MULTISPECIFIC ANTIBODIES FOR TARGETING CD28 AND PD-L1 AND METHODS OF USE THEREOF

SEQUENCE LISTING

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 Oct. 27, 2021, is named 52426-726_601_SL.txt and is 483,304 bytes in size.

SUMMARY

Disclosed herein, in certain embodiments, are multispecific antibodies comprising a CD28 binding domain and a PD-L1 binding domain, wherein when the CD28 binding domain is a single chain variable fragment (scFv), then the PD-L1 binding domain is not a scFv. In some instances, the multispecific antibody is according to the following formula:

A-L-B (Formula I)

wherein A comprises the CD28 binding domain; B comprises the PD-L1 binding domain; and L comprises a linker that connects A to B. In some instances, the CD28 binding domain comprises a single chain variable fragment, a single domain antibody, a Fab, or a Fab′. In some instances, the CD28 binding domain comprises the single chain variable fragment. In some instances, the CD28 binding domain comprises the single domain antibody. In some instances, the CD28 binding domain comprises the Fab or the Fab′. In some instances, the PD-L1 binding domain comprises a single domain antibody, a Fab, or a Fab′. In some instances, the PD-L1 binding domain comprises the Fab or the Fab′. In some instances, the PD-L1 binding domain comprises the Fab or the Fab′ and the CD28 binding domain comprises the single chain variable fragment. In some instances, the PD-L1 binding domain that comprises the Fab or the Fab′ comprises a Fab heavy chain polypeptide comprising a Fab heavy chain variable domain and a Fab light chain polypeptide comprising a Fab light chain variable domain. In some instances, the CD28 binding domain that comprises the single chain variable fragment comprises a scFv heavy chain variable domain and a scFv light chain variable domain. In some instances, the linker connects the C-terminus of A to an N-terminus of B. In some instances, the linker connects the N-terminus of A to a C-terminus of B. In some instances, the linker connects the C-terminus of A to the N-terminus of the Fab heavy chain polypeptide. In some instances, the linker connects the N-terminus of A to the C-terminus of the Fab heavy chain polypeptide. In some instances, the linker connects the C-terminus of A to the N-terminus of the Fab light chain polypeptide. In some instances, the linker connects the N-terminus of A to the C-terminus of the Fab light chain polypeptide. In some instances, the linker connects the Fab light chain polypeptide to the scFv light chain variable domain. In some instances, the linker connects the Fab light chain polypeptide to the scFv heavy chain variable domain. In some instances, the linker connects the Fab heavy chain polypeptide to the scFv light chain variable domain. In some instances, the linker connects the Fab heavy chain polypeptide to the scFv heavy chain variable domain. In some instances, the linker connects the Fab light chain polypeptide to the N-terminus of the scFv light chain variable domain. In some instances, the linker connects the Fab light chain polypeptide to the C-terminus of the scFv light chain variable domain. In some instances, the linker connects the Fab light chain polypeptide to the N-terminus of the scFv heavy chain variable domain. In some instances, the linker connects the Fab light chain polypeptide to the C-terminus of the scFv heavy chain variable domain. In some instances, the linker connects the Fab heavy chain polypeptide to the N-terminus of the scFv light chain variable domain. In some instances, the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain. In some instances, the linker connects the Fab heavy chain polypeptide to the N-terminus of the scFv heavy chain variable domain. In some instances, the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv heavy chain variable domain. In some instances, the linker is at least 5 amino acids in length. In some instances, the linker is no more than 30 amino acids in length. In some instances, the linker is at least 5 amino acids and no more than 30 amino acids in length. In some instances, the linker is 5 amino acids in length. In some instances, the linker is 15 amino acids in length. In some instances, the linker is selected from the group consisting of (G₂S)_n, (GS)_n, (GSGGS)_n(SEQ ID NO: 58), (GGGS)_n(SEQ ID NO: 59), (GGGGS)_n(SEQ ID NO: 60), and (GSSGGS)_n(SEQ ID NO: 61), wherein n is an integer of at least 1. In some instances, L has a formula comprising (G₂S)_n(SEQ ID NO: 233), wherein n is an integer from 1 to 3. In some instances, the linker comprises an amino acid sequence of SEQ ID NO: 18 (GGGGSGGGGSGGGGS) or SEQ ID NO: 19 (GGGGS). In some instances, the scFv heavy chain variable domain comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the scFv heavy chain variable domain comprise: HC-CDR1: SEQ ID NO: 1; HC-CDR2: SEQ ID NO: 2; HC-CDR3: SEQ ID NO: 3, and wherein said CDRs comprise from 0-2 amino acid modifications in at least one of said HC-CDR1, HC-CDR2, or HC-CDR3. In some instances, the scFv light chain variable domain comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the scFv light chain variable domain comprise: LC-CDR1: SEQ ID NO: 4; LC-CDR2: SEQ ID NO: 5; and LC-CDR3: SEQ ID NO: 6, and wherein the CDRs comprise from 0-2 amino acid modifications in at least one of said LC-CDR1, LC-CDR2, or LC-CDR3. In some instances, the Fab heavy chain variable domain comprises complementarity determining region (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the Fab heavy chain variable domain comprise: HC-CDR1: SEQ ID NO: 10; HC-CDR2: SEQ ID NO: 11; HC-CDR3: SEQ ID NO: 12; HC-CDR1: SEQ ID NO: 24; HC-CDR2: SEQ ID NO: 25; HC-CDR3: SEQ ID NO: 26; HC-CDR1: SEQ ID NO: 27; HC-CDR2: SEQ ID NO: 28; HC-CDR3: SEQ ID NO: 29; or HC-CDR1: SEQ ID NO: 30; HC-CDR2: SEQ ID NO: 31; HC-CDR3: SEQ ID NO: 32 and wherein said CDRs comprise from 0-2 amino acid modifications in at least one of said HC-CDR1, HC-CDR2, or HC-CDR3. In some instances, the Fab light chain variable domain comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the Fab light chain variable domain comprise:LC-CDR1: SEQ ID NO: 13; LC-CDR2: SEQ ID NO: 14; and LC-CDR3: SEQ ID NO: 15; LC-CDR1: SEQ ID NO: 33; LC-CDR2: SEQ ID NO: 34; and LC-CDR3: SEQ ID NO: 35; LC-CDR1: SEQ ID NO: 36; LC-CDR2: SEQ ID NO: 37; and LC-CDR3: SEQ ID NO: 38; or LC-CDR1: SEQ ID NO: 39; LC-CDR2: SEQ ID NO: 40; and LC-CDR3: SEQ ID NO: 41 and wherein the CDRs comprise from 0-2 amino acid modifications in at least one of said LC-CDR1, LC-CDR2, or LC-CDR3. In some instances, the scFv heavy chain variable domain comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 7. In some instances, the scFv heavy chain variable domain comprises an amino acid sequence of at least 75 consecutive amino acid residues of SEQ ID NO: 7 In some instances, the scFv heavy chain variable domain comprises an amino acid sequence of at least 110 consecutive amino acid residues of SEQ ID NO: 7. In some instances, the scFv heavy chain variable domain comprises an amino acid sequence of at least 110 consecutive amino acid residues of SEQ ID NO: 7 and has at least 80% sequence identity to the at least 110 consecutive amino acid residues of SEQ ID NO: 7. In some instances, the scFv heavy chain variable domain comprises an amino acid sequence according to SEQ ID NO: 7. In some instances, the scFv light chain variable domain comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 8. In some instances, the scFv light chain variable domain comprises an amino acid sequence of at least 75 consecutive amino acid residues of SEQ ID NO: 8. In some instances, the scFv light chain variable domain comprises an amino acid sequence of at least 100 consecutive amino acid residues of SEQ ID NO: 8. In some instances, the scFv light chain variable domain comprises an amino acid sequence of at least 100 consecutive amino acid residues of SEQ ID NO: 8 and has at least 80% sequence identity to the at least 100 consecutive amino acid residues of SEQ ID NO: 8. In some instances, the scFv light chain variable domain comprises an amino acid sequence according to SEQ ID NO: 8. In some instances, the scFv comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 9. In some instances, the scFv comprises an amino acid sequence of at least 175 consecutive amino acid residues of SEQ ID NO: 9. In some instances, the scFv comprises an amino acid sequence of at least 210 consecutive amino acid residues of SEQ ID NO: 9. In some instances, the scFv comprises an amino acid sequence of at least 210 consecutive amino acid residues of SEQ ID NO: 9 and has at least 80% sequence identity to the at least 210 consecutive amino acid residues of SEQ ID NO: 9. In some instances, the scFv comprises an amino acid sequence according to SEQ ID NO: 9. In some instances, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 17, 43, 45, or 47. In some instances, the Fab heavy chain polypeptide comprises an amino acid sequence of at least 175 consecutive amino acid residues of SEQ ID NO: 17, 43, 45, or 47. In some instances, the Fab heavy chain polypeptide comprises an amino acid sequence of at least 215 consecutive amino acid residues of SEQ ID NO: 17, 43, 45, or 47. In some instances, the Fab heavy chain polypeptide comprises an amino acid sequence of at least 215 consecutive amino acid residues of SEQ ID NO: 17 and has at least 80% sequence identity to the at least 215 consecutive amino acid residues of SEQ ID NO: 17, 43, 45, or 47. In some instances, the Fab heavy chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 17, 43, 45, or 47. In some instances, the Fab light chain polypeptide comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 16, 42, 44, or 46. In some instances, the Fab light chain polypeptide comprises an amino acid sequence of at least 175 consecutive amino acid residues of SEQ ID NO: 16, 42, 44, or 46. In some instances, the Fab light chain polypeptide comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 16, 42, 44, or 46. In some instances, the Fab light chain polypeptide comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 16 and has at least 80% sequence identity to the at least 200 consecutive amino acid residues of SEQ ID NO: 16, 42, 44, or 46. In some instances, the Fab light chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 16, 42, 44, or 46. In some instances, the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab light chain polypeptide comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 20, and an amino acid sequence of the Fab heavy chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 21. In some instances, the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab light chain polypeptide comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 20, and an amino acid sequence of the Fab heavy chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence of at least 450 consecutive amino acid residues of SEQ ID NO: 21. In some instances, the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab light chain polypeptide comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 20 and has at least 80% sequence identity to the at least 200 consecutive amino acid residues of SEQ ID NO: 20 and an amino acid sequence of the Fab heavy chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence of at least 450 consecutive amino acid residues of SEQ ID NO: 21 and has at least 80% sequence identity to the at least 450 consecutive amino acid residues of SEQ ID NO: 21. In some instances, the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab light chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 20, and an amino acid sequence of the Fab heavy chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence to SEQ ID NO: 21. In some instances, the linker connects the Fab light chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 23, and an amino acid sequence of the Fab light chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 22. In some instances, the linker connects the Fab light chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab heavy chain polypeptide comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 23, and an amino acid sequence of the Fab light chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence of at least 450 consecutive amino acid residues of SEQ ID NO: 22. In some instances, the linker connects the Fab light chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab heavy chain polypeptide comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 23 and has at least 80% sequence identity to the at least 200 consecutive amino acid residues of SEQ ID NO: 23 and an amino acid sequence of the Fab light chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence of at least 450 consecutive amino acid residues of SEQ ID NO: 22 and has at least 80% sequence identity to the at least 450 consecutive amino acid residues of SEQ ID NO: 22. In some instances, the linker connects the Fab light chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab heavy chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 23, and an amino acid sequence of the Fab light chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence to SEQ ID NO: 22.

Disclosed herein, in certain embodiments, are pharmaceutical compositions comprising: the multispecific antibody described herein; and a pharmaceutically acceptable excipient.

Disclosed herein, in certain embodiments, are isolated recombinant nucleic acid molecules encoding a polypeptide of the multispecific antibody described herein.

Disclosed herein, in certain embodiments, are methods of treating cancer in a subject in need thereof comprising administering to the subject the multispecific antibody described herein or the pharmaceutical composition described herein. In some instances, the multispecific antibody is administered to the subject as a single agent therapy. In some instances, the multispecific antibody is not administered as a part of a treatment regimen with a second therapeutic agent. In some instances, the multispecific antibody is not administered as a part of a treatment regimen with a second therapeutic agent comprising a tumor binding domain. In some instances, the multispecific antibody is not administered as a part of a treatment regimen with a second therapeutic agent comprising an anti-CD19 antibody. In some instances, the multispecific antibody is not administered as a part of a treatment regimen with a second therapeutic agent comprising an antibody that has an anti-CD19 binding domain and an anti-CD3 binding domain. In some instances, the multispecific antibody induces T cell mediated cytotoxicity of tumor cells. In some instances, the administering to the subject of the multispecific antibody is sufficient to reduce or eliminate the cancer as compared to a baseline measurement of the cancer taken from the subject prior to the administering of the multispecific antibody. In some instances, the reduction is at least about 1-fold, 5-fold, 10-fold, 20-fold, 40-fold, 60-fold, 80-fold, or up to about 100-fold. In some instances, the cancer is a hematological malignancy.

In some instances, the cancer is leukemia or lymphoma. In some instances, the cancer is lymphoma, and wherein the lymphoma is B-cell lymphoma. In some instances, the cancer is a solid tumor. In some instances, the solid tumor expresses PD-L1. In some instances, the solid tumor is sarcoma, breast cancer, lung cancer, or carcinoma. In some instances, the solid tumor is lung cancer, and wherein the lung cancer is non-small cell lung cancer. Disclosed herein are methods of treating cancer in a subject in need thereof comprising administering to the subject a multispecific antibody that comprises a CD28 binding domain and a PD-L1 binding domain wherein the multispecific antibody that comprises the CD28 binding domain and the PD-L1 binding domain is not administered as part of a treatment regimen with another multispecific antibody that targets a cancer antigen different from PD-L1 or CD28. In some embodiments, the multispecific antibody that comprises the CD28 binding domain and the PD-L1 binding domain is administered to the subject as a single agent therapy. In some embodiments, the multispecific antibody that comprises the CD28 binding domain and the PD-L1 binding domain comprises an IgG framework, an IgA framework, an IgE framework, or an IgM framework. In some embodiments, the CD28 binding domain comprises a single chain variable fragment, a single domain antibody, a Fab, or a Fab′. In some embodiments, the PD-L1 binding domain comprises a single chain variable fragment, a single domain antibody, a Fab, or a Fab′. In some embodiments, the PD-L1 binding domain comprises a single chain variable fragment and the CD28 binding domain comprises a single chain variable fragment. In some embodiments, the CD28 binding domain comprises an anti-CD28 light chain polypeptide. In some embodiments, the anti-CD28 light chain polypeptide comprises a variable domain of an IgG1, IgG2, IgG3, or IgG4 light chain. In some embodiments, the CD28 binding domain comprises an anti-CD28 heavy chain polypeptide. In some embodiments, the anti-CD28 heavy chain polypeptide comprises a variable domain of an IgG1, IgG2, IgG3, or IgG4 heavy chain. In some embodiments, the PD-L1 binding domain comprises an anti-PD-L1 light chain polypeptide. In some embodiments, the anti-PD-L1 light chain polypeptide comprises a variable domain of an IgG1, IgG2, IgG3, or IgG4 light chain. In some embodiments, the PD-L1 binding domain comprises an anti-PD-L1 heavy chain polypeptide. In some embodiments, the anti-PD-L1 heavy chain polypeptide comprises a variable domain of an IgG1, IgG2, IgG3, or IgG4 heavy chain. In some embodiments, the multispecific antibody further comprises a fragment crystallizable (Fc) region. In some embodiments, the Fc region comprises an IgG CH2 domain and an IgG CH3 domain. In some embodiments, the Fc region comprises a heterodimeric Fc region. In some embodiments, the Fc region comprises at least one amino acid modification that increases the half-life of the multispecific antibody. In some embodiments, the Fc region comprises at least one amino acid modification that modulates its interaction with an Fc receptor. In some embodiments, the Fc region comprises at least one amino acid modification that increases binding of the Fc region to an Fc receptor. In some embodiments, the Fc region comprises at least one amino acid modification that decreases glycosylation of the Fe region. In some embodiments, the modification is an amino acid substitution, deletion, or addition. In some embodiments, the modification is an amino acid substitution. In some embodiments, the at least one amino acid modification that decreases glycosylation of the Fc region comprises an amino acid substitution at a position corresponding to position N297 of human IgG1, wherein the numbering is according to the EU index of Kabat. In some embodiments, the Fc region is afucosylated. In some embodiments, the anti-CD28 light chain polypeptide comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 the anti-CD28 light chain polypeptide: LC-CDR1: SEQ ID NO: 4; LC-CDR2: SEQ ID NO: 5; and LC-CDR3: SEQ ID NO: 6 and wherein the CDRs comprise from 0-2 amino acid modifications in at least one of said LC-CDR1, LC-CDR2, or LC-CDR3. In some embodiments, the anti-CD28 heavy chain polypeptide comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the anti-CD28 heavy chain polypeptide comprise: HC-CDR1: SEQ ID NO: 1; HC-CDR2: SEQ ID NO: 2; HC-CDR3: SEQ ID NO: 3, and wherein said CDRs comprise from 0-2 amino acid modifications in at least one of said HC-CDR1, HC-CDR2, or HC-CDR3. In some embodiments, the anti-PD-L1 light chain polypeptide comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the anti-PD-L1 light chain polypeptide comprise:LC-CDR1: SEQ ID NO: 13; LC-CDR2: SEQ ID NO: 14; and LC-CDR3: SEQ ID NO: 15; LC-CDR1: SEQ ID NO: 33; LC-CDR2: SEQ ID NO: 34; and LC-CDR3: SEQ ID NO: 35; LC-CDR1: SEQ ID NO: 36; LC-CDR2: SEQ ID NO: 37; and LC-CDR3: SEQ ID NO: 38; or LC-CDR1: SEQ ID NO: 39; LC-CDR2: SEQ ID NO: 40; and LC-CDR3: SEQ ID NO: 41;and wherein the CDRs comprise from 0-2 amino acid modifications in at least one of said LC-CDR1, LC-CDR2, or LC-CDR3. In some embodiments, the anti-PD-L1 heavy chain polypeptide the anti-PD-L1 heavy chain polypeptide comprises complementarity determining region (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the anti-PD-L1 heavy chain polypeptide comprises: HC-CDR1: SEQ ID NO: 10; HC-CDR2: SEQ ID NO: 11; HC-CDR3: SEQ ID NO: 12; HC-CDR1: SEQ ID NO: 24; HC-CDR2: SEQ ID NO: 25; HC-CDR3: SEQ ID NO: 26; HC-CDR1: SEQ ID NO: 27; HC-CDR2: SEQ ID NO: 28; HC-CDR3: SEQ ID NO: 29; or HC-CDR1: SEQ ID NO: 30; HC-CDR2: SEQ ID NO: 31; HC-CDR3: SEQ ID NO: 32 and wherein said CDRs comprise from 0-2 amino acid modifications in at least one of said HC-CDR1, HC-CDR2, or HC-CDR3. In some embodiments, the multispecific antibody induces T cell mediated cytotoxicity of tumor cells. In some embodiments, the administering to the subject of the multispecific antibody is sufficient to reduce or eliminate the cancer as compared to a baseline measurement of the cancer taken from the subject prior to the administering of the multispecific antibody. In some embodiments, the reduction is at least about 1-fold, 5-fold, 10-fold, 20-fold, 40-fold, 60-fold, 80-fold, or up to about 100-fold. In some embodiments, the cancer is a hematological malignancy. In some embodiments, the cancer is leukemia or lymphoma. In some embodiments, the cancer is lymphoma, and wherein the lymphoma is B-cell lymphoma.

In some embodiments, the cancer is a solid tumor. In some embodiments, the solid tumor expresses PD-L1. In some embodiments, the solid tumor is sarcoma, breast cancer, lung cancer, or carcinoma. In some embodiments, the solid tumor is lung cancer, and wherein the lung cancer is non-small cell lung cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the 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 disclosure are utilized, and the accompanying drawings of which:

FIGS. 1A-1B illustrate exemplary schemas of PDL1 x CD28 multispecific antibodies.

FIG. 2 illustrates an exemplary schema of the in vitro immune cell activation assays using target coated beads.

FIG. 3A illustrates a graph of IFNy cytokine release from PBMCs cultured with single agent PDL1 x CD28 multispecific Ab-1.

FIG. 3B illustrates a graph of TNFα cytokine release from PBMCs cultured with single agent PDL1 x CD28 multispecific Ab-1.

FIG. 3C illustrates a graph of IL-2 cytokine release from PBMCs cultured with single agent PDL1 x CD28 multispecific Ab-1.

FIG. 4A illustrates a graph of the number of live immune cells overtime in response to PBMC co-cultured with PDL1 target coated beads and Ab-1.

FIG. 4B illustrates a graph of the number of live CD3+ cells overtime in response to PBMC co-cultured with PDL1 target coated beads and Ab-1.

FIG. 4C illustrates a graph of the number of live CD4+ cells over time in response to PBMC co-cultured with PDL1 target coated beads and Ab-1.

FIG. 4D illustrates a graph of the number of live CD8+ cells over time in response to PBMC co-culture with PDL1 target coated beads and Ab-1.

FIG. 5A illustrates binding kinetics of Ab-1 to biotinylated human PD-L1.

FIG. 5B illustrates binding kinetics of anti-PD-L1 Fab 1 to biotinylated human PD-L1.

FIG. 6A illustrates binding kinetics of Ab-1 to biotinylated human CD28.

FIG. 6B illustrates binding kinetics of Ab-2 to biotinylated human CD28.

FIG. 7A illustrates binding kinetics of Ab-1 to human PD-L1 Fc.

FIG. 7B illustrates binding kinetics of Ab-1 to cynomolgus monkey PD-L1 Fc.

FIG. 8A illustrates binding of Ab-1 to Ab-6 to PD-L1 as measured by ELISA.

FIG. 8B illustrates binding of Ab-1 to Ab-6 to CD28 as measured by ELISA.

FIG. 9A illustrates a cartoon configuration of a multispecific antibody that targets CD28 and PD-L1 that is administered in combination with a T cell engager that targets a tumor associated antigen such as TROP2 and CD3 of T cell.

FIGS. 9B-9D illustrate immune cell activation as measured by cytokine release after co-culture of target coated beads and PBMCs and administration of Ab-1 and the combination of Ab-1 and TCE-1 an anti-TROP2 x CD3 T cell engager.

FIGS. 9E-9H illustrate immune cell proliferation after co-culture of target coated beads and PBMCs and administration of Ab-1 and the combination of Ab-1 and TCE-1 an anti-TROP2 x CD3 T cell engager.

FIGS. 91-9K illustrate polypeptide complexes of different orientation harboring different PD-L1 binding domains (Ab-1 through Ab-8) activate PBMCs as measured by cytokine release in combination with a T cell engager (TCE-3) against non-immunogenic beads coated with tumor associated antigen and PD-L1.

FIG. 9L illustrates polypeptide complex mediated activation of PBMCs in combination with a T cell engager.

FIG. 9M-9S illustrate polypeptide complex mediated activation of PBMCs in combination with a T cell engager is PDL1 density dependent.

FIGS. 10A-10C illustrate results of an in vitro tumor cell killing assay using the LNCaP PDL1 positive tumor cell line in which Ab-1 and TCE-2 are co-administered in the presence of human PBMCs. In vitro tumor cell killing and PBMC activation measured by cytokine release is synergized when Ab-1 is combined with an anti-PSMA x CD3 T cell engager, TCE-2.

FIG. 10D illustrates a graph of PBMC mediated in vitro killing of H292 tumor cells using Ab-1 and TCE-4.

FIG. 10E illustrates a graph of PBMC mediated in vitro killing of H292 tumor cells using Ab-1 and TRACTr-1.

FIG. 10F illustrates a graph of PBMC mediated in vitro killing of H292 tumor cells using Ab-1 and TRACTr-pre-treated with MTSP1.

FIG. 10G illustrates a graph of IL-2 cytokine release from PBMCs co-cultured with H292 cells, Ab-1, and TCE-4.

FIG. 10H illustrates a graph of IL-2 cytokine release from PBMCs co-cultured with H292 cells, Ab-1 and TRACTr-1.

FIG. 10I illustrates a graph of IL-2 cytokine release from PBMCs co-cultured with H292 cells Ab-1, and TRACTr-1 pre-treated with MTSP1.

FIG. 11 illustrates pharmacokinetics of Ab-1 in cynomolgus monkey after a single IV bolus injection.

FIG. 12A-12F illustrate cytokine release in cynomolgus monkey after a single IV bolus injection of Ab-1.

FIG. 13A-13B illustrate serum liver enzymes in cynomolgus monkey after a single IV bolus injection of Ab-1.

FIG. 14 illustrates a schematic for identifying P1 or P2 peptides that can be attached to the PD-L1 and CD28 multispecific antibodies for selective activation in tumor microenvironments. The schematic illustrates a directed evolution and phage display technology to identify peptides that block antigen recognition by antigen binding domains.

FIGS. 15A-15C exemplify schematics for the multispecific antibodies comprising a CD28 binding domain and a PD-L1 binding domain described herein can increase activation as a monotherapy. FIG. 15A shows a T cell attacking a tumor cell, and the potential secondary signals are the PD-1 (stop) and CD28 (go).

FIG. 15B shows how a tumor deactivates T cells through PD-L1. The PD-L1/PD-1 triggers the stop signal while the CD28 (go) signaling is not activated. FIG. 15C shows a reactivated T cell in which the multispecific antibody with a PD-L1 binding domain and CD28 binding domain reactivate the T cell by converting the PD-L1/PD-1 stop signal to a CD28 go signal.

FIG. 16 exemplifies a schematic of a multispecific antibody comprising a CD28 binding domain and a PD-L1 binding domain in combination with a T cell engager.

DETAILED DESCRIPTION

Bispecific antibodies for redirecting T cells for mediating cancer cell killing have shown promise in both pre-clinical and in clinical studies. Efficient T cell activation has been obtained with single chain variable fragments (scFv), notably the Bispecific T-cell Engagers (BiTEs) format, in which one scFv targets a tumor cell antigen, and the other scFv targets an epitope such as CD3 that is involved in T cell activation. One such example of a BiTE is blinatumomab that targets CD19 and CD3 which has been approved in Europe and the United States for treatment of chemotherapy-resistant CD19+ B cell acute lymphoblastic leukemia. Despite advances with T cell engagers such as blinatumomab some patients respond poorly to treatment even if the patient expresses the tumor antigen for reasons that are not entirely understood.

Strategies for increasing T cell cytotoxicity of T cell engagers have been explored through co-administration with a second antibody that targets the co-inhibitory immune checkpoint programmed death-ligand 1 (PD-L1) and/or CD28. CD28 is a protein expressed on T cells that provide co-stimulatory signals required for T cell activation and survival. It is known that stimulatory signaling through CD28 in combinations with BiTEs increase T cell-induced tumor cell cytotoxicity. However, central to obtaining T cell mediated cytotoxicity of tumor cells in prior studies required the presence of a BiTE that has a tumor binding domain, such as an anti-CD19 antibody, and a CD3 binding domain, while single agent administration of an anti-CD28 and anti-PD-L1 in a scFv-scFv format was found to not induce T cell mediated cytotoxicity against tumor cells.

Activation of T cells is a highly regulated process that typically requires two signaling events for full functionality: the first signal is initiated upon binding of the MHC-antigen complex, which helps distinguish “self” from “non-self” to the T cell receptor (TCR) and the second signal through activation of a costimulatory receptor. While the first recognition signal activates a T cell and triggers T cell mediated toxicity of the recognized cell, if the T cell does not receive a second costimulatory signal it can lead to T cell tolerance whereby the T cells continue to recognize the tumor antigen but do not mount an immune response against the tumor cell. The second costimulatory signal prevents T cell tolerance, and further activates the T cell to enhance T cell cytotoxicity towards the targeted cell.

