Antibodies targeting PSMA and CD28 and their uses

Abstract

The present invention discloses an isolated polypeptide and a polypeptide complex composition 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

Technology Field

[0001] Cross-reference

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 595,921, filed November 3, 2023, the entirety of which is incorporated herein by reference.

[0003] Sequence list

[0004] The present application has been submitted electronically in XML format and contains a sequence list whose entire contents are incorporated herein by reference. The XML copy created on October 25, 2024, is named 52426-764_601_SL.xml and has a size of 4,007,239 bytes.

[0005] In some embodiments, the present invention discloses 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). 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 a complementary determining region (CDR): HC-CDR1, HC-CDR2, and HC-CDR3, wherein HC-CDR1, HC-CDR2, and HC-CDR3 comprise the amino acid sequences of 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 a complementary determining region (CDR): LC-CDR1, LC-CDR2, and LC-CDR3, wherein LC-CDR1, LC-CDR2, and LC-CDR3 comprise the amino acid sequences of 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.

[0006] 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 a scFv. In some embodiments, the anti-CD28 antibody comprises a single-chain variable fragment (scFv) having a engineered disulfide. In some embodiments, the anti-CD28 antibody comprises a scFv, said scFv comprising 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 a scFv, said scFv comprising 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 a scFv, said scFv comprising 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 a scFv, said scFv comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO. 12.

[0007] 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.

[0008] 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.

[0009] In some embodiments, a second antigen recognition molecule that binds to PSMA includes an anti-PSMA antibody. In some embodiments, the anti-PSMA antibody comprises an anti-PSMA heavy chain variable domain comprising a complementarity determining region (CDR): HC-CDR1, HC-CDR2, and HC-CDR3, wherein HC-CDR1, HC-CDR2, and HC-CDR3 comprise the amino acid sequences of 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 a complementarity determining region (CDR): LC-CDR1, LC-CDR2, and LC-CDR3, wherein LC-CDR1, LC-CDR2, and LC-CDR3 of the light chain variable domain comprise the amino acid sequences of 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', said 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', said 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', said 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.

[0010] In some embodiments, the anti-CD28 antibody comprises scFv, and the anti-PSMA antibody comprises Fab or Fab'. In some embodiments, said scFv and Fab or Fab' are connected via a linker, said Fab or Fab' comprises a Fab heavy chain polypeptide and a Fab light chain polypeptide. In some embodiments, the linker connects scFv to the Fab heavy chain polypeptide. In some embodiments, the linker connects scFv to the Fab light chain polypeptide. In some embodiments, the linker connects scFv to the N-terminus of the Fab heavy chain polypeptide. In some embodiments, the linker connects scFv to the C-terminus of the Fab heavy chain polypeptide. In some embodiments, the linker connects scFv to the N-terminus of the Fab light chain polypeptide. In some embodiments, the linker connects scFv to the C-terminus of the Fab light chain polypeptide. In some embodiments, the linker connects scFv to the C-terminus of the Fab light chain polypeptide. In some embodiments, the linker connects the anti-CD28 heavy chain variable domain of scFv to the Fab heavy chain polypeptide. In some embodiments, the linker connects the anti-CD28 heavy chain variable domain of scFv to the Fab light chain polypeptide. In some embodiments, the linker connects the anti-CD28 heavy chain variable domain of scFv to the N-terminus of the Fab heavy chain polypeptide. In some embodiments, the linker connects the anti-CD28 heavy chain variable domain of scFv to the C-terminus of the Fab heavy chain polypeptide. In some embodiments, the linker connects the anti-CD28 heavy chain variable domain of scFv to the N-terminus of the Fab light chain polypeptide. In some embodiments, the linker connects the anti-CD28 heavy chain variable domain of scFv to the C-terminus of the Fab light chain polypeptide. In some embodiments, the linker connects the anti-CD28 light chain variable domain of scFv to the Fab heavy chain polypeptide. In some embodiments, the linker connects the anti-CD28 light chain variable domain of scFv to the Fab light chain polypeptide.In some embodiments, the linker connects the anti-CD28 light chain variable domain of scFv to the N-terminus of the Fab heavy chain polypeptide. In some embodiments, the linker connects the anti-CD28 light chain variable domain of scFv to the C-terminus of the Fab heavy chain polypeptide. In some embodiments, the linker connects the anti-CD28 light chain variable domain of scFv to the N-terminus of the Fab light chain polypeptide. In some embodiments, the linker connects the anti-CD28 light chain variable domain of scFv to the C-terminus of the Fab light chain polypeptide.

[0011] 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 connected 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. 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 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 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 connects the Fab light 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 light 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 light 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 light chain polypeptide of the anti-CD28 antibody to the C-terminus of the Fab light chain polypeptide of the anti-PSMA antibody.

[0012] In some embodiments, the anti-CD28 antibody comprises CrossFab, and the anti-PSMA antibody comprises Fab or Fab'. In some embodiments, the CrossFab and Fab or Fab' are connected 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. In some embodiments, the linker connects CrossFab to the Fab heavy chain polypeptide of the anti-PSMA antibody. In some embodiments, the linker connects CrossFab to the Fab light chain polypeptide of the anti-PSMA antibody. In some embodiments, the linker connects CrossFab to the N-terminus of the Fab heavy chain polypeptide of the anti-PSMA antibody. In some embodiments, the linker connects CrossFab to the C-terminus of the Fab heavy chain polypeptide of the anti-PSMA antibody. In some embodiments, the linker connects CrossFab to the N-terminus of the Fab light chain polypeptide of the anti-PSMA antibody. In some embodiments, the linker connects CrossFab 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 CrossFab to the Fab heavy chain polypeptide of the anti-PSMA antibody. In some embodiments, the linker connects the Fab heavy chain polypeptide of CrossFab to the Fab light chain polypeptide of the anti-PSMA antibody. In some embodiments, the linker connects the Fab heavy chain polypeptide of CrossFab 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 CrossFab 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 CrossFab 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 CrossFab 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 CrossFab to the Fab heavy chain polypeptide of the anti-PSMA antibody. In some embodiments, the linker connects the Fab light chain polypeptide of CrossFab to the Fab light chain polypeptide of the anti-PSMA antibody. In some embodiments, the linker connects the Fab light chain polypeptide of CrossFab to the N-terminus of the Fab heavy chain polypeptide of the anti-PSMA antibody. In some embodiments, the linker connects the Fab light chain polypeptide of CrossFab to the C-terminus of the Fab heavy chain polypeptide of the anti-PSMA antibody. In some embodiments, the linker connects the Fab light chain polypeptide of CrossFab to the N-terminus of the Fab light chain polypeptide of the anti-PSMA antibody. In some embodiments, the linker connects the Fab light chain polypeptide of CrossFab to the C-terminus of the Fab light chain polypeptide of the anti-PSMA antibody.

[0013] In some embodiments, the anti-CD28 antibody includes Fab or Fab' and the anti-PSMA antibody includes CrossFab.

[0014] In some embodiments, the linker has a length of at least 5 amino acids. In some embodiments, the linker has a length of 30 or fewer amino acids. In some embodiments, the linker has a length of at least 5 to 30 amino acids. In some embodiments, the linker has a length of 5 amino acids. In some embodiments, the linker has a length of 15 amino acids. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO. 21 (GGGGSGGGGSGGGGS) or SEQ ID NO. 22 (GGGGS).

[0015] 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.

[0016] In some embodiments, the isolated polypeptide or polypeptide complex is a human or humanized polypeptide or polypeptide complex. In some embodiments, the isolated polypeptide or polypeptide complex comprises a peptide linked to an anti-CD28 antibody, said peptide impairs the binding of the anti-CD28 antibody to CD28. In some embodiments, the isolated polypeptide or polypeptide complex is of Formula I: A 2 -A 1 -L 1 -P 1 -H 1 It includes a composition according to, wherein A2 includes an anti-PSMA antibody, A1 includes an anti-CD28 antibody, L1 includes a linking moiety that links A1 to P1 and is a substrate for a tumor-specific protease, P1 includes a peptide that impairs the binding of the anti-CD28 antibody to CD28, and H1 includes a half-life extending molecule.

[0017] In some embodiments, A2 further comprises P2 and L2, wherein P2 comprises a peptide that binds to A2; L2 comprises a linking moiety that links A2 to P2 and is a substrate for a tumor-specific protease. In some embodiments, the polypeptide or polypeptide complex is of formula Ia: P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1It follows. In some embodiments, the anti-CD28 antibody comprises scFv, and the anti-PSMA antibody comprises Fab or Fab', said 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 is bound to the scFv heavy chain polypeptide of A1, and L2 is bound to the Fab light chain polypeptide of A2. In some embodiments, the Fab light chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of A1, and L2 is bound to the Fab heavy chain polypeptide of A2. In some embodiments, the Fab heavy chain polypeptide of A2 is bound to the scFv light chain polypeptide of A1, and L2 is bound to the Fab light chain polypeptide of A2. In some embodiments, the Fab light chain polypeptide of A2 is bound to the scFv light chain polypeptide of A1, and L2 is bound to the Fab heavy chain polypeptide of A2.

[0018] In some embodiments, P1 binds to A1 through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, or H-bonding interactions, or a combination thereof. In some embodiments, P1 has less than 70% sequence homology with CD28. In some embodiments, P2 impairs the binding of A2 to PSMA. In some embodiments, P2 binds to A2 through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, or H-bonding interactions, or a combination 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 having a length of at least 10 amino acids. In some embodiments, P1 or P2 comprises a peptide sequence having a length of at least 10 to 20 amino acids. In some embodiments, P1 or P2 comprises a peptide sequence having a length of at least 16 amino acids. In some embodiments, P1 or P2 comprises a peptide sequence having a length of 40 or fewer amino acids. 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 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.

[0019] In some embodiments, P1 comprises an amino acid sequence according to any one of SEQ ID NOs 33 to 106 or 732 to 813. 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 compared to the amino acid sequence of SEQ ID NO 73. 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 103, or a peptide sequence having one, two, or three amino acid substitutions, additions, or deletions compared to the amino acid sequence of SEQ ID NO 103. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO 103. In some embodiments, P1 comprises X1-X2-X3-C-X4-X5-X6-X7-X8-X9-X 10 -CX 11 -X 12 It includes an amino acid sequence according to, wherein 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 is selected from E, V, L, D, Y, R, Q, H, F, K, A, M, and N; X 11 is selected from F, Y, L, W, and V; X 12is selected from N, A, F, S, Y, H, D, T, and L. In some embodiments, X1 is selected from M, I, and L; X2 is selected from D, H, N, and A; X3 is W; X4 is P; X5 is selected from R, T, I, M, S, and K; X6 is selected from E, D, Y, H, S, and F; X7 is selected from L, M, and R; X8 is W; X9 is selected from H, N, D, A, S, and V; and X 10 is selected from E, V, L, D, and H; X 11 is selected from F, Y, and L; X 12 is selected from N, A, F, S, and Y. In some embodiments, X1 is M; X2 is selected from D and H; X3 is W; X4 is P; X5 is selected from R, T, and I; X6 is selected from E, D, and Y; X7 is selected from L, M, and R; X8 is W; X9 is selected from H, N, D, and V; and X 10 is selected from E, V, L, D, and H; X 11 is F and; X 12 is selected from N, A, and F. In some embodiments, P1 comprises an 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 compared to SEQ ID NO. 41. In some embodiments, P1 comprises an 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 NOs 41, 73, 103, and 179 to 603.

[0020] In some embodiments, P1 is J1-J2-WC-J3-J4-J5-J6-J7-J8-J9-CJ 10 -J 11It includes an amino acid sequence according to, wherein 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; J4 is selected from R, L, and I; J5 is selected from D, H, Y, 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 is selected from F, Y, and L; J 11 is 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; J3 is P; J4 is R; J5 is D; J6 is L; J7 is W; J8 is V; J9 is H; and J 10 is F and; J 11 is A. In some embodiments, 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; and J 10 is F and; J 11 is A. In some embodiments, 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 is F or Y; J 11is N or H. In some embodiments, 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 is F or Y; J 11 is N.

[0021] In some embodiments, P1 is Z1-C-Z2-Z3-Z4-Z5-Z6-Z7-Z8-Z9-Z 10 -Z 11 -CZ 12 It includes an amino acid sequence according to, wherein 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; and 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 is selected from A, Q, S, W, E, L, G, D, T, M, N, V, H, R, Y, K, P, F and I; Z 11 is selected from K, Q, N, H, E, P and A; Z 12is 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 is selected from A, Q, S, W, E, L, G, D, T, M, N, V, H, R, Y, K, P, F and I; Z 11 is selected from K, Q, N, H, E, and P; Z 12is 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 is selected from A, Q, S, W, E, L, G, D, T, M, N, V, H, R, Y, K, P, F and I; Z 11 is selected from K, Q, N, H, E, and P; Z 12 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; and Z 10 is selected from A, Q, S, W, E, L, G, D, T, M, and N; Z 11 is K and; Z 12is 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; 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 is selected from A, Q, S, W, E, L, G, and D; Z 11 is K and; Z 12 is L. In some embodiments, 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; and Z 10 is selected from A, Q, S, W, E, L, G, and D; Z 11 is K and; Z 12 is L.

[0022] 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 NOs 774 to 793. In some embodiments, P1 comprises the amino acid sequence according to any one of SEQ ID NOs 764 to 773.

[0023] In some embodiments, P1 is U1-C-U2-U3-U4-U5-U6-U7-U8-U9-U 10 -U 11 -CU 12It includes an amino acid sequence according to, wherein 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 is selected from P, S, R, L, E and A; U 11 is selected from L, A, T, M, V, W, Y, E, Q, I, F, S, P, K, R, D, H, N and G; U 12is 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 is selected from P, S, R, L and E; U 11 is selected from L, A, T, M, V, W, Y, E, Q, I, F, S, P, K, R, D, H, N and G; U 12 is 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 is selected from P and S; U 11 is selected from L, A, T, M, V, W, Y, E, Q, I, F and S; U 12is 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; and U 10 is P and; U 11 is selected from L, A, T, M, V, W, and Y; U 12 is selected from L and F.

[0024] 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 NOs 756 to 763. In some embodiments, P1 comprises the amino acid sequence according to any one of SEQ ID NOs 794 to 813. In some embodiments, P1 comprises the amino acid sequence according to any one of SEQ ID NOs 732 to 2002.

[0025] In some embodiments, P2 comprises an amino acid sequence of any one of SEQ ID NOs 2003 to 4329. In some embodiments, P2 comprises an 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 an amino acid sequence according to any one of SEQ ID NOs 2087 to 2094. In some embodiments, P2 comprises an 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 an amino acid sequence according to any one of SEQ ID NOs 2097 to 2106. In some embodiments, P2 comprises an amino acid sequence according to any one of SEQ ID NOs 2107 to 2133. In some embodiments, P2 comprises an amino acid sequence of SEQ ID NO 2120. In some embodiments, P2 consists of the amino acid sequence of SEQ ID NO 2120.

[0026] In some embodiments, P2 is B1-B2-B3-B4-C-B5-P-B6-W-B7-C-B8-B9-B 10It comprises an amino acid sequence according to (SEQ 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; B 10 is selected from F, L, M, S, I, V, D, Q, T, A, N, Y, W, E, R, H, P, G and K.

[0027] In some embodiments, L1 is bound to the N-terminus of A1. In some embodiments, L1 is bound to the C-terminus of A1. In some embodiments, L2 is bound to the N-terminus of A2. In some embodiments, L2 is bound to the C-terminus of A2. In some embodiments, L1 or L2 is a peptide sequence having at least 5 to 50 amino acids. In some embodiments, L1 or L2 is a peptide sequence having at least 10 to 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 is (G2S) n It has a chemical formula including, where n is an integer from 1 to 3 (SEQ ID NO. 605). In some embodiments, L1 or L2 is (G2S) n , (GS) n , (GSGGS) n (Sequence No. 606), (GGGS) n (Sequence No. 607), (GGGGGS) n (Sequence No. 608) and (GSSGGS) n It has a chemical formula selected from the group consisting of (sequence number 609), where n is an integer of at least 1.

[0028] In some embodiments, when L1 is cleaved by a tumor-specific protease, P1 becomes unbound from A1, thereby exposing A1 to CD28. In some embodiments, when L2 is cleaved by a tumor-specific protease, P2 becomes unbound from A2, thereby exposing A2 to PSMA. In some embodiments, the tumor-specific protease is selected from the group consisting of matrix metalloproteases (MMPs), serine proteases, cysteine ​​proteases, threonine proteases, and aspartate proteases. In some embodiments, the matrix metalloprotease comprises MMP2, MMP7, MMP9, MMP13, or MMP14. In some embodiments, the serine protease comprises matriptase (MTSP1), urokinase, or hepsin. In some embodiments, L1 or L2 comprises a urokinase-cleavable amino acid sequence, a matrixtase-cleavable amino acid sequence, a matrix metalloprotease-cleavable amino acid sequence, or a legumin-cleavable amino acid sequence. In some embodiments, L1 or L2 comprises an 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 NOs 107 to 137 and 604. In some embodiments, L1 or L2 comprises the amino acid sequence of linker 25 (ISSGLLSGRSDAG) (SEQ No. 132), linker 26 (AAGLLAPPGGLSGRSDAG) (SEQ No. 133), linker 27 (SPLGLSGRSDAG) (SEQ No. 134), or linker 28 (LSGRSDAGSPLGLAG) (SEQ No. 135), or the amino acid sequence having one, two, or three amino acid substitutions, additions, or deletions compared to the amino acid sequence of linker 25, linker 26, linker 27, or linker 28.

[0029] 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, albumin is serum albumin. In some embodiments, albumin is human serum albumin. In some embodiments, H1 comprises a polypeptide, a ligand, or a small molecule. In some embodiments, said 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 a thyroxine-binding protein, transthyretin, 1-acid glycoprotein, transferrin, a transferrin receptor or its transferrin-binding portion, fibrinogen, or albumin. In some embodiments, the circulating immunoglobulin molecule comprises IgG1, IgG2, IgG3, IgG4, slgA, IgM, or IgD. In some embodiments, the serum protein is albumin. In some embodiments, the polypeptide is an antibody. In some embodiments, the antibody comprises a single-domain antibody, a single-chain variable fragment, 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 comprises a complementarity determining region (CDR): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 of the single-domain antibody comprise HC-CDR1: SEQ ID NO. 138, HC-CDR2: SEQ ID NO. 139, and HC-CDR3: SEQ ID NO. 140. In some embodiments, the single-domain antibody comprises a complementarity determining region (CDR): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 of the single-domain antibody comprise HC-CDR1: SEQ ID NO. 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 a modified amino acid, a non-natural amino acid, a modified non-natural amino acid, or a combination thereof. In some embodiments, the modified amino acid or the modified non-natural amino acid comprises a post-translational modification.

[0030] In some embodiments, H1 comprises a linking moiety (L3) that links H1 to P1. In some embodiments, L3 is a peptide sequence having at least 5 to 50 amino acids. In some embodiments, L3 is a peptide sequence having at least 10 to 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 is (G2S) n , (GS) n , (GSGGS) n (Sequence No. 606), (GGGS) n (Sequence No. 607), (GGGGGS) n (Sequence No. 608) and (GSSGGS) n It has a chemical formula selected from the group consisting of (SEQ ID NO. 609), 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).

[0031] In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 159 and 160. In some embodiments, the polypeptide or polypeptide complex comprises the amino acid sequences of SEQ ID NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 171 and 172. In some embodiments, the polypeptide or polypeptide complex comprises the amino acid sequences of SEQ ID NOs 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 NOs 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 NOs 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 NOs 177 and 178.

[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 NOs 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 NOs 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 NOs 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 NOs 4336 and 4337.

[0033] 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 4386 and 4387.

[0034] 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 4446 and 4447.

[0035] In some embodiments, the present invention is Formula II: L 1a -P 1aDisclosing an isolated polypeptide or polypeptide complex according to -H1, wherein L 1a P when not cut 1a It comprises a tumor-specific protease cleavage linkage moiety that links to an anti-CD28 antibody, wherein the anti-CD28 antibody links to an anti-PSMA antibody; P 1a includes an anti-CD28 antibody and a peptide that impairs the binding of CD28, and H 1a It includes a half-life extending molecule. In some embodiments, L 1a When P is not cut 1a It impairs the binding of the anti-CD28 antibody to CD28. In some embodiments, the anti-CD28 antibody comprises a human or humanized antibody.

[0036] In some embodiments, H 1a contains a polymer. In some embodiments, the polymer is polyethylene glycol (PEG). In some embodiments, H 1a It contains albumin. In some embodiments, H 1a includes an Fc domain. In some embodiments, albumin is serum albumin. In some embodiments, albumin is human serum albumin. In some embodiments, H 1a... comprises a polypeptide, a ligand, or a small molecule. In some embodiments, said 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 a thyroxine-binding protein, transthyretin, a 1-acid glycoprotein, transferrin, a transferrin receptor or its transferrin-binding portion, fibrinogen, or albumin. In some embodiments, the circulating immunoglobulin molecule comprises IgG1, IgG2, IgG3, IgG4, slgA, IgM, or IgD. In some embodiments, the serum protein is albumin. In some embodiments, the polypeptide is an antibody. In some embodiments, the antibody comprises a single-domain antibody, a single-chain variable fragment, 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 a complementarity determining region (CDR): 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 a complementarity determining region (CDR): 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.

