Compositions and methods related to tumor-activating antibodies targeting PSMA and effector cell antigens

Abstract

Provided herein are multispecific antibodies that selectively bind to PSMA and effector cell antigens, such as CD3, pharmaceutical compositions and nucleic acids thereof, and methods for making and discovering the same.

Description

[Technical Field]

[0001] Cross-references to related applications This application claims the benefits of U.S. Provisional Patent Application No. 63 / 123,329 filed on 9 December 2020 and U.S. Provisional Patent Application No. 63 / 187,699 filed on 12 May 2021, which are incorporated herein by reference in their entirety.

[0002] Sequence List This application includes a sequence listing submitted electronically in ASCII format and incorporated herein by reference. A copy of the ASCII file prepared on November 30, 2021, is named 52426-730_601_SL.txt and is 321,067 bytes in size. [Overview of the project]

[0003] In certain embodiments of this specification, Formula I: A2-A1-L1-P1-H1 (Formula I) Disclosed is an isolated polypeptide or polypeptide complex, wherein A1 comprises a first antigen-recognizing molecule that binds to an effector cell antigen, P1 comprises a peptide that binds to A1, L1 comprises a linking portion that connects A1 to P1 and is a substrate of a tumor-specific protease, H1 comprises a half-life extension molecule, and A2 comprises a second antigen-recognizing molecule that binds to prostate-specific membrane antigen (PSMA). In some embodiments, the first antigen-recognizing molecule comprises an antibody or antibody fragment. In some embodiments, the first antigen-recognizing molecule comprises a human or humanized antibody or antibody fragment. In some embodiments, L1 is bound to the N-terminus of the first antigen-recognizing molecule. In some embodiments, A2 is bound to the C-terminus of the first antigen-recognizing molecule. In some embodiments, L1 is bound to the C-terminus of the first antigen-recognizing molecule. In some embodiments, A2 is bound to the N-terminus of the first antigen-recognizing molecule. In some embodiments, the antibody or antibody fragment comprises a single-chain variable fragment, a single-domain antibody, or a Fab fragment. In some embodiments, A1 is a single-chain variable fragment (scFv). In some embodiments, the scFv comprises an scFv heavy chain polypeptide and an scFv light chain polypeptide. In some embodiments, A1 is a single-domain antibody; in some embodiments, the antibody or antibody fragment comprises a single-chain variable fragment (scFv), a heavy chain variable domain (VH domain), a light chain variable domain (VL domain), or a variable domain (VHH) of a camelid-derived single-domain antibody. In some embodiments, A1 comprises an anti-CD3e single-chain variable fragment. In some embodiments, A1 has a K2+ concentration of 1 μM or less relative to CD3 on CD3-expressing cells. DIt contains an anti-CD3e single-chain variable fragment having a binding. In some embodiments, the effector cell antigen contains CD3. In some embodiments, A1 contains a variable light chain and a variable heavy chain, each specifically capable of binding to human CD3. In some embodiments, A1 includes muromonab-CD3 (OKT3), otelixizumab (TRX4), teprizumab (MGA031), bicilizumab (Nuvion), SP34, X35, VIT3, BMA030 (BW264 / 56), CLB-T3 / 3, CRIS7, YTH12.5, F111-409, CLB-T3.4.2, TR-66, WT32, SP The polypeptide or polypeptide complex of formula I is included, selected from the group consisting of v-T3b, 11D8, XIII-141, XIII-46, XIII-87, 12F6, T3 / RW2-8C8, T3 / RW2-4B6, OKT3D, M-T301, SMC2, F101.01, UCHT-1, WT-31, 15865, 15865v12, 15865v16, and 15865v19. In some embodiments, if L1 is cleaved by a tumor-specific protease, the polypeptide or polypeptide complex of formula I binds to effector cells. In some embodiments, if L1 is cleaved by a tumor-specific protease and A1 binds to effector cells, the polypeptide or polypeptide complex of formula I binds to effector cells. In some embodiments, the effector cells are T cells. In some embodiments, A1 binds to a polypeptide that is part of the TCR-CD3 complex on the effector cell. In some embodiments, the polypeptide that is part of the TCR-CD3 complex is human CD3ε.In some embodiments, the effector cell antigen comprises CD3, and the scFV comprises HC-CDR1, HC-CDR2, and HC-CDR3 as complementarity-determining regions (CDRs), where HC-CDR1, HC-CDR2, and HC-CDR3 of the scFV comprises SEQ ID NO: 1 for HC-CDR1, SEQ ID NO: 2 for HC-CDR2, and SEQ ID NO: 3 for HC-CDR3. The scFV also comprises LC-CDR1, LC-CDR2, and LC-CDR3 as CDRs, where LC-CDR1, LC-CDR2, and LC-CDR3 of the scFV comprises SEQ ID NO: 4 for LC-CDR1, SEQ ID NO: 5 for LC-CDR2, and SEQ ID NO: 6 for LC-CDR3. In some embodiments, the effector cell antigen comprises CD3, and the scFv comprises the amino acid sequence according to SEQ ID NO: 7. In some embodiments, the second antigen-recognizing molecule comprises an antibody or antibody fragment. In some embodiments, the antibody or antibody fragment comprises a single-chain variable fragment, a single-domain antibody, or a Fab. In some embodiments, the antibody or antibody fragment comprises a single-chain variable fragment (scFv), a heavy-chain variable domain (VH domain), a light-chain variable domain (VL domain), or a variable domain (VHH) of a single-domain antibody derived from a camelid family. In some embodiments, the antibody or antibody fragment is humanized or human. In some embodiments, A2 is Fab. In some embodiments, Fab comprises (a) Fab light-chain polypeptide and (b) Fab heavy-chain polypeptide. In some embodiments, Fab includes HC-CDR1, HC-CDR2, and HC-CDR3 as complementarity-determining regions (CDRs), where HC-CDR1 includes SEQ ID NO: 8, HC-CDR2 includes SEQ ID NO: 9, and HC-CDR3 includes SEQ ID NO: 10. Fab also includes LC-CDR1, LC-CDR2, and LC-CDR3 as CDRs, where LC-CDR1, LC-CDR2, and LC-CDR3 include SEQ ID NO: 11, LC-CDR2 includes SEQ ID NO: 12, and LC-CDR3 includes SEQ ID NO: 13. In some embodiments, the Fab light chain polypeptide includes the amino acid sequence by SEQ ID NO: 14.In some embodiments, the Fab heavy chain polypeptide comprises the amino acid sequence given by SEQ ID NO: 15. In some embodiments, the Fab light chain polypeptide of A2 is bound to the C-terminus of the single-chain variable fragment (scFv) of A1. In some embodiments, the Fab heavy chain polypeptide of A2 is bound to the C-terminus of the single-chain variable fragment (scFv) of A1. In some embodiments, the Fab light chain polypeptide of A2 is bound to the N-terminus of the single-chain variable fragment (scFv) of A1. In some embodiments, the Fab heavy chain polypeptide of A2 is bound to the N-terminus of the single-chain variable fragment (scFv) of A1. In some embodiments, the Fab heavy chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of A1. In some embodiments, the Fab light chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of A1. In some embodiments, the Fab heavy chain polypeptide of A2 is bound to the scFv light chain polypeptide of A1. In some embodiments, the Fab light chain polypeptide of A2 is bound to the scFv light chain polypeptide of A1. In some embodiments, A2 further comprises P2 and L2, where P2 comprises a peptide that binds to A2, and L2 comprises a linking portion that connects A1 to P1 and is a substrate for a tumor-specific protease. In some embodiments, the polypeptide or polypeptide complex is of formula Ia: P2-L2-A2-A1-L1-P1-H1 (Formula Ia) This is due to the following: 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. In some embodiments, P1 impairs the binding of A1 to the effector cell antigen. In some embodiments, P1 is bound to A1 by ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bonding interactions, or a combination thereof. In some embodiments, P1 has less than 70% sequence homology to the effector cell antigen. In some embodiments, P2 impairs the binding of A2 to PSMA. In some embodiments, P2 binds to A2 by ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and 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 to PSMA. In some embodiments, P1 or P2 includes a peptide sequence of at least 10 amino acids in length. In some embodiments, P1 or P2 includes a peptide sequence of at least 10 and no more than 20 amino acids in length. In some embodiments, P1 or P2 includes a peptide sequence of at least 16 amino acids in length. In some embodiments, P1 or P2 includes a peptide sequence of no more than 40 amino acids in length. In some embodiments, P1 or P2 includes at least two cysteine ​​amino acid residues. In some embodiments, P1 or P2 includes a cyclic peptide or a linear peptide. In some embodiments, P1 or P2 includes 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. In some embodiments, P1 comprises an amino acid sequence according to one of SEQ ID NOs: 16-19 or 78. 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 an expression that includes, where n is an integer from 1 to 3 (Sequence ID 118). In some embodiments, L1 is (G2S) n , (GS) n (GSGGS) n (Sequence ID 50), (GGGS) n (Sequence ID 51), (GGGGS) n (Sequence ID 52), and (GSSGGS) nThe formula is selected from the group consisting of (SEQ ID NO: 53), where n is at least an integer of 1. In some embodiments, when L1 is cleaved by a tumor-specific protease, thereby exposing A1 to the effector cell antigen, P1 is decoupled from A1. In some embodiments, when L2 is cleaved by a tumor-specific protease, thereby exposing A2 to PSMA, P2 is decoupled from A2. In some embodiments, the tumor-specific protease is selected from the group consisting of matrix metalloproteinases (MMPs), serine proteases, cysteine ​​proteases, threonine proteases, and aspartate proteases. In some embodiments, the matrix metalloproteinase includes MMP2, MMP7, MMP9, MMP13, or MMP14. In some embodiments, the serine protease includes matryptase (MTSP1), urokinase, or hepsin. In some embodiments, L1 or L2 includes a urokinase-cleavable amino acid sequence, a matryptase-cleavable amino acid sequence, a matrix metalloproteinase-cleavable amino acid sequence, or a regmine-cleavable amino acid sequence. In some embodiments, L1 or L2 includes the amino acid sequence according to SEQ ID NO: 23. In some embodiments, L1 or L2 includes the amino acid sequence according to any one of SEQ ID NOs: 20-49. In some embodiments, L1 or L2 includes the amino acid sequence of linker 25 (ISSGLLSGRSDAG) (SEQ ID NO: 45), linker 26 (AAGLLAPPGGLSGRSDAG) (SEQ ID NO: 46), linker 27 (SPLGLSGRSDAG) (SEQ ID NO: 47), or linker 28 (LSGRSDAGSPLGLAG) (SEQ ID NO: 48), or an amino acid sequence having one, two, or three amino acid substitutions, additions, or deletions to the amino acid sequence of linker 25, linker 26, linker 27, or linker 28. In some embodiments, H1 includes a polymer. In some embodiments, the polymer is polyethylene glycol (PEG). In some embodiments, H1 contains albumin. In some embodiments, H1 contains 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, ligand, or small molecule. In some embodiments, the polypeptide, ligand, or small molecule binds to a serum protein or fragment thereof, a circulating immunoglobulin or fragment thereof, or CD35 / CR1. In some embodiments, the serum protein comprises thyroxine-binding protein, transthyretin, α1-acid glycoprotein, transferrin, a transferrin receptor or its transferrin-binding moiety, 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 antibody or a 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 HC-CDR1, HC-CDR2, and HC-CDR3 as complementarity-determining regions (CDRs), and the HC-CDR1, HC-CDR2, and HC-CDR3 of the single-domain antibody comprise SEQ ID NO: 54 for HC-CDR1, SEQ ID NO: 55 for HC-CDR2, and SEQ ID NO: 56 for HC-CDR3. In some embodiments, the single-domain antibody comprises HC-CDR1, HC-CDR2, and HC-CDR3 as complementarity-determining regions (CDRs), and the HC-CDR1, HC-CDR2, and HC-CDR3 of the single-domain antibody comprise SEQ ID NO: 58 for HC-CDR1, SEQ ID NO: 59 for HC-CDR2, and SEQ ID NO: 60 for HC-CDR3. 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 non-natural modified amino acid, or a combination thereof. In some embodiments, the modified amino acid or non-natural modified amino acid comprises a post-translational modification. In some embodiments, H1 comprises a linking moiety (L3) that connects H1 to P1. In some embodiments, L3 is a peptide sequence having at least 5 to 50 or fewer amino acids. In some embodiments, L3 is a peptide sequence having at least 10 to 30 or fewer 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 (SEQ ID NO: 50), (GGGS) n (SEQ ID NO: 51), (GGGGS) n (SEQ ID NO: 52), and (GSSGGS) nThe formula is selected from the group consisting of (SEQ ID NO: 53), where n is at least an integer of 1. In some embodiments, L3 includes the amino acid sequence according to SEQ ID NO: 22. In some embodiments, the polypeptide or polypeptide complex includes an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 62-77. In some embodiments, the polypeptide or polypeptide complex has at least 95% sequence identity to SEQ ID NO: 72. The polypeptide or polypeptide complex includes an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 73. In some embodiments, the polypeptide or polypeptide complex includes an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 62 and SEQ ID NO: 63. In some embodiments, the polypeptide or polypeptide complex includes an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 64 and SEQ ID NO: 65. In some embodiments, the polypeptide or polypeptide complex includes an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 66 and SEQ ID NO: 67. In some embodiments, the polypeptide or polypeptide complex includes an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 68 and SEQ ID NO: 69. In some embodiments, the polypeptide or polypeptide complex includes an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 70 and SEQ ID NO: 71. In some embodiments, the polypeptide or polypeptide complex includes an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 72 and SEQ ID NO: 73. In some embodiments, the polypeptide or polypeptide complex includes an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 74 and SEQ ID NO: 75. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 76 and SEQ ID NO: 77.