Multispecific antibodies comprising a CD28 binding domain and PD-L1 binding domain as described herein are designed to act both as an antagonist of PD-L1 and a conditional agonist of C28. While CD28 agonism has shown some clinical promise, the efficacy seen with this approach has been limited due to dose-limiting toxicities that result from systemic activation of CD28. The multispecific antibodies comprising a CD28 binding domain and PD-L1 binding domain, described herein, are designed to conditionally agonize CD28 only in the presence of PD-L1, which is often overexpressed by tumors to avoid T cell mediated killing. In addition, engagement of PD-L1 is designed to block PD-1 binding and provide checkpoint inhibition. This combination provides a mechanism of action that enhances anti-tumor responses and limits the systemic toxicity of CD28 agonism. As shown in FIG. 11, studies of multispecific antibodies described herein demonstrate a lack of systemic immune system activation, as evidenced by the lack of cytokine release. Despite unprecedented clinical response rates, most patients fail to respond to therapies targeting PD-1 and PD-L1, which is due in part because T cells require costimulation for full functionality. As such, checkpoint inhibition alone is likely insufficient to fully enable the immune system to attack a tumor. Further benefit can be derived by the addition of the multispecific antibodies as described herein.

Disclosed herein are antibodies that bind specifically to PD-L1 and CD28 which are able to induce T cell mediated cytotoxicity of tumor cells as a single agent (FIG. 15C) or in combination with a T cell engager (FIG. 16). Significantly, such antibodies that target PD-L1 and CD28 are able to induce T cell mediated cytotoxicity of tumor cells as a single agent, even when not administered with a second agent that specifically targets a tumor cell antigen as exemplified in the schematics of FIGS. 15A-15C. Such antibodies that bind specifically to PD-L1 and CD28 are not in a scFv-scFv format.

Disclosed herein are multispecific antibodies that comprise a CD28 binding domain and a PD-L1 binding domain, wherein when the CD28 binding domain is a single chain variable fragment (scFv), then the PD-L1 binding domain is not a scFv. In some embodiments, the multispecific antibody is according to the formula:

A-L-B (Formula I)

wherein A comprises the CD28 binding domain; B comprises the PD-L1 binding domain; and L comprises a linker that connects A to B. In some embodiments, the multispecific antibody comprises the formula:

A-L-B (Formula I)

wherein A is the CD28 binding domain; B is the PD-L1 binding domain; and L is the linker that connects A to B. In some embodiments, the multispecific antibody is according to the formula:

A-L-B (Formula I)

wherein A is the CD28 binding domain; B is the PD-L1 binding domain; and L is the linker that connects A to B.

While preferred embodiments of the present disclosure 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 disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the disclosure. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Definitions

The terminology used herein is for the purpose of describing particular cases only and is not intended to be limiting. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”

The term “antibody” is used in the broadest sense and covers fully assembled antibodies, antibody fragments that can bind antigen, for example, Fab, F(ab′)2, Fv, single chain antibodies (scFv), diabodies, antibody chimeras, hybrid antibodies, bispecific antibodies, and the like.

The term “complementarity determining region” or “CDR” is a segment of the variable region of an antibody that is complementary in structure to the epitope to which the antibody binds and is more variable than the rest of the variable region. Accordingly, a CDR is sometimes referred to as hypervariable region. A variable region comprises three CDRs. CDR peptides can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick et al., Methods: A Companion to Methods in Enzymology 2: 106 (1991); Courtenay-Luck, “Genetic Manipulation of Monoclonal Antibodies,” in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al. (eds.), pages 166-179 (Cambridge University Press 1995); and Ward et al., “Genetic Manipulation and Expression of Antibodies,” in Monoclonal Antibodies: Principles and Applications, Birch et al., (eds.), pages 137-185 (Wiley-Liss, Inc. 1995).

In some instances, the CDRs of an antibody are determined according to (i) the Kabat numbering system (Kabat et al. (197) Ann. NY Acad. Sci. 190:382-391 and, Kabat et al. (1991) Sequences of Proteins of Immunological Interest Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242); or (ii) the Chothia numbering scheme, which will be referred to herein as the “Chothia CDRs” (see, e.g., Chothia and Lesk, 1987, J. Mol. Biol., 196:901-917; Al-Lazikani et al., 1997, J. Mol. Biol., 273 :927-948; Chothia et al., 1992, J. Mol. Biol., 227:799-817; Tramontano A et al., 1990, J. Mol. Biol. 215(1): 175-82; and U.S. Pat. No. 7,709,226); or (iii) the ImMunoGeneTics (IMGT) numbering system, for example, as described in Lefranc, M.-P., 1999, The Immunologist, 7: 132-136 and Lefranc, M.-P. et al, 1999, Nucleic Acids Res., 27:209-212 (“IMGT CDRs”); or (iv) MacCallum et al, 1996, J. Mol. Biol., 262:732-745. See also, e.g., Martin, A., “Protein Sequence and Structure Analysis of Antibody Variable Domains,” in Antibody Engineering, Kontermann and Diibel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001).

With respect to the Kabat numbering system, CDRs within an antibody heavy chain molecule are typically present at amino acid positions 31 to 35, which optionally can include one or two additional amino acids, following 35 (referred to in the Kabatnumbering scheme as 35 A and 35B) (CDRl), amino acid positions 50 to 65 (CDR2), and amino acid positions 95 to 102 (CDR3). Using the Kabat numbering system, CDRs within an antibody light chain molecule are typically present at amino acid positions 24 to 34 (CDRl), amino acid positions 50 to 56 (CDR2), and amino acid positions 89 to 97 (CDR3). As is well known to those of skill in the art, using the Kabat numbering system, the actual linear amino acid sequence of the antibody variable domain can contain fewer or additional amino acids due to a shortening or lengthening of a FR and/or CDR and, as such, an amino acid's Kabat number is not necessarily the same as its linear amino acid number.

The term “Fab” refers to a protein that contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab fragments differ from Fab′ fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab′-SH is the designation herein for Fab′ in which the cysteine residue(s) of the constant domains bear a free thiol group. Fab′ fragments are produced by reducing the F(ab′)2 fragment's heavy chain disulfide bridge. Other chemical couplings of antibody fragments are also known.

A “single-chain variable fragment (scFv)” is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of an antibody, connected with a short linker peptide of ten to about 25 amino acids. The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa. This protein retains the specificity of the original antibody, despite removal of the constant regions and the introduction of the linker. scFv antibodies are, e.g. described in Houston, J. S., Methods in Enzymol. 203 (1991) 46-96). In addition, antibody fragments comprise single chain polypeptides having the characteristics of a VH domain, namely being able to assemble together with a VL domain, or of a VL domain, namely being able to assemble together with a VH domain to a functional antigen binding site and thereby providing the antigen binding property of full length antibodies.

The term “multispecific” means that the antibody is able to specifically bind to two or more distinct antigenic determinants for example two or more binding sites each formed by a pair of an antibody heavy chain variable domain (VH) and an antibody light chain variable domain (VL), or in the case of a single domain antibody a single variable domain, binding to different antigens.

As used herein, the term “percent (%) amino acid sequence identity” with respect to a sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific sequence, 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. 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 EMBOSS MATCHER, EMBOSS WATER, EMBOSS STRETCHER, EMBOSS NEEDLE, EMBOSS LALIGN, 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 maximal alignment over the full length of the sequences being compared.

In situations where ALIGN-2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X/Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

The terms “individual(s)”, “subject(s)” and “patient(s)” are used interchangeably herein and refer to any mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is a non-human. None of the terms require or are limited to situations characterized by the supervision (e.g. constant or intermittent) of a health care worker (e.g. a doctor, a registered nurse, a nurse practitioner, a physician's assistant, an orderly or a hospice worker).

CD28 Binding Domains

Disclosed herein are multispecific antibodies that comprise a CD28 binding domain. In some embodiments, the CD28 binding domain comprises an antibody or antigen binding fragment. In some embodiments, the antibody or antigen binding fragment is a monoclonal antibody. In some embodiments, the antibody or antigen binding fragment is a human antibody, a murine antibody, a humanized antibody, or a chimeric antibody. In some embodiments, antibody or antigen binding fragment that binds specifically to CD28 comprises an anti-CD28 heavy chain polypeptide and an anti-CD28 light chain polypeptide.

In some embodiments, the anti-CD28 heavy chain polypeptide comprises an anti-CD28 heavy chain variable domain. In some embodiments, the anti-CD28 heavy chain variable domain comprises an IgG framework, an IgA framework, an IgE framework, or an IgM framework. In some embodiments, the anti-CD28 heavy chain variable domain comprises a variable domain of an IgG1, IgG2, IgG3, or IgG4 heavy chain. In some embodiments, the anti-CD28 light chain polypeptide comprises an anti-CD28 light chain variable domain. In some embodiments, the anti-CD28 light chain variable domain comprises an IgG framework, an IgA framework, an IgE framework, or an IgM framework. In some embodiments, the anti-CD28 light chain variable domain comprises a variable domain of an IgG1, IgG2, IgG3, or IgG4 light chain.

In some embodiments the CD28 binding domain comprises a single chain variable fragment, a single domain antibody, a Fab, or a Fab′. In some embodiments, the CD28 binding domain comprises the single chain variable fragment. In some embodiments, the CD28 binding domain comprises the single domain antibody. In some embodiments, the CD28 binding domain comprises the Fab or the Fab′. In some embodiments, the CD28 binding domain that comprises the single chain variable fragment comprises a scFv heavy chain variable domain and a scFv light chain variable domain. In some embodiments, the anti-CD28 heavy chain polypeptide comprises the scFv heavy chain variable domain. In some embodiments, the anti-CD28 light chain polypeptide comprises the scFv light chain variable domain.

In some embodiments, the anti-CD28 heavy chain polypeptide comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the anti-CD28 heavy chain polypeptide comprise: HC-CDR1: SEQ ID NO: 1; HC-CDR2: SEQ ID NO: 2; HC-CDR3: SEQ ID NO: 3, and wherein said CDRs comprise from 0-2 amino acid modifications in at least one of said HC-CDR1, HC-CDR2, or HC-CDR3. In some embodiments, the anti-CD28 heavy chain polypeptide comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the anti-CD28 heavy chain polypeptide comprise: HC-CDR1: SEQ ID NO: 1; HC-CDR2: SEQ ID NO: 2; HC-CDR3: SEQ ID NO: 3.

In some embodiments, the anti-CD28 light chain polypeptide comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 the anti-CD28 light chain polypeptide: LC-CDR1: SEQ ID NO: 4; LC-CDR2: SEQ ID NO: 5; and LC-CDR3: SEQ ID NO: 6, and wherein the CDRs comprise from 0-2 amino acid modifications in at least one of said LC-CDR1, LC-CDR2, or LC-CDR3. In some embodiments, the anti-CD28 light chain polypeptide comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the anti-CD28 light chain polypeptide: LC-CDR1: SEQ ID NO: 4; LC-CDR2: SEQ ID NO: 5; and LC-CDR3: SEQ ID NO: 6.

In some embodiments, anti-CD28 heavy chain polypeptide comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the anti-CD28 heavy chain polypeptide comprise: HC-CDR1: SEQ ID NO: 1; HC-CDR2: SEQ ID NO: 2; HC-CDR3: SEQ ID NO: 3, and wherein said CDRs comprise from 0-2 amino acid modifications in at least one of said HC-CDR1, HC-CDR2, or HC-CDR3; and the anti-CD28 light chain polypeptide comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the anti-CD28 light chain polypeptide comprise: LC-CDR1: SEQ ID NO: 4; LC-CDR2: SEQ ID NO: 5; and LC-CDR3: SEQ ID NO: 6, and wherein the CDRs comprise from 0-2 amino acid modifications in at least one of said LC-CDR1, LC-CDR2, or LC-CDR3.

In some embodiments, the anti-CD28 heavy chain polypeptide comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the anti-CD28 heavy chain polypeptide comprise: HC-CDR1: SEQ ID NO: 1; HC-CDR2: SEQ ID NO: 2; HC-CDR3: SEQ ID NO: 3; and the anti-CD28 light chain polypeptide comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the anti-CD28 light chain polypeptide comprise: LC-CDR1: SEQ ID NO: 4; LC-CDR2: SEQ ID NO: 5; and LC-CDR3: SEQ ID NO: 6.

TABLE 1

anti-CD28 heavy chain polypeptide complemen-

tarity determining regions (CDR)s as

determined by IMGT definition.

Amino Acid
SEQ

Construct
Sequence
ID

Description
(N to C)
NO:

anti-CD28: HC: CDR1
GYTFTSYY
1

anti-CD28: HC: CDR2
IYPGNVNT
2

anti-CD28: HC: CDR3
TRSHYGLDWNFDV
3

TABLE 2

anti-CD28 light chain polypeptide complemen-

tarity determining regions (CDR)s as

determined by IMGT definition.

Amino Acid
SEQ

Construct
Sequence
ID

Description
(N to C)
NO:

anti-CD28: LC: CDR1
QNIYVW
4

anti-CD28: LC: CDR2
KA
5

anti-CD28: LC: CDR3
QQGQTYPYT
6

In some embodiments, the scFv heavy chain variable domain comprises an amino acid sequence that has at least 70% sequence identity to the amino acid sequence according to SEQ ID NO: 7. In some embodiments, the scFv heavy chain variable domain comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 7. In some embodiments, the scFv heavy chain variable domain comprises an amino acid sequence that has at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 7. In some embodiments, the scFv heavy chain variable domain comprises an amino acid sequence that has at least 90% sequence identity to the amino acid sequence according to SEQ ID NO: 7. In some embodiments, the scFv heavy chain variable domain comprises an amino acid sequence that has at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 7. In some embodiments, the scFv heavy chain variable domain comprises an amino acid sequence that has at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 7. In some embodiments, the scFv heavy chain variable domain comprises an amino acid sequence that has at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 7. In some embodiments, the scFv heavy chain variable domain comprises an amino acid sequence that has at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 7. In some embodiments, the scFv heavy chain variable domain comprises an amino acid sequence that has at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 7. In some embodiments, the scFv heavy chain variable domain comprises an amino acid sequence according to SEQ ID NO: 7.

In some embodiments, the scFv heavy chain variable domain comprises an amino acid sequence of at least 75 consecutive amino acid residues of SEQ ID NO: 7. In some embodiments, the scFv heavy chain variable domain comprises an amino acid sequence of at least 100 consecutive amino acid residues of SEQ ID NO: 7 In some embodiments, the scFv heavy chain variable domain comprises an amino acid sequence of at least 105 consecutive amino acid residues of SEQ ID NO: 7 In some embodiments, the scFv heavy chain variable domain comprises an amino acid sequence of at least 110 consecutive amino acid residues of SEQ ID NO: 7. In some embodiments, the scFv heavy chain variable domain comprises an amino acid sequence of at least 115 consecutive amino acid residues of SEQ ID NO: 7.

In some embodiments, the scFv heavy chain variable domain comprises an amino acid sequence of at least 75 consecutive amino acid residues of SEQ ID NO: 7, and has at least 80% sequence identity to the at least 75 consecutive amino acid residues of SEQ ID NO: 7. In some embodiments, the scFv heavy chain variable domain comprises an amino acid sequence of at least 100 consecutive amino acid residues of SEQ ID NO: 7, and has at least 80% sequence identity to the at least 100 consecutive amino acid residues of SEQ ID NO: 7. In some embodiments, the scFv heavy chain variable domain comprises an amino acid sequence of at least 105 consecutive amino acid residues of SEQ ID NO: 7, and has at least 80% sequence identity to the at least 105 consecutive amino acid residues of SEQ ID NO: 7. In some embodiments, the scFv heavy chain variable domain comprises an amino acid sequence of at least 110 consecutive amino acid residues of SEQ ID NO: 7, and has at least 80% sequence identity to the at least 110 consecutive amino acid residues of SEQ ID NO: 7. In some embodiments, the scFv heavy chain variable domain comprises an amino acid sequence of at least 115 consecutive amino acid residues of SEQ ID NO: 7, and has at least 80% sequence identity to the at least 115 consecutive amino acid residues of SEQ ID NO: 7.

In some embodiments, the scFv heavy chain variable domain comprises an amino acid sequence of at least 75 consecutive amino acid residues of SEQ ID NO: 7, and has at least 90% sequence identity to the at least 75 consecutive amino acid residues of SEQ ID NO: 7. In some embodiments, the scFv heavy chain variable domain comprises an amino acid sequence of at least 100 consecutive amino acid residues of SEQ ID NO: 7, and has at least 90% sequence identity to the at least 100 consecutive amino acid residues of SEQ ID NO: 7. In some embodiments, the scFv heavy chain variable domain comprises an amino acid sequence of at least 105 consecutive amino acid residues of SEQ ID NO: 7, and has at least 90% sequence identity to the at least 105 consecutive amino acid residues of SEQ ID NO: 7. In some embodiments, the scFv heavy chain variable domain comprises an amino acid sequence of at least 110 consecutive amino acid residues of SEQ ID NO: 7, and has at least 90% sequence identity to the at least 110 consecutive amino acid residues of SEQ ID NO: 7. In some embodiments, the scFv heavy chain variable domain comprises an amino acid sequence of at least 115 consecutive amino acid residues of SEQ ID NO: 7, and has at least 90% sequence identity to the at least 115 consecutive amino acid residues of SEQ ID NO: 7.

In some embodiments, the scFv heavy chain variable domain comprises an amino acid sequence of at least 75 consecutive amino acid residues of SEQ ID NO: 7, and has at least 95% sequence identity to the at least 75 consecutive amino acid residues of SEQ ID NO: 7. In some embodiments, the scFv heavy chain variable domain comprises an amino acid sequence of at least 100 consecutive amino acid residues of SEQ ID NO: 7, and has at least 95% sequence identity to the at least 100 consecutive amino acid residues of SEQ ID NO: 7. In some embodiments, the scFv heavy chain variable domain comprises an amino acid sequence of at least 105 consecutive amino acid residues of SEQ ID NO: 7, and has at least 95% sequence identity to the at least 105 consecutive amino acid residues of SEQ ID NO: 7. In some embodiments, the scFv heavy chain variable domain comprises an amino acid sequence of at least 110 consecutive amino acid residues of SEQ ID NO: 7, and has at least 95% sequence identity to the at least 110 consecutive amino acid residues of SEQ ID NO: 7. In some embodiments, the scFv heavy chain variable domain comprises an amino acid sequence of at least 115 consecutive amino acid residues of SEQ ID NO: 7, and has at least 95% sequence identity to the at least 115 consecutive amino acid residues of SEQ ID NO: 7.

In some embodiments, the scFv heavy chain variable domain comprises an amino acid sequence of at least 75 consecutive amino acid residues of SEQ ID NO: 7, and has at least 99% sequence identity to the at least 75 consecutive amino acid residues of SEQ ID NO: 7. In some embodiments, the scFv heavy chain variable domain comprises an amino acid sequence of at least 100 consecutive amino acid residues of SEQ ID NO: 7, and has at least 99% sequence identity to the at least 100 consecutive amino acid residues of SEQ ID NO: 7. In some embodiments, the scFv heavy chain variable domain comprises an amino acid sequence of at least 105 consecutive amino acid residues of SEQ ID NO: 7, and has at least 99% sequence identity to the at least 105 consecutive amino acid residues of SEQ ID NO: 7. In some embodiments, the scFv heavy chain variable domain comprises an amino acid sequence of at least 110 consecutive amino acid residues of SEQ ID NO: 7, and has at least 99% sequence identity to the at least 110 consecutive amino acid residues of SEQ ID NO: 7. In some embodiments, the scFv heavy chain variable domain comprises an amino acid sequence of at least 115 consecutive amino acid residues of SEQ ID NO: 7, and has at least 99% sequence identity to the at least 115 consecutive amino acid residues of SEQ ID NO: 7.

In some embodiments, the scFv light chain variable domain comprises an amino acid sequence that has at least 70% sequence identity to the amino acid sequence according to SEQ ID NO: 8. In some embodiments, the scFv light chain variable domain comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 8. In some embodiments, the scFv light chain variable domain comprises an amino acid sequence that has at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 8. In some embodiments, the scFv light chain variable domain comprises an amino acid sequence that has at least 90% sequence identity to the amino acid sequence according to SEQ ID NO: 8. In some embodiments, the scFv light chain variable domain comprises an amino acid sequence that has at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 8. In some embodiments, the scFv light chain variable domain comprises an amino acid sequence that has at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 8. In some embodiments, the scFv light chain variable domain comprises an amino acid sequence that has at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 8. In some embodiments, the scFv light chain variable domain comprises an amino acid sequence that has at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 8. In some embodiments, the scFv light chain variable domain comprises an amino acid sequence that has at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 8. In some embodiments, the scFv light chain variable domain comprises an amino acid sequence according to SEQ ID NO: 8.

In some embodiments, the scFv light chain variable domain comprises an amino acid sequence of at least 75 consecutive amino acid residues of SEQ ID NO: 8. In some embodiments, the scFv light chain variable domain comprises an amino acid sequence of at least 80 consecutive amino acid residues of SEQ ID NO: 8. In some embodiments, the scFv light chain variable domain comprises an amino acid sequence of at least 90 consecutive amino acid residues of SEQ ID NO: 8. In some embodiments, the scFv light chain variable domain comprises an amino acid sequence of at least 95 consecutive amino acid residues of SEQ ID NO: 8. In some embodiments, the scFv light chain variable domain comprises an amino acid sequence of at least 100 consecutive amino acid residues of SEQ ID NO: 8. In some embodiments, the scFv light chain variable domain comprises an amino acid sequence of at least 105 consecutive amino acid residues of SEQ ID NO: 8.

In some embodiments, the scFv light chain variable domain comprises an amino acid sequence of at least 75 consecutive amino acid residues of SEQ ID NO: 8, and has at least 80% sequence identity to the at least 75 consecutive amino acid residues of SEQ ID NO: 8. In some embodiments, the scFv light chain variable domain comprises an amino acid sequence of at least 80 consecutive amino acid residues of SEQ ID NO: 8, and has at least 80% sequence identity to the at least 80 consecutive amino acid residues of SEQ ID NO: 8. In some embodiments, the scFv light chain variable domain comprises an amino acid sequence of at least 90 consecutive amino acid residues of SEQ ID NO: 8, and has at least 80% sequence identity to the at least 90 consecutive amino acid residues of SEQ ID NO: 8. In some embodiments, the scFv light chain variable domain comprises an amino acid sequence of at least 95 consecutive amino acid residues of SEQ ID NO: 8, and has at least 80% sequence identity to the at least 95 consecutive amino acid residues of SEQ ID NO: 8. In some embodiments, the scFv light chain variable domain comprises an amino acid sequence of at least 100 consecutive amino acid residues of SEQ ID NO: 8, and has at least 80% sequence identity to the at least 100 consecutive amino acid residues of SEQ ID NO: 8. In some embodiments, the scFv light chain variable domain comprises an amino acid sequence of at least 105 consecutive amino acid residues of SEQ ID NO: 8, and has at least 80% sequence identity to the at least 105 consecutive amino acid residues of SEQ ID NO: 8.

In some embodiments, the scFv light chain variable domain comprises an amino acid sequence of at least 75 consecutive amino acid residues of SEQ ID NO: 8, and has at least 90% sequence identity to the at least 75 consecutive amino acid residues of SEQ ID NO: 8. In some embodiments, the scFv light chain variable domain comprises an amino acid sequence of at least 80 consecutive amino acid residues of SEQ ID NO: 8, and has at least 90% sequence identity to the at least 80 consecutive amino acid residues of SEQ ID NO: 8. In some embodiments, the scFv light chain variable domain comprises an amino acid sequence of at least 90 consecutive amino acid residues of SEQ ID NO: 8, and has at least 90% sequence identity to the at least 90 consecutive amino acid residues of SEQ ID NO: 8. In some embodiments, the scFv light chain variable domain comprises an amino acid sequence of at least 95 consecutive amino acid residues of SEQ ID NO: 8, and has at least 90% sequence identity to the at least 95 consecutive amino acid residues of SEQ ID NO: 8. In some embodiments, the scFv light chain variable domain comprises an amino acid sequence of at least 100 consecutive amino acid residues of SEQ ID NO: 8, and has at least 90% sequence identity to the at least 100 consecutive amino acid residues of SEQ ID NO: 8. In some embodiments, the scFv light chain variable domain comprises an amino acid sequence of at least 105 consecutive amino acid residues of SEQ ID NO: 8, and has at least 90% sequence identity to the at least 105 consecutive amino acid residues of SEQ ID NO: 8.

In some embodiments, the scFv light chain variable domain comprises an amino acid sequence of at least 75 consecutive amino acid residues of SEQ ID NO: 8, and has at least 95% sequence identity to the at least 75 consecutive amino acid residues of SEQ ID NO: 8. In some embodiments, the scFv light chain variable domain comprises an amino acid sequence of at least 80 consecutive amino acid residues of SEQ ID NO: 8, and has at least 95% sequence identity to the at least 80 consecutive amino acid residues of SEQ ID NO: 8. In some embodiments, the scFv light chain variable domain comprises an amino acid sequence of at least 90 consecutive amino acid residues of SEQ ID NO: 8, and has at least 95% sequence identity to the at least 90 consecutive amino acid residues of SEQ ID NO: 8. In some embodiments, the scFv light chain variable domain comprises an amino acid sequence of at least 95 consecutive amino acid residues of SEQ ID NO: 8, and has at least 95% sequence identity to the at least 95 consecutive amino acid residues of SEQ ID NO: 8. In some embodiments, the scFv light chain variable domain comprises an amino acid sequence of at least 100 consecutive amino acid residues of SEQ ID NO: 8, and has at least 95% sequence identity to the at least 100 consecutive amino acid residues of SEQ ID NO: 8. In some embodiments, the scFv light chain variable domain comprises an amino acid sequence of at least 105 consecutive amino acid residues of SEQ ID NO: 8, and has at least 95% sequence identity to the at least 105 consecutive amino acid residues of SEQ ID NO: 8.

In some embodiments, the scFv light chain variable domain comprises an amino acid sequence of at least 75 consecutive amino acid residues of SEQ ID NO: 8, and has at least 99% sequence identity to the at least 75 consecutive amino acid residues of SEQ ID NO: 8. In some embodiments, the scFv light chain variable domain comprises an amino acid sequence of at least 80 consecutive amino acid residues of SEQ ID NO: 8, and has at least 99% sequence identity to the at least 80 consecutive amino acid residues of SEQ ID NO: 8. In some embodiments, the scFv light chain variable domain comprises an amino acid sequence of at least 90 consecutive amino acid residues of SEQ ID NO: 8, and has at least 99% sequence identity to the at least 90 consecutive amino acid residues of SEQ ID NO: 8. In some embodiments, the scFv light chain variable domain comprises an amino acid sequence of at least 95 consecutive amino acid residues of SEQ ID NO: 8, and has at least 99% sequence identity to the at least 95 consecutive amino acid residues of SEQ ID NO: 8. In some embodiments, the scFv light chain variable domain comprises an amino acid sequence of at least 100 consecutive amino acid residues of SEQ ID NO: 8, and has at least 99% sequence identity to the at least 100 consecutive amino acid residues of SEQ ID NO: 8. In some embodiments, the scFv light chain variable domain comprises an amino acid sequence of at least 105 consecutive amino acid residues of SEQ ID NO: 8, and has at least 99% sequence identity to the at least 105 consecutive amino acid residues of SEQ ID NO: 8.

In some embodiments, the scFv comprises an amino acid sequence that has at least 70% sequence identity to the amino acid sequence according to SEQ ID NO: 9. In some embodiments, the scFv comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 9. In some embodiments, the scFv comprises an amino acid sequence that has at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 9. In some embodiments, the scFv comprises an amino acid sequence that has at least 90% sequence identity to the amino acid sequence according to SEQ ID NO: 9. In some embodiments, the scFv comprises an amino acid sequence that has at least 91% sequence identity to the amino acid sequence according to SEQ ID NO: 9. In some embodiments, the scFv comprises an amino acid sequence that has at least 92% sequence identity to the amino acid sequence according to SEQ ID NO: 9. In some embodiments, the scFv comprises an amino acid sequence that has at least 93% sequence identity to the amino acid sequence according to SEQ ID NO: 9. In some embodiments, the scFv comprises an amino acid sequence that has at least 94% sequence identity to the amino acid sequence according to SEQ ID NO: 9. In some embodiments, the scFv comprises an amino acid sequence that has at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 9. In some embodiments, the scFv comprises an amino acid sequence that has at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 9. In some embodiments, the scFv comprises an amino acid sequence that has at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 9. In some embodiments, the scFv comprises an amino acid sequence that has at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 9. In some embodiments, the scFv comprises an amino acid sequence that has at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 9. In some embodiments, the scFv comprises an amino acid sequence according to SEQ ID NO: 9.