[0037] In some embodiments, H 1a is H 1a ul P 1a The connection moiety (L) connecting to 2a Includes ). In some embodiments, L 2a is a peptide sequence having at least 5 to 50 amino acids. In some embodiments, L 2a is a peptide sequence having at least 10 to 30 or fewer amino acids. In some embodiments, L 2a is a peptide sequence having at least 10 amino acids. In some embodiments, L 2a is a peptide sequence having at least 18 amino acids. In some embodiments, L 2ais a peptide sequence having at least 26 amino acids. In some embodiments, L 2a is (G2S) n , (GS) n , (GSGGS) n (Sequence No. 606), (GGGS) n (Sequence No. 607), (GGGGGS) n (Sequence No. 608) and (GSSGGS) n It has a chemical formula selected from the group consisting of (SEQ ID NO. 609), where n is an integer of at least 1. In some embodiments, L 2a It includes the amino acid sequence according to SEQ ID NO. 110.

[0038] In some embodiments, P 1a has less than 70% sequence homology with CD28. In some embodiments, P 1a comprises a peptide sequence having a length of at least 10 amino acids. In some embodiments, P 1a comprises a peptide sequence having a length of at least 10 to 20 amino acids. In some embodiments, P 1a comprises a peptide sequence having a length of at least 16 amino acids. In some embodiments, P 1a comprises a peptide sequence having a length of 40 or fewer amino acids. In some embodiments, P 1a contains at least two cysteine ​​amino acid residues. In some embodiments, P 1a comprises a cyclic peptide or a linear peptide. In some embodiments, P 1a includes a cyclic peptide. In some embodiments, P 1a includes a linear peptide. In some embodiments, P 1a comprises an amino acid sequence according to any one of SEQ ID NOs 33 to 106 or 732 to 813. In some embodiments, P 1aincludes the amino acid sequence of SEQ ID NO. 73. In some embodiments, P 1a includes the amino acid sequence of SEQ ID NO. 784. In some embodiments, P 1a 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 compared to the amino acid sequence of SEQ ID NO. 73. In some embodiments, P 1a includes the amino acid sequence of SEQ ID NO. 73. In some embodiments, P 1a 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 compared to the amino acid sequence of SEQ ID NO. 103. In some embodiments, P 1a includes the amino acid sequence of SEQ ID NO. 103. In some embodiments, P 1a is X1-X2-X3-C-X4-X5-X6-X7-X8-X9-X 10 -CX 11 -X 12 It includes an amino acid sequence according to, wherein 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 is selected from E, V, L, D, Y, R, Q, H, F, K, A, M, and N; X 11 is selected from F, Y, L, W, and V; X 12is selected from N, A, F, S, Y, H, D, T, and L. In some embodiments, X1 is selected from M, I, and L; X2 is selected from D, H, N, and A; X3 is W; X4 is P; X5 is selected from R, T, I, M, S, and K; X6 is selected from E, D, Y, H, S, and F; X7 is selected from L, M, and R; X8 is W; X9 is selected from H, N, D, A, S, and V; and X 10 is selected from E, V, L, D, and H; X 11 is selected from F, Y, and L; X 12 is selected from N, A, F, S, and Y. In some embodiments, X1 is M; X2 is selected from D and H; X3 is W; X4 is P; X5 is selected from R, T, and I; X6 is selected from E, D, and Y; X7 is selected from L, M, and R; X8 is W; X9 is selected from H, N, D, and V; and X 10 is selected from E, V, L, D, and H; X 11 is F and; X 12 is selected from N, A, and F. In some embodiments, P 1a comprises an 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 compared to SEQ ID NO. 41. In some embodiments, P 1a includes the amino acid sequence according to SEQ ID NO. 41. In some embodiments, P 1a It includes an amino acid sequence selected from any one of SEQ ID NOs 41, 73, 103 and 179 to 603.

[0039] In some embodiments, P 1a is J1-J2-WC-J3-J4-J5-J6-J7-J8-J9-CJ 10 -J 11It includes an amino acid sequence according to, wherein 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; J4 is selected from R, L, and I; J5 is selected from D, H, Y, 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 is selected from F, Y, and L; J 11 is 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; J3 is P; J4 is R; J5 is D; J6 is L; J7 is W; J8 is V; J9 is H; and J 10 is F and; J 11 is A. In some embodiments, 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; and J 10 is F and; J 11 is A. In some embodiments, 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 is F or Y; J 11is N or H. In some embodiments, 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 is F or Y; J 11 is N.

[0040] In some embodiments, P 1a is Z1-C-Z2-Z3-Z4-Z5-Z6-Z7-Z8-Z9-Z 10 -Z 11 -CZ 12 It includes an amino acid sequence according to, wherein 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; and 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 is selected from A, Q, S, W, E, L, G, D, T, M, N, V, H, R, Y, K, P, F and I; Z 11 is selected from K, Q, N, H, E, P and A; Z 12is 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 is selected from A, Q, S, W, E, L, G, D, T, M, N, V, H, R, Y, K, P, F and I; Z 11 is selected from K, Q, N, H, E, and P; Z 12is 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 is selected from A, Q, S, W, E, L, G, D, T, M, N, V, H, R, Y, K, P, F and I; Z 11 is selected from K, Q, N, H, E, and P; Z 12 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; and Z 10 is selected from A, Q, S, W, E, L, G, D, T, M, and N; Z 11 is K and; Z 12is 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; 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 is selected from A, Q, S, W, E, L, G, and D; Z 11 is K and; Z 12 is L. In some embodiments, 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; and Z 10 is selected from A, Q, S, W, E, L, G, and D; Z 11 is K and; Z 12 is L.

[0041] In some embodiments, P 1a includes the amino acid sequence of SEQ ID NO. 784. In some embodiments, P 1a consists of the amino acid sequence of SEQ ID NO. 784. In some embodiments, P 1a includes the amino acid sequence of SEQ ID NO. 744. In some embodiments, P 1a consists of the amino acid sequence of SEQ ID NO. 744. In some embodiments, P 1a comprises an amino acid sequence according to any one of SEQ ID NOs 774 to 793. In some embodiments, P 1a It includes an amino acid sequence according to any one of sequence numbers 764 to 773.

[0042] In some embodiments, P 1aU1-C-U2-U3-U4-U5-U6-U7-U8-U9-U 10 -U 11 -CU 12 It includes an amino acid sequence according to, wherein 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 is selected from P, S, R, L, E and A; U 11 is selected from L, A, T, M, V, W, Y, E, Q, I, F, S, P, K, R, D, H, N and G; U 12is 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 is selected from P, S, R, L and E; U 11 is selected from L, A, T, M, V, W, Y, E, Q, I, F, S, P, K, R, D, H, N and G; U 12 is 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 is selected from P and S; U 11 is selected from L, A, T, M, V, W, Y, E, Q, I, F and S; U 12is 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; and U 10 is P and; U 11 is selected from L, A, T, M, V, W, and Y; U 12 is selected from L and F.

[0043] In some embodiments, P 1a includes the amino acid sequence of SEQ ID NO. 749. In some embodiments, P 1a includes the amino acid sequence of SEQ ID NO. 749. In some embodiments, P 1a comprises an amino acid sequence according to any one of SEQ ID NOs 756 to 763. In some embodiments, P 1a comprises an amino acid sequence according to any one of SEQ ID NOs 794 to 813. In some embodiments, P 1a It includes an amino acid sequence according to any one of sequence numbers 732 to 2002.

[0044] In some embodiments, the present invention discloses an isolated recombinant nucleic acid molecule encoding a polypeptide or polypeptide complex of any one of the embodiments disclosed herein.

[0045] In some embodiments, the present invention discloses a pharmaceutical composition comprising (a) a polypeptide or polypeptide complex of any one of the embodiments disclosed herein; and (b) a pharmaceutically acceptable excipient.

[0046] In some embodiments, the present invention discloses a method for treating cancer in a subject requiring cancer treatment, comprising the step of administering a polypeptide or polypeptide complex of any one of the embodiments disclosed herein to said subject. In some embodiments, the cancer has cells expressing PSMA. In some cases, the cancer is a solid tumor cancer. In some embodiments, the cancer is lung cancer, breast cancer (e.g., HER2+; ER / PR+; TNBC), cervical cancer, ovarian cancer, colorectal cancer, pancreatic cancer, or gastric cancer.

[0047] In some embodiments, the present invention discloses a method for treating prostate cancer in a subject requiring treatment for prostate cancer, comprising the step of administering an isolated polypeptide or polypeptide complex as described herein to said subject. In some embodiments, the present invention discloses a method for treating mCRPC in a subject requiring treatment for metastatic castration-resistant prostate cancer (mCRPC), comprising the step of administering an isolated polypeptide or polypeptide complex as described herein to said subject.

[0048] In some embodiments, the present invention discloses a method for treating cancer in a subject requiring cancer treatment, comprising the step of administering to the subject a polypeptide or polypeptide complex comprising a first antigen-recognizing molecule that binds to CD28 and a second antigen-recognizing molecule that binds to a prostate-specific membrane antigen. In some embodiments, the first antigen-recognizing 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 a complementary determining region (CDR): HC-CDR1, HC-CDR2, and HC-CDR3, wherein HC-CDR1, HC-CDR2, and HC-CDR3 comprise the amino acid sequences of 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 a complementary determining region (CDR): LC-CDR1, LC-CDR2, and LC-CDR3, wherein LC-CDR1, LC-CDR2, and LC-CDR3 comprise the amino acid sequences of 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 antibody comprises a single chain variable fragment (scFv), a single-domain antibody, Fab, or Fab'. In some embodiments, the anti-CD28 antibody comprises a scFv.In some embodiments, the anti-CD28 antibody comprises a scFv, said scFv comprising 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 a scFv, said scFv comprising 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 a scFv, said scFv comprising 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, said scFv comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO. 12.

[0049] In some embodiments, a second antigen recognition molecule that binds to PSMA includes an anti-PSMA antibody. In some embodiments, the anti-PSMA antibody comprises an anti-PSMA heavy chain variable domain comprising a complementarity determining region (CDR): HC-CDR1, HC-CDR2, and HC-CDR3, wherein HC-CDR1, HC-CDR2, and HC-CDR3 comprise the amino acid sequences of 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 a complementarity determining region (CDR): LC-CDR1, LC-CDR2, and LC-CDR3, wherein LC-CDR1, LC-CDR2, and LC-CDR3 of the light chain variable domain comprise the amino acid sequences of 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', said 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.

[0050] In some embodiments, the polypeptide or polypeptide 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 cancer. In some embodiments, the cancer is lung cancer, breast cancer, cervical cancer, ovarian cancer, colorectal cancer, pancreatic cancer, or gastric cancer.

[0051] In some embodiments, the polypeptide or polypeptide complex is administered in combination with anticancer therapy. In some embodiments, the polypeptide or polypeptide complex and the anticancer therapy are administered as the same pharmaceutical composition. In some embodiments, the polypeptide or polypeptide complex and the anticancer therapy are administered as separate pharmaceutical compositions. In some embodiments, the anticancer therapy includes small molecule, cell-based therapy, or antibody-based therapy.

[0052] In some embodiments, the antibody-based therapy is a T cell engager. In some embodiments, the T cell engager comprises a formula according to 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 linker that connects 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.

[0053] In some embodiments, the effector cell binding domain is Muromonab-CD3 (OKT3), Otelixizumab (TRX4), Teflizumab (MGA031), Bicilizumab (Nuvion), SP34, X35, VIT3, 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, It includes a complementarity determining region (CDR) selected from the group consisting of 15865v16 and 15865v19. In some embodiments, the effector cell binding domain includes amino acid sequences according to SEQ ID NOs 611 to 624.

[0054] 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 herein as TROP2). In some embodiments, the tumor antigen comprises EGFR. In some embodiments, the tumor antigen binding domain comprises amino acid sequences according to SEQ ID NOs 629 to 638. In some embodiments, the tumor antigen comprises EGFR, and the tumor binding domain comprises complementarity determining region (CDR): 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; ​​and HC-CDR3: SEQ ID NO. 634; 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 engager comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NOs. 639 and 640. In some embodiments, the tumor antigen comprises EGFR, and the T cell engager comprises an amino acid sequence according to SEQ ID NOs. 641 and 642.

[0055] In some embodiments, the tumor antigen comprises TROP2. In some embodiments, the tumor antigen comprises TROP2, and the tumor binding domain comprises a complementarity determining region (CDR): HC-CDR1, HC-CDR2 and HC-CDR3, and LC-CDR1, LC-CDR2 and LC-CDR3, wherein HC-CDR1, HC-CDR2 and HC-CDR3 comprises HC-CDR1: SEQ ID NO. 659; HC-CDR2: SEQ ID NO. 660; and HC-CDR3: SEQ ID NO. 661, 662 or 663; and LC-CDR1, LC-CDR2 and LC-CDR3 comprises 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 engager comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NOs 673 and 674. In some embodiments, the tumor antigen comprises TROP2, and the T cell engager comprises an amino acid sequence according to SEQ ID NOs 675 and 676, or SEQ ID NOs 677 and 678. In some embodiments, the tumor antigen binding domain comprises an amino acid sequence according to SEQ ID NOs 659 to 668.

[0056] In some embodiments, the tumor antigen comprises PSMA. In some embodiments, the tumor antigen binding domain comprises amino acid sequences according to SEQ ID NOs 647 to 654. In some embodiments, the tumor antigen comprises PSMA, and the tumor binding domain comprises complementarity determining regions (CDR): 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; and HC-CDR3: SEQ ID NO. 649; and 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 engager comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NOs 655 and 656.

[0057] In some embodiments, the T cell engager molecule is selectively activated in the tumor microenvironment. In some embodiments, the T cell engager follows the following subformation: P3-L3-D-L0-E (Formula IIa), wherein D comprises a CD3 binding domain; E comprises a tumor antigen binding domain; L0 comprises a linker connecting D to E; P3 comprises a peptide that binds to D; and L3 comprises a linking moiety that connects D to P3 and is a substrate for a tumor-specific protease. In some embodiments, the T cell engager follows the following subformation: D-L0-E-L4-P4 (Formula IIb), wherein D comprises a CD3 binding domain; E comprises a tumor antigen binding domain; L0 comprises a linker connecting D to E; P4 comprises a peptide that binds to E; and L4 comprises a linking moiety that connects E to P4 and is a substrate for a tumor-specific protease. In some embodiments, the T cell engager follows the following sub-formula: P3-L3-D-L0-E-L4-P4 (formula IIc), wherein D comprises a CD3 binding domain; E comprises a tumor antigen binding domain; L0 comprises a linker connecting D to E; P3 comprises a peptide that binds to D, L3 comprises a linking moiety that connects D to P3 and is a substrate for a tumor-specific protease; P4 comprises a peptide that binds to E, L4 comprises a linking moiety that connects E to P4 and is a substrate for a tumor-specific protease.

[0058] In some embodiments, the T cell engager 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 a complementarity determining region (CDR): 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.

[0059] In some embodiments, L3 or L4 is a peptide sequence having at least 5 to 50 amino acids. In some embodiments, L3 or L4 is a peptide sequence having at least 10 to 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 is (G2S) n It has a chemical formula including, where n is an integer from 1 to 3 (SEQ ID NO. 605). In some embodiments, L3 or L4 is (G2S) n It has a chemical formula including, where n is an integer of at least 1. In some embodiments, L3 or L4 is (G2S) n , (GS) n , (GSGGS) n (Sequence No. 606), (GGGS) n (Sequence No. 607), (GGGGGS) n (Sequence No. 608) and (GSSGGS) n It has a chemical formula selected from the group consisting of (sequence number 609), where n is an integer of at least 1.

[0060] In some embodiments, the tumor-specific protease is selected from the group consisting of metalloprotease, serine protease, cysteine ​​protease, threonine protease, and aspartate protease. In some embodiments, L3 or L4 comprises a urokinase-cleavable amino acid sequence, a matrixtase-cleavable amino acid sequence, or a matrix metalloprotease-cleavable amino acid sequence. In some embodiments, L3 or L4 comprises a sequence according to any one of SEQ ID NOs 21, 22, and 107 to 136. In some embodiments, L3 is bound to the N-terminus of D. In some embodiments, L3 is bound to the C-terminus of D. In some embodiments, L4 is bound to the N-terminus of E. In some embodiments, L4 is bound to the C-terminus of E.

[0061] In some embodiments, when L3 is cleaved by a tumor-specific protease, P3 becomes unbound from D, thereby exposing D to CD3. In some embodiments, when L4 is cleaved by a tumor-specific protease, P4 becomes unbound from E, thereby exposing E to the tumor antigen. In some embodiments, P3 impairs the binding between D and CD3. In some embodiments, P3 binds to D through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-binding interactions, or a combination 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 becomes unbound from D, thereby exposing D to CD3.

[0062] In some embodiments, P3 has less than 70% sequence identity with CD3. In some embodiments, P3 has less than 70% sequence identity with CD3. In some embodiments, P3 has less than 85% sequence identity with CD3. In some embodiments, P3 has less than 90% sequence identity with CD3. In some embodiments, P3 has less than 95% sequence identity with CD3. In some embodiments, P3 has less than 98% sequence identity with CD3. In some embodiments, P3 has less than 99% sequence identity with CD3. In some embodiments, P3 comprises an amino acid sequence according to SEQ ID NO. 625 or SEQ ID NO. 626. In some embodiments, P3 comprises a de novo amino acid sequence that shares less than 10% sequence identity with CD3.

[0063] In some embodiments, P4 impairs the binding of E to the tumor antigen. In some embodiments, P4 binds to E through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-binding interactions, or a combination 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 becomes unbound from 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 that shares less than 10% sequence identity with the tumor antigen.

[0064] In some embodiments, P3 or P4 comprises a peptide sequence having a length of at least 5 amino acids. In some embodiments, P3 or P4 comprises a peptide sequence having a length of at least 6 amino acids. In some embodiments, P3 or P4 comprises a peptide sequence having a length of at least 10 amino acids. In some embodiments, P3 or P4 comprises a peptide sequence having a length of at least 10 to 20 amino acids. In some embodiments, P3 or P4 comprises a peptide sequence having a length of at least 16 amino acids. In some embodiments, P3 or P4 comprises a peptide sequence having a length of 40 amino acids or less. In some embodiments, P3 or P4 comprises at least 2 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.

[0065] 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 engager comprises the amino acid sequences of SEQ ID NOs. 643 and 644.

[0066] In some embodiments, P4 comprises an amino acid sequence according to any one of SEQ ID NOs 693 to 695. In some embodiments, the tumor antigen comprises TROP2, and the T cell engager comprises the amino acid sequences of SEQ ID NOs 679 and 680, SEQ ID NOs 681 and 682, SEQ ID NOs 683 and 684, SEQ ID NOs 685 and 686, SEQ ID NOs 687 and 688, SEQ ID NOs 689 and 690, and SEQ ID NOs 691 and 692. In some embodiments, the tumor antigen comprises TROP2, and the T cell engager comprises the amino acid sequences of SEQ ID NOs 4452 and 4453, SEQ ID NOs 4454 and 4455, SEQ ID NOs 4456 and 4457, SEQ ID NOs 4458 and 4459, SEQ ID NOs 4460 and 4461, SEQ ID NOs 4462 and 4463, or SEQ ID NOs 4464 and 4465.