[0004] In certain embodiments herein, a pharmaceutical composition is disclosed comprising (a) an isolated polypeptide or polypeptide complex disclosed herein and (b) a pharmaceutically acceptable excipient.

[0005] In certain embodiments herein, isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes described herein are disclosed.

[0006] In certain embodiments of this specification, Formula II: L 1a -P 1a -H 1a (Formula II) A polypeptide or polypeptide complex isolated by the formula, wherein L 1a If not cleaved, P is the first antigen-recognition molecule that binds to the effector cell antigen. 1a It includes a linkage portion cleaved by a tumor-specific protease, and the first antigen-recognition molecule is linked to a second antigen-recognition molecule that binds to PSMA, P 1a L 1a If it is not cleaved, it contains a peptide that binds to the first antigen recognition molecule, H 1a The disclosure includes polypeptides or polypeptide complexes containing half-life extension molecules. In some embodiments, P 1a L 1a If the cleavage is not performed, the binding of the first antigen-recognition molecule to the effector cell antigen is impaired. In some embodiments, the first antigen-recognition molecule comprises an antibody or antibody fragment. In some embodiments, the effector cell antigen is an anti-CD3 effector cell antigen. In some embodiments, P 1a It has less than 70% sequence homology to the effector cell antigen. In some embodiments, P 1a It contains a peptide sequence with a length of at least 10 amino acids. In some embodiments, P 1a It contains a peptide sequence having a length of at least 10 amino acids and no more than 20 amino acids. In some embodiments, P 1aIt contains a peptide sequence with a length of at least 16 amino acids. In some embodiments, P 1a It contains a peptide sequence with a length of 40 amino acids or less. In some embodiments, P 1a It contains at least two cysteine ​​amino acid residues. In some embodiments, P 1a This includes cyclic peptides or linear peptides. In some embodiments, P 1a It contains a cyclic peptide. In some embodiments, P 1a It contains linear peptides. In some embodiments, P 1a This includes an amino acid sequence selected from the group consisting of any one of sequence numbers 16-19. In some embodiments, H 1a It 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 It contains an Fc domain. In some embodiments, albumin is serum albumin. In some embodiments, albumin is human serum albumin. In some embodiments, H 1aThis comprises a polypeptide, ligand, or small molecule. In some embodiments, the polypeptide, ligand, or small molecule binds to a serum protein or fragment thereof, a circulating immunoglobulin or fragment thereof, or CD35 / CR1. In some embodiments, the serum protein includes thyroxine-binding protein, transthyretin, α1-acid glycoprotein, transferrin, a transferrin receptor or its transferrin-binding moiety, fibrinogen, or albumin. In some embodiments, the circulating immunoglobulin molecule includes 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 includes a single-domain antibody, a single-chain variable fragment, or Fab. In some embodiments, the antibody includes a single-domain antibody that binds to albumin. In some embodiments, the antibody is a human antibody or a humanized antibody. In some embodiments, the single-domain antibody is 645gH1gL1. In some embodiments, the single-domain antibody is 645dsgH5gL4. In some embodiments, the single-domain antibody is 23-13-A01-sc02. In some embodiments, the single-domain antibody is A10m3 or a fragment thereof. In some embodiments, the single-domain antibody is DOM7r-31. In some embodiments, the single-domain antibody is DOM7h-11-15. In some embodiments, the single-domain antibody is Alb-1, Alb-8, or Alb-23. In some embodiments, the single-domain antibody is 10E. In some embodiments, the single-domain antibody includes HC-CDR1, HC-CDR2, and HC-CDR3 as complementarity-determining regions (CDRs), where HC-CDR1, HC-CDR2, and HC-CDR3 include SEQ ID NO: 54 for HC-CDR1, SEQ ID NO: 55 for HC-CDR2, and SEQ ID NO: 56 for HC-CDR3.In some embodiments, the single-domain antibody comprises HC-CDR1, HC-CDR2, and HC-CDR3 as complementarity-determining regions (CDRs), where HC-CDR1, HC-CDR2, and HC-CDR3 of the single-domain antibody comprises SEQ ID NO: 58 for HC-CDR1, SEQ ID NO: 59 for HC-CDR2, and SEQ ID NO: 60 for HC-CDR3. In some embodiments, the single-domain antibody is SA21. In some embodiments, H. 1a P 1a H 1a The connecting part (L 1a ) includes. In some embodiments, L 1a This is a peptide sequence having at least 5 to 50 amino acids. In some embodiments, L 1a This is a peptide sequence having at least 10 to 30 amino acids. In some embodiments, L 1a This is a peptide sequence having at least 10 amino acids. In some embodiments, L 1a This is a peptide sequence having at least 18 amino acids. In some embodiments, L 1a This is a peptide sequence having at least 26 amino acids. In some embodiments, L 1a (G2S) n , (GS) n (GSGGS) n (Sequence ID 50), (GGGS) n (Sequence ID 51), (GGGGS) n (Sequence ID 52), and (GSSGGS) n The formula has an expression selected from the group consisting of (Sequence ID 53), where n is at least an integer of 1. In some embodiments, L 1a This includes the amino acid sequence according to SEQ ID NO: 23. In some embodiments herein, stereochemistry 1:

