, the curve is usually biphasic with a rapid α-phase and longer β-phase. The typical β-phase half-life of a human antibody in humans is 21 days. Half-life can be measured using timed samples from anybody fluid, but is most typically measured in plasma samples.


The term “molecular weight” generally refers to the sum of atomic weights of the constituent atoms in a molecule. Molecular weight can be determined theoretically by summing the atomic masses of the constituent atoms in a molecule. When applied in the context of a polypeptide, the molecular weight is calculated by adding, based on amino acid composition, the molecular weight of each type of amino acid in the composition or by estimation from comparison to molecular weight standards in an SDS electrophoresis gel. The calculated molecular weight of a molecule can differ from the “apparent molecular weight” of a molecule, which generally refers to the molecular weight of a molecule as determined by one or more analytical techniques. “Apparent molecular weight factor” and “apparent molecular weight” are related terms and when used in the context of a polypeptide, the terms refer to a measure of the relative increase or decrease in apparent molecular weight exhibited by a particular amino acid or polypeptide sequence. The apparent molecular weight can be determined, for example, using size exclusion chromatography (SEC) or similar methods by comparing to globular protein standards, as measured in “apparent kD” units. The apparent molecular weight factor is the ratio between the apparent molecular weight and the “molecular weight”; the latter is calculated by adding, based on amino acid composition as described above, or by estimation from comparison to molecular weight standards in an SDS electrophoresis gel. The determination of apparent molecular weight and apparent molecular weight factor is described in U.S. Pat. No. 8,673,860.


A “defined medium” refers to a medium comprising nutritional and hormonal requirements necessary for the survival and/or growth of the cells in culture such that the components of the medium are known. Traditionally, the defined medium has been formulated by the addition of nutritional and growth factors necessary for growth and/or survival. Typically, the defined medium provides at least one component from one or more of the following categories: a) all essential amino acids, and usually the basic set of twenty amino acids plus cysteine; b) an energy source, usually in the form of a carbohydrate such as glucose; c) vitamins and/or other organic compounds required at low concentrations; d) free fatty acids; and e) trace elements, where trace elements are defined as inorganic compounds or naturally occurring elements that are typically required at very low concentrations, usually in the micromolar range. The defined medium may also optionally be supplemented with one or more components from any of the following categories: a) one or more mitogenic agents; b) salts and buffers as, for example, calcium, magnesium, and phosphate; c) nucleosides and bases such as, for example, adenosine and thymidine, hypoxanthine; and d) protein and tissue hydrolysates.


The term “agonist” is used in the broadest sense and includes any molecule that mimics a biological activity of a native polypeptide disclosed herein. Suitable agonist molecules specifically include agonist antibodies or antibody fragments, fragments or amino acid sequence variants of native polypeptides, peptides, small organic molecules, etc. Methods for identifying agonists of a native polypeptide may comprise contacting a native polypeptide with a candidate agonist molecule and measuring a detectable change in one or more biological activities normally associated with the native polypeptide.


As used herein, “treatment” or “treating,” or “palliating,” or “ameliorating” are used interchangeably herein. These terms refer to an approach for obtaining beneficial or desired results including but not limited to a therapeutic benefit and/or a prophylactic benefit. By therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms or improvement in one or more clinical parameters associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder. For prophylactic benefit, the compositions may be administered to a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease may not have been made.


A “therapeutic effect” or “therapeutic benefit,” as used herein, refers to a physiologic effect, including but not limited to the mitigation, amelioration, or prevention of disease or an improvement in one or more clinical parameters associated with the underlying disorder in a subject, or to otherwise enhance physical or mental wellbeing of a subject, resulting from administration of a polypeptide of the invention other than the ability to induce the production of an antibody against an antigenic epitope possessed by the biologically active protein. For prophylactic benefit, the compositions may be administered to a subject at risk of developing a particular disease, a recurrence of a former disease, condition or symptom of the disease, or to a subject reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease may not have been made.


The terms “therapeutically effective amount” and “therapeutically effective dose”, as used herein, refer to an amount of a drug or a biologically active protein, either alone or as a part of a 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. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.


The term “therapeutically effective and non-toxic dose” as used herein refers to a tolerable dose of the compositions as defined herein that is high enough to cause depletion of tumor or cancer cells, tumor elimination, tumor shrinkage or stabilization of disease without or essentially without major toxic effects in the subject. Such therapeutically effective and non-toxic doses may be determined by dose escalation studies described in the art and should be below the dose inducing severe adverse side effects.


The term “therapeutic index”, as used herein, refers to the ratio of the blood concentration at which a drug becomes toxic and the concentration at which the drug is effective. One exemplary ratio of therapeutic index is LD50:ED50, wherein LD50 is the dose resulting in 50% mortality in a populations of subjects and ED50 is the dose resulting in effectiveness in a population of subjects.


The term “dose regimen”, as used herein, refers to a schedule for consecutively administered multiple doses (i.e., at least two or more) of a composition, wherein the doses are given in therapeutically effective amounts to result in sustained beneficial effect on any symptom, aspect, measured parameter, endpoint, or characteristic of a disease state or condition in a subject.


As used herein, “administering” is meant a method of giving a dosage of a compound (e.g., an anti-CD3 antibody of the invention) or a composition (e.g., a pharmaceutical composition including an anti-CD3 antibody of the invention) to a subject.


A “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the subject or individual is a human.


The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth/proliferation. Examples of cancer include, but are not limited to, carcinomas, Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, B cell lymphoma, T-cell lymphoma, follicular lymphoma, mantle cell lymphoma, blastoma, breast cancer, colon cancer, prostate cancer, head and neck cancer, any form of skin cancer, melanoma, genito-urinary tract cancer, ovarian cancer, ovarian cancer with malignant ascites, peritoneal carcinomatosis, uterine serous carcinoma, endometrial cancer, cervical cancer, colorectal cancer, an epithelia intraperitoneal malignancy with malignant ascites, uterine cancer, mesothelioma in the peritoneum kidney cancers, lung cancer, small-cell lung cancer, non-small cell lung cancer, gastric cancer, esophageal cancer, stomach cancer, small intestine cancer, liver cancer, hepatocarcinoma, hepatoblastoma, liposarcoma, pancreatic cancer, gall bladder cancer, cancers of the bile duct, salivary gland carcinoma, thyroid cancer, epithelial cancer, adenocarcinoma, sarcomas of any origin, primary hematologic malignancies including acute or chronic lymphocytic leukemias, acute or chronic myelogenous leukemias, myeloproliferative neoplastic disorders, or myelodysplastic disorders, myasthenia gravis, Morbus Basedow, Hashimoto thyroiditis, or Goodpasture syndrome.


“Tumor,” as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms “cancer”, “cancerous”, “cell proliferative disorder”, “proliferative disorder,” and “tumor” are not mutually exclusive as used herein.


“Tumor-specific marker” as used herein, refers to an antigen that is found on or in a cancer cell that may be, but is not necessarily, found in higher numbers in or on the cancer cell relative to normal cells or tissues.


I). General Techniques

The practice of the present invention employs, unless otherwise indicated, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA, which are within the skill of the art. See Sambrook, J. et al., “Molecular Cloning: A Laboratory Manual,” 3rd edition, Cold Spring Harbor Laboratory Press, 2001; “Current protocols in molecular biology”, F. M. Ausubel, et al. eds., 1987; the series “Methods in Enzymology,” Academic Press, San Diego, CA.; “PCR 2: a practical approach”, M.J. MacPherson, B.D. Hames and G.R. Taylor eds., Oxford University Press, 1995; “Antibodies, a laboratory manual” Harlow, E. and Lane, D. eds., Cold Spring Harbor Laboratory, 1988; “Goodman & Gilman’s The Pharmacological Basis of Therapeutics,” 11th Edition, McGraw-Hill, 2005; and Freshney, R.I., “Culture of Animal Cells: A Manual of Basic Technique,” 4th edition, John Wiley & Sons, Somerset, NJ, 2000, the contents of which are incorporated in their entirety herein by reference.


Host cells can be cultured in a variety of media. Commercially available media such as Ham’s F10 (Sigma), Minimal Essential Medium (MEM, Sigma), RPMI-1640 (Sigma), and Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma) are suitable for culturing eukaryotic cells. In addition, animal cells can be grown in a defined medium that lacks serum but is supplemented with hormones, growth factors or any other factors necessary for the survival and/or growth of a particular cell type. Whereas a defined medium supporting cell survival maintains the viability, morphology, capacity to metabolize and potentially, capacity of the cell to differentiate, a defined medium promoting cell growth provides all chemicals necessary for cell proliferation or multiplication. The general parameters governing mammalian cell survival and growth in vitro are well established in the art. Physicochemical parameters which may be controlled in different cell culture systems are, e.g., pH, pO2, temperature, and osmolarity. The nutritional requirements of cells are usually provided in standard media formulations developed to provide an optimal environment. Nutrients can be divided into several categories: amino acids and their derivatives, carbohydrates, sugars, fatty acids, complex lipids, nucleic acid derivatives and vitamins. Apart from nutrients for maintaining cell metabolism, most cells also require one or more hormones from at least one of the following groups: steroids, prostaglandins, growth factors, pituitary hormones, and peptide hormones to proliferate in serum-free media (Sato, G. H., et al. in “Growth of Cells in Hormonally Defined Media”, Cold Spring Harbor Press, N.Y., 1982). In addition to hormones, cells may require transport proteins such as transferrin (plasma iron transport protein), ceruloplasmin (a copper transport protein), and high-density lipoprotein (a lipid carrier) for survival and growth in vitro. The set of optimal hormones or transport proteins will vary for each cell type. Most of these hormones or transport proteins have been added exogenously or, in a rare case, a mutant cell line has been found which does not require a particular factor. Those skilled in the art will know of other factors required for maintaining a cell culture without undue experimentation.


Growth media for growth of prokaryotic host cells include nutrient broths (liquid nutrient medium) or LB medium (Luria Bertani). Suitable media include defined and undefined media. In general, media contains a carbon source such as glucose needed for bacterial growth, water, and salts. Media may also include a source of amino acids and nitrogen, for example beef or yeast extract (in an undefined medium) or known quantities of amino acids (in a defined medium). In some embodiments, the growth medium is LB broth, for example LB Miller broth or LB Lennox broth. LB broth comprises peptone (enzymatic digestion product of casein), yeast extract and sodium chloride. In some embodiments, a selective medium is used which comprises an antibiotic. In this medium, only the desired cells possessing resistance to the antibiotic will grow.


II). EGFR Antigen Binding Compositions

In a first aspect, the disclosure provides polypeptides comprising a first antigen binding fragment (AF1) that binds to epidermal growth factor (EGFR) or an epitope thereof. The antigen binding fragments that bind EGFR antigens have particular utility for pairing with a second antigen binding fragment (AF2) with binding affinity to CD3 antigen (or other antigen) of an effector cell in compositions designed in specific formats in order to effect cell killing of diseased cells or tissues bearing EGFR antigens. Binding specificity 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 EGFR as compared to other reference antigens.


The origin of the antigen binding fragments contemplated by the disclosure can be derived from a naturally occurring antibody or fragment thereof, a non-naturally occurring antibody or fragment thereof, a humanized antibody or fragment thereof, a synthetic antibody or fragment thereof, a hybrid antibody or fragment thereof, or an engineered antibody or fragment thereof. Methods for generating an antibody for a given target marker are well known in the art. For example, the monoclonal antibodies may be made using the hybridoma method described by Kohler et al., Nature, 256:495 (1975), or may be made by recombinant DNA methods (U.S. Pat. No. 4,816,567). The structure of antibodies and fragments thereof, variable regions of heavy and light chains of an antibody (VH and VL), single chain variable regions (scFv), complementarity determining regions (CDR), and domain antibodies (dAbs) are well understood. Methods for generating a polypeptide having a desired EGFR antigen binding fragment are known in the art.


Various EGFR binding antigen binding fragments of the disclosure have been specifically modified to enhance their stability in the polypeptide embodiments described herein relative to EGFR antibodies and antigen binding fragments known in the art. Protein aggregation of monoclonal antibodies continues to be a significant problem in their developability and remains a major area of focus in antibody production. Antibody aggregation can be triggered by partial unfolding of its domains, leading to monomer-monomer association followed by nucleation and aggregate growth. Although the aggregation propensities of antibodies and antibody-based proteins can be affected by the external experimental conditions, they are strongly dependent on the intrinsic antibody properties as determined by their sequences and structures. Although it is well known that proteins are only marginally stable in their folded states, it is often less well appreciated that most proteins are inherently aggregation-prone in their unfolded or partially unfolded states, and the resulting aggregates can be extremely stable and long-lived. Reduction in aggregation propensity has also been shown to be accompanied by an increase in expression titer, showing that reducing protein aggregation is beneficial throughout the development process and can lead to a more efficient path to clinical studies. For therapeutic proteins, aggregates are a significant risk factor for deleterious immune responses in patients, and can form via a variety of mechanisms. Controlling aggregation can improve protein stability, manufacturability, attrition rates, safety, formulation, titers, immunogenicity, and solubility. The intrinsic properties of proteins such as size, hydrophobicity, electrostatics and charge distribution play important roles in protein solubility. Low solubility of therapeutic proteins due to surface hydrophobicity has been shown to render formulation development more difficult and may lead to poor bio-distribution, undesirable pharmacokinetics behavior and immunogenicity in vivo. Decreasing the overall surface hydrophobicity of candidate monoclonal antibodies can also provide benefits and cost savings relating to purification and dosing regimens. Individual amino acids can be identified by structural analysis as being contributory to aggregation potential in an antibody, and can be located in CDR as well as framework regions. In particular, residues can be predicted to be at high risk of causing hydrophobicity issues in a given antibody. In one embodiment, the present disclosure provides an antigen binding fragment having the capability to specifically bind EGFR in which the antigen binding fragment has at least one amino acid substitution of a hydrophobic amino acid in a framework region relative to the parental antibody or antibody fragment wherein the hydrophobic amino acid is selected from isoleucine, leucine or methionine. In another embodiment, the EGFR antigen binding fragment has at least two amino acid substitutions of hydrophobic amino acids in one or more framework regions wherein the hydrophobic amino acids are selected from isoleucine, leucine or methionine.


In the context of the subject antigen binding fragments, the isoelectric point (pI) is the pH at which the antibody fragment has no net electrical charge. If the pH is below the pI of an antibody fragment, then it will have a net positive charge. A greater positive charge tends to correlate with increased blood clearance and tissue retention, with a generally shorter half-life. If the pH is greater than the pI of an antibody fragment it will have a negative charge. A negative charge generally results in decreased tissue uptake and a longer half-life. It is possible to manipulate this charge through mutations to the framework residues. These considerations informed the design of various sequences of the antigen binding fragments of the embodiments described herein wherein individual amino acid substitutions were made relative to the parental antibody utilized as the starting point. The isoelectric point of a polypeptide can be determined mathematically or experimentally in an in vitro assay. The isoelectric point (pI) is the pH at which a protein has a net charge of zero and can be calculated using the charges for the specific amino acids in the protein sequence. Estimated values for the charges are called acid dissociation constants or pKa values and are used to calculate the pI. The pI can be determined in vitro by methods such as capillary isoelectric focusing (see Datta-Mannan, A., et al. The interplay of non-specific binding, target-mediated clearance and FcRn interactions on the pharmacokinetics of humanized antibodies. mAbs 7:1084 (2015); Li, B., et al. Framework selection can influence pharmacokinetics of a humanized therapeutic antibody through differences in molecule charge. mAbs 6, 1255-1264 (2014)) or other methods known in the art.


In some aspects of any of the embodiments disclosed herein, a subject polypeptide comprising an AF1 comprises light chain complementarity-determining regions (CDR-L) and heavy chain complementarity-determining regions (CDR-H) listed in Table 1 wherein the AF1 binds EGFR or an epitope thereof. Additionally or alternatively, a subject AFlof the disclosure can comprise a CDR-L or a CDR-H with at least 60% identity to any of the CDR-L or CDR-H listed in Table 1. In some aspects, a subject AFlof the disclosure can comprise CDR-L or CDR-H can exhibit at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or greater sequence identity to any of the SEQ ID NOs listed in Table 1. Additionally, the subject AF1 of the embodiments can further comprise light chain framework regions (FR-L) and heavy chain framework regions (FR-H) listed in Table 2. In some aspects, a subject AFlof the disclosure can comprise FR-L or FR-H that exhibit at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or greater sequence identity to any of the SEQ ID NOs listed in Table 2. In one embodiment, the AF1 of any of the subject composition embodiments described herein is a chimeric or a humanized antigen binding fragment. In another embodiment, the AF1 of any of the subject composition embodiments described herein is selected from the group consisting of Fv, Fab, Fab′, Fab′-SH, linear antibody, and single-chain variable fragment (scFv). The AF1 having CDR-H and CDR-L can be configured in a (CDR-H)-(CDR-L) or a (CDR-H)-(CDR-L) orientation, N-terminus to C-terminus.


In one embodiment, the present disclosure provides polypeptides comprising an AF1 wherein the AF1 comprises CDR-L and CDR-H, and heavy chain framework regions (FR-H), and wherein the AF1, (a) specifically binds to EGFR; (b) comprises FR-H1, FR-H2, FR-H3, and FR-H4, wherein FR-H1 has an amino acid sequence of any one of SEQ ID NOS: 14-16, FR-H2 has an amino acid sequence of SEQ ID NO:18 or SEQ ID NO:19, FR-H3 has an amino acid sequence of SEQ ID NO: 20 or SEQ ID NO:21, and FR-H4 has an amino acid sequence of any one of SEQ ID NOS: 22-24. In another embodiment, a polypeptide of a subject composition embodiment described herein comprises an AF1, wherein the AF1 comprises a CDR-H3 wherein the CDR-H3 has an amino acid sequence of SEQ ID NO: 6. In another embodiment, the AF1 comprises CDR-H1, CDR-H2, and CDR-H3, having amino acid sequences of SEQ ID NOS: 4, 5, and 6, respectively.


In another embodiment, the present disclosure provides polypeptides comprising an AF1, wherein the AF1 has a higher isoelectric point (pI) relative to that of an antigen binding fragment consisting of a sequence shown in SEQ ID NO:52, as evidenced by an in vitro assay. In one embodiment, the AF1 is incorporated into the polypeptide to form an anti-EGFR bispecific antibody wherein the polypeptide exhibits a higher pI relative to a control bispecific antibody, wherein said polypeptide comprises said AF1 and a reference antigen binding fragment that binds to a cluster of differentiation 3 T cell receptor (CD3), and wherein said control bispecific antigen binding fragment is identical to the polypeptide except that the AF1 is replaced with SEQ ID NO:52 . In the foregoing embodiment, the AF1 exhibits a pI that is 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 higher than the pI of the antigen binding fragment consisting of a sequence shown in SEQ ID NO:52. In the foregoing embodiments, the in vitro assay for determining the pI can be capillary isoelectrophoresis focusing or other assays known in the art.


In another embodiment, a polypeptide of any of the subject composition embodiments described herein comprises an AF1, wherein the AF1 comprises CDR-L, CDR-H, light chain framework regions (FR-L) and heavy chain framework regions (FR-H) and wherein the AF1 (a) is configured to specifically bind to EGFR; (b) comprises CDR-H1, CDR-H2, and CDR-H3, having amino acid sequences of SEQ ID NOS: 4, 5, and 6, respectively; (c) comprises FR-H1, FR-H2, FR-H3, and FR-H4, each exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to an amino acid of SEQ ID NOS: 14-16, SEQ ID NOS: 18 and 19, SEQ ID NOS: 20 and 21, and SEQ ID NOS: 22-24, respectively, and further comprises FR-L wherein the FR-L comprise: (a) a FR-L1 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO: 7, (b) a FR-L2 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO: 8, (c) a FR-L3 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO: 9, and (d) a FR-L4 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO: 13.


In another embodiment, a polypeptide of any of the subject composition embodiments described herein comprises an AF1, wherein the AF1 comprises CDR-L, CDR-H, light chain framework regions (FR-L) and heavy chain framework regions (FR-H) and wherein the AF1: (a) is configured to specifically bind to EGFR; (b) comprises CDR-H1, CDR-H2, and CDR-H3, having amino acid sequences of SEQ ID NOS: 4, 5, and 6, respectively; (c) comprises FR-H1, FR-H2, FR-H3, and FR-H4, each exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to an amino acid of SEQ ID NOS:14-16, SEQ ID NOS: 18 and 19, SEQ ID NOS: 18 and 19, SEQ ID NOS: 20 and 21, and SEQ ID NOS: 22-24, respectively; and (d) further comprises FR-L wherein the FR-L comprise (i) a FR-L1 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO: 7, (ii) a FR-L2 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO: 8, (iii) a FR-L3 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO: 10, and (iv) a FR-L4 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO: 13.


In another embodiment, a polypeptide of any of the subject composition embodiments described herein comprises an AF1, wherein the AF1 comprises CDR-L, CDR-H, light chain framework regions (FR-L) and heavy chain framework regions (FR-H) and wherein the AF1: (a) is configured to specifically bind to EGFR; (b) comprises CDR-H1, CDR-H2, and CDR-H3, having amino acid sequences of SEQ ID NOS: 4, 5, and 6, respectively; (c) comprises FR-H1, FR-H2, FR-H3, and FR-H4, each exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to an amino acid of SEQ ID NOS: 14-16, SEQ ID NOS: 18 and 19, SEQ ID NOS: 20 and 21, and SEQ ID NOS: 22-24, respectively; and (d) further comprises FR-L wherein the FR-L comprise: (i) a FR-L1 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO: 7, (ii) a FR-L2 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO: 8, (iii) a FR-L3 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO: 11, and (iv) a FR-L4 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO: 13.


In another embodiment, a polypeptide of any of the subject composition embodiments described herein comprises an AF1, wherein the AF1 comprises CDR-L, CDR-H, light chain framework regions (FR-L) and heavy chain framework regions (FR-H) and wherein the AF1: (a) is configured to specifically bind to EGFR; (b) comprises CDR-H1, CDR-H2, and CDR-H3, having amino acid sequences of SEQ ID NOS: 4, 5, and 6, respectively; (c) comprises FR-L1, FR-L2, FR-L3, and FR-L4, each exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to an amino acid of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NOS:9-11, SEQ ID NO: 13; and (d) comprises FR-H1, FR-H2, FR-H3, and FR-H4, wherein the FR-H1 exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to an amino acid sequence of SEQ ID NO: 14, wherein the FR-H2 exhibits exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO: 18, wherein the FR-H3 exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO:20, and wherein the FR-H4 exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO: 22 or 23.


In another embodiment, a polypeptide of any of the subject composition embodiments described herein comprises an AF1, wherein the AF1 comprises CDR-L, CDR-H, light chain framework regions (FR-L) and heavy chain framework regions (FR-H) and wherein the AF1 (a) is configured to specifically bind to EGFR; (b) comprises CDR-H1, CDR-H2, and CDR-H3, having amino acid sequences of SEQ ID NOS: 4, 5, and 6, respectively; (c) comprises FR-L1, FR-L2, FR-L3, and FR-L4, each exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to an amino acid of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NOS: 9-11, and SEQ ID NO: 13, respectively; and (d) comprises FR-H1, FR-H2, FR-H3, and FR-H4, wherein the FR-H1 exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to a FR-H1 having an amino acid sequence of SEQ ID NO: 15, wherein the FR-H2 exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO: 19, wherein the FR-H3 exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO: 21, and wherein the FR-H4 exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO: 24.


In yet another embodiment, a polypeptide of any of the subject composition embodiments described herein comprises an AF1, wherein the AF1 comprises CDR-L, CDR-H, light chain framework regions (FR-L) and heavy chain framework regions (FR-H) and wherein the AF1 (a) is configured to specifically bind to EGFR; (b) comprises CDR-H1, CDR-H2, and CDR-H3, having amino acid sequences of SEQ ID NOS: 4, 5, and 6, respectively; (c) comprises FR-L1, FR-L2, FR-L3, and FR-L4, each exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to an amino acid of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NOS: 9-11, and SEQ ID NO: 13; and (d) comprises FR-H1, FR-H2, FR-H3, and FR-H4, wherein the FR-H1 exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to a FR-H1 having an amino acid sequence of SEQ ID NO: 16, wherein the FR-H2 exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO: 19, wherein the FR-H1 exhibits FR-H3 at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO: 20, and wherein the FR-H4 exhibits at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO: 22 or 23.


In another embodiment, a polypeptide of any of the subject composition embodiments described herein comprises an AF1, wherein the AF1 is configured to specifically bind to EGFR and the AF1 comprises a variable heavy (VH) amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to an amino acid sequence of SEQ ID NO: 28-32.


In another embodiment, a polypeptide of any of the subject composition embodiments described herein comprises an AF1, wherein the AF1 is configured to specifically bind to EGFR wherein the AF1 comprises a variable light (VL) amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to an amino acid sequence of SEQ ID NOS: 25-27.


In another embodiment, a polypeptides of any of the subject composition embodiments described herein comprises an AF1, wherein the AF1 comprises a variable heavy (VH) amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to an amino acid sequence of SEQ ID NO: 28-32 and comprises a variable light (VL) amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to an amino acid sequence of SEQ ID NOS: 25-27. The AF1 can be configured in a VL-VH or VH-VL orientation, and are fused by a linker peptide.


In yet another embodiment, a polypeptide of any of the subject composition embodiments described herein comprises an AF1, wherein the AF1 comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% sequence identity or is identical to an amino acid sequence of any one of SEQ ID NOS: 37-51.


It will be understood that use of the term antigen binding fragments for the composition embodiments disclosed herein is not limiting and is intended to include portions or fragments of antibodies that retain the ability to bind the antigens that are the ligands of the corresponding intact antibody. In such embodiments, the antigen binding fragment can be, but is not limited to, CDRs and intervening framework regions, variable or hypervariable regions of light and/or heavy chains of an antibody (VL, VH), variable fragments (Fv), Fab′ fragments, F(ab′)2 fragments, Fab fragments, single chain antibodies (scAb), VHH camelid antibodies, single chain variable fragment (scFv), linear antibodies, a single domain antibody, complementarity determining regions (CDR), domain antibodies (dAbs), single domain heavy chain immunoglobulins of the BHH or BNAR type, single domain light chain immunoglobulins, or other polypeptides known in the art containing a fragment of an antibody capable of binding an antigen. The VL and VH of two antigen binding fragments can also be configured in a single chain diabody configuration; i.e., the VL and VH of the AF1 and AF2 configured with linkers of an appropriate length to permit arrangement as a diabody.


In certain 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 one embodiment, the VL and VH of any of the scFv embodiments described herein are linked by relatively long linkers of hydrophilic amino acids selected from the sequences









GSGEGSEGEGGGEGSEGEGSGEGGEGEGSG (SEQ ID NO: 790),













TGSGEGSEGEGGGEGSEGEGSGEGGEGEGSGT (SEQ ID NO: 791),













GATPPETGAETESPGETTGGSAESEPPGEG (SEQ ID NO: 792), o


r













GSAAPTAGTTPSASPAPPTGGSSAAGSPST (SEQ ID NO: 793).






In yet another embodiment, AF1 of any of the subject composition embodiments described herein specifically binds human or cynomolgus monkey (cyno) EGFR. In another embodiment, AF1 of any of the subject composition embodiments described herein specifically binds human and cynomolgus monkey (cyno) EGFR.


In another aspect, the disclosure provides AF1 with specific binding affinity to EGFR for incorporation into the subject compositions in which one or more individual amino acids of the framework regions were modified to increase the pI of the AF1 relative to the parental antigen binding fragment in order to enhance the stability of the bispecific polypeptide into which it is incorporated. In one embodiment, the polypeptides of any of the subject composition embodiments described herein comprise an AF1, wherein the AF1 exhibits a pI of about 5.4, or about 5.5, or about 5.6, or about 5.6, or about 5.7, or about 5.8, or about 5.9, or about 6.0, or about 6.1, or about 6.2, or about 6.3, or about 6.4 or about 6.5, or about 6.6, as evidenced by in an in vitro assay. In another embodiment, the polypeptides of any of the subject composition embodiments described herein comprise an AF1, wherein the AF1 exhibits a pI of between 5.4 and 6.6, inclusive, as evidenced by in an in vitro assay. In another embodiment, the polypeptides of any of the subject composition embodiments described herein comprise an AF1, wherein the AF1 exhibits a pI of between about 5.4 and 6.6, or about 5.6 and about 6.4, or about 5.8 and about 6.2, or about 6.0 and about 6.2, or about 6.1 and about 6.3, or about 6.2 and about 6.4, or about 6.3 and about 6.5, or about 6.4 and about 6.6, as determined computationally or evidenced by an in vitro assay.


In another aspect, the disclosure provides AF1 with specific binding affinity to EGFR for incorporation into the subject compositions in which the binding affinity to the EGFR antigen is within a set range. In one embodiment, the polypeptides of any of the subject composition embodiments described herein comprise an AF1, wherein the AF1 specifically binds EGFR with a Kd between about 0.1 nM and about 100 nM, as determined in an in vitro antigen-binding assay comprising the EGFR antigen. In another embodiment, the AF1 specifically binds EGFR with a binding affinity (as determined by the Kd in an in vitro binding assay) of less than about 0.1 nM, or less than about 0.5 nM, or less than about 1.0 nM, or less than about 10 nM, or less than about 50 nM, or less than about 100 nM. In another embodiment, the polypeptides of any of the subject composition embodiments described herein comprise an AF1 and an AF2, wherein the binding affinity of the AF1 to EGFR is at least 10-fold greater, or at least 100-fold greater, or at least 1000-fold greater than the binding affinity of the AF2 to CD3, as measured in an in vitro antigen-binding assay. It will be understood that a binding affinity with a lower Kd value; e.g., 1 nM, is a greater binding affinity than 10 nM. The binding affinity of the subject compositions for the target ligands can be assayed 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. 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 another aspect, the disclosure provides AF1 with specific binding affinity to EGFR for incorporation into the subject compositions in which one or more individual amino acids of the framework regions were modified to decrease the hydrophobicity of the antigen-binding framework relative to the parental antigen binding fragment in order to enhance the stability of the bispecific polypeptide into which it is incorporated. In one embodiment, the polypeptides of any of the subject composition embodiments described herein comprise an AF1 wherein the AF1 specifically binds EGFR and wherein the AF1 has at least one amino acid substitution of a hydrophobic amino acid in a framework region, relative to the amino acid sequence of SEQ ID NO:52, wherein the hydrophobic amino acid is selected from isoleucine, leucine or methionine and the substituted amino acid is selected from arginine, threonine, or glutamine. In another embodiment, the AF1 has at least two amino acid substitutions of hydrophobic amino acids in one or more framework regions, relative to the amino acid sequence of SEQ ID NO:52, wherein the hydrophobic amino acids are selected from isoleucine, leucine or methionine and the substituted amino acids are selected from arginine, threonine, or glutamine.





TABLE 1







EGFR CDR SEQUENCES


Construct
CDR REGION
Amino Acid Sequence
SEQ ID NO:




EGFR.2, EGFR.13, EGFR.14, EGFR.15, EGFR.16, EGFR.17, EGFR.18, EGFR.19, EGFR.20, EGFR.21, EGFR.22, EGFR.23, EGFR.24, EGFR.25, EGFR.26, EGFR.27
CDR-L1
QASQDISNYLN
1


EGFR.2, EGFR.13, EGFR. 14, EGFR.15, EGFR.16, EGFR.17, EGFR.18, EGFR.19, EGFR.20, EGFR.21, EGFR.22, EGFR.23, EGFR.24, EGFR.25, EGFR.26, EGFR.27
CDR-L2
DASNLET
2


EGFR.2, EGFR.13, EGFR. 14, EGFR.15, EGFR.16, EGFR.17, EGFR.18, EGFR.19, EGFR.20, EGFR.21, EGFR.22, EGFR.23, EGFR.24, EGFR.25, EGFR.26, EGFR.27
CDR-L3
QHFDHLPLA
3


EGFR.2, EGFR.13, EGFR. 14, EGFR.15, EGFR.16, EGFR.17, EGFR.18, EGFR.19, EGFR.20, EGFR.21, EGFR.22, EGFR.23,
CDR-H1
GGSVSSGDYY
4


EGFR.24, EGFR.25, EGFR.26, EGFR.27





EGFR.2, EGFR.13, EGFR. 14, EGFR.15, EGFR.16, EGFR.17, EGFR.18, EGFR.19, EGFR.20, EGFR.21, EGFR.22, EGFR.23, EGFR.24, EGFR.25, EGFR.26, EGFR.27
CDR-H2
HIYYSGNTNYNPSLKS
5


EGFR.2, EGFR.13, EGFR. 14, EGFR.15, EGFR.16, EGFR.17, EGFR.18, EGFR.19, EGFR.20, EGFR.21, EGFR.22, EGFR.23, EGFR.24, EGFR.25, EGFR.26, EGFR.27
CDR-H3
VRDRVTGAFDI
6









TABLE 2







EGFR FR SEQUENCES


Construct
FR REGION
Amino Acid Sequence
SEQ ID NO:




EGFR.2, EGFR.13, EGFR.14, EGFR.15, EGFR.16, EGFR.17, EGFR.18, EGFR.19, EGFR20, EGFR.21, EGFR.22, EGFR.23, EGFR.24, EGFR.25, EGFR.26, EGFR.27
FR-L1
DIQMTQSPSSLSASVGDRVTITC
7


EGFR.2, EGFR.13, EGFR.14, EGFR.15, EGFR.16, EGFR.17, EGFR.18, EGFR.19, EGFR20, EGFR.21, EGFR.22, EGFR.23, EGFR.24, EGFR.25, EGFR.26, EGFR.27
FR-L2
WYQQKPGKAPKLLIY
8


EGFR.13, EGFR.14, EGFR.15, EGFR.16, EGFR.17,
FR-L3
GVPSRFSGSGSGTDFTFTISSLQPEDTATYFC
9


EGFR.18, EGFR.19, EGFR.20, EGFR.21, EGFR.22
FR-L3
GVPSRFSGSGSGTDFTFTISRLQPEDIATY FC
10


EGFR.23, EGFR.24, EGFR.25, EGFR.26, EGFR.27
FR-L3
GVPSRFSGSGSGTDFTFTISRLQPEDTAT YFC
11


EGFR.2
FR-L3
GVPSRFSGSGSGTDFTFTISSLQPEDIATY FC
12


EGFR.2, EGFR.13, EGFR.14, EGFR.15, EGFR.16, EGFR.17, EGFR.18, EGFR.19, EGFR20, EGFR.21, EGFR.22, EGFR.23, EGFR.24, EGFR.25, EGFR.26, EGFR.27
FR-L4
FGGGTKVEIK
13


EGFR.13, EGFR.14, EGFR.18, EGFR.19, EGFR.23, EGFR.24
FR-H 1
QVQLQESG PGAVKPS ETLS LTCTVS
14


EGFR.15, EGFR20, EGFR.25
FR-H 1
QVQLQESG PG L VKPSQTLSL TCTVS
15


EGFR.16, EGFR.17, EGFR.21, EGFR.22, EGFR.26, EGFR.27
FR-H 1
QVQLQESG PGA VKPSQTLSL TCTVS
16


EGFR.2
FR-H 1
QVQLQESG PG LVKPS ETLS LTCTVS
17


EGFR.2, EGFR.13, EGFR.14, EGFR.18, EGFR.19, EGFR.23, EGFR.24
FR-H2
WTWIRQSPGKGLEWIG
18


EGFR.15, EGFR.16, EGFR.17, EGFR.20, EGRF.21, EGFR.22, EGFR.25, EGFR.26, EGFR.27
FR-H2
WTWIRQRPGKGLEWIG
19


EGFR.13, EGFR. 14, EGFR.16, EGFR.17, EGFR.18, EGFR.19, EGFR.21, EGFR.22, EGFR.23,
FR-H3
R LTI S I DTS KTQFS L K LSSVTAADTATYYC
20


EGFR.24, EGFR.26, EGFR.27





EGFR.2, EGFR.15, EGFR.20, EGFR25
FR-H3
RLTISIDTSKTQFSLKLSSVTAADTAIYYC
21


EGFR.13, EGFR.16, EGFR.18, EGFR.21, EGFR.23, EGFR.26
FR-H4
WGQGTAVTVSS
22


EGFR.14, EGFR.17, EGFR.19, EGFR.22, EGFR.24, EGFR.27
FR-H4
WGQGTTVTVSS
23


EGFR.2, EGFR.15, EGFR.20, EGFR25
FR-H4
WGQGTMVTVSS
24









TABLE 3







EGFR VL & VH SEQUENCES


Construct
REGION
Amino Acid Sequence
SEQ ID NO:




EGFR.13, EGFR. 14, EGFR.15, EGFR.16, EGFR.17
VL
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWY QQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFT ISSLQPEDTATYFCQHFDHLPLAFGGGTKVEIK
25


EGFR.18, EGFR.19, EGRF.20, EGFR.21, EGFR.22
VL
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWY QQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFT ISRLQPEDIATYFCQHFDHLPLAFGGGTKVEIK
26


