Antibodies targeting psma and cd28 and uses thereof

Abstract

This article discloses isolated peptide and peptide complex compositions comprising a first antigen recognition molecule that binds to CD28 and a second antigen recognition molecule that binds to prostate-specific membrane antigen (PSMA).

Description

[0001] Cross-referencing This application claims the benefit of U.S. Provisional Application No. 63 / 595,921, filed November 3, 2023, the entire contents of which are incorporated herein by reference.

[0002] sequence list This application contains a sequence list that has been electronically submitted in XML format, the entire contents of which are hereby incorporated by reference. The XML copy was created on October 25, 2024, named 52426-764_601_SL.xml, and is 4,007,239 bytes in size. Summary of the Invention

[0003] In some embodiments, this document discloses isolated peptides or peptide complexes comprising a first antigen recognition molecule that binds to CD28 and a second antigen recognition molecule that binds to prostate-specific membrane antigen (PSMA). In some embodiments, the first antigen recognition molecule that binds to CD28 comprises an anti-CD28 antibody. In some embodiments, the anti-CD28 antibody comprises an anti-CD28 heavy chain variable domain comprising complementarity-determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein HC-CDR1, HC-CDR2, and HC-CDR3 comprise the following amino acid sequences: HC-CDR1: SEQ ID NO: 1, HC-CDR2: SEQ ID NO: 2, and HC-CDR3: SEQ ID NO: 3; and the anti-CD28 antibody comprises an anti-CD28 light chain variable domain comprising complementarity-determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein LC-CDR1, LC-CDR2, and LC-CDR3 comprise the following amino acid sequences: LC-CDR1: SEQ ID NO: 4, LC-CDR2: (KAS), and LC-CDR3: SEQ ID NO: 6. In some embodiments, the anti-CD28 heavy chain variable domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 7, and the anti-CD28 light chain variable domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 8.

[0004] In some embodiments, the anti-CD28 antibody comprises a single-chain variable fragment (scFv), a single-domain antibody, Fab, or Fab'. In some embodiments, the anti-CD28 antibody comprises scFv. In some embodiments, the anti-CD28 antibody comprises a single-chain variable fragment (scFv) having engineered disulfide bonds. In some embodiments, the anti-CD28 antibody comprises scFv, and the scFv comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 9. In some embodiments, the anti-CD28 antibody comprises scFv, and the scFv comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 10. In some embodiments, the anti-CD28 antibody comprises scFv, and the scFv comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 11. In some embodiments, the anti-CD28 antibody comprises scFv, and the scFv comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:12.

[0005] In some embodiments, the anti-CD28 antibody comprises Fab. In some embodiments, Fab comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 696 and an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 697.

[0006] In some embodiments, the anti-CD28 antibody comprises CrossFab. In some embodiments, CrossFab comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 698 and an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 699. In some embodiments, CrossFab comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 700 and an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 701.

[0007] In some implementations, the second antigen recognition molecule that binds to PSMA contains an anti-PSMA antibody. In some embodiments, the anti-PSMA antibody comprises an anti-PSMA heavy chain variable domain comprising complementarity-determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein HC-CDR1, HC-CDR2, and HC-CDR3 comprise the following amino acid sequences: HC-CDR1: SEQ ID NO: 13, HC-CDR2: SEQ ID NO: 14, and HC-CDR3: SEQ ID NO: 15; and the anti-PSMA antibody comprises an anti-PSMA light chain variable domain comprising complementarity-determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, LC-CDR2, and LC-CDR3 of the light chain variable domain comprise the following amino acid sequences: LC-CDR1: SEQ ID NO: 16, LC-CDR2: (EA), and LC-CDR3: SEQ ID NO: 18. In some embodiments, the anti-PSMA heavy chain variable domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 20, and the anti-PSMA light chain variable domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 19. In some embodiments, the anti-PSMA antibody comprises a single-chain variable fragment (scFv), a single-domain antibody, Fab, Fab', or CrossFab. In some embodiments, the anti-PSMA antibody comprises Fab or Fab'. In some embodiments, the anti-PSMA antibody comprises Fab or Fab', and Fab or Fab' comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 19 and an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 20. In some embodiments, the anti-PSMA antibody comprises Fab or Fab', and Fab or Fab' comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 702 and an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 703.In some embodiments, the anti-PSMA antibody comprises Fab or Fab', and Fab or Fab' comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 704 and an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 705.

[0008] In some embodiments, the anti-CD28 antibody comprises scFv, and the anti-PSMA antibody comprises Fab or Fab'. In some embodiments, the scFv is linked to Fab or Fab' via a adapter, and Fab or Fab' comprises a Fab heavy chain polypeptide and a Fab light chain polypeptide. In some embodiments, the adapter links the scFv to the Fab heavy chain polypeptide. In some embodiments, the adapter links the scFv to the Fab light chain polypeptide. In some embodiments, the adapter links the scFv to the N-terminus of the Fab heavy chain polypeptide. In some embodiments, the adapter links the scFv to the C-terminus of the Fab heavy chain polypeptide. In some embodiments, the adapter links the scFv to the N-terminus of the Fab light chain polypeptide. In some embodiments, the adapter links the scFv to the C-terminus of the Fab light chain polypeptide. In some embodiments, the adapter links the anti-CD28 heavy chain variable domain of the scFv to the Fab heavy chain polypeptide. In some embodiments, the adapter links the anti-CD28 heavy chain variable domain of the scFv to the Fab light chain polypeptide. In some embodiments, the adapter links the anti-CD28 heavy chain variable domain of the scFv to the N-terminus of the Fab heavy chain polypeptide. In some embodiments, the adapter links the anti-CD28 heavy chain variable domain of the scFv to the C-terminus of the Fab heavy chain peptide. In some embodiments, the adapter links the anti-CD28 heavy chain variable domain of the scFv to the N-terminus of the Fab light chain peptide. In some embodiments, the adapter links the anti-CD28 heavy chain variable domain of the scFv to the C-terminus of the Fab light chain peptide. In some embodiments, the adapter links the anti-CD28 light chain variable domain of the scFv to the Fab heavy chain peptide. In some embodiments, the adapter links the anti-CD28 light chain variable domain of the scFv to the N-terminus of the Fab heavy chain peptide. In some embodiments, the adapter links the anti-CD28 light chain variable domain of the scFv to the C-terminus of the Fab heavy chain peptide. In some embodiments, the adapter links the anti-CD28 light chain variable domain of the scFv to the N-terminus of the Fab light chain peptide. In some implementations, the linker connects the anti-CD28 light chain variable domain of scFv to the C-terminus of the Fab light chain peptide.

[0009] In some embodiments, the anti-CD28 antibody comprises Fab or Fab', and the anti-PSMA antibody comprises Fab or Fab'. In some embodiments, the Fab or Fab' of the anti-CD28 antibody and the Fab or Fab' of the anti-PSMA antibody are linked by a linker, and the Fab or Fab' of the anti-PSMA antibody comprises a Fab heavy chain polypeptide and a Fab light chain polypeptide. In some embodiments, the linker connects the Fab or Fab' of the anti-CD28 antibody to the Fab heavy chain polypeptide of the anti-PSMA antibody. In some embodiments, the linker connects the Fab or Fab' of the anti-CD28 antibody to the Fab light chain polypeptide of the anti-PSMA antibody. In some embodiments, the linker connects the Fab or Fab' of the anti-CD28 antibody to the N-terminus of the Fab heavy chain polypeptide of the anti-PSMA antibody. In some embodiments, the linker connects the Fab or Fab' of the anti-CD28 antibody to the C-terminus of the Fab heavy chain polypeptide of the anti-PSMA antibody. In some embodiments, the linker connects the Fab or Fab' of the anti-CD28 antibody to the N-terminus of the Fab light chain polypeptide of the anti-PSMA antibody. In some embodiments, the linker connects the Fab or Fab' of the anti-CD28 antibody to the C-terminus of the Fab light chain polypeptide of the anti-PSMA antibody. In some embodiments, the linker connects the Fab heavy chain polypeptide of the anti-CD28 antibody to the Fab heavy chain polypeptide of the anti-PSMA antibody. In some embodiments, the linker connects the Fab heavy chain polypeptide of the anti-CD28 antibody to the Fab light chain polypeptide of the anti-PSMA antibody. In some embodiments, the linker connects the Fab heavy chain polypeptide of the anti-CD28 antibody to the N-terminus of the Fab heavy chain polypeptide of the anti-PSMA antibody. In some embodiments, the linker connects the Fab heavy chain polypeptide of the anti-CD28 antibody to the C-terminus of the Fab heavy chain polypeptide of the anti-PSMA antibody. In some embodiments, the linker connects the Fab heavy chain polypeptide of the anti-CD28 antibody to the N-terminus of the Fab light chain polypeptide of the anti-PSMA antibody. In some embodiments, the linker connects the Fab heavy chain polypeptide of the anti-CD28 antibody to the C-terminus of the Fab light chain polypeptide of the anti-PSMA antibody. In some embodiments, the linker connects the Fab light chain polypeptide of the anti-CD28 antibody to the Fab heavy chain polypeptide of the anti-PSMA antibody. In some embodiments, the linker ligates the Fab light chain peptide of the anti-CD28 antibody to the Fab light chain peptide of the anti-PSMA antibody. In some embodiments, the linker ligates the Fab light chain peptide of the anti-CD28 antibody to the N-terminus of the Fab heavy chain peptide of the anti-PSMA antibody. In some embodiments, the linker ligates the Fab light chain peptide of the anti-CD28 antibody to the N-terminus of the Fab light chain peptide of the anti-PSMA antibody. In some embodiments, the linker ligates the Fab light chain peptide of the anti-CD28 antibody to the C-terminus of the Fab light chain peptide of the anti-PSMA antibody.

[0010] In some embodiments, the anti-CD28 antibody comprises CrossFab, and the anti-PSMA antibody comprises Fab or Fab'. In some embodiments, CrossFab and Fab or Fab' are linked by a connector, and the Fab or Fab' of the anti-PSMA antibody comprises a Fab heavy chain polypeptide and a Fab light chain polypeptide. In some embodiments, the connector links CrossFab to the Fab heavy chain polypeptide of the anti-PSMA antibody. In some embodiments, the connector links CrossFab to the Fab light chain polypeptide of the anti-PSMA antibody. In some embodiments, the connector links CrossFab to the N-terminus of the Fab heavy chain polypeptide of the anti-PSMA antibody. In some embodiments, the connector links CrossFab to the C-terminus of the Fab heavy chain polypeptide of the anti-PSMA antibody. In some embodiments, the connector links CrossFab to the N-terminus of the Fab light chain polypeptide of the anti-PSMA antibody. In some embodiments, the connector links CrossFab to the C-terminus of the Fab light chain polypeptide of the anti-PSMA antibody. In some embodiments, the connector links the Fab heavy chain polypeptide of CrossFab to the Fab heavy chain polypeptide of the anti-PSMA antibody. In some embodiments, the adapter links the CrossFab Fab heavy chain peptide to the Fab light chain peptide of the anti-PSMA antibody. In some embodiments, the adapter links the CrossFab Fab heavy chain peptide to the N-terminus of the anti-PSMA antibody Fab heavy chain peptide. In some embodiments, the adapter links the CrossFab Fab heavy chain peptide to the C-terminus of the anti-PSMA antibody Fab heavy chain peptide. In some embodiments, the adapter links the CrossFab Fab heavy chain peptide to the N-terminus of the anti-PSMA antibody Fab light chain peptide. In some embodiments, the adapter links the CrossFab Fab heavy chain peptide to the C-terminus of the anti-PSMA antibody Fab light chain peptide. In some embodiments, the adapter links the CrossFab Fab light chain peptide to the anti-PSMA antibody Fab heavy chain peptide. In some embodiments, the adapter links the CrossFab Fab light chain peptide to the anti-PSMA antibody Fab light chain peptide. In some embodiments, the adapter links the CrossFab Fab light chain peptide to the N-terminus of the anti-PSMA antibody Fab heavy chain peptide. In some embodiments, the adapter links the CrossFab Fab light chain peptide to the C-terminus of the anti-PSMA antibody Fab heavy chain peptide. In some embodiments, the adapter links the CrossFab Fab light chain peptide to the N-terminus of the anti-PSMA antibody Fab light chain peptide. In some embodiments, the adapter links the CrossFab Fab light chain peptide to the C-terminus of the anti-PSMA antibody Fab light chain peptide.

[0011] In some implementations, the anti-CD28 antibody contains Fab or Fab', and the anti-PSMA antibody contains CrossFab.

[0012] In some embodiments, the linker is at least 5 amino acids long. In some embodiments, the linker is no more than 30 amino acids long. In some embodiments, the linker is at least 5 amino acids and no more than 30 amino acids long. In some embodiments, the linker is 5 amino acids long. In some embodiments, the linker is 15 amino acids long. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 21 (GGGGSGGGGSGGGGS) or SEQ ID NO: 22 (GGGGS).

[0013] In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 23 and SEQ ID NO: 24. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 25 and SEQ ID NO: 26. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 27 and SEQ ID NO: 28. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 29 and SEQ ID NO: 30. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 31 and SEQ ID NO: 32. In some embodiments, the isolated polypeptide or polypeptide complex comprises the amino acid sequences of SEQ ID NO: 25 and SEQ ID NO: 26. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 706 and SEQ ID NO: 707. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 708, SEQ ID NO: 709, and SEQ ID NO: 710. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 711, SEQ ID NO: 712, and SEQ ID NO: 713. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 714, SEQ ID NO: 715, and SEQ ID NO: 716.In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 717, SEQ ID NO: 718, and SEQ ID NO: 719. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 720, SEQ ID NO: 721, and SEQ ID NO: 722. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 723, SEQ ID NO: 724, and SEQ ID NO: 725. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 726, SEQ ID NO: 727, and SEQ ID NO: 728. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 729, SEQ ID NO: 730, and SEQ ID NO: 731.

[0014] In some embodiments, the isolated peptide or peptide complex is human or humanized. In some embodiments, the isolated peptide or peptide complex comprises a peptide linked to an anti-CD28 antibody, wherein the peptide weakens the binding of the anti-CD28 antibody to CD28. In some embodiments, the isolated peptide or peptide complex has a configuration according to Formula I: A2-A1-L1-P1-H1, wherein A2 comprises an anti-PSMA antibody, A1 comprises an anti-CD28 antibody, L1 comprises a linker portion connecting A1 to P1 and is a substrate of a tumor-specific protease, P1 comprises a peptide weakening the binding of the anti-CD28 antibody to CD28, and H1 comprises a half-life-extending molecule.

[0015] In some embodiments, A2 further comprises P2 and L2, wherein P2 comprises a peptide bound to A2; and L2 comprises a linker portion connecting A2 to P2 and is a substrate of a tumor-specific protease. In some embodiments, the polypeptide or polypeptide complex is according to formula Ia: P2-L2-A2-A1-L1-P1-H1. In some embodiments, the anti-CD28 antibody comprises scFv, and the anti-PSMA antibody comprises Fab or Fab', and Fab or Fab' comprises a Fab heavy chain polypeptide and a Fab light chain polypeptide. In some embodiments, the Fab heavy chain polypeptide of A2 binds to the scFv heavy chain polypeptide of A1, and L2 binds to the Fab light chain polypeptide of A2. In some embodiments, the Fab light chain polypeptide of A2 binds to the scFv heavy chain polypeptide of A1, and L2 binds to the Fab heavy chain polypeptide of A2. In some embodiments, the Fab heavy chain polypeptide of A2 binds to the scFv light chain polypeptide of A1, and L2 binds to the Fab light chain polypeptide of A2. In some implementations, the Fab light chain polypeptide of A2 binds to the scFv light chain polypeptide of A1, and L2 binds to the Fab heavy chain polypeptide of A2.

[0016] In some embodiments, P1 binds to A1 via ionic interactions, electrostatic interactions, hydrophobic interactions, π-stacking interactions, or hydrogen bonding interactions, or combinations thereof. In some embodiments, P1 has less than 70% sequence homology with CD28. In some embodiments, P2 weakens the binding of A2 to PSMA. In some embodiments, P2 binds to A2 via ionic interactions, electrostatic interactions, hydrophobic interactions, π-stacking interactions, or hydrogen bonding interactions, or combinations thereof. In some embodiments, P2 binds to A2 at or near the antigen binding site. In some embodiments, P2 has less than 70% sequence homology with PSMA. In some embodiments, P1 or P2 comprises a peptide sequence of at least 10 amino acids in length. In some embodiments, P1 or 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, P1 or P2 comprises a peptide sequence of at least 16 amino acids in length. In some embodiments, P1 or 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 comprises at least two cysteine ​​amino acid residues.

[0017] In some embodiments, P1 comprises an amino acid sequence according to any one of SEQ ID NO: 33 to 106 or 732-813. In some embodiments, P1 comprises an amino acid sequence of SEQ ID NO: 73 or a peptide sequence having one, two, or three amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 73. In some embodiments, P1 comprises an amino acid sequence of SEQ ID NO: 73. In some embodiments, P1 comprises an amino acid sequence of SEQ ID NO: 103 or a peptide sequence having one, two, or three amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 103. In some embodiments, P1 comprises an amino acid sequence of SEQ ID NO: 103. 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 12 The amino acid sequence is defined as follows: X1 is selected from M, I, L, and V; X2 is selected from D, H, N, A, F, S, T, Y, and V; X3 is selected from W, L, and F; X4 is selected from P, A, and L; X5 is selected from R, T, I, M, S, K, L, V, W, F, A, P, and D; X6 is selected from E, D, Y, H, S, F, A, N, T, I, P, and V; X7 is selected from L, M, R, S, Q, and H; X8 is selected from W and Q; X9 is selected from 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 Selected from F, Y, L, W, and V; and X 12 The X1 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 Selected from F, Y, and L; and X 12 The X1 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 It is F; and X 12The amino acid sequence is selected from N, A, and F. In some embodiments, P1 comprises the amino acid sequence according to SEQ ID NO: 41 or an amino acid sequence having one, two, or three amino acid mutations, substitutions, or deletions relative to SEQ ID NO: 41. In some embodiments, P1 comprises the amino acid sequence according to SEQ ID NO: 41. In some embodiments, P1 comprises an amino acid sequence selected from any one of SEQ ID NO: 41, 73, 103, and 179-603.

[0018] In some embodiments, P1 comprises the amino acid sequence according to the following sequence: J1-J2-WC-J3-J4-J5-J6-J7-J8-J9-CJ 10 -J 11 J1 is selected from A, R, N, D, Q, E, G, H, I, L, K, F, P, S, T, W, Y, and V; J2 is selected from D, P, and Y; J3 is selected from P and Q; J4 is selected from R, L, and I; J5 is selected from D, H, Y, and I; J6 is selected from L, S, and R; J7 is selected from W and G; J8 is selected from V, A, D, H, L, and N; J9 is selected from H, L, E, and D; J 10 Selected from F, Y, and L; and J 11 Selected from A, N, H, and T. In some embodiments, J1 is selected from A, R, N, D, Q, E, G, H, K, F, P, S, T, W, and Y. In some embodiments, J1 is selected from R, Q, E, H, I, L, K, F, T, W, Y, and V; J2 is D; J3 is P; J4 is R; J5 is D; J6 is L; J7 is W; J8 is V; J9 is H; J 10 It is F; and J 11 It is A. In some implementations, J1 is selected from R, Q, E, L, K, T, and W; J2 is D; J3 is P; J4 is R; J5 is D; J6 is L; J7 is W; J8 is V; J9 is H; J 10 It is F; and J 11 It is A. In some implementations, J1 is L or I; J2 is D or Y; J3 is P; J4 is R, L, or I; J5 is H, Y, or D; J6 is L or R; J7 is W; J8 is A, D, H, or N; J9 is L, E, or D; J 10 It is F or Y; and J 11 It is N or H. In some implementations, J1 is L or I; J2 is D or Y; J3 is P; J4 is R or I; J5 is H, Y, or D; J6 is L or R; J7 is W; J8 is A, D, or N; J9 is L, E, or D; J 10 It is F or Y; and J 11 It is N.

[0019] In some embodiments, P1 comprises an amino acid sequence according to the following sequence: Z1-C-Z2-Z3-Z4-Z5-Z6-Z7-Z8-Z9-Z 10 -Z 11 -CZ 12 Z1 is selected from Y, H, and A; Z2 is selected from D, E, A, Q, S, V, M, H, L, F, T, W, and N; Z3 is selected from G, E, S, and A; Z4 is selected from H, L, W, and A; Z5 is selected from L, S, A, T, I, V, M, R, E, D, F, H, K, Q, N, Y, W, and G; Z6 is selected from P, A, E, Q, S, L, W, G, V, D, K, I, M, R, T, F, H, N, and Y; Z7 is selected from E, Q, I, M, V, A, L, R, W, P, D, T, K, S, G, H, and N; Z8 is selected from Y, H, F, W, N, Q, and A; Z9 is selected from M, I, L, R, V, K, F, Q, Y, W, and A; Z 10 Selected from A, Q, S, W, E, L, G, D, T, M, N, V, H, R, Y, K, P, F, and I; Z 11 Selected from K, Q, N, H, E, P, and A; and Z 12 The Z1 is selected from L, M, I, F, V, Y, Q, D, T, and A. In some embodiments, Z1 is selected from Y and H; Z2 is selected from D, E, A, Q, S, V, M, H, L, F, T, W, and N; Z3 is selected from G, E, and S; Z4 is selected from H, L, and W; Z5 is selected from L, S, A, T, I, V, M, R, E, D, F, H, K, Q, N, Y, W, and G; Z6 is selected from P, A, E, Q, S, L, W, G, V, D, K, I, M, R, T, F, H, N, and Y; Z7 is selected from E, Q, I, M, V, A, L, R, W, P, D, T, K, S, G, H, and N; Z8 is selected from Y, H, F, W, N, and Q; Z9 is selected from M, I, L, R, V, K, F, Q, Y, and W; Z 10 Selected from A, Q, S, W, E, L, G, D, T, M, N, V, H, R, Y, K, P, F, and I; Z 11 Selected from K, Q, N, H, E, and P; and Z 12The numbers are selected from L, M, I, F, V, Y, Q, D, and T. In some embodiments, Z1 is selected from Y and H; Z2 is selected from D, E, A, Q, S, V, M, H, L, F, T, W, and N; Z3 is selected from G, E, and S; Z4 is selected from H, L, and W; Z5 is selected from L, S, A, T, I, V, M, R, E, D, F, H, K, Q, N, Y, W, and G; Z6 is selected from P, A, E, Q, S, L, W, G, V, D, K, I, M, R, T, F, H, N, and Y; Z7 is selected from E, Q, I, M, V, A, L, R, W, P, D, T, K, S, G, H, and N; Z8 is selected from Y, H, F, W, N, and Q; Z9 is selected from M, I, L, R, V, K, F, Q, Y, and W; Z 10 Selected from A, Q, S, W, E, L, G, D, T, M, N, V, H, R, Y, K, P, F, and I; Z 11 Selected from K, Q, N, H, E, and P; and Z 12 The Z1 is selected from L, M, I, F, V, Y, Q, D, and T. In some embodiments, Z1 is Y; Z2 is selected from D, E, A, and Q; Z3 is G; Z4 is H; Z5 is selected from L, S, A, T, I, V, and M; Z6 is selected from P, A, E, Q, S, L, W, G, V, and D; Z7 is selected from E, Q, I, M, V, A, and L; Z8 is selected from Y and H; Z9 is selected from M, I, and L; Z 10 Selected from A, Q, S, W, E, L, G, D, T, and N; Z 11 It is K; and Z 12 Z1 is selected from L and M. In some embodiments, Z1 is Y; Z2 is selected from D, E, and A; Z3 is G; Z4 is H; Z5 is selected from L, S, A, and T; Z6 is selected from P, A, E, Q, S, L, and W; Z7 is selected from E, Q, I, and M; Z8 is Y; Z9 is selected from M, I, and L; Z 10 Selected from A, Q, S, W, E, L, G, and D; Z 11 It is K; and Z 12 It is L. In some implementations, Z1 is Y; Z2 is selected from D and E; Z3 is G; Z4 is H; Z5 is selected from L, S and A; Z6 is selected from P, A, E, Q, S and L; Z7 is selected from E, Q and I; Z8 is Y; Z9 is selected from M, I and L; Z 10 Selected from A, Q, S, W, E, L, G, and D; Z 11 It is K; and Z 12 It is L.

[0020] In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 784. In some embodiments, P1 consists of the amino acid sequence of SEQ ID NO: 784. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 744. In some embodiments, P1 consists of the amino acid sequence of SEQ ID NO: 744. In some embodiments, P1 comprises the amino acid sequence according to any one of SEQ ID NO: 774-793. In some embodiments, P1 comprises the amino acid sequence according to any one of SEQ ID NO: 764-773.

[0021] In some embodiments, P1 comprises amino acids according to the following sequence: U1-C-U2-U3-U4-U5-U6-U7-U8-U9-U 10 -U 11 -CU 12 Among them: U1 is selected from T, S, L, M, W, Q, P, R, G, N, I, Y, D, E, V, H and A; U2 is selected from A, S, T, K, E, R, G, N, W and D; U3 is selected from A, E, P, D, G, W, Q, V and F; U4 is selected from G, V, F, D, E, R and A; U5 is selected from A, F, V, S, L, P and M; U6 is selected from T, S, H, M, A, N, P, G, Q, D, Y, V, L, I, E, K, F and R; U7 is selected from Y, W, F, L, N, T, Q, I and A; U8 is selected from P, S, T, A, E, K, Q, N, V, G, H, L, D, Y, I, M and R; U9 is selected from W, L and A; U 10 Selected from P, S, R, L, E, and A; U 11 Selected from L, A, T, M, V, W, Y, E, Q, I, F, S, P, K, R, D, H, N, and G; and U 12 The elements are selected from L, F, V, M, I, Y, E, W, and A. In some embodiments, U1 is selected from T, S, L, M, W, Q, P, R, G, N, I, Y, D, E, V, and H; U2 is selected from A, S, T, K, E, R, G, N, W, and D; U3 is selected from A, E, P, D, G, W, Q, V, and F; U4 is selected from G, V, F, D, E, and R; U5 is selected from A, F, V, S, L, P, and M; U6 is selected from T, S, H, M, A, N, P, G, Q, D, Y, V, L, I, E, K, F, and R; U7 is selected from Y, W, F, L, N, T, Q, and I; U8 is selected from P, S, T, A, E, K, Q, N, V, G, H, L, D, Y, I, M, and R; U9 is selected from W, L, and A; U 10 Selected from P, S, R, L, and E; U 11Selected from L, A, T, M, V, W, Y, E, Q, I, F, S, P, K, R, D, H, N, and G; and U 12 The elements are selected from L, F, V, M, I, Y, E, and W. In some embodiments, U1 is selected from T, S, L, M, and W; U2 is selected from A, S, and T; U3 is selected from A and E; U4 is selected from G, V, and F; U5 is selected from A, F, and V; U6 is selected from T, S, H, M, A, and N; U7 is selected from Y, W, and F; U8 is selected from P, S, T, A, E, K, Q, N, and V; U9 is selected from W and L; U 10 Selected from P and S; U 11 Selected from L, A, T, M, V, W, Y, E, Q, I, F, and S; and U 12 The elements are selected from L, F, V, and M. In some embodiments, U1 is selected from T and S; U2 is selected from A and S; U3 is A; U4 is selected from G and V; U5 is selected from A and F; U6 is selected from T, S, and H; U7 is selected from Y and W; U8 is selected from P, S, T, and A; U9 is W; U 10 It is P; U 11 Selected from L, A, T, M, V, W, and Y; and U 12 Selected from L and F.

[0022] In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 749. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 749. In some embodiments, P1 comprises the amino acid sequence according to any one of SEQ ID NO: 756-763. In some embodiments, P1 comprises the amino acid sequence according to any one of SEQ ID NO: 794-813. In some embodiments, P1 comprises the amino acid sequence according to any one of SEQ ID NO: 732-2002.

[0023] In some embodiments, P2 comprises the amino acid sequence of any one of SEQ ID NO: 2003-4329. In some embodiments, P2 comprises the amino acid sequence of SEQ ID NO: 2025. In some embodiments, P2 consists of the amino acid sequence of SEQ ID NO: 2025. In some embodiments, P2 comprises the amino acid sequence of any one of SEQ ID NO: 2087-2094. In some embodiments, P2 comprises the amino acid sequence of SEQ ID NO: 2026. In some embodiments, P2 consists of the amino acid sequence of SEQ ID NO: 2026. In some embodiments, P2 comprises the amino acid sequence of any one of SEQ ID NO: 2097-2106. In some embodiments, P2 comprises the amino acid sequence of any one of SEQ ID NO: 2107-2133. In some embodiments, P2 comprises the amino acid sequence of SEQ ID NO: 2120. In some embodiments, P2 consists of the amino acid sequence of SEQ ID NO: 2120.

[0024] In some implementations, P2 includes components based on B1-B2-B3-B4-C-B5-P-B6-W-B7-C-B8-B9-B 10 The amino acid sequence of (SEQ IDNO: 4470), wherein: B1 is selected from V, E, L, D, I, G, M, S, P, T, A, F, W, Y, Q, H, N, K and R; B2 is selected from E, V, D, T, S, L, G, P, A, M, I, Q, H, F, Y, N, W, R and K; B3 is selected from K, P, R, I, N, H, V, M, A, L, Q, T, S, G, F, Y, E, W and D; B4 is selected from W, L, M, R, V, Y, A, K, I, S, Q, F, H, E, T, N, G and D; B5 is selected from I, V, T, K, R, E, S, Q, M, L, F, A, N, and H; B6 is selected from A, E, S, P, Q, T, L, D, M, V, R, K, N, I, H, W, Y, F, and G; B7 is selected from M, L, I, A, V, F, G, and K; B8 is selected from E, S, T, A, V, D, Q, L, N, I, M, Y, H, F, W, G, K, R, and P; B9 is selected from G, V, A, S, W, E, D, M, T, L, F, H, Q, N, R, I, Y, P, and K; and B 10 Selected from F, L, M, S, I, V, D, Q, T, A, N, Y, W, E, R, H, P, G, and K.

[0025] 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 A2. In some embodiments, L2 binds to the C-terminus of A2. 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 contains (G2S). n The formula is given, where n is an integer from 1 to 3 (SEQ ID NO: 605). In some embodiments, L1 or L2 has a value selected from (G2S). n (GS) n (GSGGS) n (SEQ ID NO: 606), (GGGS) n (SEQ ID NO: 607), (GGGGS) n (SEQ ID NO:608) and (GSSGGS) n The expression (SEQ ID NO: 609) where n is an integer of at least 1.

