Tumor-activating multispecific antibodies for targeting CD28 and PD-L1 and methods of use thereof

Abstract

Provided herein are antibodies that selectively bind to CD28 and PD-L1, pharmaceutical compositions and nucleic acids thereof, methods of use, and methods for making and discovering the same.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 338,115, filed May 4, 2022, which is incorporated by reference in its entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing, which is submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML copy was created on April 13, 2023 and is named 52426-741_601_SL.xml and is 578,045 bytes in size. Summary of the Invention

[0005] Disclosed herein are isolated multispecific antibodies according to the formula: P1-L1-A1-LB (Formula I), wherein A1 comprises a CD28 binding domain; B comprises a PD-L1 binding domain; L comprises a linker connecting A1 and B; P1 comprises a peptide that binds to A1, and L1 comprises a linker connecting A1 to P1 and is a substrate for a tumor-specific protease, wherein P1 comprises an amino acid sequence according to any one of SEQ ID NOs: 24-53, 128-148, and any of the amino acid sequences of Table 20, or an amino acid sequence having 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 24-53, 128-148, and any of the amino acid sequences of Table 20. In some embodiments, the multispecific antibody is according to the formula: P1-L1-A1-LB-L2-P2 (Formula Ia), wherein P2 comprises a peptide that binds to B, and L2 comprises a linker connecting B to P2 and is a substrate for a tumor-specific protease. In some embodiments, P1 comprises an amino acid sequence according to any one of SEQ ID NOs: 24-53, 128-148, and the amino acid sequences of Table 20. In some embodiments, P1 comprises an amino acid sequence according to any one of SEQ ID NOs: 42-53, or an amino acid sequence having 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 42-53. In some embodiments, P1 comprises an amino acid sequence according to any one of SEQ ID NOs: 42-53. In some embodiments, P1 comprises an amino acid sequence according to any one of Table 20, or an amino acid sequence having 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of the amino acid sequences of Table 20. In some embodiments, P1 comprises an amino acid sequence according to any one of SEQ ID NOs: 128-147, or an amino acid sequence having 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 128-147. In some embodiments, P1 comprises an amino acid sequence according to any one of SEQ ID NOs: 128-147. In some embodiments, P1 comprises an amino acid sequence according to X1-X2-X3-C-X4-X5-X6-X7-X8-X9-X 10 -CX 11 -X 12an amino acid sequence wherein X1 is selected from the group consisting of M, I, L, and V; X2 is selected from the group consisting of D, H, N, A, F, S, T, Y, and V; X3 is selected from the group consisting of W, L, and F; X4 is selected from the group consisting of P, A, and L; X5 is selected from the group consisting of R, T, I, M, S, K, L, V, W, F, A, P, and D; X6 is selected from the group consisting of E, D, Y, H, S, F, A, N, T, I, P, and V; X7 is selected from the group consisting of L, M, R, S, Q, and H; X8 is selected from the group consisting of W and Q; X9 is selected from the group consisting of H, N, D, A, S, Y, T, F, V, L, and I; X 10 Selected from E, V, L, D, Y, R, Q, H, F, K, A, M and N; X 11 is selected from F, Y, L, W, and V; and X 12 is selected from N, A, F, S, Y, H, D, T, and L. In some embodiments, X1 is selected from M, I, and L; X2 is selected from D, H, N, and A; X3 is W; X4 is P; X5 is selected from R, T, I, M, S, and K; X6 is selected from E, D, Y, H, S, and F; X7 is selected from L, M, and R; X8 is W; X9 is selected from H, N, D, A, S, and V; X 10 Selected from E, V, L, D and H; X 11 is selected from F, Y and L; and X 12 is selected from N, A, F, S, and Y. In some embodiments, X1 is M; X2 is selected from D and H; X3 is W; X4 is P; X5 is selected from R, T, and I; X6 is selected from E, D, and Y; X7 is selected from L, M, and R; X8 is W; X9 is selected from H, N, D, and V; X 10 Selected from E, V, L, D and H; X 11 is F; and X 12Selected from N, A and F. In some embodiments, P1 comprises an amino acid sequence according to SEQ ID NO: 32 or an amino acid sequence having 1, 2 or 3 amino acid mutations, substitutions or deletions relative to SEQ ID NO: 32. In some embodiments, P1 comprises an amino acid sequence according to SEQ ID NO: 32. In some embodiments, P1 comprises an amino acid sequence according to SEQ ID NO: 138 or an amino acid sequence having 1, 2 or 3 amino acid mutations, substitutions or deletions relative to SEQ ID NO: 138. In some embodiments, P1 comprises an amino acid sequence according to SEQ ID NO: 138. In some embodiments, P1 weakens the binding of A1 to CD28. In some embodiments, P1 binds to A1 through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bonding interactions, or a combination thereof. In some embodiments, P1 binds to A1 at or near the antigen binding site. In some embodiments, when L1 is cleaved by a tumor-specific protease, P1 dissociates from A1, thereby exposing A1 to CD28. In some embodiments, P1 has less than 75% sequence identity with CD28. In some embodiments, P1 has less than 80% sequence identity with CD28. In some embodiments, P1 has less than 85% sequence identity with CD28. In some embodiments, P1 has less than 90% sequence identity with CD28. In some embodiments, P1 has less than 95% sequence identity with CD28. In some embodiments, P1 comprises a de novo amino acid sequence that shares less than 10% sequence identity with CD28. In some embodiments, P2 weakens the binding of B to PD-L1. In some embodiments, P2 binds to B through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bond interactions, or a combination thereof. In some embodiments, P2 binds to B at or near the antigen binding site. In some embodiments, when L2 is cleaved by a tumor-specific protease, P2 unbinds from B, thereby exposing B to PD-L1. In some embodiments, P2 has less than 70% sequence identity with PD-L1. In some embodiments, P2 has less than 75% sequence identity with PD-L1. In some embodiments, P2 has less than 80% sequence identity with PD-L1. In some embodiments, P2 has less than 85% sequence identity with PD-L1. In some embodiments, P2 has less than 90% sequence identity with PD-L1. In some embodiments, P2 has less than 95% sequence identity with PD-L1. In some embodiments, P2 comprises a de novo generated amino acid sequence that shares less than 10% sequence identity with PD-L1. In some embodiments, P2 comprises a peptide sequence that is at least 5 amino acids in length.In some embodiments, P2 comprises a peptide sequence of at least 6 amino acids in length. In some embodiments, P2 comprises a peptide sequence of at least 10 amino acids in length. In some embodiments, P2 comprises a peptide sequence of at least 10 amino acids in length and no more than 20 amino acids in length. In some embodiments, P2 comprises a peptide sequence of at least 16 amino acids in length. In some embodiments, P2 comprises a peptide sequence of no more than 40 amino acids in length. In some embodiments, P1 or P2 comprises at least two cysteine ​​amino acid residues. In some embodiments, P1 or P2 comprises a cyclic peptide or a linear peptide. In some embodiments, P1 or P2 comprises a cyclic peptide. In some embodiments, P1 or P2 comprises a linear peptide. In some embodiments, P1 or P2 comprises a modified amino acid or a non-natural amino acid or a modified non-natural amino acid, or a combination thereof. In some embodiments, P1 or P2 does not comprise albumin or an albumin fragment. In some embodiments, P1 or P2 does not comprise an albumin binding domain. In some embodiments, L1 or L2 is a peptide sequence of at least 5 and no more than 50 amino acids. In some embodiments, L1 or L2 is a peptide sequence having at least 10 to no more than 30 amino acids. In some embodiments, L1 or L2 is a peptide sequence having at least 10 amino acids. In some embodiments, L1 or L2 is a peptide sequence having at least 18 amino acids. In some embodiments, L1 or L2 is a peptide sequence having at least 26 amino acids. In some embodiments, L1 or L2 comprises a formula comprising (G2S)n, wherein n is an integer from 1 to 3 (SEQ ID NO: 228). In some embodiments, L1 or L2 comprises a formula comprising (G2S)n, wherein n is an integer of at least 1. In some embodiments, L1 or L2 comprises a formula selected from the following: (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 a metalloprotease, a serine protease, a cysteine ​​protease, a threonine protease, and an aspartic protease. In some embodiments, L1 or L2 comprises an amino acid sequence cleavable by urokinase, an amino acid sequence cleavable by membrane-type serine protease (matriptase), or an amino acid sequence cleavable by matrix metalloprotease. In some embodiments, L1 or L2 comprises a sequence according to SEQ ID NOs:18-19, 62-88. In some embodiments, L1 binds to the N-terminus of A1. In some embodiments, L1 binds to the C-terminus of A1. In some embodiments, L2 binds to the N-terminus of B. In some embodiments, L2 binds to the C-terminus of B. In some embodiments, the CD28 binding domain comprises a single-chain variable fragment, a single-domain antibody, Fab, or Fab′. In some embodiments, the CD28 binding domain comprises a single-chain variable fragment. In some embodiments, the CD28 binding domain comprises a single-domain antibody. In some embodiments, the CD28 binding domain comprises a Fab or Fab′. In some embodiments, the PD-L1 binding domain comprises a single-chain variable fragment, a single-domain antibody, a Fab or Fab′. In some embodiments, the PD-L1 binding domain comprises a Fab or Fab′. In some embodiments, the PD-L1 binding domain comprises a Fab or Fab′ and the CD28 binding domain comprises a single-chain variable fragment. In some embodiments, the PD-L1 binding domain comprising a Fab or Fab′ comprises a Fab heavy chain polypeptide and a Fab light chain polypeptide, the Fab heavy chain polypeptide comprising a Fab heavy chain variable domain, and the Fab light chain polypeptide comprising a Fab light chain variable domain. In some embodiments, the CD28 binding domain comprising a single-chain variable fragment comprises a scFv heavy chain variable domain and a scFv light chain variable domain. In some embodiments, a linker connects the C-terminus of A1 to the N-terminus of B. In some embodiments, a linker connects the N-terminus of A1 to the C-terminus of B. In some embodiments, a linker connects the C-terminus of A1 to the N-terminus of Fab heavy chain polypeptide. In some embodiments, a linker connects the N-terminus of A1 to the C-terminus of the Fab heavy chain polypeptide. In some embodiments, a linker connects the C-terminus of A1 to the N-terminus of the Fab light chain polypeptide. In some embodiments, a linker connects the N-terminus of A1 to the C-terminus of the Fab light chain polypeptide. In some embodiments, a linker connects the Fab light chain polypeptide to the scFv light chain variable domain. In some embodiments, a linker connects the Fab light chain polypeptide to the scFv heavy chain variable domain. In some embodiments, a linker connects the Fab heavy chain polypeptide to the scFv light chain variable domain. In some embodiments, a linker connects the Fab heavy chain polypeptide to the scFv heavy chain variable domain.In some embodiments, a linker connects the Fab light chain polypeptide to the N-terminus of the scFv light chain variable domain. In some embodiments, a linker connects the Fab light chain polypeptide to the C-terminus of the scFv light chain variable domain. In some embodiments, a linker connects the Fab light chain polypeptide to the N-terminus of the scFv heavy chain variable domain. In some embodiments, a linker connects the Fab light chain polypeptide to the C-terminus of the scFv heavy chain variable domain. In some embodiments, a linker connects the Fab heavy chain polypeptide to the N-terminus of the scFv light chain variable domain. In some embodiments, a linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain. In some embodiments, a linker connects the Fab heavy chain polypeptide to the N-terminus of the scFv heavy chain variable domain. In some embodiments, a linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv heavy chain variable domain. 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 in length and no more than 30 amino acids in length. In some embodiments, the length of the linker is 5 amino acids. In some embodiments, the length of the linker is 15 amino acids. In some embodiments, the linker comprises (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, L comprises the formula comprising (G2S)n, wherein n is an integer of 1 to 3 (SEQ ID NO: 228). In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 18 (GGGGSGGGGSGGGGS) or SEQ ID NO: 19 (GGGGS). 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, HC-CDR2, and 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 the CDRs comprise 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, LC-CDR2, and LC-CDR3 of the scFv light chain variable domain comprise: LC-CDR1: SEQ ID NO: 4; LC-CDR2: SEQ ID NO: 5(KA); and LC-CDR3: SEQ ID NO: 6, and wherein the CDRs comprise 0-2 amino acid modifications in at least one of said LC-CDR1, LC-CDR2, or LC-CDR3. In some embodiments, A1 comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, LC-CDR2, and LC-CDR3 of A1 comprise: LC-CDR1: SEQ ID NO: 4; LC-CDR2: SEQ ID NO: 5(KA); and LC-CDR3: SEQ ID NO: 6; wherein A1 comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 of A1 comprise: HC-CDR1: SEQ ID NO: 1; HC-CDR2: SEQ ID NO: 2; HC-CDR3: SEQ ID NO: 3. In some embodiments, the Fab heavy chain variable domain comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein HC-CDR1, HC-CDR2, and 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 the CDRs comprise 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, LC-CDR2, and LC-CDR3 of the Fab light chain variable domain comprise: LC-CDR1: SEQ ID NO: 13; LC-CDR2: SEQ ID NO: 14 (DA); and LC-CDR3: SEQ ID NO: 15; and wherein the CDRs comprise 0-2 amino acid modifications in at least one of said LC-CDR1, LC-CDR2, or LC-CDR3.In some embodiments, B comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 of B comprise: HC-CDR1: SEQ ID NO: 10; HC-CDR2: SEQ ID NO: 11; HC-CDR3: SEQ ID NO: 12; and wherein B comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, LC-CDR2, and LC-CDR3 of B comprise: LC-CDR1: SEQ ID NO: 13; LC-CDR2: SEQ ID NO: 14 (DA); and LC-CDR3: SEQ ID NO: 15. In some embodiments, the scFv heavy chain variable domain comprises an amino acid sequence having 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 with 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 with at least 80% sequence identity with 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 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 contiguous amino acid residues of SEQ ID NO: 8 and has at least 80% sequence identity with at least 100 contiguous 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 having at least 80% sequence identity with an amino acid sequence according to SEQ ID NO: 9. In some embodiments, the scFv comprises an amino acid sequence of at least 175 contiguous amino acid residues of SEQ ID NO: 9.In some embodiments, the scFv comprises an amino acid sequence of at least 210 contiguous amino acid residues of SEQ ID NO: 9. In some embodiments, the scFv comprises an amino acid sequence of at least 210 contiguous amino acid residues of SEQ ID NO: 9 and has at least 80% sequence identity with at least 210 contiguous 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 having at least 80% sequence identity with 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 contiguous 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 contiguous 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 contiguous amino acid residues of SEQ ID NO: 17 and has at least 80% sequence identity with at least 215 contiguous 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 having at least 80% sequence identity to 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 contiguous 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 contiguous 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 contiguous amino acid residues of SEQ ID NO: 16 and has at least 80% sequence identity to at least 200 contiguous 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 having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO:20, and the amino acid sequence of the Fab heavy chain polypeptide connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO:21.In some embodiments, a 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 the amino acid sequence according to SEQ ID NO: 20, and the amino acid sequence of the Fab heavy chain polypeptide connected to the C-terminus of the scFv light chain variable domain comprises the amino acid sequence according to SEQ ID NO: 21. In some embodiments, a 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 an amino acid sequence according to SEQ ID NO: 23, and the amino acid sequence of the Fab light chain polypeptide 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 an amino acid sequence according to SEQ ID NO: 22. In some embodiments, a 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 the amino acid sequence of the Fab light chain polypeptide connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence according to SEQ ID NO: 22. In some embodiments, the multispecific antibody further comprises a half-life extending molecule (H1). In some embodiments, H1 is connected to P1. In some embodiments, H1 is connected to P2. In some embodiments, H1 does not block A1 from binding to CD28. In some embodiments, H1 does not block B from binding to PD-L1. In some embodiments, H1 comprises a linker portion (L5) connecting H1 to P1 or H1 to P2. In some embodiments, the half-life extending molecule (H1) does not have binding affinity for PD-L1. In some embodiments, the half-life extending molecule (H1) does not have binding affinity for CD28. In some embodiments, the half-life extending molecule (H1) does not protect the multispecific antibody from the effects of CD28. In some embodiments, H1 comprises a sequence according to SEQ ID NOs: 54-57. In some embodiments, H1 comprises an amino acid sequence having a repeating sequence motif. In some embodiments, H1 comprises an amino acid sequence having a highly ordered secondary structure. In some embodiments, H1 comprises a polymer. The isolated multispecific antibody of claim 148, wherein the polymer is polyethylene glycol (PEG). In some embodiments, H1 comprises albumin. In some embodiments, H1 comprises an Fc domain. In some embodiments, the albumin is serum albumin. In some embodiments, the albumin is human serum albumin. In some embodiments, H1 comprises a polypeptide, ligand, or small molecule. In some embodiments, the polypeptide, ligand, or small molecule binds to a serum protein or fragment thereof, a circulating immunoglobulin or fragment thereof, or CD35 / CR1.In some embodiments, serum proteins include thyroxine binding protein, transthyretin, 1-acid glycoprotein, transferrin, transferrin receptor or its transferrin binding portion, fibrinogen or albumin. In some embodiments, circulating immunoglobulin molecules include IgG1, IgG2, IgG3, IgG4, slgA, IgM or IgD. In some embodiments, serum proteins are albumin. In some embodiments, polypeptides are antibodies. In some embodiments, antibodies include single domain antibodies, single chain variable fragments, Fab or Fab'. In some embodiments, single domain antibodies include single domain antibodies that bind to albumin. In some embodiments, single domain antibodies are human antibodies or humanized antibodies. In some embodiments, the single domain antibody is selected from 645gH1gL1, 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 a complementary determining region (CDR): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and 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 CDR comprises 0-2 amino acid modifications in at least one of HC-CDR1, HC-CDR2, or HC-CDR3. In some embodiments, H1 comprises an amino acid sequence according to SEQ ID NO: 57. In some embodiments, H1 comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 57. In some embodiments, H1 comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 57. In some embodiments, H1 comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 57. In some embodiments, H1 comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 57. In some embodiments, H1 comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 57. In some embodiments, H1 comprises a modified amino acid or a non-natural amino acid or a modified non-natural amino acid, or a combination thereof. In some embodiments, the modified amino acid or modified non-natural amino acid comprises a post-translational modification. In some embodiments, H1 comprises a linker (L5) connecting H1 to P1 or P2. In some embodiments, L5 is a peptide sequence having at least 5 to no more than 50 amino acids. In some embodiments, L5 is a peptide sequence having at least 10 and no more than 30 amino acids.In some embodiments, L5 is a peptide sequence having at least 10 amino acids. In some embodiments, L5 is a peptide sequence having at least 18 amino acids. In some embodiments, L5 is a peptide sequence having at least 26 amino acids. In some embodiments, L5 comprises 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 isolated multispecific antibody comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NOs: 149-170. In some embodiments, the isolated multispecific antibody comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NOs: 149-170. In some embodiments, the isolated multispecific antibody comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NOs: 149-170. In some embodiments, the isolated multispecific antibody comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 149-170. In some embodiments, the isolated multispecific antibody comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NOs: 149-170. In some embodiments, the isolated multispecific antibody comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 149 and 150. In some embodiments, the isolated multispecific antibody comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NOs: 149 and 150. In some embodiments, the isolated multispecific antibody comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NOs: 151 and 152. In some embodiments, the isolated multispecific antibody comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NOs: 151 and 152. In some embodiments, the isolated multispecific antibody comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NOs: 153 and 154. In some embodiments, the isolated multispecific antibody comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NOs: 153 and 154. In some embodiments, the isolated multispecific antibody comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NOs: 155 and 156. In some embodiments, the isolated multispecific antibody comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NOs: 155 and 156. In some embodiments, the isolated multispecific antibody comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NOs: 157 and 158. In some embodiments, the isolated multispecific antibody comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NOs: 157 and 158. In some embodiments, the isolated multispecific antibody comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NOs: 159 and 160. In some embodiments, the isolated multispecific antibody comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NOs: 159 and 160.In some embodiments, the isolated multispecific antibody comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NOs: 161 and 162. In some embodiments, the isolated multispecific antibody comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NOs: 161 and 162. In some embodiments, the isolated multispecific antibody comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NOs: 163 and 164. In some embodiments, the isolated multispecific antibody comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NOs: 163 and 164. In some embodiments, the isolated multispecific antibody comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NOs: 165 and 166. In some embodiments, the isolated multispecific antibody comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NOs: 165 and 166. In some embodiments, the isolated multispecific antibody comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NOs: 167 and 168. In some embodiments, the isolated multispecific antibody comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NOs: 167 and 168. In some embodiments, the isolated multispecific antibody comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NOs: 169 and 170. In some embodiments, the isolated multispecific antibody comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NOs: 169 and 170. In some embodiments, the isolated multispecific antibody comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NOs: 208 and 209. In some embodiments, the isolated multispecific antibody comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NOs: 208 and 209.

[0006] Disclosed herein are isolated recombinant nucleic acid molecules encoding the polypeptide of the isolated multispecific antibody of any of the above embodiments.

[0007] Disclosed herein are pharmaceutical compositions comprising: (a) the isolated multispecific antibody of any one of the above embodiments; and (b) a pharmaceutically acceptable excipient.

[0008] Disclosed herein are pharmaceutical compositions comprising: (a) an isolated multispecific antibody of any one of the above embodiments, (b) an anticancer therapy, and (c) a pharmaceutically acceptable excipient. In some embodiments, the anticancer therapy comprises a small molecule, a cell-based therapy, or an antibody-based therapy. In some embodiments, the antibody-based therapy is a T cell adapter. In some embodiments, the T cell adapter comprises a formula according to: D1-L0-E1 (Formula II), wherein D1 comprises an effector cell binding domain that binds to an effector cell antigen, E1 comprises a tumor antigen binding domain that binds to a tumor antigen, and L0 comprises a linker connecting D1 to E1. In some embodiments, D1 comprises a single-chain variable fragment, a single-domain antibody, a Fab fragment, or Fab'. In some embodiments, D1 comprises a single-chain variable fragment. In some embodiments, E1 comprises a single-chain variable fragment, a single-domain antibody, a Fab fragment, or Fab'. In some embodiments, E1 comprises a Fab fragment. In some embodiments, the effector cell antigen comprises CD3. In some embodiments, the effector cell binding domain comprises a complementarity determining region (CDR) selected from muromonab-CD3 (OKT3), otelixizumab (TRX4), teplizumab (MGA031), visilizumab (Nuvion), SP34, X35, VIT3, BMA030 (BW264 / 56), CLB-T3 / 3, In some embodiments, the effector cell binding domain comprises an amino acid sequence according to SEQ ID NOs: 89-101. In some embodiments, the tumor antigen comprises epidermal growth factor receptor (EGFR), prostate-specific membrane antigen (PSMA), or tumor-associated calcium signal transducer 2 (referred to herein as TROP2). In some embodiments, the tumor antigen comprises EGFR. In some embodiments, the tumor antigen binding domain comprises an amino acid sequence according to SEQ ID NOs: 102-111.In some embodiments, the tumor antigen comprises EGFR, and the tumor binding domain comprises the complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, and LC-CDR1, LC-CDR2, and LC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 comprise: HC-CDR1: SEQ ID NO: 105; HC-CDR2: SEQ ID NO: 106; HC-CDR3: SEQ ID NO: 107; and wherein the LC-CDR1, LC-CDR2, and LC-CDR3 comprise: LC-CDR1: SEQ ID NO: 102; LC-CDR2: SEQ ID NO: 103 (YAS); and LC-CDR3: SEQ ID NO: 104. In some embodiments, the tumor antigen comprises EGFR, and the T cell engager comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 181 and 182. In some embodiments, the tumor antigen comprises EGFR, and the T cell engager comprises an amino acid sequence according to SEQ ID NOs: 181 and 182. In some embodiments, the tumor antigen comprises EGFR, and the T cell engager comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 181 and 182. In some embodiments, the tumor antigen comprises EGFR, and the T cell engager comprises an amino acid sequence according to SEQ ID NOs: 214 and 215. In some embodiments, the tumor antigen comprises TROP2. In some embodiments, the tumor antigen comprises TROP2, and the tumor binding domain comprises the complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, and LC-CDR1, LC-CDR2, and LC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 comprise HC-CDR1: SEQ ID NO: 112; HC-CDR2: SEQ ID NO: 113; HC-CDR3: SEQ ID NO: 114; and wherein the LC-CDR1, LC-CDR2, and LC-CDR3 comprise: LC-CDR1: SEQ ID NO: 115; LC-CDR2: SEQ ID NO: 116 (SAS); and LC-CDR3: SEQ ID NO: 117. In some embodiments, the tumor antigen comprises TROP2, and the T cell engager comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 187-192. In some embodiments, the tumor antigen comprises TROP2, and the T cell engager comprises the amino acid sequence according to any one of SEQ ID NOs: 187-192.In some embodiments, the tumor antigen binding domain comprises an amino acid sequence according to SEQ ID NOs: 112-119. In some embodiments, the tumor antigen comprises PSMA. In some embodiments, the tumor antigen binding domain comprises an amino acid sequence according to SEQ ID NOs: 120-127. In some embodiments, the tumor antigen comprises PSMA, and the tumor binding domain comprises the complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, and LC-CDR1, LC-CDR2, and LC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 comprise HC-CDR1: SEQ ID NO: 120; HC-CDR2: SEQ ID NO: 121; HC-CDR3: SEQ ID NO: 122; and wherein the LC-CDR1, LC-CDR2, and LC-CDR3 comprise: LC-CDR1: SEQ ID NO: 123; LC-CDR2: SEQ ID NO: 124(EA); and LC-CDR3: SEQ ID NO: 125. In some embodiments, the tumor antigen comprises PSMA, and the T cell engager comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 173 and 174. In some embodiments, the tumor antigen comprises PSMA, and the T cell engager comprises an amino acid sequence according to SEQ ID NOs: 173 and 174. In some embodiments, the T cell engager molecule is selectively activated in the tumor microenvironment. In some embodiments, the T cell engager is according to the following formula: P3-L3-D1-L0-E1 (Formula IIa), wherein D1 comprises a CD3 binding domain; E1 comprises a tumor antigen binding domain; L0 comprises a linker connecting D1 and E1; P3 comprises a peptide that binds to D1, and L3 comprises a linker connecting D1 and P3 and is a substrate for a tumor-specific protease. In some embodiments, the T cell engager is according to the following formula: D1-L0-E1-L4-P4 (Formula IIb), wherein D1 comprises a CD3 binding domain; E1 comprises a tumor antigen binding domain; L0 comprises a linker connecting D1 and E1; P4 comprises a peptide that binds to E1, and L4 comprises a linker connecting E1 and P4 and is a substrate for a tumor-specific protease. In some embodiments, the T cell engager is according to the following subformula: P3-L3-D1-L0-E1-L4-P4 (Formula IIc), wherein D1 comprises a CD3 binding domain; E1 comprises a tumor antigen binding domain; L0 comprises a linker connecting D1 and E1; P3 comprises a peptide that binds to D1, and L3 comprises a linker that connects D1 to P3 and is a substrate for a tumor-specific protease; P4 comprises a peptide that binds to E1, and L4 comprises a linker that connects E1 to P4 and is a substrate for a tumor-specific protease.In some embodiments, the T cell engager comprises H1. In some embodiments, H1 comprises a sequence according to SEQ ID NOs: 54-57. In some embodiments, H1 comprises a single domain antibody. In some embodiments, the single domain antibody comprises complementary determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and 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, L3 or L4 is a peptide sequence having at least 5 and no more than 50 amino acids. In some embodiments, L3 or L4 is a peptide sequence having at least 10 and no more than 30 amino acids. In some embodiments, L3 or L4 is a peptide sequence having at least 10 amino acids. In some embodiments, L3 or L4 is a peptide sequence having at least 18 amino acids. In some embodiments, L3 or L4 is a peptide sequence having at least 26 amino acids. In some embodiments, L3 or L4 comprises a formula comprising (G2S)n, wherein n is an integer from 1 to 3 (SEQ ID NO: 228). In some embodiments, L3 or L4 comprises a formula comprising (G2S)n, wherein n is an integer of at least 1. In some embodiments, L3 or L4 comprises 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 a metalloprotease, a serine protease, a cysteine ​​protease, a threonine protease, and an aspartic protease. In some embodiments, L3 or L4 comprises an amino acid sequence cleavable by urokinase, an amino acid sequence cleavable by a membrane-type serine protease, or an amino acid sequence cleavable by a matrix metalloprotease. In some embodiments, L3 or L4 comprises a sequence according to SEQ ID NOs:18-19, 62-88. In some embodiments, L3 binds to the N-terminus of D1. In some embodiments, L3 binds to the C-terminus of D1. In some embodiments, L4 binds to the N-terminus of E1. In some embodiments, L4 binds to the C-terminus of E1. In some embodiments, when L3 is cleaved by a tumor-specific protease, P3 dissociates from D1, thereby exposing D1 to CD3. In some embodiments, when L4 is cleaved by a tumor-specific protease, P4 dissociates from E1, thereby exposing E1 to tumor antigens. In some embodiments, P3 weakens the binding of D1 to CD3. In some embodiments, P3 binds to D1 through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bonding interactions, or a combination thereof. In some embodiments, P3 binds to D1 at or near the antigen binding site. In some embodiments, when L3 is cleaved by a tumor-specific protease, P3 dissociates from D1, thereby exposing D1 to CD3. In some embodiments, P3 has less than 70% sequence identity with CD3. In some embodiments, P3 has less than 85% sequence identity with CD3. In some embodiments, P3 has less than 90% sequence identity with CD3. In some embodiments, P3 has less than 95% sequence identity with CD3. In some embodiments, P3 has less than 98% sequence identity with CD3. In some embodiments, P3 has less than 99% sequence identity with CD3. In some embodiments, P3 comprises an amino acid sequence according to SEQ ID NOs: 177-180. In some embodiments, P3 comprises a de novo generated amino acid sequence that shares less than 10% sequence identity with CD3. In some embodiments, P4 weakens the binding of E1 to a tumor antigen. In some embodiments, P4 binds to E1 through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bond interactions, or a combination thereof. In some embodiments, P4 binds to E1 at or near the antigen binding site. In some embodiments, when L4 is cleaved by a tumor-specific protease, P4 debinds from E1, thereby exposing E1 to the tumor antigen. In some embodiments, P4 has less than 70% sequence identity with the tumor antigen. In some embodiments, P4 has less than 80% sequence identity with the tumor antigen.In some embodiments, P4 has less than 85% sequence identity with a tumor antigen. In some embodiments, P4 has less than 90% sequence identity with a tumor antigen. In some embodiments, P4 has less than 95% sequence identity with a tumor antigen. In some embodiments, P4 comprises a de novo generated amino acid sequence that shares less than 10% sequence identity with a tumor antigen. In some embodiments, P3 or P4 comprises a peptide sequence of at least 5 amino acids in length. In some embodiments, P3 or P4 comprises a peptide sequence of at least 6 amino acids in length. In some embodiments, P3 or P4 comprises a peptide sequence of at least 10 amino acids in length. In some embodiments, P3 or P4 comprises a peptide sequence of at least 10 amino acids in length and no more than 20 amino acids in length. In some embodiments, P3 or P4 comprises a peptide sequence of at least 16 amino acids in length. In some embodiments, P3 or P4 comprises a peptide sequence of no more than 40 amino acids in length. In some embodiments, P3 or P4 comprises at least two cysteine ​​amino acid residues. In some embodiments, P3 or P4 comprises a cyclic peptide or a linear peptide. In some embodiments, P3 or P4 comprises a cyclic peptide. In some embodiments, P3 or P4 comprises a linear peptide. In some embodiments, P4 comprises an amino acid sequence according to SEQ ID NO: 185 or 186. In some embodiments, the tumor antigen comprises EGFR, and the T cell engager comprises the amino acid sequence of SEQ ID NO: 183 and 184. In some embodiments, P4 comprises an amino acid sequence according to SEQ ID NO: 199-201. In some embodiments, the tumor antigen comprises TROP2, and the T cell engager comprises any one of the amino acid sequences of SEQ ID NO: 193-198. In some embodiments, the tumor antigen comprises PSMA, and the T cell engager comprises the amino acid sequence of SEQ ID NO: 175 and 176.