EGFR.23, EGFR.24, EGFR.25, EGFR.26, EGFR.27
VL
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWY QQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFT ISRLQPEDTATYFCQHFDHLPLAFGGGTKVEIK
27


EGFR.13, EGFR.18, EGFR.23
VH
QVQLQESGPGAVKPSETLSLTCTVSGGSVSSGDYYW TWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISID TSKTQFSLKLSSVTAADTATYYCVRDRVTGAFDIWGQ GTAVTVSS
28


EGFR.14, EGFR.19, EGFR.24
VH
QVQLQESGPGAVKPSETLSLTCTVSGGSVSSGDYYW TWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISID TSKTQFSLKLSSVTAADTATYYCVRDRVTGAFDIWGQ GTTVTVSS
29


EGFR.15, EGFR.20, EGFR.25
VH
QVQLQESGPGLVKPSQTLSLTCTVSGGSVSSGDYYW TWIRQRPGKGLEWIGHIYYSGNTNYNPSLKSRLTISID TSKTQFSLKLSSVTAADTAIYYCVRDRVTGAFDIWGQ GTMVTVSS
30


EGFR.16, EGFR.21, EGFR.26
VH
QVQLQESGPGAVKPSQTLSLTCTVSGGSVSSGDYYW TWIRQRPGKGLEWIGHIYYSGNTNYNPSLKSRLTISID TSKTQFSLKLSSVTAADTATYYCVRDRVTGAFDIWGQ GTAVTVSS
31


EGFR.17, EGFR.22, EGFR.27
VH
QVQLQESGPGAVKPSQTLSLTCTVSGGSVSSGDYYW TWIRQRPGKGLEWIGHIYYSGNTNYNPSLKSRLTISID TSKTQFSLKLSSVTAADTATYYCVRDRVTGAFDIWGQ GTTVTVSS
32


EGFR.2
VL
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWY QQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFT ISSLQPEDIATYFCQHFDHLPLAFGGGTKVEIK
35


EGFR.2
VH
QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWT WIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDT SKTQFSLKLSSVTAADTAIYYCVRDRVTGAFDIWGQG TMVTVSS
36









TABLE 4






EGFR scFv sequences


Construct
Amino Acid Sequence
SEQ ID NO:




EGFR.13
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLET GVPSRFSGSGSGTDFTFTISSLQPEDTATYFCOHFDHLPLAFGGGTKVEIKGATPPET GAETESPGETTGGSAESEPPGEGQVQLQESGPGAVKPSETLSLTCTVSGGSVSSGD YYWTWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAAD TATYYCVRDRVTGAFDIWGQGTAVTVSS
37


EGFR.14
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLET GVPSRFSGSGSGTDFTFTISSLQPEDTATYFCOHFDHLPLAFGGGTKVEIKGATPPET GAETESPGETTGGSAESEPPGEGQVQLQESGPGAVKPSETLSLTCTVSGGSVSSGD YYWTWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAAD TATYYCVRDRVTGAFDIWGQGTTVTVSS
38


EGFR.15
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLET GVPSRFSGSGSGTDFTFTISSLQPEDTATYFCOHFDHLPLAFGGGTKVEIKGATPPET GAETESPGETTGGSAESEPPGEGQVQLQESGPGLVKPSQTLSLTCTVSGGSVSSGD YYWTWIRQRPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAAD TAIYYCVRDRVTGAFDIWGQGTMVTVSS
39


EGFR.16
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLET GVPSRFSGSGSGTDFTFTISSLQPEDTATYFCOHFDHLPLAFGGGTKVEIKGATPPET GAETESPGETTGGSAESEPPGEGQVQLQESGPGAVKPSQTLSLTCTVSGGSVSSGD YYWTWIRQRPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAAD TATYYCVRDRVTGAFDIWGQGTAVTVSS
40


EGFR.17
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLET GVPSRFSGSGSGTDFTFTISSLQPEDTATYFCOHFDHLPLAFGGGTKVEIKGATPPET GAETESPGETTGGSAESEPPGEGQVQLQESGPGAVKPSQTLSLTCTVSGGSVSSGD YYWTWIRQRPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAAD TATYYCVRDRVTGAFDIWGQGTTVTVSS
41


EGFR.18
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLET GVPSRFSGSGSGTDFTFTISRLQPEDIATYFCQHFDHLPLAFGGGTKVEIKGATPPET GAETESPGETTGGSAESEPPGEGQVQLQESGPGAVKPSETLSLTCTVSGGSVSSGD YYWTWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAAD TATYYCVRDRVTGAFDIWGQGTAVTVSS
42


EGFR.19
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLET GVPSRFSGSGSGTDFTFTISRLQPEDIATYFCQHFDHLPLAFGGGTKVEIKGATPPET GAETESPGETTGGSAESEPPGEGQVQLQESGPGAVKPSETLSLTCTVSGGSVSSGD YYWTWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAAD TATYYCVRDRVTGAFDIWGQGTTVTVSS
43


EGFR.20
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLET GVPSRFSGSGSGTDFTFTISRLQPEDIATYFCQHFDHLPLAFGGGTKVEIKGATPPET GAETESPGETTGGSAESEPPGEGQVQLQESGPGLVKPSQTLSLTCTVSGGSVSSGD YYWTWIRQRPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAAD TAIYYCVRDRVTGAFDIWGQGTMVTVSS
44


EGFR.21
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLET GVPSRFSGSGSGTDFTFTISRLQPEDIATYFCQHFDHLPLAFGGGTKVEIKGATPPET GAETESPGETTGGSAESEPPGEGQVQLQESGPGAVKPSQTLSLTCTVSGGSVSSGD YYWTWIRQRPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAAD TATYYCVRDRVTGAFDIWGQGTAVTVSS
45


EGFR.22
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLET GVPSRFSGSGSGTDFTFTISRLQPEDIATYFCQHFDHLPLAFGGGTKVEIKGATPPET GAETESPGETTGGSAESEPPGEGQVQLQESGPGAVKPSQTLSLTCTVSGGSVSSGD YYWTWIRQRPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAAD TATYYCVRDRVTGAFDIWGQGTTVTVSS
46


EGFR.23
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLET GVPSRFSGSGSGTDFTFTISRLQPEDTATYFCQHFDHLPLAFGGGTKVEIKGATPPE TGAETESPGETTGGSAESEPPGEGQVQLQESGPGAVKPSETLSLTCTVSGGSVSSG DYYWTWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVRAA DTATYYCVRDRVTGAFDIWGQGTAVTVSS
47


EGFR.24
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLET GVPSRFSGSGSGTDFTFTISRLQPEDTATYFCQHFDHLPLAFGGGTKVEIKGATPPE TGAETESPGETTGGSAESEPPGEGQVQLQESGPGAVKPSETLSLTCTVSGGSVSSG DYYWTWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVRAA DTATYYCVRDRVTGAFDIWGQGTTVTVSS
48


EGFR.25
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLET GVPSRFSGSGSGTDFTFTISRLQPEDTATYFCQHFDHLPLAFGGGTKVEIKGATPPE TGAETESPGETTGGSAESEPPGEGQVQLQESGPGLVKPSQTLSLTCTVSGGSVSSG DYYWTWIRQRPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAA DTAIYYCVRDRVTGAFDIWGQGTMVTVSS
49


EGFR.26
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLET GVPSRFSGSGSGTDFTFTISRLQPEDTATYFCQHFDHLPLAFGGGTKVEIKGATPPE TGAETESPGETTGGSAESEPPGEGQVQLQESGPGAVKPSQTLSLTCTVSGGSVSSG DYYWTWIRQRPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAA DTATYYCVRDRVTGAFDIWGQGTAVTVSS
50


EGFR.27
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLET GVPSRFSGSGSGTDFTFTISRLQPEDTATYFCQHFDHLPLAFGGGTKVEIKGATPPE TGAETESPGETTGGSAESEPPGEGQVQLQESGPGAVKPSQTLSLTCTVSGGSVSSG DYYWTWIRQRPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAA DTATYYCVRDRVTGAFDIWGQGTTVTVSS
51


EGFR.2
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLET GVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQHFDHLPLAFGGGTKVEIKGATPPET GAETESPGETTGGSAESEPPGEGQVQLQESGPGLVKPSETLSLTCTVSGGSVSSGD YYWTWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQFSLKLSSVTAAD TAIYYCVRDRVTGAFDIWGQGTMVTVSS
52






III). Release Segments

In another aspect, the disclosure relates to release segment (RS) peptides suitable for inclusion in the subject compositions described herein 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. The RS are useful for, amongst other things, conferring a prodrug format on the subject compositions that can be activated by the cleavage of the RS by mammalian proteases. As described herein, the RS are incorporated into the subject composition embodiments described herein, linking the incorporated antigen binding fragment to the XTEN (the configurations of which are described more fully, below) such that upon cleavage of the RS by action of the one or more proteases for which the RS are substrates, the antigen binding fragments and XTEN are released from the composition and the antigen binding fragments, no longer shielded by the XTEN, increase their binding potential to their respective ligands. In a particular feature, the RS serve as substrates for proteases found in close association with or are co-localized with disease tissues or cells, such as but not limited to tumors, cancer cells, and inflammatory tissues, and upon cleavage of the RS, the antigen binding fragments that are otherwise shielded by the XTEN of the subject compositions (and thus have a lower binding affinity for their respective ligands) are released from the composition and regain their increased potential to bind the target and/or effector cell ligands. In another embodiment, the RS of the subject polypeptide compositions comprise an amino acid sequence that is a substrate for a cellular protease located within a targeted cell. In another particular feature of the subject compositions described herein, the RS that are substrates for two or three classes of proteases were designed with sequences that are capable of being cleaved in different locations of the RS sequence by the different proteases, with a representative example depicted in FIG. 6. Thus, the RS that are substrates for two, three, or more classes of proteases have two, three, or a plurality of distinct cleavage sites in the RS sequence, but cleavage by a single protease nevertheless results in the release of the antigen binding fragments and the XTEN from the composition comprising the RS.


In one embodiment, the disclosure provides an activatable polypeptide comprising one or more release segments wherein the release segment is a substrate for cleavage by one or more mammalian proteases. In another embodiment, the present disclosure provides a polypeptide comprising a first release segment (RS1) sequence wherein the RS1 is a substrate for cleavage by a mammalian protease wherein the RS1 is a substrate for a protease selected from the group consisting of legumain, MMP-2, MMP-7, MMP-9, MMP-11, MMP-14, uPA, and matriptase. In other cases, the polypeptides of any of the subject composition embodiments described herein comprise a first release segment (RS1) sequence wherein the RS1 is a substrate for cleavage by one or more mammalian proteases selected from the group consisting of 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 another embodiment, the present disclosure provides polypeptides comprising a first release segment (RS1) sequence for incorporation into the subject polypeptide compositions described herein wherein the RS1 is a substrate for cleavage by one or more mammalian proteases wherein the RS1 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOS:53-671. In another embodiment, the RS1 comprises an amino acid sequence selected from the sequences of RSR-2089, RSR-2295, RSR-2298, RSR-2488, RSR-2599, RSR-2485, RSR-2486, RSR-2728, RSN-2089, RSN-2295, RSN-2298, RSN-2488, RSN-2599, RSN-2485, RSN-2486, RSN-2728, RSC-2089, RSC-2295, RSC-2298, RSC-2488, RSC-2599, RSC-2485, RSC-2486, and RSC-2728, each of which being forth in Table 5. As described more fully in descriptions of the configurations and properties of the subject polypeptide compositions, below, the release segment is fused between the antigen binding fragment and an XTEN polypeptide such that upon cleavage of the release segment, the XTEN is released from the composition.


In other embodiments, the disclosure provides polypeptides comprising a first release segment (RS1) sequence and a second release segment (RS2) for incorporation into the subject polypeptide compositions described herein wherein the RS1 and the RS2 are identical. In another embodiment, the present disclosure provides polypeptides comprising a first release segment (RS1) sequence and a second release segment (RS2) for incorporation into the subject polypeptide compositions wherein the RS1 and the RS2 are different. In some cases of the foregoing embodiments, theRS1 and the RS2 are each a substrate for cleavage by a mammalian protease selected from the group consisting of legumain, MMP-2, MMP-7, MMP-9, MMP-11, MMP-14, uPA, and matriptase. In another embodiment, the disclosure provides polypeptides comprising an RS1 and an RS2 sequence for incorporation into the subject polypeptide compositions described herein wherein the RS1 and RS2 are each a substrate for cleavage by one or more mammalian proteases wherein the RS1 and RS2 each comprise an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOS:53-671. In another embodiment, the RS1 and RS2 each comprise an amino acid sequence selected from the sequences of RSR-2089, RSR-2295, RSR-2298, RSR-2488, RSR-2599, RSR-2485, RSR-2486, RSR-2728, RSN-2089, RSN-2295, RSN-2298, RSN-2488, RSN-2599, RSN-2485, RSN-2486, RSN-2728, RSC-2089, RSC-2295, RSC-2298, RSC-2488, RSC-2599, RSC-2485, RSC-2486, and RSC-2728, each of which being set forth in Table 5. As described more fully in paragraphs related to the descriptions of the configurations and properties of the subject polypeptide compositions, below, the release segments are fused between the antigen binding fragment and an XTEN polypeptide such that upon cleavage of each release segment, the adjoining XTEN is released from the composition.





TABLE 5






Release Segments and Amino Acid Sequences


Name
Amino Acid Sequence
SEQ ID NO:




RSR-1517
EAGRSANHEPLGLVAT
53


BSRS-A1
ASGRSTNAGPSGLAGP
54


BSRS-A2
ASGRSTNAGPQGLAGQ
55


BSRS-A3
ASGRSTNAGPPGLTGP
56


VP-1
ASSRGTNAGPAGLTGP
57


RSR-1752
ASSRTTNTGPSTLTGP
58


RSR-1512
AAGRSDNGTPLELVAP
59


RSR-1517
EAGRSANHEPLGLVAT
53


VP-2
ASGRGTNAGPAGLTGP
60


RSR-1018
LFGRNDNHEPLELGGG
61


RSR-1053
TAGRSDNLEPLGLVFG
62


RSR-1059
LDGRSDNFHPPELVAG
63


RSR-1065
LEGRSDNEEPENLVAG
64


RSR-1167
LKGRSDNNAPLALVAG
65


RSR-1201
VYSRGTNAGPHGLTGR
66


RSR-1218
ANSRGTNKGFAGLIGP
67


RSR-1226
ASSRLTNEAPAGLTIP
68


RSR-1254
DQSRGTNAGPEGLTDP
69


RSR-1256
ESSRGTNIGQGGLTGP
70


RSR-1261
SSSRGTNQDPAGLTIP
71


RSR-1293
ASSRGQNHSPMGLTGP
72


RSR-1309
AYSRGPNAGPAGLEGR
73


RSR-1326
ASERGNNAGPANLTGF
74


RSR-1345
ASHRGTNPKPAILTGP
75


RSR-1354
MSSRRTNANPAQLTGP
76


RSR-1426
GAGRTDNHEPLELGAA
77


RSR-1478
LAGRSENTAPLELTAG
78


RSR-1479
LEGRPDNHEPLALVAS
79


RSR-1496
LSGRSDNEEPLALPAG
80


RSR-1508
EAGRTDNHEPLELSAP
81


RSR-1513
EGGRSDNHGPLELVSG
82


RSR-1516
LSGRSDNEAPLELEAG
83


RSR-1524
LGGRADNHEPPELGAG
84


RSR-1622
PPSRGTNAEPAGLTGE
85


RSR-1629
ASTRGENAGPAGLEAP
86


RSR-1664
ESSRGTNGAPEGLTGP
87


RSR-1667
ASSRATNESPAGLTGE
88


RSR-1709
ASSRGENPPPGGLTGP
89


RSR-1712
AASRGTNTGPAELTGS
90


RSR-1727
AGSRTTNAGPGGLEGP
91


RSR-1754
APSRGENAGPATLTGA
92


RSR-1819
ESGRAANTGPPTLTAP
93


RSR-1832
NPGRAANEGPPGLPGS
94


RSR-1855
ESSRAANLTPPELTGP
95


RSR-1911
ASGRAANETPPGLTGA
96


RSR-1929
NSGRGENLGAPGLTGT
97


RSR-1951
TTGRAANLTPAGLTGP
98


RSR-2295
EAGRSANHTPAGLTGP
99


RSR-2298
ESGRAANTTPAGLTGP
100


RSR-2038
TTGRATEAANLTPAGLTGP
101


RSR-2072
TTGRAEEAANLTPAGLTGP
102


RSR-2089
TTGRAGEAANLTPAGLTGP
103


RSR-2302
TTGRATEAANATPAGLTGP
104


RSR-3047
TTGRAGEAEGATSAGATGP
105


RSR-3052
TTGEAGEAANATSAGATGP
106


RSR-3043
TTGEAGEAAGLTPAGLTGP
107


RSR-3041
TTGAAGEAANATPAGLTGP
108


RSR-3044
TTGRAGEAAGLTPAGLTGP
109


RSR-3057
TTGRAGEAANATSAGATGP
110


RSR-3058
TTGEAGEAAGATSAGATGP
111


RSR-2485
ESGRAANTEPPELGAG
112


RSR-2486
ESGRAANTAPEGLTGP
113


RSR-2488
EPGRAANHEPSGLTEG
114


RSR-2599
ESGRAANHTGAPPGGLTGP
115


RSR-2706
TTGRTGEGANATPGGLTGP
116


RSR-2707
RTGRSGEAANETPEGLEGP
117


RSR-2708
RTGRTGESANETPAGLGGP
118


RSR-2709
STGRTGEPANETPAGLSGP
119


RSR-2710
TTGRAGEPANATPTGLSGP
120


RSR-2711
RTGRPGEGANATPTGLPGP
121


RSR-2712
RTGRGGEAANATPSGLGGP
122


RSR-2713
STGRSGESANATPGGLGGP
123


RSR-2714
RTGRTGEEANATPAGLPGP
124


RSR-2715
ATGRPGEPANTTPEGLEGP
125


RSR-2716
STGRSGEPANATPGGLTGP
126


RSR-2717
PTGRGGEGANTTPTGLPGP
127


RSR-2718
PTGRSGEGANATPSGLTGP
128


RSR-2719
TTGRASEGANSTPAPLTEP
129


RSR-2720
TYGRAAEAANTTPAGLTAP
130


RSR-2721
TTGRATEGANATPAELTEP
131


RSR-2722
TVGRASEEANTTPASLTGP
132


RSR-2723
TTGRAPEAANATPAPLTGP
133


RSR-2724
TWGRATEPANATPAPLTSP
134


RSR-2725
TVGRASESANATPAELTSP
135


RSR-2726
TVGRAPEGANSTPAGLTGP
136


RSR-2727
TWGRATEAPNLEPATLTTP
137


RSR-2728
TTGRATEAPNLTPAPLTEP
138


RSR-2729
TQGRATEAPNLSPAALTSP
139


RSR-2730
TQGRAAEAPNLTPATLTAP
140


RSR-2731
TSGRAPEATNLAPAPLTGP
141


RSR-2732
TQGRAAEAANLTPAGLTEP
142


RSR-2733
TTGRAGSAPNLPPTGLTTP
143


RSR-2734
TTGRAGGAENLPPEGLTAP
144


RSR-2735
TTSRAGTATNLTPEGLTAP
145


RSR-2736
TTGRAGTATNLPPSGLTTP
146


RSR-2737
TTARAGEAENLSPSGLTAP
147


RSR-2738
TTGRAGGAGNLAPGGLTEP
148


RSR-2739
TTGRAGTATNLPPEGLTGP
149


RSR-2740
TTGRAGGAANLAPTGLTEP
150


RSR-2741
TTGRAGTAENLAPSGLTTP
151


RSR-2742
TTGRAGSATNLGPGGLTGP
152


RSR-2743
TTARAGGAENLTPAGLTEP
153


RSR-2744
TTARAGSAENLSPSGLTGP
154


RSR-2745
TTARAGGAGNLAPEGLTTP
155


RSR-2746
TTSRAGAAENLTPTGLTGP
156


RSR-2747
TYGRTTTPGNEPPASLEAE
157


RSR-2748
TYSRGESGPNEPPPGLTGP
158


RSR-2749
AWGRTGASENETPAPLGGE
159


RSR-2750
RWGRAETTPNTPPEGLETE
160


RSR-2751
ESGRAANHTGAEPPELGAG
161


RSR-2754
TTGRAGEAANLTPAGLTES
162


RSR-2755
TTGRAGEAANLTPAALTES
163


RSR-2756
TTGRAGEAANLTPAPLTES
164


RSR-2757
TTGRAGEAANLTPEPLTES
165


RSR-2758
TTGRAGEAANLTPAGLTGA
166


RSR-2759
TTGRAGEAANLTPEGLTGA
167


RSR-2760
TTGRAGEAANLTPEPLTGA
168


RSR-2761
TTGRAGEAANLTPAGLTEA
169


RSR-2762
TTGRAGEAANLTPEGLTEA
170


RSR-2763
TTGRAGEAANLTPAPLTEA
171


RSR-2764
TTGRAGEAANLTPEPLTEA
172


RSR-2765
TTGRAGEAANLTPEPLTGP
173


RSR-2766
TTGRAGEAANLTPAGLTGG
174


RSR-2767
TTGRAGEAANLTPEGLTGG
175


RSR-2768
TTGRAGEAANLTPEALTGG
176


RSR-2769
TTGRAGEAANLTPEPLTGG
177


RSR-2770
TTGRAGEAANLTPAGLTEG
178


RSR-2771
TTGRAGEAANLTPEGLTEG
179


RSR-2772
TTGRAGEAANLTPAPLTEG
180


RSR-2773
TTGRAGEAANLTPEPLTEG
181


RSN-0001
GSAPGSAGGYAELRMGGAIATSGSETPGT
182


RSN-0002
GSAPGTGGGYAPLRMGGGAATSGSETPGT
183


RSN-0003
GSAPGAEGGYAALRMGGEIATSGSETPGT
184


RSN-0004
GSAPGGPGGYALLRMGGPAATSGSETPGT
185


RSN-0005
GSAPGEAGGYAFLRMGGSIATSGSETPGT
186


RSN-0006
GSAPGPGGGYASLRMGGTAATSGSETPGT
187


RSN-0007
GSAPGSEGGYATLRMGGAIATSGSETPGT
188


RSN-0008
GSAPGTPGGYANLRMGGGAATSGSETPGT
189


RSN-0009
GSAPGASGGYAHLRMGGEIATSGSETPGT
190


RSN-0010
GSAPGGTGGYGELRMGGPAATSGSETPGT
191


RSN-0011
GSAPGEAGGYPELRMGGSIATSGSETPGT
192


RSN-0012
GSAPGPGGGYVELRMGGTAATSGSETPGT
193


RSN-0013
GSAPGSEGGYLELRMGGAIATSGSETPGT
194


RSN-0014
GSAPGTPGGYSELRMGGGAATSGSETPGT
195


RSN-0015
GSAPGASGGYTELRMGGEIATSGSETPGT
196


RSN-0016
GSAPGGTGGYQELRMGGPAATSGSETPGT
197


RSN-0017
GSAPGEAGGYEELRMGGSIATSGSETPGT
198


RSN-0018
GSAPGPGIGPAELRMGGTAATSGSETPGT
199


RSN-0019
GSAPGSEIGAAELRMGGAIATSGSETPGT
200


RSN-0020
GSAPGTPIGSAELRMGGGAATSGSETPGT
201


RSN-0021
GSAPGASIGTAELRMGGEIATSGSETPGT
202


RSN-0022
GSAPGGTIGNAELRMGGPAATSGSETPGT
203


RSN-0023
GSAPGEAIGQAELRMGGSIATSGSETPGT
204


RSN-0024
GSAPGPGGPYAELRMGGTAATSGSETPGT
205


RSN-0025
GSAPGSEGAYAELRMGGAIATSGSETPGT
206


RSN-0026
GSAPGTPGVYAELRMGGGAATSGSETPGT
207


RSN-0027
GSAPGASGLYAELRMGGEIATSGSETPGT
208


RSN-0028
GSAPGGTGIYAELRMGGPAATSGSETPGT
209


RSN-0029
GSAPGEAGFYAELRMGGSIATSGSETPGT
210


RSN-0030
GSAPGPGGYYAELRMGGTAATSGSETPGT
211


RSN-0031
GSAPGSEGSYAELRMGGAIATSGSETPGT
212


RSN-0032
GSAPGTPGNYAELRMGGGAATSGSETPGT
213


RSN-0033
GSAPGASGEYAELRMGGEIATSGSETPGT
214


RSN-0034
GSAPGGTGHYAELRMGGPAATSGSETPGT
215


RSN-0035
GSAPGEAGGYAEARMGGSIATSGSETPGT
216


RSN-0036
GSAPGPGGGYAEVRMGGTAATSGSETPGT
217


RSN-0037
GSAPGSEGGYAEIRMGGAIATSGSETPGT
218


RSN-0038
GSAPGTPGGYAEFRMGGGAATSGSETPGT
219


RSN-0039
GSAPGASGGYAEYRMGGEIATSGSETPGT
220


RSN-0040
GSAPGGTGGYAESRMGGPAATSGSETPGT
221


RSN-0041
GSAPGEAGGYAETRMGGSIATSGSETPGT
222


RSN-0042
GSAPGPGGGYAELAMGGTRATSGSETPGT
223


RSN-0043
GSAPGSEGGYAELVMGGARATSGSETPGT
224


RSN-0044
GSAPGTPGGYAELLMGGGRATSGSETPGT
225


RSN-0045
GSAPGASGGYAELIMGGERATSGSETPGT
226


RSN-0046
GSAPGGTGGYAELWMGGPRATSGSETPGT
227


RSN-0047
GSAPGEAGGYAELSMGGSRATSGSETPGT
228


RSN-0048
GSAPGPGGGYAELTMGGTRATSGSETPGT
229


RSN-0049
GSAPGSEGGYAELQMGGARATSGSETPGT
230


RSN-0050
GSAPGTPGGYAELNMGGGRATSGSETPGT
231


RSN-0051
GSAPGASGGYAELEMGGERATSGSETPGT
232


RSN-0052
GSAPGGTGGYAELRPGGPIATSGSETPGT
233


RSN-0053
GSAPGEAGGYAELRAGGSAATSGSETPGT
234


RSN-0054
GSAPGPGGGYAELRLGGTIATSGSETPGT
235


RSN-0055
GSAPGSEGGYAELRIGGAAATSGSETPGT
236


RSN-0056
GSAPGTPGGYAELRSGGGIATSGSETPGT
237


RSN-0057
GSAPGASGGYAELRNGGEAATSGSETPGT
238


RSN-0058
GSAPGGTGGYAELRQGGPIATSGSETPGT
239


RSN-0059
GSAPGEAGGYAELRDGGSAATSGSETPGT
240


RSN-0060
GSAPGPGGGYAELREGGTIATSGSETPGT
241


RSN-0061
GSAPGSEGGYAELRHGGAAATSGSETPGT
242


RSN-0062
GSAPGTPGGYAELRMPGGIATSGSETPGT
243


RSN-0063
GSAPGASGGYAELRMAGEAATSGSETPGT
244


RSN-0064
GSAPGGTGGYAELRMVGPIATSGSETPGT
245


RSN-0065
GSAPGEAGGYAELRMLGSAATSGSETPGT
246


RSN-0066
GSAPGPGGGYAELRMIGTIATSGSETPGT
247


RSN-0067
GSAPGSEGGYAELRMYGAIATSGSETPGT
248


RSN-0068
GSAPGTPGGYAELRMSGGAATSGSETPGT
249


RSN-0069
GSAPGASGGYAELRMNGEIATSGSETPGT
250


RSN-0070
GSAPGGTGGYAELRMQGPAATSGSETPGT
251


RSN-0071
GSAPGANHTPAGLTGPGARATSGSETPGT
252


RSN-0072
GSAPGANTAPEGLTGPSTRATSGSETPGT
253


RSN-0073
GSAPGTGAPPGGLTGPGTRATSGSETPGT
254


RSN-0074
GSAPGANHEPSGLTEGSPRATSGSETPGT
255


RSN-0075
GSAPGANTEPPELGAGTERATSGSETPGT
256


RSN-0076
GSAPGASGPPPGLTGPPGRATSGSETPGT
257


RSN-0077
GSAPGASGTPAPLGGEPGRATSGSETPGT
258


RSN-0078
GSAPGPAGPPEGLETEAGRATSGSETPGT
259


RSN-0079
GSAPGPTSGQGGLTGPESRATSGSETPGT
260


RSN-0080
GSAPGSAGGAANLVRGGAIATSGSETPGT
261


RSN-0081
GSAPGTGGGAAPLVRGGGAATSGSETPGT
262


RSN-0082
GSAPGAEGGAAALVRGGEIATSGSETPGT
263


RSN-0083
GSAPGGPGGAALLVRGGPAATSGSETPGT
264


RSN-0084
GSAPGEAGGAAFLVRGGSIATSGSETPGT
265


RSN-0085
GSAPGPGGGAASLVRGGTAATSGSETPGT
266


RSN-0086
GSAPGSEGGAATLVRGGAIATSGSETPGT
267


RSN-0087
GSAPGTPGGAAGLVRGGGAATSGSETPGT
268


RSN-0088
GSAPGASGGAADLVRGGEIATSGSETPGT
269


RSN-0089
GSAPGGTGGAGNLVRGGPAATSGSETPGT
270


RSN-0090
GSAPGEAGGAPNLVRGGSIATSGSETPGT
271


RSN-0091
GSAPGPGGGAVNLVRGGTAATSGSETPGT
272


RSN-0092
GSAPGSEGGALNLVRGGAIATSGSETPGT
273


RSN-0093
GSAPGTPGGASNLVRGGGAATSGSETPGT
274


RSN-0094
GSAPGASGGATNLVRGGEIATSGSETPGT
275


RSN-0095
GSAPGGTGGAQNLVRGGPAATSGSETPGT
276


RSN-0096
GSAPGEAGGAENLVRGGSIATSGSETPGT
277


RSN-1517
GSAPEAGRSANHEPLGLVATATSGSETPGT
278


BSRS-A1
GSAPASGRSTNAGPSGLAGPATSGSETPGT
279


BSRS-A2
GSAPASGRSTNAGPQGLAGQATSGSETPGT
280


BSRS-A3
GSAPASGRSTNAGPPGLTGPATSGSETPGT
281


VP-1
GSAPASSRGTNAGPAGLTGPATSGSETPGT
282


RSN-1752
GSAPASSRTTNTGPSTLTGPATSGSETPGT
283


RSN-1512
GSAPAAGRSDNGTPLELVAPATSGSETPGT
284


RSN-1517
GSAPEAGRSANHEPLGLVATATSGSETPGT
278


VP-2
GSAPASGRGTNAGPAGLTGPATSGSETPGT
285


RSN-1018
GSAPLFGRNDNHEPLELGGGATSGSETPGT
286


RSN-1053
GSAPTAGRSDNLEPLGLVFGATSGSETPGT
287


RSN-1059
GSAPLDGRSDNFHPPELVAGATSGSETPGT
288


RSN-1065
GSAPLEGRSDNEEPENLVAGATSGSETPGT
289


RSN-1167
GSAPLKGRSDNNAPLALVAGATSGSETPGT
290


RSN-1201
GSAPVYSRGTNAGPHGLTGRATSGSETPGT
291


RSN-1218
GSAPANSRGTNKGFAGLIGPATSGSETPGT
292


RSN-1226
GSAPASSRLTNEAPAGLTIPATSGSETPGT
293


RSN-1254
GSAPDQSRGTNAGPEGLTDPATSGSETPGT
294


RSN-1256
GSAPESSRGTNIGQGGLTGPATSGSETPGT
295


RSN-1261
GSAPSSSRGTNQDPAGLTIPATSGSETPGT
296


RSN-1293
GSAPASSRGQNHSPMGLTGPATSGSETPGT
297


RSN-1309
GSAPAYSRGPNAGPAGLEGRATSGSETPGT
298


RSN-1326
GSAPASERGNNAGPANLTGFATSGSETPGT
299


RSN-1345
GSAPASHRGTNPKPAILTGPATSGSETPGT
300


RSN-1354
GSAPMSSRRTNANPAQLTGPATSGSETPGT
301


RSN-1426
GSAPGAGRTDNHEPLELGAAATSGSETPGT
302


RSN-1478
GSAPLAGRSENTAPLELTAGATSGSETPGT
303


RSN-1479
GSAPLEGRPDNHEPLALVASATSGSETPGT
304


RSN-1496
GSAPLSGRSDNEEPLALPAGATSGSETPGT
305


RSN-1508
GSAPEAGRTDNHEPLELSAPATSGSETPGT
306


RSN-1513
GSAPEGGRSDNHGPLELVSGATSGSETPGT
307


RSN-1516
GSAPLSGRSDNEAPLELEAGATSGSETPGT
308


RSN-1524
GSAPLGGRADNHEPPELGAGATSGSETPGT
309


RSN-1622
GSAPPPSRGTNAEPAGLTGEATSGSETPGT
310


RSN-1629
GSAPASTRGENAGPAGLEAPATSGSETPGT
311


RSN-1664
GSAPESSRGTNGAPEGLTGPATSGSETPGT
312


RSN-1667
GSAPASSRATNESPAGLTGEATSGSETPGT
313


RSN-1709
GSAPASSRGENPPPGGLTGPATSGSETPGT
314


RSN-1712
GSAPAASRGTNTGPAELTGSATSGSETPGT
315


RSN-1727
GSAPAGSRTTNAGPGGLEGPATSGSETPGT
316


RSN-1754
GSAPAPSRGENAGPATLTGAATSGSETPGT
317


RSN-1819
GSAPESGRAANTGPPTLTAPATSGSETPGT
318


RSN-1832
GSAPNPGRAANEGPPGLPGSATSGSETPGT
319


RSN-1855
GSAPESSRAANLTPPELTGPATSGSETPGT
320


RSN-1911
GSAPASGRAANETPPGLTGAATSGSETPGT
321


RSN-1929
GSAPNSGRGENLGAPGLTGTATSGSETPGT
322


RSN-1951
GSAPTTGRAANLTPAGLTGPATSGSETPGT
323


RSN-2295
GSAPEAGRSANHTPAGLTGPATSGSETPGT
324


RSN-2298
GSAPESGRAANTTPAGLTGPATSGSETPGT
325


RSN-2038
GSAPTTGRATEAANLTPAGLTGPATSGSETPGT
326


RSN-2072
GSAPTTGRAEEAANLTPAGLTGPATSGSETPGT
327


RSN-2089
GSAPTTGRAGEAANLTPAGLTGPATSGSETPGT
328


RSN-2302
GSAPTTGRATEAANATPAGLTGPATSGSETPGT
329


RSN-3047
GSAPTTGRAGEAEGATSAGATGPATSGSETPGT
330


RSN-3052
GSAPTTGEAGEAANATSAGATGPATSGSETPGT
331


RSN-3043
GSAPTTGEAGEAAGLTPAGLTGPATSGSETPGT
332


RSN-3041
GSAPTTGAAGEAANATPAGLTGPATSGSETPGT
333


RSN-3044
GSAPTTGRAGEAAGLTPAGLTGPATSGSETPGT
334


RSN-3057
GSAPTTGRAGEAANATSAGATGPATSGSETPGT
335


RSN-3058
GSAPTTGEAGEAAGATSAGATGPATSGSETPGT
336


RSN-2485
GSAPESGRAANTEPPELGAGATSGSETPGT
337


RSN-2486
GSAPESGRAANTAPEGLTGPATSGSETPGT
338


RSN-2488
GSAPEPGRAANHEPSGLTEGATSGSETPGT
339


RSN-2599
GSAPESGRAANHTGAPPGGLTGPATSGSETPGT
340


RSN-2706
GSAPTTGRTGEGANATPGGLTGPATSGSETPGT
341


RSN-2707
GSAPRTGRSGEAANETPEGLEGPATSGSETPGT
342


RSN-2708
GSAPRTGRTGESANETPAGLGGPATSGSETPGT
343


RSN-2709
GSAPSTGRTGEPANETPAGLSGPATSGSETPGT
344


RSN-2710
GSAPTTGRAGEPANATPTGLSGPATSGSETPGT
345


RSN-2711
GSAPRTGRPGEGANATPTGLPGPATSGSETPGT
346


RSN-2712
GSAPRTGRGGEAANATPSGLGGPATSGSETPGT
347


RSN-2713
GSAPSTGRSGESANATPGGLGGPATSGSETPGT
348


RSN-2714
GSAPRTGRTGEEANATPAGLPGPATSGSETPGT
349


RSN-2715
GSAPATGRPGEPANTTPEGLEGPATSGSETPGT
350


RSN-2716
GSAPSTGRSGEPANATPGGLTGPATSGSETPGT
351


RSN-2717
GSAPPTGRGGEGANTTPTGLPGPATSGSETPGT
352


RSN-2718
GSAPPTGRSGEGANATPSGLTGPATSGSETPGT
353


RSN-2719
GSAPTTGRASEGANSTPAPLTEPATSGSETPGT
354


RSN-2720
GSAPTYGRAAEAANTTPAGLTAPATSGSETPGT
355


RSN-2721
GSAPTTGRATEGANATPAELTEPATSGSETPGT
356


RSN-2722
GSAPTVGRASEEANTTPASLTGPATSGSETPGT
357


RSN-2723
GSAPTTGRAPEAANATPAPLTGPATSGSETPGT
358


RSN-2724
GSAPTWGRATEPANATPAPLTSPATSGSETPGT
359


RSN-2725
GSAPTVGRASESANATPAELTSPATSGSETPGT
360


RSN-2726
GSAPTVGRAPEGANSTPAGLTGPATSGSETPGT
361


RSN-2727
GSAPTWGRATEAPNLEPATLTTPATSGSETPGT
362


RSN-2728
GSAPTTGRATEAPNLTPAPLTEPATSGSETPGT
363


RSN-2729
GSAPTQGRATEAPNLSPAALTSPATSGSETPGT
364


RSN-2730
GSAPTQGRAAEAPNLTPATLTAPATSGSETPGT
365


RSN-2731
GSAPTSGRAPEATNLAPAPLTGPATSGSETPGT
366


RSN-2732
GSAPTQGRAAEAANLTPAGLTEPATSGSETPGT
367


RSN-2733
GSAPTTGRAGSAPNLPPTGLTTPATSGSETPGT
368


RSN-2734
GSAPTTGRAGGAENLPPEGLTAPATSGSETPGT
369


RSN-2735
GSAPTTSRAGTATNLTPEGLTAPATSGSETPGT
370


RSN-2736
GSAPTTGRAGTATNLPPSGLTTPATSGSETPGT
371


RSN-2737
GSAPTTARAGEAENLSPSGLTAPATSGSETPGT
372


RSN-2738
GSAPTTGRAGGAGNLAPGGLTEPATSGSETPGT
373


RSN-2739
GSAPTTGRAGTATNLPPEGLTGPATSGSETPGT
374


RSN-2740
GSAPTTGRAGGAANLAPTGLTEPATSGSETPGT
375


RSN-2741
GSAPTTGRAGTAENLAPSGLTTPATSGSETPGT
376


RSN-2742
GSAPTTGRAGSATNLGPGGLTGPATSGSETPGT
377


RSN-2743
GSAPTTARAGGAENLTPAGLTEPATSGSETPGT
378


RSN-2744
GSAPTTARAGSAENLSPSGLTGPATSGSETPGT
379


RSN-2745
GSAPTTARAGGAGNLAPEGLTTPATSGSETPGT
380


RSN-2746
GSAPTTSRAGAAENLTPTGLTGPATSGSETPGT
381


RSN-2747
GSAPTYGRTTTPGNEPPASLEAEATSGSETPGT
382


RSN-2748
GSAPTYSRGESGPNEPPPGLTGPATSGSETPGT
383


RSN-2749
GSAPAWGRTGASENETPAPLGGEATSGSETPGT
384


RSN-2750
GSAPRWGRAETTPNTPPEGLETEATSGSETPGT
385


RSN-2751
GSAPESGRAANHTGAEPPELGAGATSGSETPGT
386


RSN-2754
GSAPTTGRAGEAANLTPAGLTESATSGSETPGT
387


RSN-2755
GSAPTTGRAGEAANLTPAALTESATSGSETPGT
388


RSN-2756
GSAPTTGRAGEAANLTPAPLTESATSGSETPGT
389


RSN-2757
GSAPTTGRAGEAANLTPEPLTESATSGSETPGT
390


RSN-2758
GSAPTTGRAGEAANLTPAGLTGAATSGSETPGT
391


RSN-2759
GSAPTTGRAGEAANLTPEGLTGAATSGSETPGT
392


RSN-2760
GSAPTTGRAGEAANLTPEPLTGAATSGSETPGT
393


RSN-2761
GSAPTTGRAGEAANLTPAGLTEAATSGSETPGT
394


RSN-2762
GSAPTTGRAGEAANLTPEGLTEAATSGSETPGT
395


RSN-2763
GSAPTTGRAGEAANLTPAPLTEAATSGSETPGT
396


RSN-2764
GSAPTTGRAGEAANLTPEPLTEAATSGSETPGT
397


RSN-2765
GSAPTTGRAGEAANLTPEPLTGPATSGSETPGT
398


RSN-2766
GSAPTTGRAGEAANLTPAGLTGGATSGSETPGT
399


RSN-2767
GSAPTTGRAGEAANLTPEGLTGGATSGSETPGT
400


RSN-2768
GSAPTTGRAGEAANLTPEALTGGATSGSETPGT
401


RSN-2769
GSAPTTGRAGEAANLTPEPLTGGATSGSETPGT
402


RSN-2770
GSAPTTGRAGEAANLTPAGLTEGATSGSETPGT
403


RSN-2771
GSAPTTGRAGEAANLTPEGLTEGATSGSETPGT
404


RSN-2772
GSAPTTGRAGEAANLTPAPLTEGATSGSETPGT
405


RSN-2773
GSAPTTGRAGEAANLTPEPLTEGATSGSETPGT
406


RSN-3047
GSAPTTGRAGEAEGATSAGATGPATSGSETPGT
330


RSN-2783
GSAPEAGRSAEATSAGATGPATSGSETPGT
407


RSN-3107
GSAPSASGTYSRGESGPGSPATSGSETPGT
408


RSN-3103
GSAPSASGEAGRTDTHPGSPATSGSETPGT
409


RSN-3102
GSAPSASGEPGRAAEHPGSPATSGSETPGT
410


RSN-3119
GSAPSPAGESSRGTTIAGSPATSGSETPGT
411


RSN-3043
GSAPTTGEAGEAAGLTPAGLTGPATSGSETPGT
332


RSN-2789
GSAPEAGESAGATPAGLTGPATSGSETPGT
412


RSN-3109
GSAPSASGAPLELEAGPGSPATSGSETPGT
413


RSN-3110
GSAPSASGEPPELGAGPGSPATSGSETPGT
414


RSN-3111
GSAPSASGEPSGLTEGPGSPATSGSETPGT
415


RSN-3112
GSAPSASGTPAPLTEPPGSPATSGSETPGT
416


RSN-3113
GSAPSASGTPAELTEPPGSPATSGSETPGT
417


RSN-3114
GSAPSASGPPPGLTGPPGSPATSGSETPGT
418


RSN-3115
GSAPSASGTPAPLGGEPGSPATSGSETPGT
419


RSN-3125
GSAPSPAGAPEGLTGPAGSPATSGSETPGT
420


RSN-3126
GSAPSPAGPPEGLETEAGSPATSGSETPGT
421


RSN-3127
GSAPSPTSGQGGLTGPGSEPATSGSETPGT
422


RSN-3131
GSAPSESAPPEGLETESTEPATSGSETPGT
423


RSN-3132
GSAPSEGSEPLELGAASETPATSGSETPGT
424


RSN-3133
GSAPSEGSGPAGLEAPSETPATSGSETPGT
425


RSN-3138
GSAPSEPTPPASLEAEPGSPATSGSETPGT
426


RSC-0001
GTAEAASASGGSAGGYAELRMGGAIPGSP
427


RSC-0002
GTAEAASASGGTGGGYAPLRMGGGAPGSP
428


RSC-0003
GTAEAASASGGAEGGYAALRMGGEIPGSP
429


RSC-0004
GTAEAASASGGGPGGYALLRMGGPAPGSP
430


RSC-0005
GTAEAASASGGEAGGYAFLRMGGSIPGSP
431


RSC-0006
GTAEAASASGGPGGGYASLRMGGTAPGSP
432


RSC-0007
GTAEAASASGGSEGGYATLRMGGAIPGSP
433


RSC-0008
GTAEAASASGGTPGGYANLRMGGGAPGSP
434


RSC-0009
GTAEAASASGGASGGYAHLRMGGEIPGSP
435


RSC-0010
GTAEAASASGGGTGGYGELRMGGPAPGSP
436


RSC-0011
GTAEAASASGGEAGGYPELRMGGSIPGSP
437


RSC-0012
GTAEAASASGGPGGGYVELRMGGTAPGSP
438


RSC-0013
GTAEAASASGGSEGGYLELRMGGAIPGSP
439


RSC-0014
GTAEAASASGGTPGGYSELRMGGGAPGSP
440


RSC-0015
GTAEAASASGGASGGYTELRMGGEIPGSP
441


RSC-0016
GTAEAASASGGGTGGYQELRMGGPAPGSP
442


RSC-0017
GTAEAASASGGEAGGYEELRMGGSIPGSP
443


RSC-0018
GTAEAASASGGPGIGPAELRMGGTAPGSP
444


RSC-0019
GTAEAASASGGSEIGAAELRMGGAIPGSP
445


RSC-0020
GTAEAASASGGTPIGSAELRMGGGAPGSP
446


RSC-0021
GTAEAASASGGASIGTAELRMGGEIPGSP
447


RSC-0022
GTAEAASASGGGTIGNAELRMGGPAPGSP
448


RSC-0023
GTAEAASASGGEAIGQAELRMGGSIPGSP
449


RSC-0024
GTAEAASASGGPGGPYAELRMGGTAPGSP
450


RSC-0025
GTAEAASASGGSEGAYAELRMGGAIPGSP
451


RSC-0026
GTAEAASASGGTPGVYAELRMGGGAPGSP
452


RSC-0027
GTAEAASASGGASGLYAELRMGGEIPGSP
453


RSC-0028
GTAEAASASGGGTGIYAELRMGGPAPGSP
454


RSC-0029
GTAEAASASGGEAGFYAELRMGGSIPGSP
455


RSC-0030
GTAEAASASGGPGGYYAELRMGGTAPGSP
456


RSC-0031
GTAEAASASGGSEGSYAELRMGGAIPGSP
457


RSC-0032
GTAEAASASGGTPGNYAELRMGGGAPGSP
458


RSC-0033
GTAEAASASGGASGEYAELRMGGEIPGSP
459


RSC-0034
GTAEAASASGGGTGHYAELRMGGPAPGSP
460


RSC-0035
GTAEAASASGGEAGGYAEARMGGSIPGSP
461


RSC-0036
GTAEAASASGGPGGGYAEVRMGGTAPGSP
462


RSC-0037
GTAEAASASGGSEGGYAEIRMGGAIPGSP
463


RSC-0038
GTAEAASASGGTPGGYAEFRMGGGAPGSP
464


RSC-0039
GTAEAASASGGASGGYAEYRMGGEIPGSP
465


RSC-0040
GTAEAASASGGGTGGYAESRMGGPAPGSP
466


RSC-0041
GTAEAASASGGEAGGYAETRMGGSIPGSP
467


RSC-0042
GTAEAASASGGPGGGYAELAMGGTRPGSP
468


RSC-0043
GTAEAASASGGSEGGYAELVMGGARPGSP
469


RSC-0044
GTAEAASASGGTPGGYAELLMGGGRPGSP
470


RSC-0045
GTAEAASASGGASGGYAELIMGGERPGSP
471


RSC-0046
GTAEAASASGGGTGGYAELWMGGPRPGSP
472


RSC-0047
GTAEAASASGGEAGGYAELSMGGSRPGSP
473


RSC-0048
GTAEAASASGGPGGGYAELTMGGTRPGSP
474


RSC-0049
GTAEAASASGGSEGGYAELQMGGARPGSP
475


RSC-0050
GTAEAASASGGTPGGYAELNMGGGRPGSP
476


RSC-0051
GTAEAASASGGASGGYAELEMGGERPGSP
477


RSC-0052
GTAEAASASGGGTGGYAELRPGGPIPGSP
478


RSC-0053
GTAEAASASGGEAGGYAELRAGGSAPGSP
479


RSC-0054
GTAEAASASGGPGGGYAELRLGGTIPGSP
480


RSC-0055
GTAEAASASGGSEGGYAELRIGGAAPGSP
481


RSC-0056
GTAEAASASGGTPGGYAELRSGGGIPGSP
482


RSC-0057
GTAEAASASGGASGGYAELRNGGEAPGSP
483


RSC-0058
GTAEAASASGGGTGGYAELRQGGPIPGSP
484


RSC-0059
GTAEAASASGGEAGGYAELRDGGSAPGSP
485


RSC-0060
GTAEAASASGGPGGGYAELREGGTIPGSP
486


RSC-0061
GTAEAASASGGSEGGYAELRHGGAAPGSP
487


RSC-0062
GTAEAASASGGTPGGYAELRMPGGIPGSP
488


RSC-0063
GTAEAASASGGASGGYAELRMAGEAPGSP
489


RSC-0064
GTAEAASASGGGTGGYAELRMVGPIPGSP
490


RSC-0065
GTAEAASASGGEAGGYAELRMLGSAPGSP
491


RSC-0066
GTAEAASASGGPGGGYAELRMIGTIPGSP
492


RSC-0067
GTAEAASASGGSEGGYAELRMYGAIPGSP
493


RSC-0068
GTAEAASASGGTPGGYAELRMSGGAPGSP
494


RSC-0069
GTAEAASASGGASGGYAELRMNGEIPGSP
495


RSC-0070
GTAEAASASGGGTGGYAELRMQGPAPGSP
496


RSC-0071
GTAEAASASGGANHTPAGLTGPGARPGSP
497


RSC-0072
GTAEAASASGGANTAPEGLTGPSTRPGSP
498


RSC-0073
GTAEAASASGGTGAPPGGLTGPGTRPGSP
499


RSC-0074
GTAEAASASGGANHEPSGLTEGSPRPGSP
500


RSC-0075
GTAEAASASGGANTEPPELGAGTERPGSP
501


RSC-0076
GTAEAASASGGASGPPPGLTGPPGRPGSP
502


RSC-0077
GTAEAASASGGASGTPAPLGGEPGRPGSP
503


RSC-0078
GTAEAASASGGPAGPPEGLETEAGRPGSP
504


RSC-0079
GTAEAASASGGPTSGQGGLTGPESRPGSP
505


RSC-0080
GTAEAASASGGSAGGAANLVRGGAIPGSP
506


RSC-0081
GTAEAASASGGTGGGAAPLVRGGGAPGSP
507


RSC-0082
GTAEAASASGGAEGGAAALVRGGEIPGSP
508


RSC-0083
GTAEAASASGGGPGGAALLVRGGPAPGSP
509


RSC-0084
GTAEAASASGGEAGGAAFLVRGGSIPGSP
510


RSC-0085
GTAEAASASGGPGGGAASLVRGGTAPGSP
511


RSC-0086
GTAEAASASGGSEGGAATLVRGGAIPGSP
512


RSC-0087
GTAEAASASGGTPGGAAGLVRGGGAPGSP
513


RSC-0088
GTAEAASASGGASGGAADLVRGGEIPGSP
514


RSC-0089
GTAEAASASGGGTGGAGNLVRGGPAPGSP
515


RSC-0090
GTAEAASASGGEAGGAPNLVRGGSIPGSP
516


RSC-0091
GTAEAASASGGPGGGAVNLVRGGTAPGSP
517


RSC-0092
GTAEAASASGGSEGGALNLVRGGAIPGSP
518


RSC-0093
GTAEAASASGGTPGGASNLVRGGGAPGSP
519


RSC-0094
GTAEAASASGGASGGATNLVRGGEIPGSP
520


RSC-0095
GTAEAASASGGGTGGAQNLVRGGPAPGSP
521


RSC-0096
GTAEAASASGGEAGGAENLVRGGSIPGSP
522


RSC-1517
GTAEAASASGEAGRSANHEPLGLVATPGSP
523


BSRS-A1
GTAEAASASGASGRSTNAGPSGLAGPPGSP
524


BSRS-A2
GTAEAASASGASGRSTNAGPQGLAGQPGSP
525


BSRS-A3
GTAEAASASGASGRSTNAGPPGLTGPPGSP
526


VP-1
GTAEAASASGASSRGTNAGPAGLTGPPGSP
527


RSC-1752
GTAEAASASGASSRTTNTGPSTLTGPPGSP
528


RSC-1512
GTAEAASASGAAGRSDNGTPLELVAPPGSP
529


RSC-1517
GTAEAASASGEAGRSANHEPLGLVATPGSP
523


VP-2
GTAEAASASGASGRGTNAGPAGLTGPPGSP
530


RSC-1018
GTAEAASASGLFGRNDNHEPLELGGGPGSP
531


RSC-1053
GTAEAASASGTAGRSDNLEPLGLVFGPGSP
532


RSC-1059
GTAEAASASGLDGRSDNFHPPELVAGPGSP
533


RSC-1065
GTAEAASASGLEGRSDNEEPENLVAGPGSP
534


RSC-1167
GTAEAASASGLKGRSDNNAPLALVAGPGSP
535


RSC-1201
GTAEAASASGVYSRGTNAGPHGLTGRPGSP
536


RSC-1218
GTAEAASASGANSRGTNKGFAGLIGPPGSP
537


RSC-1226
GTAEAASASGASSRLTNEAPAGLTIPPGSP
538


RSC-1254
GTAEAASASGDQSRGTNAGPEGLTDPPGSP
539


RSC-1256
GTAEAASASGESSRGTNIGQGGLTGPPGSP
540


RSC-1261
GTAEAASASGSSSRGTNQDPAGLTIPPGSP
541


RSC-1293
GTAEAASASGASSRGQNHSPMGLTGPPGSP
542


RSC-1309
GTAEAASASGAYSRGPNAGPAGLEGRPGSP
543


RSC-1326
GTAEAASASGASERGNNAGPANLTGFPGSP
544


RSC-1345
GTAEAASASGASHRGTNPKPAILTGPPGSP
545


RSC-1354
GTAEAASASGMSSRRTNANPAQLTGPPGSP
546


RSC-1426
GTAEAASASGGAGRTDNHEPLELGAAPGSP
547


RSC-1478
GTAEAASASGLAGRSENTAPLELTAGPGSP
548


RSC-1479
GTAEAASASGLEGRPDNHEPLALVASPGSP
549


RSC-1496
GTAEAASASGLSGRSDNEEPLALPAGPGSP
550


RSC-1508
GTAEAASASGEAGRTDNHEPLELSAPPGSP
551


RSC-1513
GTAEAASASGEGGRSDNHGPLELVSGPGSP
552


RSC-1516
GTAEAASASGLSGRSDNEAPLELEAGPGSP
553


RSC-1524
GTAEAASASGLGGRADNHEPPELGAGPGSP
554


RSC-1622
GTAEAASASGPPSRGTNAEPAGLTGEPGSP
555


RSC-1629
GTAEAASASGASTRGENAGPAGLEAPPGSP
556


RSC-1664
GTAEAASASGESSRGTNGAPEGLTGPPGSP
557


RSC-1667
GTAEAASASGASSRATNESPAGLTGEPGSP
558


RSC-1709
GTAEAASASGASSRGENPPPGGLTGPPGSP
559


RSC-1712
GTAEAASASGAASRGTNTGPAELTGSPGSP
560


RSC-1727
GTAEAASASGAGSRTTNAGPGGLEGPPGSP
561


RSC-1754
GTAEAASASGAPSRGENAGPATLTGAPGSP
562


RSC-1819
GTAEAASASGESGRAANTGPPTLTAPPGSP
563


RSC-1832
GTAEAASASGNPGRAANEGPPGLPGSPGSP
564


RSC-1855
GTAEAASASGESSRAANLTPPELTGPPGSP
565


RSC-1911
GTAEAASASGASGRAANETPPGLTGAPGSP
566


RSC-1929
GTAEAASASGNSGRGENLGAPGLTGTPGSP
567


RSC-1951
GTAEAASASGTTGRAANLTPAGLTGPPGSP
568


RSC-2295
GTAEAASASGEAGRSANHTPAGLTGPPGSP
569


RSC-2298
GTAEAASASGESGRAANTTPAGLTGPPGSP
570


RSC-2038
GTAEAASASGTTGRATEAANLTPAGLTGPPGSP
571


RSC-2072
GTAEAASASGTTGRAEEAANLTPAGLTGPPGSP
572


RSC-2089
GTAEAASASGTTGRAGEAANLTPAGLTGPPGSP
573


RSC-2302
GTAEAASASGTTGRATEAANATPAGLTGPPGSP
574


RSC-3047
GTAEAASASGTTGRAGEAEGATSAGATGPPGSP
575


RSC-3052
GTAEAASASGTTGEAGEAANATSAGATGPPGSP
576


RSC-3043
GTAEAASASGTTGEAGEAAGLTPAGLTGPPGSP
577


RSC-3041
GTAEAASASGTTGAAGEAANATPAGLTGPPGSP
578


RSC-3044
GTAEAASASGTTGRAGEAAGLTPAGLTGPPGSP
579


RSC-3057
GTAEAASASGTTGRAGEAANATSAGATGPPGSP
580


RSC-3058
GTAEAASASGTTGEAGEAAGATSAGATGPPGSP
581


RSC-2485
GTAEAASASGESGRAANTEPPELGAGPGSP
582


RSC-2486
GTAEAASASGESGRAANTAPEGLTGPPGSP
583


RSC-2488
GTAEAASASGEPGRAANHEPSGLTEGPGSP
584


RSC-2599
GTAEAASASGESGRAANHTGAPPGGLTGPPGSP
585


RSC-2706
GTAEAASASGTTGRTGEGANATPGGLTGPPGSP
586


RSC-2707
GTAEAASASGRTGRSGEAANETPEGLEGPPGSP
587


RSC-2708
GTAEAASASGRTGRTGESANETPAGLGGPPGSP
588


RSC-2709
GTAEAASASGSTGRTGEPANETPAGLSGPPGSP
589


RSC-2710
GTAEAASASGTTGRAGEPANATPTGLSGPPGSP
590


RSC-2711
GTAEAASASGRTGRPGEGANATPTGLPGPPGSP
591


RSC-2712
GTAEAASASGRTGRGGEAANATPSGLGGPPGSP
592


RSC-2713
GTAEAASASGSTGRSGESANATPGGLGGPPGSP
593


RSC-2714
GTAEAASASGRTGRTGEEANATPAGLPGPPGSP
594


RSC-2715
GTAEAASASGATGRPGEPANTTPEGLEGPPGSP
595


RSC-2716
GTAEAASASGSTGRSGEPANATPGGLTGPPGSP
596


RSC-2717
GTAEAASASGPTGRGGEGANTTPTGLPGPPGSP
597


RSC-2718
GTAEAASASGPTGRSGEGANATPSGLTGPPGSP
598


RSC-2719
GTAEAASASGTTGRASEGANSTPAPLTEPPGSP
599


RSC-2720
GTAEAASASGTYGRAAEAANTTPAGLTAPPGSP
600


RSC-2721
GTAEAASASGTTGRATEGANATPAELTEPPGSP
601


RSC-2722
GTAEAASASGTVGRASEEANTTPASLTGPPGSP
602


RSC-2723
GTAEAASASGTTGRAPEAANATPAPLTGPPGSP
603


RSC-2724
GTAEAASASGTWGRATEPANATPAPLTSPPGSP
604


RSC-2725
GTAEAASASGTVGRASESANATPAELTSPPGSP
605


RSC-2726
GTAEAASASGTVGRAPEGANSTPAGLTGPPGSP
606


RSC-2727
GTAEAASASGTWGRATEAPNLEPATLTTPPGSP
607


RSC-2728
GTAEAASASGTTGRATEAPNLTPAPLTEPPGSP
608


RSC-2729
GTAEAASASGTQGRATEAPNLSPAALTSPPGSP
609


RSC-2730
GTAEAASASGTQGRAAEAPNLTPATLTAPPGSP
610


RSC-2731
GTAEAASASGTSGRAPEATNLAPAPLTGPPGSP
611


RSC-2732
GTAEAASASGTQGRAAEAANLTPAGLTEPPGSP
612


RSC-2733
GTAEAASASGTTGRAGSAPNLPPTGLTTPPGSP
613


RSC-2734
GTAEAASASGTTGRAGGAENLPPEGLTAPPGSP
614


RSC-2735
GTAEAASASGTTSRAGTATNLTPEGLTAPPGSP
615


RSC-2736
GTAEAASASGTTGRAGTATNLPPSGLTTPPGSP
616


RSC-2737
GTAEAASASGTTARAGEAENLSPSGLTAPPGSP
617


RSC-2738
GTAEAASASGTTGRAGGAGNLAPGGLTEPPGSP
618


RSC-2739
GTAEAASASGTTGRAGTATNLPPEGLTGPPGSP
619


RSC-2740
GTAEAASASGTTGRAGGAANLAPTGLTEPPGSP
620


RSC-2741
GTAEAASASGTTGRAGTAENLAPSGLTTPPGSP
621


RSC-2742
GTAEAASASGTTGRAGSATNLGPGGLTGPPGSP
622


RSC-2743
GTAEAASASGTTARAGGAENLTPAGLTEPPGSP
623


RSC-2744
GTAEAASASGTTARAGSAENLSPSGLTGPPGSP
624


RSC-2745
GTAEAASASGTTARAGGAGNLAPEGLTTPPGSP
625


RSC-2746
GTAEAASASGTTSRAGAAENLTPTGLTGPPGSP
626


RSC-2747
GTAEAASASGTYGRTTTPGNEPPASLEAEPGSP
627


RSC-2748
GTAEAASASGTYSRGESGPNEPPPGLTGPPGSP
628


RSC-2749
GTAEAASASGAWGRTGASENETPAPLGGEPGSP
629


RSC-2750
GTAEAASASGRWGRAETTPNTPPEGLETEPGSP
630


RSC-2751
GTAEAASASGESGRAANHTGAEPPELGAGPGSP
631


RSC-2754
GTAEAASASGTTGRAGEAANLTPAGLTESPGSP
632


RSC-2755
GTAEAASASGTTGRAGEAANLTPAALTESPGSP
633


RSC-2756
GTAEAASASGTTGRAGEAANLTPAPLTESPGSP
634


RSC-2757
GTAEAASASGTTGRAGEAANLTPEPLTESPGSP
635


RSC-2758
GTAEAASASGTTGRAGEAANLTPAGLTGAPGSP
636


RSC-2759
GTAEAASASGTTGRAGEAANLTPEGLTGAPGSP
637


RSC-2760
GTAEAASASGTTGRAGEAANLTPEPLTGAPGSP
638


RSC-2761
GTAEAASASGTTGRAGEAANLTPAGLTEAPGSP
639


RSC-2762
GTAEAASASGTTGRAGEAANLTPEGLTEAPGSP
640


RSC-2763
GTAEAASASGTTGRAGEAANLTPAPLTEAPGSP
641


RSC-2764
GTAEAASASGTTGRAGEAANLTPEPLTEAPGSP
642


RSC-2765
GTAEAASASGTTGRAGEAANLTPEPLTGPPGSP
643


RSC-2766
GTAEAASASGTTGRAGEAANLTPAGLTGGPGSP
644


RSC-2767
GTAEAASASGTTGRAGEAANLTPEGLTGGPGSP
645


RSC-2768
GTAEAASASGTTGRAGEAANLTPEALTGGPGSP
646


RSC-2769
GTAEAASASGTTGRAGEAANLTPEPLTGGPGSP
647


RSC-2770
GTAEAASASGTTGRAGEAANLTPAGLTEGPGSP
648


RSC-2771
GTAEAASASGTTGRAGEAANLTPEGLTEGPGSP
649


RSC-2772
GTAEAASASGTTGRAGEAANLTPAPLTEGPGSP
650


RSC-2773
GTAEAASASGTTGRAGEAANLTPEPLTEGPGSP
651


RSC-3047
GTAEAASASGTTGRAGEAEGATSAGATGPPGSP
575


RSC-2783
GTAEAASASGEAGRSAEATSAGATGPPGSP
652


RSC-3107
GTAEAASASGSASGTYSRGESGPGSPPGSP
653


RSC-3103
GTAEAASASGSASGEAGRTDTHPGSPPGSP
654


RSC-3102
GTAEAASASGSASGEPGRAAEHPGSPPGSP
655


RSC-3119
GTAEAASASGSPAGESSRGTTIAGSPPGSP
656


RSC-3043
GTAEAASASGTTGEAGEAAGLTPAGLTGPPGSP
577


RSC-2789
GTAEAASASGEAGESAGATPAGLTGPPGSP
657


RSC-3109
GTAEAASASGSASGAPLELEAGPGSPPGSP
658


RSC-3110
GTAEAASASGSASGEPPELGAGPGSPPGSP
659


RSC-3111
GTAEAASASGSASGEPSGLTEGPGSPPGSP
660


RSC-3112
GTAEAASASGSASGTPAPLTEPPGSPPGSP
661


RSC-3113
GTAEAASASGSASGTPAELTEPPGSPPGSP
662


RSC-3114
GTAEAASASGSASGPPPGLTGPPGSPPGSP
663


RSC-3115
GTAEAASASGSASGTPAPLGGEPGSPPGSP
664


RSC-3125
GTAEAASASGSPAGAPEGLTGPAGSPPGSP
665


RSC-3126
GTAEAASASGSPAGPPEGLETEAGSPPGSP
666


RSC-3127
GTAEAASASGSPTSGQGGLTGPGSEPPGSP
667


RSC-3131
GTAEAASASGSESAPPEGLETESTEPPGSP
668


RSC-3132
GTAEAASASGSEGSEPLELGAASETPPGSP
669


RSC-3133
GTAEAASASGSEGSGPAGLEAPSETPPGSP
670


RSC-3138
GTAEAASASGSEPTPPASLEAEPGSPPGSP
671






In another aspect, the release segments (either RS1 and/or RS2) for incorporation into the polypeptides of any of the subject composition embodiments described herein can be designed to be selectively sensitive in order to have different rates of cleavage and different cleavage efficiencies to the various proteases for which they are substrates. As a given protease may be found in different concentrations in diseased tissues, including but not limited to a tumor, a blood cancer, or an inflammatory tissue or site of inflammation compared to healthy tissues or in the circulation, the disclosure provides RS that have had the individual amino acid sequences engineered to have a higher or lower cleavage efficiency for a given protease in order to ensure that the polypeptide is preferentially converted from the prodrug form to the active form (i.e., by the separation and release of the antigen binding fragments and XTEN from the polypeptide after cleavage of the release segment) when in proximity to the target cell or tissue and its co-localized proteases compared to the rate of cleavage of the release segment in healthy tissue or the circulation such that the released antigen binding fragments have a greater ability to bind to ligands in the diseased tissues compared to the prodrug 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.


As used herein cleavage efficiency is defined as the log2 value of the ratio of the percentage of the test substrate comprising the release segment cleaved to the percentage of the control substrate RSR-1517 (AC1611) cleaved when each is subjected to the protease enzyme in biochemical assays (further detailed in the Examples) in which the reaction is conducted wherein the initial substrate concentration is 6 µM, the reactions are incubated at 37° C. for 2 hours before being stopped (e.g., 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 of the release segments cleaved. The cleavage efficiency is calculated as follows:






L
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g
2





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%

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A
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1611

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n
t








Thus, a cleavage efficiency of -1 means 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. As detailed in the Examples, a control RS sequence AC1611 (RSR-1517), having the amino acid sequence EAGRSANHEPLGLVAT (SEQ ID NO: 53), 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 (detailed more fully in the Examples), resulting in profiles that were used to establish guidelines for appropriate amino acid substitutions in order to achieve RS with desired cleavage efficiencies. In making RS 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 release segment retains at least some susceptibility to cleavage by a protease.


IV). XTEN Polypeptides

In another aspect, the disclosure relates to polypeptides comprising at least a first extended recombinant polypeptide (XTEN) that is incorporated into the subject composition embodiments described herein, thereby both increasing the mass and size of the construct, and also serving to greatly reduce the ability of the antigen binding fragments to bind their ligands when the molecule is in the intact, uncleaved state, as described more fully below. In some embodiments, the disclosure provides a polypeptide comprising a single XTEN fused to the terminus of the RS that is located between the antigen binding fragment and the XTEN. In other embodiments, the disclosure provides a polypeptide comprising a first and a second XTEN (XTEN1 and XTEN2) fused to the N- and C-terminus of an RS1 and RS2, respectively, that are located between each antigen binding fragment and the XTEN.


Without being bound by theory, the incorporation of the XTEN can be incorporated into the design of the subject compositions to confer certain properties: 1) provide polypeptide compositions with an XTEN that shields the antigen binding fragments and reduces their binding affinity for the target cell markers and effector cell antigens when the composition is in its intact, prodrug form; ii) provide polypeptide compositions with an XTEN that provides enhanced half-life when administered to a subject, iii) contribute to the solubility and stability of the intact composition, thereby enhancing the pharmaceutical properties of the subject compositions; and iv) provide polypeptide compositions with an XTEN that reduces extravasation in normal tissues and organs yet permits a degree of extravasation in diseased tissues (e.g., a tumor) with larger pore sizes in the vasculature, yet could be released from the composition by action of certain mammalian proteases, thereby permitting the antigen binding fragments of the composition to more readily penetrate into the diseased tissues, e.g. a tumor, and to bind to and link together the target cell markers on the effector cell and tumor cell. To meet these needs, the disclosure provides compositions comprising one or more XTEN in which the XTEN provides increased mass and hydrodynamic radius to the resulting composition. The XTEN polypeptides of the embodiments provide certain advantages in the design of the subject compositions in that is provides not only provides increased mass and hydrodynamic radius to the composition, but its flexible, unstructured characteristics can provide a shielding effect over the antigen binding fragments of the composition, thereby reducing the binding to antigens in normal tissues or the vasculature of normal tissues that don’t express or express reduced levels of target cell markers and/or effector cell antigens. Additionally, the incorporation of XTEN into the subject compositions can enhance the solubility and proper folding of the single chain antibody binding fragments during their expression and recovery.


XTEN are polypeptides with non-naturally occurring, substantially non-repetitive sequences having a low degree or no secondary or tertiary structure under physiologic conditions, as well as one or more additional properties described in the paragraphs that follow. In some embodiments, the present disclosure provides polypeptides comprising one or more XTEN having from at least about 36, 72, 96, 100, 144, 200, 288, 292, 293, 300, 576, 584, 800, 864, 867, 868, 900, or at least about 1000 or more amino acids. In one embodiment, the present disclosure provides a polypeptide comprising an XTEN1 wherein the XTEN1 is characterized in that it has at least about 36 amino acid residues wherein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the amino acid residues of the XTEN1 sequence are selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) and it has at least 4-6 different amino acids selected from G, A, S, T, E and P. In some embodiments, the present disclosure provides polypeptides comprising an XTEN1 having at least about 36 to about 1000, or at least 100 to about 900, or at least about 144 to about 868, or at least about 288-868 amino acid residues. In other cases, the present disclosure provides polypeptides comprising an XTEN1 having at least about 36 to about 1000, or at least 100 to about 900, or at least about 144 to about 868, or at least about 288-868 amino acid residues wherein 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid residues are selected from 4-6 types of amino acids selected from the group consisting of glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P). In other cases, the present disclosure provides polypeptides comprising an XTEN1 wherein the XTEN1 is characterized in that it has at least about 36 to about 1000 amino acid residues, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the amino acid residues of the XTEN1 sequence are selected from six types of amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P).


In another embodiment, the present disclosure provides polypeptides of any of the embodiments described herein comprising an XTEN1 wherein the XTEN1 is characterized in that it has at least about 36 to about 1000, or at least about 100 to about 900, or at least 144 to about 868 amino acid residues, wherein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the amino acid residues of the XTEN1 sequence are selected from at least three of the sequences of SEQ ID NOS: 672-675. In some cases, the XTEN 1 sequence can be assembled by any combination of the 12 amino acid units of SEQ ID NOS: 672-675 such that any length of at least 36 amino acids or longer, in 12 amino acid increments, can be achieved; e.g., 36, 48, 60, 72, 84, 96 amino acids, etc. In other cases, the polypeptides of any of the subject composition embodiments described herein can comprise an XTEN1 wherein the XTEN1 is characterized in that it has at least about 36 to about 1000, or at least about 100 to about 900, or at least 144 to about 868 amino acid residues, wherein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the amino acid residues of the XTEN1 sequence are selected from the sequences of SEQ ID NOS: 676-734. In another embodiment, the XTEN of any of the subject composition embodiments described herein can have an affinity tag of HHHHHH (SEQ ID NO: 794), HHHHHHHH (SEQ ID NO: 795), or the sequence EPEA (SEQ ID NO: 796) appended to the N- or C-terminus of the XTEN of the composition to facilitate the purification of the composition to at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% purity by chromatography methods known in the art; e.g., IMAC chromatography or C-tagXL chromatography, or methods described in the Examples, below.


In another embodiment, the present disclosure provides a polypeptide comprising an XTEN1 wherein the XTEN1 comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an AE36 (comprising a sequence selected from any three of the sequences of SEQ ID NOS: 672-675), or a sequence selected from the sequences of AE144_1A, AE144_2A, AE144_2B, AE144_3A, AE144_3B, AE144_4A, AE144_4B, AE144_5A, AE144_6B, AE144_7A, AE284, AE288_1, AE288_2, AE288_3, AE292, AE293, AE576, AE584, AE864, AE864_2, AE865, AE866, AE867, and AE868, each of which being set forth in Table 7.