[0026] In some embodiments, when L1 is cleaved by a tumor-specific protease, P1 unbinds to A1, thereby exposing A1 to CD28. In some embodiments, when L2 is cleaved by a tumor-specific protease, P2 unbinds to A2, thereby exposing A2 to PSMA. In some embodiments, the tumor-specific protease is selected from matrix metalloproteinases (MMPs), serine proteases, cysteine ​​proteases, threonine proteases, and aspartic proteases. In some embodiments, the matrix metalloproteinase comprises MMP2, MMP7, MMP9, MMP13, or MMP14. In some embodiments, the serine protease comprises matrix protease (MTSP1), urokinase, or hepsin. In some embodiments, L1 or L2 comprises a urokinase-cleavable amino acid sequence, a matrix protease-cleavable amino acid sequence, a matrix metalloproteinase-cleavable amino acid sequence, or an asparagine endonuclease-cleavable amino acid sequence. In some embodiments, L1 or L2 comprises the amino acid sequence according to SEQ ID NO: 110. In some embodiments, L1 or L2 comprises an amino acid sequence according to any one of SEQ ID NO: 107-137 and 604. In some embodiments, L1 or L2 comprises an amino acid sequence of linker 25 (ISSGLLSGRSDAG) (SEQ ID NO: 132), linker 26 (AAGLLAPPGGLSGRSDAG) (SEQ ID NO: 133), linker 27 (SPLGLSGRSDAG) (SEQ ID NO: 134), or linker 28 (LSGRSDAGSPLGLAG) (SEQ ID NO: 135), or an amino acid sequence having one, two, or three amino acid substitutions, additions, or deletions relative to the amino acid sequence of linker 25, linker 26, linker 27, or linker 28.

[0027] 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 a fragment thereof, a circulating immunoglobulin or a fragment thereof, or CD35 / CR1. In some embodiments, the serum protein comprises thyroxine-binding protein, transthyroxine, 1-acid glycoprotein, transferrin, transferrin receptor or its transferrin-binding moiety, fibrinogen, or albumin. In some embodiments, the circulating immunoglobulin molecule comprises IgG1, IgG2, IgG3, IgG4, sIgA, IgM, or IgD. In some embodiments, the serum protein is albumin. In some embodiments, the polypeptide is an antibody. In some embodiments, the antibody comprises a single-domain antibody, a single-chain variable fragment, or Fab. In some embodiments, the single-domain antibody comprises a single-domain antibody that binds to albumin. In some embodiments, the single-domain antibody is a human or humanized antibody. In some embodiments, the single-domain antibody is 645gH1gL1. In some embodiments, the single-domain antibody is 645dsgH5gL4. In some embodiments, the single-domain antibody is 23-13-A01-sc02. In some embodiments, the single-domain antibody is A10m3 or a fragment thereof. In some embodiments, the single-domain antibody is DOM7r-31. In some embodiments, the single-domain antibody is DOM7h-11-15. In some embodiments, the single-domain antibody is Alb-1, Alb-8, or Alb-23. In some embodiments, the single-domain antibody is 10E. In some embodiments, the single-domain antibody includes complementarity-determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein HC-CDR1, HC-CDR2, and HC-CDR3 of the single-domain antibody comprise: HC-CDR1: SEQ ID NO: 138, HC-CDR2: SEQ ID NO: 139, and HC-CDR3: SEQ ID NO: 140. In some embodiments, the single-domain antibody includes complementarity-determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein HC-CDR1, HC-CDR2, and HC-CDR3 of the single-domain antibody comprise: HC-CDR1: SEQ ID NO: 142, HC-CDR2: SEQ ID NO: 143, and HC-CDR3: SEQ ID NO: 144. In some embodiments, the single-domain antibody comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 141.In some embodiments, the single-domain antibody comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 146. In some embodiments, the single-domain antibody comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 145. In some embodiments, the single-domain antibody comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 4448. In some embodiments, the single-domain antibody is SA21. In some embodiments, the polypeptide or polypeptide complex comprises modified amino acids, non-natural amino acids, modified non-natural amino acids, or combinations thereof. In some embodiments, the modified amino acids or modified non-natural amino acids comprise post-translational modifications.

[0028] In some embodiments, H1 includes a linker portion (L3) connecting H1 to P1. In some embodiments, L3 is a peptide sequence having at least 5 to no more than 50 amino acids. In some embodiments, L3 is a peptide sequence having at least 10 to no more than 30 amino acids. In some embodiments, L3 is a peptide sequence having at least 10 amino acids. In some embodiments, L3 is a peptide sequence having at least 18 amino acids. In some embodiments, L3 is a peptide sequence having at least 26 amino acids. In some embodiments, L3 has a component selected from (G2S). n (GS) n (GSGGS) n (SEQ ID NO: 606), (GGGS) n (SEQ ID NO: 607), (GGGGS) n (SEQ ID NO: 608) and (GSSGGS) n The formula (SEQ ID NO: 609) is used, where n is an integer of at least 1. In some embodiments, L3 comprises an amino acid sequence according to GGGGSGGGSGG (SEQ ID NO: 610).

[0029] In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 147 and 148. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 149 and 150. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 151 and 152. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 153 and 154. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 155 and 156. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 157 and 158. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 159 and 160. In some embodiments, the polypeptide or polypeptide complex comprises the amino acid sequences of SEQ ID NO: 159 and SEQ ID NO: 160. In some embodiments, the polypeptide or polypeptide complex comprises amino acid sequences having at least 95% sequence identity with SEQ ID NO: 161 and 162. In some embodiments, the polypeptide or polypeptide complex comprises amino acid sequences having at least 95% sequence identity with SEQ ID NO: 163 and 164. In some embodiments, the polypeptide or polypeptide complex comprises amino acid sequences having at least 95% sequence identity with SEQ ID NO: 165 and 166. In some embodiments, the polypeptide or polypeptide complex comprises amino acid sequences having at least 95% sequence identity with SEQ ID NO: 167 and 168. In some embodiments, the polypeptide or polypeptide complex comprises amino acid sequences having at least 95% sequence identity with SEQ ID NO: 169 and 170. In some embodiments, the polypeptide or polypeptide complex comprises amino acid sequences having at least 95% sequence identity with SEQ ID NO: 171 and 172. In some embodiments, the polypeptide or polypeptide complex comprises the amino acid sequences of SEQ ID NO: 171 and SEQ ID NO: 172. In some embodiments, the polypeptide or polypeptide complex comprises amino acid sequences having at least 95% sequence identity with SEQ ID NO: 173 and 174. In some embodiments, the polypeptide or polypeptide complex comprises amino acid sequences having at least 95% sequence identity with SEQ ID NO: 175 and 176.In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 177 and 178.

[0030] In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4330 and 4331. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4332 and 4333. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4334 and 4335. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4336 and 4337.

[0031] In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4338 and 4339. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4340 and 4341. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4342 and 4343. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4344 and 4345. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4346 and 4347. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4348 and 4349. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4350 and 4351. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4352 and 4353. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4352 and 4353. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4354 and 4355. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4356 and 4357. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4358 and 4359.In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4360 and 4361. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4362 and 4363. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4364 and 4365. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4366 and 4367. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4368 and 4369. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4370 and 4371. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4372 and 4373. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4374 and 4375. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4376 and 4377. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4378 and 4379. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4380 and 4381. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4382 and 4383.In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4384 and 4385. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4386 and 4387.

[0032] In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4386 and 4387. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4388 and 4389. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4390 and 4391. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4392 and 4393. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4394 and 4395. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4396 and 4397. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4398 and 4399. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4400 and 4401. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4402 and 4403. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4404 and 4405. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4406 and 4407.In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4408 and 4409. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4410 and 4411. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4412 and 4413. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4414 and 4415. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4416 and 4417. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4418 and 4419. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4420 and 4421. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4422 and 4423. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4424 and 4425. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4426 and 4427. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4428 and 4429. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4430 and 4431.In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4432 and 4433. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4434 and 4435. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4436 and 4437. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4438 and 4439. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4440 and 4441. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4442 and 4443. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4444 and 4445. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4446 and 4447.

[0033] In some embodiments, this document discloses polypeptides or polypeptide complexes isolated according to Formula II: L 1a -P 1a -H 1a , where: L 1a Contains a linker that is cleaved by tumor-specific proteases, which, when not cleaved, will cause P... 1a Linked to anti-CD28 antibody, and anti-CD28 antibody linked to anti-PSMA antibody; P 1a It contains peptides that weaken the binding of anti-CD28 antibodies to CD28, and H 1a It contains molecules that extend the half-life. In some embodiments, when L... 1a When not cut, P 1a This weakens the binding of anti-CD28 antibodies to CD28. In some implementations, anti-CD28 antibodies comprise human or humanized antibodies.

[0034] In some implementations, H 1a Contains a polymer. In some embodiments, the polymer is polyethylene glycol (PEG). In some embodiments, H 1a Contains albumin. In some implementations, H 1a It contains an Fc domain. In some embodiments, albumin is serum albumin. In some embodiments, albumin is human serum albumin. In some embodiments, H... 1aIt comprises a peptide, ligand, or small molecule. In some embodiments, the peptide, ligand, or small molecule binds to a serum protein or a fragment thereof, a circulating immunoglobulin or a fragment thereof, or CD35 / CR1. In some embodiments, the serum protein comprises thyroxine-binding protein, transthyroxine, 1-acid glycoprotein, transferrin, transferrin receptor or its transferrin-binding moiety, fibrinogen, or albumin. In some embodiments, the circulating immunoglobulin molecule comprises IgG1, IgG2, IgG3, IgG4, sIgA, IgM, or IgD. In some embodiments, the serum protein is albumin. In some embodiments, the peptide is an antibody. In some embodiments, the antibody comprises a single-domain antibody, a single-chain variable fragment, or Fab. In some embodiments, the antibody comprises a single-domain antibody that binds to albumin. In some embodiments, the antibody is a human or humanized antibody. In some embodiments, the single-domain antibody is 645gH1gL1. In some embodiments, the single-domain antibody is 645dsgH5gL4. In some embodiments, the single-domain antibody is 23-13-A01-sc02. In some embodiments, the single-domain antibody is A10m3 or a fragment thereof. In some embodiments, the single-domain antibody is DOM7r-31. In some embodiments, the single-domain antibody is DOM7h-11-15. In some embodiments, the single-domain antibody is Alb-1, Alb-8, or Alb-23. In some embodiments, the single-domain antibody is 10E. In some embodiments, the single-domain antibody comprises complementarity-determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein HC-CDR1, HC-CDR2, and HC-CDR3 of the single-domain antibody comprise: HC-CDR1: SEQ ID NO: 138, HC-CDR2: SEQ ID NO: 139, and HC-CDR3: SEQ ID NO: 140. In some embodiments, the single-domain antibody comprises complementarity-determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein HC-CDR1, HC-CDR2, and HC-CDR3 of the single-domain antibody comprise: HC-CDR1: SEQ ID NO: 142, HC-CDR2: SEQ ID NO: 143, and HC-CDR3: SEQ ID NO: 144. In some embodiments, the single-domain antibody comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 141. In some embodiments, the single-domain antibody comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 146.In some embodiments, the single-domain antibody comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 145. In some embodiments, the single-domain antibody comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 4448. In some embodiments, the single-domain antibody is SA21.

[0035] In some implementations, H 1a Includes H 1a Connect to P 1a Connection part (L) 2a In some implementations, L 2a It is a peptide sequence having at least 5 and no more than 50 amino acids. In some embodiments, L 2a It is a peptide sequence having at least 10 and no more than 30 amino acids. In some embodiments, L 2a It is a peptide sequence having at least 10 amino acids. In some embodiments, L 2a It is a peptide sequence having at least 18 amino acids. In some embodiments, L 2a It is a peptide sequence having at least 26 amino acids. In some embodiments, L 2a It has the characteristics selected from (G2S) n (GS) n (GSGGS) n (SEQ ID NO: 606), (GGGS) n (SEQ ID NO: 607), (GGGGS) n (SEQ ID NO: 608) and (GSSGGS) n The formula (SEQ IDNO: 609), where n is an integer of at least 1. In some implementations, L 2a It contains the amino acid sequence according to SEQ ID NO: 110.

[0036] In some implementation schemes, P 1a It has less than 70% sequence homology with CD28. In some implementations, P 1a It contains a peptide sequence of at least 10 amino acids in length. In some embodiments, P 1a It comprises a peptide sequence with a length of at least 10 amino acids and a length of no more than 20 amino acids. In some embodiments, P 1a It contains a peptide sequence of at least 16 amino acids in length. In some embodiments, P 1a It contains a peptide sequence of no more than 40 amino acids in length. In some embodiments, P1a It contains at least two cysteine ​​amino acid residues. In some embodiments, P 1a It contains cyclic or linear peptides. In some embodiments, P 1a It contains a cyclic peptide. In some embodiments, P 1a It contains a linear peptide. In some embodiments, P 1a Contains an amino acid sequence according to any one of SEQ ID NO: 33 to 106 or 732-813. In some embodiments, P 1a Contains the amino acid sequence of SEQ ID NO: 73. In some embodiments, P 1a Contains the amino acid sequence of SEQ ID NO: 784. In some embodiments, P 1a A peptide sequence comprising the amino acid sequence of SEQ ID NO: 73 or having one, two, or three amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 73. In some embodiments, P 1a Contains the amino acid sequence of SEQ ID NO: 73. In some embodiments, P 1a A peptide sequence comprising the amino acid sequence of SEQ ID NO: 103 or having one, two, or three amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 103. In some embodiments, P 1a Contains the amino acid sequence of SEQ ID NO: 103. In some embodiments, P 1a Includes X1-X2-X3-C-X4-X5-X6-X7-X8-X9-X 10 -CX 11 -X 12 The amino acid sequence is defined as follows: X1 is selected from M, I, L, and V; X2 is selected from D, H, N, A, F, S, T, Y, and V; X3 is selected from W, L, and F; X4 is selected from P, A, and L; X5 is selected from R, T, I, M, S, K, L, V, W, F, A, P, and D; X6 is selected from E, D, Y, H, S, F, A, N, T, I, P, and V; X7 is selected from L, M, R, S, Q, and H; X8 is selected from W and Q; X9 is selected from 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 Selected from F, Y, L, W, and V; and X 12The X1 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 Selected from F, Y, and L; and X 12 The X1 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 It is F; and X 12 Selected from N, A, and F. In some implementations, P 1a It comprises the amino acid sequence according to SEQ ID NO: 41 or an amino acid sequence having one, two, or three amino acid mutations, substitutions, or deletions relative to SEQ ID NO: 41. In some embodiments, P 1a It contains the amino acid sequence according to SEQ ID NO: 41. In some embodiments, P 1a It contains an amino acid sequence selected from any one of SEQ ID NO: 41, 73, 103 and 179-603.

[0037] In some implementation schemes, P 1a It contains the following amino acid sequence: J1-J2-WC-J3-J4-J5-J6-J7-J8-J9-CJ 10 -J 11 J1 is selected from A, R, N, D, Q, E, G, H, I, L, K, F, P, S, T, W, Y, and V; J2 is selected from D, P, and Y; J3 is selected from P and Q; J4 is selected from R, L, and I; J5 is selected from D, H, Y, and I; J6 is selected from L, S, and R; J7 is selected from W and G; J8 is selected from V, A, D, H, L, and N; J9 is selected from H, L, E, and D; J 10 Selected from F, Y, and L; and J 11 Selected from A, N, H, and T. In some embodiments, J1 is selected from A, R, N, D, Q, E, G, H, K, F, P, S, T, W, and Y. In some embodiments, J1 is selected from R, Q, E, H, I, L, K, F, T, W, Y, and V; J2 is D; J3 is P; J4 is R; J5 is D; J6 is L; J7 is W; J8 is V; J9 is H; J10 It is F; and J 11 It is A. In some implementations, J1 is selected from R, Q, E, L, K, T, and W; J2 is D; J3 is P; J4 is R; J5 is D; J6 is L; J7 is W; J8 is V; J9 is H; J 10 It is F; and J 11 It is A. In some implementations, J1 is L or I; J2 is D or Y; J3 is P; J4 is R, L, or I; J5 is H, Y, or D; J6 is L or R; J7 is W; J8 is A, D, H, or N; J9 is L, E, or D; J 10 It is F or Y; and J 11 It is N or H. In some implementations, J1 is L or I; J2 is D or Y; J3 is P; J4 is R or I; J5 is H, Y, or D; J6 is L or R; J7 is W; J8 is A, D, or N; J9 is L, E, or D; J 10 It is F or Y; and J 11 It is N.

[0038] In some implementation schemes, P 1a It contains the following amino acid sequence: Z1-C-Z2-Z3-Z4-Z5-Z6-Z7-Z8-Z9-Z 10 -Z 11 -CZ 12 Z1 is selected from Y, H, and A; Z2 is selected from D, E, A, Q, S, V, M, H, L, F, T, W, and N; Z3 is selected from G, E, S, and A; Z4 is selected from H, L, W, and A; Z5 is selected from L, S, A, T, I, V, M, R, E, D, F, H, K, Q, N, Y, W, and G; Z6 is selected from P, A, E, Q, S, L, W, G, V, D, K, I, M, R, T, F, H, N, and Y; Z7 is selected from E, Q, I, M, V, A, L, R, W, P, D, T, K, S, G, H, and N; Z8 is selected from Y, H, F, W, N, Q, and A; Z9 is selected from M, I, L, R, V, K, F, Q, Y, W, and A; Z 10 Selected from A, Q, S, W, E, L, G, D, T, M, N, V, H, R, Y, K, P, F, and I; Z 11 Selected from K, Q, N, H, E, P, and A; and Z 12The Z1 is selected from L, M, I, F, V, Y, Q, D, T, and A. In some embodiments, Z1 is selected from Y and H; Z2 is selected from D, E, A, Q, S, V, M, H, L, F, T, W, and N; Z3 is selected from G, E, and S; Z4 is selected from H, L, and W; Z5 is selected from L, S, A, T, I, V, M, R, E, D, F, H, K, Q, N, Y, W, and G; Z6 is selected from P, A, E, Q, S, L, W, G, V, D, K, I, M, R, T, F, H, N, and Y; Z7 is selected from E, Q, I, M, V, A, L, R, W, P, D, T, K, S, G, H, and N; Z8 is selected from Y, H, F, W, N, and Q; Z9 is selected from M, I, L, R, V, K, F, Q, Y, and W; Z 10 Selected from A, Q, S, W, E, L, G, D, T, M, N, V, H, R, Y, K, P, F, and I; Z 11 Selected from K, Q, N, H, E, and P; and Z 12 The numbers are selected from L, M, I, F, V, Y, Q, D, and T. In some embodiments, Z1 is selected from Y and H; Z2 is selected from D, E, A, Q, S, V, M, H, L, F, T, W, and N; Z3 is selected from G, E, and S; Z4 is selected from H, L, and W; Z5 is selected from L, S, A, T, I, V, M, R, E, D, F, H, K, Q, N, Y, W, and G; Z6 is selected from P, A, E, Q, S, L, W, G, V, D, K, I, M, R, T, F, H, N, and Y; Z7 is selected from E, Q, I, M, V, A, L, R, W, P, D, T, K, S, G, H, and N; Z8 is selected from Y, H, F, W, N, and Q; Z9 is selected from M, I, L, R, V, K, F, Q, Y, and W; Z 10 Selected from A, Q, S, W, E, L, G, D, T, M, N, V, H, R, Y, K, P, F, and I; Z 11 Selected from K, Q, N, H, E, and P; and Z 12 The Z1 is selected from L, M, I, F, V, Y, Q, D, and T. In some embodiments, Z1 is Y; Z2 is selected from D, E, A, and Q; Z3 is G; Z4 is H; Z5 is selected from L, S, A, T, I, V, and M; Z6 is selected from P, A, E, Q, S, L, W, G, V, and D; Z7 is selected from E, Q, I, M, V, A, and L; Z8 is selected from Y and H; Z9 is selected from M, I, and L; Z 10 Selected from A, Q, S, W, E, L, G, D, T, M, and N; Z 11 It is K; and Z 12 Z1 is selected from L and M. In some embodiments, Z1 is Y; Z2 is selected from D, E, and A; Z3 is G; Z4 is H; Z5 is selected from L, S, A, and T; Z6 is selected from P, A, E, Q, S, L, and W; Z7 is selected from E, Q, I, and M; Z8 is Y; Z9 is selected from M, I, and L; Z10 Selected from A, Q, S, W, E, L, G, and D; Z 11 It is K; and Z 12 It is L. In some implementations, Z1 is Y; Z2 is selected from D and E; Z3 is G; Z4 is H; Z5 is selected from L, S and A; Z6 is selected from P, A, E, Q, S and L; Z7 is selected from E, Q and I; Z8 is Y; Z9 is selected from M, I and L; Z 10 Selected from A, Q, S, W, E, L, G, and D; Z 11 It is K; and Z 12 It is L.

[0039] In some implementation schemes, P 1a Contains the amino acid sequence of SEQ ID NO: 784. In some embodiments, P 1a It consists of the amino acid sequence of SEQ ID NO: 784. In some embodiments, P 1a Contains the amino acid sequence of SEQ ID NO: 744. In some embodiments, P 1a It consists of the amino acid sequence of SEQ ID NO: 744. In some embodiments, P 1a Contains an amino acid sequence according to any one of SEQ ID NO: 774-793. In some embodiments, P 1a It contains an amino acid sequence according to any one of SEQ ID NO: 764-773.

[0040] In some implementation schemes, P 1a It contains amino acids according to the following sequence: U1-C-U2-U3-U4-U5-U6-U7-U8-U9-U 10 -U 11 -CU 12 Among them: U1 is selected from T, S, L, M, W, Q, P, R, G, N, I, Y, D, E, V, H and A; U2 is selected from A, S, T, K, E, R, G, N, W and D; U3 is selected from A, E, P, D, G, W, Q, V and F; U4 is selected from G, V, F, D, E, R and A; U5 is selected from A, F, V, S, L, P and M; U6 is selected from T, S, H, M, A, N, P, G, Q, D, Y, V, L, I, E, K, F and R; U7 is selected from Y, W, F, L, N, T, Q, I and A; U8 is selected from P, S, T, A, E, K, Q, N, V, G, H, L, D, Y, I, M and R; U9 is selected from W, L and A; U 10 Selected from P, S, R, L, E, and A; U 11 Selected from L, A, T, M, V, W, Y, E, Q, I, F, S, P, K, R, D, H, N, and G; and U12 The elements are selected from L, F, V, M, I, Y, E, W, and A. In some embodiments, U1 is selected from T, S, L, M, W, Q, P, R, G, N, I, Y, D, E, V, and H; U2 is selected from A, S, T, K, E, R, G, N, W, and D; U3 is selected from A, E, P, D, G, W, Q, V, and F; U4 is selected from G, V, F, D, E, and R; U5 is selected from A, F, V, S, L, P, and M; U6 is selected from T, S, H, M, A, N, P, G, Q, D, Y, V, L, I, E, K, F, and R; U7 is selected from Y, W, F, L, N, T, Q, and I; U8 is selected from P, S, T, A, E, K, Q, N, V, G, H, L, D, Y, I, M, and R; U9 is selected from W, L, and A; U 10 Selected from P, S, R, L, and E; U 11 Selected from L, A, T, M, V, W, Y, E, Q, I, F, S, P, K, R, D, H, N, and G; and U 12 The elements are selected from L, F, V, M, I, Y, E, and W. In some embodiments, U1 is selected from T, S, L, M, and W; U2 is selected from A, S, and T; U3 is selected from A and E; U4 is selected from G, V, and F; U5 is selected from A, F, and V; U6 is selected from T, S, H, M, A, and N; U7 is selected from Y, W, and F; U8 is selected from P, S, T, A, E, K, Q, N, and V; U9 is selected from W and L; U 10 Selected from P and S; U 11 Selected from L, A, T, M, V, W, Y, E, Q, I, F, and S; and U 12 The elements are selected from L, F, V, and M. In some embodiments, U1 is selected from T and S; U2 is selected from A and S; U3 is A; U4 is selected from G and V; U5 is selected from A and F; U6 is selected from T, S, and H; U7 is selected from Y and W; U8 is selected from P, S, T, and A; U9 is W; U 10 It is P; U 11 Selected from L, A, T, M, V, W, and Y; and U 12 Selected from L and F.

[0041] In some implementation schemes, P 1a Contains the amino acid sequence of SEQ ID NO: 749. In some embodiments, P 1a Contains the amino acid sequence of SEQ ID NO: 749. In some embodiments, P 1a Contains an amino acid sequence according to any one of SEQ ID NO: 756-763. In some embodiments, P 1a Contains an amino acid sequence according to any one of SEQ ID NO: 794-813. In some embodiments, P 1aIt contains an amino acid sequence according to any one of SEQ ID NO: 732-2002.

[0042] In some embodiments, this document discloses isolated recombinant nucleic acid molecules encoding any of the embodiments disclosed herein, such as polypeptides or polypeptide complexes.

[0043] In some embodiments, pharmaceutical compositions are disclosed herein comprising: (a) a polypeptide or polypeptide complex of any of the embodiments disclosed herein; and (b) a pharmaceutically acceptable excipient.

[0044] In some embodiments, this document discloses methods for treating cancer in a subject of need, comprising administering to the subject a polypeptide or polypeptide complex of any of the embodiments disclosed herein. In some embodiments, the cancer has cells expressing PSMA. In some cases, the cancer is a solid tumor. In some embodiments, the cancer is lung cancer, breast cancer (e.g., HER2-positive). + ;ER / PR + (TNBC), cervical cancer, ovarian cancer, colorectal cancer, pancreatic cancer, or stomach cancer.

[0045] In some embodiments, this document discloses methods for treating prostate cancer in subjects of need, comprising administering to the subject the isolated polypeptide or polypeptide complex described herein. In some embodiments, this document discloses methods for treating metastatic castration-resistant prostate cancer (mCRPC) in subjects of need, comprising administering to the subject the isolated polypeptide or polypeptide complex described herein.

[0046] In some embodiments, this document discloses a method of treating cancer in a subject of need, comprising administering to the subject a polypeptide or polypeptide complex comprising a first antigen recognition molecule that binds to CD28 and a second antigen recognition molecule that binds to a prostate-specific membrane antigen. In some embodiments, the first antigen recognition molecule that binds to CD28 comprises an anti-CD28 antibody. In some embodiments, the anti-CD28 antibody comprises an anti-CD28 heavy chain variable domain comprising complementarity-determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein HC-CDR1, HC-CDR2, and HC-CDR3 comprise the following amino acid sequences: HC-CDR1: SEQ ID NO: 1, HC-CDR2: SEQ ID NO: 2, and HC-CDR3: SEQ ID NO: 3; and the anti-CD28 antibody comprises an anti-CD28 light chain variable domain comprising complementarity-determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein LC-CDR1, LC-CDR2, and LC-CDR3 comprise the following amino acid sequences: LC-CDR1: SEQ ID NO: 4, LC-CDR2: (KAS), and LC-CDR3: SEQ ID NO: 6. In some embodiments, the anti-CD28 heavy chain variable domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 7, and wherein the anti-CD28 light chain variable domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 8. In some embodiments, the anti-CD28 antibody comprises a single-chain variable fragment (scFv), a single-domain antibody, Fab, or Fab'. In some embodiments, the anti-CD28 antibody comprises scFv. In some embodiments, the anti-CD28 antibody comprises scFv, and the scFv comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 9. In some embodiments, the anti-CD28 antibody comprises an scFv, and the scFv comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 10. In some embodiments, the anti-CD28 antibody comprises an scFv, and the scFv comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 11.In some embodiments, the anti-CD28 antibody comprises scFv, and the scFv comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 12.

[0047] In some implementations, the second antigen recognition molecule that binds to PSMA contains an anti-PSMA antibody. In some embodiments, the anti-PSMA antibody comprises an anti-PSMA heavy chain variable domain comprising complementarity-determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein HC-CDR1, HC-CDR2, and HC-CDR3 comprise the following amino acid sequences: HC-CDR1: SEQ ID NO: 13, HC-CDR2: SEQ ID NO: 14, and HC-CDR3: SEQ ID NO: 15; and the anti-PSMA antibody comprises an anti-PSMA light chain variable domain comprising complementarity-determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, LC-CDR2, and LC-CDR3 of the light chain variable domain comprise the following amino acid sequences: LC-CDR1: SEQ ID NO: 16, LC-CDR2: (EA), and LC-CDR3: SEQ ID NO: 18. In some embodiments, the anti-PSMA heavy chain variable domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 20, and the anti-PSMA light chain variable domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 19. In some embodiments, the anti-PSMA antibody comprises a single-chain variable fragment (scFv), a single-domain antibody, Fab, or Fab'. In some embodiments, the anti-PSMA antibody comprises Fab or Fab'. In some embodiments, the anti-PSMA antibody comprises Fab or Fab', and Fab or Fab' comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 19 and an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 20.

[0048] In some embodiments, the peptide or peptide complex induces T cell-mediated cytotoxicity in tumor cells. In some embodiments, the cancer has cells expressing PSMA. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is lung cancer, breast cancer, cervical cancer, ovarian cancer, colorectal cancer, pancreatic cancer, or gastric cancer.

[0049] In some embodiments, the peptide or peptide complex is administered in combination with an anticancer therapy. In some embodiments, the peptide or peptide complex is administered with the anticancer therapy in the same pharmaceutical composition. In some embodiments, the peptide or peptide complex and the anticancer therapy are administered as a separate pharmaceutical composition. In some embodiments, the anticancer therapy comprises a small molecule, a cell-based therapy, or an antibody-based therapy.

[0050] In some embodiments, the antibody-based therapy is a T-cell connector. In some embodiments, the T-cell connector comprises according to the formula: D-L0-E (Formula II), wherein D comprises an effector cell binding domain that binds to an effector cell antigen, E comprises a tumor antigen binding domain that binds to a tumor antigen, and L0 comprises a connector linking D to E. In some embodiments, D comprises a single-chain variable fragment, a single-domain antibody, or a Fab fragment. In some embodiments, D comprises a single-chain variable fragment. In some embodiments, E comprises a single-chain variable fragment, a single-domain antibody, or a Fab fragment. In some embodiments, E comprises a Fab fragment.

[0051] In some implementations, the effector cell binding domain includes a complementarity-determining region (CDR) selected from: moromumab-CD3 (OKT3), ostalizumab (TRX4), telizumab (MGA031), vexizumab (Nuvion), SP34, X35, VIT3, and BMA030. (BW264 / 56), CLB-T3 / 3, CRIS7, YTH12.5, F111-409, 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. In some embodiments, the effector cell binding domain comprises the amino acid sequence according to SEQ ID NO: 611-624.

[0052] In some embodiments, the tumor antigen comprises epidermal growth factor receptor (EGFR), prostate-specific membrane antigen (PSMA), or tumor-associated calcium signaling transducer 2 (hereinafter referred to as TROP2). In some embodiments, the tumor antigen comprises EGFR. In some embodiments, the tumor antigen-binding domain comprises the amino acid sequence according to SEQ ID NO: 629-638. In some embodiments, the tumor antigen comprises EGFR, and the tumor-binding domain comprises 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: 632; HC-CDR2: SEQ ID NO: 633; ​​HC-CDR3: SEQ ID NO: 634; and wherein LC-CDR1, LC-CDR2, and LC-CDR3 comprise: LC-CDR1: SEQ ID NO: 629; LC-CDR2: (YAS); and LC-CDR3: SEQ ID NO: 631. In some embodiments, the tumor antigen comprises EGFR, and the T-cell adaptor comprises an amino acid sequence having at least 95% sequence identity with respect to SEQ ID NOs: 639 and 640. In some implementations, the tumor antigen comprises EGFR, and the T-cell connector comprises the amino acid sequence according to SEQ ID NO: 641 and 642.