[0009] Disclosed herein are isolated polypeptides or polypeptide complexes comprising a CD28 binding domain linked to a peptide that impairs binding of the CD28 binding domain to CD28, wherein the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 24-53, 128-148, and the amino acid sequences of Table 20, or an amino acid sequence having 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 24-53, 128-148, and the amino acid sequences of Table 20. In some embodiments, the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 24-53, 128-148, and the amino acid sequences of Table 20. In some embodiments, the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 42-53, or an amino acid sequence having 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 42-53. In some embodiments, the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 42-53. In some embodiments, the peptide comprises an amino acid sequence according to any one of the amino acid sequences of Table 20 or an amino acid sequence having 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of the amino acid sequences of Table 20. In some embodiments, the peptide comprises an amino acid sequence according to any one of the amino acid sequences of Table 20. In some embodiments, the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 128-147 or an amino acid sequence having 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 128-147. In some embodiments, the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 128-147. In some embodiments, the peptide comprises an amino acid sequence according to X1-X2-X3-C-X4-X5-X6-X7-X8-X9-X 10 -CX 11 -X 12 an amino acid sequence wherein X1 is selected from the group consisting of M, I, L, and V; X2 is selected from the group consisting of D, H, N, A, F, S, T, Y, and V; X3 is selected from the group consisting of W, L, and F; X4 is selected from the group consisting of P, A, and L; X5 is selected from the group consisting of R, T, I, M, S, K, L, V, W, F, A, P, and D; X6 is selected from the group consisting of E, D, Y, H, S, F, A, N, T, I, P, and V; X7 is selected from the group consisting of L, M, R, S, Q, and H; X8 is selected from the group consisting of W and Q; X9 is selected from the group consisting of H, N, D, A, S, Y, T, F, V, L, and I; X 10 Selected from E, V, L, D, Y, R, Q, H, F, K, A, M and N; X 11 is selected from F, Y, L, W, and V; and X 12is selected from N, A, F, S, Y, H, D, T, and L. In some embodiments, X1 is selected from M, I, and L; X2 is selected from D, H, N, and A; X3 is W; X4 is P; X5 is selected from R, T, I, M, S, and K; X6 is selected from E, D, Y, H, S, and F; X7 is selected from L, M, and R; X8 is W; X9 is selected from H, N, D, A, S, and V; X 10 Selected from E, V, L, D and H; X 11 is selected from F, Y and L; and X 12 is selected from N, A, F, S, and Y. In some embodiments, X1 is M; X2 is selected from D and H; X3 is W; X4 is P; X5 is selected from R, T, and I; X6 is selected from E, D, and Y; X7 is selected from L, M, and R; X8 is W; X9 is selected from H, N, D, and V; X 10 Selected from E, V, L, D and H; X 11 is F; and X 12Selected from N, A and F. In some embodiments, the peptide comprises an amino acid sequence according to SEQ ID NO: 32 or an amino acid sequence having 1, 2 or 3 amino acid mutations, substitutions or deletions relative to SEQ ID NO: 32. In some embodiments, the peptide comprises an amino acid sequence according to SEQ ID NO: 32. In some embodiments, the peptide comprises an amino acid sequence according to SEQ ID NO: 138 or an amino acid sequence having 1, 2 or 3 amino acid mutations, substitutions or deletions relative to SEQ ID NO: 138. In some embodiments, the peptide comprises an amino acid sequence according to SEQ ID NO: 138. In some embodiments, the CD28 binding domain comprises a single-chain variable fragment, a single-domain antibody, Fab or Fab′. In some embodiments, the CD28 binding domain comprises a single-chain variable fragment and the single-chain variable fragment comprises an scFv heavy chain variable domain and an scFv light chain variable domain. In some embodiments, the CD28 binding domain comprises a single-domain antibody. The isolated polypeptide or polypeptide complex of claim 313, wherein the CD28 binding domain comprises Fab or Fab′. 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, HC-CDR2, and 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 the CDRs comprise 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 a complementarity determining region (CDR): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, LC-CDR2, and LC-CDR3 of the scFv light chain variable domain comprise: LC-CDR1: SEQ ID NO: 4; LC-CDR2: SEQ ID NO: 5 (KA); and LC-CDR3: SEQ ID NO: 6, and wherein the CDR comprises 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 having at least 80% sequence identity with 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 with 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 with at least 80% sequence identity with 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 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 with 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 having at least 80% sequence identity with 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 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 with 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 CD28 binding domain is linked to a peptide via a linker portion (L1). In some embodiments, L1 is a substrate for a tumor-specific protease. In some embodiments, L1 is a peptide sequence having at least 5 and no more than 50 amino acids. In some embodiments, L1 is a peptide sequence having at least 10 to no more than 30 amino acids. In some embodiments, L1 is a peptide sequence having at least 10 amino acids. In some embodiments, L1 is a peptide sequence having at least 18 amino acids. In some embodiments, L1 is a peptide sequence having at least 26 amino acids. In some embodiments, L1 comprises (G2S). n wherein n is an integer from 1 to 3 (SEQ ID NO: 228). In some embodiments, L1 comprises (G2S)n wherein n is an integer of at least 1. In some embodiments, L1 comprises 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 a metalloprotease, a serine protease, a cysteine ​​protease, a threonine protease, and an aspartic protease. In some embodiments, L1 comprises an amino acid sequence cleavable by urokinase, an amino acid sequence cleavable by a membrane-type serine protease, or an amino acid sequence cleavable by a matrix metalloprotease. In some embodiments, L1 comprises a sequence according to SEQ ID NOs:18-19, 62-88. In some embodiments, L1 is bound to the N-terminus of A1. In some embodiments, L1 is bound to the C-terminus of A1. In some embodiments, when L1 is cleaved by a tumor-specific protease, P1 dissociates from A1, thereby exposing A1 to CD28. In some embodiments, L1 comprises a modified amino acid or a non-natural amino acid or a modified non-natural amino acid, or a combination thereof. In some embodiments, the modified amino acid or modified non-natural amino acid comprises a post-translational modification. In some embodiments, the isolated polypeptide or polypeptide complex further comprises a half-life extending molecule (H1). In some embodiments, H1 is linked to the peptide. In some embodiments, H1 does not block the CD28 binding domain from CD28. In some embodiments, H1 comprises a linker portion (L5) connecting H1 to a peptide. In some embodiments, the half-life extending molecule (H1) does not have binding affinity for CD28. In some embodiments, the half-life extending molecule (H1) does not protect an isolated polypeptide or polypeptide complex from CD28. In some embodiments, H1 comprises a sequence according to SEQ ID NOs: 54-57. In some embodiments, H1 comprises an amino acid sequence having a repeating sequence motif. In some embodiments, H1 comprises an amino acid sequence having a highly ordered secondary structure. In some embodiments, H1 comprises a polymer. In some embodiments, the polymer is polyethylene glycol (PEG). In some embodiments, H1 comprises albumin. In some embodiments, H1 comprises an Fc domain. In some embodiments, the albumin is serum albumin. In some embodiments, the albumin is human serum albumin. In some embodiments, H1 comprises a polypeptide, a ligand, or a small molecule. In some embodiments, the polypeptide, ligand, or small molecule binds to a serum protein or fragment thereof, a circulating immunoglobulin or fragment thereof, or CD35 / CR1. In some embodiments, the serum protein comprises thyroxine binding protein, transthyretin, 1-acid glycoprotein, transferrin, transferrin receptor or a transferrin-binding portion thereof, fibrinogen, or 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, a Fab, 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 antibody or a humanized antibody. In some embodiments, the single domain antibody is selected from 645gH1gL1, 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 a complementarity determining region (CDR): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and 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 0-2 amino acid modifications in at least one of HC-CDR1, HC-CDR2, or HC-CDR3. In some embodiments, H1 comprises an amino acid sequence according to SEQ ID NO: 57. In some embodiments, H1 comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 57. In some embodiments, H1 comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 57. In some embodiments, H1 comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 57. In some embodiments, H1 comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 57. In some embodiments, H1 comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 57. In some embodiments, H1 comprises a modified amino acid or a non-natural amino acid or a modified non-natural amino acid, or a combination thereof. In some embodiments, the modified amino acid or the modified non-natural amino acid comprises a post-translational modification. In some embodiments, H1 comprises a linker (L5) connecting H1 to P1 or P2. In some embodiments, L5 is a peptide sequence having at least 5 to no more than 50 amino acids. In some embodiments, L5 is a peptide sequence having at least 10 to no more than 30 amino acids. In some embodiments, L5 is a peptide sequence having at least 10 amino acids. In some embodiments, L5 is a peptide sequence having at least 18 amino acids. In some embodiments, L5 is a peptide sequence having at least 26 amino acids.In some embodiments, L5 comprises 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.

[0010] Disclosed herein are methods for treating cancer in a subject in need thereof, comprising administering to the subject a multispecific antibody of any one of the above embodiments. In some embodiments, the multispecific antibody induces T cell-mediated cytotoxicity of tumor cells. In some embodiments, the cancer is a hematological malignancy. In some embodiments, the cancer is a leukemia or lymphoma. In some embodiments, the cancer is a lymphoma, and wherein the lymphoma is a 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 a 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 is administered in combination with an anticancer therapy. In some embodiments, the multispecific antibody and the anticancer therapy are administered in the same pharmaceutical composition. In some embodiments, the multispecific antibody and the anticancer therapy are administered as separate pharmaceutical compositions. 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 anticancer therapy includes small molecules, cell-based therapies, or antibody-based therapies. In some embodiments, the antibody-based therapy is a T cell adapter. In some embodiments, the T cell adapter comprises a formula according to: D1-L0-E1 (Formula II), wherein D1 comprises an effector cell binding domain that binds to an effector cell antigen, E1 comprises a tumor antigen binding domain that binds to a tumor antigen, and L0 comprises a linker connecting D1 and E1. In some embodiments, D1 comprises a single-chain variable fragment, a single-domain antibody, a Fab fragment, or Fab'. In some embodiments, D1 comprises a single-chain variable fragment. In some embodiments, E1 comprises a single-chain variable fragment, a single-domain antibody, a Fab fragment, or Fab'. In some embodiments, E1 comprises a Fab fragment. In some embodiments, the effector cell binding domain comprises a complementary determining region (CDR) selected from the group consisting of: muromonab-CD3 (OKT3), oxicitumomab (TRX4), tilizumab (MGA031), visilizumab (Nuvion), SP34, X35, VIT3, BMA030 (BW264 / 56), CLB-T3 / 3, CRIS7, YTH12.5, F111-409, CL B-T3.4.2, TR-66, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII-87, 12F6, T3 / RW2-8C8, T3 / R W2-4B6, OKT3D, M-T301, SMC2, F101.01, UCHT-1, WT-31, 15865, 15865v12, 15865v16 and 15865v19.In some embodiments, the effector cell binding domain comprises an amino acid sequence according to SEQ ID NOs: 89-101. In some embodiments, the tumor antigen comprises epidermal growth factor receptor (EGFR), prostate-specific membrane antigen (PSMA), or tumor-associated calcium signal transducer 2 (referred to herein as TROP2). In some embodiments, the tumor antigen comprises EGFR. In some embodiments, the tumor antigen binding domain comprises an amino acid sequence according to SEQ ID NOs: 102-111. In some embodiments, the tumor antigen comprises EGFR, and the tumor binding domain comprises the complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, and LC-CDR1, LC-CDR2, and LC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 comprise HC-CDR1: SEQ ID NO: 105; HC-CDR2: SEQ ID NO: 106; HC-CDR3: SEQ ID NO: 107; and wherein the LC-CDR1, LC-CDR2, and LC-CDR3 comprise: LC-CDR1: SEQ ID NO: 102; LC-CDR2: SEQ ID NO: 103 (YAS); and LC-CDR3: SEQ ID NO: 104. In some embodiments, the tumor antigen comprises EGFR, and the T cell engager comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 181 and 182. In some embodiments, the tumor antigen comprises EGFR, and the T cell engager comprises an amino acid sequence according to SEQ ID NOs: 181 and 182. In some embodiments, the tumor antigen comprises EGFR, and the T cell engager comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 181 and 182. In some embodiments, the tumor antigen comprises EGFR, and the T cell engager comprises an amino acid sequence according to SEQ ID NOs: 214 and 215. In some embodiments, the cancer is colorectal cancer (CRC), squamous cell carcinoma of the head and neck (SCCHN), non-small cell lung cancer (NSCLC), prostate cancer, breast cancer, colon / rectal cancer, head and neck cancer, esophagogastric cancer, liver cancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, kidney cancer, or pancreatic cancer. In some embodiments, the tumor antigen comprises TROP2.In some embodiments, the tumor antigen comprises TROP2, and the tumor binding domain comprises the complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, and LC-CDR1, LC-CDR2, and LC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 comprise HC-CDR1: SEQ ID NO: 112; HC-CDR2: SEQ ID NO: 113; HC-CDR3: SEQ ID NO: 114; and wherein the LC-CDR1, LC-CDR2, and LC-CDR3 comprise: LC-CDR1: SEQ ID NO: 115; LC-CDR2: SEQ ID NO: 116 (SAS); and LC-CDR3: SEQ ID NO: 117. In some embodiments, the tumor antigen comprises TROP2, and the T cell engager comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 187-192. In some embodiments, the tumor antigen comprises TROP2, and the T cell engager comprises an amino acid sequence according to any one of SEQ ID NOs: 187-192. In some embodiments, the tumor antigen binding domain comprises an amino acid sequence according to SEQ ID NOs: 112-119. The method of claim 416, wherein the cancer is lung cancer, breast cancer (e.g., HER2+; ER / PR+; TNBC), cervical cancer, ovarian cancer, colorectal cancer, pancreatic cancer, gastric cancer, triple-negative breast cancer (TNBC), urothelial carcinoma (UC), non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), gastric cancer, esophageal cancer, head and neck cancer, prostate cancer, or endometrial cancer. In some embodiments, the tumor antigen comprises PSMA. In some embodiments, the tumor antigen binding domain comprises an amino acid sequence according to SEQ ID NOs: 120-127. In some embodiments, the tumor antigen comprises PSMA, and the tumor binding domain comprises the complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, and LC-CDR1, LC-CDR2, and LC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 comprise HC-CDR1: SEQ ID NO: 120; HC-CDR2: SEQ ID NO: 121; HC-CDR3: SEQ ID NO: 122; and wherein the LC-CDR1, LC-CDR2, and LC-CDR3 comprise: LC-CDR1: SEQ ID NO: 123; LC-CDR2: SEQ ID NO: 124(EA); and LC-CDR3: SEQ ID NO: 125.In some embodiments, the tumor antigen comprises PSMA and the T cell engager comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 173 and 174. In some embodiments, the tumor antigen comprises PSMA and the T cell engager comprises an amino acid sequence according to SEQ ID NOs: 173 and 174. In some embodiments, the cancer is lung cancer, breast cancer (e.g., HER2+; ER / PR+; TNBC), cervical cancer, ovarian cancer, colorectal cancer, pancreatic cancer, or gastric cancer. In some embodiments, the T cell engager molecule is selectively activated in the tumor microenvironment.

[0011] In some embodiments, the T cell engager is according to the following subformula: P3-L3-D1-L0-E1 (Formula IIa), wherein D1 comprises a CD3 binding domain; E1 comprises a tumor antigen binding domain; L0 comprises a linker connecting D1 and E1; P3 comprises a peptide that binds to D1, and L3 comprises a linker portion that connects D1 and P3 and is a substrate for a tumor-specific protease.

[0012] In some embodiments, the T cell engager is according to the following formula: D1-L0-E1-L4-P4 (Formula IIb), wherein D1 comprises a CD3 binding domain; E1 comprises a tumor antigen binding domain; L0 comprises a linker connecting D1 and E1; P4 comprises a peptide that binds to E1, and L4 comprises a linker connecting E1 and P4 and is a substrate for a tumor-specific protease. In some embodiments, the T cell engager is according to the following formula: P3-L3-D1-L0-E1-L4-P4 (Formula IIc), wherein D1 comprises a CD3 binding domain; E1 comprises a tumor antigen binding domain; L0 comprises a linker connecting D1 and E1; P3 comprises a peptide that binds to D1, and L3 comprises a linker connecting D1 and P3 and is a substrate for a tumor-specific protease; P4 comprises a peptide that binds to E1, and L4 comprises a linker connecting E1 and P4 and is a substrate for a tumor-specific protease. In some embodiments, the T cell engager comprises H1. In some embodiments, H1 comprises a sequence according to SEQ ID NOs: 54-57. In some embodiments, H1 comprises a single domain antibody. In some embodiments, the single domain antibody comprises the complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and 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, L3 or L4 is a peptide sequence having at least 5 and no more than 50 amino acids. In some embodiments, L3 or L4 is a peptide sequence having at least 10 and no more than 30 amino acids. In some embodiments, L3 or L4 is a peptide sequence having at least 10 amino acids. In some embodiments, L3 or L4 is a peptide sequence having at least 18 amino acids. In some embodiments, L3 or L4 is a peptide sequence having at least 26 amino acids. In some embodiments, L3 or L4 comprises (G2S) n wherein n is an integer from 1 to 3 (SEQ ID NO: 228). In some embodiments, L3 or L4 comprises (G2S) n wherein n is an integer of at least 1. In some embodiments, L3 or L4 comprises 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 a metalloprotease, a serine protease, a cysteine ​​protease, a threonine protease, and an aspartic protease. In some embodiments, L3 or L4 comprises an amino acid sequence cleavable by urokinase, an amino acid sequence cleavable by a membrane-type serine protease, or an amino acid sequence cleavable by a matrix metalloprotease. In some embodiments, L3 or L4 comprises a sequence according to SEQ ID NOs:18-19, 62-88. In some embodiments, L3 binds to the N-terminus of D1. In some embodiments, L3 binds to the C-terminus of D1. In some embodiments, L4 binds to the N-terminus of E1. In some embodiments, L4 binds to the C-terminus of E1. In some embodiments, when L3 is cleaved by a tumor-specific protease, P3 dissociates from D1, thereby exposing D1 to CD3. In some embodiments, when L4 is cleaved by a tumor-specific protease, P4 dissociates from E1, thereby exposing E1 to tumor antigens. In some embodiments, P3 weakens the binding of D1 to CD3. In some embodiments, P3 binds to D1 through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bonding interactions, or a combination thereof. In some embodiments, P3 binds to D1 at or near the antigen binding site. In some embodiments, when L3 is cleaved by a tumor-specific protease, P3 dissociates from D1, thereby exposing D1 to CD3. In some embodiments, P3 has less than 70% sequence identity with CD3. In some embodiments, P3 has less than 85% sequence identity with CD3. In some embodiments, P3 has less than 90% sequence identity with CD3. In some embodiments, P3 has less than 95% sequence identity with CD3. In some embodiments, P3 has less than 98% sequence identity with CD3. In some embodiments, P3 has less than 99% sequence identity with CD3. In some embodiments, P3 comprises an amino acid sequence according to SEQ ID NOs: 177-180. In some embodiments, P3 comprises a de novo generated amino acid sequence that shares less than 10% sequence identity with CD3. In some embodiments, P4 weakens the binding of E1 to a tumor antigen. In some embodiments, P4 binds to E1 through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bond interactions, or a combination thereof. In some embodiments, P4 binds to E1 at or near the antigen binding site. In some embodiments, when L4 is cleaved by a tumor-specific protease, P4 debinds from E1, thereby exposing E1 to the tumor antigen. In some embodiments, P4 has less than 70% sequence identity with the tumor antigen. In some embodiments, P4 has less than 80% sequence identity with the tumor antigen.In some embodiments, P4 has less than 85% sequence identity with a tumor antigen. In some embodiments, P4 has less than 90% sequence identity with a tumor antigen. In some embodiments, P4 has less than 95% sequence identity with a tumor antigen. In some embodiments, P4 comprises a de novo generated amino acid sequence that shares less than 10% sequence identity with a tumor antigen. In some embodiments, P3 or P4 comprises a peptide sequence of at least 5 amino acids in length. In some embodiments, P3 or P4 comprises a peptide sequence of at least 6 amino acids in length. In some embodiments, P3 or P4 comprises a peptide sequence of at least 10 amino acids in length. In some embodiments, P3 or P4 comprises a peptide sequence of at least 10 amino acids in length and no more than 20 amino acids in length. In some embodiments, P3 or P4 comprises a peptide sequence of at least 16 amino acids in length. In some embodiments, P3 or P4 comprises a peptide sequence of no more than 40 amino acids in length. In some embodiments, P3 or P4 comprises at least two cysteine ​​amino acid residues. In some embodiments, P3 or P4 comprises a cyclic peptide or a linear peptide. In some embodiments, P3 or P4 comprises a cyclic peptide. In some embodiments, P3 or P4 comprises a linear peptide. In some embodiments, P4 comprises an amino acid sequence according to SEQ ID NO: 185 or 186. In some embodiments, the tumor antigen comprises EGFR, and the T cell engager comprises the amino acid sequence of SEQ ID NO: 183 and 184. In some embodiments, P4 comprises an amino acid sequence according to SEQ ID NO: 199-201. In some embodiments, the tumor antigen comprises TROP2, and the T cell engager comprises any one of the amino acid sequences of SEQ ID NO: 193-198. In some embodiments, the tumor antigen comprises PSMA, and the T cell engager comprises the amino acid sequence of SEQ ID NO: 175 and 176. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The novel features of the present disclosure are particularly set forth in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by referring to the following detailed description of illustrative embodiments utilizing the principles of the present disclosure and the accompanying drawings, in which:

[0014] Figures 1A-1B Exemplary formats of anti-PDL1 x CD28 multispecific antibodies are illustrated. Figure 1A The "Vh" format of an antibody Fab-scFv format is illustrated. Figure 1B The "Vl" format of a Fab-scFv antibody format is illustrated.

[0015] Figure 2A schematic diagram illustrating the identification of peptides that can be attached to anti-PD-L1 and anti-CD28 multispecific antibodies for selective activation in the tumor microenvironment. This schematic illustrates directed evolution and phage display techniques to identify peptides that block antigen recognition via their antigen-binding domains.

[0016] Figure 3A Binding of anti-CD28 scFv to peptides measured by ELISA is shown.

[0017] Figure 3B The binding of Ab-12 to the peptide as measured by ELISA is shown.

[0018] Figure 3C Binding of anti-CD28 scFv to peptides measured by ELISA is shown.

[0019] Figure 3D The binding of Ab-12 to the peptide as measured by ELISA is shown.

[0020] Figures 3E-3F It was demonstrated that the peptides inhibited binding of the anti-CD28 scFv to the CD28 antigen as measured by ELISA.

[0021] Figure 3G It was demonstrated that the peptide inhibited the binding of Ab-12 to the CD28 antigen as measured by ELISA.

[0022] Figures 4A-4D The kinetic binding of anti-CD28 scFv or Ab-12 to peptide-9 and peptide-12 by Octet is illustrated.

[0023] Figures 5A-5B Binding of anti-CD28 scFv to the Ala scanning peptide of peptide-9 is demonstrated.

[0024] Figures 6A-6B Inhibition of anti-CD28 scFv by Ala scanning peptide of peptide-9 is demonstrated.

[0025] Figure 7 The core sequence motif of the optimized anti-CD28 scFv peptide-9 sequence generated using WebLogo 3.7.4 is illustrated.

[0026] Figures 8A-8C Peptides that inhibit binding of anti-CD28 scFv to CD28 antigen as measured by ELISA are illustrated.

[0027] Figures 9A-9C Peptides that inhibit the binding of Ab-12 to the CD28 antigen by ELISA are shown.

[0028] Figures 10A-10UIllustrated are the kinetic binding of anti-CD28 scFv to peptides as measured by Octet.

[0029] Figure 11A Depicted are the binding of Ab-12 and anti-PD-L1 Fab 1 (SEQ ID NOs: 16 and 17) to PD-L1 as measured by ELISA.

[0030] Figure 11B Depicted are the binding of Ab-12 and anti-CD28 scFv (SEQ ID NO: 9) to CD28 as measured by ELISA.

[0031] Figure 11C Binding of Ab-12 and Ab-13 to PD-L1 as measured by ELISA is illustrated.

[0032] Figure 11D Binding of Ab-12 and Ab-13 to CD28 as measured by ELISA is illustrated.

[0033] Figure 11E Depicted are binding of Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, and Ab-12 to PD-L1 as measured by ELISA. In some cases, the antibodies were incubated with MTSP1.

[0034] Figure 11F Binding of Ab-12, Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, and Ab-7 to CD28 as measured by ELISA is illustrated. In some cases, the antibodies were incubated with MTSP1.

[0035] Figure 11G Depicted are binding of Ab-12, Ab-1, Ab-2, Ab-5, and Ab-6 to PD-L1 as measured by ELISA. In some cases, the antibodies were incubated with MMP9.

[0036] Figure 11H Binding of Ab-12, Ab-1, Ab-2, Ab-5, and Ab-6 to CD28 as measured by ELISA is illustrated. In some cases, the antibodies were incubated with MMP9.

[0037] Figure 11I Binding of Ab-12, Ab-8, Ab-9, Ab-10, and Ab-11 to CD28 as measured by ELISA is illustrated. In some cases, the antibodies were incubated with MTSP1.

[0038] Figure 11JDepicted is the binding of Ab-12, Ab-5, Ab-1 and Ab-9 to CD28 as measured by ELISA.

[0039] Figure 11K Depicted are the binding of Ab-12, Ab-5, Ab-1 and Ab-9 to PD-L1 as measured by ELISA.

[0040] Figure 11L Depicted are the binding of Ab-12, Ab-9, and Ab-9+MTSP1 to PD-L1 as measured by ELISA.

[0041] Figure 11M Depicted are the binding of Ab-12, Ab-9, and Ab-9+MTSP1 to CD28 as measured by ELISA.

[0042] Figures 12A-12D Illustrated are immune cell activation as measured by cytokine release following co-culture of PBMCs with target-coated beads coated with TROP2 and PD-L1 and administration of antibody constructs targeting CD28 and PD-L1 and an anti-TROP2 x CD3 T cell engager (Ab-14).

[0043] Figure 12E Illustrated is a cartoon configuration of an antibody construct targeting CD28 and PD-L1 administered in combination with a T cell engager (TCE) targeting tumor-associated antigens (TAAs) such as TROP2 and CD3 on T cells.

[0044] Figures 13A-13B The results show that the target-coated beads and human PBMC ( Figure 13A ) or cynomolgus monkey PSMC ( Figure 13B ) After co-culture, immune cell activation as measured by IL-2 release. Beads were treated with biotinylated PD-L1 and soluble biotinylated TROP2, and antibody constructs targeting CD28 and PD-L1 were administered as single agents or in combination.

[0045] Figures 14A-14C Described are the results of an in vitro PBMC activation assay using the LNCaP PD-L1 positive tumor cell line, in which various antibody constructs targeting CD28 and PD-L1 were co-administered with Ab-15 in the presence of human PBMCs. In vitro PBMC activation, as measured by cytokine release, was synergistic when various antibody constructs targeting CD28 and PD-L1 were combined with the anti-PSMA x CD3 T cell engager (Ab-15).

[0046] Figures 14D-14FThe results of an in vitro tumor cell killing assay using the LNCaP PDL1-positive tumor cell line in the presence of human PBMCs are presented. In vitro tumor cell killing was enhanced when various antibody constructs targeting CD28 and PD-L1 were combined with an anti-PSMA x CD3 T cell engager (Ab-15) or a masked PSMA x CD3 T cell engager (Ab-16). Tumor cell killing was mask-dependent, with removal of the mask via cleavage of MTSP1 leading to enhanced tumor cell killing.

[0047] Figure 15A Illustrated is a cartoon configuration of a multispecific antibody targeting CD28 and PD-L1 administered in combination with a T cell engager targeting tumor-associated antigens (TAAs) such as TROP2 and CD3 on T cells.

[0048] Figure 15B Illustrated are immune cell activation measured via IL-2 induction following co-culture of PBMCs with MDAMB231 tumor cells and the indicated antibodies.

[0049] Figure 16A and 16C Illustrated are immune cell activation measured via IL-2 induction following co-culture of PBMCs with MDAMB231 tumor cells and the indicated antibodies. Figure 16B Illustrated is a cartoon configuration of a multispecific antibody targeting CD28 and PD-L1 administered in combination with a T cell engager targeting tumor-associated antigens (TAAs) such as TROP2 and CD3 on T cells.

[0050] Figure 17 The pharmacokinetics of Ab-12 and Ab-9 in cynomolgus monkeys following a single IV bolus injection are demonstrated.

[0051] Figures 18A-18C Cytokine release in cynomolgus monkeys following a single IV bolus injection of Ab-12 and Ab-9 is illustrated.

[0052] Figures 19A-19D Depicted are serum liver enzymes in cynomolgus monkeys following a single IV bolus injection of Ab-12 and Ab-9.

[0053] Figures 20A-20D Depicted are the binding results of Ab-12 (unmasked antibody that binds to PD-L1 and CD28 in Vh format), Ab-9 (antibody that binds to PD-L1 and CD28 in cleavable masked Vh format), and Ab-19 (antibody that binds to PD-L1 and CD28 in uncleavable masked Vh format) to human PBMCs or cynomolgus monkey PBMCs by flow cytometry.

[0054] Figure 21 Depicted are the results of PD-1 reporter assays for Ab-12, Ab-9, Pembrolizumab, Atezolizumab, and Nivolumab.

[0055] Figure 22 Depicted are the results of CD28 reporter assays for Ab-12, Ab-9, Ab-19, and TGN1412.

[0056] Figure 23A Depicted are the results of in vitro IL-2 induction by Ab-12, Ab-9, and Ab-19 from mixed lymphocyte reactions of human PBMCs and tumor cells. Also shown are cleaved Ab-9 using MTSP1 and MMP9. Figure 23B Depicted are the results of Ab-12 in combination with pembrolizumab, Ab-9 in combination with pembrolizumab, MMP9-cleaved Ab-9 in combination with pembrolizumab, and MTSP1-cleaved Ab-9 in combination with pembrolizumab.

[0057] Figure 24 Depicted are the results of Ab-12, Ab-9, and Ab-19 binding to PD-L1 on the PD-L1 expressing MDA MB231 tumor cell line.

[0058] Figure 25A Illustrated is a cartoon configuration of a multispecific antibody targeting CD28 and PD-L1 administered in combination with a T cell engager targeting tumor-associated antigens (TAAs) such as EGFR and CD3 on T cells.

[0059] Figures 25B-25C Tumor cell killing of CAL27 tumor cells by Ab-12, Ab-9, Ab-18 alone or in combination with 1 pM of Ab-20 (EGFR T cell engager) is illustrated. The results of the graph are also summarized in Table 28.

[0060] Figures 25D-25F Depicted are cytokine induction (IFNγ, TNF, and IL-2) from human PBMC co-cultured with Cal27 tumor cells in the presence of titrated Ab-12 or titrated Ab-12 in combination with 1 pM Ab-20 in human serum-supplemented medium.

[0061] Figures 25G-25IDepicted are cytokine induction (IFNγ, TNF, and IL-2) from human PBMCs co-cultured with Cal27 tumor cells in the presence of titrated Ab-9 or titrated Ab-9 in combination with 1 pM Ab-20, and also titrated Ab-18 or titrated Ab-18 in combination with 1 pM Ab-20 in medium supplemented with human serum.

[0062] Figure 26 Depicted are the in vivo tumor growth kinetics (mean tumor volume) of MDAMB231 in immunocompromised mice following treatment with Ab-22 in combination with Ab-18, or Ab-21 in combination with Ab-17, or Ab-17 alone, or Ab-21 alone.

[0063] Figure 27 The pharmacokinetics of non-human primates dosed with 15 mg / kg, 5 mg / kg, and 1 mg / kg of Ab-9 are demonstrated.

[0064] Figures 28A-28E Cytokine release (IFNγ, TNF, IL-2, IL-6, and IL-10) in non-human primates following administration of 15 mg / kg, 5 mg / kg, and 1 mg / kg of Ab-9 is demonstrated.