In some embodiments, the scFv comprises an amino acid sequence of at least 175 consecutive amino acid residues of SEQ ID NO: 9. In some embodiments, the scFv comprises an amino acid sequence of at least 190 consecutive amino acid residues of SEQ ID NO: 9. In some embodiments, the scFv comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 9.In some embodiments, the scFv comprises an amino acid sequence of at least 205 consecutive amino acid residues of SEQ ID NO: 9. In some embodiments, the scFv comprises an amino acid sequence of at least 210 consecutive amino acid residues of SEQ ID NO: 9. In some embodiments, the scFv comprises an amino acid sequence of at least 215 consecutive amino acid residues of SEQ ID NO: 9.

In some embodiments, the scFv comprises an amino acid sequence of at least 175 consecutive amino acid residues of SEQ ID NO: 9, and has at least 80% sequence identity to the at least 175 consecutive amino acid residues of SEQ ID NO: 9. In some embodiments, the scFv comprises an amino acid sequence of at least 190 consecutive amino acid residues of SEQ ID NO: 9, and has at least 80% sequence identity to the at least 190 consecutive amino acid residues of SEQ ID NO: 9. In some embodiments, the scFv comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 9, and has at least 80% sequence identity to the at least 200 consecutive amino acid residues of SEQ ID NO: 9. In some embodiments, the scFv comprises an amino acid sequence of at least 205 consecutive amino acid residues of SEQ ID NO: 9, and has at least 80% sequence identity to the at least 205 consecutive amino acid residues of SEQ ID NO: 9. In some embodiments, the scFv comprises an amino acid sequence of at least 210 consecutive amino acid residues of SEQ ID NO: 9, and has at least 80% sequence identity to the at least 210 consecutive amino acid residues of SEQ ID NO: 9. In some embodiments, the scFv comprises an amino acid sequence of at least 215 consecutive amino acid residues of SEQ ID NO: 9, and has at least 80% sequence identity to the at least 215 consecutive amino acid residues of SEQ ID NO: 9.

In some embodiments, the scFv comprises an amino acid sequence of at least 175 consecutive amino acid residues of SEQ ID NO: 9, and has at least 90% sequence identity to the at least 175 consecutive amino acid residues of SEQ ID NO: 9. In some embodiments, the scFv comprises an amino acid sequence of at least 190 consecutive amino acid residues of SEQ ID NO: 9, and has at least 90% sequence identity to the at least 190 consecutive amino acid residues of SEQ ID NO: 9. In some embodiments, the scFv comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 9, and has at least 90% sequence identity to the at least 200 consecutive amino acid residues of SEQ ID NO: 9. In some embodiments, the scFv comprises an amino acid sequence of at least 205 consecutive amino acid residues of SEQ ID NO: 9, and has at least 90% sequence identity to the at least 205 consecutive amino acid residues of SEQ ID NO: 9. In some embodiments, the scFv comprises an amino acid sequence of at least 210 consecutive amino acid residues of SEQ ID NO: 9, and has at least 90% sequence identity to the at least 210 consecutive amino acid residues of SEQ ID NO: 9. In some embodiments, the scFv comprises an amino acid sequence of at least 215 consecutive amino acid residues of SEQ ID NO: 9, and has at least 90% sequence identity to the at least 215 consecutive amino acid residues of SEQ ID NO: 9.

In some embodiments, the scFv comprises an amino acid sequence of at least 175 consecutive amino acid residues of SEQ ID NO: 9, and has at least 95% sequence identity to the at least 175 consecutive amino acid residues of SEQ ID NO: 9. In some embodiments, the scFv comprises an amino acid sequence of at least 190 consecutive amino acid residues of SEQ ID NO: 9, and has at least 95% sequence identity to the at least 190 consecutive amino acid residues of SEQ ID NO: 9. In some embodiments, the scFv comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 9, and has at least 95% sequence identity to the at least 200 consecutive amino acid residues of SEQ ID NO: 9. In some embodiments, the scFv comprises an amino acid sequence of at least 205 consecutive amino acid residues of SEQ ID NO: 9, and has at least 95% sequence identity to the at least 205 consecutive amino acid residues of SEQ ID NO: 9. In some embodiments, the scFv comprises an amino acid sequence of at least 210 consecutive amino acid residues of SEQ ID NO: 9, and has at least 95% sequence identity to the at least 210 consecutive amino acid residues of SEQ ID NO: 9. In some embodiments, the scFv comprises an amino acid sequence of at least 215 consecutive amino acid residues of SEQ ID NO: 9, and has at least 95 sequence identity to the at least 215 consecutive amino acid residues of SEQ ID NO: 9.

In some embodiments, the scFv comprises an amino acid sequence of at least 175 consecutive amino acid residues of SEQ ID NO: 9, and has at least 99% o sequence identity to the at least 175 consecutive amino acid residues of SEQ ID NO: 9. In some embodiments, the scFv comprises an amino acid sequence of at least 190 consecutive amino acid residues of SEQ ID NO: 9, and has at least 99% o sequence identity to the at least 190 consecutive amino acid residues of SEQ ID NO: 9. In some embodiments, the scFv comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 9, and has at least 99% o sequence identity to the at least 200 consecutive amino acid residues of SEQ ID NO: 9. In some embodiments, the scFv comprises an amino acid sequence of at least 205 consecutive amino acid residues of SEQ ID NO: 9, and has at least 99% o sequence identity to the at least 205 consecutive amino acid residues of SEQ ID NO: 9. In some embodiments, the scFv comprises an amino acid sequence of at least 210 consecutive amino acid residues of SEQ ID NO: 9, and has at least 99% o sequence identity to the at least 210 consecutive amino acid residues of SEQ ID NO: 9. In some embodiments, the scFv comprises an amino acid sequence of at least 215 consecutive amino acid residues of SEQ ID NO: 9, and has at least 99% o sequence identity to the at least 215 consecutive amino acid residues of SEQ ID NO: 9.

TABLE 3

anti-CD28 light chain variable domain, heavy

chain variable domain sequences, and full

length sequence. CDR sequences are underlined

and were determined using IMGT definition.

Amino Acid
SEQ

Construct
Sequence
ID

Description
(N to C)
NO:

anti-CD28:
QVQLVQSGAEVKKPGASVKV
7

HC
SCKASGYTFTSYYIHWVRQAP

GQGLEWIGSIYPGNVNTNYNE

KFKDRATLTVDTSISTAYMEL

SRLRSDDTAVYFCTRSHYGLD

WNFD
VWGQGTTVTVSS

anti-CD28:
DIQMTQSPSSLSASVGDRVTIT
8

LC
CHASQNIYVWLNWYQQKPG

KAPKLLIYKASNLHTGVPSRFS

GSGSGTDFTLTISSLQPEDFAT

YYCQQGQTYPYTFGGGTKVE

IK

Anti-CD28
QVQLVQSGAEVKKPGASVKV
9

scFv (VH -
SCKASGYTFTSYYIHWVRQAP

linker 1 -
GQGLEWIGSIYPGNVNTNYNE

VL)
KFKDRATLTVDTSISTAYMEL

SRLRSDDTAVYFCTRSHYGLD

WNFDV
WGQGTTVTVSSGGG

GSGGGGSGGGGSDIQMTQSPS

SLSASVGDRVTITCHASQNIYV

W
LNWYQQKPGKAPKLLIYKA

SNLHTGVPSRFSGSGSGTDFTL

TISSLQPEDFATYYCQQGQTY

PYT
FGGGTKVEIK

In some embodiments, the CD28 binding domain comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the CD28 binding domain comprise: HC-CDR1: SEQ ID NO: 1; HC-CDR2: SEQ ID NO: 2; HC-CDR3: SEQ ID NO: 3, and wherein said CDRs comprise from 0-2 amino acid modifications in at least one of said HC-CDR1, HC-CDR2, or HC-CDR3. In some embodiments, the CD28 binding domain comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the CD28 binding domain comprise: HC-CDR1: SEQ ID NO: 1; HC-CDR2: SEQ ID NO: 2; HC-CDR3: SEQ ID NO: 3.

In some embodiments, the CD28 binding domain comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 the anti-CD28 light chain polypeptide: LC-CDR1: SEQ ID NO: 4; LC-CDR2: SEQ ID NO: 5; and LC-CDR3: SEQ ID NO: 6, and wherein the CDRs comprise from 0-2 amino acid modifications in at least one of said LC-CDR1, LC-CDR2, or LC-CDR3. In some embodiments, the CD28 binding domain comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the anti-CD28 light chain polypeptide: LC-CDR1: SEQ ID NO: 4; LC-CDR2: SEQ ID NO: 5; and LC-CDR3: SEQ ID NO: 6.

In some embodiments, CD28 binding domain comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the CD28 binding domain comprise: HC-CDR1: SEQ ID NO: 1; HC-CDR2: SEQ ID NO: 2; HC-CDR3: SEQ ID NO: 3, and wherein said CDRs comprise from 0-2 amino acid modifications in at least one of said HC-CDR1, HC-CDR2, or HC-CDR3; and the CD28 binding domain comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the CD28 binding domain comprise: LC-CDR1: SEQ ID NO: 4; LC-CDR2: SEQ ID NO: 5; and LC-CDR3: SEQ ID NO: 6, and wherein the CDRs comprise from 0-2 amino acid modifications in at least one of said LC-CDR1, LC-CDR2, or LC-CDR3.

In some embodiments, the CD28 binding domain comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the CD28 binding domain comprise: HC-CDR1: SEQ ID NO: 1; HC-CDR2: SEQ ID NO: 2; HC-CDR3: SEQ ID NO: 3; and the CD28 binding domain comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the CD28 binding domain comprise: LC-CDR1: SEQ ID NO: 4; LC-CDR2: SEQ ID NO: 5; and LC-CDR3: SEQ ID NO: 6.

In some embodiments, the CD28 binding domain comprises an amino acid sequence that has at least 70% sequence identity to the amino acid sequence according to SEQ ID NO: 7. In some embodiments, the CD28 binding domain comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 7. In some embodiments, the CD28 binding domain comprises an amino acid sequence that has at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 7. In some embodiments, the CD28 binding domain comprises an amino acid sequence that has at least 90% sequence identity to the amino acid sequence according to SEQ ID NO: 7. In some embodiments, the CD28 binding domain comprises an amino acid sequence that has at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 7. In some embodiments, the CD28 binding domain comprises an amino acid sequence that has at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 7. In some embodiments, the CD28 binding domain comprises an amino acid sequence that has at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 7. In some embodiments, the CD28 binding domain comprises an amino acid sequence that has at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 7. In some embodiments, the CD28 binding domain comprises an amino acid sequence that has at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 7. In some embodiments, the CD28 binding domain comprises an amino acid sequence according to SEQ ID NO: 7.

In some embodiments, the CD28 binding domain comprises an amino acid sequence of at least 75 consecutive amino acid residues of SEQ ID NO: 7. In some embodiments, the CD28 binding domain comprises an amino acid sequence of at least 100 consecutive amino acid residues of SEQ ID NO: 7 In some embodiments, the CD28 binding domain comprises an amino acid sequence of at least 105 consecutive amino acid residues of SEQ ID NO: 7 In some embodiments, the CD28 binding domain comprises an amino acid sequence of at least 110 consecutive amino acid residues of SEQ ID NO: 7. In some embodiments, the CD28 binding domain comprises an amino acid sequence of at least 115 consecutive amino acid residues of SEQ ID NO: 7.

In some embodiments, the CD28 binding domain comprises an amino acid sequence of at least 75 consecutive amino acid residues of SEQ ID NO: 7, and has at least 80% sequence identity to the at least 75 consecutive amino acid residues of SEQ ID NO: 7. In some embodiments, the CD28 binding domain comprises an amino acid sequence of at least 100 consecutive amino acid residues of SEQ ID NO: 7, and has at least 80% sequence identity to the at least 100 consecutive amino acid residues of SEQ ID NO: 7. In some embodiments, the CD28 binding domain comprises an amino acid sequence of at least 105 consecutive amino acid residues of SEQ ID NO: 7, and has at least 80% sequence identity to the at least 105 consecutive amino acid residues of SEQ ID NO: 7. In some embodiments, the CD28 binding domain comprises an amino acid sequence of at least 110 consecutive amino acid residues of SEQ ID NO: 7, and has at least 80% sequence identity to the at least 110 consecutive amino acid residues of SEQ ID NO: 7. In some embodiments, the CD28 binding domain comprises an amino acid sequence of at least 115 consecutive amino acid residues of SEQ ID NO: 7, and has at least 80% sequence identity to the at least 115 consecutive amino acid residues of SEQ ID NO: 7.

In some embodiments, the CD28 binding domain comprises an amino acid sequence of at least 75 consecutive amino acid residues of SEQ ID NO: 7, and has at least 90% sequence identity to the at least 75 consecutive amino acid residues of SEQ ID NO: 7. In some embodiments, the CD28 binding domain comprises an amino acid sequence of at least 100 consecutive amino acid residues of SEQ ID NO: 7, and has at least 90% sequence identity to the at least 100 consecutive amino acid residues of SEQ ID NO: 7. In some embodiments, the CD28 binding domain comprises an amino acid sequence of at least 105 consecutive amino acid residues of SEQ ID NO: 7, and has at least 90% sequence identity to the at least 105 consecutive amino acid residues of SEQ ID NO: 7. In some embodiments, the CD28 binding domain comprises an amino acid sequence of at least 110 consecutive amino acid residues of SEQ ID NO: 7, and has at least 90% sequence identity to the at least 110 consecutive amino acid residues of SEQ ID NO: 7. In some embodiments, the CD28 binding domain comprises an amino acid sequence of at least 115 consecutive amino acid residues of SEQ ID NO: 7, and has at least 90% sequence identity to the at least 115 consecutive amino acid residues of SEQ ID NO: 7.

In some embodiments, the CD28 binding domain comprises an amino acid sequence of at least 75 consecutive amino acid residues of SEQ ID NO: 7, and has at least 95% sequence identity to the at least 75 consecutive amino acid residues of SEQ ID NO: 7. In some embodiments, the CD28 binding domain comprises an amino acid sequence of at least 100 consecutive amino acid residues of SEQ ID NO: 7, and has at least 95% sequence identity to the at least 100 consecutive amino acid residues of SEQ ID NO: 7. In some embodiments, the CD28 binding domain comprises an amino acid sequence of at least 105 consecutive amino acid residues of SEQ ID NO: 7, and has at least 95% sequence identity to the at least 105 consecutive amino acid residues of SEQ ID NO: 7. In some embodiments, the CD28 binding domain comprises an amino acid sequence of at least 110 consecutive amino acid residues of SEQ ID NO: 7, and has at least 95% sequence identity to the at least 110 consecutive amino acid residues of SEQ ID NO: 7. In some embodiments, the CD28 binding domain comprises an amino acid sequence of at least 115 consecutive amino acid residues of SEQ ID NO: 7, and has at least 95% sequence identity to the at least 115 consecutive amino acid residues of SEQ ID NO: 7.

In some embodiments, the CD28 binding domain comprises an amino acid sequence of at least 75 consecutive amino acid residues of SEQ ID NO: 7, and has at least 99% sequence identity to the at least 75 consecutive amino acid residues of SEQ ID NO: 7. In some embodiments, the CD28 binding domain comprises an amino acid sequence of at least 100 consecutive amino acid residues of SEQ ID NO: 7, and has at least 99% sequence identity to the at least 100 consecutive amino acid residues of SEQ ID NO: 7. In some embodiments, the CD28 binding domain comprises an amino acid sequence of at least 105 consecutive amino acid residues of SEQ ID NO: 7, and has at least 99% sequence identity to the at least 105 consecutive amino acid residues of SEQ ID NO: 7. In some embodiments, the CD28 binding domain comprises an amino acid sequence of at least 110 consecutive amino acid residues of SEQ ID NO: 7, and has at least 99% sequence identity to the at least 110 consecutive amino acid residues of SEQ ID NO: 7. In some embodiments, the CD28 binding domain comprises an amino acid sequence of at least 115 consecutive amino acid residues of SEQ ID NO: 7, and has at least 99% sequence identity to the at least 115 consecutive amino acid residues of SEQ ID NO: 7.

In some embodiments, the CD28 binding domain comprises an amino acid sequence that has at least 70% sequence identity to the amino acid sequence according to SEQ ID NO: 8. In some embodiments, the CD28 binding domain comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 8. In some embodiments, the CD28 binding domain comprises an amino acid sequence that has at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 8. In some embodiments, the CD28 binding domain comprises an amino acid sequence that has at least 90% sequence identity to the amino acid sequence according to SEQ ID NO: 8. In some embodiments, the CD28 binding domain comprises an amino acid sequence that has at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 8. In some embodiments, the CD28 binding domain comprises an amino acid sequence that has at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 8. In some embodiments, the CD28 binding domain comprises an amino acid sequence that has at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 8. In some embodiments, the CD28 binding domain comprises an amino acid sequence that has at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 8. In some embodiments, the CD28 binding domain comprises an amino acid sequence that has at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 8. In some embodiments, the CD28 binding domain comprises an amino acid sequence according to SEQ ID NO: 8.

In some embodiments, the CD28 binding domain comprises an amino acid sequence of at least 75 consecutive amino acid residues of SEQ ID NO: 8. In some embodiments, the CD28 binding domain comprises an amino acid sequence of at least 80 consecutive amino acid residues of SEQ ID NO: 8. In some embodiments, the CD28 binding domain comprises an amino acid sequence of at least 90 consecutive amino acid residues of SEQ ID NO: 8. In some embodiments, the CD28 binding domain comprises an amino acid sequence of at least 95 consecutive amino acid residues of SEQ ID NO: 8. In some embodiments, the CD28 binding domain comprises an amino acid sequence of at least 100 consecutive amino acid residues of SEQ ID NO: 8. In some embodiments, the CD28 binding domain comprises an amino acid sequence of at least 105 consecutive amino acid residues of SEQ ID NO: 8.

In some embodiments, the CD28 binding domain comprises an amino acid sequence of at least 75 consecutive amino acid residues of SEQ ID NO: 8, and has at least 80% sequence identity to the at least 75 consecutive amino acid residues of SEQ ID NO: 8. In some embodiments, the CD28 binding domain comprises an amino acid sequence of at least 80 consecutive amino acid residues of SEQ ID NO: 8, and has at least 80% sequence identity to the at least 80 consecutive amino acid residues of SEQ ID NO: 8. In some embodiments, the CD28 binding domain comprises an amino acid sequence of at least 90 consecutive amino acid residues of SEQ ID NO: 8, and has at least 80% sequence identity to the at least 90 consecutive amino acid residues of SEQ ID NO: 8. In some embodiments, the CD28 binding domain comprises an amino acid sequence of at least 95 consecutive amino acid residues of SEQ ID NO: 8, and has at least 80% sequence identity to the at least 95 consecutive amino acid residues of SEQ ID NO: 8. In some embodiments, the CD28 binding domain comprises an amino acid sequence of at least 100 consecutive amino acid residues of SEQ ID NO: 8, and has at least 80% sequence identity to the at least 100 consecutive amino acid residues of SEQ ID NO: 8. In some embodiments, the CD28 binding domain comprises an amino acid sequence of at least 105 consecutive amino acid residues of SEQ ID NO: 8, and has at least 80% sequence identity to the at least 105 consecutive amino acid residues of SEQ ID NO: 8.

In some embodiments, the CD28 binding domain comprises an amino acid sequence of at least 75 consecutive amino acid residues of SEQ ID NO: 8, and has at least 90% sequence identity to the at least 75 consecutive amino acid residues of SEQ ID NO: 8. In some embodiments, the CD28 binding domain comprises an amino acid sequence of at least 80 consecutive amino acid residues of SEQ ID NO: 8, and has at least 90% sequence identity to the at least 80 consecutive amino acid residues of SEQ ID NO: 8. In some embodiments, the CD28 binding domain comprises an amino acid sequence of at least 90 consecutive amino acid residues of SEQ ID NO: 8, and has at least 90% sequence identity to the at least 90 consecutive amino acid residues of SEQ ID NO: 8. In some embodiments, the CD28 binding domain comprises an amino acid sequence of at least 95 consecutive amino acid residues of SEQ ID NO: 8, and has at least 90% sequence identity to the at least 95 consecutive amino acid residues of SEQ ID NO: 8. In some embodiments, the CD28 binding domain comprises an amino acid sequence of at least 100 consecutive amino acid residues of SEQ ID NO: 8, and has at least 90% sequence identity to the at least 100 consecutive amino acid residues of SEQ ID NO: 8. In some embodiments, the CD28 binding domain comprises an amino acid sequence of at least 105 consecutive amino acid residues of SEQ ID NO: 8, and has at least 90% sequence identity to the at least 105 consecutive amino acid residues of SEQ ID NO: 8.

In some embodiments, the CD28 binding domain comprises an amino acid sequence of at least 75 consecutive amino acid residues of SEQ ID NO: 8, and has at least 95% sequence identity to the at least 75 consecutive amino acid residues of SEQ ID NO: 8. In some embodiments, the CD28 binding domain comprises an amino acid sequence of at least 80 consecutive amino acid residues of SEQ ID NO: 8, and has at least 95% sequence identity to the at least 80 consecutive amino acid residues of SEQ ID NO: 8. In some embodiments, the CD28 binding domain comprises an amino acid sequence of at least 90 consecutive amino acid residues of SEQ ID NO: 8, and has at least 95% sequence identity to the at least 90 consecutive amino acid residues of SEQ ID NO: 8.

In some embodiments, the CD28 binding domain comprises an amino acid sequence of at least 95 consecutive amino acid residues of SEQ ID NO: 8, and has at least 95% sequence identity to the at least 95 consecutive amino acid residues of SEQ ID NO: 8. In some embodiments, the CD28 binding domain comprises an amino acid sequence of at least 100 consecutive amino acid residues of SEQ ID NO: 8, and has at least 95% sequence identity to the at least 100 consecutive amino acid residues of SEQ ID NO: 8. In some embodiments, the CD28 binding domain comprises an amino acid sequence of at least 105 consecutive amino acid residues of SEQ ID NO: 8, and has at least 95% sequence identity to the at least 105 consecutive amino acid residues of SEQ ID NO: 8.

In some embodiments, the CD28 binding domain comprises an amino acid sequence of at least 75 consecutive amino acid residues of SEQ ID NO: 8, and has at least 99% sequence identity to the at least 75 consecutive amino acid residues of SEQ ID NO: 8. In some embodiments, the CD28 binding domain comprises an amino acid sequence of at least 80 consecutive amino acid residues of SEQ ID NO: 8, and has at least 99% sequence identity to the at least 80 consecutive amino acid residues of SEQ ID NO: 8. In some embodiments, the CD28 binding domain comprises an amino acid sequence of at least 90 consecutive amino acid residues of SEQ ID NO: 8, and has at least 99% sequence identity to the at least 90 consecutive amino acid residues of SEQ ID NO: 8.

In some embodiments, the CD28 binding domain comprises an amino acid sequence of at least 95 consecutive amino acid residues of SEQ ID NO: 8, and has at least 99% sequence identity to the at least 95 consecutive amino acid residues of SEQ ID NO: 8. In some embodiments, the CD28 binding domain comprises an amino acid sequence of at least 100 consecutive amino acid residues of SEQ ID NO: 8, and has at least 99% sequence identity to the at least 100 consecutive amino acid residues of SEQ ID NO: 8. In some embodiments, the CD28 binding domain comprises an amino acid sequence of at least 105 consecutive amino acid residues of SEQ ID NO: 8, and has at least 99% sequence identity to the at least 105 consecutive amino acid residues of SEQ ID NO: 8.

In some embodiments, the scFv comprises an amino acid sequence of at least 175 consecutive amino acid residues of SEQ ID NO: 9, and has at least 95% sequence identity to the at least 175 consecutive amino acid residues of SEQ ID NO: 9. In some embodiments, the scFv comprises an amino acid sequence of at least 190 consecutive amino acid residues of SEQ ID NO: 9, and has at least 95% sequence identity to the at least 190 consecutive amino acid residues of SEQ ID NO: 9. In some embodiments, the scFv comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 9, and has at least 95% sequence identity to the at least 200 consecutive amino acid residues of SEQ ID NO: 9. In some embodiments, the scFv comprises an amino acid sequence of at least 205 consecutive amino acid residues of SEQ ID NO: 9, and has at least 95% sequence identity to the at least 205 consecutive amino acid residues of SEQ ID NO: 9. In some embodiments, the scFv comprises an amino acid sequence of at least 210 consecutive amino acid residues of SEQ ID NO: 9, and has at least 95% sequence identity to the at least 210 consecutive amino acid residues of SEQ ID NO: 9. In some embodiments, the scFv comprises an amino acid sequence of at least 215 consecutive amino acid residues of SEQ ID NO: 9, and has at least 95% sequence identity to the at least 215 consecutive amino acid residues of SEQ ID NO: 9.

In some embodiments, the scFv comprises an amino acid sequence of at least 175 consecutive amino acid residues of SEQ ID NO: 9, and has at least 99% sequence identity to the at least 175 consecutive amino acid residues of SEQ ID NO: 9. In some embodiments, the scFv comprises an amino acid sequence of at least 190 consecutive amino acid residues of SEQ ID NO: 9, and has at least 99% sequence identity to the at least 190 consecutive amino acid residues of SEQ ID NO: 9. In some embodiments, the scFv comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 9, and has at least 99% sequence identity to the at least 200 consecutive amino acid residues of SEQ ID NO: 9. In some embodiments, the scFv comprises an amino acid sequence of at least 205 consecutive amino acid residues of SEQ ID NO: 9, and has at least 99% sequence identity to the at least 205 consecutive amino acid residues of SEQ ID NO: 9. In some embodiments, the scFv comprises an amino acid sequence of at least 210 consecutive amino acid residues of SEQ ID NO: 9, and has at least 99% sequence identity to the at least 210 consecutive amino acid residues of SEQ ID NO: 9. In some embodiments, the scFv comprises an amino acid sequence of at least 215 consecutive amino acid residues of SEQ ID NO: 9, and has at least 99% sequence identity to the at least 215 consecutive amino acid residues of SEQ ID NO: 9.

PD-L1 Binding Domains

Disclosed here are multispecific antibodies that comprise a PD-L1 binding domain. In some embodiments, the PD-L1 binding domain comprises an antibody or antigen binding fragment. In some embodiments, the antibody or antigen binding fragment is a monoclonal antibody. In some embodiments, the antibody or antigen binding fragment is a human antibody, a murine antibody, a humanized antibody, or a chimeric antibody. In some embodiments, antibody or antigen binding fragment that binds specifically to PD-L1 comprises an anti-PD-L1 heavy chain polypeptide and an anti-PD-L1 light chain polypeptide.

In some embodiments, the PD-L1 binding domain is derived from BMS-936559. In some embodiments, the PD-L1 binding domain is derived from atezolizumab, durvalumab, and avelumab, CK-301, CS-1001, SHR-1316, CBT-502, envafolimab, or BGB-A333.In some embodiments, the anti-PD-L1 heavy chain polypeptide comprises an anti-PD-L1 heavy chain variable domain. In some embodiments, the anti-PD-L1 heavy chain variable domain comprises an IgG framework, an IgA framework, an IgE framework, or an IgM framework. In some embodiments, the anti-PD-L1 heavy chain variable domain comprises a variable domain of an IgG1, IgG2, IgG3, or IgG4 heavy chain. In some embodiments, the anti-PD-L1 light chain polypeptide comprises an anti-PD-L1 light chain variable domain. In some embodiments, the anti-PD-L1 light chain variable domain comprises an IgG framework, an IgA framework, an IgE framework, or an IgM framework. In some embodiments, the anti-PD-L1 light chain variable domain comprises a variable domain of an IgG1, IgG2, IgG3, or IgG4 light chain.

In some embodiments, the PD-L1 binding domain comprises a single domain antibody, a Fab, or a Fab′. In some embodiments, the PD-L1 binding domain comprises the Fab or the Fab′. In some embodiments, the PD-L1 binding domain comprises the Fab or the Fab′. In some embodiments, the PD-L1 binding domain that comprises the Fab or the Fab′ comprises a Fab heavy chain polypeptide comprising a Fab heavy chain variable domain and a Fab light chain polypeptide comprising a Fab light chain variable domain. In some embodiments, the anti-PD-L1 heavy chain polypeptide comprises the Fab heavy chain polypeptide comprising the Fab heavy chain variable domain. In some embodiments, the anti-PD-L1 light chain polypeptide comprises the Fab light chain polypeptide comprising the Fab light chain variable domain.