[0007] [ka] A polypeptide complex comprising a structural configuration such as , wherein the polypeptide complex comprises a single-chain variable fragment (scFv) comprising a light-chain variable domain and a heavy-chain variable domain, the scFv further comprising a peptide that impairs the binding of scFv to effector cell antigens, the peptide being linked to the heavy-chain variable domain of scFv by a linking portion which is a substrate of a tumor-specific protease, and the peptide further comprising a half-life-extending molecule, and Fab or Fab' which binds to prostate-specific membrane antigen (PSMA), the Fab or Fab' comprising a Fab light-chain polypeptide chain and a Fab heavy-chain polypeptide chain, the Fab heavy-chain polypeptide chain being linked to the C-terminus of the light-chain variable domain of scFv, is disclosed. In some embodiments herein, conformation 2:

[0008] [ka] A polypeptide complex comprising a structural configuration thereof, wherein the polypeptide complex comprises a single-chain variable fragment (scFv) comprising a light-chain variable domain and a heavy-chain variable domain, the scFv further comprising a peptide that impairs the binding of scFv to effector cell antigens, the peptide being ligated to the N-terminus of the heavy-chain variable domain of scFv by a ligation portion which is a substrate of a tumor-specific protease, and the peptide further comprising a half-life-extending molecule, and a prostate-specific membrane antigen (PSMA-binding Fab or Fab', the Fab or Fab' comprising a Fab light-chain polypeptide chain and a Fab heavy-chain polypeptide chain, the Fab light-chain polypeptide chain being ligated to the C-terminus of the light-chain variable domain of scFv, is disclosed.

[0009] Reference All publications, patents, and patent applications referenced herein are incorporated herein by reference to the same extent as each individual publication, patent, or patent application is specifically and individually referred to. [Brief explanation of the drawing]

[0010] Novel features of this disclosure are described, in particular, by the appended claims. The features and advantages of this disclosure will be better understood by referring to the following detailed description illustrating exemplary embodiments and accompanying drawings in which the principles of this disclosure are utilized.

[0011] [Figure 1A] This figure illustrates a polypeptide complex of the present disclosure in a normal orientation. [Figure 1B] This figure illustrates a polypeptide complex of the present disclosure in an inverted orientation. [Figure 2A] This figure illustrates titration data for PSMA binding in several polypeptide complexes of this disclosure. [Figure 2B] This figure illustrates titration data for PSMA binding in several polypeptide complexes of this disclosure. [Figure 2C] This figure illustrates titration data for PSMA binding in several polypeptide complexes of this disclosure. [Figure 3A] This figure illustrates titration data for CD3ε binding in several polypeptide complexes of this disclosure. [Figure 3B] This figure illustrates titration data for CD3ε binding in several polypeptide complexes of this disclosure. [Figure 3C] This figure illustrates titration data for CD3ε binding in several polypeptide complexes of this disclosure. [Figure 4] This figure illustrates cell viability data for 22Rv1 tumor cells treated with PC1 or PC2. [Figure 5A] This figure illustrates cell viability data for 22Rv1 tumor cells treated with PC1, PC5, or MTSP1 treated with PC5. [Figure 5B]This figure illustrates cell viability data for 22Rv1 tumor cells treated with PC2, PC4 (with or without MTSP1 treatment), or PC6 (with or without MTSP1 treatment). [Figure 6] This figure illustrates cell viability data for LNCaP tumor cells treated with PC1 or PC2. [Figure 7] This figure illustrates cell viability data for LNCaP tumor cells treated with PC2, PC4, or MTSP1 treated with PC4. [Figure 8A] This figure illustrates how polypeptide complexes mediated the death of 22Rv1 tumor cells in the presence of CD8+ T cells. [Figure 8B] This figure illustrates how polypeptide complexes mediated the death of 22Rv1 tumor cells in the presence of CD8+ T cells. [Figure 9A] This figure illustrates the pharmacokinetics of polypeptide (PSMA TCE) in cynomolgus monkeys after a single IV bolus injection. [Figure 9B] This figure illustrates the pharmacokinetics of polypeptides (PSMA TRACTrs) in cynomolgus monkeys after a single IV bolus injection. [Figure 10A] This figure illustrates cytokine release in cynomolgus monkeys after a single IV bolus of PSMA TCE. [Figure 10B] This figure illustrates cytokine release in cynomolgus monkeys after a single IV bolus of the PSMA polypeptide TRACTr complex. [Figure 10C] This figure illustrates cytokine release in cynomolgus monkeys after a single IV bolus of PSMA TRACTR. [Figure 11A] This figure illustrates serum liver enzymes in cynomolgus monkeys after a single IV bolus of PSMA TCE. [Figure 11B] This figure illustrates serum liver enzymes in cynomolgus monkeys after a single IV bolus of PSMA polypeptide TRACTr complex. [Figure 12A]This figure illustrates the anti-CD3 scFv binding of anti-CD3 scFv peptides A and B by alanine scanning peptides, as measured by ELISA. [Figure 12B] This figure illustrates the anti-CD3 scFv binding of anti-CD3 scFv peptides A and B by alanine scanning peptides as measured by ELISA. [Figure 12C] This figure illustrates the anti-CD3 scFv binding of anti-CD3 scFv peptides A and B by alanine scanning peptides as measured by ELISA. [Figure 12D] This figure illustrates the anti-CD3 scFv binding of anti-CD3 scFv peptides A and B by alanine scanning peptides as measured by ELISA. [Figure 12E] This figure illustrates the anti-CD3 scFv binding of anti-CD3 scFv peptides A and B by alanine scanning peptides as measured by ELISA. [Figure 12F] This figure illustrates the anti-CD3 scFv binding of anti-CD3 scFv peptides A and B by alanine scanning peptides as measured by ELISA. [Figure 13A] This figure illustrates the inhibition of anti-CD3 scFv binding to CD3 by alanine scanning peptides of anti-CD3 scFv peptide-A and peptide-B, as measured by ELISA. [Figure 13B] This figure illustrates the inhibition of anti-CD3 scFv binding to CD3 by alanine scanning peptides of anti-CD3 scFv peptide-A and peptide-B, as measured by ELISA. [Figure 13C] This figure illustrates the inhibition of anti-CD3 scFv binding to CD3 by alanine scanning peptides of anti-CD3 scFv peptide-A and peptide-B, as measured by ELISA. [Figure 13D] This figure illustrates the inhibition of anti-CD3 scFv binding to CD3 by alanine scanning peptides of anti-CD3 scFv peptide-A and peptide-B, as measured by ELISA. [Figure 13E] This figure illustrates the inhibition of anti-CD3 scFv binding to CD3 by alanine scanning peptides of anti-CD3 scFv peptide-A and peptide-B, as measured by ELISA. [Figure 13F] This figure illustrates the inhibition of anti-CD3 scFv binding to CD3 by alanine scanning peptides of anti-CD3 scFv peptide-A and peptide-B, as measured by ELISA. [Figure 14A] This figure illustrates anti-CD3 scFv binding using the sequence of the optimized anti-CD3 scFv peptide-B as measured by ELISA. [Figure 14B] This figure illustrates anti-CD3 scFv binding using the sequence of the optimized anti-CD3 scFv peptide-B as measured by ELISA. [Figure 15A] This figure illustrates the inhibition of anti-CD3 scFv binding to CD3 by the optimized anti-CD3 scFv peptide-B sequence as measured by ELISA. [Figure 15B] This figure illustrates the inhibition of anti-CD3 scFv binding to CD3 by the optimized anti-CD3 scFv peptide-B sequence as measured by ELISA. [Figure 16] This figure illustrates the core sequence motif of the optimized anti-CD3 scFv peptide-B sequence generated using WebLogo3.7.4. [Modes for carrying out the invention]

[0012] Multispecific antibodies combine the benefits of different binding specificities derived from two or more antibodies into a single composition. Multispecific antibodies for redirecting T cells to cancer have shown promise in both preclinical and clinical trials. This approach relies on the binding of one antigen-interacting moiety of an antibody to a tumor-associated antigen or marker, while another antigen-interacting moiety can bind to effector cell antigens on T cells, such as differentiation antigen group 3 (CD3), which can trigger cytotoxic activity.

[0013] One such tumor-associated antigen 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 in humans by the FOLH1 (folate hydrolase 1) gene. PSMA is a membrane-based zinc metalloenzyme. The majority of this enzyme resides in the extracellular fluid space. Human PSMA is highly expressed in the prostate, at over 100 times higher levels than in most other tissues. In some prostate cancers, PSMA is the second most upregulated gene product, at levels 8 to 12 times higher than in non-cancerous prostate cells.