[0065] Figures 29A-29E Depicted are clinical chemistry results (AST, ALT, TBIL, CRE, urea) for nonhuman primates dosed with 15 mg / kg, 5 mg / kg, and 1 mg / kg of Ab-9. DETAILED DESCRIPTION

[0066] Bispecific antibodies for redirecting T cells to mediate cancer cell killing have shown promise in both preclinical and clinical studies. Effective T cell activation has been obtained with single-chain variable fragments (scFv), particularly bispecific T cell engagers (BiTE) formats, in which one scFv targets tumor cell antigens, and another scFv targets epitopes such as CD3 involved in T cell activation. One such example of BiTE is blinatumomab targeting CD19 and CD3, which has been approved in Europe and the U.S. for the treatment of CD19+ B cell acute lymphoblastic leukemia resistant to chemotherapy. Although T cell engagers such as blinatumomab have made progress, some patients have poor responses to treatment, even if the patient expresses tumor antigens, and the reasons are not fully understood.

[0067] Strategies for increasing T cell cytotoxicity of T cell engagers have been explored by co-administering with secondary antibodies targeting the co-inhibitory immune checkpoints programmed death ligand 1 (PD-L1) and / or CD28. CD28 is a protein expressed on T cells that provides co-stimulatory signals required for T cell activation and survival. It is known that stimulatory signaling through CD28 combined with BiTEs increases T cell-induced cytotoxicity of tumor cells. However, in previous studies, obtaining T cell-mediated cytotoxicity of tumor cells mainly required the presence of BiTEs with tumor binding domains, such as anti-CD19 antibodies and CD3 binding domains, while single-agent administration of anti-CD28 and anti-PD-L1 in the form of scFv-scFv was found not to induce T cell-mediated cytotoxicity against tumor cells.

[0068] The activation of T cells is a highly regulated process that typically requires two signaling events for full functionality: the first signal is initiated after binding of the MHC-antigen complex, which helps distinguish "self" from "non-self" with the T cell receptor (TCR) and the second signal through activation of co-stimulatory receptors. Although the first recognition signal activates the T cell and triggers T cell-mediated toxicity of the recognized cell, if the T cell does not receive the second co-stimulatory signal, it can lead to T cell tolerance, whereby the T cell continues to recognize tumor antigens but does not mount an immune response against the tumor cell. The second co-stimulatory signal prevents T cell tolerance and further activates the T cell to enhance T cell cytotoxicity against the target cell.

[0069] As described herein, multispecific antibodies comprising a CD28-binding domain and a PD-L1-binding domain are designed to act as both antagonists of PD-L1 and conditional agonists of CD28. Although CD28 agonism has shown some clinical promise, the efficacy achieved with this approach has been limited by the dose-limiting toxicity caused by systemic activation of CD28. The multispecific antibodies described herein, comprising a CD28-binding domain and a PD-L1-binding domain, are designed to conditionally agonize CD28 only in the presence of PD-L1, which is often overexpressed by tumors to prevent T cell-mediated killing. Furthermore, 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. Studies of the multispecific antibodies described herein have demonstrated a lack of systemic immune system activation, as evidenced by a lack of cytokine release. Despite unprecedented clinical response rates, the majority of patients do not respond to therapies targeting PD-1 and PD-L1, in part because T cells require co-stimulation for full functionality. Therefore, checkpoint inhibition alone may not be sufficient to fully enable the immune system to attack tumors. As described herein, further benefits may be derived by adding multispecific antibodies.

[0070] Disclosed herein are antibodies that specifically bind to PD-L1 and CD28 and are capable of inducing T cell-mediated cytotoxicity of tumor cells as a single agent or in combination with a T cell engager. Notably, such antibodies targeting PD-L1 and CD28 are capable of inducing 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. Such antibodies that specifically bind to PD-L1 and CD28 are not in the form of scFv-scFv.

[0071] Disclosed herein are isolated multispecific antibodies according to the formula: P1-L1-A1-LB (Formula I), wherein A1 comprises a CD28 binding domain; B comprises a PD-L1 binding domain; L comprises a linker connecting A1 and B; P1 comprises a peptide that binds to A1, and L1 comprises a linker that connects A1 to P1 and is a substrate for a tumor-specific protease, wherein P1 comprises an amino acid sequence according to any one of SEQ ID NOs: 24-53, 128-148, and any one of the amino acid sequences of Table 20, or an amino acid sequence having 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 24-53, 128-148, and any one of the amino acid sequences of Table 20.

[0072] Disclosed herein are isolated multispecific antibodies comprising the formula: P1-L1-A1-LB (Formula I), wherein A1 comprises a CD28 binding domain; B comprises a PD-L1 binding domain; L comprises a linker connecting A1 and B; P1 comprises a peptide that binds to A1, and L1 comprises a linker portion that connects A1 to P1 and is a substrate for a tumor-specific protease, wherein P1 comprises an amino acid sequence according to any one of SEQ ID NOs: 24-53, 128-148, and any one of the amino acid sequences of Table 20, or an amino acid sequence having 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 24-53, 128-148, and any one of the amino acid sequences of Table 20.

[0073] Disclosed herein are isolated multispecific antibodies comprising the formula: P1-L1-A1-LB (Formula I), wherein A1 is a CD28 binding domain; B is a PD-L1 binding domain; L is a linker connecting A1 and B; P1 is a peptide that binds to A1, and L1 is a linker that connects A1 to P1 and is a substrate for a tumor-specific protease, wherein P1 comprises an amino acid sequence according to any one of SEQ ID NOs: 24-53, 128-148, and any one of the amino acid sequences of Table 20, or an amino acid sequence having 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 24-53, 128-148, and any one of the amino acid sequences of Table 20.

[0074] Disclosed herein are isolated multispecific antibodies according to the formula: P1-L1-A1-LB (Formula I), wherein A1 is a CD28 binding domain; B is a PD-L1 binding domain; L is a linker connecting A1 and B; P1 is a peptide that binds to A1, and L1 is a linker that connects A1 to P1 and is a substrate for a tumor-specific protease, wherein P1 comprises an amino acid sequence according to any one of SEQ ID NOs: 24-53, 128-148, and any one of the amino acid sequences of Table 20, or an amino acid sequence having 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 24-53, 128-148, and any one of the amino acid sequences of Table 20.

[0075] In some embodiments, the multispecific antibody is according to the formula: P1-L1-A1-LB-L2-P2 (Formula Ia), wherein P2 comprises a peptide that binds to B, and L2 comprises a linker portion that connects B to P2 and is a substrate for a tumor-specific protease.

[0076] In some embodiments, the multispecific antibody comprises the formula: P1-L1-A1-LB-L2-P2 (Formula Ia), wherein P2 comprises a peptide that binds to B, and L2 comprises a linker portion that connects B to P2 and is a substrate for a tumor-specific protease.

[0077] In some embodiments, the multispecific antibody comprises the formula: P1-L1-A1-LB-L2-P2 (Formula Ia), wherein P2 is a peptide that binds to B, and L2 is a linker that connects B to P2 and is a substrate for a tumor-specific protease.

[0078] In some embodiments, the multispecific antibody is according to the formula: P1-L1-A1-LB-L2-P2 (Formula Ia), wherein P2 is a peptide that binds to B, and L2 is a linker that connects B to P2 and is a substrate for a tumor-specific protease.

[0079] Although preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided as examples only. Without departing from the present disclosure, those skilled in the art will now appreciate that many variations, changes, and substitutions may be made. It should be understood that various alternatives to the disclosed embodiments described herein may be employed in practicing the present disclosure. The accompanying claims are intended to define the scope of the present disclosure, and therefore encompass methods and structures within the scope of these claims and their equivalents.

[0080] definition

[0081] The terms used herein are for the purpose of describing a particular situation only and are 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 the terms "including," "includes," "having," "has," "with," or variations thereof are used in the detailed description and / or claims, these terms are intended to be inclusive in a manner similar to the term "comprising."

[0082] The term "antibody" is used in the broadest sense to cover fully assembled antibodies; antibody fragments that can bind antigen, such as Fab, F(ab')2, Fv, single-chain antibodies (scFv), diabodies, antibody chimeras, hybrid antibodies, bispecific antibodies, etc.

[0083] The term "complementarity determining region" or "CDR" is a segment of an antibody variable region that is structurally complementary to the epitope to which the antibody binds and is more variable than the rest of the variable region. For this reason, CDRs are sometimes referred to as hypervariable regions. The variable region comprises three CDRs. CDR peptides can be obtained by constructing genes encoding the CDRs of the antibody of interest. For example, these genes are prepared by synthesizing the variable regions from RNA of antibody-producing cells using the polymerase chain reaction. See, e.g., 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.), pp. 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.), pp. 137-185 (Wiley-Liss, Inc. 1995).

[0084] In some cases, the CDRs of an antibody are identified 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 5th ed., US Department of Health and Human Services, NIH Publication No. 91-3242); or (ii) the Chothia numbering scheme, referred to herein as "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 et al., 1993, J. Mol. Biol., 234:817-821). A et al., 1990, J. Mol. Biol. 215(1):175-82; and U.S. Patent No. 7,709,226); or (iii) the ImMunoGeneTics (IMGT) numbering system, e.g., 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).

[0085] With respect to the Kabat numbering system, the CDRs within an antibody heavy chain molecule are typically present at amino acid positions 31 to 35, which may optionally include one or two additional amino acids after 35 (referred to as 35A and 35B in the Kabat numbering scheme) (CDR1), amino acid positions 50 to 65 (CDR2), and amino acid positions 95 to 102 (CDR3). Using the Kabat numbering system, the CDRs within an antibody light chain molecule are typically present at amino acid positions 24 to 34 (CDR1), amino acid positions 50 to 56 (CDR2), and amino acid positions 89 to 97 (CDR3). As is well known to those skilled in the art, using the Kabat numbering system, the actual linear amino acid sequence of an antibody variable domain may contain fewer or more amino acids due to shortening or lengthening of FRs and / or CDRs, and therefore the Kabat numbering of an amino acid is not necessarily the same as its linear amino acid numbering.

[0086] The term "Fab" refers to a protein containing a light chain constant domain and the first constant domain (CH1) of the heavy chain. Fab fragments differ from Fab' fragments by the addition of a few residues to the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH, referred to herein as Fab', wherein the cysteine ​​residues of the constant domains bear a free thiol group. Fab' fragments are generated by reducing the heavy chain disulfide bridges of the F(ab')2 fragment. Other chemical couplings of antibody fragments are also known.

[0087] "Single-chain variable fragment (scFv)" is a fusion protein of the variable regions of the heavy chain (VH) and light chain (VL) of an antibody, connected by a short linker peptide of ten to about 25 amino acids. For flexibility, the linker is usually rich in glycine, while for solubility, the linker is usually rich in serine or threonine, and the N-terminus of VH can be connected to the C-terminus of VL, and vice versa. This protein retains the specificity of the original antibody, despite the removal of the constant region and the introduction of a linker. scFv antibodies are described, for example, in Houston, JS, Methods in Enzymol. 203 (1991) 46-96). In addition, the antibody fragment comprises a single-chain polypeptide having the characteristics of a VH domain, i.e., capable of being assembled together with a VL domain, or having the characteristics of a VL domain, i.e., capable of being assembled together with a VH domain to form a functional antigen binding site, thereby providing the antigen binding properties of a full-length antibody.

[0088] The term "multispecific" refers to the ability of an antibody to specifically bind to two or more different antigenic determinants, e.g., two or more binding sites, each formed by a pair of antibody heavy chain variable domains (VH) and antibody light chain variable domains (VL), or in the case of a single domain antibody, a single variable domain that binds to a different antigen.

[0089] As used herein, term " percentage (%) amino acid sequence identity " about sequence is defined as, after comparing sequences and introducing room (if necessary) to realize maximum percentage sequence identity and any conservative substitution is not considered as a part for sequence identity, the percentage of the amino acid residue identical with the amino acid residue in the particular sequence in the candidate sequence.Comparison for determining percentage amino acid sequence identity can be realized in a variety of ways well known by persons skilled in the art, for example, 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 the appropriate parameters for measuring the comparison, are included in and realize maximum comparison required any algorithm on the total length of the sequence being compared.

[0090] In the case of amino acid sequence comparisons using ALIGN-2, the % amino acid sequence identity of a given amino acid sequence A with, and, or against a given amino acid sequence B (which can alternatively be expressed as a given amino acid sequence A having or comprising a certain % amino acid sequence identity with, and, or against a given amino acid sequence B) is calculated as follows: 100 multiplied by a fraction X / Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in the program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be understood that when 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 be equal to the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

[0091] The terms "individual," "subject," and "patient" are used interchangeably herein and refer to any mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is non-human. None of these terms require or are limited to situations characterized by supervision (e.g., continuous or intermittent) by a healthcare worker (e.g., a physician, registered nurse, licensed nurse, physician assistant, caregiver, or hospice worker).

[0092] Peptide (P1) or (P2)

[0093] In some embodiments, P1 comprises an amino acid sequence according to any one of SEQ ID NOs: 24-53, 128-148, and the amino acid sequence of Table 20.

[0094] In some embodiments, P1 comprises an amino acid sequence according to any one of SEQ ID NOs: 42-53 or an amino acid sequence having 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 42-53. In some embodiments, P1 comprises an amino acid sequence according to any one of SEQ ID NOs: 42-53.

[0095] In some embodiments, P1 comprises an amino acid sequence according to any one of the amino acid sequences in Table 20, or an amino acid sequence having 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of the amino acid sequences in Table 20. In some embodiments, P1 comprises an amino acid sequence according to any one of the amino acid sequences in Table 20.

[0096] In some embodiments, P1 comprises an amino acid sequence according to any one of SEQ ID NOs: 128-147, or an amino acid sequence having 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 128-147. In some embodiments, P1 comprises an amino acid sequence according to any one of SEQ ID NOs: 128-147.

[0097] In some embodiments, P1 comprises a sequence according to X1-X2-X3-C-X4-X5-X6-X7-X8-X9-X 10 -CX 11 -X 12 an amino acid sequence wherein X1 is selected from the group consisting of M, I, L, and V; X2 is selected from the group consisting of D, H, N, A, F, S, T, Y, and V; X3 is selected from the group consisting of W, L, and F; X4 is selected from the group consisting of P, A, and L; X5 is selected from the group consisting of R, T, I, M, S, K, L, V, W, F, A, P, and D; X6 is selected from the group consisting of E, D, Y, H, S, F, A, N, T, I, P, and V; X7 is selected from the group consisting of L, M, R, S, Q, and H; X8 is selected from the group consisting of W and Q; X9 is selected from the group consisting of H, N, D, A, S, Y, T, F, V, L, and I; X 10 Selected from E, V, L, D, Y, R, Q, H, F, K, A, M and N; X 11 is selected from F, Y, L, W, and V; and X 12 is selected from N, A, F, S, Y, H, D, T, and L. In some embodiments, X1 is selected from M, I, and L; X2 is selected from D, H, N, and A; X3 is W; X4 is P; X5 is selected from R, T, I, M, S, and K; X6 is selected from E, D, Y, H, S, and F; X7 is selected from L, M, and R; X8 is W; X9 is selected from H, N, D, A, S, and V; X 10 Selected from E, V, L, D and H; X 11 is selected from F, Y and L; and X 12is selected from N, A, F, S, and Y. In some embodiments, X1 is M; X2 is selected from D and H; X3 is W; X4 is P; X5 is selected from R, T, and I; X6 is selected from E, D, and Y; X7 is selected from L, M, and R; X8 is W; X9 is selected from H, N, D, and V; X 10 Selected from E, V, L, D and H; X 11 is F; and X 12 Selected from N, A and F.

[0098] In some embodiments, P1 comprises the amino acid sequence according to SEQ ID NO: 32 or an amino acid sequence having 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to SEQ ID NO: 32. In some embodiments, P1 comprises the amino acid sequence according to SEQ ID NO: 32.

[0099] In some embodiments, P1 comprises the amino acid sequence according to SEQ ID NO: 138, or an amino acid sequence having 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to SEQ ID NO: 138. In some embodiments, P1 comprises the amino acid sequence according to SEQ ID NO: 138.

[0100] In some embodiments, P1 weakens the binding of A1 to CD28. In some embodiments, P1 binds to A1 through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bond interactions, or a combination thereof. In some embodiments, P1 binds to A1 at or near the antigen binding site. In some embodiments, when L1 is cleaved by a tumor-specific protease, P1 unbinds from A1, thereby exposing A1 to CD28. In some embodiments, P1 has less than 75% sequence identity with CD28. In some embodiments, P1 has less than 80% sequence identity with CD28. In some embodiments, P1 has less than 85% sequence identity with CD28. In some embodiments, P1 has less than 90% sequence identity with CD28. In some embodiments, P1 has less than 95% sequence identity with CD28. In some embodiments, P1 comprises a de novo generated amino acid sequence that shares less than 10% sequence identity with CD28.

[0101] In some embodiments, P2 weakens the binding of B to PD-L1. In some embodiments, P2 binds to B through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bonding interactions, or a combination thereof.

[0102] In some embodiments, P2 binds to B at or near the antigen binding site. In some embodiments, when L2 is cleaved by a tumor-specific protease, P2 unbinds from B, thereby exposing B to PD-L1. In some embodiments, P2 has less than 70% sequence identity with PD-L1. In some embodiments, P2 has less than 75% sequence identity with PD-L1. In some embodiments, P2 has less than 80% sequence identity with PD-L1. In some embodiments, P2 has less than 85% sequence identity with PD-L1. In some embodiments, P2 has less than 90% sequence identity with PD-L1. In some embodiments, P2 has less than 95% sequence identity with PD-L1. In some embodiments, P2 comprises a de novo generated amino acid sequence that shares less than 10% sequence identity with PD-L1. In some embodiments, P2 comprises a peptide sequence that is at least 5 amino acids in length. In some embodiments, P2 comprises a peptide sequence that is at least 6 amino acids in length. In some embodiments, P2 comprises a peptide sequence of at least 10 amino acids in length. In some embodiments, P2 comprises a peptide sequence of at least 10 amino acids in length and no more than 20 amino acids in length. In some embodiments, P2 comprises a peptide sequence of at least 16 amino acids in length. In some embodiments, P2 comprises a peptide sequence of no more than 40 amino acids in length.

[0103] In some embodiments, P1 or P2 comprises at least two cysteine ​​amino acid residues. In some embodiments, P1 or P2 comprises a cyclic peptide or a linear peptide. In some embodiments, P1 or P2 comprises a cyclic peptide. In some embodiments, P1 or P2 comprises a linear peptide. In some embodiments, P1 or P2 comprises a modified amino acid or an unnatural amino acid or a modified unnatural amino acid, or a combination thereof. In some embodiments, P1 or P2 does not comprise albumin or an albumin fragment. In some embodiments, P1 or P2 does not comprise an albumin binding domain.

[0104] Table 1. P1 sequence

[0105]

[0106]

[0107] Connecting part (L1 or L2)

[0108] In some embodiments, L1 or L2 is a peptide sequence having at least 5 to no more than 50 amino acids. In some embodiments, L1 or L2 is a peptide sequence having at least 10 to no more than 30 amino acids. In some embodiments, L1 or L2 is a peptide sequence having at least 10 amino acids. In some embodiments, L1 or L2 is a peptide sequence having at least 18 amino acids. In some embodiments, L1 or L2 is a peptide sequence having at least 26 amino acids. In some embodiments, L1 or L2 comprises (G2S) n wherein n is an integer from 1 to 3 (SEQ ID NO: 228). In some embodiments, L1 or L2 comprises (G2S) n wherein n is an integer of at least 1. In some embodiments, L1 or L2 comprises 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 a metalloprotease, a serine protease, a cysteine ​​protease, a threonine protease, and an aspartic protease. In some embodiments, L1 or L2 comprises an amino acid sequence cleavable by urokinase, an amino acid sequence cleavable by a membrane-type serine protease, or an amino acid sequence cleavable by a matrix metalloprotease. In some embodiments, L1 or L2 comprises a sequence according to SEQ ID NOs:18-19, 62-88. In some embodiments, L1 is bound to the N-terminus of A1. In some embodiments, L1 is bound to the C-terminus of A1. In some embodiments, L2 is bound to the N-terminus of B. In some embodiments, L2 is bound to the C-terminus of B.

[0109] Table 2. L1 or L2

[0110]

[0111]

[0112] CD28 binding domain (A1), PD-L1 binding domain (B) and linker (L)

[0113] In some embodiments, the CD28 binding domain comprises a single-chain variable fragment, a single-domain antibody, Fab, or Fab′. In some embodiments, the CD28 binding domain comprises a single-chain variable fragment. In some embodiments, the CD28 binding domain comprises a single-domain antibody. In some embodiments, the CD28 binding domain comprises Fab or Fab′. In some embodiments, the PD-L1 binding domain comprises a single-chain variable fragment, a single-domain antibody, Fab, or Fab′. In some embodiments, the PD-L1 binding domain comprises Fab or Fab′. In some embodiments, the PD-L1 binding domain comprises Fab or Fab′ and the CD28 binding domain comprises a single-chain variable fragment. In some embodiments, the PD-L1 binding domain comprising Fab or Fab′ comprises a Fab heavy chain polypeptide and a Fab light chain polypeptide, the Fab heavy chain polypeptide comprising a Fab heavy chain variable domain, and the Fab light chain polypeptide comprising a Fab light chain variable domain. In some embodiments, the CD28 binding domain comprising a single-chain variable fragment comprises an scFv heavy chain variable domain and an scFv light chain variable domain. In some embodiments, a linker connects the C-terminus of A1 to the N-terminus of B. In some embodiments, a linker connects the N-terminus of A1 to the C-terminus of B. In some embodiments, a linker connects the C-terminus of A1 to the N-terminus of the Fab heavy chain polypeptide. In some embodiments, a linker connects the N-terminus of A1 to the C-terminus of the Fab heavy chain polypeptide. In some embodiments, a linker connects the C-terminus of A1 to the N-terminus of the Fab light chain polypeptide. In some embodiments, a linker connects the N-terminus of A1 to the C-terminus of the Fab light chain polypeptide. In some embodiments, a linker connects the Fab light chain polypeptide to the scFv light chain variable domain. In some embodiments, a linker connects the Fab light chain polypeptide to the scFv heavy chain variable domain. In some embodiments, a linker connects the Fab heavy chain polypeptide to the scFv light chain variable domain. In some embodiments, a linker connects the Fab heavy chain polypeptide to the scFv heavy chain variable domain. In some embodiments, a linker connects the Fab light chain polypeptide to the N-terminus of the scFv light chain variable domain. In some embodiments, a linker connects the Fab light chain polypeptide to the C-terminus of the scFv light chain variable domain. In some embodiments, a linker connects the Fab light chain polypeptide to the N-terminus of the scFv heavy chain variable domain. In some embodiments, a linker connects the Fab light chain polypeptide to the C-terminus of the scFv heavy chain variable domain. In some embodiments, a linker connects the Fab heavy chain polypeptide to the N-terminus of the scFv light chain variable domain. In some embodiments, a linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain. In some embodiments, a linker connects the Fab heavy chain polypeptide to the N-terminus of the scFv heavy chain variable domain.In some embodiments, a linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv heavy chain variable domain.

[0114] 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 in length 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 15 amino acids in length. In some embodiments, the linker comprises (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, L comprises (G2S) n wherein n is an integer from 1 to 3 (SEQ ID NO: 228). In some embodiments, L comprises the amino acid sequence of SEQ ID NO: 18 (GGGGSGGGGSGGGGS) or SEQ ID NO: 19 (GGGGS).

[0115] Table 3. Linker sequences

[0116]

[0117] 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, HC-CDR2, and 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 the CDRs comprise 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, LC-CDR2, and LC-CDR3 of the scFv light chain variable domain comprise: LC-CDR1: SEQ ID NO: 4; LC-CDR2: SEQ ID NO: 5(KA); and LC-CDR3: SEQ ID NO: 6, and wherein the CDRs comprise 0-2 amino acid modifications in at least one of said LC-CDR1, LC-CDR2, or LC-CDR3. In some embodiments, A1 comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, LC-CDR2, and LC-CDR3 of A1 comprise: LC-CDR1: SEQ ID NO: 4; LC-CDR2: SEQ ID NO: 5 (KA); and LC-CDR3: SEQ ID NO: 6; wherein A1 comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 of A1 comprise: HC-CDR1: SEQ ID NO: 1; HC-CDR2: SEQ ID NO: 2; HC-CDR3: SEQ ID NO: 3.

[0118] Table 4. Anti-CD28 Heavy Chain Polypeptide Complementarity Determining Regions (CDRs) as Defined by IMGT

[0119]

[0120] Table 5. Anti-CD28 light chain polypeptide complementarity determining regions (CDRs) as defined by IMGT

[0121]

[0122] In some embodiments, the Fab heavy chain variable domain comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein HC-CDR1, HC-CDR2, and 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 the CDRs comprise 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, LC-CDR2, and LC-CDR3 of the Fab light chain variable domain comprise: LC-CDR1: SEQ ID NO: 13; LC-CDR2: SEQ ID NO: 14 (DA); and LC-CDR3: SEQ ID NO: 15, and wherein the CDRs comprise 0-2 amino acid modifications in at least one of said LC-CDR1, LC-CDR2, or LC-CDR3. In some embodiments, B comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 of B comprise: HC-CDR1: SEQ ID NO: 10; HC-CDR2: SEQ ID NO: 11; HC-CDR3: SEQ ID NO: 12; and wherein B comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, LC-CDR2, and LC-CDR3 of B comprise: LC-CDR1: SEQ ID NO: 13; LC-CDR2: SEQ ID NO: 14 (DA); and LC-CDR3: SEQ ID NO: 15.

[0123] Table 6. Anti-PD-L1 Heavy Chain Polypeptide Complementarity Determining Regions (CDRs) as Defined by IMGT

[0124]

[0125] Table 7. Anti-PD-L1 light chain polypeptide complementarity determining regions (CDRs) as defined by IMGT

[0126]

[0127] In some embodiments, the scFv heavy chain variable domain comprises an amino acid sequence having at least 80% sequence identity with 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 with 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.

[0128] In some embodiments, the scFv light chain variable domain comprises an amino acid sequence having at least 80% sequence identity with 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 with 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.

[0129] In some embodiments, the scFv comprises an amino acid sequence having 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 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.

[0130] Table 8. Anti-CD28 light chain variable domain, heavy chain variable domain and full-length sequences. CDR sequences are underlined and determined using the IMGT definition.

[0131]

[0132] In some embodiments, the Fab heavy chain polypeptide comprises an amino acid sequence having at least 80% sequence identity to 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 contiguous 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 contiguous 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 contiguous amino acid residues of SEQ ID NO: 17 and has at least 80% sequence identity to at least 215 contiguous 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.

[0133] In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence having 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 contiguous 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 contiguous 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 contiguous amino acid residues of SEQ ID NO: 16 and has at least 80% sequence identity to at least 200 contiguous 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.

[0134] Table 9. Anti-PD-L1 Fab light chain polypeptide and Fab heavy chain polypeptide sequences. CDR sequences are underlined and determined using the IMGT definition.

[0135]

[0136] In some embodiments, a 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 having at least 80% sequence identity to an amino acid sequence according to SEQ ID NO: 20, and the amino acid sequence of the Fab heavy chain polypeptide connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence according to SEQ ID NO: 21. In some embodiments, a 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 the amino acid sequence of the Fab heavy chain polypeptide connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence according to SEQ ID NO: 21. In some embodiments, a 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 having at least 80% sequence identity to an amino acid sequence according to SEQ ID NO: 23, and the amino acid sequence of the Fab light chain polypeptide connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence according to SEQ ID NO: 22. In some embodiments, a 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 the amino acid sequence of the Fab light chain polypeptide connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence according to SEQ ID NO: 22.

[0137] Table 10. Antibodies that bind to CD28 and PD-L1

[0138]

[0139]

[0140]

[0141] Half-life extending molecules (H1)

[0142] In some embodiments, the multispecific antibody further comprises a half-life extension molecule (H1). In some embodiments, H1 is connected to P1. In some embodiments, H1 is connected to P2. In some embodiments, H1 does not block A1 from binding to CD28. In some embodiments, H1 does not block B from binding to PD-L1. H1 comprises a linker portion (L5) connecting H1 to P1 or H1 to P2. In some embodiments, the half-life extension molecule (H1) does not have binding affinity for PD-L1. In some embodiments, the half-life extension molecule (H1) does not have binding affinity for CD28. In some embodiments, the half-life extension molecule (H1) does not protect the multispecific antibody from CD28. In some embodiments, H1 comprises a sequence according to SEQ ID NO: 54-57. In some embodiments, H1 comprises an amino acid sequence having a repeating sequence motif. In some embodiments, H1 comprises an amino acid sequence having a highly ordered secondary structure. In some embodiments, H1 comprises a polymer. In some embodiments, the polymer is polyethylene glycol (PEG). In some embodiments, H1 comprises albumin. In some embodiments, H1 comprises an Fc domain. In some embodiments, the albumin is serum albumin. In some embodiments, the albumin is human serum albumin. In some embodiments, H1 comprises a polypeptide, ligand, or small molecule. In some embodiments, the polypeptide, ligand, or small molecule binds to a serum protein or fragment thereof, a circulating immunoglobulin or fragment thereof, or CD35 / CR1. In some embodiments, the serum protein comprises thyroxine-binding protein, transthyretin, 1-acid glycoprotein, transferrin, transferrin receptor or a transferrin-binding portion thereof, fibrinogen, or 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, Fab, or 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 antibody or a humanized antibody. In some embodiments, the single domain antibody is selected from 645gH1gL1, 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 a complementarity determining region (CDR): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and 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 0-2 amino acid modifications in at least one of HC-CDR1, HC-CDR2, or HC-CDR3. In some embodiments, H1 comprises an amino acid sequence according to SEQ ID NO: 57. In some embodiments, H1 comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 57. In some embodiments, H1 comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 57. In some embodiments, H1 comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 57. In some embodiments, H1 comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 57. In some embodiments, H1 comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 57. In some embodiments, H1 comprises a modified amino acid or a non-natural amino acid or a modified non-natural amino acid, or a combination thereof. In some embodiments, the modified amino acid or the modified non-natural amino acid comprises a post-translational modification. In some embodiments, H1 comprises a linker (L5) connecting H1 to P1 or P2. In some embodiments, L5 is a peptide sequence having at least 5 to no more than 50 amino acids. In some embodiments, L5 is a peptide sequence having at least 10 to no more than 30 amino acids. In some embodiments, L5 is a peptide sequence having at least 10 amino acids. In some embodiments, L5 is a peptide sequence having at least 18 amino acids. In some embodiments, L5 is a peptide sequence having at least 26 amino acids. In some embodiments, L5 comprises a formula selected from the following: (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.

[0143] In some embodiments, the single-domain antibody comprises a complementarity determining region (CDR): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 of the single-domain antibody comprise: HC-CDR1: SEQ ID NO: 204, HC-CDR2: SEQ ID NO: 205, and HC-CDR3: SEQ ID NO: 206; and wherein the CDRs comprise 0-2 amino acid modifications in at least one of HC-CDR1, HC-CDR2, or HC-CDR3. In some embodiments, H1 comprises an amino acid sequence according to SEQ ID NO: 207. In some embodiments, H1 comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 207. In some embodiments, H1 comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 207. In some embodiments, H1 comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 207. In some embodiments, H1 comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 207. In some embodiments, H1 comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 207. In some embodiments, H1 comprises a modified amino acid or a non-natural amino acid or a modified non-natural amino acid, or a combination thereof. In some embodiments, the modified amino acid or the modified non-natural amino acid comprises a post-translational modification. In some embodiments, H1 comprises a linker (L5) connecting H1 to P1 or P2. In some embodiments, L5 is a peptide sequence having at least 5 to no more than 50 amino acids. In some embodiments, L5 is a peptide sequence having at least 10 to no more than 30 amino acids. In some embodiments, L5 is a peptide sequence having at least 10 amino acids. In some embodiments, L5 is a peptide sequence having at least 18 amino acids. In some embodiments, L5 is a peptide sequence having at least 26 amino acids. In some embodiments, L5 comprises a formula selected from the following: (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.