In some embodiments, the PD-L1 binding domain comprises the single chain variable fragment. In some embodiments, the PD-L1 binding domain that comprises the single chain variable fragment comprises a scFv heavy chain variable domain and a scFv light chain variable domain. In some embodiments, the anti-PD-L1 heavy chain polypeptide comprises the scFv heavy chain variable domain. In some embodiments, the anti-PD-L1 light chain polypeptide comprises the scFv light chain variable domain.

In some embodiments, the anti-PD-L1 heavy chain polypeptide comprises complementarity determining region (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the anti-PD-L1 heavy chain polypeptide comprises: HC-CDR1: SEQ ID NO: 10; HC-CDR2: SEQ ID NO: 11; HC-CDR3: SEQ ID NO: 12, and wherein said CDRs comprise from 0-2 amino acid modifications in at least one of said HC-CDR1, HC-CDR2, or HC-CDR3. In some embodiments, the anti-PD-L1 heavy chain polypeptide comprises complementarity determining region (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the anti-PD-L1 heavy chain polypeptide comprises: HC-CDR1: SEQ ID NO: 10; HC-CDR2: SEQ ID NO: 11; HC-CDR3: SEQ ID NO: 12.

In some embodiments, the anti-PD-L1 light chain polypeptide comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the anti-PD-L1 light chain polypeptide comprise:LC-CDR1: SEQ ID NO: 13; LC-CDR2: SEQ ID NO: 14; and LC-CDR3: SEQ ID NO: 15, and wherein the CDRs comprise from 0-2 amino acid modifications in at least one of said LC-CDR1, LC-CDR2, or LC-CDR3. In some embodiments, the anti-PD-L1 light chain polypeptide comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the anti-PD-L1 light chain polypeptide comprise:LC-CDR1: SEQ ID NO: 13; LC-CDR2: SEQ ID NO: 14; and LC-CDR3: SEQ ID NO: 15.

In some embodiments, the anti-PD-L1 heavy chain polypeptide comprises complementarity determining region (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the anti-PD-L1 heavy chain polypeptide comprises: HC-CDR1: SEQ ID NO: 10; HC-CDR2: SEQ ID NO: 11; HC-CDR3: SEQ ID NO: 12, and wherein said CDRs comprise from 0-2 amino acid modifications in at least one of said HC-CDR1, HC-CDR2, or HC-CDR3; and the anti-PD-L1 light chain polypeptide comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the anti-PD-L1 light chain polypeptide comprise: LC-CDR1: SEQ ID NO: 13; LC-CDR2: SEQ ID NO: 14; and LC-CDR3: SEQ ID NO: 15, and wherein the CDRs comprise from 0-2 amino acid modifications in at least one of said LC-CDR1, LC-CDR2, or LC-CDR3.

In some embodiments, the anti-PD-L1 heavy chain polypeptide comprises complementarity determining region (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the anti-PD-L1 heavy chain polypeptide comprises: HC-CDR1: SEQ ID NO: 10; HC-CDR2: SEQ ID NO: 11; HC-CDR3: SEQ ID NO: 12; and the anti-PD-L1 light chain polypeptide comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the anti-PD-L1 light chain polypeptide comprise: LC-CDR1: SEQ ID NO: 13; LC-CDR2: SEQ ID NO: 14; and LC-CDR3: SEQ ID NO: 15.

In some embodiments, the anti-PD-L1 heavy chain polypeptide comprises complementarity determining region (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the anti-PD-L1 heavy chain polypeptide comprises: HC-CDR1: SEQ ID NO: 24; HC-CDR2: SEQ ID NO: 25; HC-CDR3: SEQ ID NO: 26, and wherein said CDRs comprise from 0-2 amino acid modifications in at least one of said HC-CDR1, HC-CDR2, or HC-CDR3. In some embodiments, the anti-PD-L1 heavy chain polypeptide comprises complementarity determining region (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the anti-PD-L1 heavy chain polypeptide comprises: HC-CDR1: SEQ ID NO: 24; HC-CDR2: SEQ ID NO: 25; HC-CDR3: SEQ ID NO: 26.

In some embodiments, the anti-PD-L1 light chain polypeptide comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the anti-PD-L1 light chain polypeptide comprise:LC-CDR1: SEQ ID NO: 33; LC-CDR2: SEQ ID NO: 34; and LC-CDR3: SEQ ID NO: 35, and wherein the CDRs comprise from 0-2 amino acid modifications in at least one of said LC-CDR1, LC-CDR2, or LC-CDR3. In some embodiments, the anti-PD-L1 light chain polypeptide comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the anti-PD-L1 light chain polypeptide comprise:LC-CDR1: SEQ ID NO: 33; LC-CDR2: SEQ ID NO: 34; and LC-CDR3: SEQ ID NO: 35.

In some embodiments, the anti-PD-L1 heavy chain polypeptide comprises complementarity determining region (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the anti-PD-L1 heavy chain polypeptide comprises: HC-CDR1: SEQ ID NO: 24; HC-CDR2: SEQ ID NO: 25; HC-CDR3: SEQ ID NO: 26, and wherein said CDRs comprise from 0-2 amino acid modifications in at least one of said HC-CDR1, HC-CDR2, or HC-CDR3; and the anti-PD-L1 light chain polypeptide comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the anti-PD-L1 light chain polypeptide comprise: LC-CDR1: SEQ ID NO: 33; LC-CDR2: SEQ ID NO: 34; and LC-CDR3: SEQ ID NO: 35, and wherein the CDRs comprise from 0-2 amino acid modifications in at least one of said LC-CDR1, LC-CDR2, or LC-CDR3.

In some embodiments, the anti-PD-L1 heavy chain polypeptide comprises complementarity determining region (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the anti-PD-L1 heavy chain polypeptide comprises: HC-CDR1: SEQ ID NO: 24; HC-CDR2: SEQ ID NO: 25; HC-CDR3: SEQ ID NO: 26; and the anti-PD-L1 light chain polypeptide comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the anti-PD-L1 light chain polypeptide comprise: LC-CDR1: SEQ ID NO: 33; LC-CDR2: SEQ ID NO: 34; and LC-CDR3: SEQ ID NO: 35.

In some embodiments, the anti-PD-L1 heavy chain polypeptide comprises complementarity determining region (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the anti-PD-L1 heavy chain polypeptide comprises: HC-CDR1: SEQ ID NO: 27; HC-CDR2: SEQ ID NO: 28; HC-CDR3: SEQ ID NO: 29, and wherein said CDRs comprise from 0-2 amino acid modifications in at least one of said HC-CDR1, HC-CDR2, or HC-CDR3. In some embodiments, the anti-PD-L1 heavy chain polypeptide comprises complementarity determining region (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the anti-PD-L1 heavy chain polypeptide comprises: HC-CDR1: SEQ ID NO: 27; HC-CDR2: SEQ ID NO: 28; HC-CDR3: SEQ ID NO: 29.

In some embodiments, the anti-PD-L1 light chain polypeptide comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the anti-PD-L1 light chain polypeptide comprise:LC-CDR1: SEQ ID NO: 36; LC-CDR2: SEQ ID NO: 37; and LC-CDR3: SEQ ID NO: 38, and wherein the CDRs comprise from 0-2 amino acid modifications in at least one of said LC-CDR1, LC-CDR2, or LC-CDR3. In some embodiments, the anti-PD-L1 light chain polypeptide comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the anti-PD-L1 light chain polypeptide comprise:LC-CDR1: SEQ ID NO: 36; LC-CDR2: SEQ ID NO: 37; and LC-CDR3: SEQ ID NO: 38.

In some embodiments, the anti-PD-L1 heavy chain polypeptide comprises complementarity determining region (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the anti-PD-L1 heavy chain polypeptide comprises: HC-CDR1: SEQ ID NO: 27; HC-CDR2: SEQ ID NO: 28; HC-CDR3: SEQ ID NO: 29, and wherein said CDRs comprise from 0-2 amino acid modifications in at least one of said HC-CDR1, HC-CDR2, or HC-CDR3; and the anti-PD-L1 light chain polypeptide comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the anti-PD-L1 light chain polypeptide comprise: LC-CDR1: SEQ ID NO: 36; LC-CDR2: SEQ ID NO: 37; and LC-CDR3: SEQ ID NO: 38, and wherein the CDRs comprise from 0-2 amino acid modifications in at least one of said LC-CDR1, LC-CDR2, or LC-CDR3.

In some embodiments, the anti-PD-L1 heavy chain polypeptide comprises complementarity determining region (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the anti-PD-L1 heavy chain polypeptide comprises: HC-CDR1: SEQ ID NO: 27; HC-CDR2: SEQ ID NO: 28; HC-CDR3: SEQ ID NO: 29; and the anti-PD-L1 light chain polypeptide comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the anti-PD-L1 light chain polypeptide comprise: LC-CDR1: SEQ ID NO: 36; LC-CDR2: SEQ ID NO: 37; and LC-CDR3: SEQ ID NO: 38.

In some embodiments, the anti-PD-L1 heavy chain polypeptide comprises complementarity determining region (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the anti-PD-L1 heavy chain polypeptide comprises: HC-CDR1: SEQ ID NO: 30; HC-CDR2: SEQ ID NO: 31; HC-CDR3: SEQ ID NO: 32, and wherein said CDRs comprise from 0-2 amino acid modifications in at least one of said HC-CDR1, HC-CDR2, or HC-CDR3. In some embodiments, the anti-PD-L1 heavy chain polypeptide comprises complementarity determining region (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the anti-PD-L1 heavy chain polypeptide comprises: HC-CDR1: SEQ ID NO: 30; HC-CDR2: SEQ ID NO: 31; HC-CDR3: SEQ ID NO: 32.

In some embodiments, the anti-PD-L1 light chain polypeptide comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the anti-PD-L1 light chain polypeptide comprise:LC-CDR1: SEQ ID NO: 39; LC-CDR2: SEQ ID NO: 40; and LC-CDR3: SEQ ID NO: 41, and wherein the CDRs comprise from 0-2 amino acid modifications in at least one of said LC-CDR1, LC-CDR2, or LC-CDR3. In some embodiments, the anti-PD-L1 light chain polypeptide comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the anti-PD-L1 light chain polypeptide comprise:LC-CDR1: SEQ ID NO: 39; LC-CDR2: SEQ ID NO: 40; and LC-CDR3: SEQ ID NO: 41.

In some embodiments, the anti-PD-L1 heavy chain polypeptide comprises complementarity determining region (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the anti-PD-L1 heavy chain polypeptide comprises: HC-CDR1: SEQ ID NO: 30; HC-CDR2: SEQ ID NO: 31; HC-CDR3: SEQ ID NO: 32, and wherein said CDRs comprise from 0-2 amino acid modifications in at least one of said HC-CDR1, HC-CDR2, or HC-CDR3; and the anti-PD-L1 light chain polypeptide comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the anti-PD-L1 light chain polypeptide comprise: LC-CDR1: SEQ ID NO: 39; LC-CDR2: SEQ ID NO: 40; and LC-CDR3: SEQ ID NO: 41, and wherein the CDRs comprise from 0-2 amino acid modifications in at least one of said LC-CDR1, LC-CDR2, or LC-CDR3.

In some embodiments, the anti-PD-L1 heavy chain polypeptide comprises complementarity determining region (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the anti-PD-L1 heavy chain polypeptide comprises: HC-CDR1: SEQ ID NO: 30; HC-CDR2: SEQ ID NO: 31; HC-CDR3: SEQ ID NO: 32; and the anti-PD-L1 light chain polypeptide comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the anti-PD-L1 light chain polypeptide comprise: LC-CDR1: SEQ ID NO: 39; LC-CDR2: SEQ ID NO: 40; and LC-CDR3: SEQ ID NO: 41.

TABLE 4

anti-PD-L1 heavy chain polypeptide comple-

mentarity determining regions (CDR)s as

determined by IMGT definition.

Amino Acid
SEQ

Construct
Sequence
ID

Description
(N to C)
NO:

anti-PD-L1 Fab 1:
GDTFSTYA
10

HC: CDR1

anti-PD-L1 Fab 1:
IIPIFGKA
11

HC: CDR2

anti-PD-L1 Fab 1:
ARKFHFVSGSPFGMDV
12

HC: CDR3

anti-PD-L1 Fab 2:
GFTFSDSW
24

HC: CDR1

anti-PD-L1 Fab 2:
ISPYGGST
25

HC: CDR2

anti-PD-L1 Fab 2:
ARRHWPGGFDY
26

HC: CDR3

anti-PD-L1 Fab 3:
GFTFSSYI
27

HC: CDR1

anti-PD-L1 Fab 3:
IYPSGGIT
28

HC: CDR2

anti-PD-L1 Fab 3:
ARIKLGTVTTVDY
29

HC: CDR3

anti-PD-L1 Fab 4:
GFTFSRYW
30

HC: CDR1

anti-PD-L1 Fab 4:
IKQDGSEK
31

HC: CDR2

anti-PD-L1 Fab 4:
AREGGWFGELAFDY
32

HC: CDR3

TABLE 5

anti-PD-L1 light chain polypeptide comple-

mentarity determining regions (CDR)s as

determined by IMGT definition.

Amino Acid
SEQ

Construct
Sequence
ID

Description
(N to C)
NO:

anti-PD-L1 Fab 1:
QSVSSY
13

LC: CDR1

anti-PD-L1 Fab 1:
DA
14

LC: CDR2

anti-PD-L1 Fab 1:
QQRSNWPT
15

LC: CDR3

anti-PD-L1 Fab 2:
QDVSTA
33

LC: CDR1

anti-PD-L1 Fab 2:
SA
34

LC: CDR2

anti-PD-L1 Fab 2:
QQYLYHPAT
35

LC: CDR3

anti-PD-L1 Fab 3:
SSDVGGYNY
36

LC: CDR1

anti-PD-L1 Fab 3:
DV
37

LC: CDR2

anti-PD-L1 Fab 3:
FGTGTKVTVLGQP
38

LC: CDR3

anti-PD-L1 Fab 4:
QRVSSSY
39

LC: CDR1

anti-PD-L1 Fab 4:
DA
40

LC: CDR2

anti-PD-L1 Fab 4:
QQYGSLPWT
41

LC: CDR3

In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 70% o sequence identity to the amino acid sequence according to SEQ ID NO: 17. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 17. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 85% o sequence identity to the amino acid sequence according to SEQ ID NO: 17. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 9000 sequence identity to the amino acid sequence according to SEQ ID NO: 17. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 95% o sequence identity to the amino acid sequence according to SEQ ID NO: 17. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 17. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 17. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 17. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 17. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 17.

In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence of at least 175 consecutive amino acid residues of SEQ ID NO: 17. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence of at least 190 consecutive amino acid residues of SEQ ID NO: 17. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 17. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence of at least 210 consecutive amino acid residues of SEQ ID NO: 17. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence of at least 215 consecutive amino acid residues of SEQ ID NO: 17. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence of at least 220 consecutive amino acid residues of SEQ ID NO: 17.

In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence of at least 175 consecutive amino acid residues of SEQ ID NO: 17, and has at least 80% sequence identity to the at least 175 consecutive amino acid residues of SEQ ID NO: 17. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence of at least 190 consecutive amino acid residues of SEQ ID NO: 17, and has at least 80% sequence identity to the at least 190 consecutive amino acid residues of SEQ ID NO: 17. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 17, and has at least 80% sequence identity to the at least 200 consecutive amino acid residues of SEQ ID NO: 17. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence of at least 210 consecutive amino acid residues of SEQ ID NO: 17, and has at least 80% sequence identity to the at least 210 consecutive amino acid residues of SEQ ID NO: 17. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence of at least 215 consecutive amino acid residues of SEQ ID NO: 17, and has at least 80% sequence identity to the at least 215 consecutive amino acid residues of SEQ ID NO: 17. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence of at least 220 consecutive amino acid residues of SEQ ID NO: 17, and has at least 80% sequence identity to the at least 220 consecutive amino acid residues of SEQ ID NO: 17.

In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence of at least 175 consecutive amino acid residues of SEQ ID NO: 17, and has at least 90% sequence identity to the at least 175 consecutive amino acid residues of SEQ ID NO: 17. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence of at least 190 consecutive amino acid residues of SEQ ID NO: 17, and has at least 90% sequence identity to the at least 190 consecutive amino acid residues of SEQ ID NO: 17. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 17, and has at least 90% sequence identity to the at least 200 consecutive amino acid residues of SEQ ID NO: 17. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence of at least 210 consecutive amino acid residues of SEQ ID NO: 17, and has at least 90% sequence identity to the at least 210 consecutive amino acid residues of SEQ ID NO: 17. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence of at least 215 consecutive amino acid residues of SEQ ID NO: 17, and has at least 90% sequence identity to the at least 215 consecutive amino acid residues of SEQ ID NO: 17. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence of at least 220 consecutive amino acid residues of SEQ ID NO: 17, and has at least 90% sequence identity to the at least 220 consecutive amino acid residues of SEQ ID NO: 17.

In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence of at least 175 consecutive amino acid residues of SEQ ID NO: 17, and has at least 95% sequence identity to the at least 175 consecutive amino acid residues of SEQ ID NO: 17. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence of at least 190 consecutive amino acid residues of SEQ ID NO: 17, and has at least 95% sequence identity to the at least 190 consecutive amino acid residues of SEQ ID NO: 17. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 17, and has at least 95% sequence identity to the at least 200 consecutive amino acid residues of SEQ ID NO: 17. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence of at least 210 consecutive amino acid residues of SEQ ID NO: 17, and has at least 95% sequence identity to the at least 210 consecutive amino acid residues of SEQ ID NO: 17. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence of at least 215 consecutive amino acid residues of SEQ ID NO: 17, and has at least 95% sequence identity to the at least 215 consecutive amino acid residues of SEQ ID NO: 17. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence of at least 220 consecutive amino acid residues of SEQ ID NO: 17, and has at least 95% sequence identity to the at least 220 consecutive amino acid residues of SEQ ID NO: 17.

In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence of at least 175 consecutive amino acid residues of SEQ ID NO: 17, and has at least 99% sequence identity to the at least 175 consecutive amino acid residues of SEQ ID NO: 17. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence of at least 190 consecutive amino acid residues of SEQ ID NO: 17, and has at least 99% sequence identity to the at least 190 consecutive amino acid residues of SEQ ID NO: 17. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 17, and has at least 99% sequence identity to the at least 200 consecutive amino acid residues of SEQ ID NO: 17. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence of at least 210 consecutive amino acid residues of SEQ ID NO: 17, and has at least 99% sequence identity to the at least 210 consecutive amino acid residues of SEQ ID NO: 17. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence of at least 215 consecutive amino acid residues of SEQ ID NO: 17, and has at least 99% sequence identity to the at least 215 consecutive amino acid residues of SEQ ID NO: 17. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence of at least 220 consecutive amino acid residues of SEQ ID NO: 17, and has at least 99% sequence identity to the at least 220 consecutive amino acid residues of SEQ ID NO: 17.

In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 70% sequence identity to the amino acid sequence according to SEQ ID NO: 16. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 16. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 16. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 90% sequence identity to the amino acid sequence according to SEQ ID NO: 16. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 16. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 16. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 16. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 16. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 16. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 16.

In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence of at least 175 consecutive amino acid residues of SEQ ID NO: 16. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence of at least 190 consecutive amino acid residues of SEQ ID NO: 16. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 16. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence of at least 205 consecutive amino acid residues of SEQ ID NO: 16. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence of at least 210 consecutive amino acid residues of SEQ ID NO: 16.

In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence of at least 175 consecutive amino acid residues of SEQ ID NO: 16, and has at least 80% sequence identity to the at least 175 consecutive amino acid residues of SEQ ID NO: 16. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence of at least 190 consecutive amino acid residues of SEQ ID NO: 16, and has at least 80% sequence identity to the at least 190 consecutive amino acid residues of SEQ ID NO: 16. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 16, and has at least 80% sequence identity to the at least 200 consecutive amino acid residues of SEQ ID NO: 16. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence of at least 205 consecutive amino acid residues of SEQ ID NO: 16, and has at least 80% sequence identity to the at least 205 consecutive amino acid residues of SEQ ID NO: 16. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence of at least 210 consecutive amino acid residues of SEQ ID NO: 16, and has at least 80% sequence identity to the at least 210 consecutive amino acid residues of SEQ ID NO: 16.

In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence of at least 175 consecutive amino acid residues of SEQ ID NO: 16, and has at least 90% sequence identity to the at least 175 consecutive amino acid residues of SEQ ID NO: 16. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence of at least 190 consecutive amino acid residues of SEQ ID NO: 16, and has at least 90% sequence identity to the at least 190 consecutive amino acid residues of SEQ ID NO: 16. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 16, and has at least 90% sequence identity to the at least 200 consecutive amino acid residues of SEQ ID NO: 16. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence of at least 205 consecutive amino acid residues of SEQ ID NO: 16, and has at least 90% sequence identity to the at least 205 consecutive amino acid residues of SEQ ID NO: 16. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence of at least 210 consecutive amino acid residues of SEQ ID NO: 16, and has at least 90% sequence identity to the at least 210 consecutive amino acid residues of SEQ ID NO: 16.

In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence of at least 175 consecutive amino acid residues of SEQ ID NO: 16, and has at least 95% sequence identity to the at least 175 consecutive amino acid residues of SEQ ID NO: 16. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence of at least 190 consecutive amino acid residues of SEQ ID NO: 16, and has at least 95% sequence identity to the at least 190 consecutive amino acid residues of SEQ ID NO: 16. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 16, and has at least 95% sequence identity to the at least 200 consecutive amino acid residues of SEQ ID NO: 16. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence of at least 205 consecutive amino acid residues of SEQ ID NO: 16, and has at least 95% sequence identity to the at least 205 consecutive amino acid residues of SEQ ID NO: 16. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence of at least 210 consecutive amino acid residues of SEQ ID NO: 16, and has at least 95% sequence identity to the at least 210 consecutive amino acid residues of SEQ ID NO: 16.

In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence of at least 175 consecutive amino acid residues of SEQ ID NO: 16, and has at least 99% sequence identity to the at least 175 consecutive amino acid residues of SEQ ID NO: 16. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence of at least 190 consecutive amino acid residues of SEQ ID NO: 16, and has at least 99% sequence identity to the at least 190 consecutive amino acid residues of SEQ ID NO: 16. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 16, and has at least 99% sequence identity to the at least 200 consecutive amino acid residues of SEQ ID NO: 16. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence of at least 205 consecutive amino acid residues of SEQ ID NO: 16, and has at least 99% sequence identity to the at least 205 consecutive amino acid residues of SEQ ID NO: 16. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence of at least 210 consecutive amino acid residues of SEQ ID NO: 16, and has at least 99% sequence identity to the at least 210 consecutive amino acid residues of SEQ ID NO: 16.

In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 70% sequence identity to the amino acid sequence according to SEQ ID NO: 17; and the Fab light chain polypeptide comprises an amino acid sequence that has at least 70% sequence identity to the amino acid sequence according to SEQ ID NO: 16. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 17; and the Fab light chain polypeptide comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 16. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 17; and the Fab light chain polypeptide comprises an amino acid sequence that has at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 16. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 90% sequence identity to the amino acid sequence according to SEQ ID NO: 17; and the Fab light chain polypeptide comprises an amino acid sequence that has at least 90% sequence identity to the amino acid sequence according to SEQ ID NO: 16. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 17; and the Fab light chain polypeptide comprises an amino acid sequence that has at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 16. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 17; and the Fab light chain polypeptide comprises an amino acid sequence that has at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 16. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 17; and the Fab light chain polypeptide comprises an amino acid sequence that has at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 16. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 17; and the Fab light chain polypeptide comprises an amino acid sequence that has at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 16. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 17; and the Fab light chain polypeptide comprises an amino acid sequence that has at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 16.

In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 70% sequence identity to the amino acid sequence according to SEQ ID NO: 43. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 43. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 43. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 90% sequence identity to the amino acid sequence according to SEQ ID NO: 43. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 43. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 43. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 43. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 43. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 43. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 43.

In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence of at least 175 consecutive amino acid residues of SEQ ID NO: 43, and has at least 95% sequence identity to the at least 175 consecutive amino acid residues of SEQ ID NO: 43. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence of at least 190 consecutive amino acid residues of SEQ ID NO: 43, and has at least 95% sequence identity to the at least 190 consecutive amino acid residues of SEQ ID NO: 43. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 43, and has at least 95% sequence identity to the at least 200 consecutive amino acid residues of SEQ ID NO: 43. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence of at least 210 consecutive amino acid residues of SEQ ID NO: 43, and has at least 95% sequence identity to the at least 210 consecutive amino acid residues of SEQ ID NO: 43. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence of at least 215 consecutive amino acid residues of SEQ ID NO: 43, and has at least 95% sequence identity to the at least 215 consecutive amino acid residues of SEQ ID NO: 43. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence of at least 220 consecutive amino acid residues of SEQ ID NO: 43, and has at least 95% sequence identity to the at least 220 consecutive amino acid residues of SEQ ID NO: 43.

In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 70% sequence identity to the amino acid sequence according to SEQ ID NO: 42. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 42. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 42. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 90% sequence identity to the amino acid sequence according to SEQ ID NO: 42. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 42. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 42. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 42. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 42. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 42. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 42.

In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence of at least 175 consecutive amino acid residues of SEQ ID NO: 42, and has at least 95% sequence identity to the at least 175 consecutive amino acid residues of SEQ ID NO: 42. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence of at least 190 consecutive amino acid residues of SEQ ID NO: 42, and has at least 95% sequence identity to the at least 190 consecutive amino acid residues of SEQ ID NO: 42. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 42, and has at least 95% sequence identity to the at least 200 consecutive amino acid residues of SEQ ID NO: 42. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence of at least 205 consecutive amino acid residues of SEQ ID NO: 42, and has at least 95% sequence identity to the at least 205 consecutive amino acid residues of SEQ ID NO: 42. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence of at least 210 consecutive amino acid residues of SEQ ID NO: 42, and has at least 95% sequence identity to the at least 210 consecutive amino acid residues of SEQ ID NO: 42.

In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 70% sequence identity to the amino acid sequence according to SEQ ID NO: 43; and the Fab light chain polypeptide comprises an amino acid sequence that has at least 70% sequence identity to the amino acid sequence according to SEQ ID NO: 42. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 43; and the Fab light chain polypeptide comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 42. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 43; and the Fab light chain polypeptide comprises an amino acid sequence that has at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 42. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 90% sequence identity to the amino acid sequence according to SEQ ID NO: 43; and the Fab light chain polypeptide comprises an amino acid sequence that has at least 90% sequence identity to the amino acid sequence according to SEQ ID NO: 42. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 43; and the Fab light chain polypeptide comprises an amino acid sequence that has at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 42. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 43; and the Fab light chain polypeptide comprises an amino acid sequence that has at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 42. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 43; and the Fab light chain polypeptide comprises an amino acid sequence that has at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 42. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 43; and the Fab light chain polypeptide comprises an amino acid sequence that has at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 42. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 43; and the Fab light chain polypeptide comprises an amino acid sequence that has at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 42.

In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 70% sequence identity to the amino acid sequence according to SEQ ID NO: 45. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 45. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 45. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 90% sequence identity to the amino acid sequence according to SEQ ID NO: 45. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 45. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 45. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 45. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 45. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 45. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 45.

In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence of at least 175 consecutive amino acid residues of SEQ ID NO: 45, and has at least 95% sequence identity to the at least 175 consecutive amino acid residues of SEQ ID NO: 45. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence of at least 190 consecutive amino acid residues of SEQ ID NO: 45, and has at least 95% sequence identity to the at least 190 consecutive amino acid residues of SEQ ID NO: 45. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 45, and has at least 95% sequence identity to the at least 200 consecutive amino acid residues of SEQ ID NO: 45. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence of at least 210 consecutive amino acid residues of SEQ ID NO: 45, and has at least 95% sequence identity to the at least 210 consecutive amino acid residues of SEQ ID NO: 45. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence of at least 215 consecutive amino acid residues of SEQ ID NO: 45, and has at least 95% sequence identity to the at least 215 consecutive amino acid residues of SEQ ID NO: 45. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence of at least 220 consecutive amino acid residues of SEQ ID NO: 45, and has at least 95% sequence identity to the at least 220 consecutive amino acid residues of SEQ ID NO: 45.

In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 70% sequence identity to the amino acid sequence according to SEQ ID NO: 44. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 44. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 44. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 90% sequence identity to the amino acid sequence according to SEQ ID NO: 44. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 44. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 44. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 44. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 44. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 44. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 44.