[0053] In some embodiments, the tumor antigen comprises TROP2. In some embodiments, the tumor antigen comprises TROP2, and the tumor-binding domain comprises 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: 659; HC-CDR2: SEQ ID NO: 660; HC-CDR3: SEQ ID NO: 661, 662, or 663; and wherein LC-CDR1, LC-CDR2, and LC-CDR3 comprise: LC-CDR1: SEQ ID NO: 664; LC-CDR2: (SAS); and LC-CDR3: SEQ ID NO: 666. In some embodiments, the tumor antigen comprises TROP2, and the T-cell adaptor comprises an amino acid sequence having at least 95% sequence identity with respect to SEQ ID NO: 673 and 674. In some embodiments, the tumor antigen comprises TROP2, and the T-cell adaptor comprises an amino acid sequence according to SEQ ID NO: 675 and 676, or SEQ ID NO: 677 and 678. In some embodiments, the tumor antigen-binding domain comprises an amino acid sequence according to SEQ ID NO: 659-668.

[0054] 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 NO: 647-654. In some embodiments, the tumor antigen comprises PSMA, and the tumor-binding domain comprises 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: 647; HC-CDR2: SEQ ID NO: 648; HC-CDR3: SEQ ID NO: 649; and wherein LC-CDR1, LC-CDR2, and LC-CDR3 comprise: LC-CDR1: SEQ ID NO: 650; LC-CDR2: SEQ ID NO: 651; and LC-CDR3: SEQ ID NO: 652. In some embodiments, the tumor antigen comprises PSMA, and the T-cell adaptor comprises an amino acid sequence having at least 95% sequence identity with respect to SEQ ID NO: 655 and 656.

[0055] In some embodiments, the T-cell adaptor molecule is selectively activated in the tumor microenvironment. In some embodiments, the T-cell adaptor is according to the formula: P3-L3-D-L0-E (Formula IIa), wherein D contains a CD3-binding domain; E contains a tumor antigen-binding domain; L0 contains a linker connecting D to E; P3 contains a peptide binding to D, and L3 contains a linker portion connecting D to P3 and is a substrate of a tumor-specific protease. In some embodiments, the T-cell adaptor is according to the formula: D-L0-E-L4-P4 (Formula IIb), wherein D contains a CD3-binding domain; E contains a tumor antigen-binding domain; L0 contains a linker connecting D to E; P4 contains a peptide binding to E, and L4 contains a linker portion connecting E to P4 and is a substrate of a tumor-specific protease. In some embodiments, the T-cell adaptor is based on the following formula: P3-L3-D-L0-E-L4-P4 (Formula IIc), wherein D contains a CD3-binding domain; E contains a tumor antigen-binding domain; L0 contains a linker connecting D to E; P3 contains a peptide that binds to D, and L3 contains a linker portion connecting D to P3 and is a substrate of a tumor-specific protease; P4 contains a peptide that binds to E, and L4 contains a linker portion connecting E to P4 and is a substrate of a tumor-specific protease.

[0056] In some embodiments, the T-cell adaptor comprises H1. In some embodiments, H1 comprises a sequence according to SEQ ID NO: 141 or SEQ ID NO: 146. In some embodiments, H1 comprises a single-domain antibody. In some embodiments, the single-domain antibody comprises complementarity-determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein HC-CDR1, HC-CDR2, and HC-CDR3 of the single-domain antibody comprise: HC-CDR1: SEQ ID NO: 138, HC-CDR2: SEQ ID NO: 139, and HC-CDR3: SEQ ID NO: 140.

[0057] In some embodiments, L3 or L4 is a peptide sequence having at least 5 to no more than 50 amino acids. In some embodiments, L3 or L4 is a peptide sequence having at least 10 to 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 contains (G2S). nThe formula is given, where n is an integer from 1 to 3 (SEQ ID NO: 605). In some embodiments, L3 or L4 has a subset (G2S). n The expression, where n is an integer of at least 1. In some implementations, L3 or L4 has a value selected from (G2S). n (GS) n (GSGGS) n (SEQ ID NO: 606), (GGGS) n (SEQ ID NO: 607), (GGGGS) n (SEQ ID NO:608) and (GSSGGS) n The expression (SEQ ID NO: 609) where n is an integer of at least 1.

[0058] In some embodiments, the tumor-specific protease is selected from metalloproteinases, serine proteases, cysteine ​​proteases, threonine proteases, and aspartic proteases. In some embodiments, L3 or L4 comprises a urokinase-cleavable amino acid sequence, a matrix protease-cleavable amino acid sequence, or a matrix metalloproteinase-cleavable amino acid sequence. In some embodiments, L3 or L4 comprises a sequence according to any one of SEQ ID NO: 21-22 and 107-136. In some embodiments, L3 binds to the N-terminus of D. In some embodiments, L3 binds to the C-terminus of D. In some embodiments, L4 binds to the N-terminus of E. In some embodiments, L4 binds to the C-terminus of E.

[0059] In some embodiments, when L3 is cleaved by a tumor-specific protease, P3 unbinds to D, thereby exposing D to CD3. In some embodiments, when L4 is cleaved by a tumor-specific protease, P4 unbinds to E, thereby exposing E to the tumor antigen. In some embodiments, P3 weakens the binding of D to CD3. In some embodiments, P3 binds to D through ionic interactions, electrostatic interactions, hydrophobic interactions, π-stacking interactions, and hydrogen bonding interactions, or combinations thereof. In some embodiments, P3 binds to D at or near the antigen-binding site. In some embodiments, when L3 is cleaved by a tumor-specific protease, P3 unbinds to D, thereby exposing D to CD3.

[0060] In some embodiments, P3 shares less than 70% sequence identity with CD3. In some embodiments, P3 shares less than 70% sequence identity with CD3. In some embodiments, P3 shares less than 85% sequence identity with CD3. In some embodiments, P3 shares less than 90% sequence identity with CD3. In some embodiments, P3 shares less than 95% sequence identity with CD3. In some embodiments, P3 shares less than 98% sequence identity with CD3. In some embodiments, P3 shares less than 99% sequence identity with CD3. In some embodiments, P3 comprises the amino acid sequence according to SEQ ID NO: 625 or SEQ ID NO: 626. In some embodiments, P3 comprises a de novo amino acid sequence sharing less than 10% sequence identity with CD3.

[0061] In some embodiments, P4 weakens the binding of E to the tumor antigen. In some embodiments, P4 binds to E through ionic interactions, electrostatic interactions, hydrophobic interactions, π-stacking interactions, and hydrogen bonding interactions, or combinations thereof. In some embodiments, P4 binds to E at or near the antigen-binding site. In some embodiments, when L4 is cleaved by a tumor-specific protease, P4 unbinds to E, thereby exposing E 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 amino acid sequence with less than 10% sequence identity to the tumor antigen.

[0062] 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.

[0063] In some embodiments, P4 comprises the amino acid sequence according to SEQ ID NO: 645. In some embodiments, the tumor antigen comprises EGFR, and the T-cell connective comprises the amino acid sequences of SEQ ID NO: 643 and 644.

[0064] In some embodiments, P4 comprises an amino acid sequence according to any one of SEQ ID NO: 693-695. In some embodiments, the tumor antigen comprises TROP2, and the T-cell adaptor comprises the amino acid sequences of SEQ ID NO: 679 and 680, SEQ ID NO: 681 and 682, SEQ ID NO: 683 and 684, SEQ ID NO: 685 and 686, SEQ ID NO: 687 and 688, SEQ ID NO: 689 and 690, and SEQ ID NO: 691 and 692. In some embodiments, the tumor antigen comprises TROP2, and the T-cell adaptor comprises the amino acid sequences of SEQ ID NO: 4452 and 4453, SEQ ID NO: 4454 and 4455, SEQ ID NO: 4456 and 4457, SEQ ID NO: 4458 and 4459, SEQ ID NO: 4460 and 4461, SEQ ID NO: 4462 and 4463, or SEQ ID NO: 4464 and 4465.

[0065] In some implementations, the tumor antigen comprises PSMA, and the T-cell connector comprises the amino acid sequences of SEQ ID NO: 657 and 658. Attached Figure Description

[0066] The novel features of the invention are specifically set forth in the appended claims. The features and advantages of the invention will be better understood by referring to the following detailed description of illustrative embodiments (in which the principles of the invention are utilized) and the accompanying drawings, wherein: Figure 1A Cartoon configurations of multispecific antibody constructs targeting PSMA and CD28 are shown, which are then administered in combination with T-cell connectives (TCEs) targeting CD3 and tumor-associated antigen EGFR. (Table showing...) Figure 1B The density (copy number per cell) of PSMA and EGFR on the LNCaP tumor cells used in the assay.

[0067] Figure 1BThis diagram illustrates the tumor cell killing effects on LNCaP tumor cells, measured using in vitro hPBMC co-culture assays, on EGFR TCE-2 alone, in combination with different concentrations of EGFR TCE-2 and Ab-2 (a multispecific antibody targeting PSMA and CD28), and Ab-2 alone. The table shows the EC2 cell killing parameters. 50 .

[0068] Figure 2A Cartoon configurations of multispecific antibody constructs targeting PSMA and CD28 are shown, in combination with TCEs targeting CD3 and tumor-associated antigen PSMA. The table shows... Figure 2B The density of PSMA (copy number per cell) on LNCaP tumor cells used in the assay.

[0069] Figure 2B This table shows the tumor cell killing effects of PSMA TCE-1 alone and different concentrations of PSMA TCE-1 combined with Ab-2 (a multispecific antibody targeting PSMA and CD28) on LNCaP tumor cells, as measured using in vitro hPBMC co-culture with tumor cells. The table also shows the EC50 values ​​for tumor cell killing. 50 .

[0070] Figure 3 The table shows the tumor cell killing effects of EGFR TCE-2, EGFR TCE-2 combined with unmasked Ab-2, EGFR TCE-2 combined with PC-2 containing a peptide mask targeting CD28, and EGFR TCE-2 combined with PC-2 after its peptide mask is cleaved by membrane-bound serine protease 1 (MTSP-1). The table also shows the tumor cell killing effects of ECMO (endothelial cells) on LNCaP tumor cells. 50 .

[0071] Figure 4A Cartoon configurations of multispecific antibody constructs targeting PSMA and CD28 are shown, in combination with TCE targeting CD3 and tumor-associated antigen TROP-2. (Table showing...) Figure 4B-4C The density (copy number per cell) of PSMA and TROP-2 on DU-145 cells expressing recombinant PSMA used in the assay.

[0072] Figure 4B The figure shows the production of IL-2 and IFNγ cytokines as measured by an in vitro T-cell activation assay using PBMCs from the first donor, under no-stimulation (“no stim”), TROP2 TCE-1 stimulation (“TROP2 x CD3”), and combined TROP2 TCE-1 and Ab-2 stimulation (“TROP2 x CD3 + PSMA x CD28”).

[0073] Figure 4C The results show the production of IL-2 and IFNγ cytokines as measured by an in vitro T-cell activation assay using PBMCs from a second donor, under no-stimulation (“no stim”), TROP2 TCE-1 stimulation (“TROP2 x CD3”), and combined TROP2 TCE-1 and Ab-2 stimulation (“TROP2 x CD3 + PSMA x CD28”).

[0074] Figure 5A Cartoon conformations of multispecific antibodies targeting PSMA and CD28 binding to DU-145 cells loaded with PSMA-expressing cytomegalovirus (CMV) peptides and CMV-positive PBMCs are shown in the table. Figures 5B-5C The density (copy number per cell) of PSMA on CMV peptide-loaded DU-145 cells used in the assay.

[0075] Figure 5B The use of PBMCs from the first donor showed that Ab-2 enhanced dose-dependent IL-2 production in the tumor cell / PBMC mixed lymphocyte response (MLR) system.

[0076] Figure 5C The use of PBMCs from a second donor showed a dose-dependent enhancement of Ab-2 production of IL-2 in the tumor cell / PBMC MLR system.

[0077] Figure 6 This diagram illustrates the tumor-specific activity of a multispecific antibody comprising a CD28-binding domain (CD28 BD), a PSMA-binding domain (PSMA BD), a peptide mask (CD28 mask) linked to the CD28-binding domain via a cleavable linker, a peptide mask (PSMA mask) linked to the PSMA-binding domain via a cleavable linker, and a half-life extension (e.g., an albumin-binding domain). In the tumor microenvironment (TME), cleavage of the cleavable linker by tumor proteases releases the peptide mask and the half-life extension, thereby activating CD28 stimulation of the multispecific antibody in tumor tissue and promoting rapid clearance of the multispecific antibody from healthy tissue.

[0078] Figure 7A The results show the tumor cell killing effects of EGFR TCE-1 alone, different concentrations of EGFR TCE-1 combined with Ab-1, and Ab-1 alone on LNCaP tumor cells, as measured by in vitro hPBMC co-culture with tumor cells.

[0079] Figure 7BThe results show the tumor cell killing effects of EGFR TCE-1 alone, different concentrations of EGFR TCE-1 combined with Ab-2, and Ab-2 alone on LNCaP tumor cells, as measured by in vitro hPBMC co-culture with tumor cells.

[0080] Figure 8 The study demonstrated the tumor cell killing effects of EGFR TCE-1 alone and the combination of EGFR TCE-1 and Ab-2 on LNCaP tumor cells, as measured by in vitro co-culture of hPBMCs with tumor cells.

[0081] Figure 9 The study demonstrated the tumor cell killing effects of PSMA TCE-1 alone, PSMA TCE-1 combined with Ab-2, and PSMA TCE-1 combined with Ab-4 on LNCaP tumor cells, as measured using in vitro tumor cell co-culture assays.

[0082] Figures 10A-10C The results show the killing effect of PSMA TCE-1 alone or PSMATCE-1 in the presence of Ab-2 on tumor cells of different cancer cell lines with different PSMA expression, as measured by in vitro tumor cell co-culture assay.

[0083] Figure 11A-11B The results, measured using in vitro tumor cell co-culture assays, showed that EGFR TCE-1 alone or EGFR TCE-1 in the presence of Ab-2 killed tumor cells from different cancer cell lines with different PSMA and EGFR expressions.

[0084] Figure 12 The study demonstrated the tumor cell killing effects of PSMA TCE-1 combined with Ab-4 and PSMA TCE-1 combined with PC-9 on LNCaP cancer cells, as measured using in vitro tumor cell co-culture assays.

[0085] Figure 13 The study demonstrated the tumor cell killing effects of PSMA TCE-1 combined with Ab-4, PSMA TCE-1 combined with PC-66, and PSMA TCE-1 combined with PC-66 treated with MMP9 protease on LNCaP cancer cells, as measured by in vitro tumor cell co-culture assays.

[0086] Figure 14 The results show the tumor cell killing effects of Ab-2 combined with PSMA TCE-1 and PC-7 combined with PSMA TRACTr-1 on LNCaP cancer cells, as measured by an in vitro tumor cell killing assay.

[0087] Figure 15The results show the tumor cell killing effects of PSMA TCE-1 alone and Ab-4 combined with PSMATCE-1 on LNCaP cancer cells, as measured by an in vitro tumor cell killing assay.

[0088] Figure 16 The study demonstrated the tumor cell killing effects of PSMA TCE-1 combined with Ab-4, PSMA TCE-1 combined with PC-25, PSMA TCE-1 combined with PC-33, and PSMA TCE-1 combined with PC-42 on LNCaP cancer cells.

[0089] Figure 17 The study demonstrated the tumor cell killing effects of PSMA TCE-1 combined with Ab-2, PSMA TCE-1 combined with PC-50, PSMA TCE-1 combined with PC-58, and PSMA TCE-1 combined with PC-66 on LNCaP cancer cells.

[0090] Figure 18 The study demonstrated that PSMA TCE-1 combined with Ab-2, PSMA TCE-1 combined with PC-25 treated with MMP9 protease, PSMA TCE-1 combined with PC-33 treated with MMP9 protease, and PSMA TCE-1 combined with PC-42 treated with MMP9 protease killed tumor cells in LNCaP cancer cells.

[0091] Figure 19 The study demonstrated that PSMA TCE-1 combined with Ab-2, PSMA TCE-1 combined with PC-50 treated with MMP9 protease, PSMA TCE-1 combined with PC-58 treated with MMP9 protease, and PSMA TCE-1 combined with PC-66 treated with MMP9 protease killed tumor cells in LNCaP cancer cells.

[0092] Figure 20 This shows the production of interferon-γ (IFNγ), interleukin-2 (IL-2), and tumor necrosis factor (TNF) after treatment with PSMA TCE-1+Ab-2 and PSMA-TRACTr-1+PC-7, as measured by an in vitro T-cell activation assay using PBMCs.

[0093] Figure 21 The PSMA binding of Ab-4, PC-66, and PC-66 treated with MMP9, as measured by ELISA, is shown.

[0094] Figure 22 The image shows the CD28 binding of Ab-4, PC-66, and PC-66 treated with MMP9, as measured by ELISA.

[0095] Figure 23 The binding of Ab-15 to the peptide is shown by ELI.

[0096] Figure 24 The image shows the binding of Ab-15 to the peptide as measured by ELISA.

[0097] Figure 25 The image shows the binding of Ab-15 to the peptide as measured by ELISA.

[0098] Figure 26 The data for biolayer interferometry (BLI) titration of Ab-15 binding to peptide 41 are shown.

[0099] Figure 27 The BLI titration data for Ab-15 binding to peptide 53 are shown.

[0100] Figure 28 The BLI titration data for Ab-15 binding to peptide 56 are shown.

[0101] Figure 29 The BLI titration data for Ab-15 binding to peptide 57 are shown.

[0102] Figure 30 The BLI titration data for Ab-15 binding to peptide 62 are shown.

[0103] Figure 31 The BLI titration data for Ab-15 binding to peptide 66 are shown.

[0104] Figure 32 The BLI titration data for Ab-15 binding to peptide 71 are shown.

[0105] Figure 33 The BLI titration data for Ab-15 binding to peptide 41 are shown.

[0106] Figure 34 The BLI titration data for Ab-15 binding to peptide 288 are shown.

[0107] Figure 35 The BLI titration data for Ab-15 binding to peptide 289 are shown.

[0108] Figure 36 This demonstrates the inhibition of Ab-15 binding to CD28 by the peptides of this disclosure.

[0109] Figure 37 This demonstrates the inhibition of Ab-15 binding to CD28 by the peptides of this disclosure.

[0110] Figure 38 This demonstrates the inhibition of Ab-15 binding to CD28 by the peptides of this disclosure.

[0111] Figure 39 The binding of anti-CD28 Fab to the peptide is shown by ELISA.

[0112] Figure 40 The binding of anti-CD28 Fab to the peptide is shown by ELISA.

[0113] Figure 41 The binding of anti-CD28 Fab to the peptide is shown by ELISA.

[0114] Figure 42 The binding of anti-CD28 Fab to the peptide is shown by ELISA.

[0115] Figure 43 This demonstrates the peptide of this disclosure that counteracts the inhibition of CD28 Fab binding to CD28.

[0116] Figure 44 This demonstrates the peptide of this disclosure that counteracts the inhibition of CD28 Fab binding to CD28.

[0117] Figure 45 This demonstrates the peptide of this disclosure that counteracts the inhibition of CD28 Fab binding to CD28.

[0118] Figure 46 This demonstrates the peptide of this disclosure that counteracts the inhibition of CD28 Fab binding to CD28.

[0119] Figure 47 This demonstrates the peptide of this disclosure that counteracts the inhibition of CD28 scFv disulfide bond binding to CD28.

[0120] Figure 48 The BLI titration data for anti-CD28 Fab binding to peptide 87 are shown.

[0121] Figure 49 The BLI titration data for anti-CD28 Fab binding to peptide 120 are shown.

[0122] Figure 50 The BLI titration data for anti-CD28 Fab binding to peptide 121 are shown.

[0123] Figure 51 The BLI titration data for anti-CD28 Fab binding to peptide 126 are shown.

[0124] Figure 52 The BLI titration data for anti-CD28 Fab binding to peptide 127 are shown.

[0125] Figure 53 The BLI titration data for anti-CD28 Fab binding to peptide 132 are shown.

[0126] Figure 54 The BLI titration data for anti-CD28 Fab binding to peptide 134 are shown.

[0127] Figure 55 The core sequence motif of peptide 87 generated using WebLogo 3.7.12 is shown.

[0128] Figure 56 The core sequence motif of the anti-CD28 Fab peptide 92 sequence generated using WebLogo 3.7.12 is shown.

[0129] Figure 57 This diagram illustrates the identification of peptides that can attach to anti-CD28 multispecific antibodies for selective activation in the tumor microenvironment. The diagram also illustrates directed evolution and phage display techniques used to identify peptides that block antigen recognition by blocking antigen-binding domains.

[0130] Figure 58 The binding of anti-CD28 scFv to the peptide, as measured by ELISA, is shown.

[0131] Figure 59 The binding of anti-CD28 scFv to the peptide, as measured by ELISA, is shown.

[0132] Figures 60-61 The peptide, as measured by ELISA, inhibits the binding of anti-CD28 scFv to the CD28 antigen.

[0133] Figures 62-63 The kinetics of binding between anti-CD28 scFv and peptide, as measured by Octet, are shown.

[0134] Figures 64A-64B The binding of anti-CD28 scFv to the alanine scanning peptide of peptide 9 is shown.

[0135] Figures 65A-65B The alanine-scanning peptide of peptide 9 is shown to counteract the inhibition of CD28 scFv binding to CD28.

[0136] Figure 66 The core sequence motif of the optimized anti-CD28 scFv peptide 9 sequence generated using WebLogo 3.7.4 is shown.

[0137] Figures 67-69 The peptide shown is an inhibitory peptide that inhibits the binding of anti-CD28 scFv to the CD28 antigen, as detected by ELISA.

[0138] Figure 70A-70U The kinetics of binding of anti-CD28 scFv to the peptide, as measured by Octet, are shown.

[0139] Figures 71-72The binding of PSMA Fab to peptides, as measured by ELISA, is shown.

[0140] Figures 73-74 This demonstrates the inhibition of PSMA Fab binding to PSMA by the peptides disclosed herein.

[0141] Figure 75 The BLI titration data for PSMA Fab binding to peptide 179 are shown.

[0142] Figure 76 The BLI titration data for PSMA Fab binding to peptide 219 are shown.

[0143] Figure 77 The BLI titration data for PSMA Fab binding to peptide 225 are shown.

[0144] Figure 78 The BLI titration data for PSMA Fab binding to peptide 227 are shown.

[0145] Figure 79 The BLI titration data for PSMA Fab binding to peptide 230 are shown.

[0146] Figure 80 The BLI titration data for PSMA Fab binding to peptide 232 are shown.

[0147] Figure 81 The BLI titration data for PSMA Fab binding to peptide 233 are shown.

[0148] Figure 82 The BLI titration data for PSMA Fab binding to peptide 235 are shown.

[0149] Figure 83 The BLI titration data for PSMA Fab binding to peptide 236 are shown.

[0150] Figure 84 The BLI titration data for PSMA Fab binding to peptide 239 are shown.

[0151] Figure 85 The BLI titration data for PSMA Fab binding to peptide 180 are shown.

[0152] Figure 86 The BLI titration data for PSMA Fab binding to peptide 263 are shown.

[0153] Figure 87 The BLI titration data for PSMA Fab binding to peptide 267 are shown.

[0154] Figure 88The BLI titration data for PSMA Fab binding to peptide 268 are shown.

[0155] Figure 89 The BLI titration data for PSMA Fab binding to peptide 270 are shown.

[0156] Figure 90 The BLI titration data for PSMA Fab binding to peptide 271 are shown.

[0157] Figure 91 The BLI titration data for PSMA Fab binding to peptide 274 are shown.

[0158] Figure 92 The BLI titration data for PSMA Fab binding to peptide 277 are shown.

[0159] Figure 93 The BLI titration data for PSMA Fab binding to peptide 282 are shown.

[0160] Figure 94 The BLI titration data for PSMA Fab binding to peptide 283 are shown.

[0161] Figure 95 The BLI titration data for PSMA Fab binding to peptide 284 are shown.

[0162] Figure 96 The BLI titration data for PSMA Fab binding to peptide 285 are shown.

[0163] Figure 97 The BLI titration data for PSMA Fab binding to peptide 286 are shown.

[0164] Figure 98 The core sequence motif of the anti-PSMA Fab peptide 180 sequence generated using Weblogo 3.7.12 is shown.

[0165] Figure 99 The titration data for PSMA binding by the peptide complex of this disclosure are shown.

[0166] Figure 100 The titration data for PSMA binding by the peptide complex of the present disclosure after treatment with matrix metalloproteinase 9 are shown.

[0167] Figure 101 The titration data for CD28 binding by the peptide complex of this disclosure are shown.

[0168] Figure 102 The titration data for CD28 binding of the peptide complex of this disclosure after treatment with matrix metalloproteinase 9 are shown. Detailed Implementation

[0169] Multispecific antibodies combine the advantages of different binding specificities derived from two or more antibodies into a single composition. Multispecific antibodies, such as T-cell adaptors (TCEs) for redirecting T cells to cancer, have shown promise in preclinical and clinical studies. This approach relies on one antigen-interacting portion of the antibody binding to a tumor-associated antigen or biomarker, while a second antigen-interacting portion binds to effector cell antigens on T cells (such as differentiation cluster 3 (CD3)), which subsequently triggers cytotoxic activity. Although TCEs have demonstrated potent antitumor activity in certain cancers, TCE therapeutics may face challenges such as off-target toxicity in healthy tissues and slow clearance.

[0170] One such tumor-associated antigen is PSMA. Prostate-specific membrane antigen (PSMA), also known as glutamate carboxypeptidase II (GCPII), N-acetyl-L-aspartate-L-glutamate peptidase I (NAALADase I), or NAAG peptidase, is an enzyme encoded by the FOLH1 (folate hydrolase 1) gene in humans. PSMA is a membrane-residing zinc metalloenzyme. The majority of this enzyme resides in the extracellular space. Human PSMA is highly expressed in the prostate, approximately one hundred times higher than in most other tissues. In some prostate cancers, PSMA is the second most upregulated gene product, increasing to 8 to 12 times the level found in non-cancerous prostate cells.

[0171] T cell activation is a highly regulated process that typically requires two signaling events to function fully: a first signal (signal 1) is initiated upon binding of the major histocompatibility complex (MHC)-antigen complex, which helps the T cell receptor (TCR) distinguish between "self" and "non-self" antigens; and a second co-stimulatory signal (signal 2) occurs through the activation of a co-stimulatory receptor (such as CD28). While the first signal activates T cells and triggers T cell-mediated cytotoxicity against recognized cells, failure to receive the second co-stimulatory signal can lead to T cell tolerance, where T cells continue to recognize tumor antigens but do not initiate an immune response against tumor cells. The second co-stimulatory signal prevents T cell tolerance and further activates T cells to enhance T cell cytotoxicity against target cells. The CD3-targeting T cell adaptor (TCE) generates signal 1 via TCE signaling without the need for TCR / MHC interaction.

[0172] This paper discloses a multispecific antibody comprising a CD28-binding domain and a PSMA-binding domain. The applicant discovered that co-stimulation with the disclosed multispecific antibody enhances TCE cell lysis activity targeting CD3. Peptide masks weakening the binding of the CD28-binding domain to CD28 and weakening the binding of the PSMA-binding domain to PSMA are linked to the multispecific antibody via cleavable linkers to block CD28-mediated co-stimulation. Selective cleavage of the cleavable linker by tumor-specific proteases in the tumor microenvironment restores the CD28 co-stimulatory activity of the multispecific antibody, preventing off-target toxicity in healthy tissues. A half-life extension is linked to the multispecific antibody via a cleavable linker, allowing cleavage of the linker by tumor-specific proteases to release the extended half-life for rapid clearance. The advantage of using a tumor protease cleavable linker is that it provides targeted co-stimulatory activity in the tumor microenvironment while minimizing off-target effects in healthy non-tumor tissues.

[0173] Some definitions The terminology used herein is for the purpose of describing particular situations only and is not intended to be limiting. In this document, the singular forms “a” and “the” are also intended to include the plural forms unless the context clearly indicates otherwise. Furthermore, with regard to the terms “including / includes,” “having / has,” “with,” or variations thereof used in the detailed description and / or claims, these terms are intended to be inclusive in a manner similar to the term “comprising.”

[0174] The term "antibody" is used in the broadest sense, covering fully assembled antibodies; antibody fragments that can bind to antigens, such as Fab, F(ab')2, Fv; single-chain antibodies (scFv); diabody; antibody chimeras; hybrid antibodies; bispecific antibodies, etc.

[0175] The term "complementarity-determining region" or "CDR" is a segment of the 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. Therefore, the CDR is sometimes referred to as the hypervariable region. The variable region contains three CDRs. CDR peptides can be obtained by constructing genes encoding the CDR of the antibody of interest. These genes are prepared, for example, by synthesizing the variable region from the RNA of antibody-producing cells using a polymerase chain reaction. See, for example, Larrick et al. Methods: A Companion to Methods in Enzymology 2: 106 (1991); Monoclonal Antibodies: Production, Engineering and Clinical ApplicationCourtenay-Luck, “Genetic Manipulation of Monoclonal Antibodies,” in Ritter et al. (eds.), pp. 166-179 (Cambridge University Press, 1995); and Monoclonal Antibodies: Principles and Applications See Ward et al., “Genetic Manipulation and Expression of Antibodies”, Birch et al., (eds.), pp. 137-185 (Wiley-Liss, Inc. 1995).

[0176] The term "Fab" refers to a protein containing a light chain constant domain and a heavy chain first constant domain (CH1). The Fab fragment differs from the Fab' fragment in that several residues are added to the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteine ​​residues from the antibody hinge region. Fab' fragments with cysteine ​​residues in the constant domain bearing free thiol groups are referred to herein as Fab'-SH. The Fab' fragment is generated by reducing the heavy chain disulfide bonds of the F(ab')2 fragment. Other chemical conjugations of antibody fragments are also known.

[0177] As used herein, the “CrossFab configuration” refers to a Fab molecule in which the variable or constant domains of the Fab heavy and light chains are exchanged (replaced) (see, for example, WO2009 / 080252A1 and WO2017 / 055388A2). For example, a CrossFab Fab molecule may contain a peptide chain consisting of a variable domain of the light chain and a constant domain of the heavy chain (VL-CH1 from N-terminus to C-terminus), and a peptide chain consisting of a variable domain of the heavy chain and a constant domain of the light chain (VH-CL from N-terminus to C-terminus).