[0144] Table 11. H1 sequences

[0145]

[0146] Tumor-activating multispecific antibodies that bind to CD28 and PD-L1

[0147] In some embodiments, the isolated multispecific antibody comprises an amino acid sequence that has at least 80% sequence identity to any one of SEQ ID NOs: 149-170. In some embodiments, the isolated multispecific antibody comprises an amino acid sequence that has at least 85% sequence identity to any one of SEQ ID NOs: 149-170. In some embodiments, the isolated multispecific antibody comprises an amino acid sequence that has at least 90% sequence identity to any one of SEQ ID NOs: 149-170. In some embodiments, the isolated multispecific antibody comprises an amino acid sequence that has at least 95% sequence identity to any one of SEQ ID NOs: 149-170. In some embodiments, the isolated multispecific antibody comprises an amino acid sequence that has at least 99% sequence identity to any one of SEQ ID NOs: 149-170. In some embodiments, the isolated multispecific antibody comprises an amino acid sequence of any one of SEQ ID NOs: 149-170.

[0148] In some embodiments, the isolated multispecific antibody comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NOs: 149 and 150. In some embodiments, the isolated multispecific antibody comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NOs: 149 and 150.

[0149] In some embodiments, the isolated multispecific antibody comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NOs: 151 and 152. In some embodiments, the isolated multispecific antibody comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NOs: 151 and 152.

[0150] In some embodiments, the isolated multispecific antibody comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NOs: 153 and 154. In some embodiments, the isolated multispecific antibody comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NOs: 153 and 154.

[0151] In some embodiments, the isolated multispecific antibody comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NOs: 155 and 156. In some embodiments, the isolated multispecific antibody comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NOs: 155 and 156.

[0152] In some embodiments, the isolated multispecific antibody comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NOs: 157 and 158. In some embodiments, the isolated multispecific antibody comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NOs: 157 and 158.

[0153] In some embodiments, the isolated multispecific antibody comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NOs: 159 and 160. In some embodiments, the isolated multispecific antibody comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NOs: 159 and 160.

[0154] In some embodiments, the isolated multispecific antibody comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NOs: 161 and 162. In some embodiments, the isolated multispecific antibody comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NOs: 161 and 162.

[0155] In some embodiments, the isolated multispecific antibody comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NOs: 163 and 164. In some embodiments, the isolated multispecific antibody comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NOs: 163 and 164.

[0156] In some embodiments, the isolated multispecific antibody comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NOs: 165 and 166. In some embodiments, the isolated multispecific antibody comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NOs: 165 and 166.

[0157] In some embodiments, the isolated multispecific antibody comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NOs: 167 and 168. In some embodiments, the isolated multispecific antibody comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NOs: 167 and 168.

[0158] In some embodiments, the isolated multispecific antibody comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NOs: 169 and 170. In some embodiments, the isolated multispecific antibody comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NOs: 169 and 170.

[0159] In some embodiments, the isolated multispecific antibody comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NOs: 208 and 209. In some embodiments, the isolated multispecific antibody comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NOs: 208 and 209.

[0160] Table 12. Sequences of tumor-activating multispecific antibodies that bind to CD28 and PD-L1

[0161]

[0162]

[0163]

[0164]

[0165]

[0166]

[0167]

[0168]

[0169] Polynucleotides encoding tumor-activating multispecific antibodies that bind to CD28 and PD-L1

[0170] Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding the multispecific antibodies disclosed herein.

[0171] In some embodiments, disclosed herein are isolated recombinant nucleic acid molecules encoding an isolated multispecific antibody according to the formula: P1-L1-A1-LB (Formula I), wherein A1 comprises a CD28 binding domain; B comprises a PD-L1 binding domain; L comprises a linker connecting A1 and B; P1 comprises a peptide that binds to A1, and L1 comprises a linker that connects A1 to P1 and is a substrate for a tumor-specific protease, wherein P1 comprises an amino acid sequence according to any one of SEQ ID NOs: 24-53, 128-148, and any one of the amino acid sequences of Table 20, or an amino acid sequence having 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 24-53, 128-148, and any one of the amino acid sequences of Table 20.

[0172] In some embodiments, disclosed herein are isolated recombinant nucleic acid molecules encoding an isolated multispecific antibody comprising the formula: P1-L1-A1-LB (Formula I), wherein A1 comprises a CD28 binding domain; B comprises a PD-L1 binding domain; L comprises a linker connecting A1 and B; P1 comprises a peptide that binds to A1, and L1 comprises a linker that connects A1 to P1 and is a substrate for a tumor-specific protease, wherein P1 comprises an amino acid sequence according to any one of SEQ ID NOs: 24-53, 128-148, and any one of the amino acid sequences of Table 20, or an amino acid sequence having 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 24-53, 128-148, and any one of the amino acid sequences of Table 20.

[0173] In some embodiments, disclosed herein are isolated recombinant nucleic acid molecules encoding an isolated multispecific antibody comprising the formula: P1-L1-A1-LB (Formula I), wherein A1 is a CD28 binding domain; B is a PD-L1 binding domain; L is a linker connecting A1 and B; P1 is a peptide that binds to A1, and L1 is a linker that connects A1 to P1 and is a substrate for a tumor-specific protease, wherein P1 comprises an amino acid sequence according to any one of SEQ ID NOs: 24-53, 128-148, and any one of the amino acid sequences of Table 20, or an amino acid sequence having 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 24-53, 128-148, and any one of the amino acid sequences of Table 20.

[0174] In some embodiments, disclosed herein are isolated recombinant nucleic acid molecules encoding an isolated multispecific antibody according to the formula: P1-L1-A1-LB (Formula I), wherein A1 is a CD28 binding domain; B is a PD-L1 binding domain; L is a linker connecting A1 and B; P1 is a peptide that binds to A1, and L1 is a linker that connects A1 to P1 and is a substrate for a tumor-specific protease, wherein P1 comprises an amino acid sequence according to any one of SEQ ID NOs: 24-53, 128-148, and any one of the amino acid sequences of Table 20, or an amino acid sequence having 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 24-53, 128-148, and any one of the amino acid sequences of Table 20.

[0175] In some embodiments, is an isolated recombinant nucleic acid molecule encoding an isolated multispecific antibody according to the following formula: P1-L1-A1-LB-L2-P2 (Formula Ia), wherein P2 comprises a peptide that binds to B and L2 comprises a linker portion that connects B to P2 and is a substrate for a tumor-specific protease.

[0176] In some embodiments, is an isolated recombinant nucleic acid molecule encoding an isolated multispecific antibody comprising the formula: P1-L1-A1-LB-L2-P2 (Formula Ia), wherein P2 comprises a peptide that binds to B and L2 comprises a linker portion that connects B to P2 and is a substrate for a tumor-specific protease.

[0177] In some embodiments, is an isolated recombinant nucleic acid molecule encoding an isolated multispecific antibody comprising the formula: P1-L1-A1-LB-L2-P2 (Formula Ia), wherein P2 is a peptide that binds to B and L2 is a linker portion that connects B to P2 and is a substrate for a tumor-specific protease.

[0178] In some embodiments, is an isolated recombinant nucleic acid molecule encoding an isolated multispecific antibody according to the following formula: P1-L1-A1-LB-L2-P2 (Formula Ia), wherein P2 is a peptide that binds to B and L2 is a linker portion that connects B to P2 and is a substrate for a tumor-specific protease.

[0179] In some embodiments, disclosed herein are isolated recombinant nucleic acid molecules encoding an isolated multispecific antibody comprising an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 149-170. In some embodiments, the present invention relates to an isolated recombinant nucleic acid molecule encoding an isolated multispecific antibody comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 149-170. In some embodiments, the present invention relates to an isolated recombinant nucleic acid molecule encoding an isolated multispecific antibody comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 149-170. In some embodiments, the present invention relates to an isolated recombinant nucleic acid molecule encoding an isolated multispecific antibody comprising an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NOs: 149-170. In some embodiments, the present invention is an isolated recombinant nucleic acid molecule encoding an isolated multispecific antibody comprising an amino acid sequence having at least 99% sequence identity to any one of SEQ ID NOs: 149-170. In some embodiments, the present invention is an isolated recombinant nucleic acid molecule encoding an isolated multispecific antibody comprising the amino acid sequence of any one of SEQ ID NOs: 149-170.

[0180] In some embodiments, the isolated multispecific antibody comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NOs: 149 and 150. In some embodiments, the isolated recombinant nucleic acid molecule encodes an isolated multispecific antibody comprising an amino acid sequence that has at least 99% sequence identity to SEQ ID NOs: 149 and 150.

[0181] In some embodiments, the present invention is an isolated recombinant nucleic acid molecule encoding an isolated multispecific antibody comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 151 and 152. In some embodiments, the present invention is an isolated recombinant nucleic acid molecule encoding an isolated multispecific antibody comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NOs: 151 and 152.

[0182] In some embodiments, the present invention is an isolated recombinant nucleic acid molecule encoding an isolated multispecific antibody comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 153 and 154. In some embodiments, the present invention is an isolated recombinant nucleic acid molecule encoding an isolated multispecific antibody comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NOs: 153 and 154.

[0183] In some embodiments, the present invention is an isolated recombinant nucleic acid molecule encoding an isolated multispecific antibody comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 155 and 156. In some embodiments, the present invention is an isolated recombinant nucleic acid molecule encoding an isolated multispecific antibody comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NOs: 155 and 156.

[0184] In some embodiments, the present invention is an isolated recombinant nucleic acid molecule encoding an isolated multispecific antibody comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 157 and 158. In some embodiments, the present invention is an isolated recombinant nucleic acid molecule encoding an isolated multispecific antibody comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NOs: 157 and 158.

[0185] In some embodiments, the present invention is an isolated recombinant nucleic acid molecule encoding an isolated multispecific antibody comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 159 and 160. In some embodiments, the present invention is an isolated recombinant nucleic acid molecule encoding an isolated multispecific antibody comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NOs: 159 and 160.

[0186] In some embodiments, the present invention is an isolated recombinant nucleic acid molecule encoding an isolated multispecific antibody comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 161 and 162. In some embodiments, the present invention is an isolated recombinant nucleic acid molecule encoding an isolated multispecific antibody comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NOs: 161 and 162.

[0187] In some embodiments, the present invention is an isolated recombinant nucleic acid molecule encoding an isolated multispecific antibody comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 163 and 164. In some embodiments, the present invention is an isolated recombinant nucleic acid molecule encoding an isolated multispecific antibody comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NOs: 163 and 164.

[0188] In some embodiments, the present invention is an isolated recombinant nucleic acid molecule encoding an isolated multispecific antibody comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 165 and 166. In some embodiments, the present invention is an isolated recombinant nucleic acid molecule encoding an isolated multispecific antibody comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NOs: 165 and 166.

[0189] In some embodiments, the present invention is an isolated recombinant nucleic acid molecule encoding an isolated multispecific antibody comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 167 and 168. In some embodiments, the present invention is an isolated recombinant nucleic acid molecule encoding an isolated multispecific antibody comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NOs: 167 and 168.

[0190] In some embodiments, the present invention is an isolated recombinant nucleic acid molecule encoding an isolated multispecific antibody comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 169 and 170. In some embodiments, the present invention is an isolated recombinant nucleic acid molecule encoding an isolated multispecific antibody comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NOs: 169 and 170.

[0191] In some embodiments, the present invention is an isolated recombinant nucleic acid molecule encoding an isolated multispecific antibody comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 208 and 209. In some embodiments, the present invention is an isolated recombinant nucleic acid molecule encoding an isolated multispecific antibody comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NOs: 208 and 209.

[0192] Pharmaceutical composition

[0193] In some embodiments, disclosed herein are pharmaceutical compositions comprising: (a) a multispecific antibody as disclosed herein; and (b) a pharmaceutically acceptable excipient.

[0194] In some embodiments, the pharmaceutical composition comprises (a) an isolated multispecific antibody according to the formula: P1-L1-A1-LB (Formula I), wherein A1 comprises a CD28 binding domain; B comprises a PD-L1 binding domain; L comprises a linker connecting A1 and B; P1 comprises a peptide that binds to A1, and L1 comprises a linker that connects A1 to P1 and is a substrate for a tumor-specific protease, wherein P1 comprises an amino acid sequence according to any one of SEQ ID NOs: 24-53, 128-148, and any one of the amino acid sequences of Table 20, or an amino acid sequence having 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 24-53, 128-148, and any one of the amino acid sequences of Table 20; and (b) a pharmaceutically acceptable excipient.

[0195] In some embodiments, a pharmaceutical composition comprises (a) an isolated multispecific antibody comprising the formula: P1-L1-A1-LB (Formula I), wherein A1 comprises a CD28 binding domain; B comprises a PD-L1 binding domain; L comprises a linker connecting A1 and B; P1 comprises a peptide that binds to A1, and L1 comprises a linker that connects A1 to P1 and is a substrate for a tumor-specific protease, wherein P1 comprises an amino acid sequence according to any one of SEQ ID NOs: 24-53, 128-148, and any one of the amino acid sequences of Table 20, or an amino acid sequence having 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 24-53, 128-148, and any one of the amino acid sequences of Table 20; and (b) a pharmaceutically acceptable excipient.

[0196] In some embodiments, the pharmaceutical composition comprises (a) an isolated multispecific antibody comprising the formula: P1-L1-A1-LB (Formula I), wherein A1 is a CD28 binding domain; B is a PD-L1 binding domain; L is a linker connecting A1 and B; P1 is a peptide that binds to A1, and L1 is a linker moiety that connects A1 to P1 and is a substrate for a tumor-specific protease, wherein P1 comprises an amino acid sequence according to any one of SEQ ID NOs: 24-53, 128-148, and any one of the amino acid sequences of Table 20, or an amino acid sequence having 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 24-53, 128-148, and any one of the amino acid sequences of Table 20; and (b) a pharmaceutically acceptable excipient.

[0197] In some embodiments, the pharmaceutical composition comprises (a) an isolated multispecific antibody according to the formula: P1-L1-A1-LB (Formula I), wherein A1 is a CD28 binding domain; B is a PD-L1 binding domain; L is a linker connecting A1 and B; P1 is a peptide that binds to A1, and L1 is a linker that connects A1 to P1 and is a substrate for a tumor-specific protease, wherein P1 comprises an amino acid sequence according to any one of SEQ ID NOs: 24-53, 128-148, and any one of the amino acid sequences of Table 20, or an amino acid sequence having 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 24-53, 128-148, and any one of the amino acid sequences of Table 20; and (b) a pharmaceutically acceptable excipient.

[0198] In some embodiments, the pharmaceutical composition comprises (a) an isolated multispecific antibody according to the formula: P1-L1-A1-LB-L2-P2 (Formula Ia), wherein P2 comprises a peptide that binds to B and L2 comprises a linker portion that connects B to P2 and is a substrate for a tumor-specific protease; and (b) a pharmaceutically acceptable excipient.

[0199] In some embodiments, the pharmaceutical composition comprises (a) an isolated multispecific antibody comprising the formula: P1-L1-A1-LB-L2-P2 (Formula Ia), wherein P2 comprises a peptide that binds to B and L2 comprises a linker portion that connects B to P2 and is a substrate for a tumor-specific protease; and (b) a pharmaceutically acceptable excipient.

[0200] In some embodiments, the pharmaceutical composition comprises (a) an isolated multispecific antibody comprising the formula: P1-L1-A1-LB-L2-P2 (Formula Ia), wherein P2 is a peptide that binds to B and L2 is a linker that connects B to P2 and is a substrate for a tumor-specific protease; and (b) a pharmaceutically acceptable excipient.

[0201] In some embodiments, the pharmaceutical composition comprises (a) an isolated multispecific antibody according to the formula: P1-L1-A1-LB-L2-P2 (Formula Ia), wherein P2 is a peptide that binds to B and L2 is a linker that connects B to P2 and is a substrate for a tumor-specific protease; and (b) a pharmaceutically acceptable excipient.

[0202] In some embodiments, a pharmaceutical composition comprises (a) an isolated multispecific antibody comprising an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 149-170; and (b) a pharmaceutically acceptable excipient.

[0203] In some embodiments, the pharmaceutical composition comprises (a) an isolated multispecific antibody comprising an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 149-170; and (b) a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises (a) an isolated multispecific antibody comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 149-170; and (b) a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises (a) an isolated multispecific antibody comprising an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NOs: 149-170; and (b) a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises (a) an isolated multispecific antibody comprising an amino acid sequence having at least 99% sequence identity to any one of SEQ ID NOs: 149-170; and (b) a pharmaceutically acceptable excipient. In some embodiments, a pharmaceutical composition comprises (a) an isolated multispecific antibody comprising the amino acid sequence of any one of SEQ ID NOs: 149-170; and (b) a pharmaceutically acceptable excipient.

[0204] In some embodiments, the pharmaceutical composition comprises (a) an isolated multispecific antibody comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 149 and 150; and (b) a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises (a) an isolated multispecific antibody comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NOs: 149 and 150; and (b) a pharmaceutically acceptable excipient.

[0205] In some embodiments, the pharmaceutical composition comprises (a) an isolated multispecific antibody comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 151 and 152; and (b) a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises (a) an isolated multispecific antibody comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NOs: 151 and 152; and (b) a pharmaceutically acceptable excipient.

[0206] In some embodiments, the pharmaceutical composition comprises (a) an isolated multispecific antibody comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 153 and 154; and (b) a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises (a) an isolated multispecific antibody comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NOs: 153 and 154; and (b) a pharmaceutically acceptable excipient.

[0207] In some embodiments, the pharmaceutical composition comprises (a) an isolated multispecific antibody comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 155 and 156; and (b) a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises (a) an isolated multispecific antibody comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NOs: 155 and 156; and (b) a pharmaceutically acceptable excipient.

[0208] In some embodiments, the pharmaceutical composition comprises (a) an isolated multispecific antibody comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 157 and 158; and (b) a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises (a) an isolated multispecific antibody comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NOs: 157 and 158; and (b) a pharmaceutically acceptable excipient.

[0209] In some embodiments, the pharmaceutical composition comprises (a) an isolated multispecific antibody comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 159 and 160; and (b) a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises (a) an isolated multispecific antibody comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NOs: 159 and 160; and (b) a pharmaceutically acceptable excipient.

[0210] In some embodiments, the pharmaceutical composition comprises (a) an isolated multispecific antibody comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 161 and 162; and (b) a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises (a) an isolated multispecific antibody comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NOs: 161 and 162; and (b) a pharmaceutically acceptable excipient.

[0211] In some embodiments, the pharmaceutical composition comprises (a) an isolated multispecific antibody comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 163 and 164; and (b) a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises (a) an isolated multispecific antibody comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NOs: 163 and 164; and (b) a pharmaceutically acceptable excipient.

[0212] In some embodiments, the pharmaceutical composition comprises (a) an isolated multispecific antibody comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 165 and 166; and (b) a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises (a) an isolated multispecific antibody comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NOs: 165 and 166; and (b) a pharmaceutically acceptable excipient.

[0213] In some embodiments, the pharmaceutical composition comprises (a) an isolated multispecific antibody comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 167 and 168; and (b) a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises (a) an isolated multispecific antibody comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NOs: 167 and 168; and (b) a pharmaceutically acceptable excipient.

[0214] In some embodiments, the pharmaceutical composition comprises (a) an isolated multispecific antibody comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 169 and 170; and (b) a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises (a) an isolated multispecific antibody comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NOs: 169 and 170; and (b) a pharmaceutically acceptable excipient.

[0215] In some embodiments, the pharmaceutical composition comprises (a) an isolated multispecific antibody comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 208 and 209; and (b) a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises (a) an isolated multispecific antibody comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NOs: 208 and 209; and (b) a pharmaceutically acceptable excipient.

[0216] Disclosed herein are pharmaceutical compositions comprising: (a) an isolated multispecific antibody described herein, (b) an anti-cancer therapy, and (c) a pharmaceutically acceptable excipient. In some embodiments, the anti-cancer therapy comprises a small molecule, a cell-based therapy, or an antibody-based therapy.

[0217] In some embodiments, the antibody-based therapy is a T cell adapter. In some embodiments, the T cell adapter comprises a formula according to: D1-L0-E1 (Formula II), wherein D1 comprises an effector cell binding domain that binds to an effector cell antigen, E1 comprises a tumor antigen binding domain that binds to a tumor antigen, and L0 comprises a linker connecting D1 and E1. In some embodiments, D1 comprises a single-chain variable fragment, a single-domain antibody, a Fab fragment, or Fab'. In some embodiments, D1 comprises a single-chain variable fragment. In some embodiments, E1 comprises a single-chain variable fragment, a single-domain antibody, a Fab fragment, or Fab'. In some embodiments, E1 comprises a Fab fragment. In some embodiments, the effector cell antigen comprises CD3. In some embodiments, the effector cell binding domain comprises a complementary determining region (CDR) selected from the group consisting of: muromonab-CD3 (OKT3), oxicitumomab (TRX4), tilizumab (MGA031), visilizumab (Nuvion), SP34, X35, VIT3, BMA030 (BW264 / 56), CLB-T3 / 3, CRIS7, YTH12.5, F111-409, CL In some embodiments, the effector cell binding domain comprises an amino acid sequence according to SEQ ID NOs: 89-101.

[0218] Table 13. Effector cell binding domain amino acid sequences

[0219]

[0220]

[0221] In some embodiments, the tumor antigen comprises epidermal growth factor receptor (EGFR), prostate-specific membrane antigen (PSMA), or tumor-associated calcium signal transducer 2 (referred to herein as TROP2). In some embodiments, the tumor antigen comprises EGFR. In some embodiments, the tumor antigen binding domain comprises an amino acid sequence according to SEQ ID NO: 102-111. In some embodiments, the tumor antigen comprises EGFR, and the tumor binding domain comprises the complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, and LC-CDR1, LC-CDR2, and LC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 comprise: HC-CDR1: SEQ ID NO: 105; HC-CDR2: SEQ ID NO: 106; HC-CDR3: SEQ ID NO: 107; and wherein the LC-CDR1, LC-CDR2, and LC-CDR3 comprise: LC-CDR1: SEQ ID NO: 102; LC-CDR2: SEQ ID NO: 103 (YAS); and LC-CDR3: SEQ ID NO: 104. In some embodiments, the tumor antigen comprises EGFR, and the T cell engager comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 181 and 182. In some embodiments, the tumor antigen comprises EGFR, and the T cell engager comprises an amino acid sequence according to SEQ ID NOs: 181 and 182. In some embodiments, the tumor antigen comprises EGFR, and the T cell engager comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 214 and 215. In some embodiments, the tumor antigen comprises EGFR, and the T cell engager comprises an amino acid sequence according to SEQ ID NOs: 214 and 215.

[0222] Table 14. Amino acid sequences of tumor antigen binding domains - anti-EGFR

[0223]

[0224]

[0225]

[0226] In some embodiments, the tumor antigen comprises TROP2. In some embodiments, the tumor antigen comprises TROP2, and the tumor binding domain comprises the complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, and LC-CDR1, LC-CDR2, and LC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 comprise HC-CDR1: SEQ ID NO: 112; HC-CDR2: SEQ ID NO: 113; HC-CDR3: SEQ ID NO: 114; and wherein the LC-CDR1, LC-CDR2, and LC-CDR3 comprise: LC-CDR1: SEQ ID NO: 115; LC-CDR2: SEQ ID NO: 116 (SAS); and LC-CDR3: SEQ ID NO: 117. In some embodiments, the tumor antigen comprises TROP2, and the T cell engager comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 187-192. In some embodiments, the tumor antigen comprises TROP2, and the T cell engager comprises an amino acid sequence according to any one of SEQ ID NOs: 187- 192. In some embodiments, the tumor antigen binding domain comprises an amino acid sequence according to SEQ ID NOs: 112-119.

[0227] Table 15. Amino acid sequence of tumor antigen binding domain - anti-TROP2

[0228]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235] In some embodiments, the tumor antigen comprises PSMA.In some embodiments, the tumor antigen binding domain comprises an amino acid sequence according to SEQ ID NOs: 120-127.

[0236] In some embodiments, the tumor antigen comprises PSMA, and the tumor binding domain comprises the complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, and LC-CDR1, LC-CDR2, and LC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 comprise HC-CDR1: SEQ ID NO: 120; HC-CDR2: SEQ ID NO: 121; HC-CDR3: SEQ ID NO: 122; and wherein the LC-CDR1, LC-CDR2, and LC-CDR3 comprise: LC-CDR1: SEQ ID NO: 123; LC-CDR2: SEQ ID NO: 124(EA); and LC-CDR3: SEQ ID NO: 125. In some embodiments, the tumor antigen comprises PSMA, and the T cell engager comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 173 and 174. In some embodiments, the tumor antigen comprises PSMA and the T cell engager comprises the amino acid sequence according to SEQ ID NOs: 173 and 174.

[0237] Table 16. Tumor antigen binding domain amino acid sequence - anti-PSMA

[0238]

[0239]

[0240]

[0241] In some embodiments, the T cell engager molecule is selectively activated in the tumor microenvironment. In some embodiments, the T cell engager is according to the following formula: P3-L3-D1-L0-E1 (Formula IIa), wherein D1 comprises a CD3 binding domain; E1 comprises a tumor antigen binding domain; L0 comprises a linker connecting D1 and E1; P3 comprises a peptide that binds to D1, and L3 comprises a linker portion that connects D1 and P3 and is a substrate for a tumor-specific protease.

[0242] In some embodiments, the T cell engager is according to the following subformula: D1-L0-E1-L4-P4 (Formula IIb), wherein D1 comprises a CD3 binding domain; E1 comprises a tumor antigen binding domain; L0 comprises a linker connecting D1 and E1; P4 comprises a peptide that binds to E1, and L4 comprises a linker portion that connects E1 and P4 and is a substrate for a tumor-specific protease.

[0243] In some embodiments, the T cell engager is according to the following subformula: P3-L3-D1-L0-E1-L4-P4 (Formula IIc), wherein D1 comprises a CD3 binding domain; E1 comprises a tumor antigen binding domain; L0 comprises a linker connecting D1 and E1; P3 comprises a peptide that binds to D1, and L3 comprises a linker that connects D1 to P3 and is a substrate for a tumor-specific protease; P4 comprises a peptide that binds to E1, and L4 comprises a linker that connects E1 to P4 and is a substrate for a tumor-specific protease.

[0244] In some embodiments, the T cell engager comprises H1. In some embodiments, H1 comprises a sequence according to SEQ ID NOs: 54-57. In some embodiments, H1 comprises a single domain antibody.

[0245] In some embodiments, the single domain antibody comprises the complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and 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, L3 or L4 is a peptide sequence having at least 5 and no more than 50 amino acids. In some embodiments, L3 or L4 is a peptide sequence having at least 10 and no more than 30 amino acids. In some embodiments, L3 or L4 is a peptide sequence having at least 10 amino acids. In some embodiments, L3 or L4 is a peptide sequence having at least 18 amino acids. In some embodiments, L3 or L4 is a peptide sequence having at least 26 amino acids. In some embodiments, L3 or L4 comprises (G2S) n wherein n is an integer from 1 to 3 (SEQ ID NO: 228). In some embodiments, L3 or L4 comprises (G2S) n wherein n is an integer of at least 1. In some embodiments, L3 or L4 comprises 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 a metalloprotease, a serine protease, a cysteine ​​protease, a threonine protease, and an aspartic protease.

[0246] In some embodiments, L3 or L4 comprises an amino acid sequence cleavable by urokinase, a membrane-type serine protease, or a matrix metalloproteinase. In some embodiments, L3 or L4 comprises a sequence according to SEQ ID NOs: 18-19, 62-88. In some embodiments, L3 binds to the N-terminus of D1. In some embodiments, L3 binds to the C-terminus of D1. In some embodiments, L4 binds to the N-terminus of E1. In some embodiments, L4 binds to the C-terminus of E1.

[0247] In some embodiments, when L3 is cleaved by a tumor-specific protease, P3 unbinds from D1, thereby exposing D1 to CD3. In some embodiments, when L4 is cleaved by a tumor-specific protease, P4 unbinds from E1, thereby exposing E1 to tumor antigens. In some embodiments, P3 weakens the binding of D1 to CD3. In some embodiments, P3 binds to D1 through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, H-bonding interactions, or a combination thereof. In some embodiments, P3 binds to D1 at or near the antigen binding site. In some embodiments, when L3 is cleaved by a tumor-specific protease, P3 unbinds from D1, thereby exposing D1 to CD3. In some embodiments, P3 has less than 70% sequence identity with CD3. In some embodiments, P3 has less than 85% sequence identity with CD3. In some embodiments, P3 has less than 90% sequence identity with CD3. In some embodiments, P3 has less than 95% sequence identity with CD3. In some embodiments, P3 has less than 98% sequence identity with CD3. In some embodiments, P3 has less than 99% sequence identity with CD3. In some embodiments, P3 comprises an amino acid sequence according to SEQ ID NOs: 177-180. In some embodiments, P3 comprises a de novo generated amino acid sequence that shares less than 10% sequence identity with CD3. In some embodiments, P4 weakens the binding of E1 to a tumor antigen. In some embodiments, P4 binds to E1 through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bonding interactions, or a combination thereof. In some embodiments, P4 binds to E1 at or near the antigen binding site. In some embodiments, when L4 is cleaved by a tumor-specific protease, P4 unbinds from E1, thereby exposing E1 to the tumor antigen. In some embodiments, P4 has less than 70% sequence identity with the tumor antigen. In some embodiments, P4 has less than 80% sequence identity with the tumor antigen. In some embodiments, P4 has less than 85% sequence identity with the tumor antigen. In some embodiments, P4 has less than 90% sequence identity with a tumor antigen. In some embodiments, P4 has less than 95% sequence identity with a tumor antigen. In some embodiments, P4 comprises a de novo generated amino acid sequence that shares less than 10% sequence identity with a tumor antigen. In some embodiments, P3 or P4 comprises a peptide sequence of at least 5 amino acids in length. In some embodiments, P3 or P4 comprises a peptide sequence of at least 6 amino acids in length. In some embodiments, P3 or P4 comprises a peptide sequence of at least 10 amino acids in length. In some embodiments, P3 or P4 comprises a peptide sequence of at least 10 amino acids in length and no more than 20 amino acids in length.In some embodiments, P3 or P4 comprises a peptide sequence of at least 16 amino acids in length. In some embodiments, P3 or P4 comprises a peptide sequence of no more than 40 amino acids in length. In some embodiments, P3 or P4 comprises at least two cysteine ​​amino acid residues. In some embodiments, P3 or P4 comprises a cyclic peptide or a linear peptide. In some embodiments, P3 or P4 comprises a cyclic peptide. In some embodiments, P3 or P4 comprises a linear peptide. In some embodiments, P4 comprises an amino acid sequence according to SEQ ID NO: 185 or 186.

[0248] In some embodiments, the tumor antigen comprises EGFR, and the T cell engager comprises the amino acid sequence of SEQ ID NOs: 183 and 184. In some embodiments, P4 comprises the amino acid sequence according to SEQ ID NOs: 199-201. In some embodiments, the tumor antigen comprises TROP2, and the T cell engager comprises any one of the amino acid sequences of SEQ ID NOs: 193-198. In some embodiments, the tumor antigen comprises PSMA, and the T cell engager comprises the amino acid sequence of SEQ ID NOs: 175 and 176.

[0249] 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 radioactive label, an enzyme, a nucleic acid probe, or a contrast agent.

[0250] For administration to a subject, the multispecific antibodies disclosed herein can 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 is non-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, and the like. Such carriers can be formulated by conventional methods and can be administered to a subject at an appropriate dose. Preferably, the composition is sterile. These compositions may also contain adjuvants such as preservatives, emulsifiers, and dispersants. Protection against the action of microorganisms can be ensured by including various antibacterial and antifungal agents.

[0251] The pharmaceutical composition can be in any suitable form (depending on the desired method of administration). It can be provided in unit dosage form, can be provided in a sealed container and can be provided as part of a kit. Such a kit can include instructions for use. It can include a plurality of such unit dosage forms.

[0252] The pharmaceutical composition can be adapted for administration by any suitable route, including parenteral (e.g., subcutaneous, intramuscular or intravenous) routes. Such compositions can be prepared by any method known in the pharmaceutical art, for example, by mixing the active ingredient with a carrier or excipient under sterile conditions.

[0253] The dosage of the substances of the present disclosure may vary widely, depending on the disease or disorder to be treated, the age and condition of the individual to be treated, etc., and the physician will ultimately determine the appropriate dosage to be used.