[0067] In some embodiments, the tumor antigen comprises PSMA, and the T cell engager comprises the amino acid sequences of SEQ ID NOs 657 and 658. Brief explanation of the drawing

[0068] The novel features of the present invention are specifically set forth in the appended claims. The features and advantages of the present invention will be better understood by referring to the following detailed description, which presents exemplary embodiments in which the principles of the present invention are utilized, and the attached drawings. Fig. 1a This exemplifies a cartoon composition of a multispecific antibody construct targeting PSMA and CD28, administered in combination with a T cell engager (TCE) targeting CD3 and the tumor-associated antigen EGFR. Fig. 1b The PSMA and EGFR densities (copy per cell) on LNCaP tumor cells used in the assay are presented in the table. Fig. 1bThis shows the tumor cell death of LNCaP tumor cells induced by EGFR TCE-2 alone, various concentrations of EGFR TCE-2 combined with Ab-2 (a multispecific antibody targeting PSMA and CD28), and Ab-2 alone, as measured using an in vitro hPBMC and tumor cell co-culture assay. EC for tumor cell death 50 It is presented in this table. Fig. 2a This exemplifies a cartoon composition of a multispecific antibody construct targeting PSMA and CD28, administered in combination with TCE targeting CD3 and the tumor-associated antigen PSMA. Fig. 2b The PSMA density (copy per cell) on LNCaP tumor cells used in the assay is presented in the table. Fig. 2b This shows the tumor cell death of LNCaP tumor cells induced by various concentrations of PSMA TCE-1, alone, and in combination with Ab-2 (a multispecific antibody targeting PSMA and CD28), as measured using an in vitro hPBMC and tumor cell co-culture assay. EC2 for tumor cell death 50 It is presented in this table. Fig. 3 Figure 1 shows tumor cell death of LNCaP tumor cells by EGFR TCE-2, EGFR TCE-2 combined with unshielded Ab-2, EGFR TCE-2 combined with PC-2 having a peptide mask for CD28, and EGFR TCE-2 combined with PC-2 after cleavage of the peptide mask with membrane serine protease 1 (MTSP-1). EC for tumor cell death 50 It is presented in this table. Fig. 4a This exemplifies a cartoon composition of a multispecific antibody construct targeting PSMA and CD28, administered in combination with TCE targeting CD3 and the tumor-associated antigen TROP-2. Fig. 4b and 4cThe PSMA and TROP-2 densities (copy per cell) on recombinant PSMA-expressing DU-145 cells used in the assay are presented in the table. Fig. 4b It shows the production of IL-2 and IFNγ cytokines when measured using an in vitro T cell activation assay with first donor-derived PBMCs, with no stimulation ("no stimulation"), stimulation with TROP2 TCE-1 ("TROP2xCD3"), and stimulation with a combination of TROP2 TCE-1 and Ab-2 ("TROP2xCD3 + PSMAxCD28"). Fig. 4c It shows the production of IL-2 and IFNγ cytokines when measured using an in vitro T cell activation assay with second donor-derived PBMCs, with no stimulation ("no stimulation"), stimulation with TROP2 TCE-1 ("TROP2xCD3"), and stimulation with a combination of TROP2 TCE-1 and Ab-2 ("TROP2xCD3 + PSMAxCD28"). Fig. 5a This exemplifies a cartoon configuration of a combination of a multispecific antibody targeting PSMA and CD28, a cytomegalovirus (CMV) peptide-loaded DU-145 cell expressing PSMA, and a CMV-positive PBMC. Fig. 5b and 5c The PSMA density (copy per cell) on CMV peptide-loaded DU-145 cells used in the assay is presented in the table. Fig. 5b It shows a dose-dependent enhancement of IL-2 production using Ab-2 in a tumor cell / PBMC mixed lymphocyte response (MLR) system using first donor-derived PBMCs. Fig. 5c It shows a dose-dependent enhancement of IL-2 production using Ab-2 in a tumor cell / PBMC MLR system using second donor-derived PBMCs. Fig. 6Figure [] shows a schematic representation of the tumor-specific activity of the multispecific antibody of the present disclosure having a CD28 binding domain (CD28 BD), a PSMA binding domain (PSMA BD), a peptide mask (CD28 mask) connected to the CD28 binding domain by a cleavable linker, a peptide mask (PSMA mask) connected to the PSMA binding domain by a cleavable linker, and a half-life extension moiety (e.g., an albumin binding domain). In the tumor microenvironment (TME), cleavage of the cleavable linker by a tumor protease releases the peptide mask and the half-life extension moiety, thereby activating the multispecific antibody for CD28 stimulation in tumor tissue and promoting the rapid clearance of the multispecific antibody in healthy tissue. Fig. 7a It shows tumor cell death in LNCaP tumor cells caused by EGFR TCE-1 alone, various concentrations of EGFR TCE-1 combined with Ab-1, and Ab-1 alone, when measured using an in vitro hPBMC and tumor cell co-culture assay. Fig. 7b It shows tumor cell death in LNCaP tumor cells caused by EGFR TCE-1 alone, various concentrations of EGFR TCE-1 combined with Ab-2, and Ab-2 alone, when measured using an in vitro hPBMC and tumor cell co-culture assay. Fig. 8 It shows tumor cell death in LNCaP tumor cells induced by EGFR TCE-1 alone and EGFR TCE-1 combined with Ab-2, as measured using an in vitro hPBMC and tumor cell co-culture assay. Fig. 9 It shows the tumor cell death of LNCaP tumor cells by PSMA TCE-1 alone, PSMA TCE-1 combined with Ab-2, and PSMA TCE-1 combined with Ab-4 when measured using an in vitro tumor cell co-culture assay. Fig. 10a inside 10cWhen measured using an in vitro tumor cell co-culture assay, it shows tumor cell death in various cancer cell lines with diverse PSMA expression when using PSMA TCE-1 alone or PSMA TCE-1 in the presence of Ab-2. Fig. 11a and 11b When measured using an in vitro tumor cell co-culture assay, it shows tumor cell death in various cancer cell lines with varying PSMA and EGFR expression when using EGFR TCE-1 alone or EGFR TCE-1 in the presence of Ab-2. Fig. 12 It shows tumor cell death in LNCaP cancer cells when using PSMA TCE-1 combined with Ab-4 and PSMA TCE-1 combined with PC-9, as measured using an in vitro tumor cell co-culture assay. Fig. 13 When measured using an in vitro tumor cell co-culture assay, it shows tumor cell death in LNCaP cancer cells when using PSMA TCE-1 combined with Ab-4, PSMA TCE-1 combined with PC-66, and PSMA TCE-1 combined with PC-66 after treatment with MMP9 protease. Fig. 14 It shows tumor cell death in LNCaP cancer cells when using the combination of Ab-2 and PSMA TCE-1, and the combination of PC-7 and PSMA TRACTr-1, as measured using an in vitro tumor cell death assay. Fig. 15 It shows tumor cell death in LNCaP cancer cells when PSMA TCE-1 alone and when the combination of Ab-4 and PSMA TCE-1 is used, as measured using an in vitro tumor cell death assay. Fig. 16 It shows tumor cell death in LNCaP cancer cells when using the combination of PSMA TCE-1 and Ab-4, the combination of PSMA TCE-1 and PC-25, the combination of PSMA TCE-1 and PC-33, and the combination of PSMA TCE-1 and PC-42. Fig. 17It shows tumor cell death in LNCaP cancer cells when using the combination of PSMA TCE-1 and Ab-2, the combination of PSMA TCE-1 and PC-50, the combination of PSMA TCE-1 and PC-58, and the combination of PSMA TCE-1 and PC-66. Fig. 18 It shows tumor cell death in LNCaP cancer cells when using the combination of PSMA TCE-1 and Ab-2, the combination of PSMA TCE-1 treated with MMP9 protease and PC-25, the combination of PSMA TCE-1 treated with MMP9 protease and PC-33, and the combination of PSMA TCE-1 treated with MMP9 protease and PC-42. Fig. 19 It shows tumor cell death in LNCaP cancer cells when using the combination of PSMA TCE-1 and Ab-2, the combination of PSMA TCE-1 treated with MMP9 protease and PC-50, the combination of PSMA TCE-1 treated with MMP9 protease and PC-58, and the combination of PSMA TCE-1 treated with MMP9 protease and PC-66. Fig. 20 It shows the production of interferon gamma (IFNγ), interleukin-2 (IL-2), and tumor necrosis factor (TNF) after treatment with PSMA TCE-1 + Ab-2 and PSMA-TRACTr-1 + PC-7 when measured using an in vitro T cell activation assay with PBMC. Fig. 21 This exemplifies PSMA binding by Ab-4, PC-66, and PC-66 treated with MMP9 when measured by ELISA. Fig. 22 This exemplifies CD28 binding by Ab-4, PC-66, and PC-66 treated with MMP9 when measured by ELISA. Fig. 23 This exemplifies the binding of Ab-15 and the peptide when measured by ELI. Fig. 24 This exemplifies the binding of Ab-15 and the peptide when measured by ELISA. Fig. 25 This exemplifies the binding of Ab-15 and the peptide when measured by ELISA. Fig. 26This exemplifies biolayer interferometer (BLI) titration data for the binding of Ab-15 and peptide-41. Fig. 27 ... exemplifies BLI titration data for the binding of Ab-15 and peptide-53. Fig. 28 ... exemplifies BLI titration data for the binding of Ab-15 and peptide-56. Fig. 29 This exemplifies BLI titration data for the binding of Ab-15 and peptide-57. Fig. 30 ... exemplifies BLI titration data for the binding of Ab-15 and peptide-62. Fig. 31 ... exemplifies BLI titration data for the binding of Ab-15 and peptide-66. Fig. 32 This exemplifies BLI titration data for the binding of Ab-15 and peptide-71. Fig. 33 ... exemplifies BLI titration data for the binding of Ab-15 and peptide-41. Fig. 34 This exemplifies BLI titration data for the binding of Ab-15 and peptide-288. Fig. 35 This exemplifies BLI titration data for the binding of Ab-15 and peptide-289. Fig. 36 This exemplifies the inhibition of binding between Ab-15 and CD28 by the peptide of the present disclosure. Fig. 37 This exemplifies the inhibition of binding between Ab-15 and CD28 by the peptide of the present disclosure. Fig. 38 This exemplifies the inhibition of binding between Ab-15 and CD28 by the peptide of the present disclosure. Fig. 39 This exemplifies the binding of anti-CD28 Fab and peptide when measured by ELISA. Fig. 40 This exemplifies the binding of anti-CD28 Fab and peptide when measured by ELISA. Fig. 41 This exemplifies the binding of anti-CD28 Fab and peptide when measured by ELISA. Fig. 42 This exemplifies the binding of anti-CD28 Fab and peptide when measured by ELISA. Fig. 43This exemplifies the inhibition of binding between anti-CD28 Fab and CD28 by the peptide of the present disclosure. Fig. 44 This exemplifies the inhibition of binding between anti-CD28 Fab and CD28 by the peptide of the present disclosure. Fig. 45 This exemplifies the inhibition of binding between anti-CD28 Fab and CD28 by the peptide of the present disclosure. Fig. 46 This exemplifies the inhibition of binding between anti-CD28 Fab and CD28 by the peptide of the present disclosure. Fig. 47 This exemplifies the inhibition of binding between anti-CD28 scFv disulfide and CD28 by the peptide of the present disclosure. Fig. 48 This exemplifies BLI titration data for the binding of anti-CD28 Fab and peptide-87. Fig. 49 This exemplifies BLI titration data for the binding of anti-CD28 Fab and peptide-120. Fig. 50 This exemplifies BLI titration data for the binding of anti-CD28 Fab and peptide-121. Fig. 51 This exemplifies BLI titration data for the binding of anti-CD28 Fab and peptide-126. Fig. 52 This exemplifies BLI titration data for the binding of anti-CD28 Fab and peptide-127. Fig. 53 This exemplifies BLI titration data for the binding of anti-CD28 Fab and peptide-132. Fig. 54 This exemplifies BLI titration data for the binding of anti-CD28 Fab and peptide-134. Fig. 55 is an example of a core sequence motif of a peptide-87 sequence generated using WebLogo 3.7.12. Fig. 56 This exemplifies the core sequence motif of an anti-CD28 Fab peptide-92 sequence generated using WebLogo 3.7.12. Fig. 57The figure illustrates a schematic diagram for identifying peptides that can attach to anti-CD28 multispecific antibodies for selective activation in the tumor microenvironment. The schematic diagram illustrates directed evolution and phage display techniques for identifying peptides that block antigen recognition by antigen-binding domains. Fig. 58 This exemplifies the binding of anti-CD28 scFv and peptide as measured by ELISA. Fig. 59 This exemplifies the binding of anti-CD28 scFv and peptide as measured by ELISA. Fig. 60 and 61 This exemplifies that the peptide inhibits the binding of anti-CD28 scFv and CD28 antigen when measured by ELISA. Fig. 62 and 63 This exemplifies the kinetic binding of anti-CD28 scFv and peptide when measured with Octet. Fig. 64a and 64b This exemplifies the binding of anti-CD28 scFv and the Ala scan peptide of peptide-9. Fig. 65a and 65b This exemplifies the inhibition of binding between anti-CD28 scFv and CD28 by the Ala scan peptide of peptide-9. Fig. 66 This exemplifies the core sequence motif of an optimized anti-CD28 scFv peptide-9 sequence generated using WebLogo 3.7.4. Fig. 67 inside 69 exemplifies a peptide that inhibits the binding of anti-CD28 scFv to the CD28 antigen when measured by ELISA. Fig. 70a inside 70u This exemplifies the kinetic binding of anti-CD28 scFv to a peptide when measured by Octet. Fig. 71 and 72 This exemplifies the binding of PSMA Fab and peptide when measured by ELISA. Fig. 73 and 74 This exemplifies the inhibition of binding between PSMA Fab and PSMA by the peptide of the present disclosure. Fig. 75This exemplifies BLI titration data for the binding of PSMA Fab and peptide-179. Fig. 76 ... exemplifies BLI titration data for the binding of PSMA Fab and peptide-219. Fig. 77 ... exemplifies BLI titration data for the binding of PSMA Fab and peptide-225. Fig. 78 ... exemplifies BLI titration data for the binding of PSMA Fab and peptide-227. Fig. 79 This exemplifies BLI titration data for the binding of PSMA Fab and peptide-230. Fig. 80 ... exemplifies BLI titration data for the binding of PSMA Fab and peptide-232. Fig. 81 This exemplifies BLI titration data for the binding of PSMA Fab and peptide-233. Fig. 82 This exemplifies BLI titration data for the binding of PSMA Fab and peptide-235. Fig. 83 ... exemplifies BLI titration data for the binding of PSMA Fab and peptide-236. Fig. 84 This exemplifies BLI titration data for the binding of PSMA Fab and peptide-239. Fig. 85 This exemplifies BLI titration data for the binding of PSMA Fab and peptide-180. Fig. 86 ... exemplifies BLI titration data for the binding of PSMA Fab and peptide-263. Fig. 87 ... exemplifies BLI titration data for the binding of PSMA Fab and peptide-267. Fig. 88 ... exemplifies BLI titration data for the binding of PSMA Fab and peptide-268. Fig. 89 This exemplifies BLI titration data for the binding of PSMA Fab and peptide-270. Fig. 90 ... exemplifies BLI titration data for the binding of PSMA Fab and peptide-271. Fig. 91 ... exemplifies BLI titration data for the binding of PSMA Fab and peptide-274. Fig. 92This exemplifies BLI titration data for the binding of PSMA Fab and peptide-277. Fig. 93 ... exemplifies BLI titration data for the binding of PSMA Fab and peptide-282. Fig. 94 This exemplifies BLI titration data for the binding of PSMA Fab and peptide-283. Fig. 95 This exemplifies BLI titration data for the binding of PSMA Fab and peptide-284. Fig. 96 ... exemplifies BLI titration data for the binding of PSMA Fab and peptide-285. Fig. 97 ... exemplifies BLI titration data for the binding of PSMA Fab and peptide-286. Fig. 98 This exemplifies the core sequence motif of the anti-PSMA Fab peptide-180 sequence generated using WebLogo 3.7.12. Figure 99 This exemplifies appropriate data for PSMA binding by the peptide complex of the present disclosure. Figure 100 The present disclosure exemplifies titration data for PSMA binding by the peptide complex of the present disclosure after treatment with matrix metalloproteinase 9. Fig. 101 This exemplifies appropriate data for CD28 binding by the peptide complex of the present disclosure. Fig. 102 This exemplifies titration data for CD28 binding by the peptide complex of the present disclosure after treatment with matrix metalloproteinase 9. Specific details for implementing the invention

[0069] Multispecific antibodies combine the benefits of diverse binding specificities derived from two or more antibodies into a single composition. Multispecific antibodies, such as T cell engagers (TCEs) designed to redirect T cells toward cancer, have demonstrated potential in both preclinical and clinical studies. This approach relies on the fact that one antigen-interacting portion of the antibody binds to a tumor-associated antigen or marker, while a second antigen-interacting portion can bind to effector cell antigens on T cells, such as differentiation cluster 3 (CD3), which triggers cytotoxic activity. Although TCEs have exhibited potent antitumor activity in some cancers, TCE therapeutics may face challenges such as off-target toxicity and slow elimination in healthy tissues.

[0070] One of these tumor-associated antigens is PSMA. Prostate-specific membrane antigen (PSMA), also known as glutamate carboxypeptidase II (GCPII), N-acetyl-L-aspartyl-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 zinc metalloenzyme present in the membrane. Most of the enzyme exists in the extracellular space. Human PSMA is highly expressed in the prostate at levels approximately 100 times higher than in most other tissues. In some prostate cancers, PSMA is the second most upregulated gene product, increasing by 8 to 12 times compared to levels within non-cancerous prostate cells.

[0071] T cell activation is a highly regulated process that typically requires two signaling events for full function: the first signal (Signal 1) is initiated upon the binding of the major histocompatibility complex (MHC)-antigen complex, which helps distinguish between "self" and "non-self," to the T cell receptor (TCR), and the second co-stimulatory signal (Signal 2) occurs through the activation of co-stimulatory receptors such as CD28. The first signal activates T cells and induces T cell-mediated cytotoxicity of recognized cells; however, if T cells do not receive the second co-stimulatory signal, it can lead to T cell tolerance, whereby T cells continue to recognize tumor antigens but do not trigger 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 toward target cells. CD3-targeted T cell engagers (TCEs) generate Signal 1 via TCE signaling without requiring TCR / MHC interactions.

[0072] The present invention discloses a multispecific antibody comprising a CD28 binding domain and a PSMA binding domain. The applicant has discovered that co-stimulation using the multispecific antibody of the present disclosure enhances CD3-targeted TCE cell lysis activity. A peptide mask that impairs the binding of the CD28 binding domain to CD28 and a peptide mask that impairs the binding of the PSMA binding domain to PSMA are attached to the multispecific antibody via a cleavable linker 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, thereby preventing off-target toxicity in healthy tissues. A half-life extension moiety is attached to the multispecific antibody via the cleavable linker so that cleavage of the cleavable linker by tumor-specific proteases releases the half-life extension moiety to promote rapid elimination. The use of tumor protease cleavage linkers provides the benefits of targeted co-stimulatory activity in the tumor microenvironment while minimizing off-target effects in healthy non-tumor tissues.

[0073] specific definition

[0074] The technical terms used herein are intended to describe specific cases only and are not intended to be limiting. As used herein, the singular form is intended to include the plural form unless the context otherwise indicates. Furthermore, where the terms "including," "includes," "having," "possesses," "possesses," or their derivatives are used in the detailed description and / or claims, these terms are intended to encompass in a manner similar to that of the term "including."

[0075] The term "antibody" is used in the broadest sense and encompasses fully assembled antibodies, antibody fragments capable of binding to antigens, such as Fab, F(ab')2, Fv, single-stranded antibodies (scFv), diabodies, antibody chimeras, hybrid antibodies, bispecific antibodies, etc.

[0076] The term "complementary determining region" or "CDR" refers to a segment of an antibody variable region that is structurally complementary to the epitope to which the antibody binds and is more variable than the rest of the variable region. Therefore, the CDR is sometimes referred to as the hypervariable region. The variable region contains three CDRs. CDR peptides can be obtained by constructing a gene encoding the CDR of the antibody of interest. Such genes are prepared, for example, by synthesizing the variable region from the RNA of antibody-producing cells using polymerase chain reaction. For example, the literature [Larrick et al., Methods: A Companion to Methods in Enzymology 2: 106 (1991); Courtenay-Luck, “Genetic Manipulation of Monoclonal Antibodies,” in Monoclonal Antibodies: Production, Engineering and Clinical Application , Ritter et al. (eds.), pages 166-179 (Cambridge University Press 1995)]; and Ward et al., “Genetic Manipulation and Expression of Antibodies,” in Monoclonal Antibodies: Principles and Applications See Birch et al., (eds.), pages 137-185 (Wiley-Liss, Inc. 1995)].

[0077] The term "Fab" refers to a protein comprising a constant domain of the light chain and a first constant domain (CH1) of the heavy chain. The Fab fragment is distinguished from the Fab' fragment by the addition of several residues, including one or more cystes derived from the antibody hinge region, to the carboxyl terminus of the heavy chain CH1 domain. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residue(s) of the constant domain have free thiol groups. The Fab' fragment is generated by reducing the heavy chain disulfide crosslink of the F(ab')2 fragment. Other chemical couplings of antibody fragments are also known.

[0078] As used herein, a “CrossFab configuration” is a Fab molecule in which the variable domain or constant domain of the Fab heavy chain and the light chain are exchanged (substituted with one another) (see, for example, WO2009 / 080252A1 and WO2017 / 055388A2). For example, a Fab molecule within a CrossFab configuration may comprise a peptide chain (VL-CH1 from N-terminus to C-terminus) composed of a variable light chain domain and a heavy chain constant domain, and a peptide chain (VH-CL from N-terminus to C-terminus) composed of a variable heavy chain domain and a light chain constant domain.

[0079] 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 connected by a short linker peptide having 10 to about 25 amino acids. The linker is typically glycine-rich for flexibility and serine or threonine-rich for solubility, and can connect the N-terminus of VH to the C-terminus of VL or vice versa. This protein retains the specificity of the original antibody despite the removal of the constant region and the introduction of the linker. scFv antibodies are described, for example, in the literature [Houston, JS, Methods in Enzymol. 203 (1991) 46-96]. In addition, the antibody fragment includes a single-strand polypeptide that provides the antigen-binding properties of a full-length antibody by forming a functional antigen-binding site having the characteristics of a VH domain, namely, the ability to be assembled with a VL domain, or the characteristics of a VL domain, namely, the ability to be assembled with a VH domain.

[0080] The term “multispecific” means that an antibody can specifically bind to two or more binding sites formed by two or more distinct antigenic determinants, for example, a pair of antibody heavy chain variable domains (VH) and antibody light chain variable domains (VL), or, in the case of a single-domain antibody, a single variable domain that binds to various antigens.

[0081] As used herein, the term “percent (%) amino acid sequence identity” for a sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a specific sequence after aligning the sequences, introducing gaps where necessary to achieve maximum percentage sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment to determine percentage amino acid sequence identity may be achieved in various ways within the art, for example, by 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. A person skilled in the art may determine appropriate parameters for measuring alignment, including any algorithm necessary to achieve maximum alignment over the entire length of the sequences being compared.

[0082] In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A with respect to a given amino acid sequence B (which may be otherwise expressed as a given amino acid sequence A having or containing a specific % amino acid sequence identity with respect to a given amino acid sequence B) is calculated as follows: 100 multiplied by the fraction X / Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in the program alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that if 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 respect to B will not be equal to the % amino acid sequence identity of B with respect to A. Unless otherwise specifically stated, all % amino acid sequence identity values ​​used herein will be obtained by using the ALIGN-2 computer program as described in the preceding paragraph.