[0178] A “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, linked by a short linker peptide of 10 to approximately 25 amino acids. For flexibility, the linker is typically glycine-rich, while for solubility, it is typically serine or threonine-rich, and the N-terminus of the VH can be linked to the C-terminus of the VL, and vice versa. This protein retains the specificity of the original antibody, although the constant region has been removed and the linker introduced. scFv antibodies are described, for example, in Houston, JS, Methods in Enzymol. 203 (1991) 46-96. Furthermore, antibody fragments contain single-chain polypeptides that are characterized by a VH domain, i.e., capable of assembling with a VL domain, or vice versa, capable of assembling with a VH domain to form a functional antigen-binding site, thereby providing the antigen-binding properties of the full-length antibody.

[0179] The term "multispecific" refers to the ability of an antibody to specifically bind to two or more different antigenic determinants, such as two or more binding sites, each formed by an antibody heavy chain variable domain (VH) and an antibody light chain variable domain (VL), or, in the case of a single-domain antibody, a single variable domain binding to a different antigen.

[0180] As used herein, the term "percentage (%) amino acid sequence identity" is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a given sequence after sequence alignment and the introduction of vacancies (if necessary) to achieve maximum percentage sequence identity without considering any conserved substitutions as part of sequence identity. Alignments used to determine percentage amino acid sequence identity can be performed in various ways known to those 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 appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0181] When using ALIGN-2 for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A with respect to, or for a given amino acid sequence B (which can be alternatively expressed as a given amino acid sequence A having or containing, or for a given amino acid sequence B, a certain % amino acid sequence identity) is calculated as follows: 100 multiplied by the score X / Y, where X is the number of amino acid residues that are scored as identical matches by the sequence alignment program ALIGN-2 in the procedural alignment of A and B, and where Y is the total number of amino acid residues in B. It should 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 with B will not be equal to the % amino acid sequence identity of B with A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein were obtained using the ALIGN-2 computer program as described in the preceding paragraph.

[0182] The terms “complementarity-determining region” and “CDR,” synonyms with “hypervariant region” or “HVR,” are known in the art to refer to a discontinuous sequence of amino acids within the variable region of an antibody that confers antigen specificity and / or binding affinity. Typically, there are three CDRs (CDR-H1, CDR-H2, CDR-H3) in each heavy chain variable region and three CDRs (CDR-L1, CDR-L2, CDR-L3) in each light chain variable region. The terms “framework region” and “FR” are known in the art to refer to the non-CDR portions of the heavy and light chain variable regions. Typically, there are four FRs (FR-H1, FR-H2, FR-H3, and FR-H4) in each full-length heavy chain variable region and four FRs (FR-L1, FR-L2, FR-L3, and FR-L4) in each full-length light chain variable region.The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of many well-known schemes, including those described in Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding site topography,” J. Mol. Biol. 262, 732-745. (“Contact” numbering scheme); Lefranc MP et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev CompImmunol, 2003 Jan;27 (1):55-77 (“IMGT” numbering scheme); Honegger A and Plückthun A, “Yetanother numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J Mol Biol, 2001 Jun 8;309 (3):657-70, (“Aho” numbering scheme); and Whitelegg NR and Rees AR, “WAM: an improved algorithm for modelling antibodies on the WEB,” Protein Eng. 2000 Dec;13 (12):819-24 (“AbM” numbering scheme). In some embodiments, the CDR of the antibodies described herein can be defined by a method selected from Kabat, Chothia, IMGT, Aho, AbM, or combinations thereof.

[0183] The boundaries of a given CDR or FR can vary depending on the scheme used for identification. For example, the Kabat scheme is based on structure alignment, while the Chothia scheme is based on structural information. Both the Kabat and Chothia schemes number based on the most common antibody region sequence lengths, where insertions are accommodated by an insertion letter, such as "30a," and deletions occur in some antibodies. These two schemes place certain insertions and deletions ("indels") in different positions, resulting in different numbering. The Contact scheme is based on the analysis of complex crystal structures and is similar to the Chothia numbering scheme in many ways.

[0184] Isolated polypeptides or polypeptide complexes In some embodiments, this document discloses isolated peptides or peptide complexes comprising a first antigen recognition molecule that binds to CD28 and a second antigen recognition molecule that binds to prostate-specific membrane antigen (PSMA). In some embodiments, the first antigen recognition molecule that binds to CD28 comprises an anti-CD28 antibody. In some embodiments, the anti-CD28 antibody comprises an anti-CD28 heavy chain variable domain comprising complementarity-determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein HC-CDR1, HC-CDR2, and HC-CDR3 comprise the following amino acid sequences: HC-CDR1: SEQ ID NO: 1, HC-CDR2: SEQ ID NO: 2, and HC-CDR3: SEQ ID NO: 3; and the anti-CD28 antibody comprises an anti-CD28 light chain variable domain comprising complementarity-determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein LC-CDR1, LC-CDR2, and LC-CDR3 comprise the following amino acid sequences: LC-CDR1: SEQ ID NO: 4, LC-CDR2: (KAS), and LC-CDR3: SEQ ID NO: 6. In some embodiments, the anti-CD28 heavy chain variable domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 7, and the anti-CD28 light chain variable domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 8. In some embodiments, the anti-CD28 heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 7, and the anti-CD28 light chain variable domain comprises the amino acid sequence of SEQ ID NO: 8.

[0185] Table 1. Anti-CD28 sequences. CDR sequences are underlined and identified using the IMGT definition. In some embodiments, the anti-CD28 antibody comprises a single-chain variable fragment (scFv), a single-domain antibody, Fab, Fab', or CrossFab. In some embodiments, the anti-CD28 antibody comprises an scFv. In some embodiments, the anti-CD28 antibody comprises a single-chain variable fragment (scFv) having engineered disulfide bonds. In some embodiments, the anti-CD28 antibody comprises an scFv, and the scFv comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 9. In some embodiments, the anti-CD28 antibody comprises an scFv, and the scFv comprises the amino acid sequence of SEQ ID NO: 9. In some embodiments, the anti-CD28 antibody comprises an scFv, and the scFv comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 10. In some embodiments, the anti-CD28 antibody comprises an scFv, and the scFv comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, the anti-CD28 antibody comprises an scFv, and the scFv comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 11. In some embodiments, the anti-CD28 antibody comprises an scFv, and the scFv comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 12. In some embodiments, the anti-CD28 antibody comprises an scFv, and the scFv comprises the amino acid sequence of SEQ ID NO: 12.

[0186] In some embodiments, the anti-CD28 antibody comprises Fab. In some embodiments, Fab comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 696 and an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 697. In some embodiments, the anti-CD28 antibody comprises CrossFab. In some embodiments, CrossFab comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 698 and an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 699. In some embodiments, CrossFab comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 700 and an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 701.

[0187] In some implementations, the second antigen recognition molecule that binds to PSMA contains an anti-PSMA antibody. In some embodiments, the anti-PSMA antibody comprises an anti-PSMA heavy chain variable domain comprising complementarity-determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein HC-CDR1, HC-CDR2, and HC-CDR3 comprise the following amino acid sequences: HC-CDR1: SEQ ID NO: 13, HC-CDR2: SEQ ID NO: 14, and HC-CDR3: SEQ ID NO: 15; and the anti-PSMA antibody comprises an anti-PSMA light chain variable domain comprising complementarity-determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, LC-CDR2, and LC-CDR3 of the light chain variable domain comprise the following amino acid sequences: LC-CDR1: SEQ ID NO: 16, LC-CDR2: (EA), and LC-CDR3: SEQ ID NO: 18. In some embodiments, the anti-PSMA heavy chain variable domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 20, and the anti-PSMA light chain variable domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 19. In some embodiments, the anti-PSMA heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 20, and the anti-PSMA light chain variable domain comprises the amino acid sequence of SEQ ID NO: 19.

[0188] Table 2. Anti-PSMA sequences. CDR sequences are underlined and identified using the IMGT definition. In some embodiments, the anti-PSMA antibody comprises a single-chain variable fragment (scFv), a single-domain antibody, Fab, Fab', or CrossFab. In some embodiments, the anti-PSMA antibody comprises Fab or Fab'. In some embodiments, the anti-PSMA antibody comprises Fab or Fab', and Fab or Fab' comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 19 and an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 20. In some embodiments, the anti-PSMA antibody comprises Fab or Fab', and Fab or Fab' comprises the amino acid sequences of SEQ ID NO: 19 and SEQ ID NO: 20. In some embodiments, the anti-PSMA antibody comprises Fab or Fab', and Fab or Fab' comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 702 and an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 703. In some embodiments, the anti-PSMA antibody comprises Fab or Fab', and Fab or Fab' comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 704 and an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 705.

[0189] In some embodiments, the anti-CD28 antibody comprises an scFv, and the anti-PSMA antibody comprises Fab or Fab'. In some embodiments, the scFv is linked to Fab or Fab' via a adapter, and Fab or Fab' comprises a Fab heavy chain polypeptide and a Fab light chain polypeptide. In some embodiments, the adapter links the scFv to the Fab heavy chain polypeptide. In some embodiments, the adapter links the scFv to the Fab light chain polypeptide. In some embodiments, the adapter links the scFv to the N-terminus of the Fab heavy chain polypeptide. In some embodiments, the adapter links the scFv to the C-terminus of the Fab heavy chain polypeptide. In some embodiments, the adapter links the scFv to the N-terminus of the Fab light chain polypeptide. In some embodiments, the adapter links the scFv to the C-terminus of the Fab light chain polypeptide. In some embodiments, the adapter links the anti-CD28 heavy chain variable domain of the scFv to the Fab heavy chain polypeptide. In some embodiments, the adapter links the anti-CD28 heavy chain variable domain of the scFv to the Fab light chain polypeptide.

[0190] In some embodiments, the adapter links the anti-CD28 heavy chain variable domain of the scFv to the N-terminus of the Fab heavy chain peptide. In some embodiments, the adapter links the anti-CD28 heavy chain variable domain of the scFv to the C-terminus of the Fab heavy chain peptide. In some embodiments, the adapter links the anti-CD28 heavy chain variable domain of the scFv to the N-terminus of the Fab light chain peptide. In some embodiments, the adapter links the anti-CD28 heavy chain variable domain of the scFv to the C-terminus of the Fab light chain peptide. In some embodiments, the adapter links the anti-CD28 light chain variable domain of the scFv to the Fab heavy chain peptide. In some embodiments, the adapter links the anti-CD28 light chain variable domain of the scFv to the N-terminus of the Fab heavy chain peptide. In some embodiments, the adapter links the anti-CD28 light chain variable domain of the scFv to the C-terminus of the Fab heavy chain peptide. In some embodiments, the adapter links the anti-CD28 light chain variable domain of the scFv to the N-terminus of the Fab light chain peptide. In some implementations, the linker connects the anti-CD28 light chain variable domain of scFv to the C-terminus of the Fab light chain peptide.

[0191] In some embodiments, the anti-CD28 antibody comprises Fab or Fab', and the anti-PSMA antibody comprises Fab or Fab'. In some embodiments, the Fab or Fab' of the anti-CD28 antibody and the Fab or Fab' of the anti-PSMA antibody are linked by a linker, and the Fab or Fab' of the anti-PSMA antibody comprises a Fab heavy chain polypeptide and a Fab light chain polypeptide. In some embodiments, the linker connects the Fab or Fab' of the anti-CD28 antibody to the Fab heavy chain polypeptide of the anti-PSMA antibody. In some embodiments, the linker connects the Fab or Fab' of the anti-CD28 antibody to the Fab light chain polypeptide of the anti-PSMA antibody. In some embodiments, the linker connects the Fab or Fab' of the anti-CD28 antibody to the N-terminus of the Fab heavy chain polypeptide of the anti-PSMA antibody. In some embodiments, the linker connects the Fab or Fab' of the anti-CD28 antibody to the C-terminus of the Fab heavy chain polypeptide of the anti-PSMA antibody. In some embodiments, the linker connects the Fab or Fab' of the anti-CD28 antibody to the N-terminus of the Fab light chain polypeptide of the anti-PSMA antibody. In some embodiments, the linker connects the Fab or Fab' of the anti-CD28 antibody to the C-terminus of the Fab light chain polypeptide of the anti-PSMA antibody. In some embodiments, the linker connects the Fab heavy chain polypeptide of the anti-CD28 antibody to the Fab heavy chain polypeptide of the anti-PSMA antibody. In some embodiments, the linker connects the Fab heavy chain polypeptide of the anti-CD28 antibody to the Fab light chain polypeptide of the anti-PSMA antibody. In some embodiments, the linker connects the Fab heavy chain polypeptide of the anti-CD28 antibody to the N-terminus of the Fab heavy chain polypeptide of the anti-PSMA antibody. In some embodiments, the linker connects the Fab heavy chain polypeptide of the anti-CD28 antibody to the C-terminus of the Fab heavy chain polypeptide of the anti-PSMA antibody. In some embodiments, the linker connects the Fab heavy chain polypeptide of the anti-CD28 antibody to the N-terminus of the Fab light chain polypeptide of the anti-PSMA antibody. In some embodiments, the linker connects the Fab heavy chain polypeptide of the anti-CD28 antibody to the C-terminus of the Fab light chain polypeptide of the anti-PSMA antibody. In some embodiments, the linker connects the Fab light chain polypeptide of the anti-CD28 antibody to the Fab heavy chain polypeptide of the anti-PSMA antibody. In some embodiments, the linker ligates the Fab light chain peptide of the anti-CD28 antibody to the Fab light chain peptide of the anti-PSMA antibody. In some embodiments, the linker ligates the Fab light chain peptide of the anti-CD28 antibody to the N-terminus of the Fab heavy chain peptide of the anti-PSMA antibody. In some embodiments, the linker ligates the Fab light chain peptide of the anti-CD28 antibody to the N-terminus of the Fab light chain peptide of the anti-PSMA antibody. In some embodiments, the linker ligates the Fab light chain peptide of the anti-CD28 antibody to the C-terminus of the Fab light chain peptide of the anti-PSMA antibody.

[0192] In some embodiments, the anti-CD28 antibody comprises CrossFab, and the anti-PSMA antibody comprises Fab or Fab'. In some embodiments, CrossFab and Fab or Fab' are linked by a connector, and the Fab or Fab' of the anti-PSMA antibody comprises a Fab heavy chain polypeptide and a Fab light chain polypeptide. In some embodiments, the connector links CrossFab to the Fab heavy chain polypeptide of the anti-PSMA antibody. In some embodiments, the connector links CrossFab to the Fab light chain polypeptide of the anti-PSMA antibody. In some embodiments, the connector links CrossFab to the N-terminus of the Fab heavy chain polypeptide of the anti-PSMA antibody. In some embodiments, the connector links CrossFab to the C-terminus of the Fab heavy chain polypeptide of the anti-PSMA antibody. In some embodiments, the connector links CrossFab to the N-terminus of the Fab light chain polypeptide of the anti-PSMA antibody. In some embodiments, the connector links CrossFab to the C-terminus of the Fab light chain polypeptide of the anti-PSMA antibody. In some embodiments, the connector links the Fab heavy chain polypeptide of CrossFab to the Fab heavy chain polypeptide of the anti-PSMA antibody. In some embodiments, the adapter links the CrossFab Fab heavy chain peptide to the Fab light chain peptide of the anti-PSMA antibody. In some embodiments, the adapter links the CrossFab Fab heavy chain peptide to the N-terminus of the anti-PSMA antibody Fab heavy chain peptide. In some embodiments, the adapter links the CrossFab Fab heavy chain peptide to the C-terminus of the anti-PSMA antibody Fab heavy chain peptide. In some embodiments, the adapter links the CrossFab Fab heavy chain peptide to the N-terminus of the anti-PSMA antibody Fab light chain peptide. In some embodiments, the adapter links the CrossFab Fab heavy chain peptide to the C-terminus of the anti-PSMA antibody Fab light chain peptide. In some embodiments, the adapter links the CrossFab Fab light chain peptide to the anti-PSMA antibody Fab heavy chain peptide. In some embodiments, the adapter links the CrossFab Fab light chain peptide to the anti-PSMA antibody Fab light chain peptide. In some embodiments, the adapter links the CrossFab Fab light chain peptide to the N-terminus of the anti-PSMA antibody Fab heavy chain peptide. In some embodiments, the adapter links the CrossFab Fab light chain peptide to the C-terminus of the anti-PSMA antibody Fab heavy chain peptide. In some embodiments, the adapter links the CrossFab Fab light chain peptide to the N-terminus of the anti-PSMA antibody Fab light chain peptide. In some embodiments, the adapter links the CrossFab Fab light chain peptide to the C-terminus of the anti-PSMA antibody Fab light chain peptide.

[0193] In some implementations, the anti-CD28 antibody contains Fab or Fab', and the anti-PSMA antibody contains CrossFab.

[0194] In some embodiments, the linker is at least 5 amino acids long. In some embodiments, the linker is no more than 30 amino acids long. In some embodiments, the linker is at least 5 amino acids long and no more than 30 amino acids long. In some embodiments, the linker is 5 amino acids long. In some embodiments, the linker is 15 amino acids long. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 21 (GGGGSGGGGSGGGGS) or SEQ ID NO: 22 (GGGGS).

[0195] Table 3. Connector Sequence Peptide sequences that bind to CD28 and PSMA In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 23 and SEQ ID NO: 24. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 25 and SEQ ID NO: 26. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 27 and SEQ ID NO: 28. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 29 and SEQ ID NO: 30. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 31 and SEQ ID NO: 32. In some embodiments, the isolated polypeptide or polypeptide complex comprises the amino acid sequences of SEQ ID NO: 25 and SEQ ID NO: 26. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 706 and SEQ ID NO: 707. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 708, SEQ ID NO: 709, and SEQ ID NO: 710. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 711, SEQ ID NO: 712, and SEQ ID NO: 713. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 714, SEQ ID NO: 715, and SEQ ID NO: 716.In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 717, SEQ ID NO: 718, and SEQ ID NO: 719. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 720, SEQ ID NO: 721, and SEQ ID NO: 722. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 723, SEQ ID NO: 724, and SEQ ID NO: 725. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 726, SEQ ID NO: 727, and SEQ ID NO: 728. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 729, SEQ ID NO: 730, and SEQ ID NO: 731.

[0196] Table 4. Peptide complexes binding to CD28 and PSMA In some embodiments, the isolated peptide or peptide complex is human or humanized. In some embodiments, the isolated peptide or peptide complex comprises a peptide linked to an anti-CD28 antibody, wherein the peptide weakens the binding of the anti-CD28 antibody to CD28. In some embodiments, the isolated peptide or peptide complex comprises a configuration according to Formula I: A2-A1-L1-P1-H1, wherein A2 comprises an anti-PSMA antibody, A1 comprises an anti-CD28 antibody, L1 comprises a linker portion linking A1 to P1 and is a substrate of a tumor-specific protease, P1 comprises a peptide weakening the binding of the anti-CD28 antibody to CD28, and H1 comprises a half-life-extending molecule. In some embodiments, A2 further comprises P2 and L2, wherein P2 comprises a peptide bound to A2; and L2 comprises a linker portion linking A2 to P2 and is a substrate of a tumor-specific protease. In some embodiments, the peptide or peptide complex comprises according to Formula Ia: P2-L2-A2-A1-L1-P1-H1.

[0197] In some embodiments, the anti-CD28 antibody comprises scFv, and the anti-PSMA antibody comprises Fab or Fab', wherein Fab or Fab' comprises a Fab heavy chain polypeptide and a Fab light chain polypeptide. In some embodiments, the Fab heavy chain polypeptide of A2 binds to the scFv heavy chain polypeptide of A1, and L2 binds to the Fab light chain polypeptide of A2. In some embodiments, the Fab light chain polypeptide of A2 binds to the scFv heavy chain polypeptide of A1, and L2 binds to the Fab heavy chain polypeptide of A2. In some embodiments, the Fab heavy chain polypeptide of A2 binds to the scFv light chain polypeptide of A1, and L2 binds to the Fab light chain polypeptide of A2. In some embodiments, the Fab light chain polypeptide of A2 binds to the scFv light chain polypeptide of A1, and L2 binds to the Fab heavy chain polypeptide of A2.

[0198] P 1 and P 2 In some embodiments, P1 binds to A1 via ionic interactions, electrostatic interactions, hydrophobic interactions, π-stacking interactions, or hydrogen bonding interactions, or combinations thereof. In some embodiments, P1 has less than 70% sequence homology with CD28. In some embodiments, P2 weakens the binding of A2 to PSMA. In some embodiments, P2 binds to A2 via ionic interactions, electrostatic interactions, hydrophobic interactions, π-stacking interactions, or hydrogen bonding interactions, or combinations thereof. In some embodiments, P2 binds to A2 at or near the antigen binding site. In some embodiments, P2 has less than 70% sequence homology with PSMA. In some embodiments, P1 or P2 comprises a peptide sequence of at least 10 amino acids in length. In some embodiments, P1 or 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, P1 or P2 comprises a peptide sequence of at least 16 amino acids in length. In some embodiments, P1 or 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 comprises at least two cysteine ​​amino acid residues.

[0199] In some embodiments, P1 comprises the amino acid sequence according to any one of SEQ ID NO: 33 to 106 or 732-813. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 33. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 34. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 35. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 36. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 37. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 38. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 39. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 40. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 41. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 42. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 43. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 44. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 45. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 46. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 47. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 48. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 49. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 50. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 51. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 52. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 53. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 54. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 55. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 56. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 57. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 58. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 59. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 60.

[0200] In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 61. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 62. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 63. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 64. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 65. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 66. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 67. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 68. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 69. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 70. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 71. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 72. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 73. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 74. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 75. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 76. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 77. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 78. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 79. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 80. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 81. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 82. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 83. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 84. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 85. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 86. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 87. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 88. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 89. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 90.In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 91. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 92. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 93. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 94. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 95. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 96. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 97. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 98. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 99. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 100. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 101. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 102. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 103. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 104. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 105. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 106.

[0201] Table 5. CD28 peptide masking sequence 1 Met(O) is methionine sulfoxide; Met(O)2 is methionine sulfone.

[0202] In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 73 or a peptide sequence having one, two, or three amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 73. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 103 or a peptide sequence having one, two, or three amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 103. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 103.

[0203] In some implementations, P1 includes components based on X1-X2-X3-C-X4-X5-X6-X7-X8-X9-X 10 -CX 11 -X 12 The amino acid sequence is defined as follows: X1 is selected from M, I, L, and V; X2 is selected from D, H, N, A, F, S, T, Y, and V; X3 is selected from W, L, and F; X4 is selected from P, A, and L; X5 is selected from R, T, I, M, S, K, L, V, W, F, A, P, and D; X6 is selected from E, D, Y, H, S, F, A, N, T, I, P, and V; X7 is selected from L, M, R, S, Q, and H; X8 is selected from W and Q; X9 is selected from 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 Selected from F, Y, L, W, and V; and X 12 The X1 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 Selected from F, Y, and L; and X 12 The X1 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 It is F; and X 12 Selected from N, A, and F.

[0204] In some embodiments, P1 comprises the amino acid sequence according to SEQ ID NO: 41 or an amino acid sequence having one, two, or three amino acid mutations, substitutions, or deletions relative to SEQ ID NO: 41. In some embodiments, P1 comprises the amino acid sequence according to SEQ ID NO: 41. In some embodiments, P1 comprises an amino acid sequence selected from any one of SEQ ID NO: 41, 73, 103, and 179-603.

[0205] Table 6. Phage panning results of the anti-CD28 scFv peptide 9 library sequence. (-) indicates the amino acid at the same position as the anti-CD28 scFv peptide 9. Peptide sequence (P1 or P...) 1a ) Table 7. Phage panning results of the peptide 87 library sequence. (-) indicates the amino acid position that corresponds to peptide 87. Table 8. Phage panning results of the peptide 92 library sequence. (-) indicates the amino acid position that corresponds to peptide 92. In some embodiments, P1 comprises the amino acid sequence according to the following sequence: J1-J2-WC-J3-J4-J5-J6-J7-J8-J9-CJ 10 -J 11 J1 is selected from A, R, N, D, Q, E, G, H, I, L, K, F, P, S, T, W, Y, and V; J2 is selected from D, P, and Y; J3 is selected from P and Q; J4 is selected from R, L, and I; J5 is selected from D, H, Y, and I; J6 is selected from L, S, and R; J7 is selected from W and G; J8 is selected from V, A, D, H, L, and N; J9 is selected from H, L, E, and D; J 10 Selected from F, Y, and L; and J 11 Selected from A, N, H, and T. In some embodiments, J1 is selected from A, R, N, D, Q, E, G, H, K, F, P, S, T, W, and Y. In some embodiments, J1 is selected from R, Q, E, H, I, L, K, F, T, W, Y, and V; J2 is D; J3 is P; J4 is R; J5 is D; J6 is L; J7 is W; J8 is V; J9 is H; J 10 It is F; and J 11It is A. In some implementations, J1 is selected from R, Q, E, L, K, T, and W; J2 is D; J3 is P; J4 is R; J5 is D; J6 is L; J7 is W; J8 is V; J9 is H; J 10 It is F; and J 11 It is A. In some implementations, J1 is L or I; J2 is D or Y; J3 is P; J4 is R, L, or I; J5 is H, Y, or D; J6 is L or R; J7 is W; J8 is A, D, H, or N; J9 is L, E, or D; J 10 It is either F or Y; and J 11 It is N or H. In some implementations, J1 is L or I; J2 is D or Y; J3 is P; J4 is R or I; J5 is H, Y, or D; J6 is L or R; J7 is W; J8 is A, D, or N; J9 is L, E, or D; J 10 It is either F or Y; and J 11 It is N.

[0206] In some embodiments, P1 comprises an amino acid sequence according to the following sequence: Z1-C-Z2-Z3-Z4-Z5-Z6-Z7-Z8-Z9-Z 10 -Z 11 -CZ 12 Z1 is selected from Y, H, and A; Z2 is selected from D, E, A, Q, S, V, M, H, L, F, T, W, and N; Z3 is selected from G, E, S, and A; Z4 is selected from H, L, W, and A; Z5 is selected from L, S, A, T, I, V, M, R, E, D, F, H, K, Q, N, Y, W, and G; Z6 is selected from P, A, E, Q, S, L, W, G, V, D, K, I, M, R, T, F, H, N, and Y; Z7 is selected from E, Q, I, M, V, A, L, R, W, P, D, T, K, S, G, H, and N; Z8 is selected from Y, H, F, W, N, Q, and A; Z9 is selected from M, I, L, R, V, K, F, Q, Y, W, and A; Z 10 Selected from A, Q, S, W, E, L, G, D, T, M, N, V, H, R, Y, K, P, F, and I; Z 11 Selected from K, Q, N, H, E, P, and A; and Z 12The Z1 is selected from L, M, I, F, V, Y, Q, D, T, and A. In some embodiments, Z1 is selected from Y and H; Z2 is selected from D, E, A, Q, S, V, M, H, L, F, T, W, and N; Z3 is selected from G, E, and S; Z4 is selected from H, L, and W; Z5 is selected from L, S, A, T, I, V, M, R, E, D, F, H, K, Q, N, Y, W, and G; Z6 is selected from P, A, E, Q, S, L, W, G, V, D, K, I, M, R, T, F, H, N, and Y; Z7 is selected from E, Q, I, M, V, A, L, R, W, P, D, T, K, S, G, H, and N; Z8 is selected from Y, H, F, W, N, and Q; Z9 is selected from M, I, L, R, V, K, F, Q, Y, and W; Z 10 Selected from A, Q, S, W, E, L, G, D, T, M, N, V, H, R, Y, K, P, F, and I; Z 11 Selected from K, Q, N, H, E, and P; and Z 12 The numbers are selected from L, M, I, F, V, Y, Q, D, and T. In some embodiments, Z1 is selected from Y and H; Z2 is selected from D, E, A, Q, S, V, M, H, L, F, T, W, and N; Z3 is selected from G, E, and S; Z4 is selected from H, L, and W; Z5 is selected from L, S, A, T, I, V, M, R, E, D, F, H, K, Q, N, Y, W, and G; Z6 is selected from P, A, E, Q, S, L, W, G, V, D, K, I, M, R, T, F, H, N, and Y; Z7 is selected from E, Q, I, M, V, A, L, R, W, P, D, T, K, S, G, H, and N; Z8 is selected from Y, H, F, W, N, and Q; Z9 is selected from M, I, L, R, V, K, F, Q, Y, and W; Z 10 Selected from A, Q, S, W, E, L, G, D, T, M, N, V, H, R, Y, K, P, F, and I; Z 11 Selected from K, Q, N, H, E, and P; and Z 12 The Z1 is selected from L, M, I, F, V, Y, Q, D, and T. In some embodiments, Z1 is Y; Z2 is selected from D, E, A, and Q; Z3 is G; Z4 is H; Z5 is selected from L, S, A, T, I, V, and M; Z6 is selected from P, A, E, Q, S, L, W, G, V, and D; Z7 is selected from E, Q, I, M, V, A, and L; Z8 is selected from Y and H; Z9 is selected from M, I, and L; Z 10 Selected from A, Q, S, W, E, L, G, D, T, M, and N; Z 11 It is K; and Z 12 Z1 is selected from L and M. In some embodiments, Z1 is Y; Z2 is selected from D, E, and A; Z3 is G; Z4 is H; Z5 is selected from L, S, A, and T; Z6 is selected from P, A, E, Q, S, L, and W; Z7 is selected from E, Q, I, and M; Z8 is Y; Z9 is selected from M, I, and L; Z10 Selected from A, Q, S, W, E, L, G, and D; Z 11 It is K; and Z 12 It is L. In some implementations, Z1 is Y; Z2 is selected from D and E; Z3 is G; Z4 is H; Z5 is selected from L, S and A; Z6 is selected from P, A, E, Q, S and L; Z7 is selected from E, Q and I; Z8 is Y; Z9 is selected from M, I and L; Z 10 Selected from A, Q, S, W, E, L, G, and D; Z 11 It is K; and Z 12 It is L.

[0207] In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 784. In some embodiments, P1 consists of the amino acid sequence of SEQ ID NO: 784. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 744. In some embodiments, P1 consists of the amino acid sequence of SEQ ID NO: 744. In some embodiments, P1 comprises the amino acid sequence according to any one of SEQ ID NO: 774-793. In some embodiments, P1 comprises the amino acid sequence according to any one of SEQ ID NO: 764-773.