[0014] T-cell engager (TCE) therapy offers several advantages, including the fact that it is not a cell therapy, and can therefore be presented as an off-the-shelf treatment to counter chimeric antigen receptor T-cell (CAR T-cell) therapy. While TCE therapy has shown promising antitumor activity against hematological malignancies, the development of TCEs for treating solid tumors faces challenges due to limitations of previous TCE technologies, namely (i) hyperactivation of the immune system leading to cytokine release syndrome (CRS), (ii) on-target healthy tissue toxicity, and (iii) poor tissue pharmacokinetics (PK) resulting in short half-lives. CRS arises from systemic activation of T cells and can lead to life-threatening increases in inflammatory cytokines such as interleukin-6 (IL-6). Severe acute CRS, resulting in dose-limiting toxicity and death, has been observed with the administration of T-cell engagers developed using other platforms to treat cancer patients in inadequate clinical trials. This toxicity limits the maximum blood concentration of T-cell engagers that can be safely administered. The efficacy of T-cell promoters is also limited by on-target toxicity to healthy tissue. T-cell promoters developed using platforms not designed for tumor-specific activation have been halted from clinical trials, resulting in targeted expression in healthy tissue. T-cell promoters are also limited by their short half-lives. Due to their short exposure half-lives, T-cell promoters are rapidly excreted from the body, quickly reaching sub-therapeutic levels after administration. Therefore, to overcome the challenges of short half-lives and maintain therapeutic drug levels in the body, T-cell promoters such as blinatumomab are typically administered at low doses via continuous infusion pumps over several weeks. Continuous dosing regimens present a significant burden on patients.

[0015] To overcome these challenges associated with the efficacy of T cell engagers, polypeptides or polypeptide complexes comprising binding domains that selectively bind to effector cell antigens and PSMA are described herein, wherein one or more binding domains are selectively activated in the tumor microenvironment, and the polypeptide or polypeptide complex contains a half-life extension molecule. Such modifications reduce the risk of CRS and on-target healthy tissue toxicity and improve pre-activation blood flow and serum half-life stability. The polypeptides or polypeptide complexes described herein are active at low levels of targeted expression and are readily manufactured.

[0016] In some embodiments, the polypeptides or polypeptide complexes described herein are used in methods of treating cancer. In some embodiments, the cancer has cells expressing PSMA. In some embodiments, the polypeptides or polypeptide complexes described herein are used in methods of treating prostate cancer. In some embodiments, the prostate cancer is metastatic castration-resistant prostate cancer (mCRPC). Prostate cancer is the second most common cancer in men worldwide, with more than 3 million men living with it in the United States alone. Early diagnosis and effective treatment mean that the majority of prostate cancer patients have a prognosis of approximately 98 percent average five-year survival rate. However, an estimated 6 percent of prostate cancer patients develop metastatic disease, which accounts for approximately 30 percent of the five-year survival rate. In 2020, an estimated 33,000 people died from prostate cancer in the United States.

[0017] In some examples, the polypeptides or polypeptide complexes described herein are used to treat solid tumor cancers. In some embodiments, the cancers are lung, breast (e.g., HER2+, ER / PR+, TNBC), cervix, ovary, colon, pancreas, or stomach cancers. Some embodiments describe a method for treating cancer, wherein the target requiring treatment of the cancer is given Formula I: A2-A1-L1-P1-H1 (Formula I) The process includes administering an isolated polypeptide or polypeptide complex, In this method, A1 comprises a first antigen-recognizing molecule that binds to an effector cell antigen, P1 comprises a peptide that binds to A1, L1 comprises a ligation portion that connects A1 to P1 and is a substrate for a tumor-specific protease, H1 comprises a half-life extension molecule, and A2 comprises another antigen-recognizing molecule that binds to PSMA.

[0018] In some embodiments of this specification, Formula I: A2-A1-L1-P1-H1 (Formula I) A peptide or peptide complex isolated by, An isolated peptide or peptide complex is disclosed, wherein A1 comprises a first antigen-recognizing molecule that binds to an effector cell antigen, P1 comprises a peptide that binds to A1, L1 comprises a ligation portion that connects A1 to P1 and is a substrate for a tumor-specific protease, H1 comprises a half-life extension molecule, and A2 comprises a second antigen-recognizing molecule that binds to prostate-specific membrane antigen (PSMA).

[0019] In some embodiments of this specification, Formula I: A2-A1-L1-P1-H1 (Formula I) A peptide or peptide complex isolated by, An isolated peptide or peptide complex is disclosed, wherein A1 is a first antigen-recognizing molecule that binds to effector cell antigens, P1 is a peptide that binds to A1, L1 is a ligation portion that connects A1 to P1 and is a substrate for tumor-specific proteases, H1 is a half-life extension molecule, and A2 is a second antigen-recognizing molecule that binds to prostate-specific membrane antigen (PSMA).

[0020] In some embodiments of this specification, Formula I: A2-A1-L1-P1-H1 (Formula I) An isolated peptide or peptide complex comprising, An isolated peptide or peptide complex is disclosed, wherein A1 comprises a first antigen-recognizing molecule that binds to an effector cell antigen, P1 comprises a peptide that binds to A1, L1 comprises a ligation portion that connects A1 to P1 and is a substrate for a tumor-specific protease, H1 comprises a half-life extension molecule, and A2 comprises a second antigen-recognizing molecule that binds to prostate-specific membrane antigen (PSMA).

[0021] In some embodiments of this specification, Formula I: A2-A1-L1-P1-H1 (Formula I) An isolated peptide or peptide complex comprising, An isolated peptide or peptide complex is disclosed, wherein A1 is a first antigen-recognizing molecule that binds to effector cell antigens, P1 is a peptide that binds to A1, L1 is a ligation portion that connects A1 to P1 and is a substrate for tumor-specific proteases, H1 is a half-life extension molecule, and A2 is a second antigen-recognizing molecule that binds to prostate-specific membrane antigen (PSMA).

[0022] In some embodiments of this specification, Formula I: A2-A1-L1-P1-H1 (Formula I) A peptide or peptide complex isolated by, An isolated peptide or peptide complex is disclosed, wherein A1 comprises a first antigen-recognizing molecule that binds to prostate-specific membrane antigen (PSMA), P1 comprises a peptide that binds to A1, L1 comprises a ligation portion that connects A1 to P1 and is a substrate of a tumor-specific protease, H1 comprises a half-life extension molecule, and A2 comprises a second antigen-recognizing molecule that binds to effector cell antigens.

[0023] In some embodiments of this specification, Formula I: A2-A1-L1-P1-H1 (Formula I) A peptide or peptide complex isolated by, An isolated peptide or peptide complex is disclosed, wherein A1 is a first antigen-recognizing molecule that binds to prostate-specific membrane antigen (PSMA), P1 is a peptide that binds to A1, L1 is a ligation portion that connects A1 to P1 and is a substrate for tumor-specific protease, H1 is a half-life extension molecule, and A2 is a second antigen-recognizing molecule that binds to effector cell antigen.

[0024] In some embodiments of this specification, Formula I: A2-A1-L1-P1-H1 (Formula I) An isolated peptide or peptide complex comprising, An isolated peptide or peptide complex is disclosed, wherein A1 comprises a first antigen-recognizing molecule that binds to prostate-specific membrane antigen (PSMA), P1 comprises a peptide that binds to A1, L1 comprises a ligation portion that connects A1 to P1 and is a substrate of a tumor-specific protease, H1 comprises a half-life extension molecule, and A2 comprises a second antigen-recognizing molecule that binds to effector cell antigens.

[0025] In some embodiments of this specification, Formula I: A2-A1-L1-P1-H1 (Formula I) An isolated peptide or peptide complex comprising, An isolated peptide or peptide complex is disclosed, wherein A1 is a first antigen-recognizing molecule that binds to prostate-specific membrane antigen (PSMA), P1 is a peptide that binds to A1, L1 is a ligation portion that connects A1 to P1 and is a substrate for tumor-specific protease, H1 is a half-life extension molecule, and A2 is a second antigen-recognizing molecule that binds to effector cell antigen.

[0026] In some embodiments herein, Formula Ia: P2-L2-A2-A1-L1-P1-H1 (Formula Ia) A peptide or peptide complex isolated by, In the formula, A2 further includes P2 and L2, P2 includes a peptide that binds to A2, and L2 includes a linking portion that connects A1 to P1 and is a substrate for a tumor-specific protease. An isolated peptide or peptide complex is disclosed.

[0027] In some embodiments herein, formula Ia: P2-L2-A2-A1-L1-P1-H1 (formula Ia) An isolated peptide or peptide complex according to In the formula, A2 further includes P2 and L2, P2 is a peptide that binds to A2, and L1 is a linking portion that connects A1 to P1 and is a substrate for a tumor-specific protease. An isolated peptide or peptide complex is disclosed.

[0028] In some embodiments herein, formula Ia: P2-L2-A2-A1-L1-P1-H1 (formula Ia) An isolated peptide or peptide complex comprising In the formula, A2 further includes P2 and L2, P2 includes a peptide that binds to A2, and L2 includes a linking portion that connects A1 to P1 and is a substrate for a tumor-specific protease. An isolated peptide or peptide complex is disclosed.

[0029] In some embodiments herein, formula Ia: P2-L2-A2-A1-L1-P1-H1 (formula Ia) An isolated peptide or peptide complex comprising In the formula, A2 further includes P2 and L2, P2 is a peptide that binds to A2, and L1 is a linking portion that connects A1 to P1 and is a substrate for a tumor-specific protease. An isolated peptide or peptide complex is disclosed.