[0083] The terms “complementary determining region” and “CDR,” synonymous with “supervariable region” or “HVR,” are known in the art to refer to discontinuous amino acid sequences within an antibody variable region that confer antigen specificity and / or binding affinity. Generally, 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. “Framework region” and “FR” are known in the art to refer to non-CDR portions of the variable regions of the heavy and light chains. Generally, 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 exact amino acid sequence boundaries of a given CDR or FR are [Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering system)], [Al-Lazikani et al., (1997) JMB 273,927-948 ("Chothia" numbering system)]; [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 system)]; [Lefranc MP et al.It can be easily determined by using any of the many well-known schemes, including those described in the literature [Honegger A and Pluckthun A, "Yet another 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 the literature [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 certain embodiments, the CDR of the antibody described herein may be defined by a method selected from Kavat, Chotia, IMGT, Aho, AbM, or a combination thereof.

[0084] The boundaries of a given CDR or FR may vary depending on the system used for identification. For example, the Kavat system is based on structural alignment, whereas the Chotia system is based on structural information. Numbering for both the Kavat and Chotia systems is based on the sequence lengths of the most common antibody regions, where insertions are accepted by insertion letters, e.g., "30a," and deletions appear in some antibodies. Both systems place specific insertions and deletions ("indels") at different locations, resulting in differential numbering. The Contact system is based on the analysis of the crystal structure of the complex and is similar to the Chotia numbering system in many respects.

[0085] Isolated polypeptide or polypeptide complex

[0086] In some embodiments, the present invention discloses 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). 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 a complementary determining region (CDR): HC-CDR1, HC-CDR2, and HC-CDR3, wherein HC-CDR1, HC-CDR2, and HC-CDR3 comprise the amino acid sequences of 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 a complementary determining region (CDR): LC-CDR1, LC-CDR2, and LC-CDR3, wherein LC-CDR1, LC-CDR2, and LC-CDR3 comprise the amino acid sequences of 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.

[0087]

[0088]

[0089] 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 a engineered disulfide. 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 the amino acid sequence of 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 the amino acid sequence of 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 scFv, and scFv comprises the amino acid sequence of SEQ ID NO. 12.

[0090] 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.

[0091] In some embodiments, a second antigen recognition molecule that binds to PSMA includes an anti-PSMA antibody. In some embodiments, the anti-PSMA antibody comprises an anti-PSMA heavy chain variable domain comprising a complementarity determining region (CDR): HC-CDR1, HC-CDR2, and HC-CDR3, wherein HC-CDR1, HC-CDR2, and HC-CDR3 comprise the amino acid sequences of HC-CDR1: SEQ ID NO. 13, HC-CDR2: SEQ ID NO. 15, and HC-CDR3: SEQ ID NO. 15, and the anti-PSMA antibody comprises an anti-PSMA light chain variable domain comprising a complementarity determining region (CDR): LC-CDR1, LC-CDR2, and LC-CDR3, wherein LC-CDR1, LC-CDR2, and LC-CDR3 of the light chain variable domain comprise the amino acid sequences of 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.

[0092]

[0093] 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', wherein 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', wherein 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.

[0094] In some embodiments, the anti-CD28 antibody comprises scFv, and the anti-PSMA antibody comprises Fab or Fab'. In some embodiments, said scFv and Fab or Fab' are connected via a linker, said Fab or Fab' comprises a Fab heavy chain polypeptide and a Fab light chain polypeptide. In some embodiments, the linker connects scFv to the Fab heavy chain polypeptide. In some embodiments, the linker connects scFv to the Fab light chain polypeptide. In some embodiments, the linker connects scFv to the N-terminus of the Fab heavy chain polypeptide. In some embodiments, the linker connects scFv to the C-terminus of the Fab heavy chain polypeptide. In some embodiments, the linker connects scFv to the N-terminus of the Fab light chain polypeptide. In some embodiments, the linker connects scFv to the C-terminus of the Fab light chain polypeptide. In some embodiments, the linker connects scFv to the C-terminus of the Fab light chain polypeptide. In some embodiments, the linker connects the anti-CD28 heavy chain variable domain of scFv to the Fab heavy chain polypeptide. In some embodiments, the linker links the anti-CD28 heavy chain variable domain of scFv to the Fab light chain polypeptide.

[0095] In some embodiments, the linker connects the anti-CD28 heavy chain variable domain of scFv to the N-terminus of the Fab heavy chain polypeptide. In some embodiments, the linker connects the anti-CD28 heavy chain variable domain of scFv to the C-terminus of the Fab heavy chain polypeptide. In some embodiments, the linker connects the anti-CD28 heavy chain variable domain of scFv to the N-terminus of the Fab light chain polypeptide. In some embodiments, the linker connects the anti-CD28 heavy chain variable domain of scFv to the C-terminus of the Fab light chain polypeptide. In some embodiments, the linker connects the anti-CD28 light chain variable domain of scFv to the Fab heavy chain polypeptide. In some embodiments, the linker connects the anti-CD28 light chain variable domain of scFv to the Fab light chain polypeptide. In some embodiments, the linker connects the anti-CD28 light chain variable domain of scFv to the N-terminus of the Fab heavy chain polypeptide. In some embodiments, the linker connects the anti-CD28 light chain variable domain of scFv to the C-terminus of the Fab heavy chain polypeptide. In some embodiments, the linker connects the anti-CD28 light chain variable domain of scFv to the N-terminus of the Fab light chain polypeptide. In some embodiments, the linker connects the anti-CD28 light chain variable domain of scFv to the C-terminus of the Fab light chain polypeptide.

[0096] 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 connected 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. 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 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 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 connects the Fab light 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 light 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 light 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 light chain polypeptide of the anti-CD28 antibody to the C-terminus of the Fab light chain polypeptide of the anti-PSMA antibody.

[0097] In some embodiments, the anti-CD28 antibody comprises CrossFab, and the anti-PSMA antibody comprises Fab or Fab'. In some embodiments, the CrossFab and Fab or Fab' are connected 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. In some embodiments, the linker connects CrossFab to the Fab heavy chain polypeptide of the anti-PSMA antibody. In some embodiments, the linker connects CrossFab to the Fab light chain polypeptide of the anti-PSMA antibody. In some embodiments, the linker connects CrossFab to the N-terminus of the Fab heavy chain polypeptide of the anti-PSMA antibody. In some embodiments, the linker connects CrossFab to the C-terminus of the Fab heavy chain polypeptide of the anti-PSMA antibody. In some embodiments, the linker connects CrossFab to the N-terminus of the Fab light chain polypeptide of the anti-PSMA antibody. In some embodiments, the linker connects CrossFab 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 CrossFab to the Fab heavy chain polypeptide of the anti-PSMA antibody. In some embodiments, the linker connects the Fab heavy chain polypeptide of CrossFab to the Fab light chain polypeptide of the anti-PSMA antibody. In some embodiments, the linker connects the Fab heavy chain polypeptide of CrossFab 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 CrossFab 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 CrossFab 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 CrossFab 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 CrossFab to the Fab heavy chain polypeptide of the anti-PSMA antibody. In some embodiments, the linker connects the Fab light chain polypeptide of CrossFab to the Fab light chain polypeptide of the anti-PSMA antibody. In some embodiments, the linker connects the Fab light chain polypeptide of CrossFab to the N-terminus of the Fab heavy chain polypeptide of the anti-PSMA antibody. In some embodiments, the linker connects the Fab light chain polypeptide of CrossFab to the C-terminus of the Fab heavy chain polypeptide of the anti-PSMA antibody. In some embodiments, the linker connects the Fab light chain polypeptide of CrossFab to the N-terminus of the Fab light chain polypeptide of the anti-PSMA antibody. In some embodiments, the linker connects the Fab light chain polypeptide of CrossFab to the C-terminus of the Fab light chain polypeptide of the anti-PSMA antibody.

[0098] In some embodiments, the anti-CD28 antibody includes Fab or Fab' and the anti-PSMA antibody includes CrossFab.

[0099] In some embodiments, the linker has a length of at least 5 amino acids. In some embodiments, the linker has a length of 30 or fewer amino acids. In some embodiments, the linker has a length of at least 5 to 30 amino acids. In some embodiments, the linker has a length of 5 amino acids. In some embodiments, the linker has a length of 15 amino acids. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO. 21 (GGGGSGGGGSGGGGS) or SEQ ID NO. 22 (GGGGS).

[0100]

[0101] polypeptide sequences binding to CD28 and PSMA

[0102] 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.

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111] In some embodiments, the isolated polypeptide or polypeptide complex is a human or humanized polypeptide or polypeptide complex. In some embodiments, the isolated polypeptide or polypeptide complex comprises a peptide linked to an anti-CD28 antibody, said peptide impairs the binding of the anti-CD28 antibody to CD28. In some embodiments, the isolated polypeptide or polypeptide complex is of Formula I: A 2 -A 1 -L 1 -P 1 -H 1 It comprises a composition according to, wherein A2 comprises an anti-PSMA antibody, A1 comprises an anti-CD28 antibody, L1 comprises a linking moiety connecting A1 to P1 and is a substrate for a tumor-specific protease, P1 comprises a peptide that impairs 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 that binds to A2; L2 comprises a linking moiety connecting A2 to P2 and is a substrate for a tumor-specific protease. In some embodiments, the polypeptide or polypeptide complex is of formula Ia: P 2-L 2 -A 2 -A 1 -L 1 -P 1 -H 1 It depends on.

[0112] 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 is bound to the scFv heavy chain polypeptide of A1, and L2 is bound to the Fab light chain polypeptide of A2. In some embodiments, the Fab light chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of A1, and L2 is bound to the Fab heavy chain polypeptide of A2. In some embodiments, the Fab heavy chain polypeptide of A2 is bound to the scFv light chain polypeptide of A1, and L2 is bound to the Fab light chain polypeptide of A2. In some embodiments, the Fab light chain polypeptide of A2 is bound to the scFv light chain polypeptide of A1, and L2 is bound to the Fab heavy chain polypeptide of A2.

[0113] P 1 and P 2

[0114] In some embodiments, P1 binds to A1 through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, or H-bonding interactions, or a combination thereof. In some embodiments, P1 has less than 70% sequence homology with CD28. In some embodiments, P2 impairs the binding of A2 to PSMA. In some embodiments, P2 binds to A2 through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, or H-bonding interactions, or a combination 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 having a length of at least 10 amino acids. In some embodiments, P1 or P2 comprises a peptide sequence having a length of at least 10 to 20 amino acids. In some embodiments, P1 or P2 comprises a peptide sequence having a length of at least 16 amino acids. In some embodiments, P1 or P2 comprises a peptide sequence having a length of 40 or fewer amino acids. 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.

[0115] In some embodiments, P1 comprises an amino acid sequence according to any one of SEQ ID NOs 33 to 106 or 732 to 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.

[0116] In some embodiments, P1 includes the amino acid sequence of SEQ ID NO. 61. In some embodiments, P1 includes the amino acid sequence of SEQ ID NO. 62. In some embodiments, P1 includes the amino acid sequence of SEQ ID NO. 63. In some embodiments, P1 includes the amino acid sequence of SEQ ID NO. 64. In some embodiments, P1 includes the amino acid sequence of SEQ ID NO. 65. In some embodiments, P1 includes the amino acid sequence of SEQ ID NO. 66. In some embodiments, P1 includes the amino acid sequence of SEQ ID NO. 67. In some embodiments, P1 includes the amino acid sequence of SEQ ID NO. 68. In some embodiments, P1 includes the amino acid sequence of SEQ ID NO. 69. In some embodiments, P1 includes the amino acid sequence of SEQ ID NO. 70. In some embodiments, P1 includes the amino acid sequence of SEQ ID NO. 71. In some embodiments, P1 includes the amino acid sequence of SEQ ID NO. 72. In some embodiments, P1 includes the amino acid sequence of SEQ ID NO. 73. In some embodiments, P1 includes 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 includes the amino acid sequence of SEQ ID NO. 86. In some embodiments, P1 includes the amino acid sequence of SEQ ID NO. 87. In some embodiments, P1 includes the amino acid sequence of SEQ ID NO. 88. In some embodiments, P1 includes the amino acid sequence of SEQ ID NO. 89. In some embodiments, P1 includes the amino acid sequence of SEQ ID NO. 90. In some embodiments, P1 includes the amino acid sequence of SEQ ID NO. 91. In some embodiments, P1 includes the amino acid sequence of SEQ ID NO. 92. In some embodiments, P1 includes the amino acid sequence of SEQ ID NO. 93. In some embodiments, P1 includes the amino acid sequence of SEQ ID NO. 94. In some embodiments, P1 includes the amino acid sequence of SEQ ID NO. 95. In some embodiments, P1 includes the amino acid sequence of SEQ ID NO. 96. In some embodiments, P1 includes the amino acid sequence of SEQ ID NO. 97. In some embodiments, P1 includes the amino acid sequence of SEQ ID NO. 98. In some embodiments, P1 includes 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.

[0117]

[0118]

[0119]

[0120]

[0121] 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 compared to the amino acid sequence of SEQ ID NO. 73. 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. 103, or a peptide sequence having one, two, or three amino acid substitutions, additions, or deletions compared to the amino acid sequence of SEQ ID NO. 103. In some embodiments, P1 comprises the amino acid sequence of SEQ ID NO. 103.

[0122] In some embodiments, P1 is X1-X2-X3-C-X4-X5-X6-X7-X8-X9-X 10 -CX 11 -X 12 It includes an amino acid sequence according to, wherein 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 is selected from E, V, L, D, Y, R, Q, H, F, K, A, M, and N; X 11 is selected from F, Y, L, W, and V; X 12is selected from N, A, F, S, Y, H, D, T, and L. In some embodiments, X1 is selected from M, I, and L; X2 is selected from D, H, N, and A; X3 is W; X4 is P; X5 is selected from R, T, I, M, S, and K; X6 is selected from E, D, Y, H, S, and F; X7 is selected from L, M, and R; X8 is W; X9 is selected from H, N, D, A, S, and V; and X 10 is selected from E, V, L, D, and H; X 11 is selected from F, Y, and L; X 12 is selected from N, A, F, S, and Y. In some embodiments, X1 is M; X2 is selected from D and H; X3 is W; X4 is P; X5 is selected from R, T, and I; X6 is selected from E, D, and Y; X7 is selected from L, M, and R; X8 is W; X9 is selected from H, N, D, and V; and X 10 is selected from E, V, L, D, and H; X 11 is F and; X 12 is selected from N, A, and F.

[0123] In some embodiments, P1 comprises an 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 compared to SEQ ID NO. 41. In some embodiments, P1 comprises an 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 NOs 41, 73, 103, and 179 to 603.

[0124]

[0125]

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[0175]

[0176]

[0177] In some embodiments, P1 is J1-J2-WC-J3-J4-J5-J6-J7-J8-J9-CJ10 -J 11 It includes an amino acid sequence according to, wherein 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; J4 is selected from R, L, and I; J5 is selected from D, H, Y, 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 is selected from F, Y, and L; J 11 is 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; J3 is P; J4 is R; J5 is D; J6 is L; J7 is W; J8 is V; J9 is H; and J 10 is F and; J 11 is A. In some embodiments, 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; and J 10 is F and; J 11 is A. In some embodiments, 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 is F or Y; J11 is N or H. In some embodiments, 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 is F or Y; J 11 is N.

[0178] In some embodiments, P1 is Z1-C-Z2-Z3-Z4-Z5-Z6-Z7-Z8-Z9-Z 10 -Z 11 -CZ 12 It includes an amino acid sequence according to, wherein 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 is selected from A, Q, S, W, E, L, G, D, T, M, N, V, H, R, Y, K, P, F and I; Z 11 is selected from K, Q, N, H, E, P and A; Z 12is 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 is selected from A, Q, S, W, E, L, G, D, T, M, N, V, H, R, Y, K, P, F and I; Z 11 is selected from K, Q, N, H, E, and P; Z 12is 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 is selected from A, Q, S, W, E, L, G, D, T, M, N, V, H, R, Y, K, P, F and I; Z 11 is selected from K, Q, N, H, E, and P; Z 12 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; and Z 10 is selected from A, Q, S, W, E, L, G, D, T, M, and N; Z 11 is K and; Z 12is 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; 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 is selected from A, Q, S, W, E, L, G, and D; Z 11 is K and; Z 12 is L. In some embodiments, 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; and Z 10 is selected from A, Q, S, W, E, L, G, and D; Z 11 is K and; Z 12 is L.

[0179] 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 NOs 774 to 793. In some embodiments, P1 comprises the amino acid sequence according to any one of SEQ ID NOs 764 to 773.

[0180] In some embodiments, P1 is U1-C-U2-U3-U4-U5-U6-U7-U8-U9-U 10 -U 11 -CU 12It includes an amino acid sequence according to, wherein 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 is selected from P, S, R, L, E and A; U 11 is selected from L, A, T, M, V, W, Y, E, Q, I, F, S, P, K, R, D, H, N and G; U 12is 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 is selected from P, S, R, L and E; U 11 is selected from L, A, T, M, V, W, Y, E, Q, I, F, S, P, K, R, D, H, N and G; U 12 is 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 is selected from P and S; U 11 is selected from L, A, T, M, V, W, Y, E, Q, I, F and S; U 12is 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; and U 10 is P and; U 11 is selected from L, A, T, M, V, W, and Y; U 12 is selected from L and F.

[0181] 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 NOs 756 to 763. In some embodiments, P1 comprises the amino acid sequence according to any one of SEQ ID NOs 794 to 813. In some embodiments, P1 comprises the amino acid sequence according to any one of SEQ ID NOs 732 to 2002.

[0182] In some embodiments, P2 comprises an amino acid sequence of any one of SEQ ID NOs 2003 to 4329. In some embodiments, P2 comprises an 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 an amino acid sequence according to any one of SEQ ID NOs 2087 to 2094. In some embodiments, P2 comprises an 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 an amino acid sequence according to any one of SEQ ID NOs 2097 to 2106. In some embodiments, P2 comprises an amino acid sequence according to any one of SEQ ID NOs 2107 to 2133. In some embodiments, P2 comprises an amino acid sequence of SEQ ID NO 2120. In some embodiments, P2 consists of the amino acid sequence of SEQ ID NO 2120.

[0183] In some embodiments, P2 is B1-B2-B3-B4-C-B5-P-B6-W-B7-C-B8-B9-B 10It comprises an amino acid sequence according to (SEQ 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; and 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; B 10 is selected from F, L, M, S, I, V, D, Q, T, A, N, Y, W, E, R, H, P, G and K.

[0184]

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

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[0205]

[0206]

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[0209]

[0210]

[0211]

[0212]

[0213]

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[0217]

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[0220]

[0221]

[0222]

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[0224]

[0225]

[0226]

[0227]

[0228]

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[0230]

[0231]

[0232]

[0233]

[0234]

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[0240]

[0241]

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[0250]

[0251]

[0252]

[0253]

[0254]

[0255]

[0256]

[0257]

[0258]

[0259]

[0260]

[0261]

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

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[0270]

[0271]

[0272]

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[0279]

[0280]

[0281]

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[0287]

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[0289]

[0290]

[0291]

[0292]

[0293]

[0294] L 1 and L 2

[0295] In some embodiments, L1 is bound to the N-terminus of A1. In some embodiments, L1 is bound to the C-terminus of A1. In some embodiments, L2 is bound to the N-terminus of A2. In some embodiments, L2 is bound to the C-terminus of A2. In some embodiments, L1 or L2 is a peptide sequence having at least 5 to 50 amino acids. In some embodiments, L1 or L2 is a peptide sequence having at least 10 to 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 is (G2S) n It has a chemical formula including, where n is an integer from 1 to 3 (SEQ ID NO. 605). In some embodiments, L1 is (G2S) n , (GS) n , (GSGGS) n (Sequence No. 606), (GGGS) n (Sequence No. 607), (GGGGGS) n (Sequence No. 608) and (GSSGGS) n It has a chemical formula selected from the group consisting of (sequence number 609), where n is an integer of at least 1.

[0296] In some embodiments, when L1 is cleaved by a tumor-specific protease, P1 becomes unbound from A1, thereby exposing A1 to CD28. In some embodiments, when L2 is cleaved by a tumor-specific protease, P2 becomes unbound from A2, thereby exposing A2 to PSMA. In some embodiments, the tumor-specific protease is selected from the group consisting of matrix metalloproteases (MMPs), serine proteases, cysteine ​​proteases, threonine proteases, and aspartate proteases. In some embodiments, the matrix metalloprotease comprises MMP2, MMP7, MMP9, MMP13, or MMP14. In some embodiments, the serine protease comprises matrixtase (MTSP1), urokinase, or hepsin. In some embodiments, L1 or L2 comprises a urokinase-cleavable amino acid sequence, a matrixtase-cleavable amino acid sequence, a matrix metalloprotease-cleavable amino acid sequence, or a legumine-cleavable amino acid sequence.

[0297] In some embodiments, L1 or L2 comprises an 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 NOs 107 to 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 compared to the amino acid sequences of linker 25, linker 26, linker 27, or linker 28.

[0298]

[0299]

[0300] H 1

[0301] 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, albumin is serum albumin. In some embodiments, albumin is human serum albumin. In some embodiments, H1 comprises a polypeptide, a ligand, or a small molecule. In some embodiments, said 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 a thyroxine-binding protein, transthyretin, 1-acid glycoprotein, transferrin, a transferrin receptor or its transferrin-binding portion, fibrinogen, or albumin. In some embodiments, the circulating immunoglobulin molecule comprises IgG1, IgG2, IgG3, IgG4, slgA, IgM, or IgD. In some embodiments, the serum protein is albumin. In some embodiments, the polypeptide is an antibody. In some embodiments, the antibody comprises a single-domain antibody, a single-chain variable fragment, 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 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.