[0208] In some embodiments, P1 comprises amino acids according to the following sequence: U1-C-U2-U3-U4-U5-U6-U7-U8-U9-U 10 -U 11 -CU 12 Among them: U1 is selected from T, S, L, M, W, Q, P, R, G, N, I, Y, D, E, V, H and A; U2 is selected from A, S, T, K, E, R, G, N, W and D; U3 is selected from A, E, P, D, G, W, Q, V and F; U4 is selected from G, V, F, D, E, R and A; U5 is selected from A, F, V, S, L, P and M; U6 is selected from T, S, H, M, A, N, P, G, Q, D, Y, V, L, I, E, K, F and R; U7 is selected from Y, W, F, L, N, T, Q, I and A; U8 is selected from P, S, T, A, E, K, Q, N, V, G, H, L, D, Y, I, M and R; U9 is selected from W, L and A; U 10 Selected from P, S, R, L, E, and A; U 11 Selected from L, A, T, M, V, W, Y, E, Q, I, F, S, P, K, R, D, H, N, and G; and U 12The elements are selected from L, F, V, M, I, Y, E, W, and A. In some embodiments, U1 is selected from T, S, L, M, W, Q, P, R, G, N, I, Y, D, E, V, and H; U2 is selected from A, S, T, K, E, R, G, N, W, and D; U3 is selected from A, E, P, D, G, W, Q, V, and F; U4 is selected from G, V, F, D, E, and R; U5 is selected from A, F, V, S, L, P, and M; U6 is selected from T, S, H, M, A, N, P, G, Q, D, Y, V, L, I, E, K, F, and R; U7 is selected from Y, W, F, L, N, T, Q, and I; U8 is selected from P, S, T, A, E, K, Q, N, V, G, H, L, D, Y, I, M, and R; U9 is selected from W, L, and A; U 10 Selected from P, S, R, L, and E; U 11 Selected from L, A, T, M, V, W, Y, E, Q, I, F, S, P, K, R, D, H, N, and G; and U 12 The elements are selected from L, F, V, M, I, Y, E, and W. In some embodiments, U1 is selected from T, S, L, M, and W; U2 is selected from A, S, and T; U3 is selected from A and E; U4 is selected from G, V, and F; U5 is selected from A, F, and V; U6 is selected from T, S, H, M, A, and N; U7 is selected from Y, W, and F; U8 is selected from P, S, T, A, E, K, Q, N, and V; U9 is selected from W and L; U 10 Selected from P and S; U 11 Selected from L, A, T, M, V, W, Y, E, Q, I, F, and S; and U 12 The elements are selected from L, F, V, and M. In some embodiments, U1 is selected from T and S; U2 is selected from A and S; U3 is A; U4 is selected from G and V; U5 is selected from A and F; U6 is selected from T, S, and H; U7 is selected from Y and W; U8 is selected from P, S, T, and A; U9 is W; U 10 It is P; U 11 Selected from L, A, T, M, V, W, and Y; and U 12 Selected from L and F.

[0209] In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 749. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO: 749. In some embodiments, P1 comprises the amino acid sequence according to any one of SEQ ID NO: 756-763. In some embodiments, P1 comprises the amino acid sequence according to any one of SEQ ID NO: 794-813. In some embodiments, P1 comprises the amino acid sequence according to any one of SEQ ID NO: 732-2002.

[0210] In some embodiments, P2 comprises the amino acid sequence of any one of SEQ ID NO: 2003-4329. In some embodiments, P2 comprises the amino acid sequence of SEQ ID NO: 2025. In some embodiments, P2 consists of the amino acid sequence of SEQ ID NO: 2025. In some embodiments, P2 comprises the amino acid sequence of any one of SEQ ID NO: 2087-2094. In some embodiments, P2 comprises the amino acid sequence of SEQ ID NO: 2026. In some embodiments, P2 consists of the amino acid sequence of SEQ ID NO: 2026. In some embodiments, P2 comprises the amino acid sequence of any one of SEQ ID NO: 2097-2106. In some embodiments, P2 comprises the amino acid sequence of any one of SEQ ID NO: 2107-2133. In some embodiments, P2 comprises the amino acid sequence of SEQ ID NO: 2120. In some embodiments, P2 consists of the amino acid sequence of SEQ ID NO: 2120.

[0211] In some implementations, P2 includes components based on B1-B2-B3-B4-C-B5-P-B6-W-B7-C-B8-B9-B 10 The amino acid sequence of (SEQ IDNO: 4470), wherein: B1 is selected from V, E, L, D, I, G, M, S, P, T, A, F, W, Y, Q, H, N, K and R; B2 is selected from E, V, D, T, S, L, G, P, A, M, I, Q, H, F, Y, N, W, R and K; B3 is selected from K, P, R, I, N, H, V, M, A, L, Q, T, S, G, F, Y, E, W and D; B4 is selected from W, L, M, R, V, Y, A, K, I, S, Q, F, H, E, T, N, G and D; B5 is selected from I, V, T, K, R, E, S, Q, M, L, F, A, N, and H; B6 is selected from A, E, S, P, Q, T, L, D, M, V, R, K, N, I, H, W, Y, F, and G; B7 is selected from M, L, I, A, V, F, G, and K; B8 is selected from E, S, T, A, V, D, Q, L, N, I, M, Y, H, F, W, G, K, R, and P; B9 is selected from G, V, A, S, W, E, D, M, T, L, F, H, Q, N, R, I, Y, P, and K; and B 10 Selected from F, L, M, S, I, V, D, Q, T, A, N, Y, W, E, R, H, P, G, and K.

[0212] Table 9. PSMA peptide masking sequence (P2) Table 10. Phage panning results of the peptide 180 library sequence. (-) indicates the amino acid position corresponding to peptide 180. L 1 and L 2 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 A2. In some embodiments, L2 binds to the C-terminus of A2. 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 contains (G2S). n The formula is given by (n) where n is an integer from 1 to 3 (SEQ ID NO: 605). In some embodiments, L1 has a value selected from (G2S). n (GS) n (GSGGS) n (SEQ ID NO: 606), (GGGS) n (SEQ ID NO: 607), (GGGGS) n (SEQ ID NO:608) and (GSSGGS) n The expression (SEQ ID NO: 609) where n is an integer of at least 1.

[0213] In some embodiments, when L1 is cleaved by a tumor-specific protease, P1 unbinds to A1, thereby exposing A1 to CD28. In some embodiments, when L2 is cleaved by a tumor-specific protease, P2 unbinds to A2, thereby exposing A2 to PSMA. In some embodiments, the tumor-specific protease is selected from matrix metalloproteinases (MMPs), serine proteases, cysteine ​​proteases, threonine proteases, and aspartic proteases. In some embodiments, the matrix metalloproteinases include MMP2, MMP7, MMP9, MMP13, or MMP14. In some embodiments, the serine protease includes matrix protease (MTSP1), urokinase, or hepatic protease. In some embodiments, L1 or L2 contains an amino acid sequence that can be cleaved by urokinase, a amino acid sequence that can be cleaved by matrix protease, an amino acid sequence that can be cleaved by matrix metalloproteinase, or an amino acid sequence that can be cleaved by asparagine endonuclease.

[0214] In some embodiments, L1 or L2 comprises the amino acid sequence according to SEQ ID NO: 110. In some embodiments, L1 or L2 comprises the amino acid sequence according to any one of SEQ ID NO: 107-137 and 604. In some embodiments, L1 or L2 comprises the amino acid sequence of linker 25 (ISSGLLSGRSDAG) (SEQ ID NO: 132), linker 26 (AAGLLAPPGGLSGRSDAG) (SEQ ID NO: 133), linker 27 (SPLGLSGRSDAG) (SEQ ID NO: 134), or linker 28 (LSGRSDAGSPLGLAG) (SEQ ID NO: 135), or an amino acid sequence having one, two, or three amino acid substitutions, additions, or deletions relative to the amino acid sequence of linker 25, linker 26, linker 27, or linker 28.

[0215] Table 11. L1, L2, L3 and L 1a sequence H 1 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 a fragment thereof, a circulating immunoglobulin or a fragment thereof, or CD35 / CR1. In some embodiments, the serum protein comprises thyroxine-binding protein, transthyroxine, 1-acid glycoprotein, transferrin, transferrin receptor or its transferrin-binding moiety, fibrinogen, or albumin. In some embodiments, the circulating immunoglobulin molecule comprises IgG1, IgG2, IgG3, IgG4, sIgA, IgM, or IgD. In some embodiments, the serum protein is albumin. In some embodiments, the polypeptide is an antibody. In some embodiments, the antibody comprises a single-domain antibody, a single-chain variable fragment, or Fab. In some embodiments, the single-domain antibody comprises a single-domain antibody that binds to albumin. In some embodiments, the single-domain antibody is a human or humanized antibody. In some embodiments, the single-domain antibody is 645gH1gL1. In some embodiments, the single-domain antibody is 645dsgH5gL4. In some embodiments, the single-domain antibody is 23-13-A01-sc02. In some embodiments, the single-domain antibody is A10m3 or a fragment thereof. In some embodiments, the single-domain antibody is DOM7r-31. In some embodiments, the single-domain antibody is DOM7h-11-15. In some embodiments, the single-domain antibody is Alb-1, Alb-8, or Alb-23. In some embodiments, the single-domain antibody is 10E.

[0216] In some embodiments, the single-domain antibody includes complementarity-determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein HC-CDR1, HC-CDR2, and HC-CDR3 of the single-domain antibody comprise: HC-CDR1: SEQ ID NO: 138, HC-CDR2: SEQ ID NO: 139, and HC-CDR3: SEQ ID NO: 140. In some embodiments, the single-domain antibody includes complementarity-determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein HC-CDR1, HC-CDR2, and HC-CDR3 of the single-domain antibody comprise: HC-CDR1: SEQ ID NO: 142, HC-CDR2: SEQ ID NO: 143, and HC-CDR3: SEQ ID NO: 144. In some embodiments, the single-domain antibody comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 141. In some embodiments, the single-domain antibody comprises the amino acid sequence of SEQ ID NO: 141. In some embodiments, the single-domain antibody comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 146. In some embodiments, the single-domain antibody comprises the amino acid sequence of SEQ ID NO: 146. In some embodiments, the single-domain antibody comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 145. In some embodiments, the single-domain antibody comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 4448.

[0217] Table 12. H1 or H 1a sequence In some embodiments, the single-domain antibody is SA21. In some embodiments, the peptide or peptide complex comprises modified amino acids, non-natural amino acids, modified non-natural amino acids, or combinations thereof. In some embodiments, the modified amino acids or modified non-natural amino acids comprise post-translational modifications.

[0218] L3 In some embodiments, H1 includes a linker portion (L3) connecting H1 to P1. In some embodiments, L3 is a peptide sequence having at least 5 to no more than 50 amino acids. In some embodiments, L3 is a peptide sequence having at least 10 to no more than 30 amino acids. In some embodiments, L3 is a peptide sequence having at least 10 amino acids. In some embodiments, L3 is a peptide sequence having at least 18 amino acids. In some embodiments, L3 is a peptide sequence having at least 26 amino acids. In some embodiments, L3 has a component selected from (G2S). n (GS) n (GSGGS) n (SEQ ID NO: 606), (GGGS) n (SEQ ID NO: 607), (GGGGS) n (SEQ ID NO: 608) and (GSSGGS) n The formula (SEQ ID NO: 609) is used, where n is an integer of at least 1. In some embodiments, L3 comprises an amino acid sequence according to GGGGSGGGSGG (SEQ ID NO: 610).

[0219] Tumor-activating antibodies that bind to CD28 and PSMA In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 147 and 148. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 147 and 148. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 147 and 148. In some embodiments, the polypeptide or polypeptide complex comprises the amino acid sequences of SEQ ID NO: 147 and 148.

[0220] In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 149 and 150. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 149 and 150. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 149 and 150. In some embodiments, the polypeptide or polypeptide complex comprises the amino acid sequences of SEQ ID NO: 149 and 150.

[0221] In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 151 and 152. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 151 and 152. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 151 and 152. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 151 and 152. In some embodiments, the polypeptide or polypeptide complex comprises the amino acid sequences of SEQ ID NO: 151 and 152.

[0222] In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 153 and 154. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 153 and 154. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 153 and 154. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 153 and 154. In some embodiments, the polypeptide or polypeptide complex comprises the amino acid sequences of SEQ ID NO: 153 and 154.

[0223] In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 155 and 156. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 155 and 156. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 155 and 156. In some embodiments, the polypeptide or polypeptide complex comprises the amino acid sequences of SEQ ID NO: 155 and 156.

[0224] In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 157 and 158. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 157 and 158. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 157 and 158. In some embodiments, the polypeptide or polypeptide complex comprises the amino acid sequences of SEQ ID NO: 157 and 158.

[0225] In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 159 and 160. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 159 and 160. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 159 and 160. In some embodiments, the polypeptide or polypeptide complex comprises the amino acid sequences of SEQ ID NO: 159 and 160.

[0226] In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 161 and 162. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 161 and 162. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 161 and 162. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 161 and 162. In some embodiments, the polypeptide or polypeptide complex comprises the amino acid sequences of SEQ ID NO: 161 and 162.

[0227] In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 163 and 164. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 163 and 164. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 163 and 164. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 163 and 164. In some embodiments, the polypeptide or polypeptide complex comprises the amino acid sequences of SEQ ID NO: 163 and 164.

[0228] In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 165 and 166. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 165 and 166. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 165 and 166. In some embodiments, the polypeptide or polypeptide complex comprises the amino acid sequences of SEQ ID NO: 165 and 166.

[0229] In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 167 and 168. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 167 and 168. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 167 and 168. In some embodiments, the polypeptide or polypeptide complex comprises the amino acid sequences of SEQ ID NO: 167 and 168.

[0230] In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 169 and 170. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 169 and 170. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 169 and 170. In some embodiments, the polypeptide or polypeptide complex comprises the amino acid sequences of SEQ ID NO: 169 and 170.

[0231] In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 171 and 172. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 171 and 172. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 171 and 172. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 171 and 172. In some embodiments, the polypeptide or polypeptide complex comprises the amino acid sequences of SEQ ID NO: 171 and 172.

[0232] In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 173 and 174. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 173 and 174. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 173 and 174. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 173 and 174. In some embodiments, the polypeptide or polypeptide complex comprises the amino acid sequences of SEQ ID NO: 173 and 174.

[0233] In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 175 and 176. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 175 and 176. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 175 and 176. In some embodiments, the polypeptide or polypeptide complex comprises the amino acid sequences of SEQ ID NO: 175 and 176.

[0234] In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 177 and 178. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 177 and 178. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 177 and 178. In some embodiments, the polypeptide or polypeptide complex comprises the amino acid sequences of SEQ ID NO: 177 and 178.

[0235] In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4330 and 4331. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4332 and 4333. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4334 and 4335. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4336 and 4337.

[0236] In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4338 and 4339. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4340 and 4341. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4342 and 4343. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4344 and 4345. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4346 and 4347. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4348 and 4349. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4350 and 4351. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4352 and 4353. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4352 and 4353. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4354 and 4355. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4356 and 4357. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4358 and 4359.In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4360 and 4361. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4362 and 4363. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4364 and 4365. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4366 and 4367. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4368 and 4369. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4370 and 4371. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4372 and 4373. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4374 and 4375. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4376 and 4377. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4378 and 4379. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4380 and 4381. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4382 and 4383.In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4384 and 4385. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4386 and 4387.

[0237] In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4386 and 4387. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4388 and 4389. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4390 and 4391. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4392 and 4393. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4394 and 4395. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4396 and 4397. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4398 and 4399. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4400 and 4401. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4402 and 4403. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4404 and 4405. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4406 and 4407.In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4408 and 4409. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4410 and 4411. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4412 and 4413. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4414 and 4415. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4416 and 4417. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4418 and 4419. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4420 and 4421. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4422 and 4423. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4424 and 4425. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4426 and 4427. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4428 and 4429. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4430 and 4431.In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4432 and 4433. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4434 and 4435. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4436 and 4437. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4438 and 4439. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4440 and 4441. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4442 and 4443. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4444 and 4445. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4446 and 4447.

[0238] Table 13. Tumor-activating antibodies that bind to CD28 and PSMA L 1a -P 1a -H 1a In some embodiments, this document discloses polypeptides or polypeptide complexes isolated according to Formula II: L 1a -P 1a -H 1a L 1a It contains a tumor-specific protease that can cleave the junctional region, which, when not cleaved, will release P 1a Linked to anti-CD28 antibody, and anti-CD28 antibody linked to anti-PSMA antibody, and P 1a It contains peptides that weaken the binding of anti-CD28 antibodies to CD28, and H 1aIt contains molecules that extend the half-life. In some embodiments, when L... 1a When not cut, P 1a This weakens the binding of anti-CD28 antibodies to CD28. In some implementations, anti-CD28 antibodies comprise human or humanized antibodies.

[0239] In some implementations, H 1a Contains a polymer. In some embodiments, the polymer is polyethylene glycol (PEG). In some embodiments, H 1a Contains albumin. In some implementations, H 1a It contains an Fc domain. In some embodiments, albumin is serum albumin. In some embodiments, albumin is human serum albumin. In some embodiments, H... 1a It comprises a peptide, ligand, or small molecule. In some embodiments, the peptide, ligand, or small molecule binds to a serum protein or a fragment thereof, a circulating immunoglobulin or a fragment thereof, or CD35 / CR1. In some embodiments, the serum protein comprises thyroxine-binding protein, transthyroxine, 1-acid glycoprotein, transferrin, transferrin receptor or its transferrin-binding moiety, fibrinogen, or albumin. In some embodiments, the circulating immunoglobulin molecule comprises IgG1, IgG2, IgG3, IgG4, sIgA, IgM, or IgD. In some embodiments, the serum protein is albumin. In some embodiments, the peptide is an antibody. In some embodiments, the antibody comprises a single-domain antibody, a single-chain variable fragment, or Fab. In some embodiments, the antibody comprises a single-domain antibody that binds to albumin. In some embodiments, the antibody is a human or humanized antibody. In some embodiments, the single-domain antibody is 645gH1gL1. In some embodiments, the single-domain antibody is 645dsgH5gL4. In some embodiments, the single-domain antibody is 23-13-A01-sc02. In some embodiments, the single-domain antibody is A10m3 or a fragment thereof. In some embodiments, the single-domain antibody is DOM7r-31. In some embodiments, the single-domain antibody is DOM7h-11-15. In some embodiments, the single-domain antibody is Alb-1, Alb-8, or Alb-23. In some embodiments, the single-domain antibody is 10E.

[0240] In some embodiments, the single-domain antibody includes complementarity-determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein HC-CDR1, HC-CDR2, and HC-CDR3 of the single-domain antibody comprise: HC-CDR1: SEQ ID NO: 138, HC-CDR2: SEQ ID NO: 139, and HC-CDR3: SEQ ID NO: 140. In some embodiments, the single-domain antibody includes complementarity-determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein HC-CDR1, HC-CDR2, and HC-CDR3 of the single-domain antibody comprise: HC-CDR1: SEQ ID NO: 142, HC-CDR2: SEQ ID NO: 143, and HC-CDR3: SEQ ID NO: 144. In some embodiments, the single-domain antibody comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 141. In some embodiments, the single-domain antibody comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 146. In some embodiments, the single-domain antibody comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 145. In some embodiments, the single-domain antibody comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 4448. In some embodiments, the single-domain antibody is SA21.

[0241] In some implementations, H 1a Includes H 1a Connect to P 1a Connection part (L) 2a In some implementations, L 2a It is a peptide sequence having at least 5 and no more than 50 amino acids. In some embodiments, L 2a It is a peptide sequence having at least 10 and no more than 30 amino acids. In some embodiments, L 2a It is a peptide sequence having at least 10 amino acids. In some embodiments, L 2a It is a peptide sequence having at least 18 amino acids. In some embodiments, L 2a It is a peptide sequence having at least 26 amino acids. In some embodiments, L 2a It has the characteristics selected from (G2S) n (GS) n (GSGGS) n(SEQ ID NO: 606), (GGGS) n (SEQ ID NO: 607), (GGGGS) n (SEQ ID NO: 608) and (GSSGGS) n The formula (SEQ IDNO: 609), where n is an integer of at least 1. In some implementations, L 2a It contains the amino acid sequence according to SEQ ID NO: 110.

[0242] In some implementation schemes, P 1a It has less than 70% sequence homology with CD28. In some implementations, P 1a It contains a peptide sequence of at least 10 amino acids in length. In some embodiments, P 1a It comprises a peptide sequence with a length of at least 10 amino acids and a length of no more than 20 amino acids. In some embodiments, P 1a It contains a peptide sequence of at least 16 amino acids in length. In some embodiments, P 1a It contains a peptide sequence of no more than 40 amino acids in length. In some embodiments, P 1a It contains at least two cysteine ​​amino acid residues. In some embodiments, P 1a It contains cyclic or linear peptides. In some embodiments, P 1a It contains a cyclic peptide. In some embodiments, P 1a It contains a linear peptide. In some embodiments, P 1a Contains an amino acid sequence according to any one of SEQ ID NO: 33 to 106 or 732-813. In some embodiments, P 1a Contains the amino acid sequence of SEQ ID NO: 73. In some embodiments, P 1a Contains the amino acid sequence of SEQ ID NO: 784. In some embodiments, P 1a A peptide sequence comprising the amino acid sequence of SEQ ID NO: 73 or having one, two, or three amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 73. In some embodiments, P 1a Contains the amino acid sequence of SEQ ID NO: 73. In some embodiments, P 1a A peptide sequence comprising the amino acid sequence of SEQ ID NO: 103 or having one, two, or three amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 103. In some embodiments, P 1a The amino acid sequence containing SEQ ID NO: 103.

[0243] In some implementation schemes, P 1a Includes X1-X2-X3-C-X4-X5-X6-X7-X8-X9-X 10 -CX 11 -X 12 The amino acid sequence is defined as follows: X1 is selected from M, I, L, and V; X2 is selected from D, H, N, A, F, S, T, Y, and V; X3 is selected from W, L, and F; X4 is selected from P, A, and L; X5 is selected from R, T, I, M, S, K, L, V, W, F, A, P, and D; X6 is selected from E, D, Y, H, S, F, A, N, T, I, P, and V; X7 is selected from L, M, R, S, Q, and H; X8 is selected from W and Q; X9 is selected from 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 Selected from F, Y, L, W, and V; and X 12 The X1 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 Selected from F, Y, and L; and X 12 The X1 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 It is F; and X 12 Selected from N, A, and F. In some implementations, P 1a It comprises the amino acid sequence according to SEQ ID NO: 41 or an amino acid sequence having one, two, or three amino acid mutations, substitutions, or deletions relative to SEQ ID NO: 41. In some embodiments, P 1a It contains the amino acid sequence according to SEQ ID NO: 41. In some embodiments, P 1a It contains an amino acid sequence selected from any one of SEQ ID NO: 41, 73, 103 and 179-603.

[0244] In some implementation schemes, P 1aIt contains the following amino acid sequence: J1-J2-WC-J3-J4-J5-J6-J7-J8-J9-CJ 10 -J 11 J1 is selected from A, R, N, D, Q, E, G, H, I, L, K, F, P, S, T, W, Y, and V; J2 is selected from D, P, and Y; J3 is selected from P and Q; J4 is selected from R, L, and I; J5 is selected from D, H, Y, and I; J6 is selected from L, S, and R; J7 is selected from W and G; J8 is selected from V, A, D, H, L, and N; J9 is selected from H, L, E, and D; J 10 Selected from F, Y, and L; and J 11 Selected from A, N, H, and T. In some embodiments, J1 is selected from A, R, N, D, Q, E, G, H, K, F, P, S, T, W, and Y. In some embodiments, J1 is selected from R, Q, E, H, I, L, K, F, T, W, Y, and V; J2 is D; J3 is P; J4 is R; J5 is D; J6 is L; J7 is W; J8 is V; J9 is H; J 10 It is F; and J 11 It is A. In some implementations, J1 is selected from R, Q, E, L, K, T, and W; J2 is D; J3 is P; J4 is R; J5 is D; J6 is L; J7 is W; J8 is V; J9 is H; J 10 It is F; and J 11 It is A. In some implementations, J1 is L or I; J2 is D or Y; J3 is P; J4 is R, L, or I; J5 is H, Y, or D; J6 is L or R; J7 is W; J8 is A, D, H, or N; J9 is L, E, or D; J 10 It is F or Y; and J 11 It is N or H. In some implementations, J1 is L or I; J2 is D or Y; J3 is P; J4 is R or I; J5 is H, Y, or D; J6 is L or R; J7 is W; J8 is A, D, or N; J9 is L, E, or D; J 10 It is F or Y; and J 11 It is N.

[0245] In some implementation schemes, P 1a It contains the following amino acid sequence: Z1-C-Z2-Z3-Z4-Z5-Z6-Z7-Z8-Z9-Z 10 -Z 11 -CZ 12Z1 is selected from Y, H, and A; Z2 is selected from D, E, A, Q, S, V, M, H, L, F, T, W, and N; Z3 is selected from G, E, S, and A; Z4 is selected from H, L, W, and A; Z5 is selected from L, S, A, T, I, V, M, R, E, D, F, H, K, Q, N, Y, W, and G; Z6 is selected from P, A, E, Q, S, L, W, G, V, D, K, I, M, R, T, F, H, N, and Y; Z7 is selected from E, Q, I, M, V, A, L, R, W, P, D, T, K, S, G, H, and N; Z8 is selected from Y, H, F, W, N, Q, and A; Z9 is selected from M, I, L, R, V, K, F, Q, Y, W, and A; Z 10 Selected from A, Q, S, W, E, L, G, D, T, M, N, V, H, R, Y, K, P, F, and I; Z 11 Selected from K, Q, N, H, E, P, and A; and Z 12 The Z1 is selected from L, M, I, F, V, Y, Q, D, T, and A. In some embodiments, Z1 is selected from Y and H; Z2 is selected from D, E, A, Q, S, V, M, H, L, F, T, W, and N; Z3 is selected from G, E, and S; Z4 is selected from H, L, and W; Z5 is selected from L, S, A, T, I, V, M, R, E, D, F, H, K, Q, N, Y, W, and G; Z6 is selected from P, A, E, Q, S, L, W, G, V, D, K, I, M, R, T, F, H, N, and Y; Z7 is selected from E, Q, I, M, V, A, L, R, W, P, D, T, K, S, G, H, and N; Z8 is selected from Y, H, F, W, N, and Q; Z9 is selected from M, I, L, R, V, K, F, Q, Y, and W; Z 10 Selected from A, Q, S, W, E, L, G, D, T, M, N, V, H, R, Y, K, P, F, and I; Z 11 Selected from K, Q, N, H, E, and P; and Z 12 The numbers are selected from L, M, I, F, V, Y, Q, D, and T. In some embodiments, Z1 is selected from Y and H; Z2 is selected from D, E, A, Q, S, V, M, H, L, F, T, W, and N; Z3 is selected from G, E, and S; Z4 is selected from H, L, and W; Z5 is selected from L, S, A, T, I, V, M, R, E, D, F, H, K, Q, N, Y, W, and G; Z6 is selected from P, A, E, Q, S, L, W, G, V, D, K, I, M, R, T, F, H, N, and Y; Z7 is selected from E, Q, I, M, V, A, L, R, W, P, D, T, K, S, G, H, and N; Z8 is selected from Y, H, F, W, N, and Q; Z9 is selected from M, I, L, R, V, K, F, Q, Y, and W; Z 10 Selected from A, Q, S, W, E, L, G, D, T, M, N, V, H, R, Y, K, P, F, and I; Z 11Selected from K, Q, N, H, E, and P; and Z 12 The Z1 is selected from L, M, I, F, V, Y, Q, D, and T. In some embodiments, Z1 is Y; Z2 is selected from D, E, A, and Q; Z3 is G; Z4 is H; Z5 is selected from L, S, A, T, I, V, and M; Z6 is selected from P, A, E, Q, S, L, W, G, V, and D; Z7 is selected from E, Q, I, M, V, A, and L; Z8 is selected from Y and H; Z9 is selected from M, I, and L; Z 10 Selected from A, Q, S, W, E, L, G, D, T, M, and N; Z 11 It is K; and Z 12 Z1 is selected from L and M. In some embodiments, Z1 is Y; Z2 is selected from D, E, and A; Z3 is G; Z4 is H; Z5 is selected from L, S, A, and T; Z6 is selected from P, A, E, Q, S, L, and W; Z7 is selected from E, Q, I, and M; Z8 is Y; Z9 is selected from M, I, and L; Z 10 Selected from A, Q, S, W, E, L, G, and D; Z 11 It is K; and Z 12 It is L. In some implementations, Z1 is Y; Z2 is selected from D and E; Z3 is G; Z4 is H; Z5 is selected from L, S and A; Z6 is selected from P, A, E, Q, S and L; Z7 is selected from E, Q and I; Z8 is Y; Z9 is selected from M, I and L; Z 10 Selected from A, Q, S, W, E, L, G, and D; Z 11 It is K; and Z 12 It is L.

[0246] In some implementation schemes, P 1a Contains the amino acid sequence of SEQ ID NO: 784. In some embodiments, P 1a It consists of the amino acid sequence of SEQ ID NO: 784. In some embodiments, P 1a Contains the amino acid sequence of SEQ ID NO: 744. In some embodiments, P 1a It consists of the amino acid sequence of SEQ ID NO: 744. In some embodiments, P 1a Contains an amino acid sequence according to any one of SEQ ID NO: 774-793. In some embodiments, P 1a It contains an amino acid sequence according to any one of SEQ ID NO: 764-773.

[0247] In some implementation schemes, P 1a It contains amino acids according to the following sequence: U1-C-U2-U3-U4-U5-U6-U7-U8-U9-U 10 -U 11-CU 12 Among them: U1 is selected from T, S, L, M, W, Q, P, R, G, N, I, Y, D, E, V, H and A; U2 is selected from A, S, T, K, E, R, G, N, W and D; U3 is selected from A, E, P, D, G, W, Q, V and F; U4 is selected from G, V, F, D, E, R and A; U5 is selected from A, F, V, S, L, P and M; U6 is selected from T, S, H, M, A, N, P, G, Q, D, Y, V, L, I, E, K, F and R; U7 is selected from Y, W, F, L, N, T, Q, I and A; U8 is selected from P, S, T, A, E, K, Q, N, V, G, H, L, D, Y, I, M and R; U9 is selected from W, L and A; U 10 Selected from P, S, R, L, E, and A; U 11 Selected from L, A, T, M, V, W, Y, E, Q, I, F, S, P, K, R, D, H, N, and G; and U 12 The elements are selected from L, F, V, M, I, Y, E, W, and A. In some embodiments, U1 is selected from T, S, L, M, W, Q, P, R, G, N, I, Y, D, E, V, and H; U2 is selected from A, S, T, K, E, R, G, N, W, and D; U3 is selected from A, E, P, D, G, W, Q, V, and F; U4 is selected from G, V, F, D, E, and R; U5 is selected from A, F, V, S, L, P, and M; U6 is selected from T, S, H, M, A, N, P, G, Q, D, Y, V, L, I, E, K, F, and R; U7 is selected from Y, W, F, L, N, T, Q, and I; U8 is selected from P, S, T, A, E, K, Q, N, V, G, H, L, D, Y, I, M, and R; U9 is selected from W, L, and A; U 10 Selected from P, S, R, L, and E; U 11 Selected from L, A, T, M, V, W, Y, E, Q, I, F, S, P, K, R, D, H, N, and G; and U 12 The elements are selected from L, F, V, M, I, Y, E, and W. In some embodiments, U1 is selected from T, S, L, M, and W; U2 is selected from A, S, and T; U3 is selected from A and E; U4 is selected from G, V, and F; U5 is selected from A, F, and V; U6 is selected from T, S, H, M, A, and N; U7 is selected from Y, W, and F; U8 is selected from P, S, T, A, E, K, Q, N, and V; U9 is selected from W and L; U 10 Selected from P and S; U 11 Selected from L, A, T, M, V, W, Y, E, Q, I, F, and S; and U 12 The elements are selected from L, F, V, and M. In some embodiments, U1 is selected from T and S; U2 is selected from A and S; U3 is A; U4 is selected from G and V; U5 is selected from A and F; U6 is selected from T, S, and H; U7 is selected from Y and W; U8 is selected from P, S, T, and A; U9 is W; U10 It is P; U 11 Selected from L, A, T, M, V, W, and Y; and U 12 Selected from L and F.