In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence of at least 175 consecutive amino acid residues of SEQ ID NO: 44, and has at least 95% sequence identity to the at least 175 consecutive amino acid residues of SEQ ID NO: 44. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence of at least 190 consecutive amino acid residues of SEQ ID NO: 44, and has at least 95% sequence identity to the at least 190 consecutive amino acid residues of SEQ ID NO: 44. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 44, and has at least 95% sequence identity to the at least 200 consecutive amino acid residues of SEQ ID NO: 44. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence of at least 205 consecutive amino acid residues of SEQ ID NO: 44, and has at least 95% sequence identity to the at least 205 consecutive amino acid residues of SEQ ID NO: 44. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence of at least 210 consecutive amino acid residues of SEQ ID NO: 44, and has at least 95% sequence identity to the at least 210 consecutive amino acid residues of SEQ ID NO: 44.

In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 70% sequence identity to the amino acid sequence according to SEQ ID NO: 45; and the Fab light chain polypeptide comprises an amino acid sequence that has at least 70% sequence identity to the amino acid sequence according to SEQ ID NO: 44. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 45; and the Fab light chain polypeptide comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 44. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 45; and the Fab light chain polypeptide comprises an amino acid sequence that has at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 44. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 90% sequence identity to the amino acid sequence according to SEQ ID NO: 45; and the Fab light chain polypeptide comprises an amino acid sequence that has at least 90% sequence identity to the amino acid sequence according to SEQ ID NO: 44. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 45; and the Fab light chain polypeptide comprises an amino acid sequence that has at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 44. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 45; and the Fab light chain polypeptide comprises an amino acid sequence that has at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 44. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 45; and the Fab light chain polypeptide comprises an amino acid sequence that has at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 44. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 45; and the Fab light chain polypeptide comprises an amino acid sequence that has at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 44. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 45; and the Fab light chain polypeptide comprises an amino acid sequence that has at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 44.

In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 70% sequence identity to the amino acid sequence according to SEQ ID NO: 47. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 47. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 47. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 90% sequence identity to the amino acid sequence according to SEQ ID NO: 47. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 47. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 47. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 47. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 47. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 47. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 47.

In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence of at least 175 consecutive amino acid residues of SEQ ID NO: 47, and has at least 95% sequence identity to the at least 175 consecutive amino acid residues of SEQ ID NO: 47. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence of at least 190 consecutive amino acid residues of SEQ ID NO: 47, and has at least 95% sequence identity to the at least 190 consecutive amino acid residues of SEQ ID NO: 47. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 47, and has at least 95% sequence identity to the at least 200 consecutive amino acid residues of SEQ ID NO: 47. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence of at least 210 consecutive amino acid residues of SEQ ID NO: 47, and has at least 95% sequence identity to the at least 210 consecutive amino acid residues of SEQ ID NO: 47. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence of at least 215 consecutive amino acid residues of SEQ ID NO: 47, and has at least 95% sequence identity to the at least 215 consecutive amino acid residues of SEQ ID NO: 47. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence of at least 220 consecutive amino acid residues of SEQ ID NO: 47, and has at least 95% sequence identity to the at least 220 consecutive amino acid residues of SEQ ID NO: 47.

In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 70% sequence identity to the amino acid sequence according to SEQ ID NO: 46. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 46. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 46. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 90% sequence identity to the amino acid sequence according to SEQ ID NO: 46. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 46. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 46. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 46. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 46. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 46. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 46.

In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence of at least 175 consecutive amino acid residues of SEQ ID NO: 46, and has at least 95% sequence identity to the at least 175 consecutive amino acid residues of SEQ ID NO: 46. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence of at least 190 consecutive amino acid residues of SEQ ID NO: 46, and has at least 95% sequence identity to the at least 190 consecutive amino acid residues of SEQ ID NO: 46. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 46, and has at least 95% sequence identity to the at least 200 consecutive amino acid residues of SEQ ID NO: 46. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence of at least 205 consecutive amino acid residues of SEQ ID NO: 46, and has at least 95% sequence identity to the at least 205 consecutive amino acid residues of SEQ ID NO: 46. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence of at least 210 consecutive amino acid residues of SEQ ID NO: 46, and has at least 95% sequence identity to the at least 210 consecutive amino acid residues of SEQ ID NO: 46.

In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 70% sequence identity to the amino acid sequence according to SEQ ID NO: 47; and the Fab light chain polypeptide comprises an amino acid sequence that has at least 70% sequence identity to the amino acid sequence according to SEQ ID NO: 46. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 47; and the Fab light chain polypeptide comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 46. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 47; and the Fab light chain polypeptide comprises an amino acid sequence that has at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 46. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 90% sequence identity to the amino acid sequence according to SEQ ID NO: 47; and the Fab light chain polypeptide comprises an amino acid sequence that has at least 90% sequence identity to the amino acid sequence according to SEQ ID NO: 46. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 47; and the Fab light chain polypeptide comprises an amino acid sequence that has at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 46. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 47; and the Fab light chain polypeptide comprises an amino acid sequence that has at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 46. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 47; and the Fab light chain polypeptide comprises an amino acid sequence that has at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 46. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 47; and the Fab light chain polypeptide comprises an amino acid sequence that has at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 46. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 47; and the Fab light chain polypeptide comprises an amino acid sequence that has at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 46.

TABLE 6

anti-PD-L1 Fab light chain polypeptide and Fab heavy chain

polypeptide sequences. CDR sequences are underlined and were

determined using IMGT definition

Amino Acid Sequence
SEQ ID

Construct Description
(N to C)
NO:

anti-PD-L1 Fab 1: LC
EIVLTQSPATLSLSPGERATLSC
16

RASQSVSSYLAWYQQKPGQA

PRLLIYDASNRATGIPARFSGS

GSGTDFTLTISSLEPEDFAVYY

CQQRSNWPTFGQGTKVEIKR

TVAAPSVFIFPPSDEQLKSGTA

SVVCLLNNFYPREAKVQWKV

DNALQSGNSQESVTEQDSKDS

TYSLSSTLTLSKADYEKHKVY

ACEVTHQGLSSPVTKSFNRGE

C

anti-PD-L1 Fab 1: HC
QVQLVQSGAEVKKPGSSVKVS
17

CKTSGDTFSTYAISWVRQAPG

QGLEWMGGIIPIFGKAHYAQ

KFQGRVTITADESTSTAYMEL

SSLRSEDTAVYFCARKFHFVS

GSPFGMDV
WGQGTTVTVSSA

STKGPSVFPLAPSSKSTSGGTA

ALGCLVKDYFPEPVTVSWNSG

ALTSGVHTFPAVLQSSGLYSLS

SVVTVPSSSLGTQTYICNVNH

KPSNTKVDKKVEPKSC

anti-PD-L1 Fab 2: LC
DIQMTQSPSSLSASVGDRVTIT
42

CRASQDVSTAVAWYQQKPGK

APKLLIYSASFLYSGVPSRFSG

SGSGTDFTLTISSLQPEDFATY

YCQQYLYHPATFGQGTKVEI

KRTVAAPSVFIFPPSDEQLKSG

TASVVCLLNNFYPREAKVQW

KVDNALQSGNSQESVTEQDSK

DSTYSLSSTLTLSKADYEKHK

VYACEVTHQGLSSPVTKSFNR

GEC

anti-PD-L1 Fab 2: HC
EVQLVESGGGLVQPGGSLRLS
43

CAASGFTFSDSWIHWVRQAP

GKGLEWVAWISPYGGSTYYA

DSVKGRFTISADTSKNTAYLQ

MNSLRAEDTAVYYCARRHW

PGGFDY
WGQGTLVTVSSAST

KGPSVFPLAPSSKSTSGGTAAL

GCLVKDYFPEPVTVSWNSGAL

TSGVHTFPAVLQSSGLYSLSSV

VTVPSSSLGTQTYICNVNHKPS

NTKVDKKVEPKSC

anti-PD-L1 Fab 3: LC
QSALTQPASVSGSPGQSITISCT
44

GTSSDVGGYNYVSWYQQHPG

KAPKLMIYDVSNRPSGVSNRF

SGSKSGNTASLTISGLQAEDEA

DYYCSSYTSSSTRVFGTGTKV

TVLGQP
KANPTVTLFPPSSEEL

QANKATLVCLISDFYPGAVTV

AWKADGSPVKAGVETTKPSK

QSNNKYAASSYLSLTPEQWKS

HRSYSCQVTHEGSTVEKTVAP

TECS

anti-PD-L1 Fab 3: HC
EVQLLESGGGLVQPGGSLRLS
45

CAASGFTFSSYIMMWVRQAP

GKGLEWVSSIYPSGGITFYAD

TVKGRFTISRDNSKNTLYLQM

NSLRAEDTAVYYCARIKLGT

VTTVDY
WGQGTLVTVSSAST

KGPSVFPLAPSSKSTSGGTAAL

GCLVKDYFPEPVTVSWNSGAL

TSGVHTFPAVLQSSGLYSLSSV

VTVPSSSLGTQTYICNVNHKPS

NTKVDKKVEPKSC

anti-PD-L1 Fab 4: LC
EIVLTQSPGTLSLSPGERATLSC
46

RASQRVSSSYLAWYQQKPGQ

APRLLIYDASSRATGIPDRFSG

SGSGTDFTLTISRLEPEDFAVY

YCQQYGSLPWTFGQGTKVEI

KRTVAAPSVFIFPPSDEQLKSG

TASVVCLLNNFYPREAKVQW

KVDNALQSGNSQESVTEQDSK

DSTYSLSSTLTLSKADYEKHK

VYACEVTHQGLSSPVTKSFNR

GEC

anti-PD-L1 Fab 4: HC
EVQLVESGGGLVQPGGSLRLS
47

CAASGFTFSRYWMSWVRQAP

GKGLEWVANIKQDGSEKYYV

DSVKGRFTISRDNAKNSLYLQ

MNSLRAEDTAVYYCAREGG

WFGELAFDY
WGQGTLVTVSS

ASTKGPSVFPLAPSSKSTSGGT

AALGCLVKDYFPEPVTVSWNS

GALTSGVHTFPAVLQSSGLYS

LSSVVTVPSSSLGTQTYICNVN

HKPSNTKVDKRVEPKSC

In some embodiments, the PD-L1 binding domain comprises complementarity determining region (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the PD-L1 binding domain comprises: HC-CDR1: SEQ ID NO: 10; HC-CDR2: SEQ ID NO: 11; HC-CDR3: SEQ ID NO: 12, and wherein said CDRs comprise from 0-2 amino acid modifications in at least one of said HC-CDR1, HC-CDR2, or HC-CDR3. In some embodiments, the PD-L1 binding domain comprises complementarity determining region (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the PD-L1 binding domain comprises: HC-CDR1: SEQ ID NO: 10; HC-CDR2: SEQ ID NO: 11; HC-CDR3: SEQ ID NO: 12.

In some embodiments, the PD-L1 binding domain comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the PD-L1 binding domain comprise:LC-CDR1: SEQ ID NO: 13; LC-CDR2: SEQ ID NO: 14; and LC-CDR3: SEQ ID NO: 15, and wherein the CDRs comprise from 0-2 amino acid modifications in at least one of said LC-CDR1, LC-CDR2, or LC-CDR3. In some embodiments, the PD-L1 binding domain comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the PD-L1 binding domain comprise:LC-CDR1: SEQ ID NO: 13; LC-CDR2: SEQ ID NO: 14; and LC-CDR3: SEQ ID NO: 15.

In some embodiments, the PD-L1 binding domain comprises complementarity determining region (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the PD-L1 binding domain comprises: HC-CDR1: SEQ ID NO: 10; HC-CDR2: SEQ ID NO: 11; HC-CDR3: SEQ ID NO: 12, and wherein said CDRs comprise from 0-2 amino acid modifications in at least one of said HC-CDR1, HC-CDR2, or HC-CDR3; and the PD-L1 binding domain comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the PD-L1 binding domain comprise: LC-CDR1: SEQ ID NO: 13; LC-CDR2: SEQ ID NO: 14; and LC-CDR3: SEQ ID NO: 15, and wherein the CDRs comprise from 0-2 amino acid modifications in at least one of said LC-CDR1, LC-CDR2, or LC-CDR3.

In some embodiments, the PD-L1 binding domain comprises complementarity determining region (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the PD-L1 binding domain comprises: HC-CDR1: SEQ ID NO: 10; HC-CDR2: SEQ ID NO: 11; HC-CDR3: SEQ ID NO: 12; and the PD-L1 binding domain comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the PD-L1 binding domain comprise: LC-CDR1: SEQ ID NO: 13; LC-CDR2: SEQ ID NO: 14; and LC-CDR3: SEQ ID NO: 15.

In some embodiments, the PD-L1 binding domain comprises complementarity determining region (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the PD-L1 binding domain comprises: HC-CDR1: SEQ ID NO: 24; HC-CDR2: SEQ ID NO: 25; HC-CDR3: SEQ ID NO: 26, and wherein said CDRs comprise from 0-2 amino acid modifications in at least one of said HC-CDR1, HC-CDR2, or HC-CDR3. In some embodiments, the PD-L1 binding domain comprises complementarity determining region (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the PD-L1 binding domain comprises: HC-CDR1: SEQ ID NO: 24; HC-CDR2: SEQ ID NO: 25; HC-CDR3: SEQ ID NO: 26.

In some embodiments, the PD-L1 binding domain comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the PD-L1 binding domain comprise:LC-CDR1: SEQ ID NO: 33; LC-CDR2: SEQ ID NO: 34; and LC-CDR3: SEQ ID NO: 35, and wherein the CDRs comprise from 0-2 amino acid modifications in at least one of said LC-CDR1, LC-CDR2, or LC-CDR3. In some embodiments, the PD-L1 binding domain comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the PD-L1 binding domain comprise:LC-CDR1: SEQ ID NO: 33; LC-CDR2: SEQ ID NO: 34; and LC-CDR3: SEQ ID NO: 35.

In some embodiments, the PD-L1 binding domain comprises complementarity determining region (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the PD-L1 binding domain comprises: HC-CDR1: SEQ ID NO: 24; HC-CDR2: SEQ ID NO: 25; HC-CDR3: SEQ ID NO: 26, and wherein said CDRs comprise from 0-2 amino acid modifications in at least one of said HC-CDR1, HC-CDR2, or HC-CDR3; and the PD-L1 binding domain comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the PD-L1 binding domain comprise: LC-CDR1: SEQ ID NO: 33; LC-CDR2: SEQ ID NO: 34; and LC-CDR3: SEQ ID NO: 35, and wherein the CDRs comprise from 0-2 amino acid modifications in at least one of said LC-CDR1, LC-CDR2, or LC-CDR3.

In some embodiments, the PD-L1 binding domain comprises complementarity determining region (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the PD-L1 binding domain comprises: HC-CDR1: SEQ ID NO: 24; HC-CDR2: SEQ ID NO: 25; HC-CDR3: SEQ ID NO: 26; and the PD-L1 binding domain comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the PD-L1 binding domain comprise: LC-CDR1: SEQ ID NO: 33; LC-CDR2: SEQ ID NO: 34; and LC-CDR3: SEQ ID NO: 35.

In some embodiments, the PD-L1 binding domain comprises complementarity determining region (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the PD-L1 binding domain comprises: HC-CDR1: SEQ ID NO: 27; HC-CDR2: SEQ ID NO: 28; HC-CDR3: SEQ ID NO: 29, and wherein said CDRs comprise from 0-2 amino acid modifications in at least one of said HC-CDR1, HC-CDR2, or HC-CDR3. In some embodiments, the PD-L1 binding domain comprises complementarity determining region (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the PD-L1 binding domain comprises: HC-CDR1: SEQ ID NO: 27; HC-CDR2: SEQ ID NO: 28; HC-CDR3: SEQ ID NO: 29.

In some embodiments, the PD-L1 binding domain comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the PD-L1 binding domain comprise:LC-CDR1: SEQ ID NO: 36; LC-CDR2: SEQ ID NO: 37; and LC-CDR3: SEQ ID NO: 38, and wherein the CDRs comprise from 0-2 amino acid modifications in at least one of said LC-CDR1, LC-CDR2, or LC-CDR3. In some embodiments, the PD-L1 binding domain comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the PD-L1 binding domain comprise:LC-CDR1: SEQ ID NO: 36; LC-CDR2: SEQ ID NO: 37; and LC-CDR3: SEQ ID NO: 38.

In some embodiments, the PD-L1 binding domain comprises complementarity determining region (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the PD-L1 binding domain comprises: HC-CDR1: SEQ ID NO: 27; HC-CDR2: SEQ ID NO: 28; HC-CDR3: SEQ ID NO: 29, and wherein said CDRs comprise from 0-2 amino acid modifications in at least one of said HC-CDR1, HC-CDR2, or HC-CDR3; and the PD-L1 binding domain comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the PD-L1 binding domain comprise: LC-CDR1: SEQ ID NO: 36; LC-CDR2: SEQ ID NO: 37; and LC-CDR3: SEQ ID NO: 38, and wherein the CDRs comprise from 0-2 amino acid modifications in at least one of said LC-CDR1, LC-CDR2, or LC-CDR3.

In some embodiments, the PD-L1 binding domain comprises complementarity determining region (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the PD-L1 binding domain comprises: HC-CDR1: SEQ ID NO: 27; HC-CDR2: SEQ ID NO: 28; HC-CDR3: SEQ ID NO: 29; and the PD-L1 binding domain comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the PD-L1 binding domain comprise: LC-CDR1: SEQ ID NO: 36; LC-CDR2: SEQ ID NO: 37; and LC-CDR3: SEQ ID NO: 38.

In some embodiments, the PD-L1 binding domain comprises complementarity determining region (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the PD-L1 binding domain comprises: HC-CDR1: SEQ ID NO: 30; HC-CDR2: SEQ ID NO: 31; HC-CDR3: SEQ ID NO: 32, and wherein said CDRs comprise from 0-2 amino acid modifications in at least one of said HC-CDR1, HC-CDR2, or HC-CDR3. In some embodiments, the PD-L1 binding domain comprises complementarity determining region (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the PD-L1 binding domain comprises: HC-CDR1: SEQ ID NO: 30; HC-CDR2: SEQ ID NO: 31; HC-CDR3: SEQ ID NO: 32.

In some embodiments, the PD-L1 binding domain comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the PD-L1 binding domain comprise: LC-CDR1: SEQ ID NO: 39; LC-CDR2: SEQ ID NO: 40; and LC-CDR3: SEQ ID NO: 41, and wherein the CDRs comprise from 0-2 amino acid modifications in at least one of said LC-CDR1, LC-CDR2, or LC-CDR3. In some embodiments, the PD-L1 binding domain comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the PD-L1 binding domain comprise:LC-CDR1: SEQ ID NO: 39; LC-CDR2: SEQ ID NO: 40; and LC-CDR3: SEQ ID NO: 41.

In some embodiments, the PD-L1 binding domain comprises complementarity determining region (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the PD-L1 binding domain comprises: HC-CDR1: SEQ ID NO: 30; HC-CDR2: SEQ ID NO: 31; HC-CDR3: SEQ ID NO: 32, and wherein said CDRs comprise from 0-2 amino acid modifications in at least one of said HC-CDR1, HC-CDR2, or HC-CDR3; and the PD-L1 binding domain comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the PD-L1 binding domain comprise: LC-CDR1: SEQ ID NO: 39; LC-CDR2: SEQ ID NO: 40; and LC-CDR3: SEQ ID NO: 41, and wherein the CDRs comprise from 0-2 amino acid modifications in at least one of said LC-CDR1, LC-CDR2, or LC-CDR3.

In some embodiments, the PD-L1 binding domain comprises complementarity determining region (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the PD-L1 binding domain comprises: HC-CDR1: SEQ ID NO: 30; HC-CDR2: SEQ ID NO: 31; HC-CDR3: SEQ ID NO: 32; and the PD-L1 binding domain comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the PD-L1 binding domain comprise: LC-CDR1: SEQ ID NO: 39; LC-CDR2: SEQ ID NO: 40; and LC-CDR3: SEQ ID NO: 41.

In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 70% sequence identity to the amino acid sequence according to SEQ ID NO: 17. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 17. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 17. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 90% sequence identity to the amino acid sequence according to SEQ ID NO: 17. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 17. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 17. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 17. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 17. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 17. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence according to SEQ ID NO: 17.

In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 175 consecutive amino acid residues of SEQ ID NO: 17. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 190 consecutive amino acid residues of SEQ ID NO: 17. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 17. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 210 consecutive amino acid residues of SEQ ID NO: 17. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 215 consecutive amino acid residues of SEQ ID NO: 17. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 220 consecutive amino acid residues of SEQ ID NO: 17.

In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 175 consecutive amino acid residues of SEQ ID NO: 17, and has at least 80% sequence identity to the at least 175 consecutive amino acid residues of SEQ ID NO: 17. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 190 consecutive amino acid residues of SEQ ID NO: 17, and has at least 80% sequence identity to the at least 190 consecutive amino acid residues of SEQ ID NO: 17. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 17, and has at least 80% sequence identity to the at least 200 consecutive amino acid residues of SEQ ID NO: 17. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 210 consecutive amino acid residues of SEQ ID NO: 17, and has at least 80% sequence identity to the at least 210 consecutive amino acid residues of SEQ ID NO: 17.

In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 215 consecutive amino acid residues of SEQ ID NO: 17, and has at least 80% sequence identity to the at least 215 consecutive amino acid residues of SEQ ID NO: 17. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 220 consecutive amino acid residues of SEQ ID NO: 17, and has at least 80% sequence identity to the at least 220 consecutive amino acid residues of SEQ ID NO: 17.

In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 175 consecutive amino acid residues of SEQ ID NO: 17, and has at least 90% sequence identity to the at least 175 consecutive amino acid residues of SEQ ID NO: 17. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 190 consecutive amino acid residues of SEQ ID NO: 17, and has at least 90% sequence identity to the at least 190 consecutive amino acid residues of SEQ ID NO: 17. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 17, and has at least 90% sequence identity to the at least 200 consecutive amino acid residues of SEQ ID NO: 17. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 210 consecutive amino acid residues of SEQ ID NO: 17, and has at least 90% sequence identity to the at least 210 consecutive amino acid residues of SEQ ID NO: 17.

In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 215 consecutive amino acid residues of SEQ ID NO: 17, and has at least 90% sequence identity to the at least 215 consecutive amino acid residues of SEQ ID NO: 17. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 220 consecutive amino acid residues of SEQ ID NO: 17, and has at least 90% sequence identity to the at least 220 consecutive amino acid residues of SEQ ID NO: 17.

In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 175 consecutive amino acid residues of SEQ ID NO: 17, and has at least 95% sequence identity to the at least 175 consecutive amino acid residues of SEQ ID NO: 17. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 190 consecutive amino acid residues of SEQ ID NO: 17, and has at least 95% sequence identity to the at least 190 consecutive amino acid residues of SEQ ID NO: 17. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 17, and has at least 95% sequence identity to the at least 200 consecutive amino acid residues of SEQ ID NO: 17. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 210 consecutive amino acid residues of SEQ ID NO: 17, and has at least 95% sequence identity to the at least 210 consecutive amino acid residues of SEQ ID NO: 17.

In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 215 consecutive amino acid residues of SEQ ID NO: 17, and has at least 95% sequence identity to the at least 215 consecutive amino acid residues of SEQ ID NO: 17. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 220 consecutive amino acid residues of SEQ ID NO: 17, and has at least 95% sequence identity to the at least 220 consecutive amino acid residues of SEQ ID NO: 17.

In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 175 consecutive amino acid residues of SEQ ID NO: 17, and has at least 99% sequence identity to the at least 175 consecutive amino acid residues of SEQ ID NO: 17. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 190 consecutive amino acid residues of SEQ ID NO: 17, and has at least 99% sequence identity to the at least 190 consecutive amino acid residues of SEQ ID NO: 17. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 17, and has at least 99% sequence identity to the at least 200 consecutive amino acid residues of SEQ ID NO: 17. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 210 consecutive amino acid residues of SEQ ID NO: 17, and has at least 99% sequence identity to the at least 210 consecutive amino acid residues of SEQ ID NO: 17.

In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 215 consecutive amino acid residues of SEQ ID NO: 17, and has at least 99% sequence identity to the at least 215 consecutive amino acid residues of SEQ ID NO: 17. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 220 consecutive amino acid residues of SEQ ID NO: 17, and has at least 99% sequence identity to the at least 220 consecutive amino acid residues of SEQ ID NO: 17.

In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 70% sequence identity to the amino acid sequence according to SEQ ID NO: 16. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 16. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 16. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 90% sequence identity to the amino acid sequence according to SEQ ID NO: 16. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 16. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 16. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 16. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 16. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 16. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence according to SEQ ID NO: 16.

In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 175 consecutive amino acid residues of SEQ ID NO: 16. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 190 consecutive amino acid residues of SEQ ID NO: 16. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 16. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 205 consecutive amino acid residues of SEQ ID NO: 16. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 210 consecutive amino acid residues of SEQ ID NO: 16.

In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 175 consecutive amino acid residues of SEQ ID NO: 16, and has at least 80% sequence identity to the at least 175 consecutive amino acid residues of SEQ ID NO: 16. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 190 consecutive amino acid residues of SEQ ID NO: 16, and has at least 80% sequence identity to the at least 190 consecutive amino acid residues of SEQ ID NO: 16. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 16, and has at least 80% sequence identity to the at least 200 consecutive amino acid residues of SEQ ID NO: 16. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 205 consecutive amino acid residues of SEQ ID NO: 16, and has at least 80% sequence identity to the at least 205 consecutive amino acid residues of SEQ ID NO: 16. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 210 consecutive amino acid residues of SEQ ID NO: 16, and has at least 80% sequence identity to the at least 210 consecutive amino acid residues of SEQ ID NO: 16.

In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 175 consecutive amino acid residues of SEQ ID NO: 16, and has at least 90% sequence identity to the at least 175 consecutive amino acid residues of SEQ ID NO: 16. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 190 consecutive amino acid residues of SEQ ID NO: 16, and has at least 90% sequence identity to the at least 190 consecutive amino acid residues of SEQ ID NO: 16. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 16, and has at least 90% sequence identity to the at least 200 consecutive amino acid residues of SEQ ID NO: 16. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 205 consecutive amino acid residues of SEQ ID NO: 16, and has at least 90% sequence identity to the at least 205 consecutive amino acid residues of SEQ ID NO: 16. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 210 consecutive amino acid residues of SEQ ID NO: 16, and has at least 90% sequence identity to the at least 210 consecutive amino acid residues of SEQ ID NO: 16.

In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 175 consecutive amino acid residues of SEQ ID NO: 16, and has at least 95% sequence identity to the at least 175 consecutive amino acid residues of SEQ ID NO: 16. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 190 consecutive amino acid residues of SEQ ID NO: 16, and has at least 95% sequence identity to the at least 190 consecutive amino acid residues of SEQ ID NO: 16. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 16, and has at least 95% sequence identity to the at least 200 consecutive amino acid residues of SEQ ID NO: 16. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 205 consecutive amino acid residues of SEQ ID NO: 16, and has at least 95% sequence identity to the at least 205 consecutive amino acid residues of SEQ ID NO: 16. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 210 consecutive amino acid residues of SEQ ID NO: 16, and has at least 95% sequence identity to the at least 210 consecutive amino acid residues of SEQ ID NO: 16.

In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 175 consecutive amino acid residues of SEQ ID NO: 16, and has at least 99% sequence identity to the at least 175 consecutive amino acid residues of SEQ ID NO: 16. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 190 consecutive amino acid residues of SEQ ID NO: 16, and has at least 99% sequence identity to the at least 190 consecutive amino acid residues of SEQ ID NO: 16. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 16, and has at least 99% sequence identity to the at least 200 consecutive amino acid residues of SEQ ID NO: 16. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 205 consecutive amino acid residues of SEQ ID NO: 16, and has at least 99% sequence identity to the at least 205 consecutive amino acid residues of SEQ ID NO: 16. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 210 consecutive amino acid residues of SEQ ID NO: 16, and has at least 99% sequence identity to the at least 210 consecutive amino acid residues of SEQ ID NO: 16.