[0254] CD28 binding domain linked to a peptide that impairs binding to CD28

[0255] Disclosed herein are isolated polypeptides or polypeptide complexes comprising a CD28 binding domain linked to a peptide that impairs binding of the CD28 binding domain to CD28, wherein the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 24-53, 128-148, and the amino acid sequences of Table 20, or an amino acid sequence having 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 24-53, 128-148, and the amino acid sequences of Table 20. In some embodiments, the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 24-53, 128-148, and the amino acid sequences of Table 20. In some embodiments, the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 42-53, or an amino acid sequence having 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 42-53. In some embodiments, the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 42-53. In some embodiments, the peptide comprises an amino acid sequence according to any one of the amino acid sequences of Table 20, or an amino acid sequence having 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of the amino acid sequences of Table 20. In some embodiments, the peptide comprises an amino acid sequence according to any one of the amino acid sequences of Table 20. In some embodiments, the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 128-147, or an amino acid sequence having 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 128-147. In some embodiments, the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 128-147.

[0256] In some embodiments, the peptide comprises a sequence according to X1-X2-X3-C-X4-X5-X6-X7-X8-X9-X 10 -CX 11 -X 12an amino acid sequence wherein X1 is selected from the group consisting of M, I, L, and V; X2 is selected from the group consisting of D, H, N, A, F, S, T, Y, and V; X3 is selected from the group consisting of W, L, and F; X4 is selected from the group consisting of P, A, and L; X5 is selected from the group consisting of R, T, I, M, S, K, L, V, W, F, A, P, and D; X6 is selected from the group consisting of E, D, Y, H, S, F, A, N, T, I, P, and V; X7 is selected from the group consisting of L, M, R, S, Q, and H; X8 is selected from the group consisting of W and Q; X9 is selected from the group consisting of H, N, D, A, S, Y, T, F, V, L, and I; X 10 Selected from E, V, L, D, Y, R, Q, H, F, K, A, M and N; X 11 is selected from F, Y, L, W, and V; and X 12 is selected from N, A, F, S, Y, H, D, T, and L. In some embodiments, X1 is selected from M, I, and L; X2 is selected from D, H, N, and A; X3 is W; X4 is P; X5 is selected from R, T, I, M, S, and K; X6 is selected from E, D, Y, H, S, and F; X7 is selected from L, M, and R; X8 is W; X9 is selected from H, N, D, A, S, and V; X 10 Selected from E, V, L, D and H; X 11 is selected from F, Y and L; and X 12 is selected from N, A, F, S, and Y. In some embodiments, X1 is M; X2 is selected from D and H; X3 is W; X4 is P; X5 is selected from R, T, and I; X6 is selected from E, D, and Y; X7 is selected from L, M, and R; X8 is W; X9 is selected from H, N, D, and V; X 10 Selected from E, V, L, D and H; X 11 is F; and X 12 Selected from N, A and F. In some embodiments, the peptide comprises the amino acid sequence according to SEQ ID NO: 32 or an amino acid sequence having 1, 2 or 3 amino acid mutations, substitutions or deletions relative to SEQ ID NO: 32. In some embodiments, the peptide comprises the amino acid sequence according to SEQ ID NO: 32. In some embodiments, the peptide comprises the amino acid sequence according to SEQ ID NO: 138 or an amino acid sequence having 1, 2 or 3 amino acid mutations, substitutions or deletions relative to SEQ ID NO: 138. In some embodiments, the peptide comprises the amino acid sequence according to SEQ ID NO: 138.

[0257] 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 a single-chain variable fragment. In some embodiments, the CD28 binding domain comprises a single-domain antibody. In some embodiments, the CD28 binding domain comprises a Fab or a Fab'. In some embodiments, the scFv heavy chain variable domain comprises a complementarity determining region (CDR): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and 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 the CDR comprises 0-2 amino acid modifications in at least one of the HC-CDR1, HC-CDR2, or HC-CDR3. In some embodiments, the scFv light chain variable domain comprises a complementarity determining region (CDR): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, LC-CDR2, and LC-CDR3 of the scFv light chain variable domain comprise: LC-CDR1: SEQ ID NO: 4; LC-CDR2: SEQ ID NO: 5 (KA); and LC-CDR3: SEQ ID NO: 6, and wherein the CDR comprises 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 having at least 80% sequence identity with 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 contiguous amino acid residues of SEQ ID NO: 7 and has at least 80% sequence identity with at least 110 contiguous 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 having at least 80% sequence identity with 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 contiguous 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 with 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 having at least 80% sequence identity with 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 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 with at least 210 consecutive amino acid residues of SEQ ID NO: 9. In some embodiments, the scFv comprises the amino acid sequence according to SEQ ID NO:9.

[0258] In some embodiments, the CD28 binding domain is linked to the peptide via a linker portion (L1). In some embodiments, L1 is a substrate for a tumor-specific protease. In some embodiments, L1 is a peptide sequence having at least 5 to no more than 50 amino acids. In some embodiments, L1 is a peptide sequence having at least 10 to no more than 30 amino acids. In some embodiments, L1 is a peptide sequence having at least 10 amino acids. In some embodiments, L1 is a peptide sequence having at least 18 amino acids. In some embodiments, L1 is a peptide sequence having at least 26 amino acids. In some embodiments, L1 comprises (G2S) n wherein n is an integer from 1 to 3 (SEQ ID NO: 228). In some embodiments, L1 comprises (G2S) n wherein n is an integer of at least 1. In some embodiments, L1 comprises 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 a metalloprotease, a serine protease, a cysteine ​​protease, a threonine protease, and an aspartic protease. In some embodiments, L1 comprises an amino acid sequence cleavable by urokinase, an amino acid sequence cleavable by a membrane-type serine protease, or an amino acid sequence cleavable by a matrix metalloprotease. In some embodiments, L1 comprises a sequence according to SEQ ID NOs:18-19, 62-88. In some embodiments, L1 is bound to the N-terminus of A1. In some embodiments, L1 is bound to the C-terminus of A1. In some embodiments, when L1 is cleaved by a tumor-specific protease, P1 is unbound from A1, thereby exposing A1 to CD28. In some embodiments, L1 comprises a modified amino acid or a non-natural amino acid or a modified non-natural amino acid, or a combination thereof. In some embodiments, the modified amino acid or the modified non-natural amino acid comprises a post-translational modification.

[0259] In some embodiments, the isolated polypeptide or polypeptide complex further comprises a half-life extending molecule (H1). In some embodiments, H1 is attached to the peptide. In some embodiments, H1 does not block the CD28 binding domain from CD28. In some embodiments, H1 comprises a linker portion (L5) connecting H1 to the peptide. In some embodiments, the half-life extending molecule (H1) does not have binding affinity for CD28. In some embodiments, the half-life extending molecule (H1) does not protect the isolated polypeptide or polypeptide complex from CD28. In some embodiments, H1 comprises a sequence according to SEQ ID NO: 54-57. In some embodiments, H1 comprises an amino acid sequence having a repeating sequence motif. In some embodiments, H1 comprises an amino acid sequence having a highly ordered secondary structure. In some embodiments, H1 comprises a polymer. In some embodiments, the polymer is polyethylene glycol (PEG). In some embodiments, H1 comprises albumin. In some embodiments, H1 comprises an Fc domain. In some embodiments, the albumin is serum albumin. In some embodiments, the albumin is human serum albumin. In some embodiments, H1 comprises a polypeptide, a ligand, or a small molecule. In some embodiments, the polypeptide, ligand, or small molecule binds to a serum protein or fragment thereof, a circulating immunoglobulin or fragment thereof, or CD35 / CR1. In some embodiments, the serum protein comprises thyroxine-binding protein, transthyretin, 1-acid glycoprotein, transferrin, transferrin receptor or a transferrin-binding portion thereof, fibrinogen, or 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, Fab, or 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 antibody or a humanized antibody. In some embodiments, the single domain antibody is selected from 645gH1gL1, 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 a complementarity determining region (CDR): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and 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 0-2 amino acid modifications in at least one of HC-CDR1, HC-CDR2, or HC-CDR3. In some embodiments, H1 comprises an amino acid sequence according to SEQ ID NO: 57. In some embodiments, H1 comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 57. In some embodiments, H1 comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 57. In some embodiments, H1 comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 57. In some embodiments, H1 comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 57. In some embodiments, H1 comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 57. In some embodiments, H1 comprises a modified amino acid or a non-natural amino acid or a modified non-natural amino acid, or a combination thereof. In some embodiments, the modified amino acid or the modified non-natural amino acid comprises a post-translational modification. In some embodiments, H1 comprises a linker (L5) connecting H1 to P1 or P2. In some embodiments, L5 is a peptide sequence having at least 5 to no more than 50 amino acids. In some embodiments, L5 is a peptide sequence having at least 10 to no more than 30 amino acids. In some embodiments, L5 is a peptide sequence having at least 10 amino acids. In some embodiments, L5 is a peptide sequence having at least 18 amino acids. In some embodiments, L5 is a peptide sequence having at least 26 amino acids. In some embodiments, L5 comprises a formula selected from the following: (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.

[0260] Treatment

[0261] Disclosed herein are methods for treating cancer in a subject in need thereof, comprising administering to the subject a multispecific antibody of any embodiment described herein. In some embodiments, the multispecific antibody induces T cell-mediated cytotoxicity of tumor cells. In some embodiments, the cancer is a hematological malignancy. In some embodiments, the cancer is a leukemia or lymphoma. In some embodiments, the cancer is a lymphoma, and wherein the lymphoma is a 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 a 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 is administered in combination with an anti-cancer therapy. In some embodiments, the multispecific antibody and the anti-cancer therapy are administered in the same pharmaceutical composition. In some embodiments, the multispecific antibody and the anti-cancer therapy are administered as separate pharmaceutical compositions. 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 anti-cancer therapy includes small molecules, cell-based therapies, or antibody-based therapies.

[0262] In some embodiments, administration of the multispecific antibody to a subject is sufficient to reduce or eliminate the cancer as compared to a baseline measurement of the cancer taken from the subject prior to administration of the multispecific antibody, hi 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.

[0263] In some embodiments, the antibody-based therapy is a T cell adapter. In some embodiments, the T cell adapter comprises a formula according to: D1-L0-E1 (Formula II), wherein D1 comprises an effector cell binding domain that binds to an effector cell antigen, E1 comprises a tumor antigen binding domain that binds to a tumor antigen, and L0 comprises a linker connecting D1 and E1. In some embodiments, D1 comprises a single-chain variable fragment, a single-domain antibody, a Fab fragment, or Fab'. In some embodiments, D1 comprises a single-chain variable fragment. In some embodiments, E1 comprises a single-chain variable fragment, a single-domain antibody, a Fab fragment, or Fab'. In some embodiments, E1 comprises a Fab fragment. In some embodiments, the effector cell binding domain comprises a complementary determining region (CDR) selected from the group consisting of: muromonab-CD3 (OKT3), oxicitumomab (TRX4), tilizumab (MGA031), visilizumab (Nuvion), SP34, X35, VIT3, BMA030 (BW264 / 56), CLB-T3 / 3, CRIS7, YTH12.5, F111-409, CL In some embodiments, the effector cell binding domain comprises an amino acid sequence according to SEQ ID NOs: 89-101. In some embodiments, the tumor antigen comprises epidermal growth factor receptor (EGFR), prostate-specific membrane antigen (PSMA), or tumor-associated calcium signal transducer 2 (referred to herein as TROP2).

[0264] In some embodiments, the tumor antigen comprises EGFR. In some embodiments, the tumor antigen binding domain comprises an amino acid sequence according to SEQ ID NOs: 102-111. In some embodiments, the tumor antigen comprises EGFR, and the tumor binding domain comprises the complementary determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, and LC-CDR1, LC-CDR2, and LC-CDR3, wherein HC-CDR1, HC-CDR2, and HC-CDR3 comprise: HC-CDR1: SEQ ID NO: 105; HC-CDR2: SEQ ID NO: 106; HC-CDR3: SEQ ID NO: 107; and wherein LC-CDR1, LC-CDR2, and LC-CDR3 comprise: LC-CDR1: SEQ ID NO: 102; LC-CDR2: SEQ ID NO: 103 (YAS); and LC-CDR3: SEQ ID NO: 104. In some embodiments, the tumor antigen comprises EGFR, and the T cell engager comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 181 and 182. In some embodiments, the tumor antigen comprises EGFR, and the T cell engager comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 181 and 182. In some embodiments, the tumor antigen comprises EGFR, and the T cell engager comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 214 and 215. In some embodiments, the tumor antigen comprises EGFR, and the T cell engager comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 214 and 215. In some embodiments, the cancer is colorectal cancer (CRC), squamous cell carcinoma of the head and neck (SCCHN), non-small cell lung cancer (NSCLC), prostate cancer, breast cancer, colon / rectal cancer, head and neck cancer, esophagogastric cancer, liver cancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, kidney cancer, or pancreatic cancer.

[0265] In some embodiments, the tumor antigen comprises TROP2. In some embodiments, the tumor antigen comprises TROP2, and the tumor binding domain comprises the complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, and LC-CDR1, LC-CDR2, and LC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 comprise HC-CDR1: SEQ ID NO: 112; HC-CDR2: SEQ ID NO: 113; HC-CDR3: SEQ ID NO: 114; and wherein the LC-CDR1, LC-CDR2, and LC-CDR3 comprise: LC-CDR1: SEQ ID NO: 115; LC-CDR2: SEQ ID NO: 116 (SAS); and LC-CDR3: SEQ ID NO: 117. In some embodiments, the tumor antigen comprises TROP2, and the T cell engager comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 187-192. In some embodiments, the tumor antigen comprises TROP2, and the T cell engager comprises an amino acid sequence according to any one of SEQ ID NOs: 187-192. In some embodiments, the tumor antigen binding domain comprises an amino acid sequence according to SEQ ID NOs: 112-119. In some embodiments, the cancer is lung cancer, breast cancer (e.g., HER2+; ER / PR+; TNBC), cervical cancer, ovarian cancer, colorectal cancer, pancreatic cancer, gastric cancer, triple-negative breast cancer (TNBC), urothelial carcinoma (UC), non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), gastric cancer, esophageal cancer, head and neck cancer, prostate cancer, or endometrial cancer. In some embodiments, the tumor antigen comprises PSMA. In some embodiments, the tumor antigen binding domain comprises an amino acid sequence according to SEQ ID NOs: 120-127.

[0266] In some embodiments, the tumor antigen comprises PSMA, and the tumor binding domain comprises the complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, and LC-CDR1, LC-CDR2, and LC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 comprise HC-CDR1: SEQ ID NO: 120; HC-CDR2: SEQ ID NO: 121; HC-CDR3: SEQ ID NO: 122; and wherein the LC-CDR1, LC-CDR2, and LC-CDR3 comprise: LC-CDR1: SEQ ID NO: 123; LC-CDR2: SEQ ID NO: 124(EA); and LC-CDR3: SEQ ID NO: 125. In some embodiments, the tumor antigen comprises PSMA, and the T cell engager comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 173 and 174. In some embodiments, the tumor antigen comprises PSMA, and the T cell engager comprises an amino acid sequence according to SEQ ID NOs: 173 and 174. In some embodiments, the cancer is lung cancer, breast cancer (e.g., HER2+; ER / PR+; TNBC), cervical cancer, ovarian cancer, colorectal cancer, pancreatic cancer, or gastric cancer.

[0267] In some embodiments, the T cell engager molecule is selectively activated in the tumor microenvironment. In some embodiments, the T cell engager is according to the following formula: P3-L3-D1-L0-E1 (Formula IIa), wherein D1 comprises a CD3 binding domain; E1 comprises a tumor antigen binding domain; L0 comprises a linker connecting D1 and E1; P3 comprises a peptide that binds to D1, and L3 comprises a linker connecting D1 and P3 and is a substrate for a tumor-specific protease. In some embodiments, the T cell engager is according to the following formula: D1-L0-E1-L4-P4 (Formula IIb), wherein D1 comprises a CD3 binding domain; E1 comprises a tumor antigen binding domain; L0 comprises a linker connecting D1 and E1; P4 comprises a peptide that binds to E1, and L4 comprises a linker connecting E1 and P4 and is a substrate for a tumor-specific protease. In some embodiments, the T cell engager is according to the following subformula: P3-L3-D1-L0-E1-L4-P4 (Formula IIc), wherein D1 comprises a CD3 binding domain; E1 comprises a tumor antigen binding domain; L0 comprises a linker connecting D1 and E1; P3 comprises a peptide that binds to D1, and L3 comprises a linker that connects D1 to P3 and is a substrate for a tumor-specific protease; P4 comprises a peptide that binds to E1, and L4 comprises a linker that connects E1 to P4 and is a substrate for a tumor-specific protease.

[0268] In some embodiments, the T cell engager comprises H1. In some embodiments, H1 comprises a sequence according to SEQ ID NOs: 54-57. In some embodiments, H1 comprises a single domain antibody. In some embodiments, the single domain antibody comprises complementary determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and 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, L3 or L4 is a peptide sequence having at least 5 and no more than 50 amino acids. In some embodiments, L3 or L4 is a peptide sequence having at least 10 and no more than 30 amino acids. In some embodiments, L3 or L4 is a peptide sequence having at least 10 amino acids. In some embodiments, L3 or L4 is a peptide sequence having at least 18 amino acids. In some embodiments, L3 or L4 is a peptide sequence having at least 26 amino acids. In some embodiments, L3 or L4 comprises (G2S) n wherein n is an integer from 1 to 3 (SEQ ID NO: 228). In some embodiments, L3 or L4 comprises (G2S) n wherein n is an integer of at least 1. In some embodiments, L3 or L4 comprises 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 a metalloprotease, a serine protease, a cysteine ​​protease, a threonine protease, and an aspartic protease. In some embodiments, L3 or L4 comprises an amino acid sequence cleavable by urokinase, an amino acid sequence cleavable by a membrane-type serine protease, or an amino acid sequence cleavable by a matrix metalloprotease. In some embodiments, L3 or L4 comprises a sequence according to SEQ ID NOs:18-19, 62-88. In some embodiments, L3 binds to the N-terminus of D1. In some embodiments, L3 binds to the C-terminus of D1. In some embodiments, L4 binds to the N-terminus of E1. In some embodiments, L4 binds to the C-terminus of E1. In some embodiments, when L3 is cleaved by a tumor-specific protease, P3 dissociates from D1, thereby exposing D1 to CD3. In some embodiments, when L4 is cleaved by a tumor-specific protease, P4 dissociates from E1, thereby exposing E1 to tumor antigens. In some embodiments, P3 weakens the binding of D1 to CD3. In some embodiments, P3 binds to D1 through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bonding interactions, or a combination thereof. In some embodiments, P3 binds to D1 at or near the antigen binding site. In some embodiments, when L3 is cleaved by a tumor-specific protease, P3 dissociates from D1, exposing D1 to CD3.

[0269] In some embodiments, P3 has less than 70% sequence identity with CD3. In some embodiments, P3 has less than 85% sequence identity with CD3. In some embodiments, P3 has less than 90% sequence identity with CD3. In some embodiments, P3 has less than 95% sequence identity with CD3. In some embodiments, P3 has less than 98% sequence identity with CD3. In some embodiments, P3 has less than 99% sequence identity with CD3. In some embodiments, P3 comprises an amino acid sequence according to SEQ ID NOs: 177-180. In some embodiments, P3 comprises a de novo generated amino acid sequence that shares less than 10% sequence identity with CD3.

[0270] In some embodiments, P4 weakens the binding of E1 to a tumor antigen. In some embodiments, P4 binds to E1 through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bond interactions, or a combination thereof. In some embodiments, P4 binds to E1 at or near the antigen binding site. In some embodiments, when L4 is cleaved by a tumor-specific protease, P4 dissociates from E1, thereby exposing E1 to the tumor antigen. In some embodiments, P4 has less than 70% sequence identity with the tumor antigen. In some embodiments, P4 has less than 80% sequence identity with the tumor antigen. In some embodiments, P4 has less than 85% sequence identity with the tumor antigen. In some embodiments, P4 has less than 90% sequence identity with the tumor antigen. In some embodiments, P4 has less than 95% sequence identity with the tumor antigen. In some embodiments, P4 comprises a de novo generated amino acid sequence that shares less than 10% sequence identity with the tumor antigen. In some embodiments, P3 or P4 comprises a peptide sequence of at least 5 amino acids in length. In some embodiments, P3 or P4 comprises a peptide sequence of at least 6 amino acids in length. In some embodiments, P3 or P4 comprises a peptide sequence of at least 10 amino acids in length. In some embodiments, P3 or P4 comprises a peptide sequence of at least 10 amino acids in length and no more than 20 amino acids in length. In some embodiments, P3 or P4 comprises a peptide sequence of at least 16 amino acids in length. In some embodiments, P3 or P4 comprises a peptide sequence of no more than 40 amino acids in length. In some embodiments, P3 or P4 comprises at least two cysteine ​​amino acid residues. In some embodiments, P3 or P4 comprises a cyclic peptide or a linear peptide. In some embodiments, P3 or P4 comprises a cyclic peptide. In some embodiments, P3 or P4 comprises a linear peptide. In some embodiments, P4 comprises an amino acid sequence according to SEQ ID NO: 185 or 186. In some embodiments, the tumor antigen comprises EGFR, and the T cell engager comprises the amino acid sequences of SEQ ID NO: 183 and 184. In some embodiments, P4 comprises an amino acid sequence according to SEQ ID NOs: 199-201. In some embodiments, the tumor antigen comprises TROP2, and the T cell engager comprises any one of the amino acid sequences of SEQ ID NOs: 193-198. In some embodiments, the tumor antigen comprises PSMA, and the T cell engager comprises the amino acid sequences of SEQ ID NOs: 175 and 176.

[0271] Antibody production

[0272] In some embodiments, the polypeptides described herein (e.g., antibodies and binding fragments thereof) are produced using any method known in the art for synthesizing polypeptides (e.g., antibodies), particularly by chemical synthesis or by recombinant expression, and preferably by recombinant expression technology.

[0273] In some cases, antibodies or binding fragments thereof are expressed recombinantly, and nucleic acids encoding the antibodies or binding fragments thereof are assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., 1994, BioTechniques 17:242), which involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligation of those oligonucleotides, and subsequent PCR amplification of the ligated oligonucleotides.

[0274] Alternatively, nucleic acid molecules encoding antibodies are optionally produced from a suitable source (e.g., an antibody cDNA library, or a cDNA library produced by any tissue or cell that expresses immunoglobulins) by PCR amplification using synthetic primers that hybridize to the 3' and 5' ends of the sequence or by cloning using oligonucleotide probes specific for a particular gene sequence.

[0275] In some cases, antibodies or binding partners thereof are optionally produced by immunizing an animal (e.g., a mouse) to produce polyclonal antibodies, or more preferably, by producing monoclonal antibodies, e.g., as described in Kohler and Milstein (1975, Nature 256:495-497), or in Kozbor et al. (1983, Immunology Today 4:72), or Cole et al. (1985 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Alternatively, clones encoding at least the Fab portion of an antibody are optionally obtained by screening a Fab expression library (e.g., as described in Huse et al., 1989, Science 246: 1275-1281) for clones that bind to the specific antigen or by screening antibody libraries (see, e.g., Clackson et al., 1991, Nature 352: 624; Hane et al., 1997 Proc. Natl. Acad. Sci. USA 94: 4937).

[0276] In some embodiments, a technique for producing "chimeric antibodies" (Morrison et al., 1984, Proc. Natl. Acad. Sci. 81:851-855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454) is used by splicing together genes from a mouse antibody molecule with the appropriate antigen specificity and genes from a human antibody molecule with the appropriate biological activity. Chimeric antibodies are molecules in which different portions are derived from different animal species, such as those having variable regions derived from a murine monoclonal antibody and human immunoglobulin constant regions.

[0277] In some embodiments, the technology described for producing single-chain antibodies (U.S. Patent No. 4,694,778; Bird, 1988, Science 242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989, Nature 334:544-54) is adapted to produce single-chain antibodies. The heavy chain and light chain fragments of the Fv region are connected by an amino acid bridge to form a single-chain antibody, producing a single-chain polypeptide. The technology of assembling functional Fv fragments in E. coli (Skerra et al., 1988, Science 242:1038-1041) is also optionally used.

[0278] In some embodiments, an expression vector comprising the nucleotide sequence of the antibody or the nucleotide sequence of the antibody is transferred to a host cell by conventional techniques (e.g., electroporation, liposome transfection, and calcium phosphate precipitation), and the transfected cells are then cultured by conventional techniques to produce the antibody. In specific embodiments, the expression of the antibody is regulated by a constitutive, inducible, or tissue-specific promoter.

[0279] In some embodiments, a variety of host-expression vector systems are utilized to express the antibodies or binding fragments thereof described herein. Such host-expression systems represent vehicles by which antibody coding sequences are produced and subsequently purified, but also represent cells that express the antibodies or binding fragments thereof in situ when transformed or transfected with the appropriate nucleotide coding sequences. These include, but are not limited to, microorganisms such as bacteria (e.g., Escherichia coli and Bacillus subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the antibody or its binding fragment coding sequence; yeast (e.g., Pichia pastoris) transformed with recombinant yeast expression vectors containing the antibody or its binding fragment coding sequence; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing the antibody or its binding fragment coding sequence; plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV)) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the antibody or its binding fragment coding sequence; or mammalian cell systems (e.g., COS, CHO, BH, 293, 293T, 3T3 cells) carrying a recombinant expression construct containing a promoter derived from the genome of a mammalian cell (e.g., metallothionein promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter).

[0280] In order to produce recombinant protein for a long time and in high yield, preferably stably expressed. In some cases, the cell line of through engineering approaches stably expressed antibody is optionally used. Rather than using the expression vector containing the viral replication origin, the host cell is transformed with the DNA controlled by suitable expression control elements (for example, promoter, enhancer, sequence, transcription terminator, polyadenylation site etc.) and selectable marker. After introducing foreign DNA, the cell of through engineering approaches is grown 1-2 days in enrichment medium and then proceeded to selective culture medium. The selectable marker in the recombinant plasmid gives the resistance to selection, and allows cells to stably integrate the plasmid into their chromosome and grow to form conversion foci (foci), which are cloned and expanded into cell line again. This method can be advantageously used for the cell line of through engineering approaches expressed antibody or its binding fragment.

[0281] In some cases, a number of selection systems are used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 192, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes for tk cells, hgprt cells, or aprt cells, respectively. In addition, antimetabolite resistance was used as the basis for selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Proc. Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 78: 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May 1993, TIBTECH 11(5):155-215); and hygro, which confers resistance to hygromycin (Santerre et al., 1984, Gene 30:147). Methods generally known in the art of recombinant DNA technology that can be used are described in Ausubel et al. (eds., 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY; and Dracopoli et al. (eds.), 1994, Current Protocols in Human Genetics, John Wiley & Sons, NY. Chapters 12 and 13; Colberre-Garapin et al., 1981, J. Mol. Biol. 150: 1).

[0282] In some cases, the expression level of the antibody is increased by vector amplification (for review, see Bebbington and Hentschel, the use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3 (Academic Press, New York, 1987)). When the marker in the vector system expressing the antibody is amplifiable, an increase in the inhibitor level present in the host cell culture will increase the copy number of the marker gene. Since the amplified region is related to the nucleotide sequence of the antibody, the production of the antibody will also increase (Crouse et al., 1983, Mol. Cell Biol. 3: 257).

[0283] In some cases, any method known in the art for purifying antibodies is used, for example, by chromatography (e.g., ion exchange; affinity, particularly by affinity for a specific antigen after protein A; and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for purifying proteins.

[0284] expression vector

[0285] In some embodiments, the vector includes any suitable vector derived from a eukaryotic or prokaryotic source. In some cases, the vector is obtained from bacteria (e.g., Escherichia coli), insects, yeast (e.g., Pichia pastoris), algae, or mammalian sources. Exemplary bacterial vectors include pACYC177, pASK75, pBAD vector series, pBADM vector series, pET vector series, pETM vector series, pGEX vector series, pHAT, pHAT2, pMal-c2, pMal-p2, pQE vector series, pRSETA, pRSET B, pRSET C, pTrcHis2 series, pZA31-Luc, pZE21-MCS-1, pFLAG ATS, pFLAG CTS, pFLAG MAC, pFLAG Shift-12c, pTAC-MAT-1, pFLAG CTC, or pTAC-MAT-2.

[0286] Exemplary insect vectors include pFastBac1, pFastBac DUAL, pFastBac ET, pFastBac HTa, pFastBac HTb, pFastBac HTc, pFastBac M30a, pFastBact M30b, pFastBac, M30c, pVL1392, pVL1393, pVL1393 M10, pVL1393M11, pVL1393 M12, a FLAG vector such as pPolh-FLAG1 or pPolh-MAT 2, or a MAT vector such as pPolh-MAT1 or pPolh-MAT2.

[0287] In some cases, the yeast carrier comprises pDEST TM 14 carriers, pDEST TM 15 carriers, pDEST TM 17 carriers, pDEST TM 24 carriers, pYES-DEST52 vector, pBAD-DEST49 Destination vector, pAO815 Pichia vector, pFLD1 Pichia pastoris vector, pGAPZA, B, & C Pichia pastoris vector, pPIC3.5K Pichia vector, pPIC6 A, B, & C Pichia vector, pPIC9K Pichia vector, pTEF1 / Zeo, pYES2 yeast vector, pYES2 / CT yeast vector, pYES2 / NT A, B, & C yeast vector, or pYES3 / CT yeast vector.

[0288] Exemplary algal vectors include pChlamy-4 vectors or MCS vectors.

[0289] Examples of mammalian vectors include transient expression vectors or stable expression vectors. Mammalian transient expression vectors may include pRK5, p3xFLAG-CMV 8, pFLAG-Myc-CMV 19, pFLAG-Myc-CMV 23, pFLAG-CMV 2, pFLAG-CMV 6a, b, c, pFLAG-CMV 5.1, pFLAG-CMV 5a, b, c, p3xFLAG-CMV 7.1, pFLAG-CMV 20, p3xFLAG-Myc-CMV 24, pCMV-FLAG-MAT1, pCMV-FLAG-MAT2, pBICEP-CMV 3, or pBICEP-CMV 4. Mammalian stable expression vectors can include pFLAG-CMV 3, p3xFLAG-CMV 9, p3xFLAG-CMV 13, pFLAG-Myc-CMV 21, p3xFLAG-Myc-CMV 25, pFLAG-CMV 4, p3xFLAG-CMV 10, p3xFLAG-CMV 14, pFLAG-Myc-CMV 22, p3xFLAG-Myc-CMV 26, pBICEP-CMV 1, or pBICEP-CMV 2.

[0290] In some cases, the cell-free system is a mixture of cytoplasmic and / or nuclear components from cells and is used for in vitro nucleic acid synthesis. In some cases, the cell-free system utilizes prokaryotic cell components or eukaryotic cell components. Sometimes, nucleic acid synthesis is obtained in a cell-free system based on, for example, Drosophila cells, Xenopus laevis eggs, or HeLa cells. Exemplary cell-free systems include, but are not limited to, E. coli S30 extract systems, E. coli T7 S30 systems, or

[0291] host cells

[0292] In some embodiments, host cells include any suitable cells, such as cells of natural origin or genetically modified cells. In some cases, host cells are production host cells. In some cases, host cells are eukaryotic cells. In other cases, host cells are prokaryotic cells. In some cases, eukaryotic cells include fungi (e.g., yeast cells), animal cells, or plant cells. In some cases, prokaryotic cells are bacterial cells. Examples of bacterial cells include gram-positive bacteria or gram-negative bacteria. Sometimes gram-negative bacteria are anaerobic, rod-shaped, or both.

[0293] In some cases, the gram-positive bacteria include Actinobacteria, Firmicutes, or Tenericutes. In some cases, the gram-negative bacteria include Aquificae, Deinococcus-Thermus, Fibrobacteres-Chlorobi / Bacteroidetes (FCB group), Fusobacteria, Gemmatimonadetes, Nitrospirae, Planctomycetes-Verrucomicrobia / Chlamydiae (PVC group), Proteobacteria, Spirochaetes, or Synergistetes. The other bacteria may be of the phylum Acidobacteria, Chloroflexi, Chrysiogenetes, Cyanobacteria, Deferribacteres, Dictyoglomi, Thermodesulfobacteria, or Thermotogae. The bacterial cell may be Escherichia coli, Clostridium botulinum, or Coli bacilli.