[0248] In some implementation schemes, P 1a Contains the amino acid sequence of SEQ ID NO: 749. In some embodiments, P 1a Contains the amino acid sequence of SEQ ID NO: 749. In some embodiments, P 1a Contains an amino acid sequence according to any one of SEQ ID NO: 756-763. In some embodiments, P 1a Contains an amino acid sequence according to any one of SEQ ID NO: 794-813. In some embodiments, P 1a It contains an amino acid sequence according to any one of SEQ ID NO: 732-2002.

[0249] Modified amino acids In some embodiments, the isolated polypeptide or polypeptide complex comprises modified amino acids, non-natural amino acids, modified non-natural amino acids, or combinations thereof. In some embodiments, the non-natural amino acids include D-amino acids. In some embodiments, the modified amino acids or modified non-natural amino acids comprise post-translational modifications.

[0250] Pharmaceutical Composition In some embodiments, pharmaceutical compositions comprising an isolated polypeptide or polypeptide complex of any of the embodiments disclosed herein are disclosed.

[0251] In some embodiments, this document discloses pharmaceutical compositions comprising isolated peptides or peptide complexes containing a first antigen recognition molecule that binds to CD28 and a second antigen recognition molecule that binds to a prostate-specific membrane antigen. In some embodiments, the first antigen recognition molecule that binds to CD28 comprises an anti-CD28 antibody. In some embodiments, the anti-CD28 antibody comprises an anti-CD28 heavy chain variable domain comprising complementarity-determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein HC-CDR1, HC-CDR2, and HC-CDR3 comprise the following amino acid sequences: HC-CDR1: SEQ ID NO: 1, HC-CDR2: SEQ ID NO: 2, and HC-CDR3: SEQ ID NO: 3; and the anti-CD28 antibody comprises an anti-CD28 light chain variable domain comprising complementarity-determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein LC-CDR1, LC-CDR2, and LC-CDR3 comprise the following amino acid sequences: LC-CDR1: SEQ ID NO: 4, LC-CDR2: (KAS), and LC-CDR3: SEQ ID NO: 6. In some embodiments, the anti-CD28 heavy chain variable domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 7, and wherein the anti-CD28 light chain variable domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 8. In some embodiments, the anti-CD28 antibody comprises a single-chain variable fragment (scFv), a single-domain antibody, Fab, Fab', or CrossFab. In some embodiments, the anti-CD28 antibody comprises scFv. In some embodiments, the anti-CD28 antibody comprises a single-chain variable fragment (scFv) having engineered disulfide bonds. In some embodiments, the anti-CD28 antibody comprises scFv, and the scFv comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 9. In some embodiments, the anti-CD28 antibody comprises scFv, and the scFv comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 10.In some embodiments, the anti-CD28 antibody comprises scFv, and the scFv comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 11. In some embodiments, the anti-CD28 antibody comprises scFv, and the scFv comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 12. In some embodiments, the anti-CD28 antibody comprises Fab. In some embodiments, Fab comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 696 and an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 697. In some embodiments, the anti-CD28 antibody comprises CrossFab. In some embodiments, CrossFab comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 698 and an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 699. In some embodiments, CrossFab comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 700 and an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 701.

[0252] In some implementations, the second antigen recognition molecule that binds to PSMA contains an anti-PSMA antibody. In some embodiments, the anti-PSMA antibody comprises an anti-PSMA heavy chain variable domain comprising complementarity-determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein HC-CDR1, HC-CDR2, and HC-CDR3 comprise the following amino acid sequences: HC-CDR1: SEQ ID NO: 13, HC-CDR2: SEQ ID NO: 14, and HC-CDR3: SEQ ID NO: 15; and the anti-PSMA antibody comprises an anti-PSMA light chain variable domain comprising complementarity-determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, LC-CDR2, and LC-CDR3 of the light chain variable domain comprise the following amino acid sequences: LC-CDR1: SEQ ID NO: 16, LC-CDR2: (EA), and LC-CDR3: SEQ ID NO: 18. In some embodiments, the anti-PSMA heavy chain variable domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 20, and the anti-PSMA light chain variable domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 19. In some embodiments, the anti-PSMA antibody comprises a single-chain variable fragment (scFv), a single-domain antibody, Fab, Fab', or CrossFab. In some embodiments, the anti-PSMA antibody comprises Fab or Fab'. In some embodiments, the anti-PSMA antibody comprises Fab or Fab', and Fab or Fab' comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 19 and an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 20. In some embodiments, the anti-PSMA antibody comprises Fab or Fab', and Fab or Fab' comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 702 and an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 703.In some embodiments, the anti-PSMA antibody comprises Fab or Fab', and Fab or Fab' comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 704 and an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 705.

[0253] In some embodiments, the peptide or peptide complex further comprises a detectable label, a therapeutic agent, or a pharmacokinetic modification. In some embodiments, the detectable label comprises a fluorescent label, a radiolabel, an enzyme, a nucleic acid probe, or a contrast agent.

[0254] For administration to a subject, the polypeptides or polypeptide complexes disclosed herein may be provided in a pharmaceutical composition together with one or more pharmaceutically acceptable carriers or excipients. The term "pharmaceutically acceptable carrier" includes, but is not limited to, any carrier that does not interfere with the bioactivity and efficacy of the component and is non-toxic to the patient administering it. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate-buffered saline solutions, water, emulsions (e.g., oil / water emulsions), various types of wetting agents, sterile solutions, etc. Such carriers can be formulated by conventional methods and can be administered to a subject at appropriate doses. Preferably, the composition is sterile. These compositions may also contain adjuvants, such as preservatives, emulsifiers, and dispersants. Prevention of microbial action can be ensured by incorporating various antimicrobial and antifungal agents.

[0255] The pharmaceutical composition may be in any suitable form, depending on the desired method of administration. It may be provided in unit dosage forms, in sealed containers, or as part of a kit. Such kits may include instructions for use. They may include multiple unit dosage forms.

[0256] Pharmaceutical compositions may be suitable for administration via any suitable route, including parenteral (e.g., subcutaneous, intramuscular, or intravenous) routes. Such compositions may be prepared by any method known in the pharmaceutical field, such as by mixing the active ingredient with a carrier or excipient under aseptic conditions.

[0257] The dosage of the disclosed substance can vary over a wide range, depending on the disease or condition to be treated, the age and condition of the individual to be treated, and the physician will ultimately determine the appropriate dosage.

[0258] isolated recombinant nucleic acid molecules This document discloses isolated recombinant nucleic acid molecules encoding any of the embodiments disclosed herein, including isolated polypeptides or polypeptide complexes.

[0259] In some embodiments, this document discloses isolated recombinant nucleic acid molecules encoding peptides or peptide complexes comprising a first antigen recognition molecule that binds to CD28 and a second antigen recognition molecule that binds to a prostate-specific membrane antigen. In some embodiments, the first antigen recognition molecule that binds to CD28 comprises an anti-CD28 antibody. In some embodiments, the anti-CD28 antibody comprises an anti-CD28 heavy chain variable domain comprising complementarity-determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein HC-CDR1, HC-CDR2, and HC-CDR3 comprise the following amino acid sequences: HC-CDR1: SEQ ID NO: 1, HC-CDR2: SEQ ID NO: 2, and HC-CDR3: SEQ ID NO: 3; and the anti-CD28 antibody comprises an anti-CD28 light chain variable domain comprising complementarity-determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein LC-CDR1, LC-CDR2, and LC-CDR3 comprise the following amino acid sequences: LC-CDR1: SEQ ID NO: 4, LC-CDR2: (KAS), and LC-CDR3: SEQ ID NO: 6. In some embodiments, the anti-CD28 heavy chain variable domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 7, and wherein the anti-CD28 light chain variable domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 8. In some embodiments, the anti-CD28 antibody comprises a single-chain variable fragment (scFv), a single-domain antibody, Fab, or Fab'. In some embodiments, the anti-CD28 antibody comprises scFv. In some embodiments, the anti-CD28 antibody comprises a single-chain variable fragment (scFv) having engineered disulfide bonds. In some embodiments, the anti-CD28 antibody comprises scFv, and the scFv comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 9. In some embodiments, the anti-CD28 antibody comprises an scFv, and the scFv comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 10. In some embodiments, the anti-CD28 antibody comprises an scFv, and the scFv comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 11.In some embodiments, the anti-CD28 antibody comprises scFv, and the scFv comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 12. In some embodiments, the anti-CD28 antibody comprises Fab. In some embodiments, Fab comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 696 and an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 697. In some embodiments, the anti-CD28 antibody comprises CrossFab. In some embodiments, CrossFab comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 698 and an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 699. In some embodiments, CrossFab comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 700 and an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 701.

[0260] In some implementations, the second antigen recognition molecule that binds to PSMA contains an anti-PSMA antibody. In some embodiments, the anti-PSMA antibody comprises an anti-PSMA heavy chain variable domain comprising complementarity-determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein HC-CDR1, HC-CDR2, and HC-CDR3 comprise the following amino acid sequences: HC-CDR1: SEQ ID NO: 13, HC-CDR2: SEQ ID NO: 14, and HC-CDR3: SEQ ID NO: 15; and the anti-PSMA antibody comprises an anti-PSMA light chain variable domain comprising complementarity-determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, LC-CDR2, and LC-CDR3 of the light chain variable domain comprise the following amino acid sequences: LC-CDR1: SEQ ID NO: 16, LC-CDR2: (EA), and LC-CDR3: SEQ ID NO: 18. In some embodiments, the anti-PSMA heavy chain variable domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 20, and the anti-PSMA light chain variable domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 19. In some embodiments, the anti-PSMA antibody comprises a single-chain variable fragment (scFv), a single-domain antibody, Fab, Fab', or CrossFab. In some embodiments, the anti-PSMA antibody comprises Fab or Fab'. In some embodiments, the anti-PSMA antibody comprises Fab or Fab', and Fab or Fab' comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 19 and an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 20. In some embodiments, the anti-PSMA antibody comprises Fab or Fab', and Fab or Fab' comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 702 and an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 703.In some embodiments, the anti-PSMA antibody comprises Fab or Fab', and Fab or Fab' comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 704 and an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 705.

[0261] Treatment In some embodiments, this document discloses methods for treating cancer in a subject of need, comprising administering to the subject a separated polypeptide or polypeptide complex of any of the embodiments disclosed herein. In some embodiments, the cancer has cells expressing PSMA. In some cases, the cancer is a solid tumor. In some embodiments, the cancer is lung cancer, breast cancer (e.g., HER2-positive). + ;ER / PR + (TNBC), cervical cancer, ovarian cancer, colorectal cancer, pancreatic cancer, or stomach cancer.

[0262] In some embodiments, this document discloses a method for treating prostate cancer in a subject of need, comprising administering to the subject a polypeptide or polypeptide complex isolated as described herein. In some embodiments, this document discloses a method for treating metastatic castration-resistant prostate cancer (mCRPC) in a subject of need, comprising administering to the subject a polypeptide or polypeptide complex isolated as described herein.

[0263] In some embodiments, this document discloses a method of treating cancer in a subject of need, comprising administering to the subject a polypeptide or polypeptide complex comprising a first antigen recognition molecule that binds to CD28 and a second antigen recognition molecule that binds to a prostate-specific membrane antigen. In some embodiments, the first antigen recognition molecule that binds to CD28 comprises an anti-CD28 antibody. In some embodiments, the anti-CD28 antibody comprises an anti-CD28 heavy chain variable domain comprising complementarity-determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein HC-CDR1, HC-CDR2, and HC-CDR3 comprise the following amino acid sequences: HC-CDR1: SEQ ID NO: 1, HC-CDR2: SEQ ID NO: 2, and HC-CDR3: SEQ ID NO: 3; and the anti-CD28 antibody comprises an anti-CD28 light chain variable domain comprising complementarity-determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein LC-CDR1, LC-CDR2, and LC-CDR3 comprise the following amino acid sequences: LC-CDR1: SEQ ID NO: 4, LC-CDR2: (KAS), and LC-CDR3: SEQ ID NO: 6. In some embodiments, the anti-CD28 heavy chain variable domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 7, and wherein the anti-CD28 light chain variable domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 8. In some embodiments, the anti-CD28 antibody comprises a single-chain variable fragment (scFv), a single-domain antibody, Fab, Fab', or CrossFab. In some embodiments, the anti-CD28 antibody comprises scFv. In some embodiments, the anti-CD28 antibody comprises a single-chain variable fragment (scFv) having engineered disulfide bonds. In some embodiments, the anti-CD28 antibody comprises scFv, and the scFv comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 9. In some embodiments, the anti-CD28 antibody comprises scFv, and the scFv comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 10.In some embodiments, the anti-CD28 antibody comprises scFv, and the scFv comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 11. In some embodiments, the anti-CD28 antibody comprises scFv, and the scFv comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 12. In some embodiments, the anti-CD28 antibody comprises Fab. In some embodiments, Fab comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 696 and an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 697. In some embodiments, the anti-CD28 antibody comprises CrossFab. In some embodiments, CrossFab comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 698 and an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 699. In some embodiments, CrossFab comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 700 and an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 701.

[0264] In some implementations, the second antigen recognition molecule that binds to PSMA contains an anti-PSMA antibody. In some embodiments, the anti-PSMA antibody comprises an anti-PSMA heavy chain variable domain comprising complementarity-determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein HC-CDR1, HC-CDR2, and HC-CDR3 comprise the following amino acid sequences: HC-CDR1: SEQ ID NO: 13, HC-CDR2: SEQ ID NO: 14, and HC-CDR3: SEQ ID NO: 15; and the anti-PSMA antibody comprises an anti-PSMA light chain variable domain comprising complementarity-determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, LC-CDR2, and LC-CDR3 of the light chain variable domain comprise the following amino acid sequences: LC-CDR1: SEQ ID NO: 16, LC-CDR2: (EA), and LC-CDR3: SEQ ID NO: 18. In some embodiments, the anti-PSMA heavy chain variable domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 20, and the anti-PSMA light chain variable domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 19. In some embodiments, the anti-PSMA antibody comprises a single-chain variable fragment (scFv), a single-domain antibody, Fab, Fab', or CrossFab. In some embodiments, the anti-PSMA antibody comprises Fab or Fab'. In some embodiments, the anti-PSMA antibody comprises Fab or Fab', and Fab or Fab' comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 19 and an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 20. In some embodiments, the anti-PSMA antibody comprises Fab or Fab', and Fab or Fab' comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 702 and an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 703.In some embodiments, the anti-PSMA antibody comprises Fab or Fab', and Fab or Fab' comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 704 and an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 705.

[0265] Combination therapy In some embodiments, the peptide or peptide complex induces T cell-mediated cytotoxicity in tumor cells. In some embodiments, the cancer has cells expressing PSMA. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is lung cancer, breast cancer, cervical cancer, ovarian cancer, colorectal cancer, pancreatic cancer, or gastric cancer.

[0266] In some embodiments, the peptide or peptide complex is administered in combination with an anticancer therapy. In some embodiments, the peptide or peptide complex is administered with the anticancer therapy in the same pharmaceutical composition. In some embodiments, the peptide or peptide complex and the anticancer therapy are administered as a separate pharmaceutical composition. In some embodiments, the anticancer therapy comprises a small molecule, a cell-based therapy, or an antibody-based therapy.

[0267] In some embodiments, the antibody-based therapy is a T-cell connector. In some embodiments, the T-cell connector comprises according to the formula: D-L0-E (Formula II), wherein D comprises an effector cell binding domain that binds to an effector cell antigen, E comprises a tumor antigen binding domain that binds to a tumor antigen, and L0 comprises a connector linking D to E. In some embodiments, D comprises a single-chain variable fragment, a single-domain antibody, or a Fab fragment. In some embodiments, D comprises a single-chain variable fragment. In some embodiments, E comprises a single-chain variable fragment, a single-domain antibody, or a Fab fragment. In some embodiments, E comprises a Fab fragment.

[0268] In some implementations, the effector cell binding domain includes a complementarity-determining region (CDR) selected from: moromumab-CD3 (OKT3), ostalizumab (TRX4), telizumab (MGA031), vexizumab (Nuvion), SP34, X35, VIT3, and BMA030. (BW264 / 56), CLB-T3 / 3, CRIS7, YTH12.5, F111-409, 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. In some embodiments, the effector cell binding domain comprises the amino acid sequence according to SEQ ID NO: 611-624.

[0269] Table 14. Amino acid sequence of effector cell binding domain In some embodiments, the tumor antigen comprises epidermal growth factor receptor (EGFR), prostate-specific membrane antigen (PSMA), or tumor-associated calcium signaling transducer 2 (hereinafter referred to as TROP2). In some embodiments, the tumor antigen comprises EGFR. In some embodiments, the tumor antigen-binding domain comprises the amino acid sequence according to SEQ ID NO: 629-638. In some embodiments, the tumor antigen comprises EGFR, and the tumor-binding domain comprises 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: 632; HC-CDR2: SEQ ID NO: 633; ​​HC-CDR3: SEQ ID NO: 634; and wherein LC-CDR1, LC-CDR2, and LC-CDR3 comprise: LC-CDR1: SEQ ID NO: 629; LC-CDR2: (YAS); and LC-CDR3: SEQ ID NO: 631. In some embodiments, the tumor antigen comprises EGFR, and the T-cell adaptor comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NOs: 639 and 640. In some implementations, the tumor antigen comprises EGFR, and the T-cell connector comprises the amino acid sequence according to SEQ ID NO: 641 and 642.

[0270] In some embodiments, the tumor antigen comprises TROP2. In some embodiments, the tumor antigen comprises TROP2, and the tumor-binding domain comprises 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: 659; HC-CDR2: SEQ ID NO: 660; HC-CDR3: SEQ ID NO: 661, 662, or 663; and wherein LC-CDR1, LC-CDR2, and LC-CDR3 comprise: LC-CDR1: SEQ ID NO: 664; LC-CDR2: (SAS); and LC-CDR3: SEQ ID NO: 666. In some embodiments, the tumor antigen comprises TROP2, and the T-cell adaptor comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 673 and 674. In some embodiments, the tumor antigen comprises TROP2, and the T-cell adaptor comprises an amino acid sequence according to SEQ ID NO: 675 and 676 or SEQ ID NO: 677 and 678. In some embodiments, the tumor antigen-binding domain comprises an amino acid sequence according to SEQ ID NO: 659-668.

[0271] 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 NO: 647-654. In some embodiments, the tumor antigen comprises PSMA, and the tumor-binding domain comprises 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: 647; HC-CDR2: SEQ ID NO: 648; HC-CDR3: SEQ ID NO: 649; and wherein LC-CDR1, LC-CDR2, and LC-CDR3 comprise: LC-CDR1: SEQ ID NO: 650; LC-CDR2: SEQ ID NO: 651; and LC-CDR3: SEQ ID NO: 652. In some embodiments, the tumor antigen comprises PSMA, and the T-cell adaptor comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 655 and 656.

[0272] Table 15. Amino acid sequence of tumor antigen-binding domain - anti-EGFR Table 16. Amino acid sequence of tumor antigen-binding domain - anti-PSMA Table 17. Amino acid sequence of tumor antigen-binding domain - anti-TROP2 In some embodiments, the T-cell adaptor molecule is selectively activated in the tumor microenvironment. In some embodiments, the T-cell adaptor is according to the formula: P3-L3-D-L0-E (Formula IIa), wherein D contains a CD3-binding domain; E contains a tumor antigen-binding domain; L0 contains a linker connecting D to E; P3 contains a peptide binding to D, and L3 contains a linker portion connecting D to P3 and is a substrate of a tumor-specific protease. In some embodiments, the T-cell adaptor is according to the formula: D-L0-E-L4-P4 (Formula IIb), wherein D contains a CD3-binding domain; E contains a tumor antigen-binding domain; L0 contains a linker connecting D to E; P4 contains a peptide binding to E, and L4 contains a linker portion connecting E to P4 and is a substrate of a tumor-specific protease. In some embodiments, the T-cell adaptor is based on the following formula: P3-L3-D-L0-E-L4-P4 (Formula IIc), wherein D contains a CD3-binding domain; E contains a tumor antigen-binding domain; L0 contains a linker connecting D to E; P3 contains a peptide that binds to D, and L3 contains a linker portion connecting D to P3 and is a substrate of a tumor-specific protease; P4 contains a peptide that binds to E, and L4 contains a linker portion connecting E to P4 and is a substrate of a tumor-specific protease.

[0273] In some embodiments, the T-cell adaptor comprises H1. In some embodiments, H1 comprises a sequence according to SEQ ID NO: 141 or SEQ ID NO: 146. In some embodiments, H1 comprises a single-domain antibody. In some embodiments, the single-domain antibody comprises complementarity-determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein HC-CDR1, HC-CDR2, and HC-CDR3 of the single-domain antibody comprise: HC-CDR1: SEQ ID NO: 138, HC-CDR2: SEQ ID NO: 139, and HC-CDR3: SEQ ID NO: 140.

[0274] In some embodiments, L3 or L4 is a peptide sequence having at least 5 to no more than 50 amino acids. In some embodiments, L3 or L4 is a peptide sequence having at least 10 to 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 contains (G2S). nThe formula is given, where n is an integer from 1 to 3 (SEQ ID NO: 605). In some embodiments, L3 or L4 has a subset (G2S). n The expression, where n is an integer of at least 1. In some implementations, L3 or L4 has a value selected from (G2S). n (GS) n (GSGGS) n (SEQ ID NO: 606), (GGGS) n (SEQ ID NO: 607), (GGGGS) n (SEQ ID NO:608) and (GSSGGS) n The expression (SEQ ID NO: 609) where n is an integer of at least 1.

[0275] In some embodiments, the tumor-specific protease is selected from matrix metalloproteinases, serine proteases, cysteine ​​proteases, threonine proteases, and aspartic proteases. In some embodiments, L3 or L4 comprises a urokinase-cleavable amino acid sequence, a matrix protease-cleavable amino acid sequence, or a matrix metalloproteinase-cleavable amino acid sequence. In some embodiments, L3 or L4 comprises a sequence according to any one of SEQ ID NO: 21-22 and 107-136. In some embodiments, L3 binds to the N-terminus of D. In some embodiments, L3 binds to the C-terminus of D. In some embodiments, L4 binds to the N-terminus of E. In some embodiments, L4 binds to the C-terminus of E.

[0276] In some embodiments, when L3 is cleaved by a tumor-specific protease, P3 unbinds to D, thereby exposing D to CD3. In some embodiments, when L4 is cleaved by a tumor-specific protease, P4 unbinds to E, thereby exposing E to the tumor antigen. In some embodiments, P3 weakens the binding of D to CD3. In some embodiments, P3 binds to D through ionic interactions, electrostatic interactions, hydrophobic interactions, π-stacking interactions, and hydrogen bonding interactions, or combinations thereof. In some embodiments, P3 binds to D at or near the antigen-binding site. In some embodiments, when L3 is cleaved by a tumor-specific protease, P3 unbinds to D, thereby exposing D to CD3.

[0277] In some embodiments, P3 shares less than 70% sequence identity with CD3. In some embodiments, P3 shares less than 70% sequence identity with CD3. In some embodiments, P3 shares less than 85% sequence identity with CD3. In some embodiments, P3 shares less than 90% sequence identity with CD3. In some embodiments, P3 shares less than 95% sequence identity with CD3. In some embodiments, P3 shares less than 98% sequence identity with CD3. In some embodiments, P3 shares less than 99% sequence identity with CD3. In some embodiments, P3 comprises the amino acid sequence according to SEQ ID NO: 625 or SEQ ID NO: 626. In some embodiments, P3 comprises a de novo amino acid sequence sharing less than 10% sequence identity with CD3.

[0278] In some embodiments, P4 weakens the binding of E to the tumor antigen. In some embodiments, P4 binds to E through ionic interactions, electrostatic interactions, hydrophobic interactions, π-stacking interactions, and hydrogen bonding interactions, or combinations thereof. In some embodiments, P4 binds to E at or near the antigen-binding site. In some embodiments, when L4 is cleaved by a tumor-specific protease, P4 unbinds to E, thereby exposing E 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 amino acid sequence with less than 10% sequence identity to the tumor antigen.

[0279] 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 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.

[0280] In some embodiments, P4 comprises the amino acid sequence according to SEQ ID NO: 645. In some embodiments, the tumor antigen comprises EGFR, and the T-cell connective comprises the amino acid sequences of SEQ ID NO: 643 and 644.

[0281] In some embodiments, P4 comprises an amino acid sequence according to any one of SEQ ID NO: 693-695. In some embodiments, the tumor antigen comprises TROP2, and the T-cell adaptor comprises the amino acid sequences of SEQ ID NO: 679 and 680, SEQ ID NO: 681 and 682, SEQ ID NO: 683 and 684, SEQ ID NO: 685 and 686, SEQ ID NO: 687 and 688, SEQ ID NO: 689 and 690, and SEQ ID NO: 691 and 692. In some embodiments, the tumor antigen comprises TROP2, and the T-cell adaptor comprises the amino acid sequences of SEQ ID NO: 4452 and 4453, SEQ ID NO: 4454 and 4455, SEQ ID NO: 4456 and 4457, SEQ ID NO: 4458 and 4459, SEQ ID NO: 4460 and 4461, SEQ ID NO: 4462 and 4463, or SEQ ID NO: 4464 and 4465.

[0282] In some implementations, the tumor antigen comprises PSMA, and the T-cell connector comprises the amino acid sequences of SEQ ID NO: 657 and 658.

[0283] Antibody production In some embodiments, antibodies or antigen-binding fragments thereof, as described herein, are generated 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.

[0284] In some cases, recombinant expression of antibodies or their binding fragments involves assembling nucleic acids encoding antibodies or their binding fragments from chemically synthesized oligonucleotides (e.g., described in Kutmeier et al., 1994, BioTechniques 17:242), which involves the synthesis of overlapping oligonucleotides containing portions of the antibody-encoding sequence, the annealing and ligation of these oligonucleotides, and subsequent PCR amplification of the ligated oligonucleotides.

[0285] Alternatively, the nucleic acid molecule encoding the antibody may be generated from a suitable source (e.g., an antibody cDNA library, or a cDNA library produced by any tissue or cell expressing an immunoglobulin) by PCR amplification using synthetic primers that can hybridize to the 3′ and 5′ ends of the sequence, or by cloning using oligonucleotide probes that are specific to a particular gene sequence.

[0286] In some cases, antibodies or their binding fragments are optionally produced by immunizing animals (e.g., mice) to generate polyclonal antibodies, or more preferably by producing monoclonal antibodies, for example, 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 fragment of an antibody may be obtained by screening Fab expression libraries (e.g., described in Huse et al., 1989, Science 246:1275-1281) for Fab fragments that bind to a 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).

[0287] In some implementations, techniques for generating “chimeric antibodies” are used, developed by splicing genes from mouse antibody molecules with appropriate antigen specificity to genes from human antibody molecules with appropriate biological activity (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). Chimeric antibodies are molecules in which different parts are derived from different animal species, such as those having a variable region derived from mouse monoclonal antibodies and a constant region of human immunoglobulins.

[0288] In some embodiments, techniques described for generating single-chain antibodies (US 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) are adapted for generating single-chain antibodies. Single-chain peptides are formed by linking heavy and light chain fragments of the Fv region via amino acid bridges. Alternatively, techniques for assembling functional Fv fragments in *E. coli* (Skerra et al., 1988, Science 242:1038-1041) are also optionally employed.

[0289] In some implementations, an expression vector containing the antibody's nucleotide sequence or the antibody's nucleotide sequence is transferred into host cells using conventional techniques (e.g., electroporation, liposome transfection, and calcium phosphate precipitation), and the transfected cells are then cultured using conventional techniques to produce the antibody. In specific implementations, antibody expression is regulated by constitutive, inducible, or tissue-specific promoters.

[0290] In some implementations, a variety of host expression vector systems are used to express the antibodies or their binding fragments described herein. Such host expression systems represent media through which antibody-encoding sequences are generated and subsequently purified, but also represent cells that express antibodies or their binding fragments in situ when transformed or transfected with appropriate nucleotide-encoding sequences. These include, but are not limited to: microorganisms, such as bacteria (e.g., *Escherichia coli* and *Bacillus subtilis*) transformed with recombinant phage DNA, plasmid DNA, or copious DNA expression vectors containing sequences encoding antibodies or their binding fragments; yeast (e.g., *Saccharomyces pichia*) transformed with recombinant yeast expression vectors containing sequences encoding antibodies or their binding fragments; insect cell systems infected with recombinant viral expression vectors (e.g., baculoviruses); 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 containing sequences encoding antibodies or their binding fragments (e.g., Ti plasmids); or mammalian cell systems carrying recombinant expression constructs containing promoters derived from mammalian cell genomes (e.g., metallothionein promoters) or promoters derived from mammalian viruses (e.g., adenovirus late promoters; vaccinia virus 7.5K promoters) (e.g., COS, CHO, BH, 293, 293T, 3T3 cells).

[0291] To achieve long-term, high-yield production of recombinant proteins, stable expression is preferred. In some cases, cell lines stably expressing antibodies are optionally engineered. Instead of using expression vectors containing viral origins of replication, host cells are transformed with DNA and selectivity markers controlled by appropriate expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.). After introducing exogenous DNA, the engineered cells are grown in enrichment medium for 1–2 days and then transferred to selectivity medium. The selectivity markers in the recombinant plasmid confer resistance to selection and allow cells to stably integrate the plasmid into their chromosomes and grow to form transformation foci, which are then cloned and expanded into cell lines. This method can be advantageously used to engineer cell lines expressing antibodies or their binding fragments.

[0292] In some cases, numerous selection systems are used, including but not limited to those for 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. Furthermore, antimetabolite resistance serves as the basis for selection of 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 aminoglycoside G-418 (Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 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, TIB TECH 11(5):155-215); and hygro, which confers resistance to hygromycin (Santerre et al., 1984, Gene 30:147).Commonly known methods in the field of recombinant DNA technology 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). In some cases, antibody expression levels are increased by vector amplification (for the 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 a marker in an antibody-expressing vector system is amplifiable, an increase in the level of inhibitor present in the host cell culture will increase the copy number of the marker gene. Because the amplified region is associated with the antibody's nucleotide sequence, antibody production will also increase (Crouse et al., 1983, Mol. Cell Biol. 3:257).