[0302] In some embodiments, the single-domain antibody comprises a complementarity determining region (CDR): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 of the single-domain antibody comprise HC-CDR1: SEQ ID NO. 138, HC-CDR2: SEQ ID NO. 139, and HC-CDR3: SEQ ID NO. 140. In some embodiments, the single-domain antibody comprises a complementarity determining region (CDR): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 of the single-domain antibody comprise HC-CDR1: SEQ ID NO. 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.

[0303]

[0304] In some embodiments, the single-domain antibody is SA21. In some embodiments, the polypeptide or polypeptide complex comprises a modified amino acid, a non-natural amino acid, a modified non-natural amino acid, or a combination thereof. In some embodiments, the modified amino acid or the modified non-natural amino acid comprises a post-translational modification.

[0305] L3

[0306] In some embodiments, H1 comprises a linking moiety (L3) that links H1 to P1. In some embodiments, L3 is a peptide sequence having at least 5 to 50 amino acids. In some embodiments, L3 is a peptide sequence having at least 10 to 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 is (G2S) n , (GS) n , (GSGGS) n (Sequence No. 606), (GGGS) n (Sequence No. 607), (GGGGGS) n (Sequence No. 608) and (GSSGGS) n It has a chemical formula selected from the group consisting of (SEQ ID NO. 609), 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).

[0307] Tumor-activating antibodies that bind to CD28 and PSMA

[0308] In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NOs 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 NOs 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 NOs 147 and 148. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NOs 147 and 148. In some embodiments, the polypeptide or polypeptide complex comprises the amino acid sequences of SEQ ID NOs 147 and 148.

[0309] In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NOs 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 NOs 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 NOs 149 and 150. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NOs 149 and 150. In some embodiments, the polypeptide or polypeptide complex comprises the amino acid sequences of SEQ ID NOs 149 and 150.

[0310] In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NOs 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 NOs 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 NOs 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 NOs 151 and 152. In some embodiments, the polypeptide or polypeptide complex comprises the amino acid sequences of SEQ ID NOs 151 and 152.

[0311] In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NOs 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 NOs 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 NOs 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 NOs 153 and 154. In some embodiments, the polypeptide or polypeptide complex comprises the amino acid sequences of SEQ ID NOs 153 and 154.

[0312] In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NOs 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 NOs 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 NOs 155 and 156. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NOs 155 and 156. In some embodiments, the polypeptide or polypeptide complex comprises the amino acid sequences of SEQ ID NOs 155 and 156.

[0313] In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NOs 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 NOs 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 NOs 157 and 158. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NOs 157 and 158. In some embodiments, the polypeptide or polypeptide complex comprises the amino acid sequences of SEQ ID NOs 157 and 158.

[0314] In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NOs 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 NOs 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 NOs 159 and 160. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NOs 159 and 160. In some embodiments, the polypeptide or polypeptide complex comprises the amino acid sequences of SEQ ID NOs 159 and 160.

[0315] In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NOs 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 NOs 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 NOs 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 NOs 161 and 162. In some embodiments, the polypeptide or polypeptide complex comprises the amino acid sequences of SEQ ID NOs 161 and 162.

[0316] In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NOs 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 NOs 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 NOs 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 NOs 163 and 164. In some embodiments, the polypeptide or polypeptide complex comprises the amino acid sequences of SEQ ID NOs 163 and 164.

[0317] In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NOs 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 NOs 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 NOs 165 and 166. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NOs 165 and 166. In some embodiments, the polypeptide or polypeptide complex comprises the amino acid sequences of SEQ ID NOs 165 and 166.

[0318] In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NOs 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 NOs 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 NOs 167 and 168. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NOs 167 and 168. In some embodiments, the polypeptide or polypeptide complex comprises the amino acid sequences of SEQ ID NOs 167 and 168.

[0319] In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NOs 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 NOs 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 NOs 169 and 170. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NOs 169 and 170. In some embodiments, the polypeptide or polypeptide complex comprises the amino acid sequences of SEQ ID NOs 169 and 170.

[0320] In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NOs 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 NOs 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 NOs 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 NOs 171 and 172. In some embodiments, the polypeptide or polypeptide complex comprises the amino acid sequences of SEQ ID NOs 171 and 172.

[0321] In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NOs 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 NOs 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 NOs 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 NOs 173 and 174. In some embodiments, the polypeptide or polypeptide complex comprises the amino acid sequences of SEQ ID NOs 173 and 174.

[0322] In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NOs 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 NOs 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 NOs 175 and 176. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NOs 175 and 176. In some embodiments, the polypeptide or polypeptide complex comprises the amino acid sequences of SEQ ID NOs 175 and 176.

[0323] In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NOs 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 NOs 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 NOs 177 and 178. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NOs 177 and 178. In some embodiments, the polypeptide or polypeptide complex comprises the amino acid sequences of SEQ ID NOs 177 and 178.

[0324] 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 NOs 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 NOs 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 NOs 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 NOs 4336 and 4337.

[0325] 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 4386 and 4387.

[0326] 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 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 NOs 4446 and 4447.

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338]

[0339]

[0340]

[0341]

[0342]

[0343]

[0344]

[0345]

[0346]

[0347]

[0348]

[0349]

[0350]

[0351]

[0352]

[0353]

[0354]

[0355]

[0356]

[0357]

[0358]

[0359]

[0360]

[0361]

[0362]

[0363]

[0364] L 1a -P 1a -H 1a

[0365] In some embodiments, the present invention is Formula II: L 1a -P 1a -H 1a Disclosing an isolated polypeptide or polypeptide complex according to, wherein L 1a P when not cut 1a It includes a tumor-specific protease cleavage linkage moiety that links to an anti-CD28 antibody, wherein the anti-CD28 antibody links to an anti-PSMA antibody, and P 1a includes an anti-CD28 antibody and a peptide that impairs the binding of CD28, and H 1a It includes a half-life extending molecule. In some embodiments, L 1a When it is not cut, P 1a It impairs the binding of the anti-CD28 antibody to CD28. In some embodiments, the anti-CD28 antibody comprises a human or humanized antibody.

[0366] In some embodiments, H 1acontains a polymer. In some embodiments, the polymer is polyethylene glycol (PEG). In some embodiments, H 1a It contains albumin. In some embodiments, H 1a includes an Fc domain. In some embodiments, albumin is serum albumin. In some embodiments, albumin is human serum albumin. In some embodiments, H 1a ... comprises a polypeptide, a ligand, or a small molecule. In some embodiments, said 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 a thyroxine-binding protein, transthyretin, a 1-acid glycoprotein, transferrin, a transferrin receptor or its transferrin-binding portion, fibrinogen, or albumin. In some embodiments, the circulating immunoglobulin molecule comprises IgG1, IgG2, IgG3, IgG4, slgA, IgM, or IgD. In some embodiments, the serum protein is albumin. In some embodiments, the polypeptide is an antibody. In some embodiments, the antibody comprises a single-domain antibody, a single-chain variable fragment, 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.

[0367] In some embodiments, the single-domain antibody comprises a complementarity determining region (CDR): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 of the single-domain antibody comprise HC-CDR1: SEQ ID NO. 138, HC-CDR2: SEQ ID NO. 139, and HC-CDR3: SEQ ID NO. 140. In some embodiments, the single-domain antibody comprises a complementarity determining region (CDR): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 of the single-domain antibody comprise HC-CDR1: SEQ ID NO. 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.

[0368] In some embodiments, H 1a is H 1a ul P 1a The connection moiety (L) connecting to 2a Includes ). In some embodiments, L 2a is a peptide sequence having at least 5 to 50 amino acids. In some embodiments, L 2ais a peptide sequence having at least 10 to 30 or fewer amino acids. In some embodiments, L 2a is a peptide sequence having at least 10 amino acids. In some embodiments, L 2a is a peptide sequence having at least 18 amino acids. In some embodiments, L 2a is a peptide sequence having at least 26 amino acids. In some embodiments, L 2a is (G2S) n , (GS) n , (GSGGS) n (Sequence No. 606), (GGGS) n (Sequence No. 607), (GGGGGS) n (Sequence No. 608) and (GSSGGS) n It has a chemical formula selected from the group consisting of (SEQ ID NO. 609), where n is an integer of at least 1. In some embodiments, L 2a It includes the amino acid sequence according to SEQ ID NO. 110.

[0369] In some embodiments, P 1a has less than 70% sequence homology with CD28. In some embodiments, P 1a comprises a peptide sequence having a length of at least 10 amino acids. In some embodiments, P 1a comprises a peptide sequence having a length of at least 10 to 20 amino acids. In some embodiments, P 1a comprises a peptide sequence having a length of at least 16 amino acids. In some embodiments, P 1a comprises a peptide sequence having a length of 40 or fewer amino acids. In some embodiments, P 1a contains at least two cysteine ​​amino acid residues. In some embodiments, P 1a comprises a cyclic peptide or a linear peptide. In some embodiments, P 1aincludes a cyclic peptide. In some embodiments, P 1a includes a linear peptide. In some embodiments, P 1a comprises an amino acid sequence according to any one of SEQ ID NOs 33 to 106 or 732 to 813. In some embodiments, P 1a includes the amino acid sequence of SEQ ID NO. 73. In some embodiments, P 1a includes the amino acid sequence of SEQ ID NO. 784. In some embodiments, P 1a 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 compared to the amino acid sequence of SEQ ID NO. 73. In some embodiments, P 1a includes the amino acid sequence of SEQ ID NO. 73. In some embodiments, P 1a 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 compared to the amino acid sequence of SEQ ID NO. 103. In some embodiments, P 1a It includes the amino acid sequence of sequence number 103.

[0370] In some embodiments, P 1a is X1-X2-X3-C-X4-X5-X6-X7-X8-X9-X 10 -CX 11 -X 12It includes an amino acid sequence according to, wherein 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 is selected from E, V, L, D, Y, R, Q, H, F, K, A, M, and N; X 11 is selected from F, Y, L, W, and V; X 12 is selected from N, A, F, S, Y, H, D, T, and L. In some embodiments, X1 is selected from M, I, and L; X2 is selected from D, H, N, and A; X3 is W; X4 is P; X5 is selected from R, T, I, M, S, and K; X6 is selected from E, D, Y, H, S, and F; X7 is selected from L, M, and R; X8 is W; X9 is selected from H, N, D, A, S, and V; and X 10 is selected from E, V, L, D, and H; X 11 is selected from F, Y, and L; X 12 is selected from N, A, F, S, and Y. In some embodiments, X1 is M; X2 is selected from D and H; X3 is W; X4 is P; X5 is selected from R, T, and I; X6 is selected from E, D, and Y; X7 is selected from L, M, and R; X8 is W; X9 is selected from H, N, D, and V; and X 10 is selected from E, V, L, D, and H; X11 is F and; X 12 is selected from N, A, and F. In some embodiments, P 1a comprises an 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 compared to SEQ ID NO. 41. In some embodiments, P 1a includes the amino acid sequence according to SEQ ID NO. 41. In some embodiments, P 1a It includes an amino acid sequence selected from any one of SEQ ID NOs 41, 73, 103 and 179 to 603.

[0371] In some embodiments, P 1a is J1-J2-WC-J3-J4-J5-J6-J7-J8-J9-CJ 10 -J 11 It includes an amino acid sequence according to, wherein 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; J4 is selected from R, L, and I; J5 is selected from D, H, Y, 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 is selected from F, Y, and L; J 11is 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; J3 is P; J4 is R; J5 is D; J6 is L; J7 is W; J8 is V; J9 is H; and J 10 is F and; J 11 is A. In some embodiments, 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; and J 10 is F and; J 11 is A. In some embodiments, 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 is F or Y; J 11 is N or H. In some embodiments, 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 is F or Y; J 11 is N.

[0372] In some embodiments, P 1a is Z1-C-Z2-Z3-Z4-Z5-Z6-Z7-Z8-Z9-Z 10 -Z 11 -CZ 12It includes an amino acid sequence according to, wherein 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 is selected from A, Q, S, W, E, L, G, D, T, M, N, V, H, R, Y, K, P, F and I; Z 11 is selected from K, Q, N, H, E, P and A; Z 12is 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 is selected from A, Q, S, W, E, L, G, D, T, M, N, V, H, R, Y, K, P, F and I; Z 11 is selected from K, Q, N, H, E, and P; Z 12is 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 is selected from A, Q, S, W, E, L, G, D, T, M, N, V, H, R, Y, K, P, F and I; Z 11 is selected from K, Q, N, H, E, and P; Z 12 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; and Z 10 is selected from A, Q, S, W, E, L, G, D, T, M, and N; Z 11 is K and; Z 12is 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; 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 is selected from A, Q, S, W, E, L, G, and D; Z 11 is K and; Z 12 is L. In some embodiments, 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; and Z 10 is selected from A, Q, S, W, E, L, G, and D; Z 11 is K and; Z 12 is L.

[0373] In some embodiments, P 1a includes the amino acid sequence of SEQ ID NO. 784. In some embodiments, P 1a consists of the amino acid sequence of SEQ ID NO. 784. In some embodiments, P 1a includes the amino acid sequence of SEQ ID NO. 744. In some embodiments, P 1a consists of the amino acid sequence of SEQ ID NO. 744. In some embodiments, P 1a comprises an amino acid sequence according to any one of SEQ ID NOs 774 to 793. In some embodiments, P 1a It includes an amino acid sequence according to any one of sequence numbers 764 to 773.

[0374] In some embodiments, P 1aU1-C-U2-U3-U4-U5-U6-U7-U8-U9-U 10 -U 11 -CU 12 It includes an amino acid sequence according to, wherein 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 is selected from P, S, R, L, E and A; U 11 is selected from L, A, T, M, V, W, Y, E, Q, I, F, S, P, K, R, D, H, N and G; U 12is 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 is selected from P, S, R, L and E; U 11 is selected from L, A, T, M, V, W, Y, E, Q, I, F, S, P, K, R, D, H, N and G; U 12 is 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 is selected from P and S; U 11 is selected from L, A, T, M, V, W, Y, E, Q, I, F and S; U 12is 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; and U 10 is P and; U 11 is selected from L, A, T, M, V, W, and Y; U 12 is selected from L and F.

[0375] In some embodiments, P 1a includes the amino acid sequence of SEQ ID NO. 749. In some embodiments, P 1a includes the amino acid sequence of SEQ ID NO. 749. In some embodiments, P 1a comprises an amino acid sequence according to any one of SEQ ID NOs 756 to 763. In some embodiments, P 1a comprises an amino acid sequence according to any one of SEQ ID NOs 794 to 813. In some embodiments, P 1a It includes an amino acid sequence according to any one of sequence numbers 732 to 2002.

[0376] modified amino acids

[0377] In some embodiments, the isolated polypeptide or polypeptide complex comprises a modified amino acid, a non-natural amino acid, a modified non-natural amino acid, or a combination thereof. In some embodiments, the non-natural amino acid comprises a D-amino acid. In some embodiments, the modified amino acid or the modified non-natural amino acid comprises a post-translational modification.

[0378] Pharmaceutical composition

[0379] In some embodiments, the present invention discloses a pharmaceutical composition comprising an isolated polypeptide or polypeptide complex of any one of the embodiments disclosed herein.

[0380] In some embodiments, the present invention discloses a pharmaceutical composition comprising an isolated polypeptide or polypeptide complex comprising a first antigen-recognizing molecule that binds to CD28 and a second antigen-recognizing molecule that binds to a prostate-specific membrane antigen. In some embodiments, the first antigen-recognizing 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 a complementary determining region (CDR): HC-CDR1, HC-CDR2, and HC-CDR3, wherein HC-CDR1, HC-CDR2, and HC-CDR3 comprise the amino acid sequences of 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 a complementary determining region (CDR): LC-CDR1, LC-CDR2, and LC-CDR3, wherein LC-CDR1, LC-CDR2, and LC-CDR3 comprise the amino acid sequences of 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 antibody comprises a single chain variable fragment (scFv), a single-domain antibody, Fab, Fab', or CrossFab. In some embodiments, the anti-CD28 antibody comprises a scFv. In some embodiments, the anti-CD28 antibody comprises a single chain variable fragment (scFv) having a engineered disulfide.In some embodiments, the anti-CD28 antibody comprises a scFv, said scFv comprising 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 a scFv, said scFv comprising 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 a scFv, said scFv comprising 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, said scFv comprising 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.

[0381] In some embodiments, a second antigen recognition molecule that binds to PSMA includes an anti-PSMA antibody. In some embodiments, the anti-PSMA antibody comprises an anti-PSMA heavy chain variable domain comprising a complementarity determining region (CDR): HC-CDR1, HC-CDR2, and HC-CDR3, wherein HC-CDR1, HC-CDR2, and HC-CDR3 comprise the amino acid sequences of 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 a complementarity determining region (CDR): LC-CDR1, LC-CDR2, and LC-CDR3, wherein LC-CDR1, LC-CDR2, and LC-CDR3 of the light chain variable domain comprise the amino acid sequences of 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', said 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', said 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', said 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.

[0382] In some embodiments, the polypeptide or polypeptide complex further comprises a detectable label, a therapeutic agent, or a pharmacokinetic modification moiety. In some embodiments, the detectable label comprises a fluorescent label, a radiolabel, an enzyme, a nucleic acid probe, or a contrast agent.

[0383] For administration to a subject, the polypeptide or polypeptide complex 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 is non-toxic to the patient receiving the formulation without interfering with the efficacy of the biological activity of the component. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate-buffered saline solutions, water, emulsions such as oil / water emulsions, various types of wetting agents, sterile solutions, etc. Such carriers may be formulated by conventional methods and administered to a subject in a suitable dose. Preferably, the composition is sterile. Such compositions may also contain adjuvants such as preservatives, emulsifiers, and dispersants. Prevention of microbial action may be ensured by the inclusion of various antimicrobial and antifungal agents.

[0384] The pharmaceutical composition may exist in any suitable form depending on the desired method of administration. It may be provided as a unit formulation, provided within a sealed container, or provided as part of a kit. Such a kit may include instructions for use. It may include a plurality of the above-mentioned unit formulations.

[0385] Pharmaceutical compositions may be adapted for administration by 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, for example, by mixing an active ingredient with a carrier(s) or excipient(s) under sterile conditions.

[0386] The dosage of the substance of the present disclosure may vary within a wide range depending on the disease or disorder being treated, the age and condition of the individual being treated, etc., and the physician will ultimately determine the appropriate dosage to be used.

[0387] Isolated recombinant nucleic acid molecules

[0388] The present invention discloses an isolated recombinant nucleic acid molecule encoding an isolated polypeptide or a polypeptide complex of any one of the embodiments disclosed herein.

[0389] In some embodiments, the present invention discloses an isolated recombinant nucleic acid molecule encoding a polypeptide or a 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 a complementary determining region (CDR): HC-CDR1, HC-CDR2, and HC-CDR3, wherein HC-CDR1, HC-CDR2, and HC-CDR3 comprise the amino acid sequences of 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 a complementary determining region (CDR): LC-CDR1, LC-CDR2, and LC-CDR3, wherein LC-CDR1, LC-CDR2, and LC-CDR3 comprise the amino acid sequences of 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 antibody comprises a single chain variable fragment (scFv), a single-domain antibody, Fab, or Fab'. In some embodiments, the anti-CD28 antibody comprises a scFv. In some embodiments, the anti-CD28 antibody comprises a single chain variable fragment (scFv) having a engineered disulfide.In some embodiments, the anti-CD28 antibody comprises a scFv, said scFv comprising 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 a scFv, said scFv comprising 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 a scFv, said scFv comprising 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, said scFv comprising 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.

[0390] In some embodiments, a second antigen recognition molecule that binds to PSMA includes an anti-PSMA antibody. In some embodiments, the anti-PSMA antibody comprises an anti-PSMA heavy chain variable domain comprising a complementarity determining region (CDR): HC-CDR1, HC-CDR2, and HC-CDR3, wherein HC-CDR1, HC-CDR2, and HC-CDR3 comprise the amino acid sequences of 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 a complementarity determining region (CDR): LC-CDR1, LC-CDR2, and LC-CDR3, wherein LC-CDR1, LC-CDR2, and LC-CDR3 of the light chain variable domain comprise the amino acid sequences of 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', said 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', said 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', said 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.

[0391] Treatment methods

[0392] In some embodiments, the present invention discloses a method for treating cancer in a subject requiring cancer treatment, comprising the step of administering to the subject an isolated polypeptide or polypeptide complex of any one of the embodiments disclosed herein. In some embodiments, the cancer has cells expressing PSMA. In some cases, the cancer is a solid tumor cancer. In some embodiments, the cancer is lung cancer, breast cancer (e.g., HER2+; ER / PR+; TNBC), cervical cancer, ovarian cancer, colorectal cancer, pancreatic cancer, or gastric cancer.

[0393] In some embodiments, the present invention discloses a method for treating prostate cancer in a subject requiring treatment for prostate cancer, comprising the step of administering an isolated polypeptide or polypeptide complex as described herein to said subject. In some embodiments, the present invention discloses a method for treating mCRPC in a subject requiring treatment for metastatic castration-resistant prostate cancer (mCRPC), comprising the step of administering an isolated polypeptide or polypeptide complex as described herein to said subject.