In some aspects of any of the embodiments disclosed herein, a subject polypeptide comprises an XTEN1 and an XTEN2. The configurations of the polypeptides comprising XTEN1 and XTEN2, amongst the other components, are described herein, below. In one embodiment, the present disclosure provides a polypeptide comprising an XTEN1 and an XTEN2 wherein the XTEN2 is characterized in that it has at least about 36 to about 1000 amino acid residues, wherein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the amino acid residues of the XTEN2 sequence are selected from at least three of the sequences of SEQ ID NOS: 672-675. In another embodiment, the present disclosure provides a polypeptide comprising an XTEN1 and an XTEN2 wherein the XTEN 1 and the XTEN2 are each characterized in that it has at least about 36 to about 1000 amino acid residues, wherein at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the amino acid residues of the XTEN2 sequence are selected from the sequences of SEQ ID NOS: 676-734. In another embodiment, the polypeptides of any of the subject composition embodiments described herein can comprise an XTEN1 and an XTEN2 wherein the XTEN 1 and the XTEN2 each comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from the sequences of AE144_1A, AE144_2A, AE144_2B, AE144_3A, AE144_3B, AE144_4A, AE144_4B, AE144_5A, AE144_6B, AE144_7A, AE284, AE288_1, AE288_2, AE288_3, AE292, AE293, AE576, AE584, AE864, AE864_2, AE865, AE866, AE867, and AE868, each of which being set forth in Table 7. In some cases of the foregoing embodiments of the paragraph, the XTEN1 and XTEN 2 are identical. In other cases of the foregoing embodiments of the paragraph, the XTEN1 and XTEN2 of the foregoing embodiments of the paragraph have different amino acid sequences. In some cases, the XTEN1 of any of the polypeptide composition embodiments having 2 XTENs is fused to the C-terminus of the polypeptide and is selected from the group consisting of AE293, AE300, AE584 and AEAE868. In other cases, the XTEN2 of any of the polypeptide composition embodiments having 2 XTENs is fused to the N-terminus of the polypeptide and is selected from the group consisting of AE144_7A, AE292, AE576, and AE864. In other cases, the XTEN1 of any of the polypeptide composition embodiments having 2 XTENs is fused to the C-terminus of the polypeptide and is selected from the group consisting of AE293, AE300, AE584 and AEAE868 and the XTEN 2 is fused to the N-terminus and is selected from the group consisting of AE144_7A, AE292, AE576, and AE864.





TABLE 6






XTEN Sequence Motifs


Motif Name
Amino Acid Sequence
SEQ ID NO:




AE1
GSPAGSPTSTEE
672


AE2
GSEPATSGSETP
673


AE3
GTSESATPESGP
674


AE4
GTSTEPSEGSAP
675









TABLE 7






XTEN Sequences


XTEN Name
Amino Acid Sequence
SEQ ID NO:




AE144
GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTE PSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGS APGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAP
676


AE144_1 A
SPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEP SEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSET PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPG
677


AE144_2 A
TSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESA TPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSA PGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPG
678


AE144_2 B
TSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESA TPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSA PGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPG
679


AE144_3 A
SPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEP SEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA PGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPG
680


AE144_3 B
SPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEP SEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA PGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPG
681


AE144_4 A
TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESA TPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTE EGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPG
682


AE144_4 B
TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESA TPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTE EGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPG
683


AE144_5 A
TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESA TPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSET PGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEG
684


AE144_6 B
TSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPAT SGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSA PGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG
685


AE288_1
GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSES
686



ATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSE TPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTS ESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSG SETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPG TSESATPESGPGTSTEPSEGSAP



AE288_2
GSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTE PSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS APGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSE PATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSE GSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEG TSESATPESGPGTSTEPSEGSAP
687


AE576
GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE PSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSE TPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSP AGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSE GSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPG TSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPAT SGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTST EPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGS ETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGS PAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP
688


AE624
MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSPA GSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEG SAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGS PAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSP TSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAP GTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSES ATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSE TPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTS TEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSE GSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPG TSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGS PTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP
689


AE864
GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE PSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSE TPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSP AGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSE GSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPG TSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPAT SGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTST EPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGS ETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGS PAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESAT PESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGP GTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSES ATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPES GPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSP AGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATP ESGPGTSTEPSEGSAP
690


AE865
GGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTST EPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGS ETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGS PAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPS EGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAP GTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPA TSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS
691



APGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTS TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSG SETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEG SPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESA TPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESG PGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSE SATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPE SGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGS PAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAT PESGPGTSTEPSEGSAP



AE866
PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTST EPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGS ETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGS PAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPS EGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAP GTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPA TSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS APGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTS TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSG SETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEG SPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESA TPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESG PGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSE SATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPE SGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGS PAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAT PESGPGTSTEPSEGSAPG
692


AE1152
GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE PSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSE TPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSP AGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSE GSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPG TSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPAT SGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTST EPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGS ETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGS PAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESAT PESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGP GTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSES ATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPES GPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSP AGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATP ESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPG SEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESA TPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTE EGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEP ATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEG SAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP
693


AE144A
STEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATS GSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP GTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGS
694


AE144B
SEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEP SEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESG PGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPG
695


AE180A
TSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEE GSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSES ATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGS
696



APGTSTEPSEGSAPGSEPATS



AE216A
PESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETP GTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSES ATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSE TPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTS ESAT
697


AE252A
ESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEG TSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGS PTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESG PGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTST EPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSE
698


AE288A
TPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESG PGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSE SATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTS TEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGS EPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPS EGSAPGSEPATSGSETPGTSESA
699


AE324A
PESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGP GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPA TSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPES GPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTS TEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSG SETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPG SEPATS
700


AE360A
PESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEE GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPA TSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGS APGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSP AGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATP ESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEG TSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAT
701


AE396A
PESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGP GSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTE PSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSE TPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSE PATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSE GSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPG SEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPS
702


AE432A
EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETP GTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSES ATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPES GPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSE PATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATP ESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEG TSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPAT SGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSA PGSEPATS
703


AE468A
EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAP GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSES ATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTST EEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSE PATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSE GSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPG SPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESA TPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTE EGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAT
704


AE504A
EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAP
705



GSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSES ATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPES GPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTS TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSG SETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPG SEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEP SEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSET PGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEP ATSGSETPGTSESATPESGPGTSTEPS



AE540A
TPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESG PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTST EPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPE SGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGT STEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSP TSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETP GTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAG SPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTST EEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTS ESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSE GSAPGTSTEP
706


AE576A
TPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSET PGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTST EPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEG SAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGT SESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSP TSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGP GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTE PSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPES GPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTS ESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPT STEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESA
707


AE612A
GSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGP GSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTE PSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS APGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSE PATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSE GSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEG TSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESA TPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTE EGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPA GSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPE SGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGT STEPSEGSAPGSEPATSGSETPGTSESAT
708


AE648A
PESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAP GTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSES ATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGS APGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSP AGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATP ESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPG SPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEP SEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSET PGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEP ATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEG SAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGS EPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATS GSETPGTSESAT
709


AE684A
EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGP GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTE
710



PSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPES GPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTS TEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPT STEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPG SEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGS PTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSA PGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSE SATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGS ETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGT SESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATS GSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATS



AE720A
TSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGS APGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTS ESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSE GSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEG TSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEP SEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA PGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSE SATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPE SGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGT STEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATS GSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGP GSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTE PSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSE TPGSEPATSGSETPGSPAGSPTSTEEGTSTE
711


AE756A
TSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGS APGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTS ESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSE GSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEG TSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEP SEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA PGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSE SATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPE SGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGT STEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATS GSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGP GSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTE PSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSE TPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSE PATSGSETPGTSES
712


AE792A
EGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEE GTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE PSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPES GPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTS ESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATP ESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPG TSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESA TPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESG PGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPA GSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGS ETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGS PAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSP TSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGP GTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTE PSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPS
713


AE828A
PESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAP GTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSES ATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGS
714



APGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSE PATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATP ESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPG TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEP SEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESG PGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTST EPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTS TEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGT SESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSP TSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEE GSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSES ATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGS APGTSTEPSEGSAPGSEPATSGSETPGTSESAT



AE869
GSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGT STEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATS GSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAP GSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTE PSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGS APGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSE PATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE GSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEG TSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPAT SGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTE EGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSE SATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPE SGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGT SESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESAT PESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETP GSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSES ATPESGPGTSTEPSEGSAPGR
715


AE144_R 1
SAGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEE GTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPA TSGSETPGSPAGSPTSTEEGTSESATPESGPGTESASR
716


AE288 _R 1
SAGSPTGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPE SGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGT SESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESAT PESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETP GSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSES ATPESGPGTSTEPSEGSAPSASR
717


AE432_R 1
SAGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEE GTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPA TSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGS APGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTS TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSE GSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPG SEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEP SEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTE EGTESASR
718


AE576_R 1
SAGSPTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEG SAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGT STEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATS GSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP GTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSES ATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPES GPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTS ESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPT STEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPG SEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEP
719



SEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPSASR



AE864_R 1
SAGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEE GTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPA TSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGS APGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTS TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSE GSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPG SEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEP SEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTE EGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEP ATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTS TEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGT SESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESAT PESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETP GTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSES ATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSE TPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTS ESATPESGPGTESASR
720


AE712
PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTST EPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGS ETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGS PAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPS EGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAP GTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPA TSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS APGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTS TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSG SETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEG SPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESA TPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESG PGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSE SATPESGPGSPAGSPTSTEAHHH
721


AE864_R 2
GSPGAGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEE GTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPA TSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGS APGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTS TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSE GSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPG SEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEP SEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTE EGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEP ATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTS TEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGT SESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESAT PESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETP GTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSES ATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSE TPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTS ESATPESGPGTESASR
722


AE288_3
SPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPAT SGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSA PGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPA GSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPE SGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGT STEPSEGSAPGTSTEPSEGSAPG
723


AE284
GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSES ATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSE
724



TPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTS ESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSG SETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPG TSESATPESGPGTSTEPSE



AE292
SPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPAT SGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSA PGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPA GSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPE SGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGT STEPSEGSAPGTSTEPSEGSAPGGSAP
725


AE864_2
AGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSE GSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPG SPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGS PTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSA PGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSE SATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGS ETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGT STEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPS EGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETP GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAG SPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPES GPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS TEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATP ESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPG TSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGS PTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESG PGTSTEPSEGAAEPEA
726


AE867
GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE PSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSE TPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSP AGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSE GSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPG TSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPAT SGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTST EPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGS ETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGS PAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESAT PESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGP GTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSES ATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPES GPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSP AGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATP ESGPGTSTEPSEGAAEPEA
727


AE867_2
SPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTS TEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSG SETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPG SPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEP SEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSA PGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEP ATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEG SAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGT STEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATS GSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEE GSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSES ATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPES GPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTS ESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATP
728



ESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPG SPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESA TPESGPGTSTEPSEGSAPG



AE868
PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTST EPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGS ETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGS PAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPS EGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAP GTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPA TSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS APGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTS TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSG SETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEG SPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESA TPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESG PGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSE SATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPE SGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGS PAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAT PESGPGTSTEPSEGAAEPEA
729


AE144_7 A
GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE PSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSE TPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP
730


AE292
SPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPAT SGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSA PGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPA GSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPE SGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGT STEPSEGSAPGTSTEPSEGSAPGGSAP
731


AE293
PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTST EPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGS ETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGS PAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPS EGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAP GTSESATPESGPGTSESATPEGAAEPEA
732


AE300
PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTST EPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGS ETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGS PAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPS EGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAP GTSESATPESGPGTSESATPESGPGSPAGAAEPEA
733


AE584
PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTST EPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGS ETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGS PAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPS EGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAP GTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPA TSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGS APGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTS TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSG SETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEG SPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGAAEPE A
734






The disclosure contemplates compositions of any of the embodiments described herein comprising XTEN of intermediate lengths to those of Table 7, as well as XTEN of longer lengths than those of Table 7, such as those in which motifs of 12 amino acids of Table 6 are added to the N- or C- terminus of an XTEN of Table 7.


In another embodiment, the disclosure contemplates polypeptide compositions of any of the embodiments described herein comprising an XTEN1 and an XTEN2 that can further comprise a His tag of HHHHHH (SEQ ID NO: 794) or HHHHHHHH (SEQ ID NO: 795) at the N-terminus and/or the sequence EPEA (SEQ ID NO: 796) at the C-terminus, respectively, of the polypeptide composition to facilitate the purification of the composition to at least 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, including but not limited to those described in the Examples, below.


Additional examples of XTEN sequences that can be used according to the present disclosure and are disclosed in U.S. Pat. Publication Nos. 2010/0239554 A1, 2010/0323956 A1, 2011/0046060 A1, 2011/0046061 A1, 2011/0077199 A1, or 2011/0172146 A1, or International Pat. 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.


V). CD3 Cell Antigen Binding Fragments

In another aspect, the disclosure relates to antigen binding fragments (AF2) having specific binding affinity for an effector cell antigen that can be incorporated into any of the subject composition embodiments described herein. In some cases, the effector cell antigen is expressed on the surface of an effector cell selected from a plasma cell, a T cell, a B cell, a cytokine induced killer cell (CIK cell), a mast cell, a dendritic cell, a regulatory T cell (RegT cell), a helper T cell, a myeloid cell, and a NK cell.


Various AF2 that bind effector cell antigens have particular utility for pairing with an antigen binding fragment with binding affinity to EGFR antigens associated with a diseased cell or tissue in composition formats in order to effect cell killing of the diseased cell or tissue. Binding specificity 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, having a first antigen binding fragment (AF1) to EGFR linked by a short, flexible peptide linker to a second antigen binding fragment (AF2) 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 AF1 directed against an EGFR of a disease tissue is used in combination with a AF2 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 AF1 and AF2 are incorporated into the specifically designed polypeptides comprising cleavable release segments and XTEN in order to confer prodrug 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 one embodiment, the AF2 of the subject compositions has binding affinity for an effector cell antigen expressed on the surface of a T cell. In another embodiment, the AF2 of the subject compositions has binding affinity for CD3. In another embodiment, 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, CD3 zeta, CD3 alpha and CD3 beta. In another embodiment, the AF2 has binding affinity for CD3 epsilon, CD3 delta, CD3 gamma, CD3 zeta, CD3 alpha or CD3 beta.


The origin of the antigen binding fragments contemplated by the disclosure can be derived from a naturally occurring antibody or fragment thereof, a non-naturally occurring antibody or fragment thereof, a humanized antibody or fragment thereof, a synthetic antibody or fragment thereof, a hybrid antibody or fragment thereof, or an engineered antibody or fragment thereof. Methods for generating an antibody for a given target marker are well known in the art. For example, the monoclonal antibodies may be made using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be made by recombinant DNA methods (U.S. Pat. No. 4,816,567). The structure of antibodies and fragments thereof, variable regions of heavy and light chains of an antibody (VH and VL), single chain variable regions (scFv), complementarity determining regions (CDR), and domain antibodies (dAbs) are well understood. Methods for generating a polypeptide having a desired antigen binding fragment with binding affinity to a given antigen are known in the art.


It will be understood that use of the term antigen binding fragments for the composition embodiments disclosed herein is intended to include portions or fragments of antibodies that retain the ability to bind the antigens that are the ligands of the corresponding intact antibody. In such embodiments, the antigen binding fragment can be, but is not limited to, CDRs and intervening framework regions, variable or hypervariable regions of light and/or heavy chains of an antibody (VL, VH), variable fragments (Fv), Fab′ fragments, F(ab′)2 fragments, Fab fragments, single chain antibodies (scAb), VHH camelid antibodies, single chain variable fragment (scFv), linear antibodies, a single domain antibody, complementarity determining regions (CDR), domain antibodies (dAbs), single domain heavy chain immunoglobulins of the BHH or BNAR type, single domain light chain immunoglobulins, or other polypeptides known in the art containing a fragment of an antibody capable of binding an antigen. The antigen binding fragments having CDR-H and CDR-L can be configured in a (CDR-H)-(CDR-L) or a (CDR-H)-(CDR-L) orientation, N-terminus to C-terminus. The VL and VH of two antigen binding fragments can also be configured in a single chain diabody configuration; i.e., the VL and VH of the AF1 and AF2 configured with linkers of an appropriate length to permit arrangement as a diabody.


Various CD3 binding AF2 of the disclosure have been specifically modified to enhance their stability in the polypeptide embodiments described herein. Protein aggregation of antibodies continues to be a significant problem in their developability and remains a major area of focus in antibody production. Antibody aggregation can be triggered by partial unfolding of its domains, leading to monomer-monomer association followed by nucleation and aggregate growth. Although the aggregation propensities of antibodies and antibody-based proteins can be affected by the external experimental conditions, they are strongly dependent on the intrinsic antibody properties as determined by their sequences and structures. Although it is well known that proteins are only marginally stable in their folded states, it is often less well appreciated that most proteins are inherently aggregation-prone in their unfolded or partially unfolded states, and the resulting aggregates can be extremely stable and long-lived. Reduction in aggregation propensity has also been shown to be accompanied by an increase in expression titer, showing that reducing protein aggregation is beneficial throughout the development process and can lead to a more efficient path to clinical studies. For therapeutic proteins, aggregates are a significant risk factor for deleterious immune responses in patients, and can form via a variety of mechanisms. Controlling aggregation can improve protein stability, manufacturability, attrition rates, safety, formulation, titers, immunogenicity, and solubility. The intrinsic properties of proteins such as size, hydrophobicity, electrostatics and charge distribution play important roles in protein solubility. Low solubility of therapeutic proteins due to surface hydrophobicity has been shown to render formulation development more difficult and may lead to poor biodistribution, undesirable pharmacokinetics behavior and immunogenicity in vivo. Decreasing the overall surface hydrophobicity of candidate monoclonal antibodies can also provide benefits and cost savings relating to purification and dosing regimens. Individual amino acids can be identified by structural analysis as being contributory to aggregation potential in an antibody, and can be located in CDR as well as framework regions. In particular, residues can be predicted to be at high risk of causing hydrophobicity issues in a given antibody. In one embodiment, the present disclosure provides an AF2 having the capability to specifically bind CD3 in which the AF2 has at least one amino acid substitution of a hydrophobic amino acid in a framework region relative to the parental antibody or antibody fragment wherein the hydrophobic amino acid is selected from isoleucine, leucine or methionine. In another embodiment, the CD3 AF2 has at least two amino acid substitutions of hydrophobic amino acids in one or more framework regions wherein the hydrophobic amino acids are selected from isoleucine, leucine or methionine.


The isoelectric point (pI) is the pH at which the antibody or antibody fragment has no net electrical charge. If the pH is below the pI of an antibody or antibody fragment, then it will have a net positive charge. A greater positive charge tends to correlate with increased blood clearance and tissue retention, with a generally shorter half-life. If the pH is greater than the pI of an antibody or antibody fragment it will have a negative charge. A negative charge generally results in decreased tissue uptake and a longer half-life. It is possible to manipulate this charge through mutations to the framework residues. These considerations informed the design of the sequences of the AF2 of the embodiments described herein wherein individual amino acid substitutions were made relative to the parental antibody utilized as the starting point. The isoelectric point of a polypeptide can be determined mathematically (e.g., computationally) or experimentally in an in vitro assay. The isoelectric point (pI) is the pH at which a protein has a net charge of zero and can be calculated using the charges for the specific amino acids in the protein sequence. Estimated values for the charges are called acid dissociation constants or pKa values and are used to calculate the pI. The pI can be determined in vitro by methods such as capillary isoelectric focusing (see Datta-Mannan, A., et al. The interplay of non-specific binding, target-mediated clearance and FcRn interactions on the pharmacokinetics of humanized antibodies. mAbs 7:1084 (2015); Li, B., et al. Framework selection can influence pharmacokinetics of a humanized therapeutic antibody through differences in molecule charge. mAbs 6, 1255-1264 (2014)) or other methods known in the art. In some embodiments, the isoelectric points of the AF1 and AF2 are designed to be within a particular range of each other, thereby promoting stability.


In one embodiment, the present disclosure provides an AF2 for use in any of the polypeptide embodiments described herein comprising CDR-L and CDR-H, wherein the AF2 (a) specifically binds to cluster of differentiation 3 T cell receptor (CD3); and (b) comprises CDR-H1, CDR-H2, and CDR-H3, having amino acid sequences of SEQ ID NOS: 742, 743, and 744, respectively. In another embodiment, the present disclosure provides an AF2 for use in any of the polypeptide embodiments described herein comprising CDR-L and CDR-H, wherein the AF2 (a) specifically binds to cluster of differentiation 3 T cell receptor (CD3); (b) comprises CDR-H1, CDR-H2, and CDR-H3, having amino acid sequences of SEQ ID NOS: 742, 743, and 744, respectively; and (c) comprises CDR-L wherein the CDR-L comprises a CDR-L1 having an amino acid sequence of SEQ ID NOS: 735 or 736, a CDR-L2 having an amino acid sequence of SEQ ID NOS: 738 or 739, and a CDR-L3 having an amino acid sequence of SEQ ID NO:740. In another embodiment, the foregoing AF2 embodiments of the paragraph further comprises light chain framework regions (FR-L) and heavy chain framework regions (FR-H) wherein AF2 comprises a FR-L1 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO:746, a FR-L2 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO:747, a FR-L3 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of any one of SEQ ID NOS:748-751, a FR-L4 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO:754, a FR-H1 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO:755 or SEQ ID NO:756, a FR-H2 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO:759, a FR-H3 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO:760; and a FR-H4 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO:764. In another embodiment, the AF2 for use in any of the polypeptide embodiments described herein comprises light chain framework regions (FR-L) and heavy chain framework regions (FR-H) wherein AF2 comprises a FR-L1 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO:746, a FR-L2 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO:747, a FR-L3 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO:748, FR-L4 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO:754, a FR-H1 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO:755, a FR-H2 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO:759, a FR-H3 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO:760; and a FR-H4 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO:764. In another embodiment, the AF2 for use in any of the polypeptide embodiments described herein comprises light chain framework regions (FR-L) and heavy chain framework regions (FR-H) wherein AF2 comprises a FR-L1 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO:746, a FR-L2 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO:747, a FR-L3 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO:749, a FR-L4 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO:754, a FR-H1 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO:755, a FR-H2 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO:759, a FR-H3 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO:760; and a FR-H4 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO:764. In another embodiment, the AF2 of the subject polypeptide embodiments described herein comprises light chain framework regions (FR-L) and heavy chain framework regions (FR-H) wherein AF2 comprises a FR-L1 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO:746, a FR-L2 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO:747, a FR-L3 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO:750, a FR-L4 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO:754, a FR-H1 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO:755, a FR-H2 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO:759, a FR-H3 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO:760, and a FR-H4 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO:764. In another embodiment, the AF2 of the subject polypeptide embodiments described herein comprises light chain framework regions (FR-L) and heavy chain framework regions (FR-H) wherein AF2 comprises a FR-L1 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO:746, a FR-L2 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO:747, a FR-L3 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO:751, a FR-L4 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO:754, a FR-H1 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO:756, a FR-H2 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO:759, a FR-H3 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO:760, and a FR-H4 exhibiting at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to the amino acid sequence of SEQ ID NO:764.


In another embodiment, the present disclosure provides an AF2 for use in any of the polypeptide embodiments described herein wherein the AF2 comprises a variable heavy (VH) amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to an amino acid sequence of SEQ ID NO:766 or SEQ ID NO:769. In another embodiment, the present disclosure provides an AF2 for use in any of the polypeptide embodiments described herein wherein the AF2 comprises a variable light (VL) amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to an amino acid sequence of any one of SEQ ID NOS: 765, 767, 768, 770, or 771. In another embodiment, the present disclosure provides an AF2 for use in any of the polypeptide embodiments described herein wherein the AF2 comprises a variable heavy (VH) amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to an amino acid sequence of SEQ ID NO:766 or SEQ ID NO:769 and a variable light (VL) amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or is identical to an amino acid sequence of any one of SEQ ID NOS: 765, 767, 768, 770, or 771.


In another embodiment, the present disclosure provides an AF2 for use in any of the polypeptide embodiments described herein wherein the AF2 comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% sequence identity or is identical to an amino acid sequence of any one of SEQ ID NOS:776-780.


In another aspect, the present disclosure provides AF2 antigen binding fragments that bind to the CD3 protein complex that have enhanced stability compared to CD3 binding antibodies or antigen binding fragments known in the art. Additionally, the CD3 antigen binding fragments of the disclosure are 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 one approach, the CD3 AF2 of the present disclosure are designed to have a higher degree of thermal stability compared to certain CD3-binding antibodies and antigen binding fragments known in the art. As a result, the CD3 AF2 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). Thermal stability is determined by measuring the “melting temperature” (Tm), 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 Tm are 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.


Thermal denaturation curves of the CD3 binding fragments and the anti-CD3 bispecific antibodies comprising said anti-CD3 binding fragment and a reference binding of the present disclosure show that the constructs of the present disclosure are more resistant to thermal denaturation than the antigen binding fragment consisting of a sequence shown in SEQ ID NO:781 or a control bispecific antibody wherein said control bispecific antigen binding fragment comprises SEQ ID NO:781 and a reference antigen binding fragment that binds to an EGFR embodiment described herein. In one embodiment, the polypeptides of any of the subject composition embodiments described herein comprise an anti-CD3 AF2 of the embodiments described herein, wherein the Tm of the AF2 is at least 2° C. greater, or at least 3° C. greater, or at least 4° C. greater, or at least 5° C. greater, or at least 6° C. greater, or at least 7° C. greater, or at least 8° C. greater, or at least 9° C. greater, or at least 10° C. greater than the Tm of an antigen binding fragment consisting of a sequence of SEQ ID NO:781, as determined by an increase in melting temperature in an in vitro assay.


In another embodiment, the polypeptides of any of the subject composition embodiments described herein comprise an AF2 that specifically binds human or cyno CD3 with a dissociation constant (Kd) 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 or cyno CD3 antigen. In another embodiment, the polypeptides of any of the subject composition embodiments described herein comprise an AF2 that specifically binds human or cyno CD3 with a dissociation constant (Kd) 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 with a Kd of 400 binds its ligand more weakly than one with a Kd of 10 nM. In another embodiment, the polypeptides of any of the subject composition embodiments described herein comprise an AF2 that specifically binds human or cyno 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 SEQ ID NO: 781, as determined by the respective dissociation constants (Kd) in an in vitro antigen-binding assays. In another embodiment, the present disclosure provides bispecific polypeptides comprising an AF2 that exhibits 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, 10-fold, 20-fold, 50-fold, 100-fold, or at least 1000-fold at weaker relative to that of the AF1 EGFR embodiments described herein that are incorporated into the subject polypeptides, as determined by the respective dissociation constants (Kd) in an in vitro antigen-binding assay. The binding affinity of the subject compositions for the target ligands can be assayed 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. 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 a related aspect, the present disclosure provides AF2 that bind to CD3 and are incorporated into chimeric, bispecific polypeptide compositions that are designed to have an isoelectric point (pI) that confer enhanced stability on the compositions of the disclosure compared to corresponding compositions comprising CD3 binding antibodies or antigen binding fragments known in the art. In one embodiment, the polypeptides of any of the subject composition embodiments described herein comprise AF2 that bind to CD3 wherein the AF2 exhibits a pI that is between 6.0 and 6.6, inclusive. In another embodiment, the polypeptides of any of the subject composition embodiments described herein comprise AF2 that bind to CD3 wherein the AF2 exhibits a pI 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 pI of a reference antigen binding fragment consisting of a sequence shown in SEQ ID NO: 781. In another embodiment, the polypeptides of any of the subject composition embodiments described herein comprise an AF2 that binds to CD3 fused to an AF1 that binds to an EGFR antigen wherein the AF2 exhibits a pI 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 pI of the AF1 that binds EGFR antigen or an epitope thereof. In another embodiment, the polypeptides of any of the subject composition embodiments described herein comprise an AF2 that binds to CD3 fused to an AF1 that binds to an EGFR antigen wherein the AF2 exhibits a pI 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 pI of the AF1. It is specifically intended that by such design wherein the pI 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. State differently, having the AF2 and the AF1 within a relatively narrow pI range of may allow for the selection of a buffer or other solution in which both the AF2 and AF1 are stable, thereby promoting overall stability of the composition.


In certain embodiments, the VL and VH of the antigen binding fragments are fused by relatively long linkers, consisting 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 hydrophilic amino acids that, when joined together, have a flexible characteristic. In one embodiment, the VL and VH of any of the scFv embodiments described herein are linked by relatively long linkers of hydrophilic amino acids selected from the sequences









GSGEGSEGEGGGEGSEGEGSGEGGEGEGSG (SEQ ID NO: 790),













TGSGEGSEGEGGGEGSEGEGSGEGGEGEGSGT (SEQ ID NO: 791),













GATPPETGAETESPGETTGGSAESEPPGEG (SEQ ID NO: 792), o


r













GSAAPTAGTTPSASPAPPTGGSSAAGSPST (SEQ ID NO: 793).






In another embodiment, 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 one embodiment, the short linker sequences are selected from the group of sequences SGGGGS (SEQ ID NO: 797), GGGGS (SEQ ID NO: 798), GGSGGS (SEQ ID NO: 799), GGS, or GSP. In another embodiment, 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. As will be appreciated by one of skill in the art, the selection of the short linker and relatively long linker is to prevent the incorrect pairing of adj acent variable domains, thereby facilitating the formation of the single chain diabody configuration comprising the VL and VH of the first antigen binding fragment and the second antigen binding fragment.





TABLE 8







CD3 CDR SEQUENCES


Construct
CDR REGION
Amino Acid Sequence
SEQ ID NO:




3.23, 3.30, 3.31, 3.32
CDR-L1
RSSNGAVTSSNYAN
735


3.24
CDR-L1
RSSNGEVTTSNYAN
736


3.33, 3.9
CDR-L1
RSSTGAVTTSNYAN
737


3.23, 3.30, 3.31, 3.32, 3.9, 3.33
CDR-L2
GTNKRAP
738


3.24
CDR-L2
GTIKRAP
739


3.23, 3.24, 3.30, 3.31, 3.32
CDR-L3
ALWYPNLWVF
740


3.33, 3.9
CDR-L3
ALWYSNLWVF
741


3.23, 3.24, 3.30, 3.31, 3.32, 3.9, 3.33
CDR-H1
GFTFNTYAMN
742


3.23, 3.24, 3.30, 3.31, 3.32, 3.9, 3.33
CDR-H2
RIRSKYNNYATYYADSVKD
743


3.23, 3.24, 3.30, 3.31, 3.32
CDR-H3
HENFGNSYVSWFAH
744


3.9, 3.33
CDR-H3
HGNFGNSYVSWFAY
745









TABLE 9







CD3 FR SEQUENCES


Construct
FR REGION
Amino Acid Sequence
SEQ ID NO:




3.23, 3.24, 3.30, 3.31, 3.32, 3.9, 3.33
FR-L1
ELVVTQEPSLTVSPGGTVTLTC
746


3.23, 3.24, 3.30, 3.31, 3.32, 3.9, 3.33
FR-L2
WVQQKPGQAPRGLIG
747


3.23, 3.24
FR-L3
GTPARFSGSLLGGKAALTLSGVQPEDEAVYYC
748


3.30
FR-L3
GTPARFSGSSLGGKAALTLSGVQPEDEAVYYC
749


3.31
FR-L3
GTPARFSGSLLGGSAALTLSGVQPEDEAVYYC
750


3.32
FR-L3
GTPARFSGSSLGGSAALTLSGVQPEDEAVYYC
751


3.9
FR-L3
GTPARFSGSLLGGKAALTLSGVQPEDEAEYYC
752


3.33
FR-L3
GTPARFSGSSLGGSAALTLSGVQPEDEAEYYC
753


3.23, 3.24, 3.30, 3.31, 3.32, 3.9, 3.33
FR-L4
GGGTKLTVL
754


3.23, 3.24
FR-H1
EVQLLESGGGIVQPGGSLKLSCAAS
755


3.30, 3.31, 3.32
FR-H1
EVQLQESGGGIVQPGGSLKLSCAAS
756


3.33
FR-H1
EVQLQESGGGLVQPGGSLKLSCAAS
757


3.9
FR-H1
EVQLLESGGGLVQPGGSLKLSCAAS
758


3.23, 3.24, 3.30, 3.31, 3.32, 3.9, 3.33
FR-H2
WVRQAPGKGLEWVA
759


3.23, 3.24, 3.30, 3.31, 3.32
FR-H3
RFTISRDDSKNTVYLQMNNLKTEDTAVYYCVR
760


3.9, 3.33
FR-H3
RFTISRDDSKNTAYLQMNNLKTEDTAVYYCVR
762


3.23, 3.24, 3.30, 3.31, 3.32, 3.9, 3.33
FR-H4
WGQGTLVTVSS
764









TABLE 10







VL & VH SEQUENCES


Construct
REGION
Amino Acid Sequence
SEQ ID




3.23
VL
ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQK PGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQP EDEAVYYCALWYPNLWVFGGGTKLTVL
765


3.23, 3.24
VH
EVQLLESGGGIVQPGGSLKLSCAASGFTFNTYAMNWVRQAP GKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTVY LQMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTV SS
766


3.24
VL
ELVVTQEPSLTVSPGGTVTLTCRSSNGEVTTSNYANWVQQK PGQAPRGLIGGTIKRAPGTPARFSGSLLGGKAALTLSGVQP EDEAVYYCALWYPNLWVFGGGTKLTVL
767


3.30
VL
ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQK PGQAPRGLIGGTNKRAPGTPARFSGSSLGGKAALTLSGVQP EDEAVYYCALWYPNLWVFGGGTKLTVL
768


3.30, 3.31, 3.32
VH
EVQLQESGGGIVQPGGSLKLSCAASGFTFNTYAMNWVRQAP GKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTVY LQMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWGQGTLVTV SS
769


3.31
VL
ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQK PGQAPRGLIGGTNKRAPGTPARFSGSLLGGSAALTLSGVQP EDEAVYYCALWYPNLWVFGGGTKLTVL
770


3.32
VL
ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQK PGQAPRGLIGGTNKRAPGTPARFSGSSLGGSAALTLSGVQP EDEAVYYCALWYPNLWVFGGGTKLTVL
771


3.9
VL
ELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQK PGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQP EDEAEYYCALWYSNLWVFGGGTKLTVL
772


3.9
VH
EVQLLESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAP GKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAY LQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTV SS
773


3.33
VL
ELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQK PGQAPRGLIGGTNKRAPGTPARFSGSSLGGSAALTLSGVQP EDEAEYYCALWYSNLWVFGGGTKLTVL
774


3.33
VH
EVQLQESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAP GKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAY LQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTV SS
775









TABLE 11:






scFv sequences


Construct
Amino Acid Sequence
SEQ ID NO:




3.23
ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIG GTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGG GTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLLESGGGIVQP GGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADS VKDRFTISRDDSKNTVYLQMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWG QGTLVTVSS
776


3.24
ELVVTQEPSLTVSPGGTVTLTCRSSNGEVTTSNYANWVQQKPGQAPRGLIG GTIKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGG GTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLLESGGGIVQP GGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADS VKDRFTISRDDSKNTVYLQMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWG QGTLVTVSS
777


3.30
ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIG GTNKRAPGTPARFSGSSLGGKAALTLSGVQPEDEAVYYCALWYPNLWVFGG GTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLQESGGGIVQP GGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADS VKDRFTISRDDSKNTVYLQMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWG QGTLVTVSS
778


3.31
ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIG GTNKRAPGTPARFSGSLLGGSAALTLSGVQPEDEAVYYCALWYPNLWVFGG GTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLQESGGGIVQP GGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADS VKDRFTISRDDSKNTVYLQMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWG QGTLVTVSS
779


3.32
ELVVTQEPSLTVSPGGTVTLTCRSSNGAVTSSNYANWVQQKPGQAPRGLIG GTNKRAPGTPARFSGSSLGGSAALTLSGVQPEDEAVYYCALWYPNLWVFGG GTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLQESGGGIVQP GGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADS VKDRFTISRDDSKNTVYLQMNNLKTEDTAVYYCVRHENFGNSYVSWFAHWG QGTLVTVSS
780


3.9
ELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIG GTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGG GTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLLESGGGLVQP GGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADS VKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWG QGTLVTVSS
781


3.33
ELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIG GTNKRAPGTPARFSGSSLGGSAALTLSGVQPEDEAEYYCALWYSNLWVFGG GTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLQESGGGLVQP GGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADS VKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWG QGTLVTVSS
782


4.11
QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYR NNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLSGLWVF GGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGQVQLQQWGGGLV KPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSRINSDGSSTNYADS VKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARELRWGNWGQGTLVTVS S
783


4.12
QAGLTQPPSASGTPGQRVTLSCSGSYSNIGTYYVYWYQQLPGTAPKLLIYS NDQRLSGVPDRFSGSKSGTSASLAISGLQSEDEAAYYCAAWDDSLNGWAFG GGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGQVQLQQWGGGLVK PGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSRINSDGSSTNYADSV KGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARELRWGNWGQGTLVTVSS
784


4.13
QPGLTQPPSASGTPGQRVTLSCSGRSSNIGSYYVYWYQHLPGMAPKLLIYR NSRRPSGVPDRFSGSKSGTSASLVISGLQSDDEADYYCAAWDDSLKSWVFG GGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGQVQLQQWGGGLVK PGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSRINSDGSSTNYADSV KGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARELRWGNWGQGTLVTVSS
785


4.14
QSVLTQPPSASGTPGQRVTISCSGSSSNIGTNYVYWYQQFPGTAPKLLIYS NNQRPSGVPDRFSGSKSGTSGSLAISGLQSEDEADYSCAAWDDSLNGWVFG GGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGQVQLVQWGGGLVK PGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSRINSDGSSTNYADSV KGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARELRWGNWGQGTLVTVSS
786


4.15
QPGLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYR NNQRPSGVPDRLSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLSGWVFG GGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGQVQLVQWGGGLVK PGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSRINSDGSSTNYADSV KGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARELRWGNWGQGTLVTVSS
787


4.16
QAVLTQPPSASGTPGQRVTISCSGSSSNIGSYYVYWYQQVPGAAPKLLMRL NNQRPSGVPDRFSGAKSGTSASLVISGLRSEDEADYYCAAWDDSLSGQWVF GGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGQVQLQQWGGGLV KPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSRINSDGSSTNYADS VKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARELRWGNWGQGTLVTVS S
788


4.17
QAGLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYR NNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDASLSGWVFG GGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLVQWGGGLVK PGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSRINSDGSSTNYADSV KGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARELRWGNWGQGTLVTVSS
789






VI). Bispecific Antigen Binding Compositions - Configurations and Functional Properties

In another aspect, the present disclosure relates to novel chimeric, bispecific antigen binding compositions that bind to an antigen or epitope of the CD3 protein complex of effector cells (e.g., a T cell) and an EGFR antigen associated with a diseased cell or tissue. Thus, they can be referred to T-cell engagers. As described more fully, below, the bispecific antigen binding compositions are configured in an activatable prodrug form that confer advantages over bispecific T-cell engagers and related compounds known in the art. The compositions of the disclosure have properties that include enhanced stability during their production and purification, enhanced stability and increased half-life in circulation when administered to a subject, the ability to become activated at intended sites of therapy but not in normal, healthy tissue, and, when activated by proteolytic cleavage of the release segments and release of the fused AF1 and AF2, exhibit binding affinity to target and effector cells that is at least comparable to a corresponding conventional bispecific IgG antibody. Upon the binding of the effector cell and target cell by the fused AF1 and AF2, an immunological synapse is formed that effects activation of the effector cell and promotes the subsequent destruction of the target cell via apoptosis or cytolysis.