[0030] In some embodiments herein, formula II: L 1a -P 1a -H1a (Formula II) A peptide or peptide complex isolated by, In the formula, L 1a If not cleaved, P is the first antigen-recognition molecule that binds to the effector cell antigen. 1a The first antigen-recognition molecule is connected to a second antigen-recognition molecule that binds to prostate-specific membrane antigen (PSMA), and the first antigen-recognition molecule is connected to a second antigen-recognition molecule that binds to prostate-specific membrane antigen (PSMA). 1a L 1a If it is not cleaved, it contains a peptide that binds to the first antigen recognition molecule, H 1a This discloses isolated peptides or peptide complexes containing half-life extension molecules.

[0031] In some embodiments herein, Formula II: L 1a -P 1a -H 1a (Formula II) An isolated peptide or peptide complex comprising, In the formula, L 1a If not cleaved, P is the first antigen-recognition molecule that binds to the effector cell antigen. 1a The first antigen-recognition molecule is connected to a second antigen-recognition molecule that binds to prostate-specific membrane antigen (PSMA), and the first antigen-recognition molecule is connected to a second antigen-recognition molecule that binds to prostate-specific membrane antigen (PSMA). 1a L 1a If it is not cleaved, it contains a peptide that binds to the first antigen recognition molecule, H 1a This discloses isolated peptides or peptide complexes containing half-life extension molecules.

[0032] In some embodiments herein, Formula II: L 1a -P 1a -H 1a (Formula II) A peptide or peptide complex isolated by, In the formula, L 1a If not cleaved, P is the first antigen-recognition molecule that binds to the effector cell antigen.1a The linkage is cleaved by a tumor-specific protease, and the first antigen-recognizing molecule is connected to a second antigen-recognizing molecule that binds to prostate-specific membrane antigen (PSMA), P 1a L 1a If it is not cleaved, it is a peptide that binds to the first antigen recognition molecule, H 1a Disclosed are isolated peptides or peptide complexes that are half-life extension molecules.

[0033] In some embodiments herein, Formula II: L 1a -P 1a -H 1a (Formula II) An isolated peptide or peptide complex comprising, In the formula, L 1a If not cleaved, P is the first antigen-recognition molecule that binds to the effector cell antigen. 1a The linkage is cleaved by a tumor-specific protease, and the first antigen-recognizing molecule is connected to a second antigen-recognizing molecule that binds to prostate-specific membrane antigen (PSMA), P 1a L 1a If it is not cleaved, it is a peptide that binds to the first antigen recognition molecule, H 1a Disclosed are isolated peptides or peptide complexes that are half-life extension molecules.

[0034] First antigen-recognition molecule (A1) In some embodiments herein, isolated polypeptides or polypeptide complexes are disclosed, wherein a first antigen-recognizing molecule binds to an effector cell antigen and a second antigen-recognizing molecule binds to PSMA. In some embodiments, the effector cell antigen comprises CD3. In some embodiments, A1 comprises a first antigen-recognizing molecule that binds to the effector cell antigen.

[0035] In some embodiments, A1 comprises an antibody or antibody fragment. In some embodiments, A1 comprises a human or humanized antibody or antibody fragment. In some embodiments, L1 is bound to the N-terminus of the antibody or antibody fragment. In some embodiments, L1 is bound to the N-terminus of the antibody or antibody fragment, and A2 is bound to the other N-terminus of the antibody or antibody fragment. In some embodiments, A2 is bound to the C-terminus of the antibody or antibody fragment. In some embodiments, L1 is bound to the C-terminus of the antibody or antibody fragment. In some embodiments, A2 is bound to the N-terminus of the antibody or antibody fragment. In some embodiments, the antibody or antibody fragment comprises a single-chain variable fragment, a single-domain antibody, or a Fab fragment. In some embodiments, A1 is a single-chain variable fragment (scFv). In some embodiments, the scFv comprises an scFv heavy-chain polypeptide and an scFv light-chain polypeptide. In some embodiments, A1 is a single-domain antibody. In some embodiments, A1 comprises a variable light chain and a variable heavy chain, each specifically capable of binding to human CD3. In some embodiments, the effector cell antigen comprises CD3. In some embodiments, A1 comprises an anti-CD3e single-chain variable fragment. In some embodiments, A1 has a K2+ ratio of 1 μM or less relative to CD3 on CD3-expressing cells. DThe A1 includes anti-CD3e single-chain variable fragments having bindings. In some embodiments, A1 includes muromonab-CD3 (OKT3), otelixizumab (TRX4), teprizumab (MGA031), bicilizumab (Nuvion), SP34, X35, VIT3, BMA030 (BW264 / 56), CLB-T3 / 3, CRIS7, YTH12.5, F111-409, CLB-T3.4.2, TR-66, WT32, SP It includes a complementarity determination region (CDR) selected from the group consisting of v-T3b, 11D8, XIII-141, XIII-46, XIII-87, 12F6, T3 / RW2-8C8, T3 / RW2-4B6, OKT3D, M-T301, SMC2, F101.01, UCHT-1, WT-31, 15865, 15865v12, 15865v16, and 15865v19.

[0036] In some embodiments, A1 includes a first antigen-recognizing molecule that binds to PSMA. In some embodiments, A1 includes a variable light chain and a variable heavy chain, each capable of specifically binding to human PSMA.

[0037] In some embodiments, the scFv bound to CD3 includes an scFv light chain variable domain and an scFv heavy chain variable domain. In some embodiments, the scFv heavy chain variable domain includes at least one, two, or three complementarity-determining regions (CDRs) disclosed in Table 1, or substantially identical sequences thereto (e.g., sequences having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity). In some embodiments, the scFv light chain variable domain includes at least one, two, or three complementarity-determining regions (CDRs) disclosed in Table 1, or substantially identical sequences thereto (e.g., sequences having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity).

[0038] In some embodiments, the scFv heavy chain variable domain includes at least one, two, or three complementarity-determining regions (CDRs) disclosed in Table 1, or substantially identical sequences thereto (e.g., sequences having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity), and the scFv light chain variable domain includes at least one, two, or three complementarity-determining regions (CDRs) disclosed in Table 1, or substantially identical sequences thereto (e.g., sequences having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity).

[0039] [Table 1]

[0040] In some embodiments, the scFv heavy chain variable domain includes HC-CDR1, HC-CDR2, and HC-CDR3 as complementarity-determining regions (CDRs), where HC-CDR1 includes SEQ ID NO: 1, HC-CDR2 includes SEQ ID NO: 2, and HC-CDR3 includes SEQ ID NO: 3, and each CDR contains 0 to 2 amino acid modifications in at least one of HC-CDR1, HC-CDR2, or HC-CDR3. In some embodiments, the scFv light chain variable domain includes LC-CDR1, LC-CDR2, and LC-CDR3 as complementarity-determining regions (CDRs), where LC-CDR1 includes SEQ ID NO: 4, LC-CDR2 includes SEQ ID NO: 5, and LC-CDR3 includes SEQ ID NO: 6, and each CDR contains 0 to 2 amino acid modifications in at least one of LC-CDR1, LC-CDR2, or LC-CDR3.

[0041] In some embodiments, when L1 is cleaved by a tumor-specific protease, the polypeptide or polypeptide complex of Formula I binds to an effector cell. In some embodiments, when L1 is cleaved by a tumor-specific protease and A1 binds to an effector cell, the polypeptide or polypeptide complex of Formula I binds to an effector cell. In some embodiments, the effector cell is a T cell. In some embodiments, A1 binds to a polypeptide that is part of the TCR-CD3 complex on the effector cell. In some embodiments, the polypeptide that is part of the TCR-CD3 complex is human CD3ε. In some embodiments, the effector cell antigen comprises CD3, the scFV comprises HC-CDR1, HC-CDR2, and HC-CDR3 as complementarity determining regions (CDRs), the HC-CDR1, HC-CDR2, and HC-CDR3 of the scFV comprise SEQ ID NO: 1 for HC-CDR1, SEQ ID NO: 2 for HC-CDR2, and SEQ ID NO: 3 for HC-CDR3, the scFV comprises LC-CDR1, LC-CDR2, and LC-CDR3 as CDRs, and the LC-CDR1, LC-CDR2, and LC-CDR3 of the scFV comprise SEQ ID NO: 4 for LC-CDR1, SEQ ID NO: 5 for LC-CDR2, and SEQ ID NO: 6 for LC-CDR3. In some embodiments, the effector cell antigen comprises CD3, and the scFv comprises the amino acid sequence according to SEQ ID NO: 7.

[0042] In some embodiments, the effector cell antigen comprises CD3, and A1 comprises HC-CDR1, HC-CDR2, and HC-CDR3 as complementarity-determining regions (CDRs), where HC-CDR1, HC-CDR2, and HC-CDR3 of A1 comprises SEQ ID NO: 1 for HC-CDR1, SEQ ID NO: 2 for HC-CDR2, and SEQ ID NO: 3 for HC-CDR3. A1 comprises LC-CDR1, LC-CDR2, and LC-CDR3 as CDRs, where LC-CDR1, LC-CDR2, and LC-CDR3 of A1 comprises SEQ ID NO: 4 for LC-CDR1, SEQ ID NO: 5 for LC-CDR2, and SEQ ID NO: 6 for LC-CDR3. In some embodiments, the effector cell antigen comprises CD3, and A1 comprises the amino acid sequence according to SEQ ID NO: 7.