[0394] In some embodiments, the present invention discloses a method for treating cancer in a subject requiring cancer treatment, comprising the step of administering to the subject a polypeptide or polypeptide complex comprising a first antigen-recognizing molecule that binds to CD28 and a second antigen-recognizing molecule that binds to a prostate-specific membrane antigen. In some embodiments, the first antigen-recognizing 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 a complementary determining region (CDR): HC-CDR1, HC-CDR2, and HC-CDR3, wherein HC-CDR1, HC-CDR2, and HC-CDR3 comprise the amino acid sequences of 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 a complementary determining region (CDR): LC-CDR1, LC-CDR2, and LC-CDR3, wherein LC-CDR1, LC-CDR2, and LC-CDR3 comprise the amino acid sequences of 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 antibody comprises a single chain variable fragment (scFv), a single-domain antibody, Fab, Fab', or CrossFab. In some embodiments, the anti-CD28 antibody comprises a scFv. In some embodiments, the anti-CD28 antibody comprises a single chain variable fragment (scFv) having a engineered disulfide.In some embodiments, the anti-CD28 antibody comprises a scFv, said scFv comprising 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 a scFv, said scFv comprising 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 a scFv, said scFv comprising 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, said scFv comprising 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.

[0395] In some embodiments, a second antigen recognition molecule that binds to PSMA includes an anti-PSMA antibody. In some embodiments, the anti-PSMA antibody comprises an anti-PSMA heavy chain variable domain comprising a complementarity determining region (CDR): HC-CDR1, HC-CDR2, and HC-CDR3, wherein HC-CDR1, HC-CDR2, and HC-CDR3 comprise the amino acid sequences of 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 a complementarity determining region (CDR): LC-CDR1, LC-CDR2, and LC-CDR3, wherein LC-CDR1, LC-CDR2, and LC-CDR3 of the light chain variable domain comprise the amino acid sequences of 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', said 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', said 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', said 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.

[0396] Combination therapy

[0397] In some embodiments, the polypeptide or polypeptide 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 cancer. In some embodiments, the cancer is lung cancer, breast cancer, cervical cancer, ovarian cancer, colorectal cancer, pancreatic cancer, or gastric cancer.

[0398] In some embodiments, the polypeptide or polypeptide complex is administered in combination with anticancer therapy. In some embodiments, the polypeptide or polypeptide complex and the anticancer therapy are administered as the same pharmaceutical composition. In some embodiments, the polypeptide or polypeptide complex and the anticancer therapy are administered as separate pharmaceutical compositions. In some embodiments, the anticancer therapy includes small molecule, cell-based therapy, or antibody-based therapy.

[0399] In some embodiments, the antibody-based therapy is a T cell engager. In some embodiments, the T cell engager comprises a formula according to 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 linker connecting 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.

[0400] In some embodiments, the effector cell binding domain is Muromonab-CD3 (OKT3), Otelixizumab (TRX4), Teflizumab (MGA031), Bicilizumab (Nuvion), SP34, X35, VIT3, 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, It includes a complementarity determining region (CDR) selected from the group consisting of 15865v16 and 15865v19. In some embodiments, the effector cell binding domain includes amino acid sequences according to SEQ ID NOs 611 to 624.

[0401]

[0402] 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 herein as TROP2). In some embodiments, the tumor antigen comprises EGFR. In some embodiments, the tumor antigen binding domain comprises amino acid sequences according to SEQ ID NOs 629 to 638. In some embodiments, the tumor antigen comprises EGFR, and the tumor binding domain comprises complementarity determining region (CDR): 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; ​​and HC-CDR3: SEQ ID NO. 634; 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 engager comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NOs. 639 and 640. In some embodiments, the tumor antigen comprises EGFR, and the T cell engager comprises an amino acid sequence according to SEQ ID NOs. 641 and 642.

[0403] In some embodiments, the tumor antigen comprises TROP2. In some embodiments, the tumor antigen comprises TROP2, and the tumor binding domain comprises a complementarity determining region (CDR): HC-CDR1, HC-CDR2 and HC-CDR3, and LC-CDR1, LC-CDR2 and LC-CDR3, wherein HC-CDR1, HC-CDR2 and HC-CDR3 comprises HC-CDR1: SEQ ID NO. 659; HC-CDR2: SEQ ID NO. 660; and HC-CDR3: SEQ ID NO. 661, 662 or 633; ​​and LC-CDR1, LC-CDR2 and LC-CDR3 comprises 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 engager comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NOs 673 and 674. In some embodiments, the tumor antigen comprises TROP2, and the T cell engager comprises an amino acid sequence according to SEQ ID NOs 675 and 676, or SEQ ID NOs 677 and 678. In some embodiments, the tumor antigen binding domain comprises an amino acid sequence according to SEQ ID NOs 659 to 668.

[0404] In some embodiments, the tumor antigen comprises PSMA. In some embodiments, the tumor antigen binding domain comprises amino acid sequences according to SEQ ID NOs 647 to 654. In some embodiments, the tumor antigen comprises PSMA, and the tumor binding domain comprises complementarity determining regions (CDR): 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; and HC-CDR3: SEQ ID NO. 649; and 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 engager comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NOs 655 and 656.

[0405]

[0406]

[0407]

[0408]

[0409]

[0410]

[0411]

[0412]

[0413]

[0414]

[0415]

[0416]

[0417]

[0418]

[0419]

[0420]

[0421]

[0422]

[0423]

[0424]

[0425]

[0426]

[0427]

[0428] In some embodiments, the T cell engager molecule is selectively activated in the tumor microenvironment. In some embodiments, the T cell engager follows the following subformation: P3-L3-D-L0-E (Formula IIa), wherein D comprises a CD3 binding domain; E comprises a tumor antigen binding domain; L0 comprises a linker connecting D to E; P3 comprises a peptide that binds to D; and L3 comprises a linking moiety that connects D to P3 and is a substrate for a tumor-specific protease. In some embodiments, the T cell engager follows the following subformation: D-L0-E-L4-P4 (Formula IIb), wherein D comprises a CD3 binding domain; E comprises a tumor antigen binding domain; L0 comprises a linker connecting D to E; P4 comprises a peptide that binds to E; and L4 comprises a linking moiety that connects E to P4 and is a substrate for a tumor-specific protease. In some embodiments, the T cell engager follows the following sub-formula: P3-L3-D-L0-E-L4-P4 (formula IIc), wherein D comprises a CD3 binding domain; E comprises a tumor antigen binding domain; L0 comprises a linker connecting D to E; P3 comprises a peptide that binds to D, L3 comprises a linking moiety that connects D to P3 and is a substrate for a tumor-specific protease; P4 comprises a peptide that binds to E, L4 comprises a linking moiety that connects E to P4 and is a substrate for a tumor-specific protease.

[0429] In some embodiments, the T cell engager 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 a complementarity determining region (CDR): 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.

[0430] In some embodiments, L3 or L4 is a peptide sequence having at least 5 to 50 amino acids. In some embodiments, L3 or L4 is a peptide sequence having at least 10 to 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 is (G2S) n It has a chemical formula including, where n is an integer from 1 to 3 (SEQ ID NO. 605). In some embodiments, L3 or L4 is (G2S) n It has a chemical formula including, where n is an integer of at least 1. In some embodiments, L3 or L4 is (G2S) n , (GS) n , (GSGGS) n (Sequence No. 606), (GGGS) n (Sequence No. 607), (GGGGGS) n (Sequence No. 608) and (GSSGGS) n It has a chemical formula selected from the group consisting of (sequence number 609), where n is an integer of at least 1.

[0431] In some embodiments, the tumor-specific protease is selected from the group consisting of metalloprotease, serine protease, cysteine ​​protease, threonine protease, and aspartate protease. In some embodiments, L3 or L4 comprises a urokinase-cleavable amino acid sequence, a matrixtase-cleavable amino acid sequence, or a matrix metalloprotease-cleavable amino acid sequence. In some embodiments, L3 or L4 comprises a sequence according to any one of SEQ ID NOs 21, 22, and 107 to 136. In some embodiments, L3 is bound to the N-terminus of D. In some embodiments, L3 is bound to the C-terminus of D. In some embodiments, L4 is bound to the N-terminus of E. In some embodiments, L4 is bound to the C-terminus of E.

[0432] In some embodiments, when L3 is cleaved by a tumor-specific protease, P3 becomes unbound from D, thereby exposing D to CD3. In some embodiments, when L4 is cleaved by a tumor-specific protease, P4 becomes unbound from E, thereby exposing E to the tumor antigen. In some embodiments, P3 impairs the binding between D and CD3. In some embodiments, P3 binds to D through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-binding interactions, or a combination 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 becomes unbound from D, thereby exposing D to CD3.

[0433] In some embodiments, P3 has less than 70% sequence identity with CD3. In some embodiments, P3 has less than 70% sequence identity with CD3. In some embodiments, P3 has less than 85% sequence identity with CD3. In some embodiments, P3 has less than 90% sequence identity with CD3. In some embodiments, P3 has less than 95% sequence identity with CD3. In some embodiments, P3 has less than 98% sequence identity with CD3. In some embodiments, P3 has less than 99% sequence identity with CD3. In some embodiments, P3 comprises an amino acid sequence according to SEQ ID NO. 625 or SEQ ID NO. 626. In some embodiments, P3 comprises a de novo amino acid sequence that shares less than 10% sequence identity with CD3.

[0434] In some embodiments, P4 impairs the binding of E to the tumor antigen. In some embodiments, P4 binds to E through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-binding interactions, or a combination 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 becomes unbound from 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 that shares less than 10% sequence identity with the tumor antigen.

[0435] In some embodiments, P3 or P4 comprises a peptide sequence having a length of at least 5 amino acids. In some embodiments, P3 or P4 comprises a peptide sequence having a length of at least 6 amino acids. In some embodiments, P3 or P4 comprises a peptide sequence having a length of at least 10 amino acids. In some embodiments, P3 or P4 comprises a peptide sequence having a length of at least 10 to 20 amino acids. In some embodiments, P3 or P4 comprises a peptide sequence having a length of at least 16 amino acids. In some embodiments, P3 or P4 comprises a peptide sequence having a length of 40 amino acids or less. In some embodiments, P3 or P4 comprises at least 2 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.

[0436] 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 engager comprises the amino acid sequences of SEQ ID NOs. 643 and 644.

[0437] In some embodiments, P4 comprises an amino acid sequence according to any one of SEQ ID NOs 693 to 695. In some embodiments, the tumor antigen comprises TROP2, and the T cell engager comprises the amino acid sequences of SEQ ID NOs 679 and 680, SEQ ID NOs 681 and 682, SEQ ID NOs 683 and 684, SEQ ID NOs 685 and 686, SEQ ID NOs 687 and 688, SEQ ID NOs 689 and 690, and SEQ ID NOs 691 and 692. In some embodiments, the tumor antigen comprises TROP2, and the T cell engager comprises the amino acid sequences of SEQ ID NOs 4452 and 4453, SEQ ID NOs 4454 and 4455, SEQ ID NOs 4456 and 4457, SEQ ID NOs 4458 and 4459, SEQ ID NOs 4460 and 4461, SEQ ID NOs 4462 and 4463, or SEQ ID NOs 4464 and 4465.

[0438] In some embodiments, the tumor antigen comprises PSMA, and the T cell engager comprises the amino acid sequences of SEQ ID NOs 657 and 658.

[0439] production of antibodies

[0440] In some embodiments, the antibody or its antigen-binding fragment described herein is produced by any method known in the art to be useful for the synthesis of polypeptides (e.g., antibodies), particularly by chemical synthesis or recombinant expression, and preferably by recombinant expression techniques.

[0441] In some cases, the antibody or its binding fragment is recombinantly expressed, and the nucleic acid encoding the antibody or its binding fragment is assembled from chemically synthesized oligonucleotides (e.g., as described in the literature [Kutmeier et al., 1994, BioTechniques 17:242]), which involves the synthesis of a nested oligonucleotide containing part of the sequence encoding the antibody, the annealing and ligation of this oligonucleotide, and the subsequent amplification of the ligated oligonucleotide by PCR.

[0442] Alternatively, nucleic acid molecules encoding antibodies are generated randomly from suitable sources (e.g., antibody cDNA libraries, or cDNA libraries generated from any tissue or cell expressing 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 specific to a specific gene sequence.

[0443] In some cases, the antibody or its binding fragment is arbitrarily generated by immunizing an animal such as a mouse to produce a polyclonal antibody, or more preferably a monoclonal antibody, as described in the literature [Kohler and Milstein (1975, Nature 256:495-497)], or in the literature [Kozbor et al. (1983, Immunology Today 4:72)] or in the literature [Cole et al. (1985 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96)]. Alternatively, clones encoding at least the Fab portion of an antibody are obtained at will by screening Fab expression libraries for clones of the Fab fragment binding to a specific antigen (e.g., as described in the literature [Huse et al., 1989, Science 246:1275-1281]) or by screening antibody libraries (e.g., see Clackson et al., 1991, Nature 352:624; Hane et al., 1997 Proc. Natl. Acad. Sci. USA 94:4937]).

[0444] In some embodiments, a technique developed to generate "chimeric antibodies" by splicing a gene from a mouse antibody molecule with appropriate antigen specificity together with a gene from a human antibody molecule 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) is used. A chimeric antibody is a molecule having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region, where different parts are derived from different animal species.

[0445] In some embodiments, the technique described for the generation of single-strand antibodies (U.S. Patent No. 4,694,778; Bird, 1988, Science 242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989, Nature 334:544-54) is applied to generate single-strand antibodies. Single-strand antibodies are formed by generating a single-strand polypeptide by linking the heavy chain fragment and the light chain fragment of the Fv region through amino acid crosslinking. A technique for assembling the functional Fv fragment in E. coli is also optionally used (Skerra et al., 1988, Science 242:1038-1041).

[0446] In some embodiments, an expression vector containing the nucleotide sequence of an antibody or the nucleotide sequence of an antibody is delivered to a host cell by conventional techniques (e.g., electroporation, liposome transfection, and calcium phosphate precipitation), and the transfected cell is then cultured by conventional techniques to produce antibodies. In certain embodiments, the expression of the antibody is regulated by a congenital, inducible, or tissue-specific promoter.

[0447] In some embodiments, various host-expression vector systems are utilized to express the antibody or its binding fragment described herein. These host-expression systems represent a vehicle that is purified after the antibody coding sequence is generated, but may also represent a cell that expresses the antibody or its binding fragment in situ when transformed or transfected with an appropriate nucleotide coding sequence. These include microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with a recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vector containing the antibody or its binding fragment coding sequence; yeast (e.g., Saccharomyces pichia) transformed with a recombinant yeast expression vector containing the antibody or its binding fragment coding sequence; and insect cell systems infected with a recombinant virus expression vector (e.g., baculovirus) containing the antibody or its binding fragment coding sequence. Plant cell systems infected with a recombinant viral expression vector (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV)) or transformed with a recombinant plasmid expression vector (e.g., Ti plasmid) containing a coding sequence for an antibody or its binding fragment; or mammalian cell systems (e.g., COS, CHO, BH, 293, 293T, 3T3 cells) having a recombinant expression construct comprising a promoter derived from the genome of a mammalian cell (e.g., metallothionein promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter).

[0448] Stable expression is desirable for the long-term production of high yields of recombinant proteins. In some cases, cell lines that stably express antibodies are engineered. Rather than using an expression vector containing a viral replication origin, host cells are transformed with DNA regulated by appropriate expression regulatory elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and selectable markers. Then, after the introduction of foreign DNA, the engineered cells are grown in enrichment medium for 1 to 2 days and then switched to selection medium. Selectable markers within the recombinant plasmid confer resistance to selection, allowing the cells to stably integrate the plasmid into their chromosomes and grow, consequently forming a focus that is cloned and expanded into a cell line. This method can be advantageously used to engineer cell lines expressing antibodies or their binding fragments.

[0449] In some cases, a number of selection systems are used, including but not limited to using the genes 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) in tk-, hgprt-, or aprt- cells, respectively. In addition, resistance to anmetatars is used as a selection criterion for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Proc. Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); Neo conferring 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 conferring resistance to hygromycin (Santerre et al., 1984, Gene 30:147). Methods generally known in the field of recombinant DNA technology that can be used are described in the literature [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 in Chapters 12 and 13, Dracopoli et al. (eds), 1994, Current Protocols in Human Genetics, John Wiley & Sons, NY.; Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1)].

[0450] In some cases, the expression level of antibodies is increased by vector amplification (for review, see Bebbington and Hentschel, the use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York, 1987)). When a marker within an antibody-expressing vector system can be amplified, an increase in the level of inhibitor present in the host cell culture will increase the copy number of the marker gene. Since the amplified region is associated with the nucleotide sequence of the antibody, antibody production will also increase (Crouse et al., 1983, Mol. Cell Biol. 3:257).

[0451] In some cases, any antibody purification method known in the art, for example, chromatography (e.g., ion exchange, affinity, particularly affinity for specific antigens after protein A, and sizing column chromatography), centrifugation, differential solubility, or any other standard protein purification technique is used.

[0452] Expression vector

[0453] In some embodiments, the present invention discloses a vector comprising an isolated recombinant nucleic acid molecule encoding an isolated polypeptide or polypeptide complex of any one of the embodiments disclosed herein.

[0454] In some embodiments, the present invention discloses a vector comprising an isolated recombinant nucleic acid molecule encoding a polypeptide or a 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 a complementary determining region (CDR): HC-CDR1, HC-CDR2, and HC-CDR3, wherein HC-CDR1, HC-CDR2, and HC-CDR3 comprise the amino acid sequences of 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 a complementary determining region (CDR): LC-CDR1, LC-CDR2, and LC-CDR3, wherein LC-CDR1, LC-CDR2, and LC-CDR3 comprise the amino acid sequences of 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 antibody comprises a single chain variable fragment (scFv), a single-domain antibody, Fab, or Fab'. In some embodiments, the anti-CD28 antibody comprises a scFv. In some embodiments, the anti-CD28 antibody comprises a scFv, said scFv comprising 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 a scFv, said scFv comprising 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 a scFv, said scFv comprising 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 a scFv, said scFv comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO. 12.

[0455] In some embodiments, a second antigen recognition molecule that binds to PSMA includes an anti-PSMA antibody. In some embodiments, the anti-PSMA antibody comprises an anti-PSMA heavy chain variable domain comprising a complementarity determining region (CDR): HC-CDR1, HC-CDR2, and HC-CDR3, wherein HC-CDR1, HC-CDR2, and HC-CDR3 comprise the amino acid sequences of 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 a complementarity determining region (CDR): LC-CDR1, LC-CDR2, and LC-CDR3, wherein LC-CDR1, LC-CDR2, and LC-CDR3 of the light chain variable domain comprise the amino acid sequences of 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', said 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.

[0456] In some embodiments, the vector comprises any suitable vector derived from a eukaryotic or prokaryotic source. In some cases, the vector is obtained from a bacterium (e.g., E. coli), insect, yeast (e.g., Pichia pastoris), algae, or mammalian source. Exemplary bacterial vectors include pACYC177, pASK75, pBAD vector series, pBADM vector series, pET vector series, pETM vector series, pGEX vector series, pHAT, pHAT2, pMal-c2, pMal-p2, pQE vector series, pRSET A, pRSET B, pRSET C, pTrcHis2 series, pZA31-Luc, pZE21-MCS-1, pFLAG ATS, pFLAG CTS, pFLAG MAC, pFLAG Shift-12c, pTAC-MAT-1, pFLAG CTC, or pTAC-MAT-2.

[0457] Exemplary insect vectors include FLAG vectors such as pFastBac1, pFastBac DUAL, pFastBac ET, pFastBac HTa, pFastBac HTb, pFastBac HTc, pFastBac M30a, pFastBac M30b, pFastBac, M30c, pVL1392, pVL1393, pVL1393 M10, pVL1393 M11, pVL1393 M12, pPolh-FLAG1 or pPolh-MAT2, or MAT vectors such as pPolh-MAT1 or pPolh-MAT2.

[0458] In some cases, yeast vectors include Gateway® pDEST™ 14 vector, Gateway® pDEST™ 15 vector, Gateway® pDEST™ 17 vector, Gateway® pDEST™ 24 vector, Gateway® pYES-DEST52 vector, pBAD-DEST49 Gateway® destination vector, pAO815 Pichia vector, pFLD1 Pichia pastoris vector, pGAPZA, B & C Pichia pastoris vector, pPIC3.5K Pichia vector, pPIC6 A, B & C Pichia vector, pPIC9K Pichia vector, pTEF1 / Zeo, pYES2 yeast vector, pYES2 / CT yeast vector, pYES2 / NT A, B & C yeast vector, or pYES3 / CT yeast vector.

[0459] Exemplary algae vectors include the pChlamy-4 vector or the MCS vector.

[0460] Examples of mammalian vectors include transient expression vectors or stable expression vectors. Mammalian transient expression vectors may include pRK5, p3xFLAG-CMV 8, pFLAG-Myc-CMV 19, pFLAG-Myc-CMV 23, pFLAG-CMV 2, pFLAG-CMV 6a,b,c, pFLAG-CMV 5.1, pFLAG-CMV 5a,b,c, p3xFLAG-CMV 7.1, pFLAG-CMV 20, p3xFLAG-Myc-CMV 24, pCMV-FLAG-MAT1, pCMV-FLAG-MAT2, pBICEP-CMV 3, or pBICEP-CMV 4. The mammalian stable expression vector 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.