In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 70% sequence identity to the amino acid sequence according to SEQ ID NO: 17; and the PD-L1 binding domain comprises an amino acid sequence that has at least 70% sequence identity to the amino acid sequence according to SEQ ID NO: 16. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 17; and the PD-L1 binding domain comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 16. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 17; and the PD-L1 binding domain comprises an amino acid sequence that has at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 16. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 90% sequence identity to the amino acid sequence according to SEQ ID NO: 17; and the PD-L1 binding domain comprises an amino acid sequence that has at least 90% sequence identity to the amino acid sequence according to SEQ ID NO: 16. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 17; and the PD-L1 binding domain comprises an amino acid sequence that has at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 16. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 17; and the PD-L1 binding domain comprises an amino acid sequence that has at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 16. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 17; and the PD-L1 binding domain comprises an amino acid sequence that has at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 16. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 17; and the PD-L1 binding domain comprises an amino acid sequence that has at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 16. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 17; and the PD-L1 binding domain comprises an amino acid sequence that has at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 16.

In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 70% sequence identity to the amino acid sequence according to SEQ ID NO: 43. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 43. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 43. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 90% sequence identity to the amino acid sequence according to SEQ ID NO: 43. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 43. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 43. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 43. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 43. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 43. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence according to SEQ ID NO: 43.

In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 175 consecutive amino acid residues of SEQ ID NO: 43, and has at least 95% sequence identity to the at least 175 consecutive amino acid residues of SEQ ID NO: 43. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 190 consecutive amino acid residues of SEQ ID NO: 43, and has at least 95% sequence identity to the at least 190 consecutive amino acid residues of SEQ ID NO: 43. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 43, and has at least 95% sequence identity to the at least 200 consecutive amino acid residues of SEQ ID NO: 43. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 210 consecutive amino acid residues of SEQ ID NO: 43, and has at least 95% sequence identity to the at least 210 consecutive amino acid residues of SEQ ID NO: 43.

In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 215 consecutive amino acid residues of SEQ ID NO: 43, and has at least 95% sequence identity to the at least 215 consecutive amino acid residues of SEQ ID NO: 43. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 220 consecutive amino acid residues of SEQ ID NO: 43, and has at least 95% sequence identity to the at least 220 consecutive amino acid residues of SEQ ID NO: 43.

In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 70% sequence identity to the amino acid sequence according to SEQ ID NO: 42. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 42. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 42. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 90% sequence identity to the amino acid sequence according to SEQ ID NO: 42. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 42. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 42. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 42. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 42. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 42. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence according to SEQ ID NO: 42.

In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 175 consecutive amino acid residues of SEQ ID NO: 42, and has at least 95% sequence identity to the at least 175 consecutive amino acid residues of SEQ ID NO: 42. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 190 consecutive amino acid residues of SEQ ID NO: 42, and has at least 95% sequence identity to the at least 190 consecutive amino acid residues of SEQ ID NO: 42. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 42, and has at least 95% sequence identity to the at least 200 consecutive amino acid residues of SEQ ID NO: 42. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 205 consecutive amino acid residues of SEQ ID NO: 42, and has at least 95% sequence identity to the at least 205 consecutive amino acid residues of SEQ ID NO: 42. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 210 consecutive amino acid residues of SEQ ID NO: 42, and has at least 95% sequence identity to the at least 210 consecutive amino acid residues of SEQ ID NO: 42.

In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 70% sequence identity to the amino acid sequence according to SEQ ID NO: 43; and the PD-L1 binding domain comprises an amino acid sequence that has at least 70% sequence identity to the amino acid sequence according to SEQ ID NO: 42. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 43; and the PD-L1 binding domain comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 42. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 43; and the PD-L1 binding domain comprises an amino acid sequence that has at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 42. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 90% sequence identity to the amino acid sequence according to SEQ ID NO: 43; and the PD-L1 binding domain comprises an amino acid sequence that has at least 90% sequence identity to the amino acid sequence according to SEQ ID NO: 42. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 43; and the PD-L1 binding domain comprises an amino acid sequence that has at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 42. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 43; and the PD-L1 binding domain comprises an amino acid sequence that has at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 42. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 43; and the PD-L1 binding domain comprises an amino acid sequence that has at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 42. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 43; and the PD-L1 binding domain comprises an amino acid sequence that has at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 42. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 43; and the PD-L1 binding domain comprises an amino acid sequence that has at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 42.

In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 70% sequence identity to the amino acid sequence according to SEQ ID NO: 45. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 45. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 45. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 90% sequence identity to the amino acid sequence according to SEQ ID NO: 45. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 45. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 45. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 45. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 45. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 45. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence according to SEQ ID NO: 45.

In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 175 consecutive amino acid residues of SEQ ID NO: 45, and has at least 95% sequence identity to the at least 175 consecutive amino acid residues of SEQ ID NO: 45. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 190 consecutive amino acid residues of SEQ ID NO: 45, and has at least 95% sequence identity to the at least 190 consecutive amino acid residues of SEQ ID NO: 45. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 45, and has at least 95% sequence identity to the at least 200 consecutive amino acid residues of SEQ ID NO: 45. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 210 consecutive amino acid residues of SEQ ID NO: 45, and has at least 95% sequence identity to the at least 210 consecutive amino acid residues of SEQ ID NO: 45.

In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 215 consecutive amino acid residues of SEQ ID NO: 45, and has at least 95% sequence identity to the at least 215 consecutive amino acid residues of SEQ ID NO: 45. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 220 consecutive amino acid residues of SEQ ID NO: 45, and has at least 95% sequence identity to the at least 220 consecutive amino acid residues of SEQ ID NO: 45.

In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 70% sequence identity to the amino acid sequence according to SEQ ID NO: 44. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 44. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 44. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 90% sequence identity to the amino acid sequence according to SEQ ID NO: 44. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 44. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 44. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 44. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 44. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 44. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence according to SEQ ID NO: 44.

In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 175 consecutive amino acid residues of SEQ ID NO: 44, and has at least 95% sequence identity to the at least 175 consecutive amino acid residues of SEQ ID NO: 44. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 190 consecutive amino acid residues of SEQ ID NO: 44, and has at least 95% sequence identity to the at least 190 consecutive amino acid residues of SEQ ID NO: 44. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 44, and has at least 95% sequence identity to the at least 200 consecutive amino acid residues of SEQ ID NO: 44. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 205 consecutive amino acid residues of SEQ ID NO: 44, and has at least 95% sequence identity to the at least 205 consecutive amino acid residues of SEQ ID NO: 44. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 210 consecutive amino acid residues of SEQ ID NO: 44, and has at least 95% sequence identity to the at least 210 consecutive amino acid residues of SEQ ID NO: 44.

In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 70% sequence identity to the amino acid sequence according to SEQ ID NO: 45; and the PD-L1 binding domain comprises an amino acid sequence that has at least 70% sequence identity to the amino acid sequence according to SEQ ID NO: 44. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 45; and the PD-L1 binding domain comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 44. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 45; and the PD-L1 binding domain comprises an amino acid sequence that has at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 44. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 90% sequence identity to the amino acid sequence according to SEQ ID NO: 45; and the PD-L1 binding domain comprises an amino acid sequence that has at least 90% sequence identity to the amino acid sequence according to SEQ ID NO: 44. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 45; and the PD-L1 binding domain comprises an amino acid sequence that has at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 44. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 45; and the PD-L1 binding domain comprises an amino acid sequence that has at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 44. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 45; and the PD-L1 binding domain comprises an amino acid sequence that has at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 44. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 45; and the PD-L1 binding domain comprises an amino acid sequence that has at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 44. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 45; and the PD-L1 binding domain comprises an amino acid sequence that has at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 44.

In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 70% sequence identity to the amino acid sequence according to SEQ ID NO: 47. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 47. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 47. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 90% sequence identity to the amino acid sequence according to SEQ ID NO: 47. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 47. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 47. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 47. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 47. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 47. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence according to SEQ ID NO: 47.

In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 175 consecutive amino acid residues of SEQ ID NO: 47, and has at least 95% sequence identity to the at least 175 consecutive amino acid residues of SEQ ID NO: 47. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 190 consecutive amino acid residues of SEQ ID NO: 47, and has at least 95% sequence identity to the at least 190 consecutive amino acid residues of SEQ ID NO: 47. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 47, and has at least 95% sequence identity to the at least 200 consecutive amino acid residues of SEQ ID NO: 47. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 210 consecutive amino acid residues of SEQ ID NO: 47, and has at least 95% sequence identity to the at least 210 consecutive amino acid residues of SEQ ID NO: 47.

In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 215 consecutive amino acid residues of SEQ ID NO: 47, and has at least 95% sequence identity to the at least 215 consecutive amino acid residues of SEQ ID NO: 47. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 220 consecutive amino acid residues of SEQ ID NO: 47, and has at least 95% sequence identity to the at least 220 consecutive amino acid residues of SEQ ID NO: 47.

In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 70% sequence identity to the amino acid sequence according to SEQ ID NO: 46. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 46. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 46. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 90% sequence identity to the amino acid sequence according to SEQ ID NO: 46. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 46. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 46. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 46. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 46. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 46. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence according to SEQ ID NO: 46.

In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 175 consecutive amino acid residues of SEQ ID NO: 46, and has at least 95% sequence identity to the at least 175 consecutive amino acid residues of SEQ ID NO: 46. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 190 consecutive amino acid residues of SEQ ID NO: 46, and has at least 95% sequence identity to the at least 190 consecutive amino acid residues of SEQ ID NO: 46. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 46, and has at least 95% sequence identity to the at least 200 consecutive amino acid residues of SEQ ID NO: 46. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 205 consecutive amino acid residues of SEQ ID NO: 46, and has at least 95% sequence identity to the at least 205 consecutive amino acid residues of SEQ ID NO: 46. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence of at least 210 consecutive amino acid residues of SEQ ID NO: 46, and has at least 95% sequence identity to the at least 210 consecutive amino acid residues of SEQ ID NO: 46.

In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 70% sequence identity to the amino acid sequence according to SEQ ID NO: 47; and the PD-L1 binding domain comprises an amino acid sequence that has at least 70% sequence identity to the amino acid sequence according to SEQ ID NO: 46. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 47; and the PD-L1 binding domain comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 46. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 47; and the PD-L1 binding domain comprises an amino acid sequence that has at least 85% sequence identity to the amino acid sequence according to SEQ ID NO: 46. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 90% sequence identity to the amino acid sequence according to SEQ ID NO: 47; and the PD-L1 binding domain comprises an amino acid sequence that has at least 90% sequence identity to the amino acid sequence according to SEQ ID NO: 46. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 47; and the PD-L1 binding domain comprises an amino acid sequence that has at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 46. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 47; and the PD-L1 binding domain comprises an amino acid sequence that has at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 46. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 47; and the PD-L1 binding domain comprises an amino acid sequence that has at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 46. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 47; and the PD-L1 binding domain comprises an amino acid sequence that has at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 46. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that has at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 47; and the PD-L1 binding domain comprises an amino acid sequence that has at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 46.

Linker

In some embodiments, the linker is at least 5 amino acids in length. In some embodiments, the linker is no more than 30 amino acids in length. In some embodiments, the linker is at least 5 amino acids and no more than 30 amino acids in length. In some embodiments, the linker is 5 amino acids in length. In some embodiments, the linker is 6 amino acids in length. In some embodiments, the linker is 7 amino acids in length. In some embodiments, the linker is 8 amino acids in length. In some embodiments, the linker is 9 amino acids in length. In some embodiments, the linker is 10 amino acids in length. In some embodiments, the linker is 11 amino acids in length. In some embodiments, the linker is 12 amino acids in length. In some embodiments, the linker is 13 amino acids in length. In some embodiments, the linker is 14 amino acids in length. In some embodiments, the linker is 15 amino acids in length. In some embodiments, the linker is 16 amino acids in length. In some embodiments, the linker is 17 amino acids in length. In some embodiments, the linker is 18 amino acids in length. In some embodiments, the linker is 19 amino acids in length. In some embodiments, the linker is 20 amino acids in length. In some embodiments, the linker is 21 amino acids in length. In some embodiments, the linker is 22 amino acids in length. In some embodiments, the linker is 23 amino acids in length. In some embodiments, the linker is 24 amino acids in length. In some embodiments, the linker is 25 amino acids in length. In some embodiments, the linker is 26 amino acids in length. In some embodiments, the linker is 27 amino acids in length. In some embodiments, the linker is 28 amino acids in length. In some embodiments, the linker is 29 amino acids in length. In some embodiments, the linker is 30 amino acids in length. In some embodiments, the linker comprises an amino acid sequence of SEQ ID NO: 18 (GGGGSGGGGSGGGGS) or SEQ ID NO: 19 (GGGGS). In some embodiments, the linker comprises an amino acid sequence of SEQ ID NO: 18 (GGGGSGGGGSGGGGS). In some embodiments, the linker comprises an amino acid sequence of SEQ ID NO: 19 (GGGGS).

In some embodiments, the linker has a formula selected from the group consisting of (G₂S)_n, (GS)_n, (GSGGS)_n(SEQ ID NO: 58), (GGGS)_n(SEQ ID NO: 59), (GGGGS)_n(SEQ ID NO: 60), and (GSSGGS)_n(SEQ ID NO: 61), wherein n is an integer of at least 1. In some embodiments, the linker has a formula selected from the group consisting of (G₂S)_n, (GS)_n, (GSGGS)_n(SEQ ID NO: 234), (GGGS)_n(SEQ ID NO: 235), (GGGGS)_n(SEQ ID NO: 236), and (GSSGGS)_n(SEQ ID NO: 237), wherein n is an integer of 1. In some embodiments, the linker has a formula selected from the group consisting of (G₂S)_n(SEQ ID NO: 233), (GS)_n(SEQ ID NO: 238), (GSGGS)_n(SEQ ID NO: 239), (GGGS)_n(SEQ ID NO: 240), (GGGGS)_n(SEQ ID NO: 241), and (GSSGGS)_n(SEQ ID NO: 242), wherein n is an integer from 1 to 3. In some embodiments, the linker has a formula selected from the group consisting of (G₂S)_n(SEQ ID NO: 243), (GS)_n(SEQ ID NO: 244), (GSGGS)_n(SEQ ID NO: 245), (GGGS)_n(SEQ ID NO: 246), (GGGGS)_n(SEQ ID NO: 247), and (GSSGGS)_n(SEQ ID NO: 248), wherein n is an integer from 1 to 10.

In some embodiments, the linker has a formula of (G₂S)_n, wherein n is an integer of least 1. In some embodiments, the linker has a formula of (GS)_n, wherein n is an integer of least 1. In some embodiments, the linker has a formula of (GSGGS)_n(SEQ ID NO: 58), wherein n is an integer of least 1. In some embodiments, the linker has a formula of (GGGS)_n(SEQ ID NO: 59), wherein n is an integer of least 1. In some embodiments, the linker has a formula of (GGGGS)_n(SEQ ID NO: 60), wherein n is an integer of least 1. In some embodiments, the linker has a formula of (GSSGGS)_n(SEQ ID NO: 61), wherein n is an integer of least 1.

In some embodiments, the linker has a formula of (G₂S)_n, wherein n is an integer of 1. In some embodiments, L₁has a formula of (GS)_n, wherein n is an integer of 1. In some embodiments, the linker has a formula of (GSGGS)_n(SEQ ID NO: 234), wherein n is an integer of 1. In some embodiments, the linker has a formula of (GGGS)_n(SEQ ID NO: 235), wherein n is an integer of 1. In some embodiments, the linker has a formula of (GGGGS)_n(SEQ ID NO: 236), wherein n is an integer of 1. In some embodiments, the linker has a formula of (GSSGGS)_n(SEQ ID NO: 237), wherein n is an integer of 1.

In some embodiments, the linker has a formula of (G₂S)_n(SEQ ID NO: 233), wherein n is an integer from 1 to 3. In some embodiments, the linker has a formula of (GS)_n(SEQ ID NO: 238), wherein n is an integer from 1 to 3. In some embodiments, the linker has a formula of (GSGGS)_n(SEQ ID NO: 239), wherein n is an integer from 1 to 3. In some embodiments, the linker has a formula of (GGGS)_n(SEQ ID NO: 240), wherein n is an integer from 1 to 3. In some embodiments, the linker has a formula of (GGGGS)_n(SEQ ID NO: 241), wherein n is an integer from 1 to 3. In some embodiments, the linker has a formula of (GSSGGS)_n(SEQ ID NO: 242), wherein n is an integer from 1 to 3.

In some embodiments, the linker has a formula of (G₂S)_n(SEQ ID NO: 243), wherein n is an integer from 1 to 10. In some embodiments, the linker has a formula of (GS)_n(SEQ ID NO: 244), wherein n is an integer from 1 to 10. In some embodiments, the linker has a formula of (GSGGS)_n(SEQ ID NO: 245), wherein n is an integer from 1 to 10. In some embodiments, the linker has a formula of (GGGS)_n(SEQ ID NO: 246), wherein n is an integer from 1 to 10. In some embodiments, the linker has a formula of (GGGGS)_n(SEQ ID NO: 247), wherein n is an integer from 1 to 10. In some embodiments, the linker has a formula of (GSSGGS)_n(SEQ ID NO: 248), wherein n is an integer from 1 to 10.

TABLE 7

Linker sequences

Construct
Amino Acid Sequence
SEQ ID

Description
(N to C)
NO:

Linker 1
GGGGSGGGGSGGGGS
18

Linker 2
GGGGS
19

In some embodiments, the linker connects the C-terminus of A to the N-terminus of the Fab light chain polypeptide. In some embodiments, the linker connects the N-terminus of A to the C-terminus of the Fab light chain polypeptide. In some embodiments, the linker connects the Fab light chain polypeptide to the scFv light chain variable domain. In some embodiments, the linker connects the Fab light chain polypeptide to the scFv heavy chain variable domain. In some embodiments, the linker connects the Fab heavy chain polypeptide to the scFv light chain variable domain. In some embodiments, the linker connects the Fab heavy chain polypeptide to the scFv heavy chain variable domain. In some embodiments, the linker connects the Fab light chain polypeptide to the N-terminus of the scFv light chain variable domain. In some embodiments, the linker connects the Fab light chain polypeptide to the C-terminus of the scFv light chain variable domain. In some embodiments, the linker connects the Fab light chain polypeptide to the N-terminus of the scFv heavy chain variable domain. In some embodiments, the linker connects the Fab light chain polypeptide to the C-terminus of the scFv heavy chain variable domain. In some embodiments, the linker connects the Fab heavy chain polypeptide to the N-terminus of the scFv light chain variable domain.

In some embodiments, the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain. In some embodiments, the linker connects the Fab heavy chain polypeptide to the N-terminus of the scFv heavy chain variable domain. In some embodiments, the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv heavy chain variable domain.

Multispecific Antibodies that Bind to CD28 and PD-L1

In some embodiments, the multispecific antibody comprising a CD28 binding domain and a PD-L1 binding domain comprises a variety of multispecific antibody formats. In some embodiments, the multispecific antibody comprises a CD28 binding domain and a PD-L1 binding domain, wherein when the CD28 binding domain is a single chain variable fragment (scFv), then the PD-L1 binding domain is not a scFv.

In some embodiments, the multispecific antibody further comprises a fragment crystallizable (Fc) region. In some embodiments, the Fc region comprises an IgG CH2 domain and an IgG CH3 domain. In some embodiments, the Fc region comprises a heterodimeric Fc region. In some embodiments, the Fc region comprises at least one amino acid modification that increases the half-life of the multispecific antibody. In some embodiments, the Fc region comprises at least one amino acid modification that modulates its interaction with an Fc receptor. In some embodiments, the Fc region comprises at least one amino acid modification that increases binding of the Fc region to an Fc receptor. In some embodiments, the Fc region comprises at least one amino acid modification that decreases glycosylation of the Fc region. In some embodiments, the modification is an amino acid substitution, deletion, or addition. In some embodiments, the modification is an amino acid substitution. In some embodiments, the at least one amino acid modification that decreases glycosylation of the Fc region comprises an amino acid substitution at a position corresponding to position N297 of human IgG1, wherein the numbering is according to the EU index of Kabat. In some embodiments, the Fc region is afucosylated.

In some embodiments, the multispecific antibody is assembled from at least two different heavy and light chains expressed in the same producer cell. In some embodiments, the multispecific antibody is produced using knobs-into-holes technology to force heavy-chain heterodimerization in which mutations are introduced into the two CH3 domains.

In some embodiments, the multispecific antibody lacks a fragment crystallizable (Fc) region. In some embodiments, two or more different antibodies are linked together to form the multispecific antibody.

In some embodiments, two different antibodies are linked together to form the multispecific antibody. For example, the PD-L1 binding domain is a Fab or Fab′ and is linked to the CD28 binding domain that is a scFv, another Fab or Fab′, or a single domain antibody. In some embodiments, the CD28 binding domain is a scFv and is linked to the PD-L1 binding domain that is Fab or Fab′ or a single domain antibody. In some embodiments, the PD-L1 binding domain is a single domain antibody and is linked to the CD28 binding domain that is another single domain antibody. In some embodiments, the PD-L1 binding domain is a Fab or Fab′ and is linked to the CD28 binding domain that is another Fab or Fab′. In some embodiments, the PD-L1 binding domain is a Fab or Fab′ and is linked to the CD28 binding domain that is a single domain antibody. In some embodiments, the PD-L1 binding domain is a Fab or Fab′ and is linked to the CD28 binding domain that is a scFv. In some embodiments, the PD-L1 binding domain is a single domain antibody and is linked to the CD28 binding domain that is a Fab or Fab′. In some embodiments, the PD-L1 binding domain is a single domain antibody and is linked to the CD28 binding domain that is a scFv. In some embodiments, the PD-L1 binding domain is a scFv and is linked to the CD28 binding domain that is a Fab or Fab′. In some embodiments, the PD-L1 binding domain is a scFv and is linked to the CD28 binding domain that is a single domain antibody. In some embodiments, the PD-L1 binding domain is a scFv and is linked to the CD28 binding domain that is a scFv.

In some embodiments, the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab light chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 20, and an amino acid sequence of the Fab heavy chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence to SEQ ID NO: 21. In some embodiments, the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab light chain polypeptide comprises an amino acid sequence that has at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 20, and an amino acid sequence of the Fab heavy chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence that has at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 21. In some embodiments, the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab light chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 20, and an amino acid sequence of the Fab heavy chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence to SEQ ID NO: 21. In some embodiments, the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab light chain polypeptide comprises an amino acid sequence that has at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 20, and an amino acid sequence of the Fab heavy chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence that has at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 21. In some embodiments, the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab light chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 20, and an amino acid sequence of the Fab heavy chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence to SEQ ID NO: 21. In some embodiments, the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab light chain polypeptide comprises an amino acid sequence that has at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 20, and an amino acid sequence of the Fab heavy chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence that has at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 21.

In some embodiments, the linker connects the Fab light chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 23, and an amino acid sequence of the Fab light chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence that has at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 22. In some embodiments, the linker connects the Fab light chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 23, and an amino acid sequence of the Fab light chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence that has at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 22. In some embodiments, the linker connects the Fab light chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 23, and an amino acid sequence of the Fab light chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence that has at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 22. In some embodiments, the linker connects the Fab light chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 23, and an amino acid sequence of the Fab light chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence that has at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 22. In some embodiments, the linker connects the Fab light chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 23, and an amino acid sequence of the Fab light chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence that has at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 22. In some embodiments, the linker connects the Fab light chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab heavy chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 23, and an amino acid sequence of the Fab light chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence to SEQ ID NO: 22.

In some embodiments, the linker connects the Fab light chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab heavy chain polypeptide comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 23 and has at least 80% sequence identity to the at least 200 consecutive amino acid residues of SEQ ID NO: 23 and an amino acid sequence of the Fab light chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence of at least 450 consecutive amino acid residues of SEQ ID NO: 22 and has at least 80% sequence identity to the at least 450 consecutive amino acid residues of SEQ ID NO: 22.

In some embodiments, the linker connects the Fab light chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab heavy chain polypeptide comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 23 and has at least 90% sequence identity to the at least 200 consecutive amino acid residues of SEQ ID NO: 23 and an amino acid sequence of the Fab light chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence of at least 450 consecutive amino acid residues of SEQ ID NO: 22 and has at least 90% sequence identity to the at least 450 consecutive amino acid residues of SEQ ID NO: 22.

In some embodiments, the linker connects the Fab light chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab heavy chain polypeptide comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 23 and has at least 95% sequence identity to the at least 200 consecutive amino acid residues of SEQ ID NO: 23 and an amino acid sequence of the Fab light chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence of at least 450 consecutive amino acid residues of SEQ ID NO: 22 and has at least 95% sequence identity to the at least 450 consecutive amino acid residues of SEQ ID NO: 22.

In some embodiments, the linker connects the Fab light chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab heavy chain polypeptide comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 23 and has at least 96% sequence identity to the at least 200 consecutive amino acid residues of SEQ ID NO: 23 and an amino acid sequence of the Fab light chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence of at least 450 consecutive amino acid residues of SEQ ID NO: 22 and has at least 96% sequence identity to the at least 450 consecutive amino acid residues of SEQ ID NO: 22.

In some embodiments, the linker connects the Fab light chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab heavy chain polypeptide comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 23 and has at least 97% sequence identity to the at least 200 consecutive amino acid residues of SEQ ID NO: 23 and an amino acid sequence of the Fab light chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence of at least 450 consecutive amino acid residues of SEQ ID NO: 22 and has at least 97% sequence identity to the at least 450 consecutive amino acid residues of SEQ ID NO: 22.

In some embodiments, the linker connects the Fab light chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab heavy chain polypeptide comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 23 and has at least 98% sequence identity to the at least 200 consecutive amino acid residues of SEQ ID NO: 23 and an amino acid sequence of the Fab light chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence of at least 450 consecutive amino acid residues of SEQ ID NO: 22 and has at least 98% sequence identity to the at least 450 consecutive amino acid residues of SEQ ID NO: 22.

In some embodiments, the linker connects the Fab light chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab heavy chain polypeptide comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 23 and has at least 99% sequence identity to the at least 200 consecutive amino acid residues of SEQ ID NO: 23 and an amino acid sequence of the Fab light chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence of at least 450 consecutive amino acid residues of SEQ ID NO: 22 and has at least 99% sequence identity to the at least 450 consecutive amino acid residues of SEQ ID NO: 22.

In some embodiments, the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab light chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 42, and an amino acid sequence of the Fab heavy chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence to SEQ ID NO: 48. In some embodiments, the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab light chain polypeptide comprises an amino acid sequence that has at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 42, and an amino acid sequence of the Fab heavy chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence that has at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 48. In some embodiments, the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab light chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 42, and an amino acid sequence of the Fab heavy chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence to SEQ ID NO: 48. In some embodiments, the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab light chain polypeptide comprises an amino acid sequence that has at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 42, and an amino acid sequence of the Fab heavy chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence that has at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 48. In some embodiments, the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab light chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 42, and an amino acid sequence of the Fab heavy chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence to SEQ ID NO: 48. In some embodiments, the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab light chain polypeptide comprises an amino acid sequence that has at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 42, and an amino acid sequence of the Fab heavy chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence that has at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 48.

In some embodiments, the linker connects the Fab light chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 43, and an amino acid sequence of the Fab light chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence that has at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 49. In some embodiments, the linker connects the Fab light chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 43, and an amino acid sequence of the Fab light chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence that has at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 49. In some embodiments, the linker connects the Fab light chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 43, and an amino acid sequence of the Fab light chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence that has at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 49. In some embodiments, the linker connects the Fab light chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 43, and an amino acid sequence of the Fab light chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence that has at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 49. In some embodiments, the linker connects the Fab light chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 43, and an amino acid sequence of the Fab light chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence that has at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 49. In some embodiments, the linker connects the Fab light chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab heavy chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 43, and an amino acid sequence of the Fab light chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence to SEQ ID NO: 49.

In some embodiments, the linker connects the Fab light chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab heavy chain polypeptide comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 43 and has at least 95% sequence identity to the at least 200 consecutive amino acid residues of SEQ ID NO: 43 and an amino acid sequence of the Fab light chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence of at least 450 consecutive amino acid residues of SEQ ID NO: 49 and has at least 95% sequence identity to the at least 450 consecutive amino acid residues of SEQ ID NO: 49.