[0043] In some embodiments, A1 includes the amino acid sequence according to SEQ ID NO: 7. In some embodiments, A1 includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 7. In some embodiments, A1 includes an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 7. In some embodiments, A1 includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 7. In some embodiments, A1 includes an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 7. In some embodiments, A1 includes an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 7.

[0044] In some embodiments, the polypeptide or polypeptide complex exhibits a weaker binding affinity to tumor cell antigens compared to the binding affinity of isolated polypeptides or polypeptide complexes that lack both P1 and L1. In some embodiments, the polypeptide or polypeptide complex exhibits a weaker binding affinity to tumor cell antigens, at least 5 times higher than the binding affinity of polypeptides or polypeptide complexes in the form of polypeptides or polypeptide complexes that lack both P1 and L1. In some embodiments, the polypeptide or polypeptide complex exhibits a weaker binding affinity to tumor cell antigens, at least 8 times higher than the binding affinity of polypeptides or polypeptide complexes in the form of polypeptides or polypeptide complexes that lack both P1 and L1. In some embodiments, the polypeptide or polypeptide complex exhibits a weaker binding affinity to tumor cell antigens, at least 10 times higher than the binding affinity of polypeptides or polypeptide complexes in the form of polypeptides or polypeptide complexes that lack both P1 and L1. In some embodiments, the polypeptide or polypeptide complex exhibits a weaker binding affinity to tumor cell antigens, at least 15 times higher than the binding affinity of polypeptides or polypeptide complexes in the form of polypeptides or polypeptide complexes that lack both P1 and L1. In some embodiments, the polypeptide or polypeptide complex has a weak binding affinity to tumor cell antigens, at least 20 times higher than the binding affinity to tumor cell antigens of polypeptides or polypeptide complexes in the form of polypeptides or polypeptide complexes that do not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has a weak binding affinity to tumor cell antigens, at least 25 times higher than the binding affinity to tumor cell antigens of polypeptides or polypeptide complexes in the form of polypeptides or polypeptide complexes that do not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has a weak binding affinity to tumor cell antigens, at least 30 times higher than the binding affinity to tumor cell antigens of polypeptides or polypeptide complexes in the form of polypeptides or polypeptide complexes that do not have P1 or L1.In some embodiments, the polypeptide or polypeptide complex has a weak binding affinity to tumor cell antigens, and its binding affinity to tumor cell antigens is at least 35 times higher than that of polypeptides or polypeptide complexes in the form of polypeptides or polypeptide complexes that do not have either P1 or L1. In some embodiments, the polypeptide or polypeptide complex has a weak binding affinity to tumor cell antigens, and its binding affinity to tumor cell antigens is at least 40 times higher than that of polypeptides or polypeptide complexes in the form of polypeptides or polypeptide complexes that do not have either P1 or L1. In some embodiments, the polypeptide or polypeptide complex has a weak binding affinity to tumor cell antigens, and its binding affinity to tumor cell antigens is at least 45 times higher than that of polypeptides or polypeptide complexes in the form of polypeptides or polypeptide complexes that do not have either P1 or L1. In some embodiments, the polypeptide or polypeptide complex has a weak binding affinity to tumor cell antigens, and its binding affinity to tumor cell antigens is at least 50 times higher than that of polypeptides or polypeptide complexes in the form of polypeptides or polypeptide complexes that do not have either P1 or L1. In some embodiments, the polypeptide or polypeptide complex has a weak binding affinity to tumor cell antigens, at least 60 times higher than the binding affinity to tumor cell antigens of polypeptides or polypeptide complexes in the form of polypeptides or polypeptide complexes that do not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has a weak binding affinity to tumor cell antigens, at least 65 times higher than the binding affinity to tumor cell antigens of polypeptides or polypeptide complexes in the form of polypeptides or polypeptide complexes that do not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has a weak binding affinity to tumor cell antigens, at least 70 times higher than the binding affinity to tumor cell antigens of polypeptides or polypeptide complexes in the form of polypeptides or polypeptide complexes that do not have P1 or L1.In some embodiments, the polypeptide or polypeptide complex has a weak binding affinity to tumor cell antigens, and its binding affinity to tumor cell antigens is at least 75 times higher than that of polypeptides or polypeptide complexes in the form of polypeptides or polypeptide complexes that do not have either P1 or L1. In some embodiments, the polypeptide or polypeptide complex has a weak binding affinity to tumor cell antigens, and its binding affinity to tumor cell antigens is at least 80 times higher than that of polypeptides or polypeptide complexes in the form of polypeptides or polypeptide complexes that do not have either P1 or L1. In some embodiments, the polypeptide or polypeptide complex has a weak binding affinity to tumor cell antigens, and its binding affinity to tumor cell antigens is at least 85 times higher than that of polypeptides or polypeptide complexes in the form of polypeptides or polypeptide complexes that do not have either P1 or L1. In some embodiments, the polypeptide or polypeptide complex has a weak binding affinity to tumor cell antigens, and its binding affinity to tumor cell antigens is at least 90 times higher than that of polypeptides or polypeptide complexes in the form of polypeptides or polypeptide complexes that do not have either P1 or L1. In some embodiments, the polypeptide or polypeptide complex has a weak binding affinity to tumor cell antigens, at least 100 times higher than the binding affinity to tumor cell antigens of polypeptides or polypeptide complexes in the form of polypeptides or polypeptide complexes that do not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has a weak binding affinity to tumor cell antigens, at least 120 times higher than the binding affinity to tumor cell antigens of polypeptides or polypeptide complexes in the form of polypeptides or polypeptide complexes that do not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has a weak binding affinity to tumor cell antigens, at least 1000 times higher than the binding affinity to tumor cell antigens of polypeptides or polypeptide complexes in the form of polypeptides or polypeptide complexes that do not have P1 or L1.

[0045] In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity to tumor cell antigens compared to the binding affinity of the polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease. In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity to tumor cell antigens, at least 5 times higher than the binding affinity of the polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease. In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity to tumor cell antigens, at least 8 times higher than the binding affinity of the polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease. In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity to tumor cell antigens, at least 10 times higher than the binding affinity of the polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease. In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity to tumor cell antigens, at least 15 times higher than the binding affinity of the polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease. In some embodiments, the polypeptide or polypeptide complex has a weak binding affinity to tumor cell antigens, at least 20 times higher than the binding affinity to tumor cell antigens of polypeptides or polypeptide complexes where L1 is cleaved by a tumor-specific protease. In some embodiments, the polypeptide or polypeptide complex has a weak binding affinity to tumor cell antigens, at least 25 times higher than the binding affinity to tumor cell antigens of polypeptides or polypeptide complexes where L1 is cleaved by a tumor-specific protease. In some embodiments, the polypeptide or polypeptide complex has a weak binding affinity to tumor cell antigens, at least 30 times higher than the binding affinity to tumor cell antigens of polypeptides or polypeptide complexes where L1 is cleaved by a tumor-specific protease.In some embodiments, the polypeptide or polypeptide complex has a weak binding affinity to tumor cell antigens, at least 35 times higher than the binding affinity to tumor cell antigens of polypeptides or polypeptide complexes where L1 is cleaved by a tumor-specific protease. In some embodiments, the polypeptide or polypeptide complex has a weak binding affinity to tumor cell antigens, at least 40 times higher than the binding affinity to tumor cell antigens of polypeptides or polypeptide complexes where L1 is cleaved by a tumor-specific protease. In some embodiments, the polypeptide or polypeptide complex has a weak binding affinity to tumor cell antigens, at least 45 times higher than the binding affinity to tumor cell antigens of polypeptides or polypeptide complexes where L1 is cleaved by a tumor-specific protease. In some embodiments, the polypeptide or polypeptide complex has a weak binding affinity to tumor cell antigens, at least 50 times higher than the binding affinity to tumor cell antigens of polypeptides or polypeptide complexes where L1 is cleaved by a tumor-specific protease. In some embodiments, the polypeptide or polypeptide complex has a weak binding affinity to tumor cell antigens, at least 55 times higher than the binding affinity to tumor cell antigens of polypeptides or polypeptide complexes where L1 is cleaved by a tumor-specific protease. In some embodiments, the polypeptide or polypeptide complex has a weak binding affinity to tumor cell antigens, at least 60 times higher than the binding affinity to tumor cell antigens of polypeptides or polypeptide complexes where L1 is cleaved by a tumor-specific protease. In some embodiments, the polypeptide or polypeptide complex has a weak binding affinity to tumor cell antigens, at least 65 times higher than the binding affinity to tumor cell antigens of polypeptides or polypeptide complexes where L1 is cleaved by a tumor-specific protease. In some embodiments, the polypeptide or polypeptide complex has a weak binding affinity to tumor cell antigens, at least 70 times higher than the binding affinity to tumor cell antigens of polypeptides or polypeptide complexes where L1 is cleaved by a tumor-specific protease.In some embodiments, the polypeptide or polypeptide complex has a weak binding affinity to tumor cell antigens, at least 75 times higher than the binding affinity to tumor cell antigens of polypeptides or polypeptide complexes where L1 is cleaved by a tumor-specific protease. In some embodiments, the polypeptide or polypeptide complex has a weak binding affinity to tumor cell antigens, at least 80 times higher than the binding affinity to tumor cell antigens of polypeptides or polypeptide complexes where L1 is cleaved by a tumor-specific protease. In some embodiments, the polypeptide or polypeptide complex has a weak binding affinity to tumor cell antigens, at least 85 times higher than the binding affinity to tumor cell antigens of polypeptides or polypeptide complexes where L1 is cleaved by a tumor-specific protease. In some embodiments, the polypeptide or polypeptide complex has a weak binding affinity to tumor cell antigens, at least 90 times higher than the binding affinity to tumor cell antigens of polypeptides or polypeptide complexes where L1 is cleaved by a tumor-specific protease. In some embodiments, the polypeptide or polypeptide complex has a weak binding affinity to tumor cell antigens, at least 95 times higher than the binding affinity to tumor cell antigens of polypeptides or polypeptide complexes where L1 is cleaved by a tumor-specific protease. In some embodiments, the polypeptide or polypeptide complex has a weak binding affinity to tumor cell antigens, at least 100 times higher than the binding affinity to tumor cell antigens of polypeptides or polypeptide complexes in which L1 is cleaved by tumor-specific proteases. In some embodiments, the polypeptide or polypeptide complex has a weak binding affinity to tumor cell antigens, at least 120 times higher than the binding affinity to tumor cell antigens of polypeptides or polypeptide complexes in which L1 is cleaved by tumor-specific proteases.In some embodiments, the polypeptide or polypeptide complex has a binding affinity for tumor cell antigens that is at least 1000-fold higher compared to the binding affinity of a polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease and has a weak binding affinity for tumor cell antigens.