[0461] In some cases, cell-free systems are mixtures of cytoplasmic and / or nuclear components from cells and are used for nucleic acid synthesis in vitro. In some cases, cell-free systems utilize prokaryotic or eukaryotic cell components. Occasionally, nucleic acid synthesis is obtained in cell-free systems based, for example, Drosophila cells, Xenopus algae, 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®.

[0462] host cell

[0463] In some embodiments, the present invention discloses a host cell comprising an isolated polypeptide or polypeptide complex of any one of the embodiments disclosed herein.

[0464] In some embodiments, the present invention discloses a host cell comprising 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 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 a complementary determining region (CDR): HC-CDR1, HC-CDR2, and HC-CDR3, wherein HC-CDR1, HC-CDR2, and HC-CDR3 comprise the amino acid sequences of 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 a complementary determining region (CDR): LC-CDR1, LC-CDR2, and LC-CDR3, wherein LC-CDR1, LC-CDR2, and LC-CDR3 comprise the amino acid sequences of 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 antibody comprises a single chain variable fragment (scFv), a single-domain antibody, Fab, or Fab'. In some embodiments, the anti-CD28 antibody comprises a scFv. In some embodiments, the anti-CD28 antibody comprises a scFv, said scFv comprising 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 a scFv, said scFv comprising 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 a scFv, said scFv comprising 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 a scFv, said scFv comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO. 12.

[0465] In some embodiments, a second antigen recognition molecule that binds to PSMA includes an anti-PSMA antibody. In some embodiments, the anti-PSMA antibody comprises an anti-PSMA heavy chain variable domain comprising a complementarity determining region (CDR): HC-CDR1, HC-CDR2, and HC-CDR3, wherein HC-CDR1, HC-CDR2, and HC-CDR3 comprise the amino acid sequences of 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 a complementarity determining region (CDR): LC-CDR1, LC-CDR2, and LC-CDR3, wherein LC-CDR1, LC-CDR2, and LC-CDR3 of the light chain variable domain comprise the amino acid sequences of 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', said 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.

[0466] In some embodiments, the host cell comprises any suitable cell, such as a naturally occurring cell or a genetically modified cell. 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, the eukaryotic cell includes fungi (e.g., yeast cells), animal cells, or plant cells. In some cases, the prokaryotic cell is a bacterial cell. Examples of bacterial cells include Gram-positive bacteria or Gram-negative bacteria. Sometimes Gram-negative bacteria are anaerobic, rod-shaped, or both.

[0467] In some cases, Gram-positive bacteria include Actinobacteria, Firmicutes, or Tenericutes. In some 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 may be Acidobacteria, Chloroflexi, Chrysiogenetes, Cyanobacteria, Deferribacteres, Dictyoglomi, Thermodesulfobacteria, or Thermotogae. Bacterial cells may be Escherichia coli, Clostridium botulinum, or Colibacilli.

[0468] Exemplary prokaryotic host cells include, but are not limited to, BL21, Mach1™, DH10B™, TOP10, DH5α, DH10Bac™, OmniMax™, MegaX™, DH12S™, INV110, TOP10F', INVαF, TOP10 / P3, ccdB Survival, PIR1, PIR2, Stbl2™, Stbl3™, or Stbl4™.

[0469] In some cases, animal cells include cells derived from vertebrates or invertebrates. In some cases, animal cells include cells derived from marine invertebrates, fish, insects, amphibians, reptiles, or mammals. In some cases, fungal cells include yeast cells such as brewer's yeast, baker's yeast, or wine yeast.

[0470] Fungi include ascomycetes, such as yeast, mold, filamentous fungi, basidiomycetes, or zygomycetes. In some cases, yeast includes ascomycota or basidiomycota. In some cases, ascomycota includes saccharomycotina (true yeast, e.g., Saccharomyces cerevisiae (baking yeast)) or taprinomycotina (e.g., Schizosaccharomycetes (fission yeast)). In some cases, basidiomycota include agaricomycotina (e.g., Tremellomycetes) or pucciniomycotina (e.g., Microbotryomycetes).

[0471] 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, and Rhodotorula Rhodotorula mucilaginosa, Pichia metanolica, Pichia angusta, Pichia pastoris, Pichia anomala, Hansenula polymorpha, Kluyveromyces lactis,Zygosaccharomyces rouxii, Yarrowia lipolytica, Trichosporon pullulans, Rhodosporidium toru-Aspergillus niger, Aspergillus nidulans, Aspergillus awamori, Aspergillus oryzae, Trichoderma reesei, Yarrowia lipolytica, Brettanomyces bruxellensis, Candida stellata, Schizosaccharomyces pombe, Torulaspora Torulaspora delbrueckii, Zygosaccharomyces bailii, Cryptococcus neoformans, Cryptococcus gattii, or Saccharomyces boulardii.,

[0472] Exemplary yeast host cells include, but are not limited to, yeast strains of *Pichia pastoris* such as GS115, KM71H, SMD1168, SMD1168H, and X-33; and yeast strains of *Saccharomyces cerevisiae* such as INVSc1.

[0473] In some cases, additional animal cells include cells obtained from mollusks, arthropods, annelids, or sponges. In some cases, additional animal cells are mammalian cells derived from, for example, primates, apes, horses, bovines, pigs, canids, felines, or rodents. In some cases, rodents include mice, rats, hamsters, gerbils, chinchillas, fancy rats, or guinea pigs.

[0474] Exemplary mammalian host cells include the 293A cell line, 293FT cell line, 293F cell, 293H cell, CHO DG44 cell, CHO-S cell, CHO-K1 cell, FUT8 KO CHOK1, Expi293F™ cell, Flp-In™ T-REx™ 293 cell line, Flp-In™-293 cell line, Flp-In™-3T3 cell line, Flp-In™-BHK cell line, Flp-In™-CHO cell line, Flp-In™-CV-1 cell line, Flp-In™-Jurkat cell line, FreeStyle™ 293-F cell, FreeStyle™ CHO-S cell, GripTite™ 293 MSR cell line, GS-CHO cell line, HepaRG™ cell, T-REx™ Jurkat cell line, Per.C6 cell, T-REx™-293 cell line, Includes, but not limited to, T-REx™-CHO cell lines and T-REx™-HeLa cell lines.

[0475] In some cases, mammalian host cells are stable cell lines or cell lines capable of incorporating the genetic material of interest into their own genome and expressing the product of the genetic material after many generations of cell division. In some cases, mammalian host cells are transient cell lines or cell lines that do not incorporate the genetic material of interest into their own genome and do not have the ability to express the product of the genetic material after many generations of cell division. Exemplary insect host cells include, but are not limited to, Drosophila S2 cells, Sf9 cells, Sf21 cells, High Five™ cells, and expresSF+® cells. In some cases, plant cells include algae-derived cells. Exemplary insect cell lines include, but are not limited to, strains derived from Chlamydomonas reinhardtii 137c or Synechococcus elongatus PPC 7942.

[0476] Manufactured goods

[0477] In another aspect of the present invention, a manufactured article containing a substance useful for the treatment, prevention, and / or diagnosis of the aforementioned disorder is provided. The manufactured article comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The container may be formed from various materials such as glass or plastic. The container may contain the composition itself or a composition combined with another composition effective for the treatment, prevention, and / or diagnosis of the condition, and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper that can be punctured by a subcutaneous injection needle). At least one active agent in the composition is an isolated polypeptide or polypeptide complex of any embodiment disclosed herein.

[0478] A label or package insert indicates that the composition is used to treat a selected pathological condition. Furthermore, the manufactured article may comprise (a) a first container containing a composition comprising the bispecific antibody of the present invention; and (b) a second container containing a composition comprising an additional cytotoxic agent or other therapeutic agent. In this embodiment of the present invention, the manufactured article may further comprise a package insert indicating that the composition can be used to treat a specific pathological condition.

[0479] Alternatively or additionally, the manufactured article may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic injectable (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. The manufactured article may further comprise other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0480] Mode of implementation

[0481] Embodiment 1 comprises 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).

[0482] Embodiment 2 comprises the isolated polypeptide or polypeptide complex of Embodiment 1, wherein the first antigen recognition molecule that binds to CD28 comprises an anti-CD28 antibody.

[0483] Embodiment 3 comprises the isolated polypeptide or polypeptide complex of Embodiment 2, wherein the anti-CD28 antibody comprises an anti-CD28 heavy chain variable domain comprising a complementarity determining region (CDR): HC-CDR1, HC-CDR2, and HC-CDR3, wherein HC-CDR1, HC-CDR2, and HC-CDR3 comprise the amino acid sequences of 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 a complementarity determining region (CDR): LC-CDR1, LC-CDR2, and LC-CDR3, wherein LC-CDR1, LC-CDR2, and LC-CDR3 comprise LC-CDR1: SEQ ID NO. 4, LC-CDR2: (KAS) and LC-CDR3: Contains the amino acid sequence of SEQ ID NO. 6.

[0484] Embodiment 4 comprises the isolated polypeptide or polypeptide complex of Embodiment 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 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.

[0485] Embodiment 5 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 2 to 4, wherein the anti-CD28 antibody comprises a single-chain variable fragment (scFv), a single-domain antibody, Fab, Fab', or CrossFab.

[0486] Embodiment 6 comprises the isolated polypeptide or polypeptide complex of Embodiment 5, wherein the anti-CD28 antibody comprises scFv.

[0487] Embodiment 7 comprises the isolated polypeptide of Embodiment 5, wherein the anti-CD28 antibody comprises a single-chain variable fragment (scFv) having a engineered disulfide.

[0488] Embodiment 8 comprises the isolated polypeptide or polypeptide complex of Embodiment 5, wherein the anti-CD28 antibody comprises scFv, and said scFv comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO. 9.

[0489] Embodiment 9 comprises the isolated polypeptide or polypeptide complex of Embodiment 5, wherein the anti-CD28 antibody comprises scFv, and said scFv comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO. 10.

[0490] Embodiment 10 comprises the isolated polypeptide or polypeptide complex of Embodiment 5, wherein the anti-CD28 antibody comprises scFv, and said scFv comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO. 11.

[0491] Embodiment 11 comprises the isolated polypeptide or polypeptide complex of Embodiment 5, wherein the anti-CD28 antibody comprises scFv, and said scFv comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO. 12.

[0492] Embodiment 12 comprises the isolated polypeptide or polypeptide complex of Embodiment 5, wherein the anti-CD28 antibody comprises Fab.

[0493] Embodiment 13 comprises the isolated polypeptide or polypeptide complex of Embodiment 12, wherein 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.

[0494] Embodiment 14 comprises the isolated polypeptide or polypeptide complex of Embodiment 5, wherein the anti-CD28 antibody comprises CrossFab.

[0495] Embodiment 15 comprises the isolated polypeptide or polypeptide complex of Embodiment 14, wherein 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.

[0496] Embodiment 16 comprises the isolated polypeptide or polypeptide complex of Embodiment 14, wherein 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.

[0497] Embodiment 17 comprises an isolated polypeptide or polypeptide complex of any one of the above embodiments, wherein the second antigen recognition molecule that binds to PSMA comprises an anti-PSMA antibody.

[0498] Embodiment 18 comprises the isolated polypeptide or polypeptide complex of Embodiment 17, wherein the anti-PSMA antibody comprises an anti-PSMA heavy chain variable domain comprising a complementarity determining region (CDR): HC-CDR1, HC-CDR2, and HC-CDR3, wherein HC-CDR1, HC-CDR2, and HC-CDR3 comprise the amino acid sequences of 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 a complementarity determining region (CDR): LC-CDR1, LC-CDR2, and LC-CDR3, wherein LC-CDR1, LC-CDR2, and LC-CDR3 of the light chain variable domain comprise LC-CDR1: SEQ ID NO. 16, LC-CDR2: (EA) and LC-CDR3: Includes the amino acid sequence of SEQ ID NO. 18.

[0499] Embodiment 19 comprises the isolated polypeptide or polypeptide complex of 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 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.

[0500] Embodiment 20 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 17 to 19, wherein the anti-PSMA antibody comprises a single-chain variable fragment (scFv), a single-domain antibody, Fab, Fab', or CrossFab.

[0501] Embodiment 21 comprises the isolated polypeptide or polypeptide complex of Embodiment 20, wherein the anti-PSMA antibody comprises Fab or Fab'.

[0502] Embodiment 22 comprises an isolated polypeptide or polypeptide complex of Embodiment 20, wherein the anti-PSMA antibody comprises Fab or Fab', and said 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.

[0503] Embodiment 23 comprises the isolated polypeptide or polypeptide complex of Embodiment 20, wherein the anti-PSMA antibody comprises Fab or Fab', and said 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.

[0504] Embodiment 24 comprises the isolated polypeptide or polypeptide complex of Embodiment 20, wherein the anti-PSMA antibody comprises Fab or Fab', and said 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.

[0505] Embodiment 25 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 17 to 24, wherein the anti-CD28 antibody comprises scFv and the anti-PSMA antibody comprises Fab or Fab'.

[0506] Embodiment 26 comprises the isolated polypeptide or polypeptide complex of Embodiment 25, wherein scFv and Fab or Fab' are connected via a linker, and said Fab or Fab' comprises a Fab heavy chain polypeptide and a Fab light chain polypeptide.

[0507] Embodiment 27 comprises the isolated polypeptide or polypeptide complex of Embodiment 26, wherein the linker connects scFv to the Fab heavy chain polypeptide.

[0508] Embodiment 28 comprises the isolated polypeptide or polypeptide complex of Embodiment 26, wherein the linker connects scFv to the Fab light chain polypeptide.

[0509] Embodiment 29 comprises the isolated polypeptide or polypeptide complex of Embodiment 26, wherein the linker connects scFv to the N-terminus of the Fab heavy chain polypeptide.

[0510] Embodiment 30 comprises the isolated polypeptide or polypeptide complex of Embodiment 26, wherein the linker connects scFv to the C-terminus of the Fab heavy chain polypeptide.

[0511] Embodiment 31 comprises the isolated polypeptide or polypeptide complex of Embodiment 26, wherein the linker connects scFv to the N-terminus of the Fab light chain polypeptide.

[0512] Embodiment 32 comprises the isolated polypeptide or polypeptide complex of Embodiment 26, wherein the linker connects scFv to the C-terminus of the Fab light chain polypeptide.

[0513] Embodiment 33 comprises the isolated polypeptide or polypeptide complex of Embodiment 26, wherein the linker links the anti-CD28 heavy chain variable domain of scFv to the Fab heavy chain polypeptide.

[0514] Embodiment 34 comprises the isolated polypeptide or polypeptide complex of Embodiment 26, wherein the linker links the anti-CD28 heavy chain variable domain of scFv to the Fab light chain polypeptide.

[0515] Embodiment 35 comprises the isolated polypeptide or polypeptide complex of Embodiment 26, wherein the linker connects the CD28 heavy chain variable domain of scFv to the N-terminus of the Fab heavy chain polypeptide.

[0516] Embodiment 36 comprises the isolated polypeptide or polypeptide complex of Embodiment 26, wherein the linker connects the anti-CD28 heavy chain variable domain of scFv to the C-terminus of the Fab heavy chain polypeptide.

[0517] Embodiment 37 comprises the isolated polypeptide or polypeptide complex of Embodiment 26, wherein the linker connects the anti-CD28 heavy chain variable domain of scFv to the N-terminus of the Fab light chain polypeptide.

[0518] Embodiment 38 comprises the isolated polypeptide or polypeptide complex of Embodiment 26, wherein the linker connects the CD28 heavy chain variable domain of scFv to the C-terminus of the Fab light chain polypeptide.

[0519] Embodiment 39 comprises the isolated polypeptide or polypeptide complex of Embodiment 26, wherein the linker links the anti-CD28 light chain variable domain of scFv to the Fab heavy chain polypeptide.

[0520] Embodiment 40 comprises the isolated polypeptide or polypeptide complex of Embodiment 26, wherein the linker links the anti-CD28 light chain variable domain of scFv to the Fab light chain polypeptide.

[0521] Embodiment 41 comprises the isolated polypeptide or polypeptide complex of Embodiment 26, wherein the linker connects the CD28 light chain variable domain of scFv to the N-terminus of the Fab heavy chain polypeptide.

[0522] Embodiment 42 comprises the isolated polypeptide or polypeptide complex of Embodiment 26, wherein the linker connects the anti-CD28 light chain variable domain of scFv to the C-terminus of the Fab heavy chain polypeptide.

[0523] Embodiment 43 comprises the isolated polypeptide or polypeptide complex of Embodiment 26, wherein the linker connects the anti-CD28 light chain variable domain of scFv to the N-terminus of the Fab light chain polypeptide.

[0524] Embodiment 44 comprises the isolated polypeptide or polypeptide complex of Embodiment 26, wherein the linker connects the CD28 light chain variable domain of scFv to the C-terminus of the Fab light chain polypeptide.

[0525] Embodiment 45 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 17 to 24, wherein the anti-CD28 antibody comprises Fab or Fab' and the anti-PSMA antibody comprises Fab or Fab'.

[0526] Embodiment 46 comprises the isolated polypeptide or polypeptide complex of Embodiment 45, wherein Fab or Fab' of the anti-CD28 antibody and Fab or Fab' of the anti-PSMA antibody are connected via a linker, and Fab or Fab' of the anti-PSMA antibody comprises a Fab heavy chain polypeptide and a Fab light chain polypeptide.

[0527] Embodiment 47 comprises the isolated polypeptide or polypeptide complex of Embodiment 46, wherein the linker links Fab or Fab' of the anti-CD28 antibody to the Fab heavy chain polypeptide of the anti-PSMA antibody.

[0528] Embodiment 48 comprises the isolated polypeptide or polypeptide complex of Embodiment 46, wherein the linker links Fab or Fab' of the anti-CD28 antibody to the Fab light chain polypeptide of the anti-PSMA antibody.

[0529] Embodiment 49 comprises the isolated polypeptide or polypeptide complex of Embodiment 46, wherein the linker connects Fab or Fab' of the anti-CD28 antibody to the N-terminus of the Fab heavy chain polypeptide of the anti-PSMA antibody.

[0530] Embodiment 50 comprises the isolated polypeptide or polypeptide complex of Embodiment 46, wherein the linker connects Fab or Fab' of the anti-CD28 antibody to the C-terminus of the Fab heavy chain polypeptide of the anti-PSMA antibody.

[0531] Embodiment 51 comprises the isolated polypeptide or polypeptide complex of Embodiment 46, wherein the linker connects Fab or Fab' of the anti-CD28 antibody to the N-terminus of the Fab light chain polypeptide of the anti-PSMA antibody.

[0532] Embodiment 52 comprises the isolated polypeptide or polypeptide complex of Embodiment 46, wherein the linker connects Fab or Fab' of the anti-CD28 antibody to the C-terminus of the Fab light chain polypeptide of the anti-PSMA antibody.

[0533] Embodiment 53 comprises the isolated polypeptide or polypeptide complex of Embodiment 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.

[0534] Embodiment 54 comprises the isolated polypeptide or polypeptide complex of Embodiment 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.

[0535] Embodiment 55 comprises the isolated polypeptide or polypeptide complex of Embodiment 46, wherein 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.

[0536] Embodiment 56 comprises the isolated polypeptide or polypeptide complex of Embodiment 46, wherein 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.

[0537] Embodiment 57 comprises the isolated polypeptide or polypeptide complex of Embodiment 46, wherein 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.

[0538] Embodiment 58 comprises the isolated polypeptide or polypeptide complex of Embodiment 46, wherein 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.

[0539] Embodiment 59 comprises the isolated polypeptide or polypeptide complex of Embodiment 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.

[0540] Embodiment 60 comprises the isolated polypeptide or polypeptide complex of Embodiment 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.

[0541] Embodiment 61 comprises the isolated polypeptide or polypeptide complex of Embodiment 46, wherein the linker connects 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.

[0542] Embodiment 62 comprises the isolated polypeptide or polypeptide complex of Embodiment 46, wherein the linker connects 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.

[0543] Embodiment 63 comprises the isolated polypeptide or polypeptide complex of Embodiment 46, wherein the linker connects 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.

[0544] Embodiment 64 comprises the isolated polypeptide or polypeptide complex of Embodiment 46, wherein the linker connects 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.

[0545] Embodiment 65 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 17 to 24, wherein the anti-CD28 antibody comprises CrossFab and the anti-PSMA antibody comprises Fab or Fab'.

[0546] Embodiment 66 comprises the isolated polypeptide or polypeptide complex of Embodiment 65, wherein CrossFab and Fab or Fab' are connected via a linker, and Fab or Fab' of the anti-PSMA antibody comprises the Fab heavy chain polypeptide and the Fab light chain polypeptide.

[0547] Embodiment 67 comprises the isolated polypeptide or polypeptide complex of Embodiment 66, wherein the linker links CrossFab to the Fab heavy chain polypeptide of the anti-PSMA antibody.

[0548] Embodiment 68 comprises the isolated polypeptide or polypeptide complex of Embodiment 66, wherein the linker links CrossFab to the Fab light chain polypeptide of the anti-PSMA antibody.

[0549] Embodiment 69 comprises the isolated polypeptide or polypeptide complex of Embodiment 66, wherein the linker connects CrossFab to the N-terminus of the Fab heavy chain polypeptide of the anti-PSMA antibody.

[0550] Embodiment 70 comprises the isolated polypeptide or polypeptide complex of Embodiment 66, wherein the linker connects CrossFab to the C-terminus of the Fab heavy chain polypeptide of the anti-PSMA antibody.

[0551] Embodiment 71 comprises the isolated polypeptide or polypeptide complex of Embodiment 66, wherein the linker connects CrossFab to the N-terminus of the Fab light chain polypeptide of the anti-PSMA antibody.