Various bispecific antigen binding compositions of the disclosure described herein are specifically designed to be in a prodrug form in that the XTEN component(s) shield the antigen binding fragments, reducing their ability to bind their ligands until released from the composition by protease cleavage of any of the protease cleavage sites located within the release segments. Proteases known to be associated with diseased cells or tissues include but are not limited to serine proteases, cysteine proteases, aspartate proteases, and metalloproteases, including but not limited to the specific proteases described herein. This prodrug property of the bispecific antigen binding compositions improves the specificity of the composition towards diseased tissues or cells compared to bispecific T-cell engager therapeutics that are not in a prodrug format. In contrast, by activating the bispecific antigen binding compositions specifically in the microenvironment of the target cell or diseased tissue, where the EGFR antigen and proteases capable of cleaving the release segments are highly expressed, the bispecific antigen binding fragments and XTEN of the constructs are released upon cleavage of the release segment and the fused AF 1 and AF2 can crosslink cytotoxic effector cells with cells expressing an EGFR antigen in a highly specific fashion, thereby directing the cytotoxic potential of the T cell towards the target cell. After protease cleavage, the fused AF 1 and AF2 are no longer shielded and effectively regain their full potential to bind to target cells bearing an EGFR antigen and an effector cell such as a cytotoxic T cell via binding to the CD3 antigen, which forms part of the T cell receptor complex, causing T cell activation that mediates the subsequent lysis of the target cell expressing the particular EGFR antigen. Thus, the bispecific antigen binding compositions are contemplated to display strong, specific and efficient target cell killing. In such case, cells are eliminated selectively, thereby reducing the potential for toxic side effects.


The design of the subject compositions having a first and a second antigen binding fragment (AF1 and AF2, respectively) was driven by consideration of at least three properties: 1) compositions having bispecific antigen binding fragments with the capability to bind to and link together an effector cell and a target cell having an EGFR antigen with the resultant formation of an immunological synapse; 2) compositions with a XTEN that i) shields both of the antigen binding fragments and reduces their ability to bind the target and effector cell ligands when the composition is in an intact prodrug form, ii) provides enhanced half-life when administered to a subject, iii) reduces extravasation of the intact composition from the circulation in normal tissues and organs compared to diseased tissues (e.g., tumor), and iv) confers an increased safety profile compared to conventional bispecific cytotoxic antibody therapeutics; and 3) is activated when the RS is cleaved by one or more mammalian proteases in proximity of diseased tissues, thereby releasing the bispecific antigen binding fragments such that they regain their full binding affinity potential for the target ligands. The design of the subject compositions takes advantage of the properties of XTEN and the release segment (RS) components, and their positioning relative to the bispecific antigen binding fragments achieves the foregoing properties, as evidenced by the results in the illustrative Examples, below.


In one embodiment, the disclosure provides bispecific antigen binding compositions having two antigen binding fragments, an AF 1 and AF2 of any of the antigen binding fragment embodiments described herein, wherein the AF2 is fused to the AF1 by a flexible peptide linker. In one embodiment, the bispecific antigen binding fragment composition comprises a first antigen binding fragment (AF1) wherein the AF1 specifically binds to EGFR or an epitope thereof, and a second antigen binding fragment (AF2) wherein the AF2 specifically binds to cluster of differentiation 3 T cell receptor (CD3), wherein a difference between an isoelectric point (pI) of the second antigen binding fragment and a pI of the first antigen binding fragment is from 0 to about 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, as determined computationally or via in vitro assays. In one embodiment of the bispecific antigen binding composition, the AF1 specifically binds EGFR with a Kd between about 0.1 nM and about 100 nM, or about 0.5 nm and about 50 nM, or about 1 nm and about 20 nM, or about 2 nM and about 10 nM, as determined by an in vitro antigen-binding assay comprising EGFR or an epitope thereof. In another embodiment of the bispecific antigen binding composition, the AF2 specifically binds human or cyno CD3 with a dissociation constant (Kd) constant between about 0.1 nM and about 100 nM, or between about 0.5 nM and about 50 nM, or between about 1.0 nM and about 20 nM, or between about 2.0 nM and about 10 nM, as determined in an in vitro antigen-binding assay. In another embodiment of the bispecific antigen binding composition, the AF2 specifically binds human or cyno CD3 with a dissociation constant (Kd) constant between about 10 nM and about 400 nM, as determined in an in vitro antigen-binding assay. In yet another embodiment of the bispecific antigen binding composition, the AF2 specifically binds human or cyno CD3 with a dissociation constant (Kd) 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. In another embodiment of the bispecific antigen binding composition, the AF2 specifically binds human or cyno CD3 with a dissociation constant (Kd) weaker than about 3 nM, or 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 400 nM, as determined in an in vitro antigen-binding assay. In another embodiment of the bispecific antigen binding composition, the AF2 specifically binds human or cyno 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 antibody-binding fragment consisting of an amino acid sequence of SEQ ID NO: 781, as determined by the respective dissociation constants (Kd) in an in vitro antigen-binding assays. In another embodiment of the bispecific antigen binding composition, the AF2 exhibits 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, 10-fold, 20-fold, 50-fold, 100-fold, or at least 1000-fold at weaker relative to that of the AF1, as determined by the respective dissociation constants (Kd) in an in vitro antigen-binding assay. For clarity, an antigen binding fragment with a Kd of 400 binds its ligand more weakly than one with a Kd of 10 nM.


In another embodiment of the bispecific antigen binding composition of any of the subject embodiments described herein having two antigen binding fragments (AF1 and AF2), a single RS, and a single XTEN, the polypeptide can have, in an uncleaved state, a structural arrangement from N-terminus to C-terminus of AF2-AF1-RS1-XTEN1, AF1-AF2-RS1-XTEN1, XTEN1-RS1-AF2-AF1, XTEN1-RS1-AF1-AF2, or diabody-RS1-XTEN1, or XTEN1-RS1-diabody, wherein the diabody comprises VL and VH of the AF1 and AF2.


In another aspect, it is a feature of various designed compositions of any of the embodiments described herein that when the RS of the bispecific antigen binding composition is cleaved by a mammalian protease in the environment of the target cell and is converted from the prodrug form to the activated or apoprotein form, upon cleavage and release of the bispecific antigen binding fragments and the XTEN from the composition, the fused AF1 and AF2 bind to and link together an effector cell (e.g., a T cell bearing CD3) targeted by the AF2 and a diseased cell bearing the EGFR antigen of a target cell targeted by the AF1, whereupon the effector cell is activated. In one embodiment, wherein RS of the bispecific antigen binding composition is cleaved and the antigen binding fragments are released, the subsequent concurrent binding of the effector cell and the target cell results in at least a 3-fold, or a 10-fold, or a 30-fold, or a 100-fold, or a 300-fold, or a 1000-fold activation of the effector cell, wherein the activation is assessed by the production of cytokines, cytolytic proteins, or lysis of the target cell, assessed in an in vitro cell-based assay. In another embodiment, the concurrent binding of a T cell bearing the CD3 antigen and a target cell bearing the EGFR antigen by the released antigen binding fragments forms an immunologic synapse, wherein the binding results in the release of T cell-derived effector molecules capable of lysing the diseased cell. Non-limiting examples of the in vitro assay for measuring effector cell activation and/or cytolysis include cell membrane integrity assay, mixed cell culture assay, FACS based propidium Iodide assay, trypan Blue influx assay, photometric enzyme release assay, ELISA, radiometric 51Cr release assay, fluorometric Europium release assay, CalceinAM release assay, photometric MTT assay, XTT assay, WST-1 assay, alamar Blue assay, radiometric 3H-Thd incorporation assay, clonogenic assay measuring cell division activity, fluorometric Rhodamine123 assay measuring mitochondrial transmembrane gradient, apoptosis assay monitored by FACS-based phosphatidylserine exposure, ELISA-based TUNEL test assay, caspase activity assay, and cell morphology assay, or other assays known in the art for the assay of cytokines, cytolytic proteins, or lysis of cells, or the methods described in the Examples, below.


In other embodiments, the disclosure provides bispecific antigen binding compositions having two antigen binding fragments of any of the embodiments described herein, two RS of any of the embodiments described herein, and two XTEN of any of the embodiments described herein. The design of these compositions was driven by considerations of further reducing the binding affinity of the uncleaved compositions to the respective ligands of the AF1 and AF2 antibody fragments by the addition of the second XTEN in order to further reduce the unintended binding of the compositions to healthy tissues or cells when administered to a subject, thereby further improving the therapeutic index of the subject compositions compared to compositions having only one RS and one XTEN. The addition of the second RS and second XTEN resulted in a surprising reduction of binding affinity of the intact, uncleaved polypeptide to the respective ligands of the AF1 and AF2 antibody fragments relative to those compositions having a single RS and XTEN, when assayed in vitro, and also resulted in reduced toxicity in animal models of disease when administered as therapeutically-effective doses, as described in the Examples, below. In embodiments of compositions having a two antigen binding fragments, two RS, and two XTEN, the compositions can have, in an uncleaved state, a structural arrangement from N-terminus to C-terminus of XTEN1-RS1-AF2-AF1-RS2-XTEN2, XTEN1-RS1-AF1-AF2-RS2-XTEN2, XTEN2-RS2-AF2-AF1-RS1-XTEN1, XTEN2-RS2-AF1-AF2-RS1-XTEN1, XTEN2-RS2-diabody-RS1-XTEN1, wherein the diabody comprises VL and VH of the AF1 and AF2, or XTEN1-RS1-diabody-RS2-XTEN2, wherein the diabody comprises VL and VH of the AF1 and AF2.


Without being bound to a particular theory, it is believed that using the bispecific antigen binding composition formats as described above, upon cleavage of the RS, the released fused AF1 and AF2 are capable of killing target cells by recruitment of cytotoxic effector cells without any need for pre- and/or co-stimulation. Further, the independence from pre- and/or co-stimulation of the effector cell may substantially contribute to the exceptionally high cytotoxicity mediated by the released, fused AF1 and AF2 antigen binding fragments. In some embodiments, the released AF1 and AF2, wherein the AF1 remains fused to the AF2 by a linker peptide, is designed with binding specificities such that it has the capability to bind and link together in close proximity cytotoxic effector cells (e.g., T cells, NK cells, cytokine induced killer cell (CIK cell)) to preselected EGFR antigens by the AF1 that has binding specificity to EGFR antigens associated with tumor cells, cancer cells, or cells associated with diseased tissues, thereby effecting an immunological synapse and a selective, directed, and localized effect of released cytokines and effector molecules against the target disease or cancer cell, with the result that disease or cancer cells are damaged or destroyed, resulting in therapeutic benefit to a subject. The released AF2 that binds to an effector cell antigen is capable of modulating one or more functions of an effector cell, resulting in or contributing to the cytolytic effect on the target tumor cell. The effector cell antigen can by expressed by the effector cell or other cells. In one embodiment, the effector cell antigen is expressed on cell surface of the effector cell. Non-limiting examples of effector cell antigens are CD3, CD4, CD8, CD16, CD25, CD38, CD45RO, CD56, CD57, CD69, CD95, CD107, and CD154. Thus, it will be understood by one of skill in the art that the configurations of the subject compositions are intended to selectively or disproportionately deliver the active form of the composition to the target tumor tissue or cancer cell, compared to healthy tissue or healthy cells in a subject in which the composition is administered, with resultant therapeutic benefit. As is evident from the foregoing, the disclosure provides a large family of polypeptides in designed configurations to effect the desired properties.


It is an object of the disclosure that the design of the subject bispecific antigen binding compositions, with the shielding effect imparted by the XTEN of the intact, circulating composition and the concomitant reduced potential to bind to effector cells and target tissues, results in reduced production of Th1 T-cell associated cytokines or other proinflammatory mediators during systemic exposure when administered to a subject such that the overall side-effect and safety profile (e.g., the therapeutic index) is improved compared to bispecific antigen binding compositions not linked to a shielding moiety such as an XTEN. As an important component of cellular immunity, the production of IL-2, TNF-alpha, and IFN-gamma are hallmarks of a Th1 response (Romagnani S. T-cell subsets (Th1 versus Th2). Ann Allergy Asthma Immunol. 2000. 85(1):9-18), particularly in T cells stimulated by anti-CD3 (Yoon, S.H. Selective addition of CXCR3+CCR4-CD4+ Th1 cells enhances generation of cytotoxic T cells by dendritic cells in vitro. Exp Mol Med. 2009. 41(3):161-170), and Il-4, IL-6, and IL-10 are also proinflammatory cytokines important in a cytotoxic response for bispecific antibody compositions (Zimmerman, Z., et al. Unleashing the clinical power of T cells: CD19/CD3 bispecific T cell engager (BiTE®) antibody composition blinatumomab as a potential therapy. Int. Immunol. (2015) 27(1): 31-37). In one embodiment, an intact, uncleaved bispecific antigen binding composition of the embodiments described herein can exhibit at least 3-fold, or at least 4-fold, or at least 5-fold, or at least 6-fold, or at least 7-fold, or at least 8-fold, or at least 9-fold, or at least 10-fold, or at least 20-fold, or at least 30-fold, or at least 50-fold, or at least 100-fold, or at least 1000-fold reduced potential to result in the production of Th1 and/or proinflammatory cytokines when the intact, uncleaved polypeptide is in contact with the effector cell and a target cell in an in vitro cell-based cytokine stimulation assay compared to the Th1 and/or cytokine levels stimulated by the corresponding released AF1 and AF2 (which remain fused together after release by proteolysis of the RS) of a corresponding protease-treated composition in the in vitro cell-based stimulation cytokine assay performed under comparable conditions, e.g., equivalent molar concentrations. Non-limiting examples of Th1 and/or proinflammatory cytokines are IL-2, IL-4, IL-6, IL-10, TNF-alpha and IFN-gamma. In one embodiment of the foregoing, the production of the Th1 cytokine is assayed in an in vitro assay comprising effector cells such as PBMC or CD3+ T cells and target cells having an EGFR antigen disclosed herein. In another embodiment, the cytokines can be assessed from a blood, fluid, or tissue sample removed from a subject in which the polypeptide composition has been administered. In the foregoing embodiment, the subject can be mouse, rat, monkey, and human. In an advantage of the subject bispecific antigen binding compositions of the embodiments described herein, however, it has been discovered that the cytolytic properties of the compositions do not require prestimulation by cytokines; that formation of the immunological synapse of the effector cell bound to the target cell by the antigen binding fragments is sufficient to effect cytolysis or apoptosis in the target cell. Nevertheless, the production of proinflammatory cytokines are useful markers to assess the potency or the effects of the subject polypeptide compositions; whether by in vitro assay or in the monitoring of treatment of a subject with a tumor.


In the context of use of the bispecific antigen binding fragment compositions in a subject, it is an object of the disclosure that the subject bispecific antigen binding compositions were designed to take advantage of the differential in pore size of the vasculature in tumor or inflamed tissues compared to healthy vasculature by the addition of the XTEN, such that extravasation of the intact bispecific antigen binding composition in normal tissue is reduced, but in the leaky environment of the tumor vasculature or other areas of inflammation, the intact assembly can extravasate and be activated by the proteases in the diseased cell environment, releasing the antigen binding fragments to the effector and target cells (see, e.g., FIG. 5). In the case of the RS of the bispecific antigen binding compositions, the design takes advantage of the circumstance that when a bispecific antigen binding composition is in proximity to diseased tissues; e.g., a tumor, that elaborates one or more proteases, the RS sequences that are susceptible to the one or more proteases expressed by the tumor are capable of being cleaved by the proteases (described more fully, above). The action of the protease cleaves the release segment (RS) of the composition, separating the antigen binding fragments from the XTEN, resulting in components with reduced molecular weight and hydrodynamic radii, particularly for the released fused AF 1 and AF2. As will be appreciated, the decrease in molecular weight and hydrodynamic radius of the composition also confers the property that the released, fused AF 1 and AF2 are able to more freely move in solution, move through smaller pore spaces in tissue and tumors, and extravasate more readily from the larger pores of the tumor vasculature and more readily penetrate into the tumor, resulting in an increased ability to attach to and link together the effector cell and the tumor cell. Such property can be measured by different assays. Thus, it will be appreciated by one of skill in the art that in the context of treatment of a subject using the subject compositions, the bispecific antigen binding compositions are present in a prodrug form and are converted to a more active form when entering a certain cellular environment by the action of proteases co-localized with the disease tissue or cell. Upon release from the composition by the action of the protease(s) in the target tissue, the AF2 with binding specificity to an effector cell antigen and the linked AF1 with binding specificity to an antigen of a target cell regain their full capability to bind to and link together the effector cell to the target cell, forming an immunological synapse. The formation of the immunological synapse causes the effector cell to become activated, with various signal pathways turning on new gene transcription and the release, by exocytosis, the effector molecule contents of its vesicles. Depending on the type of effector cell, different cytokines and lymphokines are released; e.g., Type 1 helper T cells (Th1) release cytokines like IFN-gamma, IL-2 and TNF-alpha while Type 2 helper T cells (Th2) release cytokines like IL-4, IL-5, IL-10, and IL-13 that stimulate B cells, and cytotoxic T Lymphocytes (CTLs) release cytotoxic molecules like perforin and granzymes that kill the target (collectively, “effector molecules”). It is specifically contemplated that upon the concurrent binding to and linking together the effector cell to the target tumor cell by the released bispecific antigen binding fragments of the bispecific antigen binding composition, at very low effector to target (E:T) ratios the tumor cell is acted upon by the effector molecules released by the effector cell into the immunological synapse between the cells, resulting in damage, perforin-mediated lysis, granzyme B-induced cell death and/or apoptosis of the tumor cell. Thus, in another aspect, and without being bound by theory, it is a feature of the designed compositions that when the activatable bispecific antigen binding fragment composition is administered to a subject with a tumor, the prodrug form remains in the circulatory system in normal tissue but is able to extravasate in the more permeable vasculature of the tumor such that the prodrug form of the assembly is activated by the proteases co-localized with the tumor and that the released antigen binding fragments bind together and link an effector cell (e.g., a T cell) and a tumor cell expressing the EGFR antigen targeted by the AF1 of the composition, whereupon the effector cell is activated and lysis of the tumor cell is effected. Stated differently, in some cases, the more permeable vasculature in the tumor tissue may permit the bispecific antigen-binding polypeptide to extravasate into the tissue where the tumor-associated proteases can act on a release segment (RS), cleaving it and releasing the binding moieties, which in turn can bind to and link together the effector cell and the tumor associate cell. In the case of the normal tissue, the extravasation may be blocked by the tighter vasculature barriers or, in the case where the bispecific antigen binding polypeptide does extravasate to some extent, the bispecific antigen binding polypeptide may primarily remain in the “pro” form, as insufficient proteases may be present in the healthy tissue to release the binding moieties, with the net effect that an immunological synapse is not formed. In some cases, the released, fused AF1 and AF2 in the tumor of the subject bound to both a tumor cell and an effector cell exhibits an increased ability to activate effector cells of at least 10-fold, or at least 30-fold, or at least 100-fold, or at least 200-fold, or at least 300-fold, or at least 400-fold, or at least 500-fold, or at least 1000-fold compared to the corresponding intact, uncleaved bispecific antigen binding composition. In other cases, the released, fused AF1 and AF2 in the tumor of the subject bound to both a tumor cell and an effector cell exhibits an increased ability to lyse the tumor cell of at least 10-fold, or at least 30-fold, or at least 100-fold, or at least 200-fold, or at least 300-fold, or at least 400-fold, or at least 500-fold, or at least 1000-fold compared to the corresponding intact bispecific antigen binding composition that has not been cleaved in the tumor. In the foregoing embodiments, the effector cell activation and/or the cytotoxicity can be assayed by conventional methods known in the art, such as cytometric measurement of activated effector cells, assay of cytokines, measurement of tumor size, or by histopathology. In the foregoing embodiments, the subject can be mouse, rat, dog, monkey, and human. In particular, it is specifically contemplated that the subject compositions are designed such that upon administration to a subject with a disease having an EGFR antigen to which the AF2 can bind, the bispecific antigen binding composition exhibits an enhanced therapeutic index and reduced incidence of side effects, compared to conventional bispecific antibodies known in the art, achieved by a combination of the shielding effect and steric hindrance of XTEN on binding affinity over the antigen binding fragments in the prodrug form, yet are able to release the bispecific AF1 and AF2 (achieved by inclusion of the cleavage sequences in the RS) in proximity to or within a target tissue (e.g., a tumor) that produces a protease for which the RS is a substrate.


VII). Methods and Uses of Bispecific Antigen Binding Compositions

In another aspect, the present disclosure provides activatable bispecific antigen binding compositions and pharmaceutical compositions comprising a bispecific antigen binding composition that are particularly useful in medical settings; for example, in the prevention, treatment and/or the amelioration of certain cancers, tumors or inflammatory diseases. For use of treatment of diseases, bispecific antigen binding compositions of the invention would be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners.


A number of therapeutic strategies have been used to design the polypeptide compositions for use in methods of treatment of a subject with a cancerous disease, including the modulation of T cell responses by targeting TcR signaling, particularly using VL and VH portions of anti-human CD3 monoclonal antibodies that are widely used clinically in immunosuppressive regimes. The CD3-specific monoclonal OKT3 was the first such monoclonal approved for use in humans (Sgro, Toxicology 105 (1995), 23-29) and is widely used clinically as an immunosuppressive agent in transplantation (Chatenoud L: Immunologic monitoring during OKT3 therapy. Clin Transplant 7:422-430, 1993). Moreover, anti-CD3 monoclonals can induce partial T cell signaling and clonal anergy (Smith, J. Exp. Med. 185 (1997), 1413-1422). The OKT3 reacts with and blocks the function of the CD3 complex in the membrane of T cells; the CD3 complex being associated with the antigen recognition structure of T cells (TCR), which is essential for signal transduction. These and other such CD3 specific antibodies are able to induce various T cell responses, including cytokine production (Von Wussow, Human gamma interferon production by leukocytes induced with monoclonal antibodies recognizing T cells. J. Immunol. 127:1197-1200 (1981)), proliferation and suppressor T-cell induction. In cancer, attempts have been made to utilize cytotoxic T cells to lyse cancer cells. Without being bound by theory, to effect target cell lysis, cytotoxic T cells are believed to require direct cell-to-cell contact; the TCR on the cytotoxic T cell must recognize and engage the appropriate antigen on the target cell. This creates the immunologic synapse that, in turn initiates a signaling cascade within the cytotoxic T cell, causing T-cell activation and the production of a variety of cytotoxic cytokines and effector molecules. Perforin and granzymes are highly toxic molecules that are stored in preformed granules that reside in activated cytotoxic T cells. After recognition of the target cell, the cytoplasmic granules of the engaged cytotoxic T cells migrate toward the cytotoxic T-cell membrane, ultimately fusing with it and releasing their contents in directed fashion into the immunological synapse to form a pore within the membrane of the target cell, disrupting the tumor cell plasma membrane. The created pore acts as a point of entry for granzymes; a family of serine proteases that that induce apoptosis of the tumor cells.


The subject bispecific antigen binding compositions described herein, with an AF2 with specific binding affinity to the CD3 of a T cell closely fused to an AF1 with specific binding affinity to an EGFR antigen are T-cell engagers with the ability, once released from the intact prodrug form of the composition by cleavage of the release segments, regain their full potential to bind a T cell and target cell, forming an immunological synapse that promotes activation of the T-cell and promotes the subsequent destruction of the tumor cell via apoptosis or cytolysis.


The disclosure contemplates methods of use of bispecific antigen binding compositions that are engineered to target a range of malignant cells, such as tumors, in addition to the effector cells, in order to initiate target cell lysis and to effect a beneficial therapeutic outcome in that the bispecific antigen binding compositions are designed such that one antigen binding fragment binds and engages CD3 to activate the cytotoxic T cell while the second antigen binding fragment can be designed to target EGFR markers that are characteristic of specific malignancies; bridging them together for the creation of the immunological synapse. In a particular advantage of the design, the physical binding of the cytotoxic effector cell and the EGFR-bearing cell eliminates the need for antigen processing, MHCI/β2-microglobulin, as well as co-stimulatory molecules. Because of the range of cells bearing EGFR, it will be appreciated that the resulting compositions will have utility against a variety of cancers, including solid and hematological tumors. In one embodiment, the disclosure provides a method of treatment of a subject with a tumor. The tumor being treated can comprise tumor cells arising from a cell selected from the group consisting of stromal cell, fibroblasts, myofibroblasts, glial cells, epithelial cells, fat cells, lymphocytic cells, vascular cells, smooth muscle cells, mesenchymal cells, breast tissue cells, prostate cells, kidney cells, brain cells, colon cells, ovarian cells, uterine cells, bladder cells, skin cells, stomach cells, genito-urinary tract cells, cervix cells, uterine cells, small intestine cells, liver cells, pancreatic cells, gall bladder cells, bile duct cells, esophageal cells, salivary gland cells, lung cells, and thyroid cells. In a further advantage of the compositions, as the cytotoxic effector cells are not consumed during the damage/destruction of the bridged target cancer cell, after causing lysis of one target cell, an activated effector cell can release and move on through the local tissue towards other target cancer cells, bind the EGFR antigen, and initiate additional cell lysis. In addition, it is contemplated that in a localized environment like a solid tumor, the release of effector cell molecules such as perforin and granzymes will result in damage to tumor cells that are adjacent but not bound by a given molecule of the bispecific binding domains, resulting in stasis of growth or regression of the tumor.


Accordingly, a utility of the disclosure will be understood; that after administration of a therapeutically effective dose of pharmaceutical composition comprising a bispecific antigen binding composition described herein to a subject with a cancer or tumor having the EGFR antigen, the composition can be acted upon by proteases in association with or co-localized with the cancer or tumor cells, releasing the fused AF1 and AF2 such that an immunological synapse can be created by the linking of the EGFR-bearing cell and a effector cell, with the result that effector cell-derived effector molecules capable of lysing the target cell are released into the synapse, leading to apoptosis, cytolysis, or death of the target cancer or tumor cell. Furthermore, it will be appreciated by one of skill in the art that use of the bispecific antigen binding compositions can result in a sustained and more generalized beneficial therapeutic effect than a “single kill” once the immunological synapse is formed by the binding of the released binding domains to the effector cell and target cancer cell.


In one aspect, the disclosure relates to methods of treating a disease in a subject, such as a subject with a cancer. In some embodiments, the disclosure provides a method of treating a disease in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a bispecific antigen binding composition of any of the embodiments described herein. A therapeutically effective amount of the pharmaceutical composition may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the subject compositions are outweighed by the therapeutically beneficial effects. A prophylactically effective amount refers to an amount of pharmaceutical composition required for the period of time necessary to achieve the desired prophylactic result.


A therapeutically effective dose of the bispecific antigen binding compositions described herein will generally provide therapeutic benefit without causing substantial toxicity. Toxicity and therapeutic efficacy of a bispecific antigen binding composition can be determined by standard pharmaceutical procedures in cell culture or experimental animals. Cell culture assays and animal studies can be used to determine the LD50 (the dose lethal to 50% of a population) and the ED50 (the dose therapeutically effective in 50% of a population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50/ED50. Bispecific antigen binding compositions that exhibit large therapeutic indices are preferred. In one aspect, the bispecific antigen binding molecule according to the present invention exhibits a high therapeutic index. The data obtained from cell culture assays and animal studies can be used in formulating a range of dosages suitable for use in humans. The dosage lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon a variety of factors, e.g., the dosage form employed, the route of administration utilized, the condition of the subject, and the like. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient’s condition (see, e.g., Fingl et al., 1975, in: The Pharmacological Basis of Therapeutics, Ch. 1, p. 1). A skilled artisan readily recognizes that in many cases the bispecific antigen binding composition may not provide a cure but may only provide partial benefit. In some aspects, a physiological change having some benefit is also considered therapeutically beneficial. Thus, in some aspects, an amount of bispecific antigen binding composition that provides a physiological change is considered an “effective amount” or a “therapeutically effective amount”. The subject, patient, or individual in need of treatment is typically a mouse, rat, dog, monkey, or human.


The bispecific antigen binding compositions of the invention may be administered in combination with one or more other agents in therapy. For instance, a bispecific antigen binding molecule of any of the embodiments described herein may be co-administered with at least one additional therapeutic agent. The term “therapeutic agent” encompasses any agent administered to treat a symptom or disease in an individual in need of such treatment. Such additional therapeutic agent may comprise any active ingredients suitable for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. In certain aspects, an additional therapeutic agent is an immunomodulatory agent, an immuno-oncologic antibody, a cytostatic agent, an inhibitor of cell adhesion, a cytotoxic agent, an activator of cell apoptosis, or an agent that increases the sensitivity of cells to apoptotic inducers. In a particular aspect, the additional therapeutic agent is an anti-cancer agent, for example a microtubule disruptor, an antimetabolite, a topoisomerase inhibitor, a DNA intercalator, an alkylating agent, a hormonal therapy, a kinase inhibitor, a receptor antagonist, an activator of tumor cell apoptosis, or an antiangiogenic agent.


In one embodiment of the method of treating a disease in a subject, the disease for treatment can be anaplastic and medullary thyroid cancers, appendiceal cancer, arrhenoblastoma, biliary tract carcinoma, bladder cancer, breast cancer, cancers of the bile duct, carcinoid tumor, cervical cancer, cholangiocarcinoma, colon cancer, colorectal cancer, craniopharyngioma, endometrial cancer, epithelial intraperitoneal malignancy with malignant ascites, esophageal cancer, Ewing sarcoma, fallopian tube cancer, follicular cancer, gall bladder cancer, gastric cancer, gastrointestinal stromal tumor (GIST), GE-junction cancer, genito-urinary tract cancer, glioma, glioblastoma, head and neck cancer, hepatoblastoma, hepatocarcinoma, HR+ and HER2+ breast cancer, Hurthle cell cancer, inflammatory breast cancer, Kaposi sarcoma, kidney cancer, laryngeal cancer, liposarcoma, liver cancer, lung cancer, medulloblastoma, melanoma, Merkel cell carcinoma, neuroblastoma, neuroblastoma, neuroendocrine cancer, non-small cell lung cancer, osteosarcoma (bone cancer), ovarian cancer, ovarian cancer with malignant ascites, pancreatic cancer, pancreatic neuroendocrine tumor, papillary cancer, parathyroid cancer, peritoneal carcinomatosis, peritoneal mesothelioma, primitive neuroectodermal tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland carcinoma, sarcoma, skin cancer, small cell lung cancer, small intestine cancer, stomach cancer, testicular cancer, thyroid cancer, triple negative breast cancer, urothelial cancer, uterine cancer, uterine serous carcinoma, vaginal cancer, vulvar cancer, and Wilms tumor.


The therapeutically effective amount can produce a beneficial effect in helping to treat (e.g., cure or reduce the severity) or prevent (e.g., reduce the likelihood of recurrence) of a cancer or a tumor. In another embodiment of the method of treating the disease in a subject, the pharmaceutical composition is administered to the subject as two or more therapeutically effective doses administered twice weekly, once a week, every two weeks, every three weeks, every four weeks, or monthly. In another embodiment of the method, the pharmaceutical composition is administered to the subject as two or more therapeutically effective doses over a period of at least two weeks, or at least one month, or at least two months, or at least three months, or at least four months, or at least five months, or at least six months. In another embodiment of the method, a first low priming dose is administered to the subject, followed by one or more higher maintenance doses over the dosing schedule of at least two weeks, or at least one month, or at least two months, or at least three months, or at least four months, or at least five months, or at least six months. The initial priming dose administered is selected from the group consisting of at least about 0.005 mg/kg, at least about 0.01 mg/kg, at least about 0.02 mg/kg, at least about 0.04 mg/kg, at least about 0.08 mg/kg, at least about 0.1 mg/kg, and one or more subsequent maintenance dose(s) administered is selected from the group consisting of at least about 0.02 mg/kg, at least about 0.05 mg/kg, at least about 0.1 mg/kg, at least about 0.16 mg/kg, at least about 0.18 mg/kg, at least about 0.20 mg/kg, at least about 0.22 mg/kg, at least about 0.24 mg/kg, at least about 0.26 mg/kg, at least about 0.27 mg/kg, at least about 0.28 mg/kg, at least 0.3 mg/kg, at least 0.4. mg/kg, at least about 0.5 mg/kg, at least about 0.6 mg/kg, at least about 0.7 mg/kg, at least about 0.8 mg/kg, at least about 0.9 mg/kg, at least about 1.0 mg/kg, at least about 1.5 mg/kg, or at least about 2.0 mg/kg, or at least 5.0 mg/kg. In another embodiment of the method, the pharmaceutical composition is administered to the subject intradermally, subcutaneously, intravenously, intra-arterially, intra-abdominally, intraperitoneally, intrathecally, or intramuscularly. In another embodiment of the method, the pharmaceutical composition is administered to the subject as one or more therapeutically effective bolus doses or by infusion of 5 minutes to 96 hours as tolerated for maximal safety and efficacy. In another embodiment of the method, the pharmaceutical composition is administered to the subject as one or more therapeutically effective bolus doses or by infusion of 5 minutes to 96 hours, wherein the dose is selected from the group consisting of at least about 0.005 mg/kg, at least about 0.01 mg/kg, at least about 0.02 mg/kg, at least about 0.04 mg/kg, at least about 0.08 mg/kg, at least about 0.1 mg/kg, at least about 0.12 mg/kg, at least about 0.14 mg/kg, at least about 0.16 mg/kg, at least about 0.18 mg/kg, at least about 0.20 mg/kg, at least about 0.22 mg/kg, at least about 0.24 mg/kg, at least about 0.26 mg/kg, at least about 0.27 mg/kg, at least about 0.28 mg/kg, at least 0.3 mg/kg, at least 0.4. mg/kg, at least about 0.5 mg/kg, at least about 0.6 mg/kg, at least about 0.7 mg/kg, at least about 0.8 mg/kg, at least about 0.9 mg/kg, at least about 1.0 mg/kg, at least about 1.5 mg/kg, or at least about 2.0 mg/kg, or at least about 5.0 mg/kg. In another embodiment of the method, the pharmaceutical composition is administered to the subject as one or more therapeutically effective bolus doses or by infusion over a period of 5 minutes to 96 hours, wherein the administration to the subject results in a Cmax plasma concentration of the intact, uncleaved bispecific antigen binding composition of at least about 0.1 ng/mL to at least about 2 µg/mL or more in the subject that is maintained for at least about 3 days, at least about 7 days, at least about 10 days, at least about 14 days, or at least about 21 days. The therapeutically effective dose is at least about 0.005 mg/kg, at least about 0.01 mg/kg, at least about 0.02 mg/kg, at least about 0.04 mg/kg, at least about 0.08 mg/kg, at least about 0.1 mg/kg, at least about 0.12 mg/kg, at least about 0.14 mg/kg, at least about 0.16 mg/kg, at least about 0.18 mg/kg, at least about 0.20 mg/kg, at least about 0.22 mg/kg, at least about 0.24 mg/kg, at least about 0.26 mg/kg, at least about 0.27 mg/kg, at least about 0.28 mg/kg, at least 0.3 mg/kg, at least 0.4 mg/kg, at least about 0.5 mg/kg, at least about 0.6 mg/kg, at least about 0.7 mg/kg, at least about 0.8 mg/kg, at least about 0.9 mg/kg, at least about 1.0 mg/kg, at least about 1.5 mg/kg, or at least about 2.0 mg/kg. In one embodiment, an initial dose is selected from the group consisting of at least about 0.005 mg/kg, at least about 0.01 mg/kg, at least about 0.02 mg/kg, at least about 0.04 mg/kg, at least about 0.08 mg/kg, at least about 0.1 mg/kg, and a subsequent dose is selected from the group consisting of at least about 0.1 mg/kg, at least about 0.12 mg/kg, at least about 0.14 mg/kg, at least about 0.16 mg/kg, at least about 0.18 mg/kg, at least about 0.20 mg/kg, at least about 0.22 mg/kg, at least about 0.24 mg/kg, at least about 0.26 mg/kg, at least about 0.27 mg/kg, at least about 0.28 mg/kg, at least 0.3 mg/kg, at least 0.4. mg/kg, at least about 0.5 mg/kg, at least about 0.6 mg/kg, at least about 0.7 mg/kg, at least about 0.8 mg/kg, at least about 0.9 mg/kg, at least about 1.0 mg/kg, at least about 1.5 mg/kg, or at least about 2.0 mg/kg. In the foregoing embodiments, the administration to the subject results in a plasma concentration of the polypeptide of at least about 0.1 ng/mL to at least about 2 ng/mL or more in the subject for at least about 3 days, at least about 7 days, at least about 10 days, at least about 14 days, or at least about 21 days. In the foregoing embodiments of the method, the subject can be a mouse, rat, monkey, or a human.