[0293] In some cases, any method known in the art for purifying antibodies is used, such as 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 any other standard technique for purifying proteins.

[0294] expression carrier In some embodiments, vectors are disclosed herein that comprise isolated recombinant nucleic acid molecules encoding isolated polypeptides or polypeptide complexes of any of the embodiments disclosed herein.

[0295] In some embodiments, this document discloses a vector comprising an isolated recombinant nucleic acid molecule encoding a polypeptide or polypeptide complex, the polypeptide or polypeptide complex comprising a first antigen recognition molecule that binds to CD28 and a second antigen recognition molecule that binds to a prostate-specific membrane antigen. In some embodiments, the first antigen recognition molecule that binds to CD28 comprises an anti-CD28 antibody. In some embodiments, the anti-CD28 antibody comprises an anti-CD28 heavy chain variable domain comprising complementarity-determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein HC-CDR1, HC-CDR2, and HC-CDR3 comprise the following amino acid sequences: HC-CDR1: SEQ ID NO: 1, HC-CDR2: SEQ ID NO: 2, and HC-CDR3: SEQ ID NO: 3; and the anti-CD28 antibody comprises an anti-CD28 light chain variable domain comprising complementarity-determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein LC-CDR1, LC-CDR2, and LC-CDR3 comprise the following amino acid sequences: LC-CDR1: SEQ ID NO: 4, LC-CDR2: (KAS), and LC-CDR3: SEQ ID NO: 6. In some embodiments, the anti-CD28 heavy chain variable domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 7, and wherein the anti-CD28 light chain variable domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 8. In some embodiments, the anti-CD28 antibody comprises a single-chain variable fragment (scFv), a single-domain antibody, Fab, or Fab'. In some embodiments, the anti-CD28 antibody comprises scFv. In some embodiments, the anti-CD28 antibody comprises scFv, and the scFv comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 9. In some embodiments, the anti-CD28 antibody comprises an scFv, and the scFv comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 10. In some embodiments, the anti-CD28 antibody comprises an scFv, and the scFv comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 11.In some embodiments, the anti-CD28 antibody comprises scFv, and the scFv comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 12.

[0296] In some implementations, the second antigen recognition molecule that binds to PSMA contains an anti-PSMA antibody. In some embodiments, the anti-PSMA antibody comprises an anti-PSMA heavy chain variable domain comprising complementarity-determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein HC-CDR1, HC-CDR2, and HC-CDR3 comprise the following amino acid sequences: HC-CDR1: SEQ ID NO: 13, HC-CDR2: SEQ ID NO: 14, and HC-CDR3: SEQ ID NO: 15; and the anti-PSMA antibody comprises an anti-PSMA light chain variable domain comprising complementarity-determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, LC-CDR2, and LC-CDR3 of the light chain variable domain comprise the following amino acid sequences: LC-CDR1: SEQ ID NO: 16, LC-CDR2: (EA), and LC-CDR3: SEQ ID NO: 18. In some embodiments, the anti-PSMA heavy chain variable domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 20, and the anti-PSMA light chain variable domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 19. In some embodiments, the anti-PSMA antibody comprises a single-chain variable fragment (scFv), a single-domain antibody, Fab, or Fab'. In some embodiments, the anti-PSMA antibody comprises Fab or Fab'. In some embodiments, the anti-PSMA antibody comprises Fab or Fab', and Fab or Fab' comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 19 and an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 20.

[0297] In some implementations, the vector includes any suitable vector derived from eukaryotic or prokaryotic sources. In some cases, the vector is derived from bacteria (e.g., *Escherichia coli*), insects, yeast (e.g., *Pichia pastoris*), algae, or mammalian sources. Exemplary bacterial vectors include pACYC177, pASK75, the pBAD vector series, the pBADM vector series, the pET vector series, the pETM vector series, the pGEX vector series, pHAT, pHAT2, pMal-C2, pMal-p2, the pQE vector series, pRSETA, pRSET B, pRSET C, the 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.

[0298] 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, pVL1393 M11, pVL1393 M12, FLAG vectors such as pPolh-FLAG1 or pPolh-MAT2, or MAT vectors such as pPolh-MAT1 or pPolh-MAT2.

[0299] In some cases, the yeast carrier includes Gateway® pDEST. TM 14 carriers, Gateway® pDEST TM 15 carriers, Gateway® pDEST TM 17 carriers, Gateway® pDEST TM 24 vectors, Gateway® pYES-DEST52 vector, pBAD-DEST49 Gateway® target vector, pAO815 Pichia pastoris vector, pFLD1 Pichia pastoris vector, pGAPZA, B&C Pichia pastoris vector, pPIC3.5K Pichia pastoris vector, pPIC6 A, B&C Pichia pastoris vector, pPIC9K Pichia pastoris vector, pTEF1 / Zeo, pYES2 yeast vector, pYES2 / CT yeast vector, pYES2 / NT A, B&C yeast vector, or pYES3 / CT yeast vector.

[0300] Exemplary algal vectors include the pChlamy-4 vector or the MCS vector.

[0301] Examples of mammalian vectors include transient expression vectors or stable expression vectors. Transient mammalian 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. Stable mammalian expression vectors may 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.

[0302] In some cases, cell-free systems are mixtures of cytoplasmic and / or nuclear components derived from cells and are used for in vitro nucleic acid synthesis. In other cases, cell-free systems utilize prokaryotic or eukaryotic cell components. Sometimes, nucleic acid synthesis is achieved in cell-free systems based on, for example, Drosophila cells, Xenopus eggs, or HeLa cells. Exemplary cell-free systems include, but are not limited to, the E. coli S30 extract system, the E. coli T7 S30 system, or PURExpress®.

[0303] host cells In some embodiments, this document discloses a host cell comprising any of the embodiments disclosed herein, an isolated polypeptide or polypeptide complex.

[0304] In some embodiments, this document discloses a host cell comprising an isolated polypeptide or polypeptide complex containing a first antigen recognition molecule that binds to CD28 and a second antigen recognition molecule that binds to a prostate-specific membrane antigen. In some embodiments, the first antigen recognition molecule that binds to CD28 comprises an anti-CD28 antibody. In some embodiments, the anti-CD28 antibody comprises an anti-CD28 heavy chain variable domain comprising complementarity-determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein HC-CDR1, HC-CDR2, and HC-CDR3 comprise the following amino acid sequences: HC-CDR1: SEQ ID NO: 1, HC-CDR2: SEQ ID NO: 2, and HC-CDR3: SEQ ID NO: 3; and the anti-CD28 antibody comprises an anti-CD28 light chain variable domain comprising complementarity-determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein LC-CDR1, LC-CDR2, and LC-CDR3 comprise the following amino acid sequences: LC-CDR1: SEQ ID NO: 4, LC-CDR2: (KAS), and LC-CDR3: SEQ ID NO: 6. In some embodiments, the anti-CD28 heavy chain variable domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 7, and wherein the anti-CD28 light chain variable domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 8. In some embodiments, the anti-CD28 antibody comprises a single-chain variable fragment (scFv), a single-domain antibody, Fab, or Fab'. In some embodiments, the anti-CD28 antibody comprises scFv. In some embodiments, the anti-CD28 antibody comprises scFv, and the scFv comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 9. In some embodiments, the anti-CD28 antibody comprises an scFv, and the scFv comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 10. In some embodiments, the anti-CD28 antibody comprises an scFv, and the scFv comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 11.In some embodiments, the anti-CD28 antibody comprises scFv, and the scFv comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 12.

[0305] In some implementations, the second antigen recognition molecule that binds to PSMA contains an anti-PSMA antibody. In some embodiments, the anti-PSMA antibody comprises an anti-PSMA heavy chain variable domain comprising complementarity-determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein HC-CDR1, HC-CDR2, and HC-CDR3 comprise the following amino acid sequences: HC-CDR1: SEQ ID NO: 13, HC-CDR2: SEQ ID NO: 14, and HC-CDR3: SEQ ID NO: 15; and the anti-PSMA antibody comprises an anti-PSMA light chain variable domain comprising complementarity-determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, LC-CDR2, and LC-CDR3 of the light chain variable domain comprise the following amino acid sequences: LC-CDR1: SEQ ID NO: 16, LC-CDR2: (EA), and LC-CDR3: SEQ ID NO: 18. In some embodiments, the anti-PSMA heavy chain variable domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 20, and the anti-PSMA light chain variable domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 19. In some embodiments, the anti-PSMA antibody comprises a single-chain variable fragment (scFv), a single-domain antibody, Fab, or Fab'. In some embodiments, the anti-PSMA antibody comprises Fab or Fab'. In some embodiments, the anti-PSMA antibody comprises Fab or Fab', and Fab or Fab' comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 19 and an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 20.

[0306] In some implementations, the host cell includes any suitable cell, such as cells of natural origin or genetically modified cells. In some cases, the host cell is a production host cell. In some cases, the host cell is a eukaryotic cell. In other cases, the host cell is a prokaryotic cell. 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.

[0307] In some cases, Gram-positive bacteria include Actinobacteria, Firmicutes, or Tenericulates. In other cases, Gram-negative bacteria include Aquificae, Deinococcus-Thermus, Fibrobacteres-Chlorobi / Bacteroidetes (FCB group), Fusobacteria, Gemmatimonadetes, Nitrospirae, Planctomycetes-Verrucomicrobia / Chlamydiae (PVC group), Proteobacteria, Spirochaetes, or Synergistetes. Other bacteria can be from the phyla Acidobacteria, Chloroflexi, Chrysiogenetes, Cyanobacteria, Deferribacteres, Dictyoglomi, Thermodesulfobacteria, or Thermotogae. Bacterial cells can be *Escherichia coli*, *Clostridium botulinum*, or *Coli bacilli*.

[0308] Exemplary prokaryotic host cells include, but are not limited to, BL21 and Mach1. TM DH10B TM TOP10, DH5α, DH10Bac TM OmniMax TM MegaXTM DH12S TM , INV110, TOP10F', INVαF, TOP10 / P3, ccdB Survival, PIR1, PIR2, Stbl2 TM Stbl3 TM Or Stbl4 TM .

[0309] In some cases, animal cells include cells derived from vertebrates or invertebrates. In others, animal cells include cells derived from marine invertebrates, fish, insects, amphibians, reptiles, or mammals. In still others, fungal cells include yeast cells, such as brewer's yeast, baker's yeast, or wine yeast. Fungi include ascomycetes, molds, filamentous fungi, basidiomycetes, or zygomycetes, such as yeasts. In some cases, yeasts belong to the phylum Ascomycota or Basidiomycota. In some cases, the phylum Ascomycota includes the subphylum Saccharomycotina (true yeasts, such as Saccharomyces cerevisiae (baker's yeast)) or the subphylum Taphrinomycotina (e.g., Schizosaccharomycetes (splitting yeasts)). In some cases, the Basidiomycota includes the Agaricomycotina (e.g., Tremellomycetes) or the Pucciniomycotina (e.g., Microbotryomycetes).

[0310] Exemplary yeasts or filamentous fungi include, for example, the following genera: *Saccharomyces*, *Schizosaccharomyces*, *Candida*, *Pichia*, *Hansenula*, *Kluyveromyces*, *Zygosaccharomyces*, *Yarrowia*, *Trichosporon*, *Rhodosporidi*, *Aspergillus*, *Fusarium*, or *Trichoderma*. Exemplary yeasts or filamentous fungi include, for example, the following species: *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*, and *Pichia pastoris*. Pastoris, Pichia anomala, Hansenula polymorpha, Kluyveromyces lactis, Zygosaccharomyces rouxii, Yarrowiali polytica, Trichosporon pullulans, Rhodosporidium toru-Aspergillus niger, Aspergillus nidulans, Aspergillus avalancheThe following are listed: *Aspergillus oryzae*, *Trichodermareesei*, *Yarrowia lipolytica*, *Brettanomyces bruxellensis*, *Candida stellata*, *Schizostomia spp.*, *Torulaspora delbrueckii*, *Zygosaccharomyces bailii*, *Cryptococcus neoformans*, *Cryptococcus gattii*, or *Saccharomyces boulardii*.

[0311] 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.

[0312] In some cases, additional animal cells include cells derived from mollusks, arthropods, annelids, or sponges. In others, additional animal cells are mammalian cells, such as those from primates, apes, horses, cattle, pigs, dogs, cats, or rodents. In still others, rodents include mice, rats, hamsters, gerbils, chinchillas, voles, or guinea pigs.

[0313] Exemplary mammalian host cells include, but are not limited to, 293A cell line, 293FT cell line, 293F cell line, 293H cell line, CHO DG44 cell line, CHO-S cell line, CHO-K1 cell line, FUT8 KO CHOK1 cell line, and Expi293F cell line. 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 line, Flp-In TM -CV-1 cell line, Flp-In TM -Jurkat cell line, FreeStyle TM 293-F cells, FreeStyle TMCHO-S cells, GripTite TM 293 MSR 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 line and T-REx TM -HeLa cell line.

[0314] In some cases, mammalian host cells are stable cell lines, or cell lines that have integrated the genetic material of interest into their own genome and have the ability to express the genetic material product after many generations of cell division. In other cases, mammalian host cells are transient cell lines, or cell lines that have not integrated the genetic material of interest into their own genome and do not have the ability to express the genetic material product after many generations of cell division. Exemplary insect host cells include, but are not limited to, Drosophila S2 cells, Sf9 cells, Sf21 cells, and High Five cells. TM Plant cells and expresSF+® cells. In some cases, plant cells include cells derived from algae. Exemplary insect cell lines include, but are not limited to, strains from Chlamydomonas reinhardtii 137c or Synechococcus elongatus PPC 7942.

[0315] Products In another aspect of the invention, articles containing materials for treating, preventing, and / or diagnosing the aforementioned conditions are provided. The articles comprise a container and a label or packaging instruction on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers can be formed from various materials such as glass or plastic. The container contains a composition (which is effective alone or in combination with another composition for treating, preventing, and / or diagnosing the condition) and may have a sterile inlet (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be punctured by a hypodermic needle). At least one active agent in the composition is an isolated polypeptide or polypeptide complex of any embodiment disclosed herein. The label or packaging instruction indicates that the composition is for treating the selected condition. Furthermore, the articles may comprise (a) a first container containing the composition, wherein the composition contains the bispecific antibody of the present invention; and (b) a second container containing the composition, wherein the composition contains additional cytotoxic agents or other therapeutic agents. The articles in this embodiment of the invention may further include packaging instructions indicating that the composition can be used to treat a specific condition. Optionally or additionally, the article may further comprise: a second (or third) container containing pharmaceutically acceptable buffers such as bactericidal water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextran solution. It may further include other materials desired from a commercial and user perspective, including additional buffers, diluents, filters, needles, and syringes.

[0316] Implementation Plan Implementation scheme 1 includes an isolated polypeptide or polypeptide complex comprising a first antigen recognition molecule that binds to CD28 and a second antigen recognition molecule that binds to prostate-specific membrane antigen (PSMA).

[0317] Implementation scheme 2 includes the isolated polypeptide or polypeptide complex described in implementation scheme 1, wherein the first antigen recognition molecule that binds to CD28 comprises an anti-CD28 antibody.

[0318] Implementation Scheme 3 includes the isolated polypeptide or polypeptide complex described in Implementation Scheme 2, wherein the anti-CD28 antibody comprises an anti-CD28 heavy chain variable domain, the anti-CD28 heavy chain variable domain comprising complementarity-determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein HC-CDR1, HC-CDR2, and HC-CDR3 comprise the following amino acid sequences: HC-CDR1: SEQ ID NO: 1, HC-CDR2: SEQ ID NO: 2, and HC-CDR3: SEQ ID NO: 3; and the anti-CD28 antibody comprises an anti-CD28 light chain variable domain, the anti-CD28 light chain variable domain comprising complementarity-determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein LC-CDR1, LC-CDR2, and LC-CDR3 comprise the following amino acid sequences: LC-CDR1: SEQ ID NO: 4, LC-CDR2: (KAS), and LC-CDR3: SEQ ID NO: 6.

[0319] Implementation scheme 4 includes the isolated polypeptide or polypeptide complex described in implementation scheme 3, wherein the anti-CD28 heavy chain variable domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 7, and wherein the anti-CD28 light chain variable domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 8.

[0320] Implementation scheme 5 includes the isolated polypeptide or polypeptide complex of any one of implementation schemes 2-4, wherein the anti-CD28 antibody comprises a single-chain variable fragment (scFv), a single-domain antibody, Fab, Fab', or CrossFab.

[0321] Implementation scheme 6 includes the isolated polypeptide or polypeptide complex described in implementation scheme 5, wherein the anti-CD28 antibody comprises the scFv.

[0322] Implementation Scheme 7 includes the isolated polypeptide described in Implementation Scheme 5, wherein the anti-CD28 antibody comprises a single-chain variable fragment (scFv) having engineered disulfide bonds.

[0323] Implementation scheme 8 includes the isolated polypeptide or polypeptide complex described in implementation scheme 5, wherein the anti-CD28 antibody comprises the scFv, and the scFv comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 9.

[0324] Implementation scheme 9 includes the isolated polypeptide or polypeptide complex described in implementation scheme 5, wherein the anti-CD28 antibody comprises the scFv, and the scFv comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 10.

[0325] Implementation scheme 10 includes the isolated polypeptide or polypeptide complex of implementation scheme 5, wherein the anti-CD28 antibody comprises the scFv, and the scFv comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 11.

[0326] Implementation scheme 11 includes the isolated polypeptide or polypeptide complex of implementation scheme 5, wherein the anti-CD28 antibody comprises the scFv, and the scFv comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 12.

[0327] Implementation scheme 12 includes the isolated polypeptide or polypeptide complex described in implementation scheme 5, wherein the anti-CD28 antibody comprises the Fab.

[0328] Embodiment 13 includes the isolated polypeptide or polypeptide complex described in Embodiment 12, wherein the Fab comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 696 and an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 697.

[0329] Implementation scheme 14 includes the isolated polypeptide or polypeptide complex described in implementation scheme 5, wherein the anti-CD28 antibody comprises the CrossFab.

[0330] Embodiment 15 includes the isolated polypeptide or polypeptide complex described in Embodiment 14, wherein the CrossFab comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 698 and an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 699.

[0331] Embodiment 16 includes the isolated polypeptide or polypeptide complex described in Embodiment 14, wherein the CrossFab comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 700 and an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 701.

[0332] Embodiment 17 includes the isolated polypeptide or polypeptide complex of any one of the preceding embodiments, wherein the second antigen recognition molecule that binds to PSMA comprises an anti-PSMA antibody.

[0333] Embodiment 18 includes the isolated polypeptide or polypeptide complex described in Embodiment 17, wherein the anti-PSMA antibody comprises an anti-PSMA heavy chain variable domain, the anti-PSMA heavy chain variable domain comprising complementarity-determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein HC-CDR1, HC-CDR2, and HC-CDR3 comprise the following amino acid sequences: HC-CDR1: SEQ ID NO: 13, HC-CDR2: SEQ ID NO: 14, and HC-CDR3: SEQ ID NO: 15; and the anti-PSMA antibody comprises an anti-PSMA light chain variable domain, the anti-PSMA light chain variable domain comprising complementarity-determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, LC-CDR2, and LC-CDR3 of the light chain variable domain comprise the following amino acid sequences: LC-CDR1: SEQ ID NO: 16. LC-CDR2: (EA) and LC-CDR3: SEQ ID NO: 18.

[0334] Embodiment 19 includes the isolated polypeptide or polypeptide complex described in Embodiment 18, wherein the anti-PSMA heavy chain variable domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 20, and wherein the anti-PSMA light chain variable domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 19.

[0335] Embodiment 20 includes the isolated polypeptide or polypeptide complex of any one of Embodiments 17-19, wherein the anti-PSMA antibody comprises a single-chain variable fragment (scFv), a single-domain antibody, Fab, Fab', or CrossFab.

[0336] Implementation scheme 21 includes the isolated polypeptide or polypeptide complex described in implementation scheme 20, wherein the anti-PSMA antibody comprises the Fab or Fab'.

[0337] Implementation scheme 22 includes the isolated polypeptide or polypeptide complex of implementation scheme 20, wherein the anti-PSMA antibody comprises the Fab or Fab', and the Fab or Fab' comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 19 and an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 20.

[0338] Implementation scheme 23 includes the isolated polypeptide or polypeptide complex of implementation scheme 20, wherein the anti-PSMA antibody comprises the Fab or Fab', and the Fab or Fab' comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 702 and an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 703.

[0339] Embodiment 24 includes the isolated polypeptide or polypeptide complex of Embodiment 20, wherein the anti-PSMA antibody comprises the Fab or Fab', and the Fab or Fab' comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 704 and an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 705.

[0340] Embodiment 25 includes the isolated polypeptide or polypeptide complex of any one of Embodiments 17-24, wherein the anti-CD28 antibody comprises scFv and the anti-PSMA antibody comprises Fab or Fab'.

[0341] Implementation scheme 26 includes the isolated polypeptide or polypeptide complex described in implementation scheme 25, wherein the scFv is connected to the Fab or Fab' via a linker, and the Fab or Fab' comprises a Fab heavy chain polypeptide and a Fab light chain polypeptide.

[0342] Implementation scheme 27 includes the isolated polypeptide or polypeptide complex described in implementation scheme 26, wherein the linker connects the scFv to the Fab heavy chain polypeptide.

[0343] Implementation scheme 28 includes the isolated polypeptide or polypeptide complex described in implementation scheme 26, wherein the linker connects the scFv to the Fab light chain polypeptide.

[0344] Implementation scheme 29 includes the isolated polypeptide or polypeptide complex described in implementation scheme 26, wherein the linker connects the scFv to the N-terminus of the Fab heavy chain polypeptide.

[0345] Implementation scheme 30 includes the isolated polypeptide or polypeptide complex described in implementation scheme 26, wherein the linker connects the scFv to the C-terminus of the Fab heavy chain polypeptide.

[0346] Implementation scheme 31 includes the isolated polypeptide or polypeptide complex described in implementation scheme 26, wherein the linker connects the scFv to the N-terminus of the Fab light chain polypeptide.

[0347] Implementation scheme 32 includes the isolated polypeptide or polypeptide complex described in implementation scheme 26, wherein the linker connects the scFv to the C-terminus of the Fab light chain polypeptide.

[0348] Implementation scheme 33 includes the isolated polypeptide or polypeptide complex described in implementation scheme 26, wherein the linker connects the anti-CD28 heavy chain variable domain of the scFv to the Fab heavy chain polypeptide.

[0349] Implementation scheme 34 includes the isolated polypeptide or polypeptide complex described in implementation scheme 26, wherein the linker links the anti-CD28 heavy chain variable domain of the scFv to the Fab light chain polypeptide.

[0350] Implementation scheme 35 includes the isolated polypeptide or polypeptide complex described in implementation scheme 26, wherein the linker links the anti-CD28 heavy chain variable domain of the scFv to the N-terminus of the Fab heavy chain polypeptide.

[0351] Implementation scheme 36 includes the isolated polypeptide or polypeptide complex described in implementation scheme 26, wherein the linker links the anti-CD28 heavy chain variable domain of the scFv to the C-terminus of the Fab heavy chain polypeptide.

[0352] Implementation scheme 37 includes the isolated polypeptide or polypeptide complex described in implementation scheme 26, wherein the linker links the anti-CD28 heavy chain variable domain of the scFv to the N-terminus of the Fab light chain polypeptide.

[0353] Implementation scheme 38 includes the isolated polypeptide or polypeptide complex described in implementation scheme 26, wherein the linker links the anti-CD28 heavy chain variable domain of the scFv to the C-terminus of the Fab light chain polypeptide.

[0354] Implementation scheme 39 includes the isolated polypeptide or polypeptide complex described in implementation scheme 26, wherein the linker links the anti-CD28 light chain variable domain of the scFv to the Fab heavy chain polypeptide.

[0355] Implementation scheme 40 includes the isolated polypeptide or polypeptide complex described in implementation scheme 26, wherein the linker links the anti-CD28 light chain variable domain of the scFv to the Fab light chain polypeptide.

[0356] Implementation scheme 41 includes the isolated polypeptide or polypeptide complex described in implementation scheme 26, wherein the linker links the anti-CD28 light chain variable domain of the scFv to the N-terminus of the Fab heavy chain polypeptide.

[0357] Implementation scheme 42 includes the isolated polypeptide or polypeptide complex described in implementation scheme 26, wherein the linker links the anti-CD28 light chain variable domain of the scFv to the C-terminus of the Fab heavy chain polypeptide.

[0358] Implementation scheme 43 includes the isolated polypeptide or polypeptide complex described in implementation scheme 26, wherein the linker links the anti-CD28 light chain variable domain of the scFv to the N-terminus of the Fab light chain polypeptide.

[0359] Implementation scheme 44 includes the isolated polypeptide or polypeptide complex described in implementation scheme 26, wherein the linker links the anti-CD28 light chain variable domain of the scFv to the C-terminus of the Fab light chain polypeptide.

[0360] Embodiment 45 includes the isolated polypeptide or polypeptide complex of any one of Embodiments 17-24, wherein the anti-CD28 antibody comprises Fab or Fab', and the anti-PSMA antibody comprises Fab or Fab'.

[0361] Implementation scheme 46 includes the isolated polypeptide or polypeptide complex described in implementation scheme 45, wherein the Fab or Fab' of the anti-CD28 antibody is linked to the Fab or Fab' of the anti-PSMA antibody via a linker, and the Fab or Fab' of the anti-PSMA antibody comprises a Fab heavy chain polypeptide and a Fab light chain polypeptide.

[0362] Implementation scheme 47 includes the isolated polypeptide or polypeptide complex described in implementation scheme 46, wherein the linker links the Fab or Fab' of the anti-CD28 antibody to the Fab heavy chain polypeptide of the anti-PSMA antibody.

[0363] Implementation scheme 48 includes the isolated polypeptide or polypeptide complex described in implementation scheme 46, wherein the linker links the Fab or Fab' of the anti-CD28 antibody to the Fab light chain polypeptide of the anti-PSMA antibody.

[0364] Implementation scheme 49 includes the isolated polypeptide or polypeptide complex described in implementation scheme 46, wherein the linker links the Fab or Fab' of the anti-CD28 antibody to the N-terminus of the Fab heavy chain polypeptide of the anti-PSMA antibody.

[0365] Implementation scheme 50 includes the isolated polypeptide or polypeptide complex described in implementation scheme 46, wherein the linker links the Fab or Fab' of the anti-CD28 antibody to the C-terminus of the Fab heavy chain polypeptide of the anti-PSMA antibody.

[0366] Implementation scheme 51 includes the isolated polypeptide or polypeptide complex described in implementation scheme 46, wherein the linker links the Fab or Fab' of the anti-CD28 antibody to the N-terminus of the Fab light chain polypeptide of the anti-PSMA antibody.

[0367] Implementation scheme 52 includes the isolated polypeptide or polypeptide complex described in implementation scheme 46, wherein the linker links the Fab or Fab' of the anti-CD28 antibody to the C-terminus of the Fab light chain polypeptide of the anti-PSMA antibody.

[0368] Implementation scheme 53 includes the isolated polypeptide or polypeptide complex described in implementation scheme 46, wherein the linker links the Fab heavy chain polypeptide of the anti-CD28 antibody to the Fab heavy chain polypeptide of the anti-PSMA antibody.

[0369] Implementation scheme 54 includes the isolated polypeptide or polypeptide complex described in implementation scheme 46, wherein the linker links the Fab heavy chain polypeptide of the anti-CD28 antibody to the Fab light chain polypeptide of the anti-PSMA antibody.

[0370] Implementation scheme 55 includes the isolated polypeptide or polypeptide complex described in implementation scheme 46, wherein the linker links the Fab heavy chain polypeptide of the anti-CD28 antibody to the N-terminus of the Fab heavy chain polypeptide of the anti-PSMA antibody.

[0371] Implementation scheme 56 includes the isolated polypeptide or polypeptide complex described in implementation scheme 46, wherein the linker links the Fab heavy chain polypeptide of the anti-CD28 antibody to the C-terminus of the Fab heavy chain polypeptide of the anti-PSMA antibody.

[0372] Implementation scheme 57 includes the isolated polypeptide or polypeptide complex described in implementation scheme 46, wherein the linker links the Fab heavy chain polypeptide of the anti-CD28 antibody to the N-terminus of the Fab light chain polypeptide of the anti-PSMA antibody.

[0373] Implementation scheme 58 includes the isolated polypeptide or polypeptide complex described in implementation scheme 46, wherein the linker links the Fab heavy chain polypeptide of the anti-CD28 antibody to the C-terminus of the Fab light chain polypeptide of the anti-PSMA antibody.

[0374] Implementation scheme 59 includes the isolated polypeptide or polypeptide complex described in implementation scheme 46, wherein the linker links the Fab light chain polypeptide of the anti-CD28 antibody to the Fab heavy chain polypeptide of the anti-PSMA antibody.

[0375] Implementation scheme 60 includes the isolated polypeptide or polypeptide complex described in implementation scheme 46, wherein the linker links the Fab light chain polypeptide of the anti-CD28 antibody to the Fab light chain polypeptide of the anti-PSMA antibody.

[0376] Implementation scheme 61 includes the isolated polypeptide or polypeptide complex described in implementation scheme 46, wherein the linker links the Fab light chain polypeptide of the anti-CD28 antibody to the N-terminus of the Fab heavy chain polypeptide of the anti-PSMA antibody.

[0377] Implementation scheme 62 includes the isolated polypeptide or polypeptide complex described in implementation scheme 46, wherein the linker links the Fab light chain polypeptide of the anti-CD28 antibody to the N-terminus of the Fab heavy chain polypeptide of the anti-PSMA antibody.

[0378] Implementation scheme 63 includes the isolated polypeptide or polypeptide complex described in implementation scheme 46, wherein the linker links the Fab light chain polypeptide of the anti-CD28 antibody to the N-terminus of the Fab light chain polypeptide of the anti-PSMA antibody.

[0379] Implementation scheme 64 includes the isolated polypeptide or polypeptide complex described in implementation scheme 46, wherein the linker links the Fab light chain polypeptide of the anti-CD28 antibody to the C-terminus of the Fab light chain polypeptide of the anti-PSMA antibody.

[0380] Embodiment 65 includes the isolated polypeptide or polypeptide complex of any one of Embodiments 17-24, wherein the anti-CD28 antibody comprises CrossFab, and the anti-PSMA antibody comprises Fab or Fab'.

[0381] Implementation scheme 66 includes the isolated polypeptide or polypeptide complex described in implementation scheme 65, wherein the CrossFab is connected to the Fab or Fab' via the linker, and the Fab or Fab' of the anti-PSMA antibody comprises a Fab heavy chain polypeptide and a Fab light chain polypeptide.