[0552] Embodiment 72 comprises the isolated polypeptide or polypeptide complex of Embodiment 66, wherein the linker connects CrossFab to the C-terminus of the Fab light chain polypeptide of the anti-PSMA antibody.

[0553] Embodiment 73 comprises the isolated polypeptide or polypeptide complex of Embodiment 66, wherein the linker links the Fab heavy chain polypeptide of CrossFab to the Fab heavy chain polypeptide of the anti-PSMA antibody.

[0554] Embodiment 74 comprises the isolated polypeptide or polypeptide complex of Embodiment 66, wherein the linker links the Fab heavy chain polypeptide of CrossFab to the Fab light chain polypeptide of the anti-PSMA antibody.

[0555] Embodiment 75 comprises the isolated polypeptide or polypeptide complex of Embodiment 66, wherein the linker links the Fab heavy chain polypeptide of CrossFab to the N-terminus of the Fab heavy chain polypeptide of the anti-PSMA antibody.

[0556] Embodiment 76 comprises the isolated polypeptide or polypeptide complex of Embodiment 66, wherein the linker links the Fab heavy chain polypeptide of CrossFab to the C-terminus of the Fab heavy chain polypeptide of the anti-PSMA antibody.

[0557] Embodiment 77 comprises the isolated polypeptide or polypeptide complex of Embodiment 66, wherein the linker connects the Fab heavy chain polypeptide of CrossFab to the N-terminus of the Fab light chain polypeptide of the anti-PSMA antibody.

[0558] Embodiment 78 comprises the isolated polypeptide or polypeptide complex of Embodiment 66, wherein the linker connects the Fab heavy chain polypeptide of CrossFab to the C-terminus of the Fab light chain polypeptide of the anti-PSMA antibody.

[0559] Embodiment 79 comprises the isolated polypeptide or polypeptide complex of Embodiment 66, wherein the linker links the Fab light chain polypeptide of CrossFab to the Fab heavy chain polypeptide of the anti-PSMA antibody.

[0560] Embodiment 80 comprises the isolated polypeptide or polypeptide complex of Embodiment 66, wherein the linker links the Fab light chain polypeptide of CrossFab to the Fab light chain polypeptide of the anti-PSMA antibody.

[0561] Embodiment 81 comprises the isolated polypeptide or polypeptide complex of Embodiment 66, wherein the linker connects the Fab light chain polypeptide of CrossFab to the N-terminus of the Fab heavy chain polypeptide of the anti-PSMA antibody.

[0562] Embodiment 82 comprises the isolated polypeptide or polypeptide complex of Embodiment 66, wherein the linker connects the Fab light chain polypeptide of CrossFab to the C-terminus of the Fab heavy chain polypeptide of the anti-PSMA antibody.

[0563] Embodiment 83 comprises the isolated polypeptide or polypeptide complex of Embodiment 66, wherein the linker links the Fab light chain polypeptide of CrossFab to the N-terminus of the Fab light chain polypeptide of the anti-PSMA antibody.

[0564] Embodiment 84 comprises the isolated polypeptide or polypeptide complex of Embodiment 66, wherein the linker links the Fab light chain polypeptide of CrossFab to the C-terminus of the Fab light chain polypeptide of the anti-PSMA antibody.

[0565] Embodiment 85 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 17 to 20, wherein the anti-CD28 antibody comprises Fab or Fab' and the anti-PSMA antibody comprises CrossFab.

[0566] Embodiment 86 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 26 to 44, 46 to 64, and 66 to 84, wherein the linker has a length of at least 5 amino acids.

[0567] Embodiment 87 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 26 to 44, 46 to 64, and 66 to 84, wherein the linker is an amino acid with a length of 30 or fewer.

[0568] Embodiment 88 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 26 to 44, 46 to 64, and 66 to 84, wherein the linker is an amino acid with a length of at least 5 amino acids to 30 amino acids or less.

[0569] Embodiment 89 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 26 to 44, 46 to 64, and 66 to 84, wherein the linker is 5 amino acids long.

[0570] Embodiment 90 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 26 to 44, 46 to 64, and 66 to 84, wherein the linker has a length of 15 amino acids.

[0571] Embodiment 91 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 26 to 44, 46 to 64, and 66 to 84, wherein the linker comprises the amino acid sequence of SEQ ID NO. 21 (GGGGSGGGGSGGGGS) or SEQ ID NO. 22 (GGGGS).

[0572] Embodiment 92 comprises an isolated polypeptide or polypeptide complex of 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. 23 and SEQ ID NO...

Claims

Claim 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). Claim 2 In claim 1, the first antigen recognition molecule that binds to CD28 is an isolated polypeptide or polypeptide complex comprising an anti-CD28 antibody. Claim 3 In paragraph 2, the isolated polypeptide comprises an anti-CD28 heavy chain variable domain comprising a complementarity determining region (CDR): HC-CDR1, HC-CDR2, and HC-CDR3, wherein HC-CDR1, HC-CDR2, and HC-CDR3 comprise the amino acid sequences of 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 a complementarity determining region (CDR): LC-CDR1, LC-CDR2, and LC-CDR3, wherein LC-CDR1, LC-CDR2, and LC-CDR3 comprise the amino acid sequences of LC-CDR1: SEQ ID NO. 4, LC-CDR2: (KAS), and LC-CDR3: SEQ ID NO.

6. Or polypeptide complex. Claim 4 An 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 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. Claim 5 In paragraph 3, the anti-CD28 antibody is an isolated polypeptide or polypeptide complex comprising a single-chain variable fragment (scFv), a single-domain antibody, Fab, Fab', or CrossFab. Claim 6 In claim 5, the isolated polypeptide or polypeptide complex comprising an anti-CD28 antibody containing scFv, wherein 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. Claim 7 In claim 5, the isolated polypeptide or polypeptide complex comprising an anti-CD28 antibody containing scFv, wherein 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. Claim 8 In claim 5, the isolated polypeptide or polypeptide complex comprising an anti-CD28 antibody containing scFv, wherein 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. Claim 9 In claim 5, the isolated polypeptide or polypeptide complex comprising an anti-CD28 antibody containing scFv, wherein 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. Claim 10 In paragraph 5, the anti-CD28 antibody is an isolated polypeptide or polypeptide complex comprising Fab. Claim 11 In claim 10, Fab is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO. 696 and at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO.

697. Claim 12 In paragraph 5, the anti-CD28 antibody is an isolated polypeptide or polypeptide complex comprising CrossFab. Claim 13 In claim 12, CrossFab is an isolated polypeptide or polypeptide complex comprising 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. Claim 14 In claim 12, CrossFab is an isolated polypeptide or polypeptide complex comprising 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. Claim 15 In paragraph 3, the second antigen recognition molecule that binds to PSMA is an isolated polypeptide or polypeptide complex comprising an anti-PSMA antibody. Claim 16 In claim 15, the anti-PSMA antibody comprises an anti-PSMA heavy chain variable domain comprising a complementarity determining region (CDR): HC-CDR1, HC-CDR2, and HC-CDR3, wherein HC-CDR1, HC-CDR2, and HC-CDR3 comprise the amino acid sequences of 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 a complementarity determining region (CDR): LC-CDR1, LC-CDR2, and LC-CDR3, wherein LC-CDR1, LC-CDR2, and LC-CDR3 of the light chain variable domain comprise the amino acid sequences of LC-CDR1: SEQ ID NO. 16, LC-CDR2: (EA), and LC-CDR3: SEQ ID NO.

18. Isolated polypeptide or polypeptide complex. Claim 17 An 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 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. Claim 18 In paragraph 16, the anti-PSMA antibody is an isolated polypeptide or polypeptide complex comprising a single-chain variable fragment (scFv), a single-domain antibody, Fab, Fab', or CrossFab. Claim 19 An isolated polypeptide or polypeptide complex, wherein 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. Claim 20 An isolated polypeptide or polypeptide complex according to claim 18, wherein 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. Claim 21 An isolated polypeptide or polypeptide complex, wherein 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. Claim 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. Claim 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. Claim 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. Claim 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. Claim 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. Claim 27 In claim 1, the isolated polypeptide or polypeptide complex comprises the amino acid sequences of SEQ ID NO. 25 and SEQ ID NO.

26. Claim 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. Claim 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. Claim 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. Claim 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. Claim 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. Claim 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. Claim 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. Claim 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. Claim 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. Claim 37 In paragraph 16, the isolated polypeptide or polypeptide complex comprises a peptide linked to an anti-CD28 antibody, wherein the peptide impairs the binding of the anti-CD28 antibody to CD28. Claim 38 In paragraph 37, the isolated polypeptide or polypeptide complex is of formula I: A 2 -A 1 -L 1 -P 1 -H 1 An isolated polypeptide or polypeptide complex comprising a composition according to, wherein A2 comprises an anti-PSMA antibody, A1 comprises an anti-CD28 antibody, L1 comprises a linking moiety that links A1 to P1 and is a substrate for a tumor-specific protease, P1 comprises a peptide that impairs the binding of the anti-CD28 antibody to CD28, and H1 comprises a half-life extending molecule. Claim 39 An isolated polypeptide or polypeptide complex, wherein A2 further comprises P2 and L2, wherein P2 comprises a peptide that binds to A2; and L2 comprises a linking moiety that links A2 to P2 and is a substrate for a tumor-specific protease. Claim 40 In paragraph 39, the polypeptide or polypeptide complex is of formula Ia: P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 An isolated polypeptide or polypeptide complex according to Claim 41 In claim 40, P1 is an isolated polypeptide or polypeptide complex comprising an amino acid sequence according to any one of SEQ ID NOs 33 to 106 or 732 to 813. Claim 42 In claim 40, P1 is an isolated polypeptide or polypeptide complex comprising the amino acid sequence of SEQ ID NO.

73. Claim 43 In claim 40, P1 is an isolated polypeptide or polypeptide complex comprising the amino acid sequence of SEQ ID NO.

103. Claim 44 In paragraph 40, P1 is X1-X2-X3-C-X4-X5-X6-X7-X8-X9-X 10 -CX 11 -X 12 It includes an amino acid sequence according to, wherein 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 is selected from E, V, L, D, Y, R, Q, H, F, K, A, M, and N; X 11 is selected from F, Y, L, W, and V; X 12 is an isolated polypeptide or polypeptide complex selected from N, A, F, S, Y, H, D, T, and L. Claim 45 In paragraph 44, 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 is selected from E, V, L, D, and H; X 11 is selected from F, Y, and L; X 12 is an isolated polypeptide or polypeptide complex selected from N, A, F, S, and Y. Claim 46 In claim 40, P1 is an isolated polypeptide or polypeptide complex comprising the amino acid sequence according to SEQ ID NO.

41. Claim 47 In Clause 40, P1 is J1-J2-WC-J3-J4-J5-J6-J7-J8-J9-CJ 10 -J 11 Includes an amino acid sequence according to, wherein 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 is selected from F, Y, and L; J 11 is an isolated polypeptide or polypeptide complex selected from A, N, H, and T. Claim 48 In paragraph 47, J1 is an isolated polypeptide or polypeptide complex selected from A, R, N, D, Q, E, G, H, K, F, P, S, T, W, and Y. Claim 49 In paragraph 47, 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 is F and;J 11 is an isolated polypeptide or polypeptide complex of A. Claim 50 In paragraph 40, P1 is Z1-C-Z2-Z3-Z4-Z5-Z6-Z7-Z8-Z9-Z 10 -Z 11 -CZ 12 Includes an amino acid sequence according to, wherein 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, Selected from V, K, F, Q, Y, W, and A; Z 10 is selected from A, Q, S, W, E, L, G, D, T, M, N, V, H, R, Y, K, P, F, and I; Z 11 is selected from K, Q, N, H, E, P, and A; Z 12 is an isolated polypeptide or polypeptide complex selected from L, M, I, F, V, Y, Q, D, T, and A. Claim 51 In paragraph 50, 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 Selected from W; Z 10 is selected from A, Q, S, W, E, L, G, D, T, M, N, V, H, R, Y, K, P, F, and I; Z 11 is selected from K, Q, N, H, E, and P; Z 12 is an isolated polypeptide or polypeptide complex selected from L, M, I, F, V, Y, Q, D, and T. Claim 52 In claim 40, P1 is an isolated polypeptide or polypeptide complex comprising the amino acid sequence of SEQ ID NO.

784. Claim 53 In claim 40, P1 is an isolated polypeptide or polypeptide complex comprising the amino acid sequence of SEQ ID NO.

744. Claim 54 In claim 40, P1 is an isolated polypeptide or polypeptide complex consisting of the amino acid sequence of SEQ ID NO.

744. Claim 55 In claim 40, P1 is an isolated polypeptide or polypeptide complex comprising an amino acid sequence according to any one of SEQ ID NOs 774 to 793. Claim 56 In claim 40, the peptide is an isolated polypeptide or polypeptide complex comprising an amino acid sequence according to any one of SEQ ID NOs 764 to 773. Claim 57 In paragraph 40, P1 is U1-C-U2-U3-U4-U5-U6-U7-U8-U9-U 10 -U 11 -CU 12 It includes an amino acid sequence according to, wherein 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, Selected from M, and R; U9 is selected from W, L, and A; U 10 is selected from P, S, R, L, E, and A; U 11 is selected from L, A, T, M, V, W, Y, E, Q, I, F, S, P, K, R, D, H, N, and G; U 12 is an isolated polypeptide or polypeptide complex selected from L, F, V, M, I, Y, E, W, and A. Claim 58 In paragraph 57, 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 Selected; U9 is selected from W, L, and A; U 10 is selected from P, S, R, L, and E; U 11 is selected from L, A, T, M, V, W, Y, E, Q, I, F, S, P, K, R, D, H, N, and G; U 12 is an isolated polypeptide or polypeptide complex selected from L, F, V, M, I, Y, E, and W. Claim 59 In claim 40, P1 is an isolated polypeptide or polypeptide complex comprising the amino acid sequence of SEQ ID NO.

749. Claim 60 In claim 40, P1 is an isolated polypeptide or polypeptide complex comprising an amino acid sequence according to any one of SEQ ID NOs 756 to 763. Claim 61 In claim 40, P1 is an isolated polypeptide or polypeptide complex comprising an amino acid sequence according to any one of SEQ ID NOs 794 to 813. Claim 62 In claim 40, P1 is an isolated polypeptide or polypeptide complex comprising an amino acid sequence according to any one of SEQ ID NOs 732 to 2002. Claim 63 In claim 40, P2 is an isolated polypeptide or polypeptide complex comprising the amino acid sequence of any one of SEQ ID NOs 2003 to 4329. Claim 64 In claim 40, P2 is an isolated polypeptide or polypeptide complex comprising the amino acid sequence of SEQ ID NO. 2025. Claim 65 In claim 40, P2 is an isolated polypeptide or polypeptide complex comprising an amino acid sequence according to any one of SEQ ID NOs 2087 to 2094. Claim 66 In claim 40, P2 is an isolated polypeptide or polypeptide complex comprising the amino acid sequence of SEQ ID NO. 2026. Claim 67 In claim 40, P2 is an isolated polypeptide or polypeptide complex comprising an amino acid sequence according to any one of SEQ ID NOs 2097 to 2106. Claim 68 In claim 40, P2 is an isolated polypeptide or polypeptide complex comprising an amino acid sequence according to any one of SEQ ID NOs 2107 to 2133. Claim 69 In claim 40, P2 is an isolated polypeptide or polypeptide complex comprising the amino acid sequence of SEQ ID NO. 2120. Claim 70 In paragraph 40, P2 is B1-B2-B3-B4-C-B5-P-B6-W-B7-C-B8-B9-B 10 It comprises an amino acid sequence according to (SEQ 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; and B6 is selected from A, E, S, P, Q, T, Selected from L, D, M, V, R, K, N, I, H, W, Y, F, and G; B7 selected from M, L, I, A, V, F, G, and K; B8 selected from E, S, T, A, V, D, Q, L, N, I, M, Y, H, F, W, G, K, R, and P; B9 selected from G, V, A, S, W, E, D, M, T, L, F, H, Q, N, R, I, Y, P, and K; B 10 is an isolated polypeptide or polypeptide complex selected from F, L, M, S, I, V, D, Q, T, A, N, Y, W, E, R, H, P, G, and K. Claim 71 In paragraph 40, P1 is an isolated polypeptide or polypeptide complex that exposes A1 to CD28 by becoming unbound from A1 when L1 is cleaved by a tumor-specific protease. Claim 72 In paragraph 40, P2 is an isolated polypeptide or polypeptide complex that exposes A2 to PSMA by becoming unbound from A2 when L2 is cleaved by a tumor-specific protease. Claim 73 In paragraph 40, L1 or L2 is an isolated polypeptide or polypeptide complex comprising a urokinase cleavable amino acid sequence, a matriptase cleavable amino acid sequence, a matrix metalloprotease cleavable amino acid sequence, or a legumin cleavable amino acid sequence. Claim 74 In claim 40, L1 or L2 is an isolated polypeptide or polypeptide complex comprising an amino acid sequence according to any one of SEQ ID NOs 107 to 137 and 604. Claim 75 In paragraph 40, the half-life extending molecule is an isolated polypeptide or polypeptide complex comprising a single-domain antibody. Claim 76 An isolated polypeptide or polypeptide complex according to claim 75, wherein the single-domain antibody comprises complementarity determining regions (CDR): 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. Claim 77 An isolated polypeptide or polypeptide complex according to claim 75, wherein the single-domain antibody comprises complementarity determining regions (CDR): 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. Claim 78 In claim 75, the single-domain antibody is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO.

141. Claim 79 In claim 75, the single-domain antibody is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO.

146. Claim 80 In claim 75, the single-domain antibody is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO.

145. Claim 81 In claim 75, the single-domain antibody is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO. 4448. Claim 82 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NOs 147 and 148. Claim 83 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NOs 149 and 150. Claim 84 In claim 1, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NOs 151 and 152. Claim 85 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NOs 153 and 154. Claim 86 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NOs 155 and 156. Claim 87 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NOs 157 and 158. Claim 88 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NOs 159 and 160. Claim 89 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NOs 161 and 162. Claim 90 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NOs 163 and 164. Claim 91 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NOs 165 and 166. Claim 92 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NOs 167 and 168. Claim 93 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NOs 169 and 170. Claim 94 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NOs 171 and 172. Claim 95 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NOs 173 and 174. Claim 96 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NOs 175 and 176. Claim 97 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NOs 177 and 178. Claim 98 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4330 and 4331. Claim 99 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4332 and 4333. Claim 100 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4334 and 4335. Claim 101 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4336 and 4337. Claim 102 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4338 and 4339. Claim 103 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4340 and 4341. Claim 104 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4342 and 4343. Claim 105 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4344 and 4345. Claim 106 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4346 and 4347. Claim 107 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4348 and 4349. Claim 108 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4350 and 4351. Claim 109 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4352 and 4353. Claim 110 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4354 and 4355. Claim 111 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4356 and 4357. Claim 112 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4358 and 4359. Claim 113 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4360 and 4361. Claim 114 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4362 and 4363. Claim 115 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4364 and 4365. Claim 116 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4366 and 4367. Claim 117 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4368 and 4369. Claim 118 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4370 and 4371. Claim 119 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4372 and 4373. Claim 120 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4374 and 4375. Claim 121 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4376 and 4377. Claim 122 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4378 and 4379. Claim 123 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4380 and 4381. Claim 124 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4382 and 4383. Claim 125 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4384 and 4385. Claim 126 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4386 and 4387. Claim 127 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4388 and 4389. Claim 128 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4390 and 4391. Claim 129 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4392 and 4393. Claim 130 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4394 and 4395. Claim 131 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4396 and 4397. Claim 132 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4398 and 4399. Claim 133 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4400 and 4401. Claim 134 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4402 and 4403. Claim 135 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4404 and 4405. Claim 136 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4406 and 4407. Claim 137 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4408 and 4409. Claim 138 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4410 and 4411. Claim 139 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4412 and 4413. Claim 140 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4414 and 4415. Claim 141 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4416 and 4417. Claim 142 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4418 and 4419. Claim 143 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4420 and 4421. Claim 144 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4422 and 4423. Claim 145 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4424 and 4425. Claim 146 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4426 and 4427. Claim 147 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4428 and 4429. Claim 148 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4430 and 4431. Claim 149 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4432 and 4433. Claim 150 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4434 and 4435. Claim 151 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4436 and 4437. Claim 152 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4438 and 4439. Claim 153 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4440 and 4441. Claim 154 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4442 and 4443. Claim 155 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4444 and 4445. Claim 156 In claim 1, the polypeptide or polypeptide complex is an isolated polypeptide or polypeptide complex comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NOs 4446 and 4447. Claim 157 An isolated recombinant nucleic acid molecule encoding the polypeptide or polypeptide complex of claim 1. Claim 158 A pharmaceutical composition comprising (a) the polypeptide or polypeptide complex of claim 1; and (b) a pharmaceutically acceptable excipient. Claim 159 A method for treating cancer in a subject requiring cancer treatment, comprising administering the polypeptide or polypeptide complex of claim 1 to said subject. Claim 160 In paragraph 159, the polypeptide or polypeptide complex is administered in combination with anticancer therapy. Claim 161 In paragraph 160, the anticancer therapy comprises a small molecule, cell-based therapy, or antibody-based therapy. Claim 162 In paragraph 161, the antibody-based therapy is a method that is a T cell engager. Claim 163 A vector comprising the isolated recombinant nucleic acid molecule of claim 157.

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