[0046] In some embodiments, the polypeptide or polypeptide complex has an EC in an IFNγ release T cell activation assay of an isolated polypeptide or polypeptide complex that has neither P1 nor L1 50 compared to the EC in an IFNγ release T cell activation assay 50 which is increased. In some embodiments, the polypeptide or polypeptide complex has an EC in an IFNγ release T cell activation assay 50 which is increased and is at least 10-fold higher compared to the EC in an IFNγ release T cell activation assay in the form of a polypeptide or polypeptide complex that has neither P1 nor L1 50 In some embodiments, the polypeptide or polypeptide complex has an EC in an IFNγ release T cell activation assay 50 which is increased and is at least 20-fold higher compared to the EC in an IFNγ release T cell activation assay in the form of a polypeptide or polypeptide complex that has neither P1 nor L1 50 In some embodiments, the polypeptide or polypeptide complex has an EC in an IFNγ release T cell activation assay 50 which is increased and is at least 30-fold higher compared to the EC in an IFNγ release T cell activation assay in the form of a polypeptide or polypeptide complex that has neither P1 nor L1 50 In some embodiments, the polypeptide or polypeptide complex has an EC in an IFNγ release T cell activation assay 50 which is increased and is at least 30-fold higher compared to the EC in an IFNγ release T cell activation assay in the form of a polypeptide or polypeptide complex that has neither P1 nor L1 50This is at least 40 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex is used in EC in IFNγ-releasing T cell activation assays. 50 The EC in IFNγ-releasing T cell activation assays is elevated, and polypeptides or polypeptide complexes that do not possess either P1 or L1 are present. 50 It is at least 50 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex is used in EC in IFNγ-releasing T cell activation assays. 50 The EC in IFNγ-releasing T cell activation assays is elevated, and polypeptides or polypeptide complexes that do not possess either P1 or L1 are present. 50 This is at least 60 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex is used in EC in IFNγ-releasing T cell activation assays. 50 The EC in IFNγ-releasing T cell activation assays is elevated, and polypeptides or polypeptide complexes that do not possess either P1 or L1 are present. 50 This is at least 70 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex is used in EC in IFNγ-releasing T cell activation assays. 50 The EC in IFNγ-releasing T cell activation assays is elevated, and polypeptides or polypeptide complexes that do not possess either P1 or L1 are present. 50 This is at least 80 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex is used in EC in IFNγ-releasing T cell activation assays. 50 The EC in IFNγ-releasing T cell activation assays is elevated, and polypeptides or polypeptide complexes that do not possess either P1 or L1 are present. 50 This is at least 90 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex is used in EC in IFNγ-releasing T cell activation assays. 50 The EC in IFNγ-releasing T cell activation assays is elevated, and polypeptides or polypeptide complexes that do not possess either P1 or L1 are present. 50It is at least 100 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex is used in IFNγ-releasing T cell activation assays. 50 The EC in IFNγ-releasing T cell activation assays is elevated, and polypeptides or polypeptide complexes that do not possess either P1 or L1 are present. 50 It is at least 1000 times higher compared to that.

[0047] In some embodiments, the polypeptide or polypeptide complex is cleaved at L1 by a tumor-specific protease in an IFNγ-releasing T cell activation assay. 50 In comparison with EC in IFNγ-releasing T cell activation assays 50 The EC is increasing. In some embodiments, polypeptides or polypeptide complexes are used in IFNγ-releasing T cell activation assays. 50 Elevated EC in IFNγ-releasing T cell activation assays of polypeptides or polypeptide complexes cleaved by tumor-specific proteases in L1. 50 It is at least 10 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex is used in IFNγ-releasing T cell activation assays. 50 Elevated EC in IFNγ-releasing T cell activation assays of polypeptides or polypeptide complexes cleaved by tumor-specific proteases in L1. 50 It is at least 20 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex is used in EC in IFNγ-releasing T cell activation assays. 50 Elevated EC in IFNγ-releasing T cell activation assays of polypeptides or polypeptide complexes cleaved by tumor-specific proteases in L1. 50 It is at least 30 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex is used in EC in IFNγ-releasing T cell activation assays. 50Elevated EC in IFNγ-releasing T cell activation assays of polypeptides or polypeptide complexes cleaved by tumor-specific proteases in L1. 50 This is at least 40 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex is used in EC in IFNγ-releasing T cell activation assays. 50 Elevated EC in IFNγ-releasing T cell activation assays of polypeptides or polypeptide complexes cleaved by tumor-specific proteases in L1. 50 It is at least 50 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex is used in EC in IFNγ-releasing T cell activation assays. 50 Elevated EC in IFNγ-releasing T cell activation assays of polypeptides or polypeptide complexes cleaved by tumor-specific proteases in L1. 50 This is at least 60 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex is used in EC in IFNγ-releasing T cell activation assays. 50 Elevated EC in IFNγ-releasing T cell activation assays of polypeptides or polypeptide complexes cleaved by tumor-specific proteases in L1. 50 This is at least 70 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex is used in EC in IFNγ-releasing T cell activation assays. 50 Elevated EC in IFNγ-releasing T cell activation assays of polypeptides or polypeptide complexes cleaved by tumor-specific proteases in L1. 50 This is at least 80 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex is used in EC in IFNγ-releasing T cell activation assays. 50 Elevated EC in IFNγ-releasing T cell activation assays of polypeptides or polypeptide complexes cleaved by tumor-specific proteases in L1. 50 This is at least 90 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex is used in EC in IFNγ-releasing T cell activation assays.50 Elevated EC in IFNγ-releasing T cell activation assays of polypeptides or polypeptide complexes cleaved by tumor-specific proteases in L1. 50 It is at least 100 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex is used in IFNγ-releasing T cell activation assays. 50 Elevated EC in IFNγ-releasing T cell activation assays of polypeptides or polypeptide complexes cleaved by tumor-specific proteases in L1. 50 It is at least 1000 times higher compared to that.

[0048] In some embodiments, the polypeptide or polypeptide complex that does not have P1 or L1 is used in the EC of isolated polypeptides or polypeptide complexes in T cell lysis assays. 50 In comparison, EC in IFNγ-releasing T cell lysis assays 50 The EC is increasing. In some embodiments, polypeptides or polypeptide complexes are used in T cell lysis assays. 50 Elevated EC in T cell lysis assays for polypeptides or polypeptide complexes that do not possess either P1 or L1. 50 It is at least 10 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex is used in EC2 in T cell lysis assays. 50 Elevated EC in T cell lysis assays for polypeptides or polypeptide complexes that do not possess either P1 or L1. 50 It is at least 20 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex is used in EC2 in T cell lysis assays. 50 The EC2 is elevated in T cell lysis assays of polypeptides or polypeptide complexes that do not possess either P1 or L1. 50 It is at least 30 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex is used in EC2 in T cell lysis assays. 50The EC2 is elevated in T cell lysis assays of polypeptides or polypeptide complexes that do not possess either P1 or L1. 50 It is at least 40 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex is used in EC2 in T cell lysis assays. 50 The EC2 is elevated in T cell lysis assays of polypeptides or polypeptide complexes that do not possess either P1 or L1. 50 It is at least 50 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex is used in EC24 lysis assays. 50 The EC2 is elevated in T cell lysis assays of polypeptides or polypeptide complexes that do not possess either P1 or L1. 50 This is at least 60 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex is used in EC2 in T cell lysis assays. 50 The EC2 is elevated in T cell lysis assays of polypeptides or polypeptide complexes that do not possess either P1 or L1. 50 This is at least 70 times higher compared to [another factor]. In some embodiments, polypeptides or polypeptide complexes are used in EC24 lysis assays. 50 The EC2 is elevated in T cell lysis assays of polypeptides or polypeptide complexes that do not possess either P1 or L1. 50 This is at least 80 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex is used in EC2 in T cell lysis assays. 50 The EC2 is elevated in T cell lysis assays of polypeptides or polypeptide complexes that do not possess either P1 or L1. 50 This is at least 90 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex is used in EC2 in T cell lysis assays. 50 The EC2 is elevated in T cell lysis assays of polypeptides or polypeptide complexes that do not possess either P1 or L1. 50It is at least 100 times higher compared to [another factor]. In some embodiments, polypeptides or polypeptide complexes are used in EC2 in T cell lysis assays. 50 The EC2 is elevated in T cell lysis assays of polypeptides or polypeptide complexes that do not possess either P1 or L1. 50 It is at least 1000 times higher compared to that.