In some embodiments, the linker connects the Fab light chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab heavy chain polypeptide comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 43 and has at least 99% sequence identity to the at least 200 consecutive amino acid residues of SEQ ID NO: 43 and an amino acid sequence of the Fab light chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence of at least 450 consecutive amino acid residues of SEQ ID NO: 49 and has at least 99% sequence identity to the at least 450 consecutive amino acid residues of SEQ ID NO: 49.

In some embodiments, the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab light chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 44, and an amino acid sequence of the Fab heavy chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence to SEQ ID NO: 50. In some embodiments, the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab light chain polypeptide comprises an amino acid sequence that has at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 44, and an amino acid sequence of the Fab heavy chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence that has at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 50. In some embodiments, the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab light chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 44, and an amino acid sequence of the Fab heavy chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence to SEQ ID NO: 50. In some embodiments, the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab light chain polypeptide comprises an amino acid sequence that has at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 44, and an amino acid sequence of the Fab heavy chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence that has at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 50. In some embodiments, the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab light chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 44, and an amino acid sequence of the Fab heavy chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence to SEQ ID NO: 50. In some embodiments, the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab light chain polypeptide comprises an amino acid sequence that has at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 44, and an amino acid sequence of the Fab heavy chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence that has at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 50.

In some embodiments, the linker connects the Fab light chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 45, and an amino acid sequence of the Fab light chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence that has at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 51. In some embodiments, the linker connects the Fab light chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 45, and an amino acid sequence of the Fab light chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence that has at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 51. In some embodiments, the linker connects the Fab light chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 45, and an amino acid sequence of the Fab light chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence that has at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 51. In some embodiments, the linker connects the Fab light chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 45, and an amino acid sequence of the Fab light chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence that has at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 51. In some embodiments, the linker connects the Fab light chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 45, and an amino acid sequence of the Fab light chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence that has at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 51. In some embodiments, the linker connects the Fab light chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab heavy chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 45, and an amino acid sequence of the Fab light chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence to SEQ ID NO: 51.

In some embodiments, the linker connects the Fab light chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab heavy chain polypeptide comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 45 and has at least 95% sequence identity to the at least 200 consecutive amino acid residues of SEQ ID NO: 45 and an amino acid sequence of the Fab light chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence of at least 450 consecutive amino acid residues of SEQ ID NO: 51 and has at least 95% sequence identity to the at least 450 consecutive amino acid residues of SEQ ID NO: 51.

In some embodiments, the linker connects the Fab light chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab heavy chain polypeptide comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 45 and has at least 99% sequence identity to the at least 200 consecutive amino acid residues of SEQ ID NO: 45 and an amino acid sequence of the Fab light chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence of at least 450 consecutive amino acid residues of SEQ ID NO: 51 and has at least 99% sequence identity to the at least 450 consecutive amino acid residues of SEQ ID NO: 51.

In some embodiments, the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab light chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 46, and an amino acid sequence of the Fab heavy chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence to SEQ ID NO: 52. In some embodiments, the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab light chain polypeptide comprises an amino acid sequence that has at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 46, and an amino acid sequence of the Fab heavy chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence that has at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 52. In some embodiments, the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab light chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 46, and an amino acid sequence of the Fab heavy chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence to SEQ ID NO: 52. In some embodiments, the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab light chain polypeptide comprises an amino acid sequence that has at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 46, and an amino acid sequence of the Fab heavy chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence that has at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 52. In some embodiments, the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab light chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 46, and an amino acid sequence of the Fab heavy chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence to SEQ ID NO: 52. In some embodiments, the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab light chain polypeptide comprises an amino acid sequence that has at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 46, and an amino acid sequence of the Fab heavy chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence that has at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 52.

In some embodiments, the linker connects the Fab light chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 47, and an amino acid sequence of the Fab light chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence that has at least 95% sequence identity to the amino acid sequence according to SEQ ID NO: 53. In some embodiments, the linker connects the Fab light chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 47, and an amino acid sequence of the Fab light chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence that has at least 96% sequence identity to the amino acid sequence according to SEQ ID NO: 53. In some embodiments, the linker connects the Fab light chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 47, and an amino acid sequence of the Fab light chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence that has at least 97% sequence identity to the amino acid sequence according to SEQ ID NO: 53. In some embodiments, the linker connects the Fab light chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 47, and an amino acid sequence of the Fab light chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence that has at least 98% sequence identity to the amino acid sequence according to SEQ ID NO: 53. In some embodiments, the linker connects the Fab light chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 47, and an amino acid sequence of the Fab light chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence that has at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 53. In some embodiments, the linker connects the Fab light chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab heavy chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 47, and an amino acid sequence of the Fab light chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence to SEQ ID NO: 53.

In some embodiments, the linker connects the Fab light chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab heavy chain polypeptide comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 47 and has at least 95% sequence identity to the at least 200 consecutive amino acid residues of SEQ ID NO: 47 and an amino acid sequence of the Fab light chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence of at least 450 consecutive amino acid residues of SEQ ID NO: 53 and has at least 95% o sequence identity to the at least 450 consecutive amino acid residues of SEQ ID NO: 53.

In some embodiments, the linker connects the Fab light chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab heavy chain polypeptide comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 47 and has at least 9900 sequence identity to the at least 200 consecutive amino acid residues of SEQ ID NO: 47 and an amino acid sequence of the Fab light chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence of at least 450 consecutive amino acid residues of SEQ ID NO: 53 and has at least 99% o sequence identity to the at least 450 consecutive amino acid residues of SEQ ID NO: 53.

TABLE 8

Antibody sequences that bind to CD28 and PD-L1. CDR sequences are

underlined and were determined using IMGT definition

Amino Acid Sequence
SEQ ID

Construct Description
(N to C)
NO:

Ab-1 LC
EIVLTQSPATLSLSPGERATLSC
20

Anti-PDL1 Fab LC
RASQSVSSYLAWYQQKPGQA

PRLLIYDASNRATGIPARFSGS

GSGTDFTLTISSLEPEDFAVYY

CQQRSNWPTFGQGTKVEIKR

TVAAPSVFIFPPSDEQLKSGTA

SVVCLLNNFYPREAKVQWKV

DNALQSGNSQESVTEQDSKDS

TYSLSSTLTLSKADYEKHKVY

ACEVTHQGLSSPVTKSFNRGE

C

Ab-1 HC
QVQLVQSGAEVKKPGASVKV
21

Anti-CD28 scFv-Linker 2-
SCKASGYTFTSYYIHWVRQAP

anti-PDL1 Fab HC
GQGLEWIGSIYPGNVNTNYNE

KFKDRATLTVDTSISTAYMEL

SRLRSDDTAVYFCTRSHYGLD

WNFDV
WGQGTTVTVSSGGG

GSGGGGSGGGGSDIQMTQSPS

SLSASVGDRVTITCHASQNIYV

W
LNWYQQKPGKAPKLLIYKA

SNLHTGVPSRFSGSGSGTDFTL

TISSLQPEDFATYYCQQGQTY

PYT
FGGGTKVEIKGGGGSQVQ

LVQSGAEVKKPGSSVKVSCKT

SGDTESTYAISWVRQAPGQGL

EWMGGIIPIFGKAHYAQKFQ

GRVTITADESTSTAYMELSSLR

SEDTAVYFCARKFHFVSGSPF

GMDV
WGQGTTVTVSSASTKG

PSVFPLAPSSKSTSGGTAALGC

LVKDYFPEPVTVSWNSGALTS

GVHTFPAVLQSSGLYSLSSVV

TVPSSSLGTQTYICNVNHKPSN

TKVDKKVEPKSC

Ab-2 LC
QVQLVQSGAEVKKPGASVKV
22

Anti-CD28 scFv-Linker 2-
SCKASGYTFTSYYIHWVRQAP

Anti-PDL1 Fab LC
GQGLEWIGSIYPGNVNTNYNE

KFKDRATLTVDTSISTAYMEL

SRLRSDDTAVYFCTRSHYGLD

WNFDV
WGQGTTVTVSSGGG

GSGGGGSGGGGSDIQMTQSPS

SLSASVGDRVTITCHASQNIYV

W
LNWYQQKPGKAPKLLIYKA

SNLHTGVPSRFSGSGSGTDFTL

TISSLQPEDFATYYCQQGQTY

PYT
FGGGTKVEIKGGGGSEIV

LTQSPATLSLSPGERATLSCRA

SQSVSSYLAWYQQKPGQAPR

LLIYDASNRATGIPARFSGSGS

GTDFTLTISSLEPEDFAVYYCQ

QRSNWPT
FGQGTKVEIKRTV

AAPSVFIFPPSDEQLKSGTASV

VCLLNNFYPREAKVQWKVDN

ALQSGNSQESVTEQDSKDSTY

SLSSTLTLSKADYEKHKVYAC

EVTHQGLSSPVTKSFNRGEC

Ab-2 HC
QVQLVQSGAEVKKPGSSVKVS
23

Anti-PDL1 Fab HC
CKTSGDTFSTYAISWVRQAPG

QGLEWMGGIIPIFGKAHYAQ

KFQGRVTITADESTSTAYMEL

SSLRSEDTAVYFCARKFHFVS

GSPFGMDV
WGQGTTVTVSSA

STKGPSVFPLAPSSKSTSGGTA

ALGCLVKDYFPEPVTVSWNSG

ALTSGVHTFPAVLQSSGLYSLS

SVVTVPSSSLGTQTYICNVNH

KPSNTKVDKKVEPKSC

Ab-3 LC
DIQMTQSPSSLSASVGDRVTIT
42

Anti-PDL1 Fab 2 LC
CRASQDVSTAVAWYQQKPGK

APKLLIYSASFLYSGVPSRFSG

SGSGTDFTLTISSLQPEDFATY

YCQQYLYHPATFGQGTKVEI

KRTVAAPSVFIFPPSDEQLKSG

TASVVCLLNNFYPREAKVQW

KVDNALQSGNSQESVTEQDSK

DSTYSLSSTLTLSKADYEKHK

VYACEVTHQGLSSPVTKSFNR

GEC

Ab-3 HC
QVQLVQSGAEVKKPGASVKV
48

Anti-CD28 scFv-Linker 2-
SCKASGYTFTSYYIHWVRQAP

anti-PDL1 Fab 2 HC
GQGLEWIGSIYPGNVNTNYNE

KFKDRATLTVDTSISTAYMEL

SRLRSDDTAVYFCTRSHYGLD

WNFDV
WGQGTTVTVSSGGG

GSGGGGSGGGGSDIQMTQSPS

SLSASVGDRVTITCHASQNIYV

W
LNWYQQKPGKAPKLLIYKA

SNLHTGVPSRFSGSGSGTDFTL

TISSLQPEDFATYYCQQGQTY

PYT
FGGGTKVEIKGGGGSEVQ

LVESGGGLVQPGGSLRLSCAA

SGFTFSDSWIHWVRQAPGKG

LEWVAWISPYGGSTYYADSV

KGRFTISADTSKNTAYLQMNS

LRAEDTAVYYCARRHWPGG

FDY
WGQGTLVTVSSASTKGPS

VFPLAPSSKSTSGGTAALGCLV

KDYFPEPVTVSWNSGALTSGV

HTFPAVLQSSGLYSLSSVVTVP

SSSLGTQTYICNVNHKPSNTK

VDKKVEPKSC

Ab-4 LC
QVQLVQSGAEVKKPGASVKV
49

Anti-CD28 scFv-Linker 2-
SCKASGYTFTSYYIHWVRQAP

Anti-PDL1 Fab 2 LC
GQGLEWIGSIYPGNVNTNYNE

KFKDRATLTVDTSISTAYMEL

SRLRSDDTAVYFCTRSHYGLD

WNFDV
WGQGTTVTVSSGGG

GSGGGGSGGGGSDIQMTQSPS

SLSASVGDRVTITCHASQNIYV

W
LNWYQQKPGKAPKLLIYKA

SNLHTGVPSRFSGSGSGTDFTL

TISSLQPEDFATYYCQQGQTY

PYT
FGGGTKVEIKGGGGSDIQ

MTQSPSSLSASVGDRVTITCRA

SQDVSTAVAWYQQKPGKAPK

LLIYSASFLYSGVPSRFSGSGS

GTDFTLTISSLQPEDFATYYCQ

QYLYHPAT
FGQGTKVEIKRTV

AAPSVFIFPPSDEQLKSGTASV

VCLLNNFYPREAKVQWKVDN

ALQSGNSQESVTEQDSKDSTY

SLSSTLTLSKADYEKHKVYAC

EVTHQGLSSPVTKSFNRGEC

Ab-4 HC
EVQLVESGGGLVQPGGSLRLS
43

Anti-PDL1 Fab 2 HC
CAASGFTFSDSWIHWVRQAP

GKGLEWVAWISPYGGSTYYA

DSVKGRFTISADTSKNTAYLQ

MNSLRAEDTAVYYCARRHW

PGGFDY
WGQGTLVTVSSAST

KGPSVFPLAPSSKSTSGGTAAL

GCLVKDYFPEPVTVSWNSGAL

TSGVHTFPAVLQSSGLYSLSSV

VTVPSSSLGTQTYICNVNHKPS

NTKVDKKVEPKSC

Ab-5 LC
QSALTQPASVSGSPGQSITISCT
44

Anti-PDL1 Fab 3 LC
GTSSDVGGYNYVSWYQQHPG

KAPKLMIYDVSNRPSGVSNRF

SGSKSGNTASLTISGLQAEDEA

DYYCSSYTSSSTRVFGTGTKV

TVLGQP
KANPTVTLFPPSSEEL

QANKATLVCLISDFYPGAVTV

AWKADGSPVKAGVETTKPSK

QSNNKYAASSYLSLTPEQWKS

HRSYSCQVTHEGSTVEKTVAP

TECS

Ab-5 HC
QVQLVQSGAEVKKPGASVKV
50

Anti-CD28 scFv-Linker 2-
SCKASGYTFTSYYIHWVRQAP

anti-PDL1 Fab 3 HC
GQGLEWIGSIYPGNVNTNYNE

KFKDRATLTVDTSISTAYMEL

SRLRSDDTAVYFCTRSHYGLD

WNFDV
WGQGTTVTVSSGGG

GSGGGGSGGGGSDIQMTQSPS

SLSASVGDRVTITCHASQNIYV

W
LNWYQQKPGKAPKLLIYKA

SNLHTGVPSRFSGSGSGTDFTL

TISSLQPEDFATYYCQQGQTY

PYT
FGGGTKVEIKGGGGSEVQ

LLESGGGLVQPGGSLRLSCAA

SGFTFSSYIMMWVRQAPGKG

LEWVSSIYPSGGITFYADTVK

GRFTISRDNSKNTLYLQMNSL

RAEDTAVYYCARIKLGTVTT

VDY
WGQGTLVTVSSASTKGPS

VFPLAPSSKSTSGGTAALGCLV

KDYFPEPVTVSWNSGALTSGV

HTFPAVLQSSGLYSLSSVVTVP

SSSLGTQTYICNVNHKPSNTK

VDKKVEPKSC

Ab-6 LC
QVQLVQSGAEVKKPGASVKV
51

Anti-CD28 scFv-Linker 2-
SCKASGYTFTSYYIHWVRQAP

Anti-PDL1 Fab 3
GQGLEWIGSIYPGNVNTNYNE

KFKDRATLTVDTSISTAYMEL

SRLRSDDTAVYFCTRSHYGLD

WNFDV
WGQGTTVTVSSGGG

GSGGGGSGGGGSDIQMTQSPS

SLSASVGDRVTITCHASQNIYV

W
LNWYQQKPGKAPKLLIYKA

SNLHTGVPSRFSGSGSGTDFTL

TISSLQPEDFATYYCQQGQTY

PYT
FGGGTKVEIKGGGGSQSA

LTQPASVSGSPGQSITISCTGTS

SDVGGYNY
VSWYQQHPGKAP

KLMIYDVSNRPSGVSNRFSGS

KSGNTASLTISGLQAEDEADY

YCSSYTSSSTRVFGTGTKVTV

LGQP
KANPTVTLFPPSSEELQ

ANKATLVCLISDFYPGAVTVA

WKADGSPVKAGVETTKPSKQ

SNNKYAASSYLSLTPEQWKSH

RSYSCQVTHEGSTVEKTVAPT

ECS

Ab-6 HC
EVQLLESGGGLVQPGGSLRLS
45

Anti-PDL1 Fab 3 HC
CAASGFTFSSYIMMWVRQAP

GKGLEWVSSIYPSGGITFYAD

TVKGRFTISRDNSKNTLYLQM

NSLRAEDTAVYYCARIKLGT

VTTVDY
WGQGTLVTVSSAST

KGPSVFPLAPSSKSTSGGTAAL

GCLVKDYFPEPVTVSWNSGAL

TSGVHTFPAVLQSSGLYSLSSV

VTVPSSSLGTQTYICNVNHKPS

NTKVDKKVEPKSC

Ab-7 LC
EIVLTQSPGTLSLSPGERATLSC
46

Anti-PDL1 Fab 4 LC
RASQRVSSSYLAWYQQKPGQ

APRLLIYDASSRATGIPDRFSG

SGSGTDFTLTISRLEPEDFAVY

YCQQYGSLPWTFGQGTKVEI

KRTVAAPSVFIFPPSDEQLKSG

TASVVCLLNNFYPREAKVQW

KVDNALQSGNSQESVTEQDSK

DSTYSLSSTLTLSKADYEKHK

VYACEVTHQGLSSPVTKSFNR

GEC

Ab-7 HC
QVQLVQSGAEVKKPGASVKV
52

Anti-CD28 scFv-Linker 2-
SCKASGYTFTSYYIHWVRQAP

anti-PDL1 Fab 4 HC
GQGLEWIGSIYPGNVNTNYNE

KFKDRATLTVDTSISTAYMEL

SRLRSDDTAVYFCTRSHYGLD

WNFDV
WGQGTTVTVSSGGG

GSGGGGSGGGGSDIQMTQSPS

SLSASVGDRVTITCHASQNIYV

W
LNWYQQKPGKAPKLLIYKA

SNLHTGVPSRFSGSGSGTDFTL

TISSLQPEDFATYYCQQGQTY

PYT
FGGGTKVEIKGGGGSEVQ

LVESGGGLVQPGGSLRLSCAA

SGFTFSRYWMSWVRQAPGK

GLEWVANIKQDGSEKYYVDS

VKGRFTISRDNAKNSLYLQMN

SLRAEDTAVYYCAREGGWFG

ELAFDY
WGQGTLVTVSSAST

KGPSVFPLAPSSKSTSGGTAAL

GCLVKDYFPEPVTVSWNSGAL

TSGVHTFPAVLQSSGLYSLSSV

VTVPSSSLGTQTYICNVNHKPS

NTKVDKRVEPKSC

Ab-8 LC
QVQLVQSGAEVKKPGASVKV
53

Anti-CD28 scFv-Linker 2-
SCKASGYTFTSYYIHWVRQAP

Anti-PDL1 Fab 4 LC
GQGLEWIGSIYPGNVNTNYNE

KFKDRATLTVDTSISTAYMEL

SRLRSDDTAVYFCTRSHYGLD

WNFDV
WGQGTTVTVSSGGG

GSGGGGSGGGGSDIQMTQSPS

SLSASVGDRVTITCHASQNIYV

W
LNWYQQKPGKAPKLLIYKA

SNLHTGVPSRFSGSGSGTDFTL

TISSLQPEDFATYYCQQGQTY

PYT
FGGGTKVEIKGGGGSEIV

LTQSPGTLSLSPGERATLSCRA

SQRVSSSYLAWYQQKPGQAP

RLLIYDASSRATGIPDRFSGSG

SGTDFTLTISRLEPEDFAVYYC

QQYGSLPWT
FGQGTKVEIKR

TVAAPSVFIFPPSDEQLKSGTA

SVVCLLNNFYPREAKVQWKV

DNALQSGNSQESVTEQDSKDS

TYSLSSTLTLSKADYEKHKVY

ACEVTHQGLSSPVTKSFNRGE

C

Ab-8 HC
EVQLVESGGGLVQPGGSLRLS
47

Anti-PDL1 Fab 4 HC
CAASGFTFSRYWMSWVRQAP

GKGLEWVANIKQDGSEKYYV

DSVKGRFTISRDNAKNSLYLQ

MNSLRAEDTAVYYCAREGG

WFGELAFDY
WGQGTLVTVSS

ASTKGPSVFPLAPSSKSTSGGT

AALGCLVKDYFPEPVTVSWNS

GALTSGVHTFPAVLQSSGLYS

LSSVVTVPSSSLGTQTYICNVN

HKPSNTKVDKRVEPKSC

In some embodiments, the multispecific antibodies described herein comprise improved activity in tumor cell killing. In some embodiments, the multispecific antibodies comprise an IC50 of less than about 300 picomolar (pM) in a cell killing assay. In some embodiments, the multispecific antibodies comprise an IC50 of less than about 200 picomolar (pM) in a cell killing assay. In some embodiments, the multispecific antibodies comprise an IC50 of less than about 100 picomolar (pM) in a cell killing assay. In some embodiments, the multispecific antibodies comprise an IC50 of less than about 75 picomolar (pM) in a cell killing assay. In some embodiments, the multispecific antibodies comprise an IC50 of less than about 50 picomolar (pM) in a cell killing assay. In some embodiments, the multispecific antibodies comprise an IC50 of less than about 25 picomolar (pM) in a cell killing assay. In some embodiments, the multispecific antibodies comprise an IC50 of less than about 10 picomolar (pM) in a cell killing assay.

In some embodiments, the multispecific antibodies described herein trigger little or no non-specific activation of immune cells. In some embodiments, the multispecific antibodies described herein trigger little or no cytokine release. In some embodiments, the multispecific antibodies described herein trigger little or no IFNy, TNF-alpha, or IL-2 release from immune cells. In some embodiments, the multispecific antibodies described herein trigger little or no proliferation of immune cells.

Multispecific Antibodies that Bind to CD28 and PD-L1: Formats for Selective Activation in Tumor Microenvironments

In some embodiments, the multispecific antibodies described herein are selectively activated in tumor microenvironments.

In some embodiments, the multispecific antibody is according to the following subformula: P₁-L₁-A-L-B (Formula Ia) wherein A comprises the CD28 binding domain; B comprises the PD-L1 binding domain; L comprises the linker that connects A to B; P₁comprises a peptide that binds to A and L1 comprises a linking moiety that connects A to P₁and is a substrate for a tumor specific protease.

In some embodiments, the multispecific antibody is according to the following subformula: A-L-B-L₂-P₂(Formula Ib) wherein A comprises the CD28 binding domain; B comprises the PD-L1 binding domain; L comprises the linker that connects A to B; P₂comprises a peptide that binds to B and L₂comprises a linking moiety that connects B to P₂and is a substrate for a tumor specific protease.

In some embodiments, the multispecific antibody is according to the following subformula: P₁-L₁-A-L-B-L₂-P₂(Formula Ic) wherein A comprises the CD28 binding domain; B comprises the PD-L1 binding domain; L comprises the linker that connects A to B; P₁comprises a peptide that binds to A and L1 comprises a linking moiety that connects A to P₁and is a substrate for a tumor specific protease; P₂comprises a peptide that binds to B and L₂comprises a linking moiety that connects B to P₂and is a substrate for a tumor specific protease.

Half-Life Extending Molecule (H₁)

In some embodiments, the multispecific antibodies of Formula Ia, Formula Ib, Formula Ic further comprise a half-life extending molecule (H₁). In some embodiments, H₁is connected to P₁. In some embodiments, H₁is connected to P₂. In some embodiments, H₁does not block A binding to CD28. In some embodiments, H₁comprises a linking moiety (L₅) that connects H₁to P₁or H₁to P₂. In some embodiments, half-life extending molecule (H₁) does not have binding affinity to PD-L1. In some embodiments, the half-life extending molecule (H₁) does not have binding affinity to CD28. In some embodiments, the half-life extending molecule (H₁) does not shield the multispecific antibody from CD28.

In some embodiments, H₁comprises a sequence as disclosed in Table 9 or a sequence substantially identical thereto (e.g., a sequence that has at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity).

TABLE 9

H1 Sequences

Amino Acid Sequence

Construct Description
(N to C)
SEQ ID NO:

Anti-Albumin: CDR-H1
GSTFYTAV
54

Anti-Albumin: CDR-H2
IRWTALTT
55

Anti-Albumin: CDR-H3
AARGTLGLFTTADSYDY
56

Anti-albumin
EVQLVESGGGLVQPGGSLRLSCAASGSTF
57

YTAV
MGWVRQAPGKGLEWVAAIRWTA

LTTSYADSVKGRFTISRDGAKTTLYLQM

NSLRPEDTAVYYCAARGTLGLFTTADSY

DY
WGQGTLVTVSS

In some embodiments, H₁comprises an amino acid sequence that has repetitive sequence motifs. In some embodiments, H₁comprises an amino acid sequence that has highly ordered secondary structure. “Highly ordered secondary structure,” as used in this context, means that at least about 50%, or about 70%, or about 80%, or about 90%, of amino acid residues of H₁contribute to secondary structure, as measured or determined by means, including, but not limited to, spectrophotometry (e.g. by circular dichroism spectroscopy in the “far-UV” spectral region (190-250 nm), and computer programs or algorithms, such as the Chou-Fasman algorithm and the Garier-Osguthorpe-Robson (“GOR”) algorithm.

In some embodiments, H₁comprises a polymer. In some embodiments, the polymer is polyethylene glycol (PEG). In some embodiments, H₁comprises albumin. In some embodiments, H₁comprises an Fc domain. In some embodiments, the albumin is serum albumin. In some embodiments, the albumin is human serum albumin. In some embodiments, H₁comprises a polypeptide, a ligand, or a small molecule. In some embodiments, the polypeptide, the ligand or the small molecule binds serum protein or a fragment thereof, a circulating immunoglobulin or a fragment thereof, or CD35/CR1. In some embodiments, the serum protein comprises a thyroxine-binding protein, a transthyretin, a 1-acid glycoprotein, a transferrin, transferrin receptor or a transferrin-binding portion thereof, a fibrinogen, or an albumin. In some embodiments, the circulating immunoglobulin molecule comprises IgG1, IgG2, IgG3, IgG4, slgA, IgM or IgD. In some embodiments, the serum protein is albumin. In some embodiments, the polypeptide is an antibody. In some embodiments, the antibody comprises a single domain antibody, a single chain variable fragment or a Fab.

In some embodiments, the single domain antibody comprises a single domain antibody that binds to albumin. In some embodiments, the single domain antibody is a human or humanized antibody. In some embodiments, the single domain antibody is selected from the group consisting of 645gHlgL1, 645dsgH5gL4, 23-13-A01-sc02, A10m3 or a fragment thereof, DOM7r-31, DOM7h-11-15, Alb-1, Alb-8, Alb-23, 10G, 10E and SA21. In some embodiments, the single domain antibody comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the single domain antibody comprise: HC-CDR1: SEQ ID NO: 54, HC-CDR2: SEQ ID NO: 55, and HC-CDR3: SEQ ID NO: 56. In some embodiments, the single domain antibody comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the single domain antibody comprise: HC-CDR1: SEQ ID NO: 54, HC-CDR2: SEQ ID NO: 55, and HC-CDR3: SEQ ID NO: 56; and wherein the CDRs comprise from 0-2 amino acid modifications in at least one of the HC-CDR1, HC-CDR2, or HC-CDR3.

In some embodiments, H₁comprises an amino acid sequence according to SEQ ID NO: 57. In some embodiments, H₁comprises an amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 57. In some embodiments, H₁comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 57. In some embodiments, H₁comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 57. In some embodiments, H₁comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 57. In some embodiments, H₁comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NO: 57.

In some embodiments, H₁comprise a modified amino acid or non-natural amino acid, or a modified non-natural amino acid, or a combination thereof. In some embodiments, the modified amino acid or a modified non-natural amino acid comprises a post-translational modification. In some embodiments H₁comprise a modification including, but not limited to acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. Modifications are made anywhere to H₁including the peptide backbone, the amino acid side chains, and the terminus.