[0294] Exemplary prokaryotic host cells include, but are not limited to, BL21, Mach1 TM DH10B TM 、TOP10、DH5α、DH10Bac TM 、OmniMax TM 、MegaX TM DH12S TM , INV110, TOP10F', INVαF, TOP10 / P3, ccdB Survival, PIR1, PIR2, Stbl2 TM 、Stbl3 TM or Stbl4 TM .

[0295] In some cases, animal cells include cells from vertebrates or from invertebrates. In some cases, animal cells include cells from marine invertebrates, fish, insects, amphibians, reptiles, or mammals. In some cases, fungal cells include yeast cells, such as brewer's yeast, baker's yeast, or wine yeast.

[0296] In some cases, yeast includes ascomycetes, molds, filamentous fungi, basidiomycetes or zygomycetes. In some cases, yeast includes ascomycetes or basidiomycetes (Basidiomycota). In some cases, ascomycetes include Saccharomycotina (true yeast, for example Saccharomyces cerevisiae (baker's yeast)) or Taphrinomycotina (Taphrinomycotina) (for example, Schizosaccharomycetes (fission yeast)). In some cases, Basidiomycota includes Agaricomycota (Agaricomycotina) (for example, Tremellomycetes (Tremellomycetes)) or Pucciniomycotina (Pucciniomycotina) (for example, Microbotryomycetes (Microbotryomycetes)).

[0297] Exemplary yeasts or filamentous fungi include, e.g., the genera Saccharomyces, Schizosaccharomyces, Candida, Pichia, Hansenula, Kluyveromyces, Zygosaccharomyces, Yarrowia, Trichosporon, Rhodosporidi, Aspergillus, Fusarium, or Trichoderma. Exemplary yeasts or filamentous fungi include, e.g., species such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida utilis, Candida boidini, Candida albicans, Candida tropicalis, Candida stellatoidea, Candida glabrata, Candida krusei, Candida parapsilosis, Candida guilliermondii, Candida viswanathii, Candida lusitaniae, Rhodotorula mucilaginosa, Pichia metanolica, Pichia angusta, Pichia pastoris, and the like. pastoris), Pichia anomala, Hansenula polymorpha, Kluyveromyces lactis, Zygosaccharomyces rouxii, Yarrowia lipolytica, Trichosporon pullulans, Rhodosporidium toru-Aspergillus niger, Aspergillus nidulans, Aspergillus avasoniiawamori, Aspergillus oryzae, Trichoderma reesei, Yarrowia lipolytica, Brettanomyces bruxellensis, Candida stellata, Schizosaccharomyces pombe, Torulaspora delbrueckii, Zygosaccharomyces bailii, Cryptococcus neoformans, Cryptococcus gattii, or Saccharomyces boulardii.

[0298] Exemplary yeast host cells include, but are not limited to, Pichia pastoris yeast strains such as GS115, KM71H, SMD1168, SMD1168H, and X-33; and Saccharomyces cerevisiae yeast strains such as INVSc1.

[0299] In some cases, other animal cells include cells obtained from molluscs, arthropods, annelids or sponges. In some cases, other animal cells are mammalian cells, such as from primates, apes, horses, cattle, pigs, dogs, cats or rodents. In some cases, rodents include mice, rats, hamsters, gerbils, hamsters, chinchillas (chinchilla), squirrels or guinea pigs.

[0300] Exemplary mammalian host cells include, but are not limited to, 293A cell line, 293FT cell line, 293F cells, 293H cells, CHO DG44 cells, CHO-S cells, CHO-K1 cells, FUT8 KO CHOK1, Expi293F TM cells, Flp-In TM T-REx TM 293 cell line, Flp-In TM -293 cell line, Flp-In TM -3T3 cell line, Flp-In TM -BHK cell line, Flp-In TM -CHO cell lines, Flp-In TM -CV-1 cell line, Flp-In TM -Jurkat cell line, FreeStyle TM 293-F cells, FreeStyleTM CHO-S cells, GripTite TM 293MSR cell line, GS-CHO cell line, HepaRG TM cells, T-REx TM Jurkat cell line, Per.C6 cells, T-REx TM -293 cell line, T-REx TM -CHO cell lines and T-REx TM -HeLa cell line.

[0301] In some cases, the mammalian host cell is a stable cell line, or a cell line that has integrated the genetic material of interest into its own genome and has the ability to express the product of the genetic material after many generations of cell division. In some cases, the mammalian host cell is a transient cell line, or a cell line that has not integrated the genetic material of interest into its own genome and does not have the ability to express the product of the genetic material after many generations of cell division.

[0302] Exemplary insect host cells include, but are not limited to, Drosophila S2 cells, Sf9 cells, Sf21 cells, High Five TM Cells and cell.

[0303] In some cases, the plant cell comprises a cell from algae.Exemplary insect cell lines include, but are not limited to, strains from Chlamydomonas reinhardtii 137c or Synechococcuse longatus PPC 7942.

[0304] Products

[0305] In another aspect of the present disclosure, an article of manufacture containing materials useful for treating, preventing, and / or diagnosing the aforementioned conditions is provided. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container can be formed from a variety of materials, such as glass or plastic.

[0306] The label or package insert indicates that the composition is used to treat the condition of choice.The article of manufacture in this embodiment of the disclosure may further comprise a package insert indicating that the composition can be used to treat a particular condition.

[0307] Alternatively or additionally, the article of manufacture may further comprise: a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0308] Implementation Plan

[0309] Embodiment 1. An isolated multispecific antibody according to the following formula: P1-L1-A1-LB (Formula I), wherein A1 comprises a CD28 binding domain; B comprises a PD-L1 binding domain; L comprises a linker connecting A1 and B; P1 comprises a peptide that binds to A1, and L1 comprises a linker that connects A1 and P1 and is a substrate for a tumor-specific protease, wherein P1 comprises an amino acid sequence according to any one of SEQ ID NOs: 24-53, 128-148, and any one of the amino acid sequences of Table 20, or an amino acid sequence having 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 24-53, 128-148, and any one of the amino acid sequences of Table 20.

[0310] Embodiment 2. An isolated multispecific antibody as described in embodiment 1, wherein the multispecific antibody is according to the following formula: P1-L1-A1-LB-L2-P2 (Formula Ia), wherein P2 comprises a peptide that binds to B, and L2 comprises a linker portion that connects B to P2 and is a substrate for a tumor-specific protease.

[0311] Embodiment 3. The isolated multispecific antibody of embodiment 1 or 2, wherein P1 comprises an amino acid sequence according to any one of SEQ ID NOs: 24-53, 128-148, and the amino acid sequence of Table 20.

[0312] Embodiment 4. The isolated multispecific antibody of embodiment 1 or 2, wherein P1 comprises an amino acid sequence according to any one of SEQ ID NOs: 42-53 or an amino acid sequence having 1, 2 or 3 amino acid mutations, substitutions or deletions relative to any one of SEQ ID NOs: 42-53.

[0313] Embodiment 5. The isolated multispecific antibody of embodiment 1 or 2, wherein P1 comprises the amino acid sequence according to any one of SEQ ID NOs: 42-53.

[0314] Embodiment 6. The isolated multispecific antibody of embodiment 1 or 2, wherein P1 comprises an amino acid sequence according to any one of the amino acid sequences in Table 20 or an amino acid sequence having 1, 2 or 3 amino acid mutations, substitutions or deletions relative to any one of the amino acid sequences in Table 20.

[0315] Embodiment 7. The isolated multispecific antibody of embodiment 1 or 2, wherein P1 comprises an amino acid sequence according to any one of the amino acid sequences of Table 20.

[0316] Embodiment 8. The isolated multispecific antibody of embodiment 1 or 2, wherein P1 comprises an amino acid sequence according to any one of SEQ ID NOs: 128-147 or an amino acid sequence having 1, 2 or 3 amino acid mutations, substitutions or deletions relative to any one of SEQ ID NOs: 128-147.

[0317] Embodiment 9. The isolated multispecific antibody of embodiment 1 or 2, wherein P1 comprises the amino acid sequence according to any one of SEQ ID NOs: 128-147.

[0318] Embodiment 10. The isolated multispecific antibody of embodiment 1 or 2, wherein P1 comprises a sequence according to X1-X2-X3-C-X4-X5-X6-X7-X8-X9-X 10 -CX 11 -X 12 an amino acid sequence wherein X1 is selected from the group consisting of M, I, L, and V; X2 is selected from the group consisting of D, H, N, A, F, S, T, Y, and V; X3 is selected from the group consisting of W, L, and F; X4 is selected from the group consisting of P, A, and L; X5 is selected from the group consisting of R, T, I, M, S, K, L, V, W, F, A, P, and D; X6 is selected from the group consisting of E, D, Y, H, S, F, A, N, T, I, P, and V; X7 is selected from the group consisting of L, M, R, S, Q, and H; X8 is selected from the group consisting of W and Q; X9 is selected from the group consisting of H, N, D, A, S, Y, T, F, V, L, and I; X 10 Selected from E, V, L, D, Y, R, Q, H, F, K, A, M and N; X 11 is selected from F, Y, L, W, and V; and X 12 Selected from N, A, F, S, Y, H, D, T and L.

[0319] Embodiment 11. The isolated multispecific antibody of embodiment 10, wherein X1 is selected from M, I, and L; X2 is selected from D, H, N, and A; X3 is W; X4 is P; X5 is selected from R, T, I, M, S, and K; X6 is selected from E, D, Y, H, S, and F; X7 is selected from L, M, and R; X8 is W; X9 is selected from H, N, D, A, S, and V; X 10 Selected from E, V, L, D and H; X11 is selected from F, Y and L; and X 12 Selected from N, A, F, S and Y.

[0320] Embodiment 12. The isolated multispecific antibody of embodiment 11, wherein X1 is M; X2 is selected from D and H; X3 is W; X4 is P; X5 is selected from R, T, and I; X6 is selected from E, D, and Y; X7 is selected from L, M, and R; X8 is W; X9 is selected from H, N, D, and V; X 10 Selected from E, V, L, D and H; X 11 is F; and X 12 Selected from N, A and F.

[0321] Embodiment 13. The isolated multispecific antibody of any one of embodiments 1-3, 10-12, wherein P1 comprises the amino acid sequence according to SEQ ID NO: 32 or an amino acid sequence having 1, 2 or 3 amino acid mutations, substitutions or deletions relative to SEQ ID NO: 32.

[0322] Embodiment 14. The isolated multispecific antibody of any one of embodiments 1-3, 10-12, wherein P1 comprises the amino acid sequence according to SEQ ID NO: 32.

[0323] Embodiment 15. The isolated multispecific antibody of any one of embodiments 1-3, 10-12, wherein P1 comprises the amino acid sequence according to SEQ ID NO: 138 or an amino acid sequence having 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to SEQ ID NO: 138.

[0324] Embodiment 16. The isolated multispecific antibody of any one of embodiments 1-3, 10-12, wherein P1 comprises the amino acid sequence according to SEQ ID NO: 138.

[0325] Embodiment 17. The isolated multispecific antibody of any one of embodiments 1-16, wherein P1 impairs binding of A1 to CD28.

[0326] Embodiment 18. The isolated multispecific antibody of any one of embodiments 1-17, wherein P1 is bound to A1 through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bonding interactions, or a combination thereof.

[0327] Embodiment 19. The isolated multispecific antibody of any one of embodiments 1-18, wherein P1 is bound to A1 at or near the antigen binding site.

[0328] Embodiment 20. The isolated multispecific antibody of any one of embodiments 1-18, wherein when L1 is cleaved by a tumor-specific protease, P1 is unbound from A1, thereby exposing A1 to CD28.

[0329] Embodiment 21. The isolated multispecific antibody of any one of embodiments 1-20, wherein P1 has less than 75% sequence identity with CD28.

[0330] Embodiment 22. The isolated multispecific antibody of any one of embodiments 1-21, wherein P1 has less than 80% sequence identity with CD28.

[0331] Embodiment 23. The isolated multispecific antibody of any one of embodiments 1-22, wherein P1 has less than 85% sequence identity with CD28.

[0332] Embodiment 24. The isolated multispecific antibody of any one of embodiments 1-23, wherein P1 has less than 90% sequence identity with CD28.

[0333] Embodiment 25. The isolated multispecific antibody of any one of embodiments 1-24, wherein P1 has less than 95% sequence identity with CD28.

[0334] Embodiment 26. The isolated multispecific antibody of any one of embodiments 1-25, wherein P1 comprises a de novo generated amino acid sequence that shares less than 10% sequence identity with CD28.

[0335] Embodiment 27. The isolated multispecific antibody of any one of Embodiments 2-26, wherein P2 weakens the binding of B to PD-L1.

[0336] Embodiment 28. The isolated multispecific antibody of any one of embodiments 2-27, wherein P2 is bound to B through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bonding interactions, or a combination thereof.

[0337] Embodiment 29. The isolated multispecific antibody of any one of embodiments 2-28, wherein P2 binds to B at or near the antigen binding site.

[0338] Embodiment 30. The isolated multispecific antibody of any one of embodiments 2-29, wherein when L2 is cleaved by a tumor-specific protease, P2 is unbound from B, thereby exposing B to PD-L1.

[0339] Embodiment 31. The isolated multispecific antibody of any one of Embodiments 2-30, wherein P2 has less than 70% sequence identity with PD-L1.

[0340] Embodiment 32. The isolated multispecific antibody of any one of Embodiments 2-31, wherein P2 has less than 75% sequence identity to PD-L1.

[0341] Embodiment 33. The isolated multispecific antibody of any one of Embodiments 2-32, wherein P2 has less than 80% sequence identity with PD-L1.

[0342] Embodiment 34. The isolated multispecific antibody of any one of Embodiments 2-33, wherein P2 has less than 85% sequence identity to PD-L1.

[0343] Embodiment 35. The isolated multispecific antibody of any one of Embodiments 2-34, wherein P2 has less than 90% sequence identity to PD-L1.

[0344] Embodiment 36. The isolated multispecific antibody of any one of Embodiments 2-35, wherein P2 has less than 95% sequence identity to PD-L1.

[0345] Embodiment 37. The isolated multispecific antibody of any one of embodiments 2-36, wherein P2 comprises a de novo generated amino acid sequence that shares less than 10% sequence identity with PD-L1.

[0346] Embodiment 38. The isolated multispecific antibody of any one of Embodiments 2-37, wherein P2 comprises a peptide sequence of at least 5 amino acids in length.

[0347] Embodiment 39. The isolated multispecific antibody of any one of Embodiments 2-38, wherein P2 comprises a peptide sequence of at least 6 amino acids in length.

[0348] Embodiment 40. The isolated multispecific antibody of any one of Embodiments 2-39, wherein P2 comprises a peptide sequence of at least 10 amino acids in length.

[0349] Embodiment 41. The isolated multispecific antibody of any one of Embodiments 2-40, wherein P2 comprises a peptide sequence of at least 10 amino acids in length and no more than 20 amino acids in length.

[0350] Embodiment 42. The isolated multispecific antibody of any one of Embodiments 2-41, wherein P2 comprises a peptide sequence of at least 16 amino acids in length.

[0351] Embodiment 43. The isolated multispecific antibody of any one of Embodiments 2-42, wherein P2 comprises a peptide sequence of no more than 40 amino acids in length.

[0352] Embodiment 44. The isolated multispecific antibody of any one of Embodiments 1-43, wherein P1 or P2 comprises at least two cysteine ​​amino acid residues.

[0353] Embodiment 45. The isolated multispecific antibody of any one of embodiments 1-44, wherein P1 or P2 comprises a cyclic peptide or a linear peptide.

[0354] Embodiment 46. The isolated multispecific antibody of any one of Embodiments 1-45, wherein P1 or P2 comprises a cyclic peptide.

[0355] Embodiment 47. The isolated multispecific antibody of any one of Embodiments 1-46, wherein P1 or P2 comprises a linear peptide.

[0356] Embodiment 48. The isolated multispecific antibody of any one of embodiments 1-47, wherein P1 or P2 comprises a modified amino acid or a non-natural amino acid or a modified non-natural amino acid, or a combination thereof.

[0357] Embodiment 49. The isolated multispecific antibody of any one of embodiments 1-48, wherein P1 or P2 does not comprise albumin or an albumin fragment.

[0358] Embodiment 50. The isolated multispecific antibody of any one of embodiments 1-49, wherein P1 or P2 does not comprise an albumin binding domain.

[0359] Embodiment 51. The isolated multispecific antibody of any one of embodiments 1-50, wherein L1 or L2 is a peptide sequence having at least 5 and no more than 50 amino acids.

[0360] Embodiment 52. The isolated multispecific antibody of any one of embodiments 1-51, wherein L1 or L2 is a peptide sequence having at least 10 and no more than 30 amino acids.

[0361] Embodiment 53. The isolated multispecific antibody of any one of embodiments 1-52, wherein L1 or L2 is a peptide sequence of at least 10 amino acids.

[0362] Embodiment 54. The isolated multispecific antibody of any one of embodiments 1-53, wherein L1 or L2 is a peptide sequence of at least 18 amino acids.

[0363] Embodiment 55. The isolated multispecific antibody of any one of embodiments 1-54, wherein L1 or L2 is a peptide sequence of at least 26 amino acids.

[0364] Embodiment 56. The isolated multispecific antibody of any one of embodiments 1-55, wherein L1 or L2 comprises (G2S) n wherein n is an integer from 1 to 3 (SEQ ID NO: 228).

[0365] Embodiment 57. The isolated multispecific antibody of any one of embodiments 1-56, wherein L1 or L2 comprises (G2S) n wherein n is an integer of at least 1.

[0366] Embodiment 58. The isolated multispecific antibody of any one of embodiments 1-57, wherein L1 or L2 comprises a formula selected from: (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.

[0367] Embodiment 59. The isolated multispecific antibody of any one of embodiments 1-58, wherein the tumor-specific protease is selected from the group consisting of a metalloprotease, a serine protease, a cysteine ​​protease, a threonine protease, and an aspartic protease.

[0368] Embodiment 60. The isolated multispecific antibody of any one of embodiments 1-58, wherein L1 or L2 comprises an amino acid sequence cleavable by urokinase, an amino acid sequence cleavable by a membrane-type serine protease, or an amino acid sequence cleavable by a matrix metalloprotease.

[0369] Embodiment 61. The isolated multispecific antibody of any one of Embodiments 1-60, wherein L1 or L2 comprises a sequence according to SEQ ID NOs: 18-19, 62-88.

[0370] Embodiment 62. The isolated multispecific antibody of any one of embodiments 1-61, wherein L1 is bound to the N-terminus of A1.

[0371] Embodiment 63. The isolated multispecific antibody of any one of embodiments 1-61, wherein L1 is bound to the C-terminus of A1.

[0372] Embodiment 64. The isolated multispecific antibody of any one of embodiments 1-61, wherein L2 is bound to the N-terminus of B.

[0373] Embodiment 65. The isolated multispecific antibody of any one of embodiments 1-61, wherein L2 is bound to the C-terminus of B.

[0374] Embodiment 66. The isolated multispecific antibody of any one of embodiments 1-65, wherein the CD28 binding domain comprises a single-chain variable fragment, a single domain antibody, a Fab, or a Fab'.

[0375] Embodiment 67. The isolated multispecific antibody of Embodiment 66, wherein the CD28 binding domain comprises a single-chain variable fragment.

[0376] Embodiment 68. The isolated multispecific antibody of Embodiment 66, wherein the CD28 binding domain comprises a single domain antibody.

[0377] Embodiment 69. The isolated multispecific antibody of Embodiment 66, wherein the CD28 binding domain comprises Fab or Fab'.

[0378] Embodiment 70. The isolated multispecific antibody of any one of embodiments 1-69, wherein the PD-L1 binding domain comprises a single-chain variable fragment, a single domain antibody, a Fab, or a Fab′.

[0379] Embodiment 71. The isolated multispecific antibody of Embodiment 70, wherein the PD-L1 binding domain comprises Fab or Fab'.

[0380] Embodiment 72. The isolated multispecific antibody of Embodiment 70, wherein the PD-L1 binding domain comprises a Fab or Fab' and the CD28 binding domain comprises a single-chain variable fragment.

[0381] Embodiment 73. The isolated multispecific antibody of embodiment 70, wherein the PD-L1 binding domain comprising Fab or Fab' comprises a Fab heavy chain polypeptide and a Fab light chain polypeptide, the Fab heavy chain polypeptide comprises a Fab heavy chain variable domain, and the Fab light chain polypeptide comprises a Fab light chain variable domain.

[0382] Embodiment 74. The isolated multispecific antibody of embodiment 73, wherein the CD28 binding domain comprising the single chain variable fragment comprises a scFv heavy chain variable domain and a scFv light chain variable domain.

[0383] Embodiment 75. The isolated multispecific antibody of any one of embodiments 1-74, wherein a linker connects the C-terminus of A1 to the N-terminus of B.

[0384] Embodiment 76. The isolated multispecific antibody of any one of embodiments 1-74, wherein a linker connects the N-terminus of A1 to the C-terminus of B.

[0385] Embodiment 77. The isolated multispecific antibody of Embodiment 73, wherein a linker connects the C-terminus of Al to the N-terminus of the Fab heavy chain polypeptide.

[0386] Embodiment 78. The isolated multispecific antibody of Embodiment 73, wherein a linker connects the N-terminus of Al to the C-terminus of the Fab heavy chain polypeptide.

[0387] Embodiment 79. The isolated multispecific antibody of Embodiment 73, wherein a linker connects the C-terminus of Al to the N-terminus of the Fab light chain polypeptide.

[0388] Embodiment 80. The isolated multispecific antibody of Embodiment 73, wherein a linker connects the N-terminus of Al to the C-terminus of the Fab light chain polypeptide.

[0389] Embodiment 81. The isolated multispecific antibody of Embodiment 73, wherein a linker connects the Fab light chain polypeptide to the scFv light chain variable domain.

[0390] Embodiment 82. The isolated multispecific antibody of Embodiment 73, wherein a linker connects the Fab light chain polypeptide to the scFv heavy chain variable domain.

[0391] Embodiment 83. The isolated multispecific antibody of Embodiment 73, wherein a linker connects the Fab heavy chain polypeptide to the scFv light chain variable domain.

[0392] Embodiment 84. The isolated multispecific antibody of Embodiment 73, wherein a linker connects the Fab heavy chain polypeptide to the scFv heavy chain variable domain.

[0393] Embodiment 85. The isolated multispecific antibody of Embodiment 73, wherein a linker connects the Fab light chain polypeptide to the N-terminus of the scFv light chain variable domain.

[0394] Embodiment 86. The isolated multispecific antibody of Embodiment 73, wherein a linker connects the Fab light chain polypeptide to the C-terminus of the scFv light chain variable domain.

[0395] Embodiment 87. The isolated multispecific antibody of Embodiment 73, wherein a linker connects the Fab light chain polypeptide to the N-terminus of the scFv heavy chain variable domain.

[0396] Embodiment 88. The isolated multispecific antibody of Embodiment 73, wherein a linker connects the Fab light chain polypeptide to the C-terminus of the scFv heavy chain variable domain.

[0397] Embodiment 89. The isolated multispecific antibody of Embodiment 73, wherein a linker connects the Fab heavy chain polypeptide to the N-terminus of the scFv light chain variable domain.

[0398] Embodiment 90. The isolated multispecific antibody of Embodiment 73, wherein a linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain.

[0399] Embodiment 91. The isolated multispecific antibody of Embodiment 73, wherein a linker connects the Fab heavy chain polypeptide to the N-terminus of the scFv heavy chain variable domain.

[0400] Embodiment 92. The isolated multispecific antibody of Embodiment 73, wherein a linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv heavy chain variable domain.

[0401] Embodiment 93. The isolated multispecific antibody of any one of embodiments 1-92, wherein the linker is at least 5 amino acids in length.

[0402] Embodiment 94. The isolated multispecific antibody of any one of embodiments 1-93, wherein the linker is no more than 30 amino acids in length.

[0403] Embodiment 95. The isolated multispecific antibody of any one of embodiments 1-94, wherein the linker is at least 5 amino acids and no more than 30 amino acids in length.

[0404] Embodiment 96. The isolated multispecific antibody of any one of embodiments 1-95, wherein the linker is 5 amino acids in length.

[0405] Embodiment 97. The isolated multispecific antibody of any one of Embodiments 1-96, wherein the linker is 15 amino acids in length.

[0406] Embodiment 98. The isolated multispecific antibody of any one of embodiments 1-97, wherein the linker comprises (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.

[0407] Embodiment 99. The isolated multispecific antibody of any one of embodiments 1-98, wherein L comprises (G2S) n wherein n is an integer from 1 to 3 (SEQ ID NO: 228).

[0408] Embodiment 100. The isolated multispecific antibody of any one of embodiments 1-97, wherein L comprises the amino acid sequence of SEQ ID NO: 18 (GGGGSGGGGSGGGGS) or SEQ ID NO: 19 (GGGGS).

[0409] Embodiment 101. The isolated multispecific antibody of embodiment 73, wherein the scFv heavy chain variable domain comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and 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 the CDRs comprise 0-2 amino acid modifications in at least one of said HC-CDR1, HC-CDR2, or HC-CDR3.

[0410] Embodiment 102. An isolated multispecific antibody as described in Embodiment 73, wherein the scFv light chain variable domain comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, LC-CDR2, and LC-CDR3 of the scFv light chain variable domain comprise: LC-CDR1: SEQ ID NO: 4; LC-CDR2: SEQ ID NO: 5(KA); and LC-CDR3: SEQ ID NO: 6, and wherein the CDRs comprise 0-2 amino acid modifications in at least one of said LC-CDR1, LC-CDR2, or LC-CDR3.

[0411] Embodiment 103. An isolated multispecific antibody as described in any one of embodiments 1-100, wherein A1 comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, LC-CDR2, and LC-CDR3 of A1 comprise: LC-CDR1: SEQ ID NO: 4; LC-CDR2: SEQ ID NO: 5(KA); and LC-CDR3: SEQ ID NO: 6; wherein A1 comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 of A1 comprise: HC-CDR1: SEQ ID NO: 1; HC-CDR2: SEQ ID NO: 2; HC-CDR3: SEQ ID NO: 3.

[0412] Embodiment 104. The isolated multispecific antibody of embodiment 73, wherein the Fab heavy chain variable domain comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and 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 the CDRs comprise 0-2 amino acid modifications in at least one of said HC-CDR1, HC-CDR2, or HC-CDR3.

[0413] Embodiment 105. An isolated multispecific antibody as described in Embodiment 73, wherein the Fab light chain variable domain comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2 and LC-CDR3, wherein the LC-CDR1, LC-CDR2 and LC-CDR3 of the Fab light chain variable domain comprise: LC-CDR1: SEQ ID NO: 13; LC-CDR2: SEQ ID NO: 14 (DA); and LC-CDR3: SEQ ID NO: 15; and wherein the CDRs comprise 0-2 amino acid modifications in at least one of said LC-CDR1, LC-CDR2 or LC-CDR3.

[0414] Embodiment 106. The isolated multispecific antibody of any one of Embodiments 1-100, wherein B comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 of B comprise: HC-CDR1: SEQ ID NO: 10; HC-CDR2: SEQ ID NO: 11; HC-CDR3: SEQ ID NO: 12; and wherein B comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, LC-CDR2, and LC-CDR3 of B comprise: LC-CDR1: SEQ ID NO: 13; LC-CDR2: SEQ ID NO: 14 (DA); and LC-CDR3: SEQ ID NO: 15.

[0415] Embodiment 107. The isolated multispecific antibody of Embodiment 73, wherein the scFv heavy chain variable domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO:7.

[0416] Embodiment 108. The isolated multispecific antibody of Embodiment 73, wherein the scFv heavy chain variable domain comprises an amino acid sequence of at least 75 consecutive amino acid residues of SEQ ID NO:7.

[0417] Embodiment 109. The isolated multispecific antibody of Embodiment 73, wherein the scFv heavy chain variable domain comprises an amino acid sequence of at least 110 contiguous amino acid residues of SEQ ID NO:7.

[0418] Embodiment 110. The isolated multispecific antibody of Embodiment 73, wherein 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 with at least 110 consecutive amino acid residues of SEQ ID NO:7.

[0419] Embodiment 111. The isolated multispecific antibody of Embodiment 73, wherein the scFv heavy chain variable domain comprises the amino acid sequence according to SEQ ID NO:7.

[0420] Embodiment 112. The isolated multispecific antibody of Embodiment 73, wherein the scFv light chain variable domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO:8.

[0421] Embodiment 113. The isolated multispecific antibody of Embodiment 73, wherein the scFv light chain variable domain comprises an amino acid sequence of at least 75 contiguous amino acid residues of SEQ ID NO:8.

[0422] Embodiment 114. The isolated multispecific antibody of Embodiment 73, wherein the scFv light chain variable domain comprises an amino acid sequence of at least 100 contiguous amino acid residues of SEQ ID NO:8.

[0423] Embodiment 115. The isolated multispecific antibody of Embodiment 73, wherein the scFv light chain variable domain comprises an amino acid sequence of at least 100 contiguous amino acid residues of SEQ ID NO:8 and has at least 80% sequence identity with at least 100 contiguous amino acid residues of SEQ ID NO:8.

[0424] Embodiment 116. The isolated multispecific antibody of Embodiment 73, wherein the scFv light chain variable domain comprises the amino acid sequence according to SEQ ID NO:8.

[0425] Embodiment 117. The isolated multispecific antibody of Embodiment 73, wherein the scFv comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO:9.

[0426] Embodiment 118. The isolated multispecific antibody of Embodiment 73, wherein the scFv comprises an amino acid sequence of at least 175 consecutive amino acid residues of SEQ ID NO:9.

[0427] Embodiment 119. The isolated multispecific antibody of Embodiment 73, wherein the scFv comprises an amino acid sequence of at least 210 consecutive amino acid residues of SEQ ID NO:9.

[0428] Embodiment 120. The isolated multispecific antibody of embodiment 73, wherein 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 with at least 210 consecutive amino acid residues of SEQ ID NO:9.

[0429] Embodiment 121. The isolated multispecific antibody of Embodiment 73, wherein the scFv comprises the amino acid sequence according to SEQ ID NO:9.

[0430] Embodiment 122. The isolated multispecific antibody of Embodiment 73, wherein the Fab heavy chain polypeptide comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 17.

[0431] Embodiment 123. The isolated multispecific antibody of Embodiment 73, wherein the Fab heavy chain polypeptide comprises an amino acid sequence of at least 175 contiguous amino acid residues of SEQ ID NO:17.

[0432] Embodiment 124. The isolated multispecific antibody of Embodiment 73, wherein the Fab heavy chain polypeptide comprises an amino acid sequence of at least 215 contiguous amino acid residues of SEQ ID NO: 17.

[0433] Embodiment 125. An isolated multispecific antibody as described in Embodiment 73, wherein 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 with at least 215 consecutive amino acid residues of SEQ ID NO: 17.

[0434] Embodiment 126. The isolated multispecific antibody of Embodiment 73, wherein the Fab heavy chain polypeptide comprises the amino acid sequence according to SEQ ID NO: 17.

[0435] Embodiment 127. The isolated multispecific antibody of Embodiment 73, wherein the Fab light chain polypeptide comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 16.

[0436] Embodiment 128. The isolated multispecific antibody of Embodiment 73, wherein the Fab light chain polypeptide comprises an amino acid sequence of at least 175 contiguous amino acid residues of SEQ ID NO:16.

[0437] Embodiment 129. The isolated multispecific antibody of Embodiment 73, wherein the Fab light chain polypeptide comprises an amino acid sequence of at least 200 contiguous amino acid residues of SEQ ID NO:16.

[0438] Embodiment 130. An isolated multispecific antibody as described in Embodiment 73, wherein 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 with at least 200 consecutive amino acid residues of SEQ ID NO:16.