VIII). Nucleic Acid Sequences

In some embodiments, the invention provides isolated polynucleotide sequences encoding the AF1 sequences, or the AF2 sequences, or the release segment sequences (RS1 and RS2), or the XTEN sequences, or the combination of any of these component embodiments described herein, or the complement of the polynucleotide sequences. In one embodiment, the invention provides an isolated polynucleotide sequence encoding a polypeptide or bispecific antigen binding composition of any of the embodiments described herein, or the complement of the polynucleotide sequence. In one embodiment, the invention provides an isolated polynucleotide sequence encoding a polypeptide or bispecific antigen binding composition wherein the polynucleotide sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a polynucleotide sequence set forth in Table 12.


In another aspect, the disclosure relates to methods to produce polynucleotide sequences encoding the polypeptides or bispecific antigen binding compositions of any of the embodiments described herein, or sequences complementary to the polynucleotide sequences, including homologous variants thereof, as well as methods to express the proteins expressed by the polynucleotide sequences. In general, the methods include producing a polynucleotide sequence coding for the proteinaceous polypeptides or bispecific antigen binding compositions of any of the embodiments described herein and incorporating the encoding gene into an expression vector appropriate for a host cell. For production of the encoded polypeptides or bispecific antigen binding compositions of any of the embodiments described herein, the method includes transforming an appropriate host cell with the expression vector, and culturing the host cell under conditions causing or permitting the resulting polypeptide or bispecific antigen binding composition of any of the embodiments described herein to be expressed in the transformed host cell, thereby producing the polypeptide or bispecific antigen binding composition, which is recovered by methods described herein or by standard protein purification methods known in the art. Standard recombinant techniques in molecular biology are used to make the polynucleotides and expression vectors of the present disclosure.


In accordance with the disclosure, nucleic acid sequences that encode the polypeptides or bispecific antigen binding compositions of any of the embodiments described herein (or their complement) are used to generate recombinant DNA molecules that direct the expression in appropriate host cells. Several cloning strategies are suitable for performing the present disclosure, many of which are used to generate a construct that comprises a gene coding for a composition of the present disclosure, or its complement. In one embodiment, the cloning strategy is used to create a gene that encodes a construct that comprises nucleotides encoding the polypeptide or bispecific antigen binding composition that is used to transform a host cell for expression of the composition. In the foregoing embodiments hereinabove described in this paragraph, the genes can comprise nucleotides encoding the antigen binding fragments, release segments, and the XTEN in the configurations disclosed herein.


In one approach, a construct is first prepared containing the DNA sequence encoding a polypeptide or bispecific antigen binding composition construct. Exemplary methods for the preparation of such constructs are described in the Examples. The construct is then used to create an expression vector suitable for transforming a host cell, such as a prokaryotic or eukaryotic host (e.g., mammalian) cell for the expression and recovery of the polypeptide construct. Where desired, the host cell is an E. coli. In another embodiment, the host cell is selected from BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, hybridoma cells, NIH3T3 cells, COS, HeLa, CHO, or yeast cells. Exemplary methods for the creation of expression vectors, the transformation of host cells and the expression and recovery of XTEN are described in the Examples.


The gene encoding for the polypeptide or bispecific antigen binding composition construct can be made in one or more steps, either fully synthetically or by synthesis combined with enzymatic processes, such as restriction enzyme-mediated cloning, PCR and overlap extension, including methods more fully described in the Examples. The methods disclosed herein can be used, for example, to ligate sequences of polynucleotides encoding the various components (e.g., binding domains, linkers, release segments, and XTEN) genes of a desired length and sequence. Genes encoding polypeptide compositions are assembled from oligonucleotides using standard techniques of gene synthesis. The gene design can be performed using algorithms that optimize codon usage and amino acid composition appropriate for the E. coli or mammalian host cell utilized in the production of the polypeptide or bispecific antigen binding composition. In one method of the disclosure, a library of polynucleotides encoding the components of the constructs is created and then assembled, as described above. The resulting genes are then assembled and the resulting genes used to transform a host cell and produce and recover the polypeptide compositions for evaluation of its properties, as described herein.


The resulting polynucleotides encoding the polypeptide or bispecific antigen binding composition sequences can then be individually cloned into an expression vector. The nucleic acid sequence is inserted into the vector by a variety of procedures. In general, DNA is inserted into an appropriate restriction endonuclease site(s) using techniques known in the art. Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Construction of suitable vectors containing one or more of these components employs standard ligation techniques which are known to the skilled artisan. Such techniques are well known in the art and well described in the scientific and patent literature. Various vectors are publicly available. The vector may, for example, be in the form of a plasmid, cosmid, viral particle, or phage that may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e., a vector, which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated. Once introduced into a suitable host cell, expression of the antigen binding fragments or bispecific antigen binding compositions can be determined using any nucleic acid or protein assay known in the art. For example, the presence of transcribed mRNA of light chain CDRs or heavy chain CDRs, the antigen binding fragment, or the bispecific antigen binding composition can be detected and/or quantified by conventional hybridization assays (e.g. Northern blot analysis), amplification procedures (e.g. RT-PCR), SAGE (U.S. Pat. No. 5,695,937), and array-based technologies (see e.g. U.S. Pat. Nos. 5,405,783, 5,412,087 and 5,445,934), using probes complementary to any region of antigen binding unit polynucleotide.


The disclosure provides for the use of plasmid expression vectors containing replication and control sequences that are compatible with and recognized by the host cell, and are operably linked to the gene encoding the polypeptide for controlled expression of the polypeptide. The vector ordinarily carries a replication site, as well as sequences that encode proteins that are capable of providing phenotypic selection in transformed cells. Such vector sequences are well known for a variety of bacteria, yeast, and viruses. Useful expression vectors that can be used include, for example, segments of chromosomal, non-chromosomal and synthetic DNA sequences. “Expression vector” refers to a DNA construct containing a DNA sequence that is operably linked to a suitable control sequence capable of effecting the expression of the DNA encoding the polypeptide in a suitable host. The requirements are that the vectors are replicable and viable in the host cell of choice. Low- or high-copy number vectors may be used as desired.


Suitable vectors include, but are not limited to, derivatives of SV40 and pcDNA and known bacterial plasmids such as col EI, pCR1, pBR322, pMal-C2, pET, pGEX as described by Smith, et al., Gene 57:31-40 (1988), pMB9 and derivatives thereof, plasmids such as RP4, phage DNAs such as the numerous derivatives of phage I such as NM98 9, as well as other phage DNA such as M13 and filamentous single stranded phage DNA; yeast plasmids such as the 2 micron plasmid or derivatives of the 2 m plasmid, as well as centomeric and integrative yeast shuttle vectors; vectors useful in eukaryotic cells such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNAs, such as plasmids that have been modified to employ phage DNA or the expression control sequences; and the like. Yeast expression systems that can also be used in the present disclosure include, but are not limited to, the non-fusion pYES2 vector (Invitrogen), the fusion pYESHisA, B, C (Invitrogen), pRS vectors and the like. The control sequences of the vector include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences that control termination of transcription and translation. The promoter may be any DNA sequence, which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. Promoters suitable for use in expression vectors with prokaryotic hosts include the β-lactamase and lactose promoter systems [Chang et al., Nature, 275:615 (1978); Goeddel et al., Nature, 281:544 (1979)], alkaline phosphatase, a tryptophan (trp) promoter system [Goeddel, Nucleic Acids Res., 8:4057 (1980); EP 36,776], and hybrid promoters such as the tac promoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)], all is operably linked to the DNA encoding XTEN polypeptides. Promoters for use in bacterial systems can also contain a Shine-Dalgarno (S.D.) sequence, operably linked to the DNA encoding polypeptide polypeptides.


Expression of the vector can also be determined by examining the antigen binding fragment or a component of the bispecific antigen binding composition expressed. A variety of techniques are available in the art for protein analysis. They include but are not limited to radioimmunoassays, ELISA (enzyme linked immunoradiometric assays), “sandwich” immunoassays, immunoradiometric assays, in situ immunoassays (using e.g., colloidal gold, enzyme or radioisotope labels), western blot analysis, immunoprecipitation assays, immunoflourescent assays, and SDS-PAGE.


IX). Methods of Making the Polypeptides and Bispecific Antigen Binding Compositions

In another aspect, the disclosure provides methods of manufacturing the subject compositions. In one embodiment, the method comprises culturing a host cell comprising a nucleic acid construct that encodes a polypeptide or a bispecific antigen binding composition of any of the embodiments described herein under conditions that promote the expression of the polypeptide or bispecific antigen binding composition, followed by recovery of the polypeptide or bispecific antigen binding composition 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 bispecific antigen binding composition are correctly folded. In another embodiment of the method of making, the expressed polypeptide or bispecific antigen binding composition 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 or bispecific antigen binding composition is recovered in monomeric, soluble form.


In another aspect, the disclosure relates to methods of making the polypeptide and bispecific antigen binding compositions at high fermentation expression levels of functional protein using an E. coli or mammalian host cell, as well as providing expression vectors encoding the constructs useful in methods to produce the cytotoxically active polypeptide construct compositions at high expression levels. In one embodiment, the method comprises the steps of 1) preparing the polynucleotide encoding the polypeptides of any of the embodiments disclosed herein, 2) cloning the polynucleotide into an expression vector, which can be a plasmid or other vector under 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. By the method, the expression of the polypeptide results in fermentation titers of at least 0.05 g/L, or at least 0.1 g/L, or at least 0.2 g/L, or at least 0.3 g/L, or at least 0.5 g/L, or at least 0.6 g/L, or at least 0.7 g/L, or at least 0.8 g/L, or at least 0.9 g/L, or at least 1 g/L of the expression product of the host cell and 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 expressed protein are correctly folded. As used herein, the term “correctly folded” means that the antigen binding fragments component of the composition have the ability to specifically bind its target ligand. In another embodiment, the disclosure provides a method for producing a polypeptide or bispecific antigen binding composition, the method comprising culturing in a fermentation reaction a host cell that comprises a vector encoding a polypeptide comprising the polypeptide or bispecific antigen binding composition under conditions effective to express the polypeptide product at a concentration of more than about 10 milligrams/gram of dry weight host cell (mg/g), or at least about 250 mg/g, or about 300 mg/g, or about 350 mg/g, or about 400 mg/g, or about 450 mg/g, or about 500 mg/g of said polypeptide when the fermentation reaction reaches an optical density of at least 130 at a wavelength of 600 nm, and wherein the antigen binding fragments of the expressed protein are correctly folded. In another embodiment, the disclosure provides a method for producing a polypeptide or bispecific antigen binding composition, the method comprising culturing in a fermentation reaction a host cell that comprises a vector encoding the composition under conditions effective to express the polypeptide product at a concentration of more than about 10 milligrams/gram of dry weight host cell (mg/g), or at least about 250 mg/g, or about 300 mg/g, or about 350 mg/g, or about 400 mg/g, or about 450 mg/g, or about 500 mg/g of said polypeptide when the fermentation reaction reaches an optical density of at least 130 at a wavelength of 600 nm, and wherein the expressed polypeptide product is soluble.


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


EXAMPLES
Example 1: Construction of Bispecific Antigen Binding Polypeptides With Two Release Segments.

In order to generate a plasmid where the individual scFv’s can be removed by restriction digest, pCW1700, which encodes for an anti-EpCAM-anti-CD3 (UCHT1) bispecific tandem scFv, with an RSR2486 release segment, an AE866 XTEN and a 6X His tag affinity tag (SEQ ID NO: 794), was digested with SacII and BstXI, removing the 3′ end of the anti-EpCAM binding domain, the linker between the anti-EpCAM and anti-CD3 domains and the 5′ end of the anti-CD3 domain. A fragment of DNA encoding the same region was synthesized with silent point mutations at the junction between the anti-EpCAM binding domain and the linker to introduce a Bsu36I site. Synthetic DNA fragments were cloned into digested backbone using the In-Fusion kit (New England Biolabs) to assemble pJB0035. pJB0035 was subsequently digested with NheI and BsaI to remove the BSRS1 release segment sequence. Overlapping single stranded oligonucleotides encoding RSR2486 were synthesized with single stranded tails that anneal to the NheI and BsaI overhangs. The oligonucleotides were annealed together and ligated into the digested pJB0035, resulting in pCW1880, which encodes for an anti-EpCAM-anti-CD3 (UCHT1) bispecific tandem scFv, RSR2486, XTEN866 and a 6X His tag affinity tag (SEQ ID NO: 794).


In order to generate a plasmid with different CD3 binding domain variants, pCW1880 was digested with Bsu36I and NheI to remove the UCHT1 anti-CD3 scFv. A DNA fragment encoding CD3.23 was synthesized. The gene fragment included 30 nucleotides 5′ and 3′ of the restriction sites to serve as DNA overlaps for Gibson DNA Assembly. The synthetic DNA fragment was cloned into digested backbone using the Gibson Cloning Kit (SGI-DNA, Carlsbad, CA) to assemble pJB0205.


In order to generate a bispecific antigen binding polypeptide with both an N-terminal and C-terminal XTEN, the AE292 XTEN was PCR amplified from a plasmid using primers including a 17-21 bp 5′ homology region to backbone DNA on the N-terminus and to an uncleavable release segment (RSR3058, amino acid sequence TTGEAGEAAGATSAGATGP (SEQ ID NO: 111)) on the C-terminus. A second PCR product encoding the light and part of the heavy chain of the anti-EpCAM antibody 4D5MOCB was amplified using primers that included a 16-21 bp 5′ homology region to RSR3058 on the N-terminus and the heavy chain of 4D5MOCB on the C-terminus. These PCR fragments were cloned into a backbone vector digested with BsiWI-SacII that encoding the remainder of the 4D5MOCB heavy chain/anti-CD3 tandem scFv, a second copy of the RSR3058 uncleavable release segment and AE837 XTEN with a 6xHIS affinity tag (SEQ ID NO: 794) using the In-Fusion Plasmid Assembly Kit (Takara Bio). The final vector encodes the bispecific antigen binding polypeptide with the components (from N- to C-terminus) of AE292 XTEN, the uncleavable RSR3058 release segment, anti-EpCAM-anti-CD3 bispecific tandem scFv with RSR3058 fused to AE867 XTEN with a 6xHIS affinity tag (SEQ ID NO: 794) under the control of a PhoA promoter and STII secretion leader. The resulting construct is pJB0084 with the DNA sequence and encoded amino acid sequence provided in Table 12.


pJB0084 was used as a template to create a bispecific antigen binding polypeptide construct encoding AE292 XTEN, the cleavable release segment RSR2295, anti-EpCAM-anti-CD3 bispecific tandem scFv with RSR2295 fused to AE868 XTEN. The plasmid utilized two PCR products using pJB0084 as a template; the first encoding a 6xHIS affinity tag (SEQ ID NO: 794) and AE292 XTEN with an 5′ homology region to the vector backbone and the 3′ homology region encoding the first RSR2295, the second encoding the anti-EpCAM-anti-CD3 bispecific tandem scFv with 5′ and 3′ homology regions encoding the RSR2295 release segments 5′ and 3′ of the tandem scFvs. The third fragment encoded AE868 XTEN having the C-Tag affinity tag (amino acid sequence EPEA (SEQ ID NO: 796)) with a 5′ homology region encoding the second RSR2295 and a 3′ homology region to the backbone vector. The three PCR fragments were cloned into pJB0084 that had been digested with BsiWI-NotI using the In-Fusion Plasmid Assembly Kit. The final vector, pJB0169, encodes the bispecific antigen binding polypeptide molecule with the components (from N- to C-terminus) of 6xHIS affinity tag (SEQ ID NO: 794), AE292 XTEN, RSR2295 release segment, anti-EpCAM-anti-CD3 bispecific tandem scFv, RSR2295, AE868 XTEN with the C-Tag affinity tag under the control of a PhoA promoter and STII secretion leader with the DNA sequence and protein sequence in Table 12.


In order to introduce a new CD3 scFv with alterations to the isoelectric point and removal of potential aggregation sites in the amino acid sequence, pJB0244 was digested with BsaI and BbvCI to remove both the HER2 and CD3 scFvs. DNA fragments encoding anti-EGFR scFv variants paired with CD3.33 were synthesized that included 40 bp of homology to the digested vector at both the 5′ and 3′ ends to facilitate Gibson DNA Assembly. Plasmids pJB0358-pJB0372 were assembled with the structure of 6xHIS affinity tag (SEQ ID NO: 794), AE292 XTEN, RSR2295, and individually, a total of 15 anti-EGFR scFv variants paired with an anti-CD3 scFv, RSR2295, AE868 XTEN having a C-Tag affinity tag (DNA and protein sequences in Table 12).


pAH0025 and pAH0026 were created by initially digesting pJB0368 and pJB0373 with BtsI to remove the anti-CD3 scFv. DNA fragments were ordered encoding the anti-CD3.32 scFv flanked with 40 bp homology regions to the digested backbone. These fragments were introduced into pJB0368 and pJB0373 by Gibson Assembly to create plasmids encoding a 6xHIS affinity tag (SEQ ID NO: 794), AE292 XTEN, RSR2295, anti-EGFR-anti-CD3 bispecific tandem scFv, RSR2295, AE868 XTEN having a C-Tag affinity tag constructed with two different anti-EGFR binding domains, EGFR.23 and EGFR.2 to result in the pAH0025 and pAH0026 constructs (DNA and protein sequences in Table 12). Analogous methodologies would be employed to make constructs having EGFR.13, EGFR.14, EGFR.15, EGFR. 16, EGFR.17, EGFR.18, EGFR.19, EGFR.20, EGFR.21, EGFR.22, EGFR.24, EGFR.25, EGFR.26, EGFR.27, CD3.30, CD3.31, and CD3.33 scFv, in any combination or orientation (i.e., AF1-AF2 or AF2-AF1 in an N- to C-terminal orientation), the sequences of which are provided herein.









Lengthy table referenced here




US20230312729A1-20231005-T00001


Please refer to the end of the specification for access instructions.













Lengthy table referenced here




US20230312729A1-20231005-T00002


Please refer to the end of the specification for access instructions.






Example 2: Evaluation of CD3 scFv Sequence Variants in Comparison to Parental CD3 scFv

The purpose of the experiments was to evaluate 4 CD3 sequence variants to determine if the variants had enhanced properties in comparison to the CD3.9 parental scFv.


1. Determination of Melting Temperature (Tm)

The melting temperature of each scFv variant was measured to determine its thermal stability. Briefly, a uniform quantity of scFv in 200 µL of 1% BSA-PBST was aliquoted into PCR tubes. Tubes were incubated for one hour at several different temperatures (50° C., 51.4° C., 53.7° C., 57.3° C., 61.7° C., 65.5° C., and 68° C.). 50 µL of each sample was added to an ELISA plate coated with CD3Œµ target antigen (Creative Biomart) or BSA (reference to address stickiness). The wells of the ELISA plate were prefilled with 1% BSA-PBST (50 µl/well). Plates were incubated for 1 hour at room temperature. Plates were washed three times with water with 0.05% TWEEN to remove unbound scFv. Bound scFv was detected by adding an anti-YOL antibody (Thermo Scientific # MA180189) (1:500 diluted in 1% BSA-PBST (0.05%)) that detects a porcine alpha-tubulin motif in the linker between the heavy and light chain. Samples were incubated at room temperature for 1 hour. Plates were washed three times with water with 0.05% TWEEN to remove unbound scFv. The anti-YOL antibody was detected by adding an anti-rat-HRP antibody (Thermo Scientific # 31470) (1:7500 diluted in 1% BSA-PBST (0.05%)) [100 Œ°l/well) and incubating at room temperature for 1 hour. Plates were washed three times with water with 0.05% TWEEN to remove unbound antibody. Plates were developed using TMB (3,3’,5,5′-tetramethylbenzidine) substrate (100 µL/well for 6 minutes at room temperature. The reactions were stopped with H2SO4 (0.5 M, 100 µL/well). The relative activity was measured as the absorbance reading at 450 nM. The absorbance at each temperature was graphed. The melting temperature was determined to be the EC50 of each sample, the temperature at which the binding of the scFv was reduced to 50% of maximal signal. The results are presented in Table 15.


Results: The assay results demonstrate that the CD3 scFv 3.23 and 3.24 had a Tm of 5° C. higher than the parental CD3.9, while the CD3.25 and CD3.26 (sequences shown in Table 14) scFv had Tm that were equivalent to the parental CD3.9.





TABLE 14






scFv Sequences


Construct
Amino Acid Sequence
SEQ ID NO:




CD3.25
ELVVTQEPSHTVSPGGTVTLTCRSSTGAVTSSNYANWVQQKPGQAPRGLI GGTIKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYPNLWVF GGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLLESGGGI VQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATY YADSVKDRFTISRDDSKNTVYLQMNNLKTEDTAVYYCVRHENFGNSYVSW FAHWGQGTLVTVSS
938


CD3.26
ELVVTQEPSHTVSPGGTVTLTCRSSTGEVTTSNYANWVQQKPGQAPRGLI GGTIKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYPNLWVF GGGTKLTVLGATPPETGAETESPGETTGGSAESEPPGEGEVQLLESGGGI VQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATY YADSVKDRFTISRDDSKNTVYLQMNNLKTEDTAVYYCVRHENFGNSYVSW FAHWGQGTLVTVSS
939






2. Determination of Binding Affinity to CD3

The binding affinity of each scFv was measured using the ForteBio BLItz instrument. A dilution series of each scFv was prepared in PBS (300 µL/tube) starting from 1000 nM to 62.5 nM in one to one dilution steps for CD3.24-26, 400 nM to 25 nM in one to one dilution steps for CD3.23. Biotinylated CD3Œµ antigen (Creative Biomart) was diluted in PBS to a final concentration of 30 ug/ml. Streptavidin Biosensors (ForteBio) were activated in PBS for 10 minutes. To perform the measurements, the streptavidin biosensors were applied to the BLItz instrument. A tube containing 300 µL of PBS was transferred to the BLItz instrument for 30 seconds. A tube containing biotinylated CD3Œµ (30 ug/ml, 300 µL/tube) was transferred to the BLItz instrument to measure capture of antigen to sensor for 120 seconds. A tube containing 300 µL of PBS was transferred to the BLItz instrument for 30 seconds to measure the baseline signal. A tube containing test scFv (30 ug/ml, 300 µL/tube) was transferred to the BLItz instrument to measure association of the scFv to antigen-loaded biosensor for 120 seconds. A tube containing 300 µL of PBS was transferred to the BLItz instrument for 120 seconds to measure dissociation of the scFv scFv from the antigen-loaded biosensor. The protocol was repeated for each scFv dilution. The KD of each antibody was determined using the BLI software (ForteBio). The results are presented in Table 15.


Results: The assay results demonstrate that the CD3 sequence variants all had reduced binding affinity to CD3 in comparison to the parental CD3.9.





TABLE 15






TM and Binding Affinity Results


scFv Construct
Melting temp (°C)
Binding Affinity (nM)




CD3.9
57
75


CD3.23
62
175


CD3.24
62
296


CD3.25
57
215


CD3.26
57
221






Conclusions: Two new anti-CD3 scFvs have been identified that have improved thermal stability. Each new scFv has 8 to 9 mutations relative to CD3.9, residing primarily in the CDRs. These mutations have reduced affinity of the scFvs for their target (CD3) compared to the parental CD3.9 but bispecific T cell engagers utilizing CD3.23 are still efficacious in cell killing assays and in vivo.


Example 3: Fermentation and Purification of Stable Chimeric Fusion Polypeptide Comprising Bispecific Antigen Binding Fragments, Release Segments, and XTEN

The following example describes production of a chimeric bispecific antigen binding fragment composition.


Construct ID pJB0169 is a molecule having eight distinct domains. From the N-terminus to the C-terminus, the molecule consists of an N-terminal polyhistidine tag (His6) (SEQ ID NO: 794), an unstructured 292 amino acid chain (XTEN_AE293), a protease cleavable release segment (RS), an anti-EGFR scFv (aEGFR.2), an anti-CD3 scFv (aCD3.9), another protease cleavable release segment (RS), an unstructured 864 amino acid chain, and four C-terminal residues - glutamic acid, proline, glutamic acid, alanine (C-tag) (XTEN_AE868).


EXPRESSION: Molecule pJB0169 was expressed in a proprietary E. coli AmE098 strain and partitioned into the periplasm via an N-terminal secretory leader sequence (MKKNIAFLLASMFVFSIATNAYA- (SEQ ID NO: 940)), which was cleaved during translocation. Fermentation cultures were grown with animal-free complex medium at 37° C. and temperature shifted to 26° C. prior to phosphate depletion with continued fermentation for 12 hours following phosphate depletion. During harvest, fermentation whole broth was centrifuged to pellet the cells. At harvest, the total volume and the wet cell weight (WCW; ratio of pellet to supernatant) were recorded, and the pelleted cells were collected and frozen at -80° C.


CLARIFICATION: Frozen cell pellet of pJB0169 was resuspended 3-fold in lysis buffer (60 mM acetic acid, 350 mM NaCl) at pH 4.5, and the cells were lysed via homogenization. The homogenate was flocculated overnight at pH 4.5 and 2-8° C. The flocculated homogenate was centrifuged, and the supernatant was retained. The supernatant was diluted approximately 3-fold with water, then adjusted to 7±1 mS/cm with NaCl. The supernatant was then adjusted to 0.1% (m/m) diatomaceous earth and mixed via impeller. The supernatant was filtered through a filter train ending with a 0.22 µm filter. The filtrate was adjusted to pH 7.0 with sodium phosphate dibasic.


PURIFICATION: Molecule pJB0169 was initially captured from clarified lysate and purified by Protein-L Chromatography (TOYOPEARL AF-rProtein L-650F). Subsequently, IMAC chromatography (GE IMAC Sepharose 6 FF) was used to select for the N-terminal His6-tag (SEQ ID NO: 794), then C-tag affinity chromatography (CaptureSelect C-tagXL Affinity Matrix) was used to select for the C-terminal EPEA-tag (SEQ ID NO: 796). Anion exchange chromatography (BIA CIMmultus QA monolith) was used to remove HMWCs and to polish to final purity.


ANALYTICS: The aggregation state of the process intermediates was monitored by SEC-HPLC. The SEC-HPLC method was performed using a Phenomenex 3 µm SEC-4000 300 × 7.8 mm (P/N 00H-4514-K0), a 20-minute isocratic method, at 1 mL/min, while monitoring the absorbance at 220 nm. pJB0169 monomer elutes from the analytical column at 6.2 minutes, and HMWC elute from 4.8-6.0 minutes. SEC-HPLC quality was measured as the relative area under the curve at 6.2 minutes versus the total area under the curve from 4.8-6.4 minutes.


Results: Aggregation summary (SEC-HPLC % monomer) for construct pJB0169 following each unit operation are presented in Table 16. Recovery of ≥ 95% monomer was the quality threshold upon final polishing as the criterion for considering a molecule stable or processable.





TABLE 16






Analytic Results


Unit Operation Number
Unit Operation Description
pJB0169 αEGFR.2-αCD3.9




1
Clarified lysate
26.7%


2
Protein L eluate
40.7%


3
IMAC eluate
52.8%


4
C-tag eluate
71.0%


5
AEX eluate
99.9%



stable






CONCLUSIONS: Construct pJB0169 was purified to the target monomeric quality by SEC-HPLC (≥ 95% monomer), indicating that the construct is stable and compatible with both recovery and purification operations.


STABILITY IMPROVEMENT AND ASSESSMENT: New scFv’s (anti-EGFR.23 and anti-CD3.32) were designed for improved stability via (1) reduction of surface hydrophobicity and (2) reduction of isoelectric point differences between the paired scFv molecules (fused by a short peptide linker) by substitution of amino acids at select locations. Constructs pAH0025 and pAH0026 represent design iterations on pJB0169, where pAH0025 contains the anti-CD3.32 scFv variant, and pAH0026 contains both the anti-CD3.32 scFv variant and the EGFR.23 scFv variant. Constructs pAH0025 and pAH0026 would be expressed, clarified, purified, and analyzed as above; the SEC-HPLC results throughout the purification would be monitored and compared to pJB0169 or other constructs (e.g., αEGFR.2-αCD3.23) to assess relative stability. New design pairings may be more stable than a corresponding αEGFR.2-αCD3.23 construct (such as a molecule which, from the N-terminus to the C-terminus consists of an N-terminal polyhistidine tag (His6) (SEQ ID NO: 794), an unstructured 292 amino acid chain (XTEN_AE292), a protease cleavable release segment (RS), an anti-EGFR scFv (aEGFR.2), an anti-CD3 scFv (aCD3.23), another protease cleavable release segment (RS), an unstructured 864 amino acid chain, and four C-terminal residues - glutamic acid, proline, glutamic acid, alanine (C-tag) (XTEN_AE868)). The pAH0025 and pAH0026 constructs may also be expected to show concomitant improvement in percent monomer content, as measured by SEC-HPLC, following the unit operations tabulated below or a subset thereof (Table 17). Any construct that meets the purity target of ≥ 95% monomer would be considered stable or processable.





TABLE 17








Analytic Results


Unit Operation Number
Unit Operation Description
SEC-HPLC quality (% monomer)


pJB0169 αEGFR.2-αCD3.9
pAH0025 αEGFR.2-αCD3.32
pAH0026 αEGFR.23-αCD3.32




1
Clarified lysate
26.7%
TBD
TBD


2
Protein L eluate
40.7%
TBD
TBD


3
IMAC eluate
52.8%
TBD
TBD


4
C-tag eluate
71.0%
TBD
TBD


5
AEX eluate
99.9%
TBD
TBD



stable
TBD
TBD






Example 4: Binding Affinity of anti-EpCAM × anti-CD3 Bispecific Antigen Binding Polypeptide Composition.

The binding affinity of anti-EpCAM × anti-CD3 bispecific antigen binding polypeptide constructs pJB0189 and pCW1645 to human EpCAM and human CD3 was measured using flow cytometry with huEp-CHO 4-12B (CHO cell line transfected with human EpCAM) and Jurkat cells.


The binding constants for anti-EpCAM × anti-CD3 bispecific antigen binding polypeptide binding to EpCAM-expressing and CD3-expressing cells was measured by competition binding with a fluorescently-labeled, protease-treated bispecific antigen binding polypeptide. The fluorescently-labeled, protease-treated bispecific antigen binding polypeptide was made by conjugation of Alexa Fluor 647 C2 maleimide (Thermo Fisher, cat#A20347) to a cysteine-containing, protease-treated bispecific antigen binding polypeptide mutant (MMP-9 treated pCW1645). Binding experiments were performed on 10,000 cells at 4° C. for 1 hour in a total volume of 100 microL of binding buffer (2% FCS, 5 mM EDTA, HBSS). Cells were washed once with cold binding buffer, then re-suspended in 1% formaldehyde in phosphate-buffered saline and immediately analyzed on a Millipore Guava easyCyte flow cytometer. Binding of the fluorescently labeled, protease-treated pCW1645 was found to have an apparent Kd value of 1 nM to hEp-CHO 4-12B and 4 nM to CD3+ Jurkat cells.


Competition binding experiments were performed on 10,000 hEp-CHO 4-12B cells with 1.5 nM fluorescently-labeled, protease-treated pCW1645 at 4° C. for 1 hour in a total volume of 100 microL of binding buffer (2% FCS, 5 mM EDTA, HBSS). Cells were washed once with cold binding buffer, then re-suspended in 1% formaldehyde in phosphate-buffered saline and immediately analyzed on a Millipore Guava easyCyte flow cytometer. Competition binding of fluorescently-labeled, protease-treated pCW1645 to hEp-CHO 4-12B cells with cleaved bispecific antigen binding polypeptide (pJB0189 hEp.2-hCD3.9 or AC1984 hEp.2-hCD3.23) resulted in apparent binding constants of 0.5 nM for hEp.2 (panitumimab).


Competition binding experiments were performed on 10,000 Jurkat cells with 10 nM fluorescently-labeled, protease-treated pCW1645 at 4° C. for 1 hour in a total volume of 100 microL of binding buffer (2% FCS, 5 mM EDTA, HBSS). Cells were washed once with cold binding buffer, then re-suspended in 1% formaldehyde in phosphate-buffered saline and immediately analyzed on a Millipore Guava easyCyte flow cytometer. Competition binding of fluorescently-labeled, protease-treated pCW1645 to Jurkat cells with cleaved bispecific antigen binding polypeptide (pJB0189 hEp.2-hCD3.9 or AC1984 hEp.2-hCD3.23) resulted in apparent binding constants of 75 nM for hCD3.9 and 300 nM for hCD3.23 for CD3 binding and 0.5 nM for EpCAM binding.


Conclusions: The binding affinity of CD3.23 for CD3 on Jurkat cells is 300 nM, which is 4-fold weaker than the affinity of CD3.9. The binding affinity of hEp.2 for EpCAM on Jurkat cells is 0.5 nM.


Example 5: Binding Affinity of anti-EGFR × anti-CD3 Bispecific Antigen Binding Polypeptide Composition.

The binding affinity of anti-EGFR × anti-CD3 bispecific antigen binding polypeptide constructs to human EGFR and human CD3 are measured using flow cytometry with EGFR positive human cells selected from HT-29, HCT-116, NCI-H1573, NCI-H1975, and Jurkat cells for CD3.


The binding constants for anti-EGFR × anti-CD3 bispecific antigen binding polypeptide binding to EGFR-expressing and CD3-expressing cells are measured by competition binding with a fluorescently labeled, protease-treated bispecific antigen binding polypeptide. The fluorescently labeled bispecific antigen binding polypeptide is made by conjugation of Alexa Fluor 647 C2 maleimide (Thermo Fisher, cat#A20347) to a cysteine-containing bispecific antigen binding polypeptide mutant (MMP-9 treated pJB0297) with hEGFR.2-hCD3.23 and two XTEN. The fluorescently-labeled, protease-treated bispecific antigen binding polypeptide is made by conjugation of Alexa Fluor 647 C2 maleimide (Thermo Fisher, cat#A20347) to a cysteine-containing, protease-treated bispecific antigen binding polypeptide mutant (MMP-9 treated pJB0297). Binding experiments are performed on 10,000 cells at 4° C. for 1 hour in a total volume of 100 microL of binding buffer (2% FCS, 5 mM EDTA, HBSS). Cells are washed once with cold binding buffer, then re-suspended in 1% formaldehyde in phosphate-buffered saline and immediately analyzed on a Millipore Guava easyCyte flow cytometer. Binding of the fluorescently-labeled, protease-treated pJB0297 is expected to have an apparent Kd value in the low nM concentration to hEGFR bearing cells and about 300 nM to CD3+ Jurkat cells. Binding of the fluorescently-labeled pJB0297 with two XTEN is expected to have an apparent Kd value about 10- to 100-fold weaker than for fluorescently-labeled, protease-treated bispecific antigen binding polypeptide to hEGFR bearing cells and CD3+ Jurkat cells.