[0382] Implementation scheme 67 includes the isolated polypeptide or polypeptide complex described in implementation scheme 66, wherein the linker links the CrossFab to the Fab heavy chain polypeptide of the anti-PSMA antibody.

[0383] Implementation scheme 68 includes the isolated polypeptide or polypeptide complex described in implementation scheme 66, wherein the linker connects the CrossFab to the Fab light chain polypeptide of the anti-PSMA antibody.

[0384] Implementation scheme 69 includes the isolated polypeptide or polypeptide complex described in implementation scheme 66, wherein the linker connects the CrossFab to the N-terminus of the Fab heavy chain polypeptide of the anti-PSMA antibody.

[0385] Implementation scheme 70 includes the isolated polypeptide or polypeptide complex described in implementation scheme 66, wherein the linker connects the CrossFab to the C-terminus of the Fab heavy chain polypeptide of the anti-PSMA antibody.

[0386] Implementation scheme 71 includes the isolated polypeptide or polypeptide complex described in implementation scheme 66, wherein the linker connects the CrossFab to the N-terminus of the Fab light chain polypeptide of the anti-PSMA antibody.

[0387] Implementation scheme 72 includes the isolated polypeptide or polypeptide complex described in implementation scheme 66, wherein the linker connects the CrossFab to the C-terminus of the Fab light chain polypeptide of the anti-PSMA antibody.

[0388] Implementation scheme 73 includes the isolated polypeptide or polypeptide complex described in implementation scheme 66, wherein the linker links the Fab heavy chain polypeptide of the CrossFab to the Fab heavy chain polypeptide of the antiPSMA antibody.

[0389] Implementation scheme 74 includes the isolated polypeptide or polypeptide complex described in implementation scheme 66, wherein the linker links the Fab heavy chain polypeptide of the CrossFab to the Fab light chain polypeptide of the antiPSMA antibody.

[0390] Implementation scheme 75 includes the isolated polypeptide or polypeptide complex described in implementation scheme 66, wherein the linker links the CrossFab Fab heavy chain polypeptide to the N-terminus of the antiPSMA antibody Fab heavy chain polypeptide.

[0391] Implementation scheme 76 includes the isolated polypeptide or polypeptide complex described in implementation scheme 66, wherein the linker links the CrossFab Fab heavy chain polypeptide to the C-terminus of the antiPSMA antibody Fab heavy chain polypeptide.

[0392] Implementation scheme 77 includes the isolated polypeptide or polypeptide complex described in implementation scheme 66, wherein the linker links the Fab heavy chain polypeptide of the CrossFab to the N-terminus of the Fab light chain polypeptide of the antiPSMA antibody.

[0393] Implementation scheme 78 includes the isolated polypeptide or polypeptide complex described in implementation scheme 66, wherein the linker links the Fab heavy chain polypeptide of the CrossFab to the C-terminus of the Fab light chain polypeptide of the antiPSMA antibody.

[0394] Implementation scheme 79 includes the isolated polypeptide or polypeptide complex described in implementation scheme 66, wherein the linker links the Fab light chain polypeptide of the CrossFab to the Fab heavy chain polypeptide of the antiPSMA antibody.

[0395] Implementation scheme 80 includes the isolated polypeptide or polypeptide complex described in implementation scheme 66, wherein the linker links the Fab light chain polypeptide of the CrossFab to the Fab light chain polypeptide of the antiPSMA antibody.

[0396] Implementation scheme 81 includes the isolated polypeptide or polypeptide complex described in implementation scheme 66, wherein the linker links the Fab light chain polypeptide of the CrossFab to the N-terminus of the Fab heavy chain polypeptide of the anti-PSMA antibody.

[0397] Implementation scheme 82 includes the isolated polypeptide or polypeptide complex described in implementation scheme 66, wherein the linker links the Fab light chain polypeptide of the CrossFab to the C-terminus of the Fab heavy chain polypeptide of the antiPSMA antibody.

[0398] Implementation scheme 83 includes the isolated polypeptide or polypeptide complex described in implementation scheme 66, wherein the linker links the Fab light chain polypeptide of the CrossFab to the N-terminus of the Fab light chain polypeptide of the anti-PSMA antibody.

[0399] Implementation scheme 84 includes the isolated polypeptide or polypeptide complex described in implementation scheme 66, wherein the linker links the Fab light chain polypeptide of the CrossFab to the C-terminus of the Fab light chain polypeptide of the anti-PSMA antibody.

[0400] Embodiment 85 includes the isolated polypeptide or polypeptide complex of any one of Embodiments 17-20, wherein the anti-CD28 antibody comprises Fab or Fab', and the anti-PSMA antibody comprises CrossFab.

[0401] Implementation scheme 86 includes the isolated polypeptide or polypeptide complex of any one of implementation schemes 26-44, 46-64 and 66-84, wherein the length of the linker is at least 5 amino acids.

[0402] Implementation scheme 87 includes the isolated polypeptide or polypeptide complex of any one of implementation schemes 26-44, 46-64 and 66-84, wherein the length of the linker is no more than 30 amino acids.

[0403] Implementation scheme 88 includes the isolated polypeptide or polypeptide complex of any one of implementation schemes 26-44, 46-64 and 66-84, wherein the length of the linker is at least 5 amino acids and no more than 30 amino acids.

[0404] Implementation scheme 89 includes the isolated polypeptide or polypeptide complex of any one of implementation schemes 26-44, 46-64 and 66-84, wherein the length of the linker is 5 amino acids.

[0405] Implementation scheme 90 includes the isolated polypeptide or polypeptide complex of any one of implementation schemes 26-44, 46-64 and 66-84, wherein the length of the linker is 15 amino acids.

[0406] Implementation scheme 91 includes the isolated polypeptide or polypeptide complex of any one of implementation schemes 26-44, 46-64 and 66-84, wherein the linker comprises the amino acid sequence of SEQ ID NO: 21 (GGGGSGGGGSGGGGS) or SEQ ID NO: 22 (GGGGS).

[0407] Implementation scheme 92 includes the isolated polypeptide or polypeptide complex of implementation scheme 1, wherein the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 23 and SEQ ID NO: 24.

[0408] Implementation scheme 93 includes the isolated polypeptide or polypeptide complex described in implementation scheme 1, wherein the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 25 and SEQ ID NO: 26.

[0409] Implementation scheme 94 includes the isolated polypeptide or polypeptide complex described in implementation scheme 1, wherein the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 27 and SEQ ID NO: 28.

[0410] Embodiment 95 includes the isolated polypeptide or polypeptide complex described in Embodiment 1, wherein the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 29 and SEQ ID NO: 30.

[0411] Implementation scheme 96 includes the isolated polypeptide or polypeptide complex described in implementation scheme 1, wherein the isolated polypeptide or polypeptide complex comprises an amino ac...

Claims

1. An isolated polypeptide or polypeptide complex comprising a first antigen recognition molecule that binds to CD28 and a second antigen recognition molecule that binds to prostate-specific membrane antigen (PSMA).

2. The isolated polypeptide or polypeptide complex according to claim 1, wherein the first antigen recognition molecule binding to CD28 comprises an anti-CD28 antibody.

3. The isolated polypeptide or polypeptide complex according to claim 2, wherein the anti-CD28 antibody comprises an anti-CD28 heavy chain variable domain, the anti-CD28 heavy chain variable domain comprising complementarity-determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein HC-CDR1, HC-CDR2, and HC-CDR3 comprise the following amino acid sequences: HC-CDR1: SEQ ID NO: 1, HC-CDR2: SEQ ID NO: 2, and HC-CDR3: SEQ ID NO: 3; and the anti-CD28 antibody comprises an anti-CD28 light chain variable domain, the anti-CD28 light chain variable domain comprising complementarity-determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein LC-CDR1, LC-CDR2, and LC-CDR3 comprise the following amino acid sequences: LC-CDR1: SEQ ID NO: 4, LC-CDR2: (KAS), and LC-CDR3: SEQ ID NO:

4.

6.

4. The isolated polypeptide or polypeptide complex according to claim 3, wherein the anti-CD28 heavy chain variable domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 7, and wherein the anti-CD28 light chain variable domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:

8.

5. The isolated polypeptide or polypeptide complex according to claim 3, wherein the anti-CD28 antibody comprises a single-chain variable fragment (scFv), a single-domain antibody, Fab, Fab', or CrossFab.

6. The isolated polypeptide or polypeptide complex according to claim 5, wherein the anti-CD28 antibody comprises the scFv, and the scFv comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:

9.

7. The isolated polypeptide or polypeptide complex according to claim 5, wherein the anti-CD28 antibody comprises the scFv, and the scFv comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:

10.

8. The isolated polypeptide or polypeptide complex according to claim 5, wherein the anti-CD28 antibody comprises the scFv, and the scFv comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:

11.

9. The isolated polypeptide or polypeptide complex according to claim 5, wherein the anti-CD28 antibody comprises the scFv, and the scFv comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:

12.

10. The isolated polypeptide or polypeptide complex according to claim 5, wherein the anti-CD28 antibody comprises the Fab.

11. The isolated polypeptide or polypeptide complex according to claim 10, wherein the Fab comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:696 and an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:

697.

12. The isolated polypeptide or polypeptide complex according to claim 5, wherein the anti-CD28 antibody comprises the CrossFab.

13. The isolated polypeptide or polypeptide complex according to claim 12, wherein the CrossFab comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 698 and an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:

699.

14. The isolated polypeptide or polypeptide complex according to claim 12, wherein the CrossFab comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 700 and an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:

701.

15. The isolated polypeptide or polypeptide complex of claim 3, wherein the second antigen recognition molecule that binds to PSMA comprises an anti-PSMA antibody.

16. The isolated polypeptide or polypeptide complex according to claim 15, wherein the anti-PSMA antibody comprises an anti-PSMA heavy chain variable domain, the anti-PSMA heavy chain variable domain comprising complementarity-determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein HC-CDR1, HC-CDR2, and HC-CDR3 comprise the following amino acid sequence: HC-CDR1: SEQ ID NO: 13, HC-CDR2: SEQ ID NO: 14, and HC-CDR3: SEQ ID NO: 15; and the anti-PSMA antibody comprises an anti-PSMA light chain variable domain, the anti-PSMA light chain variable domain comprising complementarity-determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, LC-CDR2, and LC-CDR3 of the light chain variable domain comprises the following amino acid sequence: LC-CDR1: SEQ ID NO:

16. LC-CDR2: (EA) and LC-CDR3: SEQ ID NO:

18.

17. The isolated polypeptide or polypeptide complex according to claim 16, wherein the anti-PSMA heavy chain variable domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 20, and wherein the anti-PSMA light chain variable domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:

19.

18. The isolated polypeptide or polypeptide complex of claim 16, wherein the anti-PSMA antibody comprises a single-chain variable fragment (scFv), a single-domain antibody, Fab, Fab', or CrossFab.

19. The isolated polypeptide or polypeptide complex according to claim 18, wherein the anti-PSMA antibody comprises the Fab or Fab', and the Fab or Fab' comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 19 and an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:

20.

20. The isolated polypeptide or polypeptide complex according to claim 18, wherein the anti-PSMA antibody comprises the Fab or Fab', and the Fab or Fab' comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 702 and an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:

703.

21. The isolated polypeptide or polypeptide complex according to claim 18, wherein the anti-PSMA antibody comprises the Fab or Fab', and the Fab or Fab' comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 704 and an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO:

705.

22. The isolated polypeptide or polypeptide complex according to claim 1, wherein the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 23 and SEQ ID NO:

24.

23. The isolated polypeptide or polypeptide complex according to claim 1, wherein the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 25 and SEQ ID NO:

26.

24. The isolated polypeptide or polypeptide complex according to claim 1, wherein the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 27 and SEQ ID NO:

28.

25. The isolated polypeptide or polypeptide complex according to claim 1, wherein the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 29 and SEQ ID NO:

30.

26. The isolated polypeptide or polypeptide complex according to claim 1, wherein the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 31 and SEQ ID NO:

32.

27. The isolated polypeptide or polypeptide complex according to claim 1, wherein the isolated polypeptide or polypeptide complex comprises the amino acid sequences of SEQ ID NO: 25 and SEQ ID NO:

26.

28. The isolated polypeptide or polypeptide complex according to claim 1, wherein the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 706 and SEQ ID NO:

707.

29. The isolated polypeptide or polypeptide complex according to claim 1, wherein the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 708, SEQ ID NO: 709, and SEQ ID NO:

710.

30. The isolated polypeptide or polypeptide complex according to claim 1, wherein the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 711, SEQ ID NO: 712, and SEQ ID NO:

713.

31. The isolated polypeptide or polypeptide complex according to claim 1, wherein the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 714, SEQ ID NO: 715, and SEQ ID NO:

716.

32. The isolated polypeptide or polypeptide complex according to claim 1, wherein the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 717, SEQ ID NO: 718, and SEQ ID NO:

719.

33. The isolated polypeptide or polypeptide complex according to claim 1, wherein the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 720, SEQ ID NO: 721, and SEQ ID NO:

722.

34. The isolated polypeptide or polypeptide complex according to claim 1, wherein the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 723, SEQ ID NO: 724, and SEQ ID NO:

725.

35. The isolated polypeptide or polypeptide complex according to claim 1, wherein the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 726, SEQ ID NO: 727, and SEQ ID NO:

728.

36. The isolated polypeptide or polypeptide complex according to claim 1, wherein the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 729, SEQ ID NO: 730, and SEQ ID NO:

731.

37. The isolated polypeptide or polypeptide complex of claim 16, wherein the isolated polypeptide or polypeptide complex comprises a peptide linked to the anti-CD28 antibody, wherein the peptide weakens the binding of the anti-CD28 antibody to CD28.

38. The isolated polypeptide or polypeptide complex of claim 37, wherein the isolated polypeptide or polypeptide complex comprises a configuration according to formula I: A2-A1-L1-P1-H1, wherein A2 comprises the anti-PSMA antibody, A1 comprises the anti-CD28 antibody, L1 comprises a linker portion connecting A1 to P1 and is a substrate of a tumor-specific protease, P1 comprises a peptide that weakens the binding of the anti-CD28 antibody to CD28, and H1 comprises a half-life-extending molecule.

39. The isolated polypeptide or polypeptide complex according to claim 38, wherein A2 further comprises P2 and L2, wherein P2 comprises a peptide bound to A2; and L2 comprises a linker portion connecting A2 to P2 and is a substrate of a tumor-specific protease.

40. The isolated polypeptide or polypeptide complex according to claim 39, wherein the polypeptide or polypeptide complex is according to formula Ia: P2-L2-A2-A1-L1-P1-H1.

41. The isolated polypeptide or polypeptide complex according to claim 40, wherein P1 comprises an amino acid sequence according to any one of SEQ ID NO: 33 to 106 or 732-813.

42. The isolated polypeptide or polypeptide complex according to claim 40, wherein P1 comprises the amino acid sequence of SEQ ID NO:

73.

43. The isolated polypeptide or polypeptide complex according to claim 40, wherein P1 comprises the amino acid sequence of SEQ ID NO:

103.

44. The isolated polypeptide or polypeptide complex according to claim 40, wherein P1 comprises the following components according to X1-X2-X3-C-X4-X5-X6-X7-X8-X9-X 10 -CX 11 -X 12 The amino acid sequence is defined as follows: X1 is selected from M, I, L, and V; X2 is selected from D, H, N, A, F, S, T, Y, and V; X3 is selected from W, L, and F; X4 is selected from P, A, and L; X5 is selected from R, T, I, M, S, K, L, V, W, F, A, P, and D; X6 is selected from E, D, Y, H, S, F, A, N, T, I, P, and V; X7 is selected from L, M, R, S, Q, and H; X8 is selected from W and Q; X9 is selected from 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 Selected from F, Y, L, W, and V; and X 12 Selected from N, A, F, S, Y, H, D, T, and L.

45. The isolated polypeptide or polypeptide complex according to claim 44, 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 Selected from F, Y, and L; and X 12 Selected from N, A, F, S, and Y.

46. ​​The isolated polypeptide or polypeptide complex according to claim 40, wherein P1 comprises the amino acid sequence according to SEQ ID NO:

41.

47. The isolated polypeptide or polypeptide complex according to claim 40, wherein P1 comprises an amino acid sequence according to the following sequence: J1-J2-WC-J3-J4-J5-J6-J7-J8-J9-CJ 10 -J 11 ,in: J1 is selected from A, R, N, D, Q, E, G, H, I, L, K, F, P, S, T, W, Y, and V; J2 is selected from D, P, and Y; J3 is selected from P and Q; J4 is selected from R, L, and I; J5 is selected from D, H, Y, and I; J6 is selected from L, S, and R; J7 is selected from W and G; J8 is selected from V, A, D, H, L, and N; J9 is selected from H, L, E, and D; J 10 Selected from F, Y, and L; and J 11 Selected from A, N, H, and T.

48. The isolated polypeptide or polypeptide complex according to claim 47, wherein: J1 is selected from A, R, N, D, Q, E, G, H, K, F, P, S, T, W, and Y.

49. The isolated polypeptide or polypeptide complex according to claim 47, wherein: J1 is selected from R, Q, E, H, I, L, K, F, T, W, Y, and V; J2 is D; J3 is P; J4 is R; J5 is D; J6 is L; J7 is W; J8 is V; J9 is H; J 10 It is F; and J 11 It is A.

50. The isolated polypeptide or polypeptide complex according to claim 40, wherein P1 comprises amino acids according to the following sequence: Z1-C-Z2-Z3-Z4-Z5-Z6-Z7-Z8-Z9-Z 10 -Z 11 -CZ 12 ,in: Z1 is selected from Y, H, and A; Z2 is selected from D, E, A, Q, S, V, M, H, L, F, T, W, and N; Z3 is selected from G, E, S, and A; Z4 is selected from H, L, W, and A; Z5 is selected from L, S, A, T, I, V, M, R, E, D, F, H, K, Q, N, Y, W and G; Z6 is selected from P, A, E, Q, S, L, W, G, V, D, K, I, M, R, T, F, H, N and Y; Z7 is selected from E, Q, I, M, V, A, L, R, W, P, D, T, K, S, G, H, and N; Z8 is selected from Y, H, F, W, N, Q, and A; Z9 is selected from M, I, L, R, V, K, F, Q, Y, W, and A; Z 10 Selected from A, Q, S, W, E, L, G, D, T, M, N, V, H, R, Y, K, P, F, and I; Z 11 Selected from K, Q, N, H, E, P, and A; and Z 12 Selected from L, M, I, F, V, Y, Q, D, T, and A.

51. The isolated polypeptide or polypeptide complex according to claim 50, wherein: Z1 is selected from Y and H; Z2 is selected from D, E, A, Q, S, V, M, H, L, F, T, W, and N; Z3 is selected from G, E, and S; Z4 is selected from H, L, and W; Z5 is selected from L, S, A, T, I, V, M, R, E, D, F, H, K, Q, N, Y, W and G; Z6 is selected from P, A, E, Q, S, L, W, G, V, D, K, I, M, R, T, F, H, N and Y; Z7 is selected from E, Q, I, M, V, A, L, R, W, P, D, T, K, S, G, H, and N; Z8 is selected from Y, H, F, W, N, and Q; Z9 is selected from M, I, L, R, V, K, F, Q, Y, and W; Z 10 Selected from A, Q, S, W, E, L, G, D, T, M, N, V, H, R, Y, K, P, F, and I; Z 11 Selected from K, Q, N, H, E, and P; and Z 12 Selected from L, M, I, F, V, Y, Q, D, and T.

52. The isolated polypeptide or polypeptide complex according to claim 40, wherein P1 comprises the amino acid sequence of SEQ ID NO:

784.

53. The isolated polypeptide or polypeptide complex according to claim 40, wherein P1 comprises the amino acid sequence of SEQ ID NO:

744.

54. The isolated polypeptide or polypeptide complex according to claim 40, wherein P1 consists of the amino acid sequence of SEQ ID NO:

744.

55. The isolated polypeptide or polypeptide complex according to claim 40, wherein P1 comprises an amino acid sequence according to any one of SEQ ID NO:774-793.

56. The isolated polypeptide or polypeptide complex according to claim 40, wherein the peptide comprises an amino acid sequence according to any one of SEQ ID NO: 764-773.

57. The isolated polypeptide or polypeptide complex according to claim 40, wherein P1 comprises amino acids according to the following sequence: U1-C-U2-U3-U4-U5-U6-U7-U8-U9-U 10 -U 11 -CU 12 ,in: U1 is selected from T, S, L, M, W, Q, P, R, G, N, I, Y, D, E, V, H, and A; U2 is selected from A, S, T, K, E, R, G, N, W and D; U3 is selected from A, E, P, D, G, W, Q, V, and F; U4 is selected from G, V, F, D, E, R, and A; U5 is selected from A, F, V, S, L, P, and M; U6 is selected from T, S, H, M, A, N, P, G, Q, D, Y, V, L, I, E, K, F and R; U7 is selected from Y, W, F, L, N, T, Q, I, and A; U8 is selected from P, S, T, A, E, K, Q, N, V, G, H, L, D, Y, I, M and R; U9 is selected from W, L, and A; U 10 Selected from P, S, R, L, E, and A; U 11 Selected from L, A, T, M, V, W, Y, E, Q, I, F, S, P, K, R, D, H, N, and G; and U 12 Selected from L, F, V, M, I, Y, E, W, and A.

58. The isolated polypeptide or polypeptide complex according to claim 57, wherein: U1 is selected from T, S, L, M, W, Q, P, R, G, N, I, Y, D, E, V, and H; U2 is selected from A, S, T, K, E, R, G, N, W and D; U3 is selected from A, E, P, D, G, W, Q, V, and F; U4 is selected from G, V, F, D, E, and R; U5 is selected from A, F, V, S, L, P, and M; U6 is selected from T, S, H, M, A, N, P, G, Q, D, Y, V, L, I, E, K, F and R; U7 is selected from Y, W, F, L, N, T, Q, and I; U8 is selected from P, S, T, A, E, K, Q, N, V, G, H, L, D, Y, I, M and R; U9 is selected from W, L, and A; U 10 Selected from P, S, R, L, and E; U 11 Selected from L, A, T, M, V, W, Y, E, Q, I, F, S, P, K, R, D, H, N, and G; and U 12 Selected from L, F, V, M, I, Y, E, and W.

59. The isolated polypeptide or polypeptide complex according to claim 40, wherein P1 comprises the amino acid sequence of SEQ ID NO:

749.

60. The isolated polypeptide or polypeptide complex according to claim 40, wherein P1 comprises an amino acid sequence according to any one of SEQ ID NO:756-763.

61. The isolated polypeptide or polypeptide complex according to claim 40, wherein P1 comprises an amino acid sequence according to any one of SEQ ID NO:794-813.

62. The isolated polypeptide or polypeptide complex according to claim 40, wherein P1 comprises an amino acid sequence according to any one of SEQ ID NO:732-2002.

63. The isolated polypeptide or polypeptide complex according to claim 40, wherein P2 comprises the amino acid sequence of any one of SEQ ID NO: 2003-4329.

64. The isolated polypeptide or polypeptide complex according to claim 40, wherein P2 comprises the amino acid sequence of SEQ ID NO: 2025.

65. The isolated polypeptide or polypeptide complex according to claim 40, wherein P2 comprises an amino acid sequence according to any one of SEQ ID NO:2087-2094.

66. The isolated polypeptide or polypeptide complex according to claim 40, wherein P2 comprises the amino acid sequence of SEQ ID NO: 2026.

67. The isolated polypeptide or polypeptide complex according to claim 40, wherein P2 comprises an amino acid sequence according to any one of SEQ ID NO: 2097-2106.

68. The isolated polypeptide or polypeptide complex according to claim 40, wherein P2 comprises an amino acid sequence according to any one of SEQ ID NO: 2107-2133.

69. The isolated polypeptide or polypeptide complex according to claim 40, wherein P2 comprises the amino acid sequence of SEQ ID NO: 2120.

70. The isolated polypeptide or polypeptide complex according to claim 40, wherein P2 comprises, according to B1-B2-B3-B4-C-B5-P-B6-W-B7-C-B8-B9-B 10 The amino acid sequence of (SEQ ID NO: 4470), wherein: B1 is selected from V, E, L, D, I, G, M, S, P, T, A, F, W, Y, Q, H, N, K, and R; B2 is selected from E, V, D, T, S, L, G, P, A, M, I, Q, H, F, Y, N, W, R and K; B3 is selected from K, P, R, I, N, H, V, M, A, L, Q, T, S, G, F, Y, E, W and D; B4 is selected from W, L, M, R, V, Y, A, K, I, S, Q, F, H, E, T, N, G, and D; B5 is selected from I, V, T, K, R, E, S, Q, M, L, F, A, N, and H; B6 is selected from A, E, S, P, Q, T, L, D, M, V, R, K, N, I, H, W, Y, F, and G; B7 is selected from M, L, I, A, V, F, G, and K; B8 is selected from E, S, T, A, V, D, Q, L, N, I, M, Y, H, F, W, G, K, R, and P; B9 is selected from G, V, A, S, W, E, D, M, T, L, F, H, Q, N, R, I, Y, P, and K; and B 10 Selected from F, L, M, S, I, V, D, Q, T, A, N, Y, W, E, R, H, P, G, and K.

71. The isolated polypeptide or polypeptide complex according to claim 40, wherein when L1 is cleaved by the tumor-specific protease, P1 unbinds from A1, thereby exposing A1 to CD28.

72. The isolated polypeptide or polypeptide complex according to claim 40, wherein when L2 is cleaved by the tumor-specific protease, P2 unbinds from A2, thereby exposing A2 to PSMA.

73. The isolated polypeptide or polypeptide complex according to claim 40, wherein L1 or L2 comprises a urokinase-cleavable amino acid sequence, a matrix protease-cleavable amino acid sequence, a matrix metalloproteinase-cleavable amino acid sequence, or an asparagine endopeptidase-cleavable amino acid sequence.

74. The isolated polypeptide or polypeptide complex according to claim 40, wherein L1 or L2 comprises an amino acid sequence according to any one of SEQ ID NO: 107-137 and 604.

75. The isolated polypeptide or polypeptide complex of claim 40, wherein the half-life-extending molecule comprises a single-domain antibody.

76. The isolated polypeptide or polypeptide complex according to claim 75, wherein 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 comprises: HC-CDR1: SEQ ID NO: 138, HC-CDR2: SEQ ID NO: 139, and HC-CDR3: SEQ ID NO:

140.

77. The isolated polypeptide or polypeptide complex according to claim 75, wherein 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 comprises: HC-CDR1: SEQ ID NO: 142, HC-CDR2: SEQ ID NO: 143, and HC-CDR3: SEQ ID NO:

144.

78. The isolated polypeptide or polypeptide complex according to claim 75, wherein the single-domain antibody comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO:

141.

79. The isolated polypeptide or polypeptide complex according to claim 75, wherein the single-domain antibody comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO:

146.

80. The isolated polypeptide or polypeptide complex according to claim 75, wherein the single-domain antibody comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO:

145.

81. The isolated polypeptide or polypeptide complex according to claim 75, wherein the single-domain antibody comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 4448.

82. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 147 and 148.

83. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 149 and 150.

84. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 151 and 152.

85. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 153 and 154.

86. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 155 and 156.

87. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 157 and 158.

88. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 159 and 160.

89. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 161 and 162.

90. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 163 and 164.

91. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 165 and 166.

92. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 167 and 168.

93. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 169 and 170.

94. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 171 and 172.

95. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 173 and 174.

96. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 175 and 176.

97. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 177 and 178.

98. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4330 and 4331.

99. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4332 and 4333.

100. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4334 and 4335.

101. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4336 and 4337.

102. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99% or 100% sequence identity with SEQ ID NO: 4338 and 4339.

103. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99% or 100% sequence identity with SEQ ID NO: 4340 and 4341.

104. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4342 and 4343.

105. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4344 and 4345.

106. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4346 and 4347.

107. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99% or 100% sequence identity with SEQ ID NO: 4348 and 4349.

108. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4350 and 4351.

109. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99% or 100% sequence identity with SEQ ID NO: 4352 and 4353.

110. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4354 and 4355.

111. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99% or 100% sequence identity with SEQ ID NO: 4356 and 4357.

112. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4358 and 4359.

113. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4360 and 4361.

114. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99% or 100% sequence identity with SEQ ID NO: 4362 and 4363.

115. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4364 and 4365.

116. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4366 and 4367.

117. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99% or 100% sequence identity with SEQ ID NO: 4368 and 4369.

118. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4370 and 4371.

119. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4372 and 4373.

120. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4374 and 4375.

121. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4376 and 4377.

122. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4378 and 4379.

123. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99% or 100% sequence identity with SEQ ID NO: 4380 and 4381.

124. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99% or 100% sequence identity with SEQ ID NO: 4382 and 4383.

125. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4384 and 4385.

126. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4386 and 4387.

127. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99% or 100% sequence identity with SEQ ID NO: 4388 and 4389.

128. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99% or 100% sequence identity with SEQ ID NO: 4390 and 4391.

129. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99% or 100% sequence identity with SEQ ID NO: 4392 and 4393.

130. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4394 and 4395.

131. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99% or 100% sequence identity with SEQ ID NO: 4396 and 4397.

132. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99% or 100% sequence identity with SEQ ID NO: 4398 and 4399.

133. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99% or 100% sequence identity with SEQ ID NO: 4400 and 4401.

134. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4402 and 4403.

135. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4404 and 4405.

136. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4406 and 4407.

137. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4408 and 4409.

138. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4410 and 4411.

139. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4412 and 4413.

140. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4414 and 4415.

141. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99% or 100% sequence identity with SEQ ID NO: 4416 and 4417.

142. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4418 and 4419.

143. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99% or 100% sequence identity with SEQ ID NO: 4420 and 4421.

144. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4422 and 4423.

145. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4424 and 4425.

146. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4426 and 4427.

147. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4428 and 4429.

148. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4430 and 4431.

149. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99% or 100% sequence identity with SEQ ID NO: 4432 and 4433.

150. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4434 and 4435.

151. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4436 and 4437.

152. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99% or 100% sequence identity with SEQ ID NO: 4438 and 4439.

153. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99% or 100% sequence identity with SEQ ID NO: 4440 and 4441.

154. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4442 and 4443.

155. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4444 and 4445.

156. The isolated polypeptide or polypeptide complex according to claim 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 4446 and 4447.

157. An isolated recombinant nucleic acid molecule encoding the polypeptide or polypeptide complex of claim 1.

158. A pharmaceutical composition comprising: (a) the polypeptide or polypeptide complex of claim 1; and (b) Pharmaceutically acceptable excipients.

159. A method of treating cancer in a subject in need, comprising administering the polypeptide or polypeptide complex of claim 1 to said subject.

160. The method of claim 159, wherein the polypeptide or polypeptide complex is administered in combination with an anticancer therapy.

161. The method of claim 160, wherein the anticancer therapy comprises small molecule, cell-based, or antibody-based therapies.

162. The method of claim 161, wherein the antibody-based therapy is a T-cell connective.

163. A vector comprising the isolated recombinant nucleic acid molecule of claim 157.

Images