[0439] Embodiment 131. The isolated multispecific antibody of Embodiment 73, wherein the Fab light chain polypeptide comprises the amino acid sequence according to SEQ ID NO:16.

[0440] Embodiment 132. An isolated multispecific antibody as described in Embodiment 73, wherein a 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 having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO:20, and the amino acid sequence of the Fab heavy chain polypeptide connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO:21.

[0441] Embodiment 133. An isolated multispecific antibody as described in Embodiment 73, wherein a 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 the amino acid sequence according to SEQ ID NO:20, and the amino acid sequence of the Fab heavy chain polypeptide connected to the C-terminus of the scFv light chain variable domain comprises the amino acid sequence according to SEQ ID NO:21.

[0442] Embodiment 134. An isolated multispecific antibody as described in Embodiment 73, wherein a 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 having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 23, and the amino acid sequence of the Fab light chain polypeptide connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 22.

[0443] Embodiment 135. An isolated multispecific antibody as described in Embodiment 73, wherein a 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 the amino acid sequence according to SEQ ID NO: 23, and the amino acid sequence of the Fab light chain polypeptide connected to the C-terminus of the scFv light chain variable domain comprises the amino acid sequence according to SEQ ID NO: 22.

[0444] Embodiment 136. The isolated multispecific antibody of any one of embodiments 1-135, wherein the multispecific antibody further comprises a half-life extending molecule (H1).

[0445] Embodiment 137. The isolated multispecific antibody of Embodiment 136, wherein H1 is linked to P1.

[0446] Embodiment 138. The isolated multispecific antibody of Embodiment 136, wherein H1 is linked to P2.

[0447] Embodiment 139. The isolated multispecific antibody of any one of embodiments 136-138, wherein H1 does not block binding of A1 to CD28.

[0448] Embodiment 140. The isolated multispecific antibody of any one of Embodiments 136-139, wherein H1 does not block B binding to PD-L1.

[0449] Embodiment 141. The isolated multispecific antibody of any one of Embodiments 136-140, wherein H1 comprises a linking portion (L5) connecting H1 to P1 or H1 to P2.

[0450] Embodiment 142. The isolated multispecific antibody of any one of embodiments 136-141, wherein the half-life extending molecule (H1) has no binding affinity for PD-L1.

[0451] Embodiment 143. The isolated multispecific antibody of any one of embodiments 136-142, wherein the half-life extending molecule (H1) has no binding affinity for CD28.

[0452] Embodiment 144. The isolated multispecific antibody of any one of embodiments 136-143, wherein the half-life extending molecule (H1) does not protect the multispecific antibody from CD28.

[0453] Embodiment 145. The isolated multispecific antibody of any one of Embodiments 136-144, wherein H1 comprises a sequence according to SEQ ID NOs: 54-57.

[0454] Embodiment 146. The isolated multispecific antibody of any one of Embodiments 136-144, wherein H1 comprises an amino acid sequence having a repeating sequence motif.

[0455] Embodiment 147. The isolated multispecific antibody of any one of Embodiments 136-144, wherein H1 comprises an amino acid sequence having a highly ordered secondary structure.

[0456] Embodiment 148. The isolated multispecific antibody of any one of Embodiments 136-144, wherein H1 comprises a polymer.

[0457] Embodiment 149. The isolated multispecific antibody of Embodiment 148, wherein the polymer is polyethylene glycol (PEG).

[0458] Embodiment 150. The isolated multispecific antibody of any one of Embodiments 136-149, wherein H1 comprises albumin.

[0459] Embodiment 151. The isolated multispecific antibody of any one of Embodiments 136-150, wherein H1 comprises an Fc domain.

[0460] Embodiment 152. The isolated multispecific antibody of embodiment 150, wherein the albumin is serum albumin.

[0461] Embodiment 153. The isolated multispecific antibody of embodiment 152, wherein the albumin is human serum albumin.

[0462] Embodiment 154. The isolated multispecific antibody of any one of Embodiments 136-153, wherein H1 comprises a polypeptide, a ligand, or a small molecule.

[0463] Embodiment 155. The isolated multispecific antibody of Embodiment 153, wherein the polypeptide, ligand or small molecule binds to a serum protein or fragment thereof, a circulating immunoglobulin or fragment thereof, or CD35 / CR1.

[0464] Embodiment 156. The isolated multispecific antibody of embodiment 155, wherein the serum protein comprises thyroxine-binding protein, transthyretin, 1-acid glycoprotein, transferrin, transferrin receptor or a transferrin-binding portion thereof, fibrinogen, or albumin.

[0465] Embodiment 157. The isolated multispecific antibody of Embodiment 155, wherein the circulating immunoglobulin molecules comprise IgG1, IgG2, IgG3, IgG4, slgA, IgM, or IgD.

[0466] Embodiment 158. The isolated multispecific antibody of Embodiment 155, wherein the serum protein is albumin.

[0467] Embodiment 159. The isolated multispecific antibody of embodiment 154, wherein the polypeptide is an antibody.

[0468] Embodiment 160. The isolated multispecific antibody of embodiment 159, wherein the antibody comprises a single domain antibody, a single chain variable fragment, a Fab, or a Fab'.

[0469] Embodiment 161. The isolated multispecific antibody of Embodiment 160, wherein the single domain antibody comprises a single domain antibody that binds to albumin.

[0470] Embodiment 162. The isolated multispecific antibody of embodiment 160, wherein the single domain antibody is a human antibody or a humanized antibody.

[0471] Embodiment 163. The isolated multispecific antibody of embodiment 160, wherein the single domain antibody is selected from 645gH1gL1, 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.

[0472] Embodiment 164. The isolated multispecific antibody of embodiment 160, wherein the single domain antibody comprises complementary determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and 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 0-2 amino acid modifications in at least one of HC-CDR1, HC-CDR2, or HC-CDR3, or wherein the single domain antibody comprises complementary determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 of the single domain antibody comprise: HC-CDR1: SEQ ID NO: 204, HC-CDR2: SEQ ID NO: 205, and HC-CDR3: SEQ ID NO: NO: 206; and wherein the CDR comprises 0-2 amino acid modifications in at least one of HC-CDR1, HC-CDR2 or HC-CDR3.

[0473] Embodiment 165. The isolated multispecific antibody of Embodiment 164, wherein H1 comprises the amino acid sequence according to SEQ ID NO: 57 or SEQ ID NO: 207.

[0474] Embodiment 166. The isolated multispecific antibody of Embodiment 165, wherein H1 comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 57 or SEQ ID NO: 207.

[0475] Embodiment 167. The isolated multispecific antibody of Embodiment 165, wherein H1 comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 57 or SEQ ID NO: 207.

[0476] Embodiment 168. The isolated multispecific antibody of Embodiment 165, wherein H1 comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 57 or SEQ ID NO: 207.

[0477] Embodiment 169. The isolated multispecific antibody of Embodiment 165, wherein H1 comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 57 or SEQ ID NO: 207.

[0478] Embodiment 170. The isolated multispecific antibody of Embodiment 165, wherein H1 comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NO: 57 or SEQ ID NO: 207.

[0479] Embodiment 171. The isolated multispecific antibody of any one of embodiments 136-170, wherein H1 comprises a modified amino acid or a non-natural amino acid or a modified non-natural amino acid, or a combination thereof.

[0480] Embodiment 172. The isolated multispecific antibody of Embodiment 171, wherein the modified amino acid or modified non-natural amino acid comprises a post-translational modification.

[0481] Embodiment 173. The isolated multispecific antibody of any one of Embodiments 136-172, wherein H1 comprises a linking portion (L5) connecting H1 to P1 or P2.

[0482] Embodiment 174. The isolated multispecific antibody of Embodiment 173, wherein L5 is a peptide sequence having at least 5 and no more than 50 amino acids.

[0483] Embodiment 175. The isolated multispecific antibody of embodiment 173, wherein L5 is a peptide sequence having at least 10 and no more than 30 amino acids.

[0484] Embodiment 176. The isolated multispecific antibody of Embodiment 173, wherein L5 is a peptide sequence of at least 10 amino acids.

[0485] Embodiment 177. The isolated multispecific antibody of Embodiment 173, wherein L5 is a peptide sequence of at least 18 amino acids.

[0486] Embodiment 178. The isolated multispecific antibody of Embodiment 173, wherein L5 is a peptide sequence of at least 26 amino acids.

[0487] Embodiment 179. The isolated multispecific antibody of Embodiment 173, wherein L5 comprises 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.

[0488] Embodiment 180. The isolated multispecific antibody of any one of Embodiments 1-179, wherein the isolated multispecific antibody comprises an amino acid sequence that has at least 80% sequence identity to any one of SEQ ID NOs: 149-170.

[0489] Embodiment 181. The isolated multispecific antibody of any one of Embodiments 1-180, wherein the isolated multispecific antibody comprises an amino acid sequence that has at least 85% sequence identity to any one of SEQ ID NOs: 149-170.

[0490] Embodiment 182. The isolated multispecific antibody of any one of Embodiments 1-181, wherein the isolated multispecific antibody comprises an amino acid sequence that has at least 90% sequence identity to any one of SEQ ID NOs: 149-170.

[0491] Embodiment 183. The isolated multispecific antibody of any one of Embodiments 1-182, wherein the isolated multispecific antibody comprises an amino acid sequence that has at least 95% sequence identity to any one of SEQ ID NOs: 149-170.

[0492] Embodiment 184. The isolated multispecific antibody of any one of Embodiments 1-183, wherein the isolated multispecific antibody comprises an amino acid sequence that has at least 99% sequence identity to any one of SEQ ID NOs: 149-170.

[0493] Embodiment 185. The isolated multispecific antibody of any one of Embodiments 1-183, wherein the isolated multispecific antibody comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NOs: 149 and 150.

[0494] Embodiment 186. The isolated multispecific antibody of any one of Embodiments 1-183, wherein the isolated multispecific antibody comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NOs: 149 and 150.

[0495] Embodiment 187. The isolated multispecific antibody of any one of Embodiments 1-183, wherein the isolated multispecific antibody comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NOs: 151 and 152.

[0496] Embodiment 188. The isolated multispecific antibody of any one of Embodiments 1-183, wherein the isolated multispecific antibody comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NOs: 151 and 152.

[0497] Embodiment 189. The isolated multispecific antibody of any one of Embodiments 1-183, wherein the isolated multispecific antibody comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NOs: 153 and 154.

[0498] Embodiment 190. The isolated multispecific antibody of any one of Embodiments 1-183, wherein the isolated multispecific antibody comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NOs: 153 and 154.

[0499] Embodiment 191. The isolated multispecific antibody of any one of Embodiments 1-183, wherein the isolated multispecific antibody comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NOs: 155 and 156.

[0500] Embodiment 192. The isolated multispecific antibody of any one of Embodiments 1-183, wherein the isolated multispecific antibody comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NOs: 155 and 156.

[0501] Embodiment 193. The isolated multispecific antibody of any one of Embodiments 1-183, wherein the isolated multispecific antibody comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NOs: 157 and 158.

[0502] Embodiment 194. The isolated multispecific antibody of any one of Embodiments 1-183, wherein the isolated multispecific antibody comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NOs: 157 and 158.

[0503] Embodiment 195. The isolated multispecific antibody of any one of Embodiments 1-183, wherein the isolated multispecific antibody comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NOs: 159 and 160.

[0504] Embodiment 196. The isolated multispecific antibody of any one of Embodiments 1-183, wherein the isolated multispecific antibody comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NOs: 159 and 160.

[0505] Embodiment 197. The isolated multispecific antibody of any one of Embodiments 1-183, wherein the isolated multispecific antibody comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NOs: 161 and 162.

[0506] Embodiment 198. The isolated multispecific antibody of any one of Embodiments 1-183, wherein the isolated multispecific antibody comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NOs: 161 and 162.

[0507] Embodiment 199. The isolated multispecific antibody of any one of Embodiments 1-183, wherein the isolated multispecific antibody comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NOs: 163 and 164.

[0508] Embodiment 200. The isolated multispecific antibody of any one of Embodiments 1-183, wherein the isolated multispecific antibody comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NOs: 163 and 164.

[0509] Embodiment 201. The isolated multispecific antibody of any one of Embodiments 1-183, wherein the isolated multispecific antibody comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NOs: 165 and 166.

[0510] Embodiment 202. The isolated multispecific antibody of any one of Embodiments 1-183, wherein the isolated multispecific antibody comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NOs: 165 and 166.

[0511] Embodiment 203. The isolated multispecific antibody of any one of Embodiments 1-183, wherein the isolated multispecific antibody comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NOs: 167 and 168.

[0512] Embodiment 204. The isolated multispecific antibody of any one of Embodiments 1-183, wherein the isolated multispecific antibody comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NOs: 167 and 168.

[0513] Embodiment 205. The isolated multispecific antibody of any one of Embodiments 1-183, wherein the isolated multispecific antibody comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NOs: 169 and 170 or at least 95% sequence identity to SEQ ID NOs: 208 and 209.

[0514] Embodiment 206. The isolated multispecific antibody of any one of Embodiments 1-183, wherein the isolated multispecific antibody comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NOs: 169 and 170 or at least 99% sequence identity to SEQ ID NOs: 208 and 209.

[0515] Embodiment 207. An isolated recombinant nucleic acid molecule encoding a polypeptide of the isolated multispecific antibody of any one of embodiments 1-206.

[0516] Embodiment 208. A pharmaceutical composition comprising:

[0517] (a) the isolated multispecific antibody of any one of embodiments 1-206; and

[0518] (b) a pharmaceutically acceptable excipient.

[0519] Embodiment 209. A pharmaceutical composition comprising: (a) the isolated multispecific antibody of any one of Embodiments 1-206, (b) an anti-cancer therapy, and (c) a pharmaceutically acceptable excipient.

[0520] Embodiment 210. Embodiment 2. The pharmaceutical composition of Embodiment 209, wherein the anti-cancer therapy comprises a small molecule, a cell-based therapy, or an antibody-based therapy.

[0521] Embodiment 211. The pharmaceutical composition of embodiment 210, wherein the antibody-based therapy is a T cell engager.

[0522] Embodiment 212. A pharmaceutical composition as described in Embodiment 211, wherein the T cell engager comprises a formula according to: D1-L0-E1 (Formula II), wherein D1 comprises an effector cell binding domain that binds to an effector cell antigen, E1 comprises a tumor antigen binding domain that binds to a tumor antigen, and L0 comprises a linker connecting D1 and E1.

[0523] Embodiment 213. A pharmaceutical composition as described in embodiment 212, wherein D1 comprises a single-chain variable fragment, a single domain antibody, a Fab fragment or Fab'.

[0524] Embodiment 214. The pharmaceutical composition of embodiment 213, wherein D1 comprises a single-chain variable fragment.

[0525] Embodiment 215. The pharmaceutical composition of embodiment 212, wherein E1 comprises a single-chain variable fragment, a single domain antibody, a Fab fragment, or a Fab′.

[0526] Embodiment 216. The pharmaceutical composition of embodiment 215, wherein E1 comprises a Fab fragment.

[0527] Embodiment 217. The pharmaceutical composition of embodiment 215, wherein the effector cell antigen comprises CD3.

[0528] Embodiment 218. The pharmaceutical composition of embodiment 217, wherein the effector cell binding domain comprises a complementary determining region (CDR) selected from the group consisting of muromonab-CD3 (OKT3), otuximab (TRX4), tilizumab (MGA031), visilizumab (Nuvion), SP34, X35, VIT3, BMA030 (BW264 / 56), CLB-T3 / 3, CRIS7, YTH12.5, F1 11-409, CLB-T3.4.2, TR-66, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII-87, 12F6, T3 / RW2-8C8, T3 / RW2-4B6, OKT3D, M-T301, SMC2, F101.01, UCHT-1, WT-31, 15865, 15865v12, 15865v16 and 15865v19.

[0529] Embodiment 219. The pharmaceutical composition of embodiment 217, wherein the effector cell binding domain comprises an amino acid sequence according to SEQ ID NO: 89-101.

[0530] Embodiment 220. A pharmaceutical composition as described in any of Embodiments 212-219, wherein the tumor antigen comprises epidermal growth factor receptor (EGFR), prostate-specific membrane antigen (PSMA), or tumor-associated calcium signal transducer 2 (referred to herein as TROP2).

[0531] Embodiment 221. A pharmaceutical composition as described in Embodiment 220, wherein the tumor antigen includes EGFR.

[0532] Embodiment 222. The pharmaceutical composition of Embodiment 220, wherein the tumor antigen binding domain comprises an amino acid sequence according to SEQ ID NO: 102-111.

[0533] Embodiment 223. A pharmaceutical composition as described in Embodiment 220, wherein the tumor antigen comprises EGFR, and the tumor binding domain comprises the complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, and LC-CDR1, LC-CDR2, and LC-CDR3, wherein HC-CDR1, HC-CDR2, and HC-CDR3 comprise: HC-CDR1: SEQ ID NO: 105; HC-CDR2: SEQ ID NO: 106; HC-CDR3: SEQ ID NO: 107; and wherein LC-CDR1, LC-CDR2, and LC-CDR3 comprise: LC-CDR1: SEQ ID NO: 102; LC-CDR2: SEQ ID NO: 103 (YAS); and LC-CDR3: SEQ ID NO: 104.

[0534] Embodiment 224. A pharmaceutical composition as described in Embodiment 220, wherein the tumor antigen includes EGFR and the T cell engager comprises an amino acid sequence having at least 95% sequence identity according to SEQ ID NOs: 181 and 182 or at least 95% sequence identity according to SEQ ID NOs: 214 and 215.

[0535] Embodiment 225. A pharmaceutical composition as described in Embodiment 220, wherein the tumor antigen includes EGFR and the T cell engager comprises the amino acid sequence according to SEQ ID NOs: 181 and 182 or according to SEQ ID NOs: 214 and 215.

[0536] Embodiment 226. The pharmaceutical composition of embodiment 220, wherein the tumor antigen comprises TROP2.

[0537] Embodiment 227. A pharmaceutical composition as described in Embodiment 220, wherein the tumor antigen comprises TROP2, and the tumor binding domain comprises the complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, and LC-CDR1, LC-CDR2, and LC-CDR3, wherein HC-CDR1, HC-CDR2, and HC-CDR3 comprise HC-CDR1: SEQ ID NO: 112; HC-CDR2: SEQ ID NO: 113; HC-CDR3: SEQ ID NO: 114; and wherein LC-CDR1, LC-CDR2, and LC-CDR3 comprise: LC-CDR1: SEQ ID NO: 115; LC-CDR2: SEQ ID NO: 116 (SAS); and LC-CDR3: SEQ ID NO: 117.

[0538] Embodiment 228. The pharmaceutical composition of embodiment 220, wherein the tumor antigen comprises TROP2 and the T cell engager comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 187-192.

[0539] Embodiment 229. The pharmaceutical composition of embodiment 220, wherein the tumor antigen comprises TROP2 and the T cell engager comprises the amino acid sequence according to any one of SEQ ID NOs: 187-192.

[0540] Embodiment 230. The pharmaceutical composition of Embodiment 220, wherein the tumor antigen binding domain comprises an amino acid sequence according to SEQ ID NO: 112-119.

[0541] Embodiment 231. The pharmaceutical composition of Embodiment 220, wherein the tumor antigen comprises PSMA.

[0542] Embodiment 232. The pharmaceutical composition of Embodiment 220, wherein the tumor antigen binding domain comprises an amino acid sequence according to SEQ ID NO: 120-127.

[0543] Embodiment 233. The pharmaceutical composition of embodiment 220, wherein the tumor antigen comprises PSMA, and the tumor binding domain comprises the complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, and LC-CDR1, LC-CDR2, and LC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 comprise HC-CDR1: SEQ ID NO: 120; HC-CDR2: SEQ ID NO: 121; HC-CDR3: SEQ ID NO: 122; and wherein the LC-CDR1, LC-CDR2, and LC-CDR3 comprise: LC-CDR1: SEQ ID NO: 123; LC-CDR2: SEQ ID NO: 124(EA); and LC-CDR3: SEQ ID NO: 125.

[0544] Embodiment 234. The pharmaceutical composition of embodiment 220, wherein the tumor antigen comprises PSMA and the T cell engager comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 173 and 174.

[0545] Embodiment 235. The pharmaceutical composition of embodiment 220, wherein the tumor antigen comprises PSMA and the T cell engager comprises the amino acid sequence according to SEQ ID NO: 173 and 174.

[0546] Embodiment 236. The pharmaceutical composition of any one of Embodiments 211-235, wherein the T cell engager molecule is selectively activated in the tumor microenvironment.

[0547] Embodiment 237. A pharmaceutical composition as described in Embodiment 236, wherein the T cell engager is according to the following sub-formula: P3-L3-D1-L0-E1 (Formula IIa), wherein D1 comprises a CD3 binding domain; E1 comprises a tumor antigen binding domain; L0 comprises a linker connecting D1 and E1; P3 comprises a peptide that binds to D1, and L3 comprises a linker portion that connects D1 to P3 and is a substrate for a tumor-specific protease.

[0548] Embodiment 238. A pharmaceutical composition as described in Embodiment 236, wherein the T cell engager is according to the following sub-formula: D1-L0-E1-L4-P4 (Formula IIb), wherein D1 comprises a CD3 binding domain; E1 comprises a tumor antigen binding domain; L0 comprises a linker connecting D1 and E1; P4 comprises a peptide that binds to E1, and L4 comprises a linker portion that connects E1 and P4 and is a substrate for a tumor-specific protease.

[0549] Embodiment 239. A pharmaceutical composition as described in Embodiment 236, wherein the T cell engager is according to the following sub-formula: P3-L3-D1-L0-E1-L4-P4 (Formula IIc), wherein D1 comprises a CD3 binding domain; E1 comprises a tumor antigen binding domain; L0 comprises a linker connecting D1 and E1; P3 comprises a peptide that binds to D1, and L3 comprises a linker that connects D1 to P3 and is a substrate for a tumor-specific protease; P4 comprises a peptide that binds to E1, and L4 comprises a linker that connects E1 to P4 and is a substrate for a tumor-specific protease.

[0550] Embodiment 240. The pharmaceutical composition of any one of Embodiments 211-239, wherein the T cell engager comprises H1.

[0551] Embodiment 241. The pharmaceutical composition of embodiment 240, wherein H1 comprises a sequence according to SEQ ID NOs: 54-57.

[0552] Embodiment 242. The pharmaceutical composition of Embodiment 240, wherein H1 comprises a single domain antibody.

[0553] Embodiment 243. The pharmaceutical composition of embodiment 240, wherein the single domain antibody comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and 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.

[0554] Embodiment 244. A pharmaceutical composition as described in any of embodiments 237-243, wherein L3 or L4 is a peptide sequence having at least 5 and no more than 50 amino acids.

[0555] Embodiment 245. A pharmaceutical composition as described in any of embodiments 237-244, wherein L3 or L4 is a peptide sequence having at least 10 and no more than 30 amino acids.

[0556] Embodiment 246. A pharmaceutical composition as described in any of embodiments 237-245, wherein L3 or L4 is a peptide sequence of at least 10 amino acids.

[0557] Embodiment 247. A pharmaceutical composition as described in any of embodiments 237-246, wherein L3 or L4 is a peptide sequence of at least 18 amino acids.

[0558] Embodiment 248. A pharmaceutical composition as described in any of embodiments 237-247, wherein L3 or L4 is a peptide sequence of at least 26 amino acids.

[0559] Embodiment 249. A pharmaceutical composition as described in any of embodiments 237-243, wherein L3 or L4 comprises (G2S) n wherein n is an integer from 1 to 3 (SEQ ID NO: 228).

[0560] Embodiment 250. A pharmaceutical composition as described in any of embodiments 237-243, wherein L3 or L4 comprises (G2S) n wherein n is an integer of at least 1.

[0561] Embodiment 251. A pharmaceutical composition as described in any of Embodiments 237-243, wherein L3 or L4 comprises 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.

[0562] Embodiment 252. The pharmaceutical composition of any one of embodiments 237-243, wherein the tumor-specific protease is selected from the group consisting of a metalloprotease, a serine protease, a cysteine ​​protease, a threonine protease, and an aspartic protease.

[0563] Embodiment 253. A pharmaceutical composition as described in any of Embodiments 237-243, wherein L3 or L4 comprises an amino acid sequence cleavable by urokinase, an amino acid sequence cleavable by a membrane-type serine protease, or an amino acid sequence cleavable by a matrix metalloproteinase.

[0564] Embodiment 254. A pharmaceutical composition as described in any of Embodiments 237-243, wherein L3 or L4 comprises a sequence according to SEQ ID NO: 18-19, 62-88.

[0565] Embodiment 255. The pharmaceutical composition of any one of Embodiments 237-254, wherein L3 is bound to the N-terminus of D1.

[0566] Embodiment 256. The pharmaceutical composition of any one of Embodiments 237-254, wherein L3 is bound to the C-terminus of D1.

[0567] Embodiment 257. The pharmaceutical composition of any one of embodiments 238-254, wherein L4 is bound to the N-terminus of E1.

[0568] Embodiment 258. The pharmaceutical composition of any one of embodiments 238-254, wherein L4 is bound to the C-terminus of E1.

[0569] Embodiment 259. The pharmaceutical composition of any one of Embodiments 237-254, wherein when L3 is cleaved by a tumor-specific protease, P3 is unbound from D1, thereby exposing D1 to CD3.

[0570] Embodiment 260. The pharmaceutical composition of any one of embodiments 238-254, wherein when L4 is cleaved by a tumor-specific protease, P4 is unbound from E1, thereby exposing E1 to the tumor antigen.

[0571] Embodiment 261. The pharmaceutical composition of any one of Embodiments 237-260, wherein P3 weakens the binding of D1 to CD3.

[0572] Embodiment 262. The pharmaceutical composition of any one of embodiments 237-261, wherein P3 is bound to D1 through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bonding interactions, or a combination thereof.

[0573] Embodiment 263. The pharmaceutical composition of any one of Embodiments 237-262, wherein P3 is bound to D1 at or near the antigen binding site.

[0574] Embodiment 264. The pharmaceutical composition of any one of Embodiments 237-263, wherein when L3 is cleaved by a tumor-specific protease, P3 is unbound from D1, thereby exposing D1 to CD3.

[0575] Embodiment 265. The pharmaceutical composition of any one of Embodiments 237-264, wherein P3 has less than 70% sequence identity with CD3.

[0576] Embodiment 266. The pharmaceutical composition of any one of Embodiments 237-265, wherein P3 has less than 85% sequence identity with CD3.

[0577] Embodiment 267. The pharmaceutical composition of any one of Embodiments 237-266, wherein P3 has less than 90% sequence identity with CD3.

[0578] Embodiment 268. The pharmaceutical composition of any one of Embodiments 237-267, wherein P3 has less than 95% sequence identity with CD3.

[0579] Embodiment 269. The pharmaceutical composition of any one of Embodiments 237-268, wherein P3 has less than 98% sequence identity with CD3.

[0580] Embodiment 270. The pharmaceutical composition of any one of Embodiments 237-269, wherein P3 has less than 99% sequence identity with CD3.

[0581] Embodiment 271. The pharmaceutical composition of any one of Embodiments 237-270, wherein P3 comprises an amino acid sequence according to SEQ ID NOs: 177-180.

[0582] Embodiment 272. The pharmaceutical composition of any one of Embodiments 237-271, wherein P3 comprises a de novo generated amino acid sequence that shares less than 10% sequence identity with CD3.

[0583] Embodiment 273. The pharmaceutical composition of any one of Embodiments 238-272, wherein P4 weakens the binding of E1 to a tumor antigen.

[0584] Embodiment 274. The pharmaceutical composition of any one of embodiments 238-273, wherein P4 is bound to E1 through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bonding interactions, or a combination thereof.

[0585] Embodiment 275. The pharmaceutical composition of any one of Embodiments 238-274, wherein P4 is bound to E1 at or near the antigen binding site.

[0586] Embodiment 276. The pharmaceutical composition of any one of Embodiments 238-275, wherein when L4 is cleaved by a tumor-specific protease, P4 is unbound from E1, thereby exposing E1 to the tumor antigen.

[0587] Embodiment 277. The pharmaceutical composition of any one of Embodiments 238-276, wherein P4 has less than 70% sequence identity with the tumor antigen.

[0588] Embodiment 278. The pharmaceutical composition of any one of Embodiments 238-277, wherein P4 has less than 80% sequence identity with the tumor antigen.

[0589] Embodiment 279. The pharmaceutical composition of any one of Embodiments 238-278, wherein P4 has less than 85% sequence identity with the tumor antigen.

[0590] Embodiment 280. The pharmaceutical composition of any one of Embodiments 238-279, wherein P4 has less than 90% sequence identity with the tumor antigen.

[0591] Embodiment 281. The pharmaceutical composition of any one of Embodiments 238-280, wherein P4 has less than 95% sequence identity with the tumor antigen.

[0592] Embodiment 282. The pharmaceutical composition of any one of Embodiments 238-281, wherein P4 comprises a de novo generated amino acid sequence that shares less than 10% sequence identity with a tumor antigen.

[0593] Embodiment 283. A pharmaceutical composition as described in any of embodiments 237-282, wherein P3 or P4 comprises a peptide sequence of at least 5 amino acids in length.

[0594] Embodiment 284. The pharmaceutical composition of any one of Embodiments 237-283, wherein P3 or P4 comprises a peptide sequence of at least 6 amino acids in length.

[0595] Embodiment 285. The pharmaceutical composition of any one of Embodiments 237-284, wherein P3 or P4 comprises a peptide sequence of at least 10 amino acids in length.

[0596] Embodiment 286. The pharmaceutical composition of any one of embodiments 237-285, wherein P3 or P4 comprises a peptide sequence of at least 10 amino acids in length and no more than 20 amino acids in length.

[0597] Embodiment 287. The pharmaceutical composition of any one of Embodiments 237-286, wherein P3 or P4 comprises a peptide sequence of at least 16 amino acids in length.

[0598] Embodiment 288. The pharmaceutical composition of any one of Embodiments 237-287, wherein P3 or P4 comprises a peptide sequence of no more than 40 amino acids in length.

[0599] Embodiment 289. The pharmaceutical composition of any one of Embodiments 237-288, wherein P3 or P4 comprises at least two cysteine ​​amino acid residues.

[0600] Embodiment 290. The pharmaceutical composition of any one of Embodiments 237-289, wherein P3 or P4 comprises a cyclic peptide or a linear peptide.

[0601] Embodiment 291. The pharmaceutical composition of any one of Embodiments 237-290, wherein P3 or P4 comprises a cyclic peptide.

[0602] Embodiment 292. The pharmaceutical composition of any one of Embodiments 237-291, wherein P3 or P4 comprises a linear peptide.

[0603] Embodiment 293. A pharmaceutical composition as described in any of embodiments 238-292, wherein P4 comprises the amino acid sequence according to SEQ ID NO: 185 or 186.

[0604] Embodiment 294. The pharmaceutical composition of any one of Embodiments 237-293, wherein the tumor antigen comprises EGFR and the T cell engager comprises the amino acid sequence of SEQ ID NOs: 183 and 184.

[0605] Embodiment 295. The pharmaceutical composition of any one of Embodiments 238-292, wherein P4 comprises an amino acid sequence according to SEQ ID NOs: 199-201.

[0606] Embodiment 296. The pharmaceutical composition of any one of Embodiments 237-292, wherein the tumor antigen comprises TROP2 and the T cell engager comprises any one of the amino acid sequences of SEQ ID NOs: 193-198.

[0607] Embodiment 297. The pharmaceutical composition of any one of Embodiments 237-292, wherein the tumor antigen comprises PSMA and the T cell engager comprises the amino acid sequences of SEQ ID NOs: 175 and 176.

[0608] Embodiment 298. An isolated polypeptide or polypeptide complex comprising a CD28 binding domain linked to a peptide that impairs binding of the CD28 binding domain to CD28, wherein the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 24-53, 128-148, and any one of the amino acid sequences of Table 20, or an amino acid sequence having 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 24-53, 128-148, and any one of the amino acid sequences of Table 20.

[0609] Embodiment 299. The isolated polypeptide or polypeptide complex of embodiment 298, wherein the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 24-53, 128-148, and the amino acid sequence of Table 20.