Competition binding experiments are performed on 10,000 hEGFR cells at a concentration of fluorescently-labeled, protease-treated pJB0297 close to the Kd from the previously described binding experiment at 4° C. for 1 hour in a total volume of 100 microL of binding buffer (2% FCS, 5 mM EDTA, HBSS). Cells are washed once with cold binding buffer, then re-suspended in 1% formaldehyde in phosphate-buffered saline and immediately analyzed on a Millipore Guava easyCyte flow cytometer. Competition binding of fluorescently-labeled, protease-treated pJB0297 to hEGFR cells with pJB0244 bispecific antigen binding polypeptide is expected to have an apparent binding constant similar to the direct binding constant of fluorescently-labeled pJB0297.


Competition binding experiments are performed on 10,000 Jurkat cells with about 300 nM (or a concentration close to the Kd from the previously described binding experiment) of fluorescently-labeled, protease-treated pJB0297 at 4° C. for 1 hour in a total volume of 100 microL of binding buffer (2% FCS, 5 mM EDTA, HBSS). Cells are washed once with cold binding buffer, then re-suspended in 1% formaldehyde in phosphate-buffered saline and immediately analyzed on a Millipore Guava easyCyte flow cytometer. Competition binding of fluorescently-labeled, protease-treated pJB0297 to Jurkat cells with pJB0244 bispecific antigen binding polypeptide is expected to have an apparent binding constant similar to the direct binding constant of fluorescently-labeled pJB0297, which is expected to be in the low micromolar to nanomolar concentration range.


Example 6: Enzyme Activation, Storage and Digestion of RSR-1517-containing XTEN AC1611 (RSR-1517).

This example demonstrates that RSR-1517-containing XTEN construct AC1611, can be cleaved by various tumor-associated proteases including recombinant human uPA, matriptase, legumain, MMP-2, MMP-7, MMP-9, and MMP-14, in test tubes. The amino acid sequence of AC1611 is presented in Table 18, below.


1. Enzyme Activation

All enzymes used were obtained from R&D Systems. Recombinant human u-plasminogen activator (uPA) and recombinant human matriptase were provided as activated enzymes and stored at -80° C. until use. Recombinant mouse MMP-2, recombinant human MMP-7, and recombinant mouse MMP-9 were supplied as zymogens and required activation by 4-aminophenylmercuric acetate (APMA). APMA was first dissolved in 0.1 M NaOH to a final concentration of 10 mM before the pH was readjusted to neutral using 0.1 M HCl. Further dilution of the APMA stock to 2.5 mM was done in 50 mM Tris pH 7.5, 150 mM NaCl, 10 mM CaCl2. To activate pro-MMP, 1 mM APMA and 100 µg/mL of pro-MMP in 50 mM Tris pH 7.5, 150 mM NaCl, 10 mM CaCl2 were incubated at 37° C. for 1 hour (MMP-2, MMP-7) or 24 hours (MMP-9). To activate MMP-14, 0.86 µg/mL recombinant human furin and 40 µg/mL pro-MMP-14 in 50 mM Tris pH 9, 1 mM CaCl2 were incubated at 37° C. for 1.5 hours. To activate legumain, 100 µg/mL pro-legumain in 50 mM sodium acetate pH 4, 100 mM NaCl were incubated at 37° C. for 2 hours. 100% Ultrapure glycerol were added to all activated enzymes (including uPA and MTSP1) to a final concentration of 50% glycerol, then be stored at -20° C. for several weeks.


2. Enzymatic Digestion

A panel of enzymes was tested to determine cleavage efficiency of each enzyme for AC1611. 6 µM of the substrate was incubated with each enzyme in the following enzyme-to-substrate molar ratios and conditions: uPA (1:25 in 50 mM Tris pH 8.5), matriptase (1:25 in 50 mM Tris pH 9, 50 mM NaCl), legumain (1:20 in 50 mM MES pH 5, 250 mM NaCl), MMP-2 (1:1200 in 50 mM Tris pH 7.5, 150 mM NaCl, 10 mM CaCl2), MMP-7 (1:1200 in 50 mM Tris pH 7.5, 150 mM NaCl, 10 mM CaCl2), MMP-9 (1:2000 in 50 mM Tris pH 7.5, 150 mM NaCl, 10 mM CaCl2), and MMP-14 (1:30 in 50 mM Tris pH 8.5, 3 mM CaCl2, 1 µM ZnCl2) in 20 uL reactions. Reactions were incubated at 37° C. for two hours before stopped by adding EDTA to 20 mM in the case of MMP reactions, heating at 85° C. for 15 minutes in the case of uPA and matriptase reactions, and adjusting pH to 8.5 in the case of legumain.


3. Analysis of Cleavage Efficiency.

Analysis of the samples to determine percentage of cleaved product was performed by loading 2 µL of undigested substrate (at 12 µM) and 4 µL digested (at 6 µM) reaction mixture on SDS-PAGE and staining with Stains-All (Sigma Aldrich), as shown on FIG. 75. ImageJ software was used to analyze corresponding band intensity and determine percent of cleavage. Upon cleavage by various proteases at the release segment, the substrate RSR-1517-containing XTEN would yield two fragments, and the larger fragment was utilized in % cleavage calculations (quantity of reaction product divided by total initial substrate went into the reaction) while band intensity of the smaller product is too low to quantify. The percentage of cleavage of AC1611 under the current standard experimental conditions is 31%, 14%, 16%, 40%, 51%, 38%, 30%, for uPA, matriptase, legumain, MMP-2, MMP-7, MMP-9, MMP-14, respectively.


Conclusions: We selected a particular release segment RSR-1517 (amino acid sequence EAGRSANHEPLGLVAT (SEQ ID NO: 53)) and determined its cleavage profile as defined by percentage of cleavage under current standard experimental condition for all seven enzymes. This release segment has intermediate cleavage efficiency for all enzymes so that during screening, cleavage of faster or slower variants will fall within the assay window to allow accurate ranking.





TABLE 18





Amino acid sequence of AC1611 with Release Segment RSR-1617


Construct Name
Amino Acid Sequence*




AC1611
MKNPEQAEEQAEEQREETGKPIPNPLLGLDSTEGTSTEPSEGSAPGTSESATPESGPGT SESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEG TSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTAEAASASGEA GRSANHEPLGLVATPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPT STEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATS GSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGS PTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSES ATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSE SATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS TEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGT STEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPG TSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGP GSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSA PGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPES GPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPE SGPGSPAGSPTSTEEGSPAHHHHHHHH (SEQ ID NO: 941)






Example 7: Screening Release Segment Using RSR-1517 (AC1611) as Control

Here we select uPA as the example to show how the release segment screening was performed. The same procedure was applied to all seven tumor-associated proteases to define the relative cleavage profile for each substrate, which is a seven number array to describe how well it can be cleaved for each enzyme, when compared to the control substrate RSR-1517. All polypeptides of Table 19 had the amino acid sequence of AC1611, but with the substitution of the release segment peptide of the indicated construct swapped in for the EAGRSANHEPLGLVAT sequence of AC1611 (SEQ ID NO: 53); e.g., BSRS-4 has a release segment sequence of LAGRSDNHSPLGLAGS (SEQ ID NO: 945) but otherwise has complete sequence identity to AC1611.


1. Enzymatic Digestion

All release segment-containing XTEN variants and the control AC1611 were diluted to 12 µM in 50 mM Tris pH 7.5, 150 mM NaCl, 10 mM CaCl2 in individual Eppendorf tubes. A master mix of uPA was prepared so that after 1:1 mixing with each substrate, the total reaction volume is 20 µL, the initial substrate concentration is 6 µM, and the enzyme-to-substrate ratio was varied between 1:20 to 1:3000, depending on the enzyme, in order to have reaction products and uncleaved substrate that could be visualized at the endpoint. All reactions were incubated at 37° C. for two hours before stopped by adding EDTA to a final concentration of 20 mM. All products were analyzed by non-reducing SDS-PAGE followed by Stains-All. For each gel, AC1611 digestion product was always included as the staining control to normalize differential staining between different gels.


2. Relative Cleavage Efficiency Calculation

Percentage of cleavage for individual substrate was analyzed by ImageJ software and calculated as described before. For each variant, the relative cleavage efficiency is calculated as follows:






L
o

g
2





%

C
l
e
a
v
e
d

f
o
r

s
u
b
s
t
r
a
t
e

o
f

i
n
t
e
r
e
s
t


%

c
l
e
a
v
e
d

f
o
r

A
C
1611

i
n

t
h
e

s
a
m
e

e
x
p
e
r
i
m
e
n
t








A +1 value in relative cleavage efficiency indicates the substrate yielded twice as much product when compared to the AC1611 control while a -1 value in relative cleavage efficiency indicates the substrate yielded only 50% as much product when compared to the AC1611 control, under the experimental condition specified above.


In this experiment, the percentage of cleavage (% cleaved) for AC1611 is 20%, as quantified by ImageJ. The substrates being screened in this experiment demonstrated 21%, 39%, 1%, 58%, 24%, 6%, 15%, 1%, 1%, and 25% cleavage, where 1% essentially represents below detection limit and does not indicate accurate values. The relative cleavage efficiencies calculated based on the formula above were 0.08, 0.95, -4.34, 1.51, 0.26, -1.76, -0.47, -4.34, -4.34, and 0.32, respectively.


Conclusions: We determined relative cleavage efficiencies of 10 release segment variants when subject to uPA when compared to AC1611 in the same experiment. Following similar procedures, we determined the cleavage profiles of 134 release segments, the results of which are listed in Table 19, using RSR1517 (AC1611) as the reference control. These release segments covers a wide range of cleavage efficiency for individual enzyme as well as combinations. For example, RSR-1478 has a -2.00 value for MMP-14, meaning that this substrate yielded only 25% of product compared to the reference control RSR-1517 when digested with MMP-14. Certain release segments, such as RSR-1951, appear to be better substrate for all seven proteases tested. These faster release segments may prove to be useful in the clinic if the systemic toxicity is low/manageable while efficacy (partially depending on how fast cleavage happens to render bispecific antigen binding composition as the activated form) needs improvement.





TABLE 19














Cleavage profiles of Release Segment when subjected to seven human proteases using RSR-1517 as control


RS ID
AC#
AA Sequence
SEQ ID NO:
uPA
Matriptase
Leguman
MMP-2
MMP-7
MMP-9
MMP-14




RSR-1517
AC1611
EAGRSANHEPLGLVAT
53
0.00
0.00
0.00
0.00
0.00
0.00
0.00


BSRS-4
AC1602
LAGRSDNHSPLGLAGS
945
-0.99
1.69
0.48
0.09
-0.49
-0.04
-0.58


BSRS-5
AC1603
LAGRSDNHVPLSLSMG
942
-1.40
1.76
0.56
-0.52
0.00
-0.75
-0.21


BSRS-6
AC1604
LAGRSDNHEPLELVAG
943
-2.71
0.47
0.23
-1.26
0.00
-1.16
-2.79


BSRS-A1
AC1605
ASGRSTNAGPSGLAGP
54
1.43
2.77
0.09
-0.16
-2.18
0.03
-1.24


BSRS-A2
AC1606
ASGRSTNAGPQGLAGQ
55
1.36
2.77
-0.14
0.09
-2.64
0.03
-1.03


BSRS-A3
AC1607
ASGRSTNAGPPGLTGP
56
1.49
2.77
0.05
-1.07
-3.47
-1.82
-3.59


VP-1
AC1608
ASSRGTNAGPAGLTGP
57
-2.19
1.16
0.90
0.09
-1.22
0.23
0.00


RSR-1752
AC1609
ASSRTTNTGPSTLTGP
58
-0.55
0.70
0.29
-0.34
-1.29
-0.94
-5.38


RSR-1512
AC1610
AAGRSDNGTPLELVAP
59
-2.96
1.51
0.56
-1.43
-0.45
-1.09
-3.91


RSR-1517
AC1611
EAGRSANHEPLGLVAT
53
0.00
0.00
0.00
0.00
0.00
0.00
0.00


VP-2
AC1612
ASGRGTNAGPAGLTGP
60
-0.70
1.38
1.12
0.00
-0.58
0.23
-0.15


RSR-1018
AC1613
LFGRNDNHEPLELGGG
61
-4.62
-0.53
1.36
-0.73
-0.43
-2.56
-1.79


RSR-1053
AC1614
TAGRSDNLEPLGLVFG
62
-3.21
-0.12
-0.13
0.09
-0.03
0.25
-0.19


RSR-1059
AC1615
LDGRSDNFHPPELVAG
63
-4.62
-0.89
0.56
-3.10
-2.62
-5.14
-6.49


RSR-1065
AC1616
LEGRSDNEEPENLVAG
64
-4.62
-2.70
0.43
-1.84
-1.00
-3.14
-1.85


RSR-1167
AC1617
LKGRSDNNAPLALVAG
65
-4.62
3.35
1.32
0.09
0.22
1.18
0.06


RSR-1201
AC1618
VYSRGTNAGPHGLTGR
66
-3.02
2.35
1.25
0.09
-1.30
0.79
-0.30


RSR-1218
AC1619
ANSRGTNKGFAGLIGP
67
-0.52
2.66
1.74
-0.30
0.00
-1.33
-1.60


RSR-1226
AC1620
ASSRLTNEAPAGLTIP
68
-0.98
0.29
0.58
0.07
-0.43
-2.33
-0.42


RSR-1254
AC1621
DQSRGTNAGPEGLTDP
69
-1.27
-1.17
1.00
-3.10
-2.32
-4.14
-2.92


RSR-1256
AC 1622
ESSRGTNIGQGGLTGP
70
-1.65
-0.58
0.27
-2.26
-3.32
-5.14
-5.51


RSR-1261
AC1623
SSSRGTNQDPAGLTIP
71
-1.77
-0.36
0.62
-1.14
-1.25
-3.56
-0.98


RSR-1293
AC1624
ASSRGQNHSPMGLTGP
72
-4.69
2.15
0.91
-0.70
-0.01
1.30
-0.67


RSR-1309
AC1625
AYSRGPNAGPAGLEGR
73
-4.69
0.53
0.74
-0.70
-2.25
0.86
0.02


RSR-1326
AC1626
ASERGNNAGPANLTGF
74
-0.27
1.27
1.64
-0.85
-0.74
0.28
-0.13


RSR-1345
AC1627
ASHRGTNPKPAILTGP
75
0.42
ND
ND
ND
ND
-0.50
ND


RSR-1354
AC1628
MSSRRTNANPAQLTGP
76
1.07
2.82
0.36
-0.77
-0.64
-1.82
-1.87


RSR-1426
AC1629
GAGRTDNHEPLELGAA
77
-2.36
-0.65
-0.19
-2.82
-0.18
-0.11
-4.07


RSR-1478
AC1630
LAGRSENTAPLELTAG
78
-2.06
1.18
0.54
-0.82
0.00
1.73
-2.00


RSR-1479
AC1631
LEGRPDNHEPLALVAS
79
-3.47
-3.46
0.12
-0.74
0.00
2.05
0.02


RSR-1496
AC1632
LSGRSDNEEPLALPAG
80
-3.48
-1.46
0.22
-2.81
-5.06
-3.20
-3.22


RSR-1508
AC1633
EAGRTDNHEPLELSAP
81
-2.74
-1.46
-0.26
-1.48
0.00
0.56
-2.93


RSR-1513
AC1634
EGGRSDNHGPLELVSG
82
-2.81
-1.87
0.27
-0.90
0.00
1.29
-3.81


RSR-1516
AC1635
LSGRSDNEAPLELEAG
83
-3.71
-1.87
0.70
-1.69
-0.09
-1.39
-3.22


RSR-1524
AC1636
LGGRADNHEPPELGAG
84
-0.84
1.12
0.95
-1.22
-2.84
-0.74
-2.57


RSR-1622
AC1637
PPSRGTNAEPAGLTGE
85
-4.66
-3.46
0.62
-0.70
-1.09
0.93
-0.78


RSR-1629
AC1638
ASTRGENAGPAGLEAP
86
-4.66
-1.14
1.09
-0.70
-1.74
0.19
-0.25


RSR-1664
AC1639
ESSRGTNGAPEGLTGP
87
-4.66
-3.46
0.32
-1.18
-0.76
-0.76
-2.31


RSR-1667
AC1640
ASSRATNESPAGLTGE
88
-3.05
2.00
0.46
-0.93
-1.25
-0.97
-0.83


RSR-1709
AC1641
ASSRGENPPPGGLTGP
89
-2.64
0.77
-1.00
-0.93
-2.06
-0.76
-1.72


RSR-1712
AC1642
AASRGTNTGPAELTGS
90
-4.07
-0.51
0.66
-0.93
-0.64
0.29
-0.19


RSR-1727
AC1643
AGSRTTNAGPGGLEGP
91
-3.55
-0.51
0.32
-1.58
-4.84
-3.08
-1.78


RSR-1754
AC1644
APSRGENAGPATLTGA
92
-4.68
-3.32
1.06
0.19
-1.40
-1.50
-0.17


RSR-1819
AC1645
ESGRAANTGPPTLTAP
93
1.20
0.79
-0.70
-3.41
-5.64
-4.67
-6.92


RSR-1832
AC1646
NPGRAANEGPPGLPGS
94
-3.62
0.58
0.81
-4.39
-6.64
-4.67
-6.48


RSR-1855
AC1647
ESSRAANLTPPELTGP
95
-0.08
-1.62
0.77
-3.07
-3.47
-4.67
-2.92


RSR-1911
AC1648
ASGRAANETPPGLTGA
96
0.99
2.20
0.56
-1.29
-3.84
-1.39
-3.11


RSR-1929
AC1649
NSGRGENLGAPGLTGT
97
-1.68
ND
ND
ND
ND
-3.08
ND


RSR-1951
AC1650
TTGRAANLTPAGLTGP
98
1.94
2.57
0.39
0.09
-0.09
0.13
-0.42


RSR-2295
AC1761
EAGRSANHTPAGLTGP
99
0.40
1.48
0.01
1.20
0.35
0.13
0.97


RSR-2298
AC1762
ESGRAANTTPAGLTGP
100
1.01
0.86
0.55
1.24
0.24
0.07
1.03


RSR-2038
AC1679
TTGRATEAANLTPAGLTGP
101
4.75
1.00
0.81
0.86
0.10
0.15
0.27


RSR-2072
AC1680
TTGRAEEAANLTPAGLTGP
102
0.00
-0.49
1.00
0.86
0.11
-0.12
0.27


RSR-2089
AC1681
TTGRAGEAANLTPAGLTGP
103
3.91
2.05
0.32
0.85
0.02
-0.04
0.27


RSR-2302
AC1682
TTGRATEAANATPAGLTGP
104
4.73
0.65
0.00
0.74
-0.48
-0.35
0.10


RSR-3047
AC1697
TTGRAGEAEGATSAGATGP
105









RSR-3052
AC1698
TTGEAGEAANATSAGATGP
106









RSR-3043
AC1699
TTGEAGEAAGLTPAGLTGP
107









RSR-3041
AC1700
TTGAAGEAANATPAGLTGP
108









RSR-3044
AC1701
TTGRAGEAAGLTPAGLTGP
109









RSR-3057
AC1702
TTGRAGEAANATSAGATGP
110









RSR-3058
AC1703
TTGEAGEAAGATSAGATGP
111









RSR-2485
AC1763
ESGRAANTEPPELGAG
112
0.61
-0.90
0.15
-5.82
-6.27
-5.36
-5.64


RSR-2486
AC1764
ESGRAANTAPEGLTGP
113
1.03
0.24
0.95
0.30
0.37
-0.33
-0.74


RSR-2488
AC1688
EPGRAANHEPSGLTEG
114
-3.27
-1.21
-1.30
-0.73
-0.91
-1.86
-0.88


RSR-2599
AC1706
ESGRAANHTGAPPGGLTGP
115
1.70
1.02
0.36
0.68
-1.49
-0.71
-2.04


RSR-2706
AC1716
TTGRTGEGANATPGGLTGP
116
0.07
0.83
1.17
-0.04
-2.25
-2.25
0.00


RSR-2707
AC1717
RTGRSGEAANETPEGLEGP
117
1.95
3.25
0.96
-1.96
-2.75
-5.00
-1.39


RSR-2708
AC1718
RTGRTGESANETPAGLGGP
118
1.24
3.25
0.88
-0.37
-3.55
-4.00
-0.49


RSR-2709
AC1719
STGRTGEPANETPAGLSGP
119
-0.14
0.38
0.40
0.35
-1.03
-1.68
1.86


RSR-2710
AC1720
TTGRAGEPANATPTGLSGP
120
-0.21
2.04
0.56
0.15
-3.23
-1.83
-0.07


RSR-2711
AC1721
RTGRPGEGANATPTGLPGP
121
0.58
3.22
1.45
-6.04
-5.55
-5.00
-4.39


RSR-2712
AC1722
RTGRGGEAANATPSGLGGP
122
0.86
3.15
1.21
-0.34
-3.97
-2.68
-1.58


RSR-2713
AC1723
STGRSGESANATPGGLGGP
123
0.96
2.22
0.78
-5.04
-5.25
-5.25
-3.32


RSR-2714
AC 1724
RTGRTGEEANATPAGLPGP
124
0.83
3.23
0.96
-4.46
-5.55
-5.00
-4.39


RSR-2715
AC1725
ATGRPGEPANTTPEGLEGP
125
-4.32
-3.17
0.46
-1.34
-1.93
-1.93
-1.32


RSR-2716
AC1726
STGRSGEPANATPGGLTGP
126
1.00
2.41
0.51
-0.46
-3.55
-2.68
-1.22


RSR-2717
AC1727
PTGRGGEGANTTPTGLPGP
127
-0.21
1.54
1.28
-6.04
-5.55
-5.00
-4.39


RSR-2718
AC1728
PTGRSGEGANATPSGLTGP
128
1.54
3.40
1.29
1.30
-0.20
-0.20
1.63


RSR-2719
AC1729
TTGRASEGANSTPAPLTEP
129
0.26
1.15
1.30
-1.46
-0.16
-0.16
1.68


RSR-2720
AC1730
TYGRAAEAANTTPAGLTAP
130
-1.65
2.14
1.21
0.56
0.45
0.21
2.25


RSR-2721
AC1731
TTGRATEGANATPAELTEP
131
0.77
-0.85
1.25
-2.44
0.00
-4.91
-3.75


RSR-2722
AC1732
TVGRASEEANTTPASLTGP
132
-1.74
-1.17
0.39
1.08
1.00
1.00
2.14


RSR-2723
AC1733
TTGRAPEAANATPAPLTGP
133
-0.42
-3.17
1.32
0.76
0.66
0.66
2.17


RSR-2724
AC1734
TWGRATEPANATPAPLTSP
134
-4.32
1.00
0.55
0.81
0.42
0.42
2.58


RSR-2725
AC1735
TVGRASESANATPAELTSP
135
-4.32
-0.17
0.86
-0.02
0.45
-1.74
-2.17


RSR-2726
AC1736
TVGRAPEGANSTPAGLTGP
136
-4.32
-3.17
1.39
1.22
0.24
0.24
2.10


RSR-2727
AC1737
TWGRATEAPNLEPATLTTP
137
-4.32
0.00
-0.30
-0.50
0.17
-3.91
-1.95


RSR-2728
AC1738
TTGRATEAPNLTPAPLTEP
138
0.32
0.83
-0.61
-0.80
0.45
0.45
2.00


RSR-2729
AC1739
TQGRATEAPNLSPAALTSP
139
-4.52
1.73
0.37
1.75
0.93
0.93
2.85


RSR-2730
AC1740
TQGRAAEAPNLTPATLTAP
140
-2.20
2.73
0.22
1.19
0.51
0.51
1.29


RSR-2731
AC1741
TSGRAPEATNLAPAPLTGP
141
-1.72
-2.70
1.22
1.57
0.92
0.92
2.32


RSR-2732
AC1742
TQGRAAEAANLTPAGLTEP
142
-2.52
2.49
1.44
0.32
-0.21
-0.21
2.29


RSR-2733
AC1743
TTGRAGSAPNLPPTGLTTP
143
1.09
2.91
0.32
0.48
-2.32
-2.32
-3.17


RSR-2734
AC1744
TTGRAGGAENLPPEGLTAP
144
0.83
2.00
0.66
0.55
0.55
0.55
1.83


RSR-2735
AC1745
TTSRAGTATNLTPEGLTAP
145
0.38
2.34
0.32
0.48
0.26
0.26
2.12


RSR-2736
AC1746
TTGRAGTATNLPPSGLTTP
146
1.03
2.91
0.17
1.34
-1.10
-1.10
1.42


RSR-2737
AC1747
TTARAGEAENLSPSGLTAP
147
-0.20
0.30
0.37
1.57
-0.03
-0.03
2.35


RSR-2738
AC1748
TTGRAGGAGNLAPGGLTEP
148
1.68
3.37
1.03
-1.32
-1.65
-2.10
-1.05


RSR-2739
AC1749
TTGRAGTATNLPPEGLTGP
149
1.49
3.43
0.31
-0.12
0.71
-0.58
-0.67


RSR-2740
AC1750
TTGRAGGAANLAPTGLTEP
150
1.77
3.38
1.49
-1.02
-0.75
-1.32
-0.43


RSR-2741
AC1751
TTGRAGTAENLAPSGLTTP
151
0.68
3.10
0.56
0.58
-0.51
-0.91
0.42


RSR-2742
AC1752
TTGRAGSATNLGPGGLTGP
152
1.43
3.42
0.51
-0.27
-3.23
-2.32
-0.17


RSR-2743
AC1753
TTARAGGAENLTPAGLTEP
153
1.63
2.19
0.78
-0.50
-0.13
-2.58
1.18


RSR-2744
AC1754
TTARAGSAENLSPSGLTGP
154
1.04
2.32
0.65
0.59
0.00
-0.15
0.49


RSR-2745
AC1755
TTARAGGAGNLAPEGLTTP
155
1.12
2.77
0.40
-0.77
-0.58
-2.28
-1.00


RSR-2746
AC1756
TTSRAGAAENLTPTGLTGP
156
-0.81
1.54
0.18
0.42
-0.85
-1.50
-0.26


RSR-2747
AC1757
TYGRTTTPGNEPPASLEAE
157
-1.49
1.26
0.06
-0.20
-0.36
-2.77
-2.10


RSR-2748
AC1758
TYSRGESGPNEPPPGLTGP
158
-4.81
-2.32
-0.76
-0.28
-2.68
-2.28
-2.91


RSR-2749
AC1759
AWGRTGASENETPAPLGGE
159
-4.81
3.15
0.24
-1.28
-3.91
-5.09
-2.58


RSR-2750
AC1760
RWGRAETTPNTPPEGLETE
160
-1.49
3.28
-0.29
-3.17
-3.91
-5.09
-4.91


RSR-2751
AC1765
ESGRAANHTGAEPPELGAG
161
1.04
0.37
0.40
-1.59
-5.67
-5.26
-4.93


RSR-2754
AC1801
TTGRAGEAANLTPAGLTES
162



-0.15
-0.82
-3.61
0.45


RSR-2755
AC1802
TTGRAGEAANLTPAALTES
163



0.06
0.29
-2.91
0.62


RSR-2756
AC1803
TTGRAGEAANLTPAPLTES
164



-0.58
-0.39
-2.58
0.49


RSR-2757
AC1804
TTGRAGEAANLTPEPLTES
165



-1.59
-0.27
-1.89
-0.52


RSR-2758
AC1805
TTGRAGEAANLTPAGLTGA
166



0.70
-0.43
0.17
0.85


RSR-2759
AC1806
TTGRAGEAANLTPEGLTGA
167



0.04
-0.72
-1.06
-0.18


RSR-2760
AC1807
TTGRAGEAANLTPEPLTGA
168



-0.06
-0.12
-1.90
-0.15


RSR-2761
AC1808
TTGRAGEAANLTPAGLTEA
169



-0.06
-0.55
-3.71
0.69


RSR-2762
AC1809
TTGRAGEAANLTPEGLTEA
170



-2.14
-0.69
-4.30
-0.59


RSR-2763
AC1810
TTGRAGEAANLTPAPLTEA
171



-0.76
-0.31
-5.28
0.64


RSR-2764
AC1811
TTGRAGEAANLTPEPLTEA
172



-2.18
-0.06
-5.28
-0.11


RSR-2765
AC1812
TTGRAGEAANLTPEPLTGP
173



-0.31
0.07
-5.28
-5.63


RSR-2766
AC1813
TTGRAGEAANLTPAGLTGG
174



0.77
-0.61
-5.28
-5.63


RSR-2767
AC1814
TTGRAGEAANLTPEGLTGG
175



-0.20
-0.85
-1.26
-0.25


RSR-2768
AC1815
TTGRAGEAANLTPEALTGG
176



-0.50
0.13
-1.80
-0.43


RSR-2769
AC1816
TTGRAGEAANLTPEPLTGG
177



-0.44
-0.26
-2.40
-0.39


RSR-2770
AC1817
TTGRAGEAANLTPAGLTEG
178



-0.07
-0.47
-3.18
0.40


RSR-2771
AC1818
TTGRAGEAANLTPEGLTEG
179



-3.05
-0.93
-5.28
-0.99


RSR-2772
AC1819
TTGRAGEAANLTPAPLTEG
180



-0.53
-0.24
-2.19
0.39


RSR-2773
AC1820
TTGRAGEAANLTPEPLTEG
181



-3.80
-0.42
-5.28
-0.81


BSRS-1
AC1601
LSGRSDNHSPLGLAGS
944
0.89
1.94
0.10
-0.67
-2.12
-0.50
-1.92


ND=not determined






Example 7: Competitive Digestion Using RSR-1517 as Internal Control

This competitive assay is developed to minimize any variability in enzyme concentration or reaction condition between reactions in different vials within the same experiment. In order to resolve both the control substrate and the RS of interest in the same example, new control plasmids are constructed.


1. Molecular Cloning of RSR-1517-containing Internal Control

Two internal control plasmids, AC1830 (HD2-V5-AE144-RSR-1517-XTEN288) and AC1840 (HD2-V5-AE144-RSR-1517-XTEN432), are constructed in a similar fashion as AC1611 described in Example 6, with the only difference in the length of the C-terminal XTEN.


2. Enzymatic Digestion

2× substrate solution is prepared by mixing and diluting purified AC1830 or AC1840 and the RS of interest in assay buffer so that the final concentrations of individual substrates are 6 µM. An enzyme master mix is prepared so that after 1:1 mixing with 2× substrate solution, the total reaction volume is 20 µL, the final substrate concentration of each component is 3 µM, and the enzyme-to-substrate ratio is as selected in assay development. The reaction is incubated at 37° C. for 2 hours before stopped by procedures as described above.


3. Relative Cleavage Efficiency Calculation

The reaction mixture is analyzed by non-reducing 4-12% SDS-PAGE. Since the internal control and the substrate of interest have different molecular weight, once cleaved, four bands should be visible in the same sample lane. Percentage of cleavage for both can be calculated and the relative cleavage efficiency can be derived from the same formula in Example 6:






L
o

g
2





%

C
l
e
a
v
e
d

f
o
r

s
u
b
s
t
r
a
t
e

o
f

i
n
t
e
r
e
s
t


%

c
l
e
a
v
e
d

f
o
r

A
C
1611

i
n

t
h
e

s
a
m
e

e
x
p
e
r
i
m
e
n
t








The only difference is now both values are calculated from the reaction mixture in the same vial, while previously from two reactions sharing the same enzyme mix.


Conclusions: We expect this competitive digestion assay with RSR-1517 as internal control to have less assay-to-assay variability when compared to the assay described in Example 6 We anticipate to adopt this method for future release segment screening.


Example 9: In Vitro Caspase 3/7 Assay of anti-EGFR X anti-CD3 Bispecific Antigen Binding Composition

Redirected cellular cytotoxicity of unmasked (with XTEN removed by proteolysis), masked (having 2 XTEN and 2 release segments cleavable by proteolysis), and uncleavable (with 2 XTEN and the release segments replaced by a peptide not susceptible to proteolysis) anti-EGFR x anti-CD3 bispecific antigen binding polypeptide compositions was assessed in an in vitro cell-based assay of caspase 3/7 activities of apoptotic cells. Similar to the caspase cytotoxicity assay described in the Examples, above, PBMC were mixed with EGFR positive tumor target cells in a ratio of 10 effector cells to 1 target cell. All anti-EGFR x anti-CD3 bispecific antigen binding polypeptide compositions were tested using a 10-point, 5x serial dilution of dose concentrations. The unmasked anti-EGFR x anti-CD3 composition was evaluated at a final dose range of 0.000012 to 10 nM. The masked and uncleavable bispecific antigen binding polypeptide compositions were analyzed at a final dose range of 0.00064 to 250 nM. Appropriate EGFR positive human tumor target cell lines included FaDu (squamous cell carcinoma of the head and neck, SCCHN), SCC-9 (SCCHN), HCT-116 (colorectal bearing KRAS mutation), NCI-H1573 (colorectal bearing KRAS mutation), HT-29 (colorectal bearing BRAF mutation) and NCI-H1975 (EGFR T790M mutation). The cell lines were selected to represent colorectal and SCCHN tumors with wild type EGFR and T790M, KRAS and BRAF mutations.


Upon cell lysis, released caspase 3/7 in culture supernatants was measured by the amount of luminogenic caspase 3/7 substrate cleavage by caspase 3/7 to generate the “glow-type” luminescent signal (Promega Caspase-Glo 3/7 cat#G8091). The amount of luminescence is proportional to the amount of caspase activities.


Results: As shown in Table 20, when evaluated in EGFR KRAS mutant HCT-116 cell line, the EC50 activity of the masked anti-EGFR x anti-CD3 bispecific antigen binding polypeptide was 3,408 pM. The EC50 of the uncleavable variant activity was >100,000 pM and the unmasked EC50 activity of the unmasked compositions was 0.8 pM.


When evaluated in EGFR BRAF mutant HT-29 cell line, the EC50 activity of the masked anti-EGFR x anti-CD3 bispecific antigen binding polypeptide was 10,930 pM. The EC50 activity of the uncleavable and unmasked compositions was >100,000 pM and 0.8 pM respectively.


The masked anti-EGFR x anti-CD3 bispecific antigen binding polypeptide was ~4,000 to 14,000-fold less active than the unmasked anti-EGFR x anti-CD3 bispecific antigen binding polypeptide in the two EGFR mutant cell lines tested. As expected, the activity of the uncleavable variant was the least active of the 3 versions evaluated, with an EC50 of greater than 100,000 pM.


Conclusions: The results demonstrated that anti-EGFR x anti-CD3 bispecific antigen binding polypeptide are cytotoxically active against EGFR KRAS- and BRAF-mutant cell lines. Masked anti-EGFR x anti-CD3 bispecific antigen binding polypeptide bearing two XTEN offered strong blocking of cytotoxicity activity, with 4000- to 14,000-fold less cytotoxicity compared to the unmasked form.





TABLE 20






In vitro cytotoxicity activity of unmasked, masked/cleavable and uncleavable anti-EGFR × anti-CD3 variants in HT-29 and HCT-116 human cell lines


ProTIA
EC50 (pM)


HCT-116
HT-29




Unmasked pJB0169
0.8
0.8


Masked pJB0169
3408
10930


Uncleavable pJB0169
>100000
>100000






Example 10: Anti-tumor Properties of anti-EGFR × anti-CD3 Bispecific Antigen Binding Polypeptide Compositions in Early Treatment HT-29 In Vivo Model.

An in vivo efficacy experiment was performed to evaluate an EGFR-CD3 bispecific antigen binding polypeptide composition based on the pJB0169 construct in immunodeficient NOD/SCID mice, characterized by the deficiency of T and B cells and impaired natural killer cells. Mice were maintained in sterile, standardized environmental conditions and the experiment was performed in accordance with US Institutional Animal Care Association for Assessment and Use Committee (IACUC Accreditation of Laboratory Animal Care (AAALAC)) guidelines. The efficacy of protease-treated and protease-untreated anti-EGFR × anti-CD3 bispecific antigen binding polypeptide (e.g. pJB0169) was evaluated using the EGFR BRAF mutant human HT-29 adenocarcinoma xenograft model. Briefly, on day 0, 6 NOD/SCID mice were subcutaneously implanted in the right flank with 3 × 106 HT-29 cells per mouse (Cohort 1). On the same day, cohort 2 to 7 each consisting of 6 NOD/SCID mice per group were subcutaneously injected in the right flank with a mixture of 6 × 106 human PBMC and 3 × 106 HT-29 cells per mouse. Four hours after HT-29 or HT-29/PBMC mixture inoculation, treatments were initiated. Cohort 1 and 2 were injected intravenously with vehicle (PBS+0.05% Tween 80), cohort 3 and 4 were injected with 0.05 mg/kg of the intact anti-EGFR × anti-CD3 bispecific construct and 0.5 mg/kg of the anti-EGFR × anti-CD3 bispecific construct treated with protease to remove the XTEN from the polypeptide, respectively, cohort 5 and 6 were injected with 0.143 mg/kg and 1.43 mg/kg intact anti-EGFR × anti-CD3 bispecific construct, and cohort 7 were injected with 50 mg/kg cetuximab as the positive control. Cohorts 1 to 6 further received seven additional doses administered daily from day 1 to day 7 (total 8 doses). Cohort 7 was dosed with cetuximab twice/week for 4 weeks for a total of 8 doses.