In some embodiments, H₁comprises a linking moiety (L₅) that connects H₁to P₁or P₂.In some embodiments, L₅is a peptide sequence having at least 5 to no more than 50 amino acids. In some embodiments, L₅is a peptide sequence having at least 10 to no more than 30 amino acids. In some embodiments, L₅is a peptide sequence having at least 10 amino acids. In some embodiments, L₅is a peptide sequence having at least 18 amino acids. In some embodiments, L₅is a peptide sequence having at least 26 amino acids. In some embodiments, L₅has a formula selected from the group consisting of (G₂S)_n, (GS)_n, (GSGGS)_n(SEQ ID NO: 58), (GGGS)_n(SEQ ID NO: 59), (GGGGS)_n(SEQ ID NO: 60), and (GSSGGS)_n(SEQ ID NO: 61), wherein n is an integer of at least 1.

Linking Moiety (L₁or L₂)

In some embodiments, L₁or L₂is a peptide sequence having at least 5 to no more than 50 amino acids. In some embodiments L₁or L₂is a peptide sequence having at least 10 to no more than 30 amino acids. In some embodiments, L₁or L₂is a peptide sequence having at least 10 amino acids. In some embodiments, L₁or L₂is a peptide sequence having at least 18 amino acids. In some embodiments, L₁or L₂is a peptide sequence having at least 26 amino acids. In some embodiments, L₁or L₂has a formula comprising (G₂S)_n(SEQ ID NO: 233), wherein n is an integer from 1 to 3. In some embodiments, L₁or L₂has a formula comprising (G₂S)_n, wherein n is an integer of at least 1. In some embodiments, L₁or L₂has a formula selected from the group consisting of (G₂S)_n, (GS)_n, (GSGGS)_n(SEQ ID NO: 58), (GGGS)_n(SEQ ID NO: 59), (GGGGS)_n(SEQ ID NO: 60), and (GSSGGS)_n(SEQ ID NO: 61), wherein n is an integer of at least 1. In some embodiments, the tumor specific protease is selected from the group consisting of metalloprotease, serine protease, cysteine protease, threonine protease, and aspartic protease. In some embodiments L₁or L₂comprises a urokinase cleavable amino acid sequence, a matriptase cleavable amino acid sequence, a legumain cleavable amino acid sequence, or a matrix metalloprotease cleavable amino acid sequence.

In some embodiments, L₁or L₂comprises a sequence as disclosed in Table 10 or a sequence substantially identical thereto (e.g., a sequence that has 0, 1, or 2 amino acid modifications).

TABLE 10

L1 or L2

Construct
Amino Acid Sequence
SEQ ID

Description
(N to C)
NO:

Linker 1
GGGGSGGGGSGGGGS
18

Linker 2
GGGGS
19

Linker 3
GGGGSGGGS
62

Cleavable linker
GGGGSGGGLSGRSDAGSPLGL
63

AGSGGGS

Linker 4
GGGGSLSGRSDNHGSSGT
64

Linker 5
GGGGSSGGSGGSGLSGRSDNH
65

GSSGT

Linker 6
ASGRSDNH
66

Linker 7
LAGRSDNH
67

Linker 8
ISSGLASGRSDNH
68

Linker 9
ISSGLLAGRSDNH
69

Linker 10
LSGRSDNH
70

Linker 11
ISSGLLSGRSDNP
71

Linker 12
ISSGLLSGRSDNH
72

Linker 13
LSGRSDNHSPLGLAGS
73

Linker 14
SPLGLAGSLSGRSDNH
74

Linker 15
SPLGLSGRSDNH
75

Linker 16
LAGRSDNHSPLGLAGS
76

Linker 17
LSGRSDNHVPLSLKMG
77

Linker 18
LSGRSDNHVPLSLSMG
78

Linker 19
GSSGGSGGSGGSGISSGLLSGR
79

SDNHGSSGT

Linker 20
GSSGGSGGSGGISSGLLSGRSD
80

NHGGGS

Linker 21
ASGRSDNH
81

Linker 22
LAGRSDNH
82

Linker 23
ISSGLASGRSDNH
83

Linker 24
LSGRSDAG
84

Linker 25
ISSGLLSGRSDAG
85

Linker 26
AAGLLAPPGGLSGRSDAG
86

Linker 27
SPLGLSGRSDAG
87

Linker 28
LSGRSDAGSPLGLAG
88

In some embodiments, L₁is bound to N-terminus of A. In some embodiments, L₁is bound to C-terminus of A. In some embodiments, L₂is bound to N-terminus of B. In some embodiments, L₂is bound to C-terminus of B. In some embodiments, P₁becomes unbound from A when L₁is cleaved by the tumor specific protease thereby exposing A to CD28. In some embodiments, P₂becomes unbound from B when L₂is cleaved by the tumor specific protease thereby exposing B to PD-L1.

In some embodiments, L₁or L₂, comprise a modified amino acid or non-natural amino acid, or a modified non-natural amino acid, or a combination thereof. In some embodiments, the modified amino acid or a modified non-natural amino acid comprises a post-translational modification. In some embodiments, L₁or L₂comprise a modification including, but not limited, to acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. Modifications are made anywhere to L₁or L₂including the peptide backbone, or the amino acid side chains.

Peptide (P₁and P₂)

P₁or P₂is designed to be optimized for a specific antigen-binding domain through an iterative process of phage display and quantitative binding assays designed to select for those peptides that are able to prevent binding to CD28 or PD-L1. A directed evolution-based process that includes phage libraries is used for identifying P₁or P₂. Multiple cycles of selection and amplification of potential inhibitory peptides that are capable of blocking the antigen binding domain from binding to its target of CD28 or PD-L1 with the goal of optimizing masked PD-L1 x CD28 antibodies in serum and limiting cleavage, thereby reducing toxicity. Discovery of P₁or P₂is depicted in FIG. 18.

In some embodiments, P₁impairs binding of A to CD28. In some embodiments, P₁is bound to A through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bonding interactions, or a combination thereof. In some embodiments, P₁is bound to A at or near an antigen binding site. In some embodiments, P₁becomes unbound from A when L₁is cleaved by the tumor specific protease thereby exposing A to CD28. In some embodiments, P₁has less than 70% sequence identity to CD28. In some embodiments, P₁has less than 75% sequence identity to CD28. In some embodiments, P₁has less than 80% sequence identity to CD28. In some embodiments, P₁has less than 85% sequence identity to CD28. In some embodiments, P₁has less than 90% sequence identity to CD28. In some embodiments, P₁has less than 95% sequence identity to CD28. In some embodiments, P₁has less than 98% sequence identity to CD28. In some embodiments, P₁has less than 99% sequence identity to CD28. In some embodiments, P₁comprises a de novo amino acid sequence that shares less than 10% sequence identity to CD28.

In some embodiments, P₂impairs binding of B to PD-L1. In some embodiments, P₂is bound to B through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bonding interactions, or a combination thereof. In some embodiments, P₂is bound to B at or near an antigen binding site. In some embodiments, P₂becomes unbound from B when L₂is cleaved by the tumor specific protease thereby exposing B to the PD-L1. In some embodiments, P₂has less than 70% sequence identity to the PD-L1. In some embodiments, P₂has less than 75% sequence identity to the PD-L1. In some embodiments, P₂has less than 80% sequence identity to the PD-L1. In some embodiments, P₂has less than 85% sequence identity to the PD-L1. In some embodiments, P₂has less than 90% sequence identity to the PD-L1. In some embodiments, P₂has less than 95% sequence identity to the PD-L1. In some embodiments, P₂has less than 98% sequence identity to the PD-L1. In some embodiments, P₂has less than 99% sequence identity to the PD-L1. In some embodiments, P₂comprises a de novo amino acid sequence that shares less than 10% sequence identity to the PD-L1.

In some embodiments, P₁or P₂comprises a peptide sequence of at least 5 amino acids in length. In some embodiments, P₁or P₂comprises a peptide sequence of at least 6 amino acids in length. In some embodiments, P₁or P₂comprises a peptide sequence of at least 10 amino acids in length. In some embodiments, P₁or P₂comprises a peptide sequence of at least 10 amino acids in length and no more than 20 amino acids in length. In some embodiments, P₁or P₂comprises a peptide sequence of at least 16 amino acids in length. In some embodiments, P₁or P₂comprises a peptide sequence of no more than 40 amino acids in length. In some embodiments, P₁or P₂comprises at least two cysteine amino acid residues. In some embodiments, P₁or P₂comprises a cyclic peptide or a linear peptide. In some embodiments, P₁or P₂comprises a cyclic peptide. In some embodiments, P₁or P₂comprises a linear peptide.

In some embodiments, P₁or P₂comprise a modified amino acid or non-natural amino acid, or a modified non-natural amino acid, or a combination thereof. In some embodiments, the modified amino acid or a modified non-natural amino acid comprises a post-translational modification. In some embodiments P₁or P₂comprise a modification including, but not limited to acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. Modifications are made anywhere to P₁or P₂including the peptide backbone, the amino acid side chains, and the terminus.

In some embodiments, P₁or P₂does not comprise albumin or an albumin fragment. In some embodiments, P₁or P₂does not comprise an albumin binding domain.

Polynucleotides Encoding Multispecific Antibodies that Bind to CD28 and PD-L1

Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding the multispecific antibodies disclosed herein. Described herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding multispecific antibodies that comprise a CD28 binding domain and a PD-L1 binding domain, wherein when the CD28 binding domain is a single chain variable fragment (scFv), then the PD-L1 binding domain is not a scFv.

In some embodiments, the isolated recombinant nucleic acid molecules encode multispecific antibodies according to the formula:

A-L-B (Formula I)

wherein A comprises the CD28 binding domain; B comprises the PD-L1 binding domain; and L comprises a linker that connects A to B. In some embodiments, the isolated recombinant nucleic acid molecules encode multispecific antibodies comprising the formula:

A-L-B (Formula I)

wherein A is the CD28 binding domain; B is the PD-L1 binding domain; and L is a linker that connects A to B. In some embodiments, the isolated recombinant nucleic acid molecules encode multispecific antibodies according to the formula:

A-L-B (Formula I)

wherein A is the CD28 binding domain; B is the PD-L1 binding domain; and L is a linker that connects A to B.

Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding an isolated polypeptide comprising an amino acid according to SEQ ID NO: 20.

Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding an isolated polypeptide comprising an amino acid according to SEQ ID NO: 21.

Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding an isolated polypeptide comprising an amino acid according to SEQ ID NO: 22.

Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding an isolated polypeptide comprising an amino acid according to SEQ ID NO: 23.

Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding an isolated polypeptide comprising an amino acid according to SEQ ID NO: 42.

Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding an isolated polypeptide comprising an amino acid according to SEQ ID NO: 48.

Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding an isolated polypeptide comprising an amino acid according to SEQ ID NO: 49.

Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding an isolated polypeptide comprising an amino acid according to SEQ ID NO: 43.

Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding an isolated polypeptide comprising an amino acid according to SEQ ID NO: 44.

Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding an isolated polypeptide comprising an amino acid according to SEQ ID NO: 50.

Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding an isolated polypeptide comprising an amino acid according to SEQ ID NO: 51.

Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding an isolated polypeptide comprising an amino acid according to SEQ ID NO: 45.

Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding an isolated polypeptide comprising an amino acid according to SEQ ID NO: 46.

Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding an isolated polypeptide comprising an amino acid according to SEQ ID NO: 52.

Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding an isolated polypeptide comprising an amino acid according to SEQ ID NO: 53.

Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding an isolated polypeptide comprising an amino acid according to SEQ ID NO: 47.

Pharmaceutical Compositions

Disclosed herein, in some embodiments, are pharmaceutical compositions comprising: (a) multispecific antibodies as disclosed herein; and (b) a pharmaceutically acceptable excipient.

In some embodiments, the pharmaceutical composition comprises (a) multispecific antibodies that comprise a CD28 binding domain and a PD-L1 binding domain, wherein when the CD28 binding domain is a single chain variable fragment (scFv), then the PD-L1 binding domain is not a scFv; and (b) a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises (a) multispecific antibodies according to the formula: A-L-B (Formula I) wherein A comprises the CD28 binding domain; B comprises the PD-L1 binding domain; and L comprises a linker that connects A to B; and (b) a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises (a) a multispecific antibodies comprising the formula: A-L-B (Formula I) wherein A comprises the CD28 binding domain; B comprises the PD-L1 binding domain; and L comprises a linker that connects A to B; and (b) a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises (a) a multispecific antibodies comprising the formula: A-L-B (Formula I) wherein A is the CD28 binding domain; B is the PD-L1 binding domain; and L is a linker that connects A to B; and (b) a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises (a) multispecific antibodies according to the formula: A-L-B (Formula I) wherein A is the CD28 binding domain; B is the PD-L1 binding domain; and L is a linker that connects A to B; and (b) a pharmaceutically acceptable excipient.

In some embodiments, the multispecific antibody further comprises a detectable label, a therapeutic agent, or a pharmacokinetic modifying moiety. In some embodiments, the detectable label comprises a fluorescent label, a radiolabel, an enzyme, a nucleic acid probe, or a contrast agent.

For administration to a subject, the multispecific antibody as disclosed herein, may be provided in a pharmaceutical composition together with one or more pharmaceutically acceptable carriers or excipients. The term “pharmaceutically acceptable carrier” includes, but is not limited to, any carrier that does not interfere with the effectiveness of the biological activity of the ingredients and that is not toxic to the patient to whom it is administered. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Such carriers can be formulated by conventional methods and can be administered to the subject at a suitable dose. Preferably, the compositions are sterile. These compositions may also contain adjuvants such as preservative, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents.

The pharmaceutical composition may be in any suitable form, (depending upon the desired method of administration). It may be provided in unit dosage form, may be provided in a sealed container and may be provided as part of a kit. Such a kit may include instructions for use. It may include a plurality of said unit dosage forms.

The pharmaceutical composition may be adapted for administration by any appropriate route, including a parenteral (e.g., subcutaneous, intramuscular, or intravenous) route. Such compositions may be prepared by any method known in the art of pharmacy, for example by mixing the active ingredient with the carrier(s) or excipient(s) under sterile conditions.

Dosages of the substances of the present disclosure can vary between wide limits, depending upon the disease or disorder to be treated, the age and condition of the individual to be treated, etc. and a physician will ultimately determine appropriate dosages to be used.

Methods of Use

In some embodiments, are methods of treating cancer in a subject in need thereof comprising administering to the subject a multispecific antibody as disclosed herein that binds to CD28 and PD-L1.

In some embodiments, the cancer comprises cancer cells that express PD-L1.

In some embodiments, the cancer is a hematological malignancy. In some embodiments, wherein the cancer is leukemia or lymphoma. In some embodiments, the cancer is lymphoma, and wherein the lymphoma is B-cell lymphoma. In some embodiments, the cancer is a solid tumor. In some embodiments, the solid tumor is sarcoma, breast cancer, lung cancer, or carcinoma. In some embodiments, the solid tumor is lung cancer, and wherein the lung cancer is non-small cell lung cancer.

In some embodiments, the multispecific antibody induces T cell mediated cytotoxicity of tumor cells. In some embodiments, the administering to the subject of the multispecific antibody is sufficient to reduce or eliminate the cancer as compared to a baseline measurement of the cancer taken from the subject prior to the administering of the multispecific antibody. In some embodiments, the reduction is at least about 1-fold, 5-fold, 10-fold, 20-fold, 40-fold, 60-fold, 80-fold, or up to about 100 fold.

In some embodiments, the multispecific antibody is administered to the subject as a single agent therapy.

In some embodiments, the subject is refractory to checkpoint inhibitor therapy.

In some embodiments, the subject has relapsed from checkpoint inhibitor therapy.

In some embodiments, the multispecific antibody is not administered as a part of a treatment regimen with a second therapeutic agent. In some embodiments, the multispecific antibody is not administered as a part of a treatment regimen with a second therapeutic agent comprising a tumor binding domain. In some embodiments, the multispecific antibody is not administered as a part of a treatment regimen with a second therapeutic agent comprising an anti-CD19 antibody. In some embodiments, the multispecific antibody is not administered as a part of a treatment regimen with a second therapeutic agent comprising an antibody that has an anti-CD19 binding domain and an anti-CD3 binding domain.

In some embodiments, the multispecific antibody is any multispecific antibody as disclosed herein that binds to CD28 and PD-L1.

In some embodiments, the subject is refractory to checkpoint inhibitor therapy.

In some embodiments, the subject has relapsed from checkpoint inhibitor therapy.

In some embodiments, the linker connects the C-terminus of A to an N-terminus of B. In some embodiments, the linker connects the N-terminus of A to a C-terminus of B. In some embodiments, the linker connects the C-terminus of A to the N-terminus of the Fab heavy chain polypeptide. In some embodiments, the linker connects the N-terminus of A to the C-terminus of the Fab heavy chain polypeptide. In some embodiments, the linker connects the C-terminus of A to the N-terminus of the Fab light chain polypeptide. In some embodiments, the linker connects the N-terminus of A to the C-terminus of the Fab light chain polypeptide. In some embodiments, the linker connects the Fab light chain polypeptide to the scFv light chain variable domain. In some embodiments, the linker connects the Fab light chain polypeptide to the scFv heavy chain variable domain. In some embodiments, the linker connects the Fab heavy chain polypeptide to the scFv light chain variable domain. In some embodiments, the linker connects the Fab heavy chain polypeptide to the scFv heavy chain variable domain. In some embodiments, the linker connects the Fab light chain polypeptide to the N-terminus of the scFv light chain variable domain. In some embodiments, the linker connects the Fab light chain polypeptide to the C-terminus of the scFv light chain variable domain. In some embodiments, the linker connects the Fab light chain polypeptide to the N-terminus of the scFv heavy chain variable domain. In some embodiments, the linker connects the Fab light chain polypeptide to the C-terminus of the scFv heavy chain variable domain. In some embodiments, the linker connects the Fab heavy chain polypeptide to the N-terminus of the scFv light chain variable domain.

In some embodiments, the scFv heavy chain variable domain comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the scFv heavy chain variable domain comprise: HC-CDR1: SEQ ID NO: 1; HC-CDR2: SEQ ID NO: 2; HC-CDR3: SEQ ID NO: 3, and wherein said CDRs comprise from 0-2 amino acid modifications in at least one of said HC-CDR1, HC-CDR2, or HC-CDR3.In some embodiments, the scFv light chain variable domain comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the scFv light chain variable domain comprise: LC-CDR1: SEQ ID NO: 4; LC-CDR2: SEQ ID NO: 5; and LC-CDR3: SEQ ID NO: 6, and wherein the CDRs comprise from 0-2 amino acid modifications in at least one of said LC-CDR1, LC-CDR2, or LC-CDR3. In some embodiments, the Fab heavy chain variable domain comprises complementarity determining region (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the Fab heavy chain variable domain comprise: HC-CDR1: SEQ ID NO: 10; HC-CDR2: SEQ ID NO: 11; HC-CDR3: SEQ ID NO: 12, and wherein said CDRs comprise from 0-2 amino acid modifications in at least one of said HC-CDR1, HC-CDR2, or HC-CDR3. In some embodiments, the Fab light chain variable domain comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the Fab light chain variable domain comprise:LC-CDR1: SEQ ID NO: 13; LC-CDR2: SEQ ID NO: 14; and LC-CDR3: SEQ ID NO: 15, and wherein the CDRs comprise from 0-2 amino acid modifications in at least one of said LC-CDR1, LC-CDR2, or LC-CDR3.

In some embodiments, the scFv heavy chain variable domain comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 7. In some embodiments, the scFv heavy chain variable domain comprises an amino acid sequence of at least 75 consecutive amino acid residues of SEQ ID NO: 7. In some embodiments, the scFv heavy chain variable domain comprises an amino acid sequence of at least 110 consecutive amino acid residues of SEQ ID NO: 7.

In some embodiments, the scFv heavy chain variable domain comprises an amino acid sequence of at least 110 consecutive amino acid residues of SEQ ID NO: 7 and has at least 80% sequence identity to the at least 110 consecutive amino acid residues of SEQ ID NO: 7. In some embodiments, the scFv heavy chain variable domain comprises an amino acid sequence according to SEQ ID NO: 7. In some embodiments, the scFv light chain variable domain comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 8. In some embodiments, the scFv light chain variable domain comprises an amino acid sequence of at least 75 consecutive amino acid residues of SEQ ID NO: 8. In some embodiments, the scFv light chain variable domain comprises an amino acid sequence of at least 100 consecutive amino acid residues of SEQ ID NO: 8. In some embodiments, the scFv light chain variable domain comprises an amino acid sequence of at least 100 consecutive amino acid residues of SEQ ID NO: 8 and has at least 80% sequence identity to the at least 100 consecutive amino acid residues of SEQ ID NO: 8. In some embodiments, the scFv light chain variable domain comprises an amino acid sequence according to SEQ ID NO: 8. In some embodiments, the scFv comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 9. In some embodiments, the scFv comprises an amino acid sequence of at least 175 consecutive amino acid residues of SEQ ID NO: 9. In some embodiments, the scFv comprises an amino acid sequence of at least 210 consecutive amino acid residues of SEQ ID NO: 9. In some embodiments, the scFv comprises an amino acid sequence of at least 210 consecutive amino acid residues of SEQ ID NO: 9 and has at least 80% sequence identity to the at least 210 consecutive amino acid residues of SEQ ID NO: 9. In some embodiments, the scFv comprises an amino acid sequence according to SEQ ID NO: 9. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 17. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence of at least 175 consecutive amino acid residues of SEQ ID NO: 17.In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence of at least 215 consecutive amino acid residues of SEQ ID NO: 17. In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence of at least 215 consecutive amino acid residues of SEQ ID NO: 17 and has at least 80% sequence identity to the at least 215 consecutive amino acid residues of SEQ ID NO: 17.

In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 17. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 16. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence of at least 175 consecutive amino acid residues of SEQ ID NO: 16. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 16. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 16 and has at least 80% sequence identity to the at least 200 consecutive amino acid residues of SEQ ID NO: 16. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 16. In some embodiments, the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab light chain polypeptide comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 20, and an amino acid sequence of the Fab heavy chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 21.

In some embodiments, the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab light chain polypeptide comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 20, and an amino acid sequence of the Fab heavy chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence of at least 450 consecutive amino acid residues of SEQ ID NO: 21. In some embodiments, the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab light chain polypeptide comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 20 and has at least 80% sequence identity to the at least 200 consecutive amino acid residues of SEQ ID NO: 20 and an amino acid sequence of the Fab heavy chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence of at least 450 consecutive amino acid residues of SEQ ID NO: 21 and has at least 80% sequence identity to the at least 450 consecutive amino acid residues of SEQ ID NO: 21. In some embodiments, the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab light chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 20, and an amino acid sequence of the Fab heavy chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence to SEQ ID NO: 21. In some embodiments, the linker connects the Fab light chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab heavy chain polypeptide comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 23, and an amino acid sequence of the Fab light chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence that has at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 22. In some embodiments, the linker connects the Fab light chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab heavy chain polypeptide comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 23, and an amino acid sequence of the Fab light chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence of at least 450 consecutive amino acid residues of SEQ ID NO: 22. In some embodiments, the linker connects the Fab light chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab heavy chain polypeptide comprises an amino acid sequence of at least 200 consecutive amino acid residues of SEQ ID NO: 23 and has at least 80% sequence identity to the at least 200 consecutive amino acid residues of SEQ ID NO: 23 and an amino acid sequence of the Fab light chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence of at least 450 consecutive amino acid residues of SEQ ID NO: 22 and has at least 80% sequence identity to the at least 450 consecutive amino acid residues of SEQ ID NO: 22. In some embodiments, the linker connects the Fab light chain polypeptide to the C-terminus of the scFv light chain variable domain and wherein the Fab heavy chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 23, and an amino acid sequence of the Fab light chain polypeptide that is connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence to SEQ ID NO: 22.

Disclosed herein are methods of treating cancer in a subject in need thereof comprising administering to the subject a multispecific antibody that comprises a CD28 binding domain and a PD-L1 binding domain wherein the multispecific antibody that comprises the CD28 binding domain and the PD-L1 binding domain is not administered as part of a treatment regimen with another multispecific antibody that targets a cancer antigen different from PD-L1 or CD28. In some embodiments, the multispecific antibody that comprises the CD28 binding domain and the PD-L1 binding domain is administered to the subject as a single agent therapy. In some embodiments, the multispecific antibody that comprises the CD28 binding domain and the PD-L1 binding domain comprises an IgG framework, an IgA framework, an IgE framework, or an IgM framework. In some embodiments, the CD28 binding domain comprises a single chain variable fragment, a single domain antibody, a Fab, or a Fab′. In some embodiments, the PD-L1 binding domain comprises a single chain variable fragment, a single domain antibody, a Fab, or a Fab′. In some embodiments, the PD-L1 binding domain comprises a single chain variable fragment and the CD28 binding domain comprises a single chain variable fragment. In some embodiments, the CD28 binding domain comprises an anti-CD28 light chain polypeptide. In some embodiments, the anti-CD28 light chain polypeptide comprises a variable domain of an IgG1, IgG2, IgG3, or IgG4 light chain. In some embodiments, the CD28 binding domain comprises an anti-CD28 heavy chain polypeptide. In some embodiments, the anti-CD28 heavy chain polypeptide comprises a variable domain of an IgG1, IgG2, IgG3, or IgG4 heavy chain. In some embodiments, the PD-L1 binding domain comprises an anti-PD-L1 light chain polypeptide. In some embodiments, the anti-PD-L1 light chain polypeptide comprises a variable domain of an IgG1, IgG2, IgG3, or IgG4 light chain. In some embodiments, the PD-L1 binding domain comprises an anti-PD-L1 heavy chain polypeptide. In some embodiments, the anti-PD-L1 heavy chain polypeptide comprises a variable domain of an IgG1, IgG2, IgG3, or IgG4 heavy chain. In some embodiments, the multispecific antibody further comprises a fragment crystallizable (Fc) region. In some embodiments, the Fc region comprises an IgG CH2 domain and an IgG CH3 domain. In some embodiments, the Fc region comprises a heterodimeric Fc region. In some embodiments, the Fc region comprises at least one amino acid modification that increases the half-life of the multispecific antibody. In some embodiments, the Fc region comprises at least one amino acid modification that modulates its interaction with an Fc receptor. In some embodiments, the Fc region comprises at least one amino acid modification that increases binding of the Fc region to an Fc receptor. In some embodiments, the Fc region comprises at least one amino acid modification that decreases glycosylation of the Fc region. In some embodiments, the modification is an amino acid substitution, deletion, or addition. In some embodiments, the modification is an amino acid substitution. In some embodiments, the at least one amino acid modification that decreases glycosylation of the Fc region comprises an amino acid substitution at a position corresponding to position N297 of human IgG1, wherein the numbering is according to the EU index of Kabat. In some embodiments, the Fc region is afucosylated.

In some embodiments, the anti-CD28 light chain polypeptide comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 the anti-CD28 light chain polypeptide: LC-CDR1: SEQ ID NO: 4; LC-CDR2: SEQ ID NO: 5; and LC-CDR3: SEQ ID NO: 6, and wherein the CDRs comprise from 0-2 amino acid modifications in at least one of said LC-CDR1, LC-CDR2, or LC-CDR3. In some embodiments, the anti-CD28 heavy chain polypeptide comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the anti-CD28 heavy chain polypeptide comprise: HC-CDR1: SEQ ID NO: 1; HC-CDR2: SEQ ID NO: 2; HC-CDR3: SEQ ID NO: 3, and wherein said CDRs comprise from 0-2 amino acid modifications in at least one of said HC-CDR1, HC-CDR2, or HC-CDR3.

In some embodiments, the anti-PD-L1 light chain polypeptide comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the anti-PD-L1 light chain polypeptide comprise:LC-CDR1: SEQ ID NO: 13; LC-CDR2: SEQ ID NO: 14; and LC-CDR3: SEQ ID NO: 15, and wherein the CDRs comprise from 0-2 amino acid modifications in at least one of said LC-CDR1, LC-CDR2, or LC-CDR3. In some embodiments, the anti-PD-L1 heavy chain polypeptide the anti-PD-L1 heavy chain polypeptide comprises complementarity determining region (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the anti-PD-L1 heavy chain polypeptide comprises: HC-CDR1: SEQ ID NO: 10; HC-CDR2: SEQ ID NO: 11; HC-CDR3: SEQ ID NO: 12, and wherein said CDRs comprise from 0-2 amino acid modifications in at least one of said HC-CDR1, HC-CDR2, or HC-CDR3.