[0610] Embodiment 300. The isolated polypeptide or polypeptide complex of embodiment 298, wherein the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 42-53 or an amino acid sequence having 1, 2 or 3 amino acid mutations, substitutions or deletions relative to any one of SEQ ID NOs: 42-53.

[0611] Embodiment 301. The isolated polypeptide or polypeptide complex of embodiment 298, wherein the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 42-53.

[0612] Embodiment 302. An isolated polypeptide or polypeptide complex as described in embodiment 298, wherein the peptide comprises an amino acid sequence according to any one of the amino acid sequences in Table 20 or an amino acid sequence having 1, 2 or 3 amino acid mutations, substitutions or deletions relative to any one of the amino acid sequences in Table 20.

[0613] Embodiment 303. An isolated polypeptide or polypeptide complex as described in embodiment 298, wherein the peptide comprises an amino acid sequence according to any one of the amino acid sequences in Table 20.

[0614] Embodiment 304. The isolated polypeptide or polypeptide complex of embodiment 298, wherein the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 128-147 or an amino acid sequence having 1, 2 or 3 amino acid mutations, substitutions or deletions relative to any one of SEQ ID NOs: 128-147.

[0615] Embodiment 305. The isolated polypeptide or polypeptide complex of embodiment 298, wherein the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 128-147.

[0616] Embodiment 306. The isolated polypeptide or polypeptide complex of embodiment 298, wherein the peptide comprises a peptide according to X1-X2-X3-C-X4-X5-X6-X7-X8-X9-X 10 -CX 11 -X 12 an amino acid sequence wherein X1 is selected from the group consisting of M, I, L, and V; X2 is selected from the group consisting of D, H, N, A, F, S, T, Y, and V; X3 is selected from the group consisting of W, L, and F; X4 is selected from the group consisting of P, A, and L; X5 is selected from the group consisting of R, T, I, M, S, K, L, V, W, F, A, P, and D; X6 is selected from the group consisting of E, D, Y, H, S, F, A, N, T, I, P, and V; X7 is selected from the group consisting of L, M, R, S, Q, and H; X8 is selected from the group consisting of W and Q; X9 is selected from the group consisting of H, N, D, A, S, Y, T, F, V, L, and I; X 10 Selected from E, V, L, D, Y, R, Q, H, F, K, A, M and N; X 11 is selected from F, Y, L, W, and V; and X 12 Selected from N, A, F, S, Y, H, D, T and L.

[0617] Embodiment 307. The isolated polypeptide or polypeptide complex of embodiment 306, wherein X1 is selected from M, I, and L; X2 is selected from D, H, N, and A; X3 is W; X4 is P; X5 is selected from R, T, I, M, S, and K; X6 is selected from E, D, Y, H, S, and F; X7 is selected from L, M, and R; X8 is W; X9 is selected from H, N, D, A, S, and V; X 10 Selected from E, V, L, D and H; X 11 is selected from F, Y and L; and X 12 Selected from N, A, F, S and Y.

[0618] Embodiment 308. The isolated polypeptide or polypeptide complex of embodiment 307, wherein X1 is M; X2 is selected from D and H; X3 is W; X4 is P; X5 is selected from R, T and I; X6 is selected from E, D and Y; X7 is selected from L, M and R; X8 is W; X9 is selected from H, N, D and V; X10 Selected from E, V, L, D and H; X 11 is F; and X 12 Selected from N, A and F.

[0619] Embodiment 309. An isolated polypeptide or polypeptide complex as described in any one of embodiments 298-302 or 306-308, wherein the peptide comprises an amino acid sequence according to SEQ ID NO: 32 or an amino acid sequence having 1, 2 or 3 amino acid mutations, substitutions or deletions relative to SEQ ID NO: 32.

[0620] Embodiment 310. The isolated polypeptide or polypeptide complex of any one of embodiments 298-302 or 306-308.

[0621] Embodiment 311. The isolated polypeptide or polypeptide complex of embodiment 298, wherein the peptide comprises the amino acid sequence according to SEQ ID NO: 32.

[0622] Embodiment 312. An isolated polypeptide or polypeptide complex as described in any one of embodiments 298-302 or 306-308, wherein the peptide comprises an amino acid sequence according to SEQ ID NO: 138 or an amino acid sequence having 1, 2 or 3 amino acid mutations, substitutions or deletions relative to SEQ ID NO: 138.

[0623] Embodiment 313. The isolated polypeptide or polypeptide complex of any one of embodiments 298-302 or 306-308, wherein the peptide comprises the amino acid sequence according to SEQ ID NO: 138.

[0624] Embodiment 314. The isolated polypeptide or polypeptide complex of any one of embodiments 298-312, wherein the CD28 binding domain comprises a single-chain variable fragment, a single-domain antibody, a Fab, or a Fab'.

[0625] Embodiment 315. An isolated polypeptide or polypeptide complex as described in embodiment 314, wherein the CD28 binding domain comprises a single chain variable fragment and the single chain variable fragment comprises an scFv heavy chain variable domain and an scFv light chain variable domain.

[0626] Embodiment 316. The isolated polypeptide or polypeptide complex of embodiment 314, wherein the CD28 binding domain comprises a single domain antibody.

[0627] Embodiment 317. The isolated polypeptide or polypeptide complex of embodiment 314, wherein the CD28 binding domain comprises Fab or Fab'.

[0628] Embodiment 318. An isolated polypeptide or polypeptide complex as described in embodiment 315, wherein the scFv heavy chain variable domain comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2 and HC-CDR3, wherein the HC-CDR1, HC-CDR2 and 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 the CDRs comprise 0-2 amino acid modifications in at least one of the HC-CDR1, HC-CDR2 or HC-CDR3.

[0629] Embodiment 319. An isolated polypeptide or polypeptide complex as described in embodiment 315, wherein the scFv light chain variable domain comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2 and LC-CDR3, wherein the LC-CDR1, LC-CDR2 and LC-CDR3 of the scFv light chain variable domain comprise: LC-CDR1: SEQ ID NO: 4; LC-CDR2: SEQ ID NO: 5(KA); and LC-CDR3: SEQ ID NO: 6, and wherein the CDRs comprise 0-2 amino acid modifications in at least one of said LC-CDR1, LC-CDR2 or LC-CDR3.

[0630] Embodiment 320. The isolated polypeptide or polypeptide complex of embodiment 315, wherein the scFv heavy chain variable domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO:7.

[0631] Embodiment 321. An isolated polypeptide or polypeptide complex as described in embodiment 315, wherein the scFv heavy chain variable domain comprises an amino acid sequence of at least 75 consecutive amino acid residues of SEQ ID NO:7.

[0632] Embodiment 322. An isolated polypeptide or polypeptide complex as described in embodiment 315, wherein the scFv heavy chain variable domain comprises an amino acid sequence of at least 110 consecutive amino acid residues of SEQ ID NO:7.

[0633] Embodiment 323. An isolated polypeptide or polypeptide complex as described in embodiment 315, wherein 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 with at least 110 consecutive amino acid residues of SEQ ID NO:7.

[0634] Embodiment 324. An isolated polypeptide or polypeptide complex as described in embodiment 315, wherein the scFv heavy chain variable domain comprises the amino acid sequence according to SEQ ID NO:7.

[0635] Embodiment 325. An isolated polypeptide or polypeptide complex as described in embodiment 315, wherein the scFv light chain variable domain comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence according to SEQ ID NO:8.

[0636] Embodiment 326. An isolated polypeptide or polypeptide complex as described in embodiment 315, wherein the scFv light chain variable domain comprises an amino acid sequence of at least 75 consecutive amino acid residues of SEQ ID NO:8.

[0637] Embodiment 327. An isolated polypeptide or polypeptide complex as described in embodiment 315, wherein the scFv light chain variable domain comprises an amino acid sequence of at least 100 consecutive amino acid residues of SEQ ID NO:8.

[0638] Embodiment 328. An isolated polypeptide or polypeptide complex as described in embodiment 315, wherein 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 with at least 100 consecutive amino acid residues of SEQ ID NO:8.

[0639] Embodiment 329. An isolated polypeptide or polypeptide complex as described in Embodiment 315, wherein the sc...

Claims

1. An isolated multispecific antibody according to the following formula: P1-L1-A1-LB (Formula I), wherein A1 comprises a CD28 binding domain; B comprises a PD-L1 binding domain; L comprises a linker connecting A1 and B; P1 comprises a peptide that binds to A1, and L1 comprises a linker portion that connects A1 and P1 and is a substrate for a tumor-specific protease, wherein P1 comprises a polypeptide according to X1-X2-X3-C-X4-X5-X6-X7-X8-X9-X 10 -CX 11 -X 12 an amino acid sequence wherein X1 is selected from the group consisting of M, I, L and V; X2 is selected from the group consisting of D, H, N, A, F, S, T, Y and V; X3 is selected from the group consisting of W, L and F; X4 is selected from the group consisting of P, A and L; X5 is selected from the group consisting of R, T, I, M, S, K, L, V, W, F, A, P and D; X6 is selected from the group consisting of E, D, Y, H, S, F, A, N, T, I, P and V; X7 is selected from the group consisting of L, M, R, S, Q and H; X8 is selected from the group consisting of W and Q; X9 is selected from the group consisting of H, N, D, A, S, Y, T, F, V, L and I; X 10 Selected from E, V, L, D, Y, R, Q, H, F, K, A, M and N; X 11 is selected from F, Y, L, W and V; and X 12 Selected from N, A, F, S, Y, H, D, T and L.

2. The isolated multispecific antibody of claim 1, wherein X1 is selected from M, I and L; X2 is selected from D, H, N and A; X3 is W; X4 is P; X5 is selected from R, T, I, M, S and K; X6 is selected from E, D, Y, H, S and F; X7 is selected from L, M and R; X8 is W; X9 is selected from H, N, D, A, S and V; X 10 Selected from E, V, L, D and H; X 11 is selected from F, Y and L; and X 12 Selected from N, A, F, S and Y.

3. The isolated multispecific antibody of claim 2, wherein X1 is M; X2 is selected from D and H; X3 is W; X4 is P; X5 is selected from R, T and I; X6 is selected from E, D and Y; X7 is selected from L, M and R; X8 is W; X9 is selected from H, N, D and V; X 10 Selected from E, V, L, D and H; X 11 is F; and X 12 Selected from N, A and F.

4. The isolated multispecific antibody of claim 1, wherein P1 comprises the amino acid sequence according to SEQ ID NO: 32 or an amino acid sequence having 1, 2 or 3 amino acid mutations, substitutions or deletions relative to SEQ ID NO:

32.

5. The isolated multispecific antibody of claim 1, wherein P1 comprises the amino acid sequence according to SEQ ID NO:

32.

6. The isolated multispecific antibody of claim 1, wherein P1 comprises the amino acid sequence according to SEQ ID NO: 138 or an amino acid sequence having 1, 2 or 3 amino acid mutations, substitutions or deletions relative to SEQ ID NO:

138.

7. The isolated multispecific antibody of claim 1, wherein P1 comprises the amino acid sequence according to SEQ ID NO:

138.

8. The isolated multispecific antibody of claim 1, wherein P1 impairs binding of A1 to CD28.

9. The isolated multispecific antibody of claim 1, wherein P1 is bound to A1 through ionic interaction, electrostatic interaction, hydrophobic interaction, Pi-stacking interaction, and H-bonding interaction, or a combination thereof.

10. The isolated multispecific antibody of claim 1, wherein P1 is bound to A1 at or near the antigen binding site.

11. The isolated multispecific antibody of claim 1, wherein when L1 is cleaved by the tumor-specific protease, P1 is unbound from A1, thereby exposing A1 to CD28.

12. The isolated multispecific antibody of claim 1, wherein P1 has less than 75% sequence identity with CD28.

13. The isolated multispecific antibody of claim 1, wherein P1 comprises a de novo generated amino acid sequence that shares less than 10% sequence identity with CD28.

14. The isolated multispecific antibody of claim 1, wherein P1 comprises a modified amino acid or an unnatural amino acid or a modified unnatural amino acid, or a combination thereof.

15. The isolated multispecific antibody of claim 1, wherein P1 does not comprise albumin or an albumin fragment.

16. The isolated multispecific antibody of claim 1, wherein P1 does not comprise an albumin binding domain.

17. The isolated multispecific antibody of claim 1, wherein L1 is a peptide sequence having at least 5 and no more than 50 amino acids.

18. The isolated multispecific antibody of claim 1, wherein L1 is a peptide sequence having at least 10 and no more than 30 amino acids.

19. The isolated multispecific antibody of claim 1, wherein L1 is a peptide sequence having at least 10 amino acids.

20. The isolated multispecific antibody of claim 1, wherein L1 is a peptide sequence having at least 18 amino acids.

21. The isolated multispecific antibody of claim 1, wherein L1 is a peptide sequence having at least 26 amino acids.

22. The isolated multispecific antibody of claim 1, wherein L1 comprises (G2S) n wherein n is an integer from 1 to 3 (SEQ ID NO: 228).

23. The isolated multispecific antibody of claim 1, wherein L1 comprises (G2S) n wherein n is an integer of at least 1.

24. The isolated multispecific antibody of claim 1, wherein L1 comprises 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.

25. The isolated multispecific antibody of claim 1, wherein the tumor-specific protease is selected from the group consisting of a metalloprotease, a serine protease, a cysteine ​​protease, a threonine protease, and an aspartic protease.

26. The isolated multispecific antibody of claim 1, wherein L1 comprises an amino acid sequence cleavable by urokinase, an amino acid sequence cleavable by a membrane-type serine protease, or an amino acid sequence cleavable by a matrix metalloprotease.

27. The isolated multispecific antibody of claim 1, wherein L1 comprises a sequence according to SEQ ID NOs: 18-19, 62-88.

28. The isolated multispecific antibody of claim 1, wherein L1 is bound to the N-terminus of A1.

29. The isolated multispecific antibody of claim 1, wherein L1 is bound to the C-terminus of A1.

30. The isolated multispecific antibody of claim 1, wherein the CD28 binding domain comprises a single chain variable fragment, a single domain antibody, Fab, or Fab'.

31. The isolated multispecific antibody of claim 30, wherein the CD28 binding domain comprises the single chain variable fragment.

32. The isolated multispecific antibody of claim 1, wherein the PD-L1 binding domain comprises a single chain variable fragment, a single domain antibody, Fab, or Fab'.

33. The isolated multispecific antibody of claim 32, wherein the PD-L1 binding domain comprises the Fab or the Fab'.

34. The isolated multispecific antibody of claim 32, wherein the PD-L1 binding domain comprises the Fab or the Fab' and the CD28 binding domain comprises the single chain variable fragment.

35. The isolated multispecific antibody of claim 32, wherein the PD-L1 binding domain comprising the Fab or the Fab' comprises a Fab heavy chain polypeptide and a Fab light chain polypeptide, the Fab heavy chain polypeptide comprising a Fab heavy chain variable domain, and the Fab light chain polypeptide comprising a Fab light chain variable domain.

36. The isolated multispecific antibody of claim 35, wherein the CD28 binding domain comprising the single chain variable fragment comprises a scFv heavy chain variable domain and a scFv light chain variable domain.

37. The isolated multispecific antibody of claim 1, wherein the linker connects the C-terminus of A1 to the N-terminus of B.

38. The isolated multispecific antibody of claim 1, wherein the linker connects the N-terminus of A1 to the C-terminus of B.

39. The isolated multispecific antibody of claim 36, wherein the linker connects the C-terminus of A1 to the N-terminus of the Fab heavy chain polypeptide.

40. The isolated multispecific antibody of claim 36, wherein the linker connects the N-terminus of A1 to the C-terminus of the Fab heavy chain polypeptide.

41. The isolated multispecific antibody of claim 36, wherein the linker connects the C-terminus of A1 to the N-terminus of the Fab light chain polypeptide.

42. The isolated multispecific antibody of claim 36, wherein the linker connects the N-terminus of A1 to the C-terminus of the Fab light chain polypeptide.

43. The isolated multispecific antibody of claim 36, wherein the linker connects the Fab light chain polypeptide to the scFv light chain variable domain.

44. The isolated multispecific antibody of claim 36, wherein the linker connects the Fab light chain polypeptide to the scFv heavy chain variable domain.

45. The isolated multispecific antibody of claim 36, wherein the linker connects the Fab heavy chain polypeptide to the scFv light chain variable domain.

46. ​​The isolated multispecific antibody of claim 36, wherein the linker connects the Fab heavy chain polypeptide to the scFv heavy chain variable domain.

47. The isolated multispecific antibody of claim 36, wherein the linker connects the Fab light chain polypeptide to the N-terminus of the scFv light chain variable domain.

48. The isolated multispecific antibody of claim 36, wherein the linker connects the Fab light chain polypeptide to the C-terminus of the scFv light chain variable domain.

49. The isolated multispecific antibody of claim 36, wherein the linker connects the Fab light chain polypeptide to the N-terminus of the scFv heavy chain variable domain.

50. The isolated multispecific antibody of claim 36, wherein the linker connects the Fab light chain polypeptide to the C-terminus of the scFv heavy chain variable domain.

51. The isolated multispecific antibody of claim 36, wherein the linker connects the Fab heavy chain polypeptide to the N-terminus of the scFv light chain variable domain.

52. The isolated multispecific antibody of claim 36, wherein the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv light chain variable domain.

53. The isolated multispecific antibody of claim 36, wherein the linker connects the Fab heavy chain polypeptide to the N-terminus of the scFv heavy chain variable domain.

54. The isolated multispecific antibody of claim 36, wherein the linker connects the Fab heavy chain polypeptide to the C-terminus of the scFv heavy chain variable domain.

55. The isolated multispecific antibody of claim 1, wherein the linker is at least 5 amino acids in length.

56. The isolated multispecific antibody of claim 1, wherein the linker comprises (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.

57. The isolated multispecific antibody of claim 1, wherein L comprises a formula comprising (G2S)n, wherein n is an integer from 1 to 3 (SEQ ID NO: 228).

58. The isolated multispecific antibody of claim 1, wherein the L comprises the amino acid sequence of SEQ ID NO: 18 (GGGGSGGGGSGGGGS) or SEQ ID NO: 19 (GGGGS).

59. The isolated multispecific antibody of claim 36, wherein 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 the CDRs comprise 0-2 amino acid modifications in at least one of the HC-CDR1, HC-CDR2, or HC-CDR3.

60. An isolated multispecific antibody as described in claim 36, wherein 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(KA); and LC-CDR3: SEQ ID NO:6, and wherein the CDRs comprise 0-2 amino acid modifications in at least one of the LC-CDR1, LC-CDR2 or LC-CDR3.

61. The isolated multispecific antibody of claim 1, wherein A1 comprises complementary determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of A1 comprise: LC-CDR1: SEQ ID NO:4; LC-CDR2: SEQ ID NO:5(KA); and LC-CDR3: SEQ ID NO:6; wherein A1 comprises complementary determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of A1 comprise: HC-CDR1: SEQ ID NO:1; HC-CDR2: SEQ ID NO:2; HC-CDR3: SEQ ID NO:

3.

62. The isolated multispecific antibody of claim 36, wherein the Fab 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 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 the CDRs comprise 0-2 amino acid modifications in at least one of the HC-CDR1, HC-CDR2, or HC-CDR3.

63. An isolated multispecific antibody as described in claim 36, wherein 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 (DA); and LC-CDR3: SEQ ID NO: 15; and wherein the CDRs comprise 0-2 amino acid modifications in at least one of the LC-CDR1, LC-CDR2 or LC-CDR3.

64. The isolated multispecific antibody of claim 1, wherein B comprises complementary determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of B comprise: HC-CDR1: SEQ ID NO: 10; HC-CDR2: SEQ ID NO: 11; HC-CDR3: SEQ ID NO: 12; and wherein B comprises complementary determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of B comprise: LC-CDR1: SEQ ID NO: 13; LC-CDR2: SEQ ID NO: 14 (DA); and LC-CDR3: SEQ ID NO:

15.

65. The isolated multispecific antibody of claim 36, wherein the scFv heavy chain variable domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO:

7.

66. The isolated multispecific antibody of claim 36, wherein the scFv heavy chain variable domain comprises an amino acid sequence of at least 75 consecutive amino acid residues of SEQ ID NO:

7.

67. The isolated multispecific antibody of claim 36, wherein the scFv heavy chain variable domain comprises an amino acid sequence of at least 110 consecutive amino acid residues of SEQ ID NO:

7.

68. The isolated multispecific antibody of claim 36, wherein 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 with the at least 110 consecutive amino acid residues of SEQ ID NO:

7.

69. The isolated multispecific antibody of claim 36, wherein the scFv heavy chain variable domain comprises the amino acid sequence according to SEQ ID NO:

7.

70. The isolated multispecific antibody of claim 36, wherein the scFv light chain variable domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO:

8.

71. The isolated multispecific antibody of claim 36, wherein the scFv light chain variable domain comprises an amino acid sequence of at least 75 consecutive amino acid residues of SEQ ID NO:

8.

72. The isolated multispecific antibody of claim 36, wherein the scFv light chain variable domain comprises an amino acid sequence of at least 100 consecutive amino acid residues of SEQ ID NO:

8.

73. The isolated multispecific antibody of claim 36, wherein 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 with the at least 100 consecutive amino acid residues of SEQ ID NO:

8.

74. The isolated multispecific antibody of claim 36, wherein the scFv light chain variable domain comprises the amino acid sequence according to SEQ ID NO:

8.

75. The isolated multispecific antibody of claim 36, wherein the scFv comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO:

9.

76. The isolated multispecific antibody of claim 36, wherein the scFv comprises an amino acid sequence of at least 175 consecutive amino acid residues of SEQ ID NO:

9.

77. The isolated multispecific antibody of claim 36, wherein the scFv comprises an amino acid sequence of at least 210 consecutive amino acid residues of SEQ ID NO:

9.

78. The isolated multispecific antibody of claim 36, wherein 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 with the at least 210 consecutive amino acid residues of SEQ ID NO:

9.

79. The isolated multispecific antibody of claim 36, wherein the scFv comprises the amino acid sequence according to SEQ ID NO:

9.

80. The isolated multispecific antibody of claim 36, wherein the Fab heavy chain polypeptide comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO:

17.

81. The isolated multispecific antibody of claim 36, wherein the Fab heavy chain polypeptide comprises an amino acid sequence of at least 175 consecutive amino acid residues of SEQ ID NO:

17.

82. The isolated multispecific antibody of claim 36, wherein the Fab heavy chain polypeptide comprises an amino acid sequence of at least 215 consecutive amino acid residues of SEQ ID NO:

17.

83. The isolated multispecific antibody of claim 36, wherein 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 with the at least 215 consecutive amino acid residues of SEQ ID NO:

17.

84. The isolated multispecific antibody of claim 36, wherein the Fab heavy chain polypeptide comprises the amino acid sequence according to SEQ ID NO:

17.

85. The isolated multispecific antibody of claim 36, wherein the Fab light chain polypeptide comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO:

16.

86. The isolated multispecific antibody of claim 36, wherein the Fab light chain polypeptide comprises an amino acid sequence of at least 175 consecutive amino acid residues of SEQ ID NO:

16.

87. The isolated multispecific antibody of claim 36, wherein the Fab light chain polypeptide comprises an amino acid sequence of at least 200 contiguous amino acid residues of SEQ ID NO:

16.

88. The isolated multispecific antibody of claim 36, wherein 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 with the at least 200 consecutive amino acid residues of SEQ ID NO:

16.

89. The isolated multispecific antibody of claim 36, wherein the Fab light chain polypeptide comprises an amino acid sequence according to SEQ ID NO:

16.

90. An isolated multispecific antibody as described in claim 36, wherein 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 having at least 80% sequence identity with the amino acid sequence according to SEQ ID NO:20, and the amino acid sequence of the Fab heavy chain polypeptide connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence according to SEQ ID NO:

21.

91. An isolated multispecific antibody as described in claim 36, wherein 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 the amino acid sequence according to SEQ ID NO:20, and the amino acid sequence of the Fab heavy chain polypeptide connected to the C-terminus of the scFv light chain variable domain comprises the amino acid sequence according to SEQ ID NO:

21.

92. An isolated multispecific antibody as described in claim 36, wherein 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 having at least 80% sequence identity with the amino acid sequence according to SEQ ID NO:23, and the amino acid sequence of the Fab light chain polypeptide connected to the C-terminus of the scFv light chain variable domain comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence according to SEQ ID NO:

22.

93. An isolated multispecific antibody as described in claim 36, wherein 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 the amino acid sequence according to SEQ ID NO:23, and the amino acid sequence of the Fab light chain polypeptide connected to the C-terminus of the scFv light chain variable domain comprises the amino acid sequence according to SEQ ID NO:

22.

94. The isolated multispecific antibody of claim 1, wherein the multispecific antibody further comprises a half-life extending molecule (H1).

95. The isolated multispecific antibody of claim 94, wherein H1 is linked to P1.

96. The isolated multispecific antibody of claim 94, wherein H1 does not block binding of A1 to CD28.

97. The isolated multispecific antibody of claim 94, wherein H1 does not block B binding to PD-L1.

98. The isolated multispecific antibody of claim 94, wherein H1 comprises a linker portion (L5) connecting H1 to P1.

99. The isolated multispecific antibody of claim 94, wherein the half-life extending molecule (H1) has no binding affinity for PD-L1.

100. The isolated multispecific antibody of claim 94, wherein the half-life extending molecule (H1) has no binding affinity for CD28.

101. The isolated multispecific antibody of claim 94, wherein the half-life extending molecule (H1) does not protect the multispecific antibody from CD28.

102. The isolated multispecific antibody of claim 94, wherein H1 comprises a sequence according to SEQ ID NOs: 54-57.

103. The isolated multispecific antibody of claim 94, wherein H1 comprises an amino acid sequence having a repeating sequence motif.

104. The isolated multispecific antibody of claim 94, wherein H1 comprises an amino acid sequence having a highly ordered secondary structure.

105. The isolated multispecific antibody of claim 94, wherein H1 comprises a polymer.

106. The isolated multispecific antibody of claim 105, wherein the polymer is polyethylene glycol (PEG).

107. The isolated multispecific antibody of claim 94, wherein H1 comprises albumin.

108. The isolated multispecific antibody of claim 94, wherein H1 comprises an Fc domain.

109. The isolated multispecific antibody of claim 107, wherein the albumin is serum albumin.

110. The isolated multispecific antibody of claim 107, wherein the albumin is human serum albumin.

111. The isolated multispecific antibody of claim 94, wherein H1 comprises a polypeptide, a ligand, or a small molecule.

112. The isolated multispecific antibody of claim 111, wherein the polypeptide, the ligand, or the small molecule binds to a serum protein or fragment thereof, a circulating immunoglobulin or fragment thereof, or CD35 / CR1.

113. The isolated multispecific antibody of claim 112, wherein the serum protein comprises thyroxine binding protein, transthyretin, 1-acid glycoprotein, transferrin, transferrin receptor or a transferrin binding portion thereof, fibrinogen, or albumin.

114. The isolated multispecific antibody of claim 112, wherein the circulating immunoglobulin molecules comprise IgG1, IgG2, IgG3, IgG4, slgA, IgM, or IgD.

115. The isolated multispecific antibody of claim 112, wherein the serum protein is albumin.

116. The isolated multispecific antibody of claim 112, wherein the polypeptide is an antibody.

117. The isolated multispecific antibody of claim 112, wherein the antibody comprises a single domain antibody, a single chain variable fragment, Fab, or Fab'.

118. The isolated multispecific antibody of claim 117, wherein the single domain antibody comprises a single domain antibody that binds to albumin.

119. The isolated multispecific antibody of claim 118, wherein the single domain antibody is a human antibody or a humanized antibody.

120. The isolated multispecific antibody of claim 117, wherein the single domain antibody is selected from 645gH1gL1, 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.

121. The isolated multispecific antibody of claim 117, wherein 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 0-2 amino acid modifications in at least one of the HC-CDR1, HC-CDR2, or HC-CDR3.

122. The isolated multispecific antibody of claim 94, wherein H1 comprises the amino acid sequence according to SEQ ID NO:

57.

123. The isolated multispecific antibody of claim 94, wherein H1 comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

57.

124. The isolated multispecific antibody of claim 94, wherein H1 comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:

57.

125. The isolated multispecific antibody of claim 94, wherein H1 comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:

57.

126. The isolated multispecific antibody of claim 94, wherein H1 comprises a modified amino acid or a non-natural amino acid or a modified non-natural amino acid, or a combination thereof.

127. The isolated multispecific antibody of claim 126, wherein the modified amino acid or modified non-natural amino acid comprises a post-translational modification.

128. The isolated multispecific antibody of claim 94, wherein H1 comprises a linker portion (L5) connecting H1 to P1.

129. The isolated multispecific antibody of claim 128, wherein L5 is a peptide sequence having at least 5 and no more than 50 amino acids.

130. The isolated multispecific antibody of claim 128, wherein L5 is a peptide sequence of at least 26 amino acids.

131. The isolated multispecific antibody of claim 128, wherein L5 comprises 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.

132. The isolated multispecific antibody of claim 1, wherein the isolated multispecific antibody comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NOs: 149-170.

133. The isolated multispecific antibody of claim 1, wherein the isolated multispecific antibody comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 149-170.

134. The isolated multispecific antibody of claim 1, wherein the isolated multispecific antibody comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NOs: 149-170.

135. The isolated multispecific antibody of claim 1, wherein the isolated multispecific antibody comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 149 and 150.

136. The isolated multispecific antibody of claim 1, wherein the isolated multispecific antibody comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NOs: 149 and 150.

137. The isolated multispecific antibody of claim 1, wherein the isolated multispecific antibody comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 151 and 152.

138. The isolated multispecific antibody of claim 1, wherein the isolated multispecific antibody comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NOs: 151 and 152.

139. The isolated multispecific antibody of claim 1, wherein the isolated multispecific antibody comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 153 and 154.

140. The isolated multispecific antibody of claim 1, wherein the isolated multispecific antibody comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NOs: 153 and 154.

141. The isolated multispecific antibody of claim 1, wherein the isolated multispecific antibody comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 155 and 156.

142. The isolated multispecific antibody of claim 1, wherein the isolated multispecific antibody comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NOs: 155 and 156.

143. The isolated multispecific antibody of claim 1, wherein the isolated multispecific antibody comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 157 and 158.

144. The isolated multispecific antibody of claim 1, wherein the isolated multispecific antibody comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NOs: 157 and 158.

145. The isolated multispecific antibody of claim 1, wherein the isolated multispecific antibody comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 159 and 160.

146. The isolated multispecific antibody of claim 1, wherein the isolated multispecific antibody comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NOs: 159 and 160.

147. The isolated multispecific antibody of claim 1, wherein the isolated multispecific antibody comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 161 and 162.

148. The isolated multispecific antibody of claim 1, wherein the isolated multispecific antibody comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NOs: 161 and 162.

149. The isolated multispecific antibody of claim 1, wherein the isolated multispecific antibody comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 163 and 164.

150. The isolated multispecific antibody of claim 1, wherein the isolated multispecific antibody comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NOs: 163 and 164.

151. The isolated multispecific antibody of claim 1, wherein the isolated multispecific antibody comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 165 and 166.

152. The isolated multispecific antibody of claim 1, wherein the isolated multispecific antibody comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NOs: 165 and 166.

153. The isolated multispecific antibody of claim 1, wherein the isolated multispecific antibody comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 167 and 168.

154. The isolated multispecific antibody of claim 1, wherein the isolated multispecific antibody comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NOs: 167 and 168.

155. The isolated multispecific antibody of claim 1, wherein the isolated multispecific antibody comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 169 and 170.

156. The isolated multispecific antibody of claim 1, wherein the isolated multispecific antibody comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NOs: 169 and 170.

157. The isolated multispecific antibody of claim 1, wherein the isolated multispecific antibody comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 208 and 209.

158. The isolated multispecific antibody of claim 1, wherein the isolated multispecific antibody comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NOs: 208 and 209.

159. An isolated recombinant nucleic acid molecule encoding a polypeptide of the isolated multispecific antibody of claim 1.

160. A pharmaceutical composition comprising: (a) the isolated multispecific antibody of claim 1; and (b) a pharmaceutically acceptable excipient.

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