Tumors in the mice were measured twice per week for a projected 33 days with a caliper in two perpendicular dimensions and tumor volumes were calculated by applying the (width2 × length) / 2 formula. Body weight, general appearance and clinical observations such as seizures, tremors, lethargy, hyper-reactivity, pilo-erection, labored/rapid breathing, coloration and ulceration of tumor and death were also closely monitored as a measure of treatment related toxicity. Percent tumor growth inhibition index (%TGI) was calculated for each of the treatment group by applying the formula: ((Mean tumor volume of Cohort 2 vehicle control - Mean tumor volume of test article treatment)/mean tumor volume of Cohort 2 vehicle control) × 100. Treatment results with a %TGI ≥60% is considered therapeutically active.


Results: At day 33, vehicle-treated cohort 1 mice bearing tumor cells only had an average tumor burden of 250±113 mm3. Cohort 2 mice treated with vehicle in the presence of human effector cells did not inhibit tumor progression, having an average tumor burden of 238±228 mm3, demonstrating that human effector cells alone, as such, could not elicit an anti-tumor effect. Treatment with the protease-treated anti-EGFR × anti-CD3 construct at 0.05 mg/kg and 0.5 mg/kg (cohort 3 and 4 respectively) in the presence of human effector cells exhibited clear tumor growth inhibition with a %TGI of 99% for both treatment groups. Importantly, treatment with anti-EGRF × anti-CD3 XPAT at 0.143 mg/kg and 1.43 mg/kg (cohort 5 and 6 respectively) in the presence of human effector cells also inhibited tumor growth in a dose-dependent manner with %TGI of 70% for the 0.143 mg/kg dose group and 96% in the 1.43 mg/kg cohort. The data suggest that at 0.143 mg/kg and 1.43 mg/kg dosages, sufficient amounts of the anti-EGRF × anti-CD3 constructs were effectively cleaved by proteases in the in vivo tumor environment into the more active, unXTENylated anti-EGFR × anti-CD3 bispecific antigen binding fragments to yield the observed efficacy. Significantly, cohort 7 treated with 50 mg/kg of cetuximab did not induce tumor regression, with a %TGI of -20%.


Conclusions: The results suggest that the anti-EGFR × anti-CD3 bispecific construct can be effectively cleaved in vivo into the active form and is efficacious in the EGFR BRAF mutant HT-29 tumor environment to inhibit tumor progression. In addition, the anti-EGFR × anti-CD3 bispecific construct was superior to the cetuximab control in anti-tumor activity under the conditions of the experiment. Of note, no significant body weight loss was observed in all test article treatment groups and vehicle control indicating that all treatments were well tolerated.


Example 11: Cell Binding Assessed by Flow Cytometry

Bispecific binding of the anti-EGFR × anti-CD3 bispecific antigen binding composition is also evaluated by flow cytometry-based assays utilizing CD3 positive human Jurkat cells and EGFR positive human cells selected from HT-29, HCT-116, NCI-H1573, NCI-H1975, FaDu, and SCC-9 or a stable CHO cell line expressing EGFR. CD3+ and EGFR+ cells are incubated with a dose range of untreated anti-EGFR × anti-CD3 bispecific antigen binding composition (PJB0169, comprising 2 XTEN and 2 RS), protease-treated PJB0169, and anti-CD3 scFv and anti-EGFR scFv positive controls for 30 min at 4° C. in binding buffer containing HBSS with 2% BSA and 5 mM EDTA. After washing with binding buffer to remove unbound test material, cells are incubated with FITC-conjugated anti-His tag antibody (Abcam cat #ab1206) for 30 min at 4° C. Unbound FITC-conjugated antibody is removed by washing with binding buffer and cells resuspended in binding buffer for acquisition on a FACS Calibur flow cytometer (Becton Dickerson) or equivalent instrument. All flow cytometry data are analyzed with FlowJo software (FlowJo LLC) or equivalent.


While anti-EGFR scFv is not expected to bind to Jurkat cells, anti-CD3 scFv, untreated PJB0169 and protease-treated PJB0169 are all expected to bind to Jurkat cells as indicated by an increase in fluorescence intensity when compared to Jurkat cells incubated with FITC-conjugated anti-His tag antibody alone. Similarly, anti-EGFR scFv, protease-treated and untreated PJB0169 are all expected to bind to EGFR positive cells, while anti-CD3 scFv is not expected to bind to EGFR positive cells. It is expected that these data will reflect the bispecific binding ability of the anti-EGFR × anti-CD3 bispecific antigen binding composition to recognize both the CD3 and EGFR antigen expressed respectively on Jurkat and the panel of EGFR expressing human cell lines. Furthermore, due to the XTEN polymer providing some interference in surface binding, the untreated anti-EGFR × anti-CD3 bispecific antigen binding composition is expected to bind at a lower affinity than the protease-treated bispecific antigen binding composition for both the CD3 and EpCAM antigens.


Example 12: Cell Lysis Assessed by Flow Cytometry

Cell lysis by the anti-EGFR × anti-CD3 bispecific antigen binding composition is evaluated by flow cytometry utilizing human PBMCs and an EGFR positive cell line. EGFR positive HCT-116 target cells (or target cells selected from HT-29, NCI-H1573, NCI-H1975, FaDu, and SCC-9 or a stable CHO cell line expressing EGFR) are labeled with the fluorescent membrane dye CellVue Maroon dye (Affymetrix/eBioscience, cat #88-0870-16) according to manufacturer’s instructions. Alternatively PKH26 (Sigma, cat #MINI26 and PKH26GL) can also be used. In brief, HCT-116 cells are washed twice with PBS followed by resuspension of 2 × 106 cells in 0.1 mL Diluent C provided with the CellVue Maroon labeling kit. In a separate tube, 2 microL of CellVue Maroon dye is mixed with 0.5 mL diluent C, and then 0.1 mL added to the HCT-116 cell suspension. The cell suspension and CellVue Maroon dye are mixed and incubated for 2 min at room temperature. The labeling reaction is then quenched by the addition of 0.2 mL of fetal bovine serum (FCS). Labeled cells are washed twice with complete cell culture medium (RPMI-1640 containing 10% FCS) and the total number of viable cells determined by trypan blue exclusion. For an effector to target ratio of 10:1 in a total volume of 200 microL per well, 1×105 PBMC are co-cultured with 1 × 104 CellVue Maroon-labeled HCT-116 cells per well in a 96-well round-bottom plate in the absence or presence of the indicated dose range concentration of protease-treated and untreated anti-EGFR × anti-CD3 bispecific antigen binding composition (PJB0169, comprising 2 XTEN and 2 RS) samples. After 24 h, cells are harvested with Accutase (Innovative Cell Technologies, cat #AT104) and washed with 2% FCS/PBS. Before cell acquisition on a Guava easyCyte flow cytometer (Millipore), cells are resuspended in 100 microL 2% FCS/PBS supplemented with 2.5 micrograms/mL 7-AAD (Affymetrix/eBioscience, cat #00-6993-50) to discriminate between alive (7-AAD-negative) and dead (7-AAD-positive) cells. FACS data are analyzed with guavaSoft software (Millipore); and percentage of dead target cells is calculated by the number of 7-AAD-positive/CellVue Maroon-positive cells divided by the total number of CellVue Maroon-positive cells.


Dose response kill curves of percent cytotoxicity against bispecific antigen binding composition concentration are analyzed by 4 parameter-logistic regression equation using GraphPad Prism; and the concentration of bispecific antigen binding composition that induced half maximal percent cell cytotoxicity is thus determined.


Cytotoxicity results utilizing flow cytometry are expected to be in-line with results obtained with other cytotoxicity assays, including LDH and caspase. Exposure of HCT-116 cells to protease-cleaved and uncleaved anti-EGFR × anti-CD3 bispecific antigen binding compositions in the absence of PBMC are expected to have no effect. Similarly, PBMC are not expected to be activated in the presence of bispecific antigen binding composition without target cells. These results are expected to indicate that bispecific antigen binding compositions need to be clustered on the surface of target cells in order to stimulate PBMC for cytotoxicity activity. In the presence of PBMC and target cells, there would be a concentration-dependent cytotoxic effect due to bispecific antigen binding composition pretreated or untreated with protease. Further, results are expected to show that exposure of HCT-116 cells to untreated bispecific antigen binding composition (no protease) in the presence of PBMC would show reduced cytotoxicity as compared to protease-cleaved bispecific antigen binding composition.


Example 13: T-cell Activation Marker Assays of anti-EGFR × anti-CD3 Bispecific Antigen Binding Composition.

To measure the anti-EGFR × anti-CD3 bispecific antigen binding composition induced activation markers (CD69 and CD25), 1 × 105 PBMC or purified CD3+ cells are co-cultured in RPMI-1640 containing 10% FCS with 1 × 104 HCT-116 or HT-29 cells per assay well (i.e., effector to target ratio of 10:1) in the presence of anti-EGFR × anti-CD3 bispecific antigen binding composition (PJB0169, comprising 2 XTEN and 2 RS) in a 96-well round-bottom plate with total final volume of 200 microL. After 20 h incubation in a 37° C., 5% CO2 humidified incubator, cells are stained with PECy5-conjugated anti-CD4, APC-conjugated anti-CD8, PE-conjugated anti-CD25, and FITC-conjugated anti-CD69 (all antibodies from BioLegend) in FACS buffer (1% BSA/PBS) at 4° C., washed twice with FACS buffer, and then re-suspended in FACS buffer for acquisition on a Guava easyCyte flow cytometer (Millipore).


The T-cell activation marker expression trend of the three bispecific antigen binding composition molecules is expected to be similar to that observed by cytotoxicity assays, including LDH and caspase. Activation of CD69 on CD8 and CD4 populations of PBMC or CD3+ cells by untreated anti-EGFR × anti-CD3 bispecific antigen binding composition (pJB0169) is expected to be less active than protease-treated pJB0169 bispecific antigen binding composition; and the non-cleavable anti-EGFR × anti-CD3 bispecific antigen binding composition (pJB0172) is expected to be less active than the untreated pJB0169.


Example 14: Cytometric Bead Array Analysis for Human Th1/Th2 Cytokines Using Stimulated Normal Healthy Human PBMCs and Intact and Protease-treated anti-EGFR × anti-CD3 Bispecific Antigen Binding Composition

As a safety assessment of the ability of intact versus cleaved anti-EGFR × anti-CD3 bispecific antigen binding composition (pJB0169, comprising 2 XTEN and 2 RS) to stimulate release of T-cell related cytokines in a cell-based in vitro assay, a panel of cytokines including IL-2, IL-4, IL-6, IL-10, TNF-alpha, IFN-gamma are analyzed using the cytometric bead array (CBA) on supernatants from cultured human PBMC stimulated with protease-treated and untreated anti-EGFR × anti-CD3 bispecific antigen binding composition samples. The antihuman CD3 antibody, OKT3, is used as positive control and untreated wells serve as negative control.


Briefly, OKT3 (0, 10 nM, 100 nM and 1000 nM) and protease-treated and untreated anti-EGFR × anti-CD3 bispecific antigen binding composition (pJB0169 at 10 nM, 100 nM, 1000 nM and 2000 nM) are dry-coated onto a 96-well flat bottomed plate by allowing the wells to evaporate overnight in the biosafety hood. Wells are then washed once gently with PBS and 1X106 PBMC in 200 microL were added to each well. The plate is then incubated at 37° C., 5% CO2 for 24 h, after which tissue culture supernatant is collected from each well and analyzed for cytokine released using the validated commercial CBA kit (BD CBA human Th1/Th2 cytokine kit, cat # 551809) by flow cytometry following manufacturer’s instructions.


OKT3, but not untreated wells, is expected to induce robust secretion of all cytokines (IL-2, IL-4, IL-6, IL-10, TNF-alpha, IFN-gamma) evaluated, thereby confirming the performance of the CBA cytokine assay. Stimulation with protease-treated anti-EGFR × anti-CD3 bispecific antigen binding composition is expected to trigger significant cytokine expression, especially at concentrations higher than 100 nM for all of the cytokines tested. In contrast, baseline levels of IL-2, IL-6, IL-10, TNF-alpha and IFN-gamma are expected when the intact non-cleaved anti-EGFR × anti-CD3 bispecific antigen binding composition molecule is the stimulant at a concentration range of 10 to 2000 nM. These data support that the XTEN polymer of the intact bispecific antigen binding composition provides considerable shielding effect and hinders PBMC stimulated cytokine responses compared to the protease-treated bispecific antigen binding composition in which the EGFR × anti-CD3 portion is released from the composition.


Example 15: Cytotoxicity Assays of anti-EGFR × anti-CD3 Bispecific Antigen Binding Composition in the Presence of Purified CD3 Positive T cells.

To demonstrate that cytotoxic activity of bispecific antigen binding composition molecules is mediated by CD3 positive T cells, non-cleavable anti-EGFR × anti-CD3 bispecific antigen binding composition without the release segment (pJB0172, comprising 2 XTEN) and protease-treated and untreated anti-EGFR × anti-CD3 bispecific antigen binding composition (pJB0169, comprising 2 XTEN and 2 RS) are evaluated in EGFR+ human cell lines (e.g. HCT-116 or HT-29) in the presence of purified human CD3 positive T cells. Purified human CD3 positive T cells are purchased from BioreclamationIV, where they are isolated by negative selection using MagCellect Human CD3+ T cell isolation kit from whole blood of healthy donors. In this experiment, purified human CD3 positive T cells are mixed with an EGFR+ cell line in a ratio of about 10:1 and all three bispecific antigen binding composition molecules were tested as a 12-point, 5x serial dilution dose curve in the LDH assay as described above. The activity trend of the three bispecific antigen binding composition molecules profiled with CD3+ cells is expected to be similar to the profile of the same cell line with PBMCs. Untreated pJB0169 is expected to be less active than protease-treated pJB0169; and the non-cleavable pJB0172 is expected to be less active than untreated pJB0169. Such results would demonstrate that cytotoxic activity of bispecific antigen binding composition molecules is indeed mediated by CD3 positive T cells. The susceptibility of the release segment contained within the cleavable anti-EGFR × anti-CD3 bispecific antigen binding composition molecule to proteases postulated to be released from the tumor cells and/or activated CD3 positive T cells in the assay mixture is likely to differ between cell lines.


Example 16: T-cell Activation Marker and Cytokine Release Assays of anti-EGFR × anti-CD3 Bispecific Antigen Binding Composition.

To measure the anti-EGFR × anti-CD3 bispecific antigen binding composition induced expression of cytokines, purified CD3+ cells are co-cultured with HCT-116 cells per assay well (i.e., effector to target ratio of about 10:1) in the presence of anti-EGFR × anti-CD3 bispecific antigen binding composition (pJB0169, comprising 2 XTEN and 2 RS) in a 96-well round-bottom plate with total final volume of 200 microL. After 20 h incubation in a 37° C., 5% CO2 humidified incubator, cell supernatant is harvested for cytokine measurements. This assay can also be performed with other target cells selected from HT-29, NCI-H1573, NCI-H1975, FaDu, and SCC-9 as well as PBMC in place of purified CD3+ cells.


Cytokine analysis of interleukin (IL)-2, IL-4, IL-6, IL-10, tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma secreted into the cell culture supernatant is quantitated using the Human Th1/Th2 Cytokine Cytometric Bead Array (CBA) kit (BD Biosciences cat #550749) following manufacturer’s instruction. In the absence of bispecific antigen binding composition, no cytokine secretion above background is expected from purified CD3+ cells. pJB0169 in the presence of EGFR-positive target cells and purified CD3+ cells is expected to activate T cells and secrete a pattern of T cell cytokines with a high proportion of Th1 cytokines such as IFN-gamma and TNF-alpha. Compared to intact pJB0169, lower concentrations of protease-treated pJB0169 are expected to active T cells and secrete T cell cytokines, supporting the shielding effect of the XTEN polymer in the bispecific antigen binding composition.


Example 17: Single- and Multi-dose Pharmacokinetic Determination of anti-EGFR × anti-CD3 Bispecific Antigen Binding Polypeptide in Non-Human Primates

The pharmacokinetics (PK) and general tolerability of anti-EGFR × anti-CD3 bispecific antigen binding polypeptide bearing 2 XTEN polymers (i.e., pJB0169) following single and multiple intravenous administrations was evaluated in naïve, healthy non-human primates (NHP) (e.g., cynomolgus monkeys). Briefly, one female and one male monkey was intravenously infused with 8.5 µg/kg of the composition via the cephalic vein. Both animals were monitored for two weeks. Following no observable adverse events, animals were subjected to a multi-dose regimen initiated as one dose every three days for three weeks (total 9 doses in study). The multi-dose phase began with Day 15 and ended on Day 36. At specific time points throughout the study, blood was collected for assay of pharmacokinetics, cytokines, hematology and serum chemistries.


Animal monitoring included body weight, body temperature and cage-side observations once or twice daily during the duration of the study. Animals were monitored for general health and appearance; signs of pain and distress, fever, chills, nauseas, vomiting and skin integrity. On dosing days, animals were checked for injection site reactions before and after administration of the compositions. Hematology and serum chemistry were determined at predose and 24 hour after first single dose. Cytokines were evaluated at pre-dose and at appropriate intervals within 72 hours post first single dose and in the multi-dose phase.


The amount of pJB0169 present in plasma was quantitated on a sandwich ELISA using EGFR-biotin captured on an electrochemiluminescence streptavidin plate with sulfo-tagged anti-XTEN-antibody as detection. Pharmacokinetic parameters including Cmax, Tmax, area under the curve, half-life and exposure profile were analyzed using the WinNonLin software.


The cytokine panel included measurement of IFN-gamma, IL-1beta, TNF-alpha, IL-1beta, IL-2, IL-4, IL-6, and IL-10 using the Meso-Scale Discovery platform following manufacturer’s instructions. The lower limit of detection for these cytokines are 2.0 pg/mL, 0.32 pg/mL, 0.11 pg/mL, 0.68 pg/mL, 0.04 pg/mL, 0.23 pg/mL and 0.10 pg/mL respectively. The hematology panel included measurement of white blood cells, red blood cells, hemoglobin, hematocrit, mean corpuscular hemoglobin volume, mean corpuscular hemoglobin concentration, red blood cell distribution width, platelet, mean platelet volume, % neutrophils, % lymphocytes, % monocytes, % eosinophils and % basophils.The serum chemistry panel included measurement of alanine aminotransferase, aspartate aminotransferase, total protein, albumin, alkaline phosphatase, globulin, albumin/globulin ratio, γ-glutamyltransferase, glucose, urea, creatinine, calcium, total cholesterol, triglycerides, total bilirubin, sodium, potassium, chlorine and creatine kinase.


Results: pJB0169 was well tolerated at a dose of 8.5 µg/kg. There was no loss in body weight. No chills, fever, nausea, vomiting, skin rash, test artile injection site reaction were observed. All measured cytokine levels except IL-6 were below the limits of detection. Although detectable in the single-dose and multi-dose phase, the level of IL-6 detected is considered to be background with the highest level not exceeding 51 pg/mL in male and 19 pg/mL in female animals in the range of time points evaluated. Hematology and clinical panel were within normal range. Following Day 1 administration, at 8.5 µg/kg, the average Cmax value was 372 ng/mL, the averaged AUC0-168h was 15839 ng*h/mL, the averaged AUC0-inf was 16342 ng*h/mL, the averaged CL value was 0.00886 mL/min/kg and the averaged T½ value was 24.2 hours. The volume of distribution (Vd) was 0.0238 L/kg. Following Day 36 administration, average Cmax value was 410 ng/mL, the averaged AUC0-168h was 22985 ng*h/mL, the averaged AUC0-inf was 24663 ng*h/mL, the averaged CL value was 0.00578 mL/min/kg and the averaged T½ value was 44.0 hours. The volume of distribution (Vd) was 0.0196 L/kg. The accumulative index of Cmax and AUC0-168h in monkey following single or multiple IV infusion administration of pJB0169 at 8.5 µg/kg were 1.10 and 1.45. There was no significant difference in systemic exposure between Day 1 and Day 36 administration. Data also suggest no emergence of anti-drug antibodies.


Example 18: Dose Range Finding of anti-EGFR × anti-CD3 Bispecific Antigen Binding Polypeptide in Non-Human Primates

The dose range finding study of pJB0169 bispecific antigen binding polypeptide in non-human primates was carried out in healthy, naïve cynomolgus monkeys with one female and one male monkey per cohort. Briefly, one female and one male monkey was intravenously infused with pJB0169 via the cephalic vein. Both animals were monitored for two weeks. Following no observable adverse events, animals were subjected to a multi-dose regimen initiated as one dose every three days for three weeks (total 9 doses in study). The multi-dosing phase began with Day 15 and ended on Day 36. At specific time points throughout the study, blood was collected for assay of pharmacokinetics, cytokines, hematology and serum chemistries. Twenty-four hours after the last dose (i.e., Day 37), animals were necropsied for histopathology evaluation. When no adverse events were observed one week after the first dose in a cohort, pJB0169 was dose escalated 2- or 3-fold in the next cohort. Dose escalation will proceed until adverse events are observed.


Animal monitoring included body weight, food consumption, body temperature and cage-side observations once or twice daily during the duration of the study. Animals were monitored for general health and appearance; signs of pain and distress; fever, chills, nauseas, vomiting and skin integrity. On dosing days, animals were checked for injection site reaction before and after XPAT administration.


The amount of pJB0169 present in plasma will be quantitated on a sandwich ELISA using EGFR-biotin captured on an electrochemiluminescence streptavidin plate with sulfo-tagged anti-XTEN-antibody as detection. Pharmacokinetic parameters including Cmax, Tmax, area under the curve, half-life and exposure profile will be analyzed using WinNonLin software.


The cytokine panel includes measurement of IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, IFN-γ and TNFα using Beckon Dickinson Cytometric Bead Array.


The hematology panel included measurement of white blood cells, red blood cells, hemoglobin, hematocrit, mean corpuscular hemoglobin volume, mean corpuscular hemoglobin concentration, red blood cell distribution width, platelet, mean platelet volume, % neutrophils, % lymphocytes, % monocytes, % eosinophils and % basophils.


The serum chemistry panel included measurement of alanine aminotransferase, aspartate aminotransferase, total protein, albumin, alkaline phosphatase, globulin, albumin/globulin ratio, γ-glutamyltransferase, glucose, urea, creatinine, calcium, total cholesterol, triglycerides, total bilirubin, sodium, potassium, chlorine and creatine kinase.


Histopathology evaluation with H&E staining were performed on a panel of tissues including adrenal glands, aorta, bone, brain, epididymides, esophagus, eyes, fallopian tubes (female only), heart, kidney, large intestines, liver with gall bladder, lungs, lymph nodes, mammary glands (female only), ovaries (female only), pancreas, pituitary gland, prostate gland, salivary glands, skeletal muscles, skin, small intestines, spinal cord, spleen, stomach, testes (male only), thymus, thyroid glands, trachea, urinary bladder, uterus and injection site.


Interim results: The starting dose in this dose range finding study was Cohort 1 at 25.5 µg/kg of pJB0169. No observable adverse events such as fever, chills, skin rash, nausea, vomiting, abnormal hematology and serum chemistry were observed in the single-dose and multi-dose phase. pJB0169 was therefore dose-escalated 3-fold to 76.5 µg/kg in Cohort 2. No observable adverse events were observed in Cohort 2 and pJB0169 was dose-escalated 3-fold to 230 µg/kg in Cohort 3. Other than a reversible increase in AST, ALT and total bilirubin readings above the normal range, no other adverse events were observed and pJB0169 was next dose-escalated 2-fold to 460 µg/kg in Cohort 4. No observable adverse events were observed in Cohort 4. Further dose escalation of pJB0169 is ongoing.


There were no found dead and moribund animals during the whole study period. There were no test article-related organ weight changes in any treatment groups. There were no observed gross lesions in all the tested animals. Microscopically, the major findings were subcutaneous hemorrhage, tissue necrosis, neutrophilic infiltration, venous necrosis or thrombosis, and skin crust at the injection sites of some animals. These changes were likely attributed to the intravenous infusion procedure.


Interim conclusions: pJB0169 is well tolerated in non-human primates at doses up to 460 µg/kg. No test article-related organ weight and pathologic changes were observed in all tested dose groups.


Example 19: Determination of Isoelectric Point (pI) of Antigen Binding Fragments

To determine the isoelectric point of each CD3 and EGFR variant antigen binding fragment, each was analyzed using the Protein Titration Curve Panel in the Biologics suite of Maestro (Schrödinger, Germany). The titration curve for a protein is calculated from the pKa values of titratable groups—individual ionizable residues and termini- by summing the fractional charges of each such group at intervals in the pH value. The pKa values are generated with ProPKA (Sondergaard, C. et al. Toxicol Lett. 205(2):116 (2011); Olsson, M. et. al. Proteins 79:3333 (2011)). The titration curves were plotted and the isoelectric point (pI) was determined for each curve, with the results presented in the tables, below.





TABLE 21






Isoelectric points for CD3 variants


Antibody
Variant
Isoelectric Point (pI)




CD3
3.9
6.8


CD3
CD3.30
6.8


CD3
CD3.31
6.2


CD3
CD3.32
6.2


CD3
CD3.33
6.2









TABLE 22






Isoelectric points for EGFR variants


Antibody
Variant
Isoelectric Point (pI)




EGFR
EGFR.2
5.0


EGFR
EGFR.13
5.0


EGFR
EGFR.18
5.1


EGFR
EGFR.23
5.1


EGFR
EGFR.14
5.0


EGFR
EGFR.19
5.1


EGFR
EGFR.24
5.1


EGFR
EGFR.15
5.3


EGFR
EGFR.20
5.5


EGFR
EGFR.25
5.5


EGFR
EGFR.16
5.3


EGFR
EGFR.21
5.5


EGFR
EGFR.26
5.5


EGFR
EGFR.17
5.3


EGFR
EGFR.22
5.5


EGFR
EGFR.27
5.5





Claims
  • 1. A polypeptide comprising an antibody binding fragment (AF1), wherein the AF1 comprises light chain complementarity-determining regions (CDR-L), heavy chain complementarity-determining regions (CDR-H), light chain framework regions (FR-L), and heavy chain framework regions (FR-H), and wherein the AF1: a. specifically binds to epidermal growth factor receptor (EGFR);b. comprises a variable heavy (VH) amino acid sequence having at least 90% sequence identity to an amino acid sequence of SEQ ID NO: 28-32; andc. comprises a variable light (VL) amino acid sequence having at least 90% sequence identity to an amino acid sequence of SEQ ID NO: 25- 27.
  • 2. (canceled)
  • 3. (canceled)
  • 4. The polypeptide of claim 1, wherein the AF1 exhibits an isoelectric point (pI) that is at least 0.1 pH units higher the pI of the antigen binding fragment consisting of a sequence shown in SEQ ID NO:52.
  • 5. The polypeptide of claim 1, wherein the AF1 is incorporated into the polypeptide to form an anti-EGFR bispecific antibody, wherein the polypeptide exhibits a higher pI relative to a control bispecific antibody, wherein said polypeptide comprises said AF1 and a reference antigen binding fragment that binds to a cluster of differentiation 3 T cell receptor (CD3), and wherein said control bispecific antigen binding fragment is identical to the polypeptide except that the AF1 is replaced with SEQ ID NO:52-.
  • 6-25. (canceled)
  • 26. The polypeptide of any one of the preceding claims, further comprising a first release segment peptide (RS1), wherein the RS1 is a substrate for cleavage by a mammalian protease selected from the group consisting of legumain, MMP-2, MMP-7, MMP-9, MMP-11, MMP-14, uPA, and matriptase and has an amino acid sequence having at least 90% sequence identity to a sequence selected form any one of SEQ ID NOs: 53-671.
  • 27-29. (canceled)
  • 30. The polypeptide of any one of the preceding claims, further comprising a first extended recombinant polypeptide (XTEN1) wherein the XTEN1 is characterized in that it has at least about 36 amino acids;at least 90% of its the amino acid residues are selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P);it has at least 4-6 different amino acids selected from G, A, S, T, E and P; andit comprises at least three of the amino acid sequences of SEQ ID NOS:672-675 and/or comprises an amino acid sequence at least about 90% identical to a sequence selected from any one of SEQ ID NOS: 676-734 .
  • 31-33. (canceled)
  • 34. The polypeptide of any one of the preceding claims, wherein the AF1 is a chimeric or a humanized antigen binding fragment and optionally is selected from the group consisting of Fv, Fab, Fab′, Fab′-SH, linear antibody, and single-chain variable fragment (scFv).
  • 35. (canceled)
  • 36. The polypeptide of any one of the preceding claims expressed as a fusion protein, wherein the fusion protein, in an uncleaved state, has a structural arrangement from N-terminus to C-terminus of AF1-RS1-XTEN1 or XTEN1-RS1-AF1.
  • 37. The polypeptide of any one of the preceding claims, further comprising a second antigen binding fragment (AF2) that specifically binds to cluster of differentiation 3 T cell receptor (CD3) or a CD3 complex subunit selected from any one of CD3 epsilon, CD3 delta, CD3 gamma, CD3 zeta, CD3 alpha and CD3 beta epsilon.
  • 38-40. (canceled)
  • 41. The polypeptide of claim 37, wherein the AF2 comprises light chain complementarity-determining regions (CDR-L) and heavy chain complementarity-determining regions (CDR-H), and wherein the AF2 comprises: a CDR-H1 having the amino acid sequence of SEQ ID NO: 742,a CDR-H2 having the amino acid sequence of SEQ ID NO: 743,a CDR-H3 having the amino acid sequence of SEQ ID NO: 744,a CDR-L1 having an amino acid sequence of SEQ ID NOS: 735 or 736,a CDR-L2 having an amino acid sequence of SEQ ID NOS: 738 or 739, anda CDR-L3 having an amino acid sequence of SEQ ID NO:740.
  • 42. (canceled)
  • 43. The polypeptide of claim 41, wherein the AF2 further comprises light chain framework regions (FR-L) and heavy chain framework regions (FR-H) wherein AF2 comprises: a FR-L1 having an amino acid sequence of SEQ ID NO:746;a FR-L2 having an amino acid sequence of SEQ ID NO:747;a FR-L3 having an amino acid sequence of any one of SEQ ID NOS:748-751;a FR-L4 having an amino acid sequence of SEQ ID NO:754;a FR-H1 having an amino acid sequence of SEQ ID NO:755 or SEQ ID NO:756;a FR-H2 having an amino acid sequence of SEQ ID NO:759;a FR-H3 having an amino acid sequence of SEQ ID NO:760; anda FR-H4 having an amino acid sequence of any one of SEQ ID NO:764.
  • 44-47. (canceled)
  • 48. The polypeptide of claim 27, wherein the AF2 comprises a variable heavy (VH) amino acid sequence having at least 90% sequence identity to an amino acid sequence of SEQ ID NOs: 766, 769, 773, or 775 and wherein the AF2 comprises a variable light (VL) amino acid sequence having at least 90%, sequence identity or is identical to an amino acid sequence of any one of SEQ ID NOs: 765, 767, 768, 770, 771, 772, or 774.
  • 49. (canceled)
  • 50. The polypeptide of claim 37, wherein the AF2 comprises an amino acid sequence having at least 95% sequence identity to an amino acid sequence of any one of SEQ ID NOS:776-780.
  • 51-55. (canceled)
  • 56. The polypeptide of claim 37, wherein the AF2 is fused to the AF1 by a flexible peptide linker, and wherein the flexible linker comprises 2 or 3 amino acids selected from the group consisting of glycine, serine, and proline.
  • 57. (canceled)
  • 58. The polypeptide of claim 37, wherein (1) the AF2 fragment is selected from the group consisting of Fv, Fab, Fab′, Fab′-SH, linear antibody, a single domain antibody, and single-chain variable fragment (scFv), or (2) the AF1 and AF2 are configured as an (Fab′)2 or a single chain diabody.
  • 59-64. (canceled)
  • 65. The polypeptide of claim 37, further comprising a second release segment (RS2), wherein the RS2 is a substrate for cleavage by a mammalian protease selected from the group consisting of legumain, MMP-2, MMP-7, MMP-9, MMP-11, MMP-14, uPA, and matriptase.
  • 66-70. (canceled)
  • 71. The polypeptide of claim 65, further comprising a second extended recombinant polypeptide (XTEN2) wherein the XTEN2 is characterized in that it has at least about 36 amino acids;at least 90% of the amino acid residues of its sequence are selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P); andit has at least 4-6 different amino acids selected from G, A, S, T, E and P.
  • 72. The polypeptide of claim 71, wherein the XTEN2 comprises an amino acid sequence that comprises at least three of the amino acid sequences selected from SEQ ID NOs: 672-675 and/or wherein the XTEN2 comprises an amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NOS: 676-734.
  • 73. (canceled)
  • 74. (canceled)
  • 75. The polypeptide of claim 71, wherein the polypeptide has a structural arrangement from N-terminus to C-terminus as follows: XTEN1-RS1-AF1-AF2-RS2-XTEN2, XTEN1-RS1-AF2-AF1-RS2-XTEN2, XTEN2-RS2-AF2-AF1-RS1-XTEN1, XTEN2-RS2-AF1-AF2-RS1-XTEN1, XTEN2-RS2-diabody-RS1-XTEN1, or XTEN1-RS1-diabody-RS2-XTEN2, wherein the diabody comprises VL and VH of the AF1 and AF2, wherein the AF2 specifically binds CD3 and AF1 specifically binds EGFR, and wherein XTEN 1 and XTEN2 are of the same or different amino acid length or sequence.
  • 76. (canceled)
  • 77. A pharmaceutical composition comprising the polypeptide of any one of the preceding claims and one or more pharmaceutically suitable excipients.
  • 78-81. (canceled)
  • 82. Use of the polypeptide comprising the polypeptide of any one of the preceding claims in the preparation of a medicament for the treatment of a disease in a subject, wherein the disease is selected from the group of cancers consisting of anaplastic and medullary thyroid cancers, appendiceal cancer, arrhenoblastoma, biliary tract carcinoma, bladder cancer, breast cancer, cancers of the bile duct, carcinoid tumor, cervical cancer, cholangiocarcinoma, colon cancer, colorectal cancer, craniopharyngioma, endometrial cancer, epithelial intraperitoneal malignancy with malignant ascites, esophageal cancer, Ewing sarcoma, fallopian tube cancer, follicular cancer, gall bladder cancer, gastric cancer, gastrointestinal stromal tumor (GIST), GE-junction cancer, genito-urinary tract cancer, glioma, glioblastoma, head and neck cancer, hepatoblastoma, hepatocarcinoma, HR+ and HER2+ breast cancer, Hurthle cell cancer, Inflammatory breast cancer, Kaposi sarcoma, kidney cancer, laryngeal cancer, liposarcoma, liver cancer, lung cancer, medulloblastoma, melanoma, Merkel cell carcinoma, neuroblastoma, neuroblastoma, neuroendocrine cancer, non-small cell lung cancer, osteosarcoma (bone cancer), ovarian cancer, ovarian cancer with malignant ascites, pancreatic cancer, pancreatic neuroendocrine tumor, papillary cancer, parathyroid cancer, peritoneal carcinomatosis, peritoneal mesothelioma, primitive neuroectodermal tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland carcinoma, sarcoma, skin cancer, small cell lung cancer, small intestine cancer, stomach cancer, testicular cancer, thyroid cancer, triple negative breast cancer, urothelial cancer, uterine cancer, uterine serous carcinoma, vaginal cancer, vulvar cancer, and Wilms tumor.
  • 83-89. (canceled)
  • 90. An isolated nucleic acid, the nucleic acid comprising (a) a polynucleotide encoding a polypeptide of any one of the preceding claims; or (b) the complement of the polynucleotide of (a).
  • 91-147. (canceled)
CROSS-REFERENCE

This This application is a U.S. National Phase of International Application No. PCT/US2020/039682, filed Jun. 25, 2020, which claims priority to U.S. Provisional Application No. 63/043,486, filed Jun. 24, 2020 and U.S. Provisional Application No. 62/866,749, filed Jun. 26, 2019, all of which are incorporated by reference herein in their entirety.

PCT Information
Filing Document Filing Date Country Kind
PCT/US2020/039682 6/25/2020 WO
Provisional Applications (2)
Number Date Country
63043486 Jun 2020 US
62866749 Jun 2019 US