[0049] In some embodiments, the polypeptide or polypeptide complex is cleaved by a tumor-specific protease in an EC T cell lysis assay. 50 In comparison with EC in T cell lysis assays 50 The EC is increasing. In some embodiments, polypeptides or polypeptide complexes are used in T cell lysis assays. 50 Elevated EC in T cell lysis assays of polypeptides or polypeptide complexes cleaved by tumor-specific proteases in L1. 50 It is at least 10 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex is used in EC2 in T cell lysis assays. 50 Elevated EC in T cell lysis assays of polypeptides or polypeptide complexes cleaved by tumor-specific proteases in L1. 50 It is at least 20 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex is used in EC2 in T cell lysis assays. 50 Elevated EC in T cell lysis assays of polypeptides or polypeptide complexes cleaved by tumor-specific proteases in L1. 50 It is at least 30 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex is used in EC2 in T cell lysis assays. 50 Elevated EC in T cell lysis assays of polypeptides or polypeptide complexes cleaved by tumor-specific proteases in L1. 50It is at least 40 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex is used in EC2 in T cell lysis assays. 50 Elevated EC in T cell lysis assays of polypeptides or polypeptide complexes cleaved by tumor-specific proteases in L1. 50 It is at least 50 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex is used in EC24 lysis assays. 50 Elevated EC in T cell lysis assays of polypeptides or polypeptide complexes cleaved by tumor-specific proteases in L1. 50 This is at least 60 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex is used in EC2 in T cell lysis assays. 50 Elevated EC in T cell lysis assays of polypeptides or polypeptide complexes cleaved by tumor-specific proteases in L1. 50 This is at least 70 times higher compared to [another factor]. In some embodiments, polypeptides or polypeptide complexes are used in EC24 lysis assays. 50 Elevated EC in T cell lysis assays of polypeptides or polypeptide complexes cleaved by tumor-specific proteases in L1. 50 This is at least 80 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex is used in EC2 in T cell lysis assays. 50 Elevated EC in T cell lysis assays of polypeptides or polypeptide complexes cleaved by tumor-specific proteases in L1. 50 This is at least 90 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex is used in EC2 in T cell lysis assays. 50 Elevated EC in T cell lysis assays of polypeptides or polypeptide complexes cleaved by tumor-specific proteases in L1. 50 It is at least 100 times higher compared to [another factor]. In some embodiments, polypeptides or polypeptide complexes are used in EC2 in T cell lysis assays.50 Elevated EC in T cell lysis assays of polypeptides or polypeptide complexes cleaved by tumor-specific proteases in L1. 50 It is at least 1000 times higher compared to that.

[0050] In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits weaker binding affinity to tumor cell antigens compared to the binding affinity of isolated polypeptides or polypeptide complexes of Formula Ia that do not have P1, L1, P2, or L2. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits weaker binding affinity to tumor cell antigens, at least 10 times higher than the binding affinity of polypeptides or polypeptide complexes of Formula Ia that do not have P1, L1, P2, or L2. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits weaker binding affinity to tumor cell antigens, at least 50 times higher than the binding affinity of polypeptides or polypeptide complexes of Formula Ia that do not have P1, L1, P2, or L2. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weak binding affinity to tumor cell antigens, at least 75 times higher than the binding affinity to tumor cell antigens in the form of a polypeptide or polypeptide complex of Formula Ia that does not have P1, L1, P2, or L2. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weak binding affinity to tumor cell antigens, at least 100 times higher than the binding affinity to tumor cell antigens in the form of a polypeptide or polypeptide complex of Formula Ia that does not have P1, L1, P2, or L2. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weak binding affinity to tumor cell antigens, at least 120 times higher than the binding affinity to tumor cell antigens in the form of a polypeptide or polypeptide complex of Formula Ia that does not have P1, L1, P2, or L2.In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weak binding affinity to tumor cell antigens, at least 200 times higher than the binding affinity to tumor cell antigens in the form of a polypeptide or polypeptide complex of Formula Ia that does not have P1, L1, P2, or L2. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weak binding affinity to tumor cell antigens, at least 300 times higher than the binding affinity to tumor cell antigens in the form of a polypeptide or polypeptide complex of Formula Ia that does not have P1, L1, P2, or L2. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weak binding affinity to tumor cell antigens, at least 400 times higher than the binding affinity to tumor cell antigens in the form of a polypeptide or polypeptide complex of Formula Ia that does not have P1, L1, P2, or L2. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits a weak binding affinity to tumor cell antigens, at least 500 times higher than the binding affinity to tumor cell antigens of the polypeptide or polypeptide complex of Formula Ia in the form of a polypeptide or polypeptide complex that does not have P1, L1, P2, or L2. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits a weak binding affinity to tumor cell antigens, at least 600 times higher than the binding affinity to tumor cell antigens of the polypeptide or polypeptide complex of Formula Ia in the form of a polypeptide or polypeptide complex that does not have P1, L1, P2, or L2. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits a weak binding affinity to tumor cell antigens, which is at least 700 times higher than the binding affinity to tumor cell antigens of the polypeptide or polypeptide complex of Formula Ia in the form of a polypeptide or polypeptide complex that does not have P1, L1, P2, or L2.In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits a weak binding affinity to tumor cell antigens, at least 800 times higher than the binding affinity to tumor cell antigens of the polypeptide or polypeptide complex of Formula Ia in the form of a polypeptide or polypeptide complex that does not have P1, L1, P2, or L2. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits a weak binding affinity to tumor cell antigens, at least 900 times higher than the binding affinity to tumor cell antigens of the polypeptide or polypeptide complex of Formula Ia in the form of a polypeptide or polypeptide complex that does not have P1, L1, P2, or L2. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weak binding affinity to tumor cell antigens, at least 1000 times higher than the binding affinity to tumor cell antigens of the polypeptide or polypeptide complex of Formula Ia in the form of not having P1, L1, P2, or L2. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weak binding affinity to tumor cell antigens, at least 10000 times higher than the binding affinity to tumor cell antigens of the polypeptide or polypeptide complex of Formula Ia in the form of not having P1, L1, P2, or L2.

[0051] In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity to tumor cell antigens compared to the binding affinity to tumor cell antigens of the polypeptide or polypeptide complex of Formula Ia in which L1 and L2 are cleaved by a tumor-specific protease. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity to tumor cell antigens, at least 10 times higher than the binding affinity to tumor cell antigens of the polypeptide or polypeptide complex of Formula Ia in which L1 and L2 are cleaved by a tumor-specific protease. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity to tumor cell antigens, at least 50 times higher than the binding affinity to tumor cell antigens of the polypeptide or polypeptide complex of Formula Ia in which L1 and L2 are cleaved by a tumor-specific protease. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weak binding affinity to tumor cell antigens, at least 75 times higher than the binding affinity to tumor cell antigens of the polypeptide or polypeptide complex of Formula Ia in which L1 and L2 are cleaved by tumor-specific proteases. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weak binding affinity to tumor cell antigens, at least 100 times higher than the binding affinity to tumor cell antigens of the polypeptide or polypeptide complex of Formula Ia in which L1 and L2 are cleaved by tumor-specific proteases. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weak binding affinity to tumor cell antigens, at least 120 times higher than the binding affinity to tumor cell antigens of the polypeptide or polypeptide complex of Formula Ia in which L1 and L2 are cleaved by tumor-specific proteases.In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weak binding affinity to tumor cell antigens, at least 200 times higher than the binding affinity to tumor cell antigens of the polypeptide or polypeptide complex of Formula Ia in which L1 and L2 are cleaved by tumor-specific proteases. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weak binding affinity to tumor cell antigens, at least 300 times higher than the binding affinity to tumor cell antigens of the polypeptide or polypeptide complex of Formula Ia in which L1 and L2 are cleaved by tumor-specific proteases. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weak binding affinity to tumor cell antigens, at least 400 times higher than the binding affinity to tumor cell antigens of the polypeptide or polypeptide complex of Formula Ia in which L1 and L2 are cleaved by tumor-specific proteases. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weak binding affinity to tumor cell antigens, which is at least 500 times higher than the binding affinity to tumor cell antigens of the polypeptide or polypeptide complex of Formula Ia in which L1 and L2 are cleaved by tumor-specific proteases. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weak binding affinity to tumor cell antigens, which is at least 600 times higher than the binding affinity to tumor cell antigens of the polypeptide or polypeptide complex of Formula Ia in which L1 and L2 are cleaved by tumor-specific proteases. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weak binding affinity to tumor cell antigens, which is at least 700 times higher than the binding affinity to tumor cell antigens of the polypeptide or polypeptide complex of Formula Ia in which L1 and L2 are cleaved by tumor-specific proteases.In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weak binding affinity to tumor cell antigens, which is at least 800 times higher than the binding affinity to tumor cell antigens of the polypeptide or polypeptide complex of Formula Ia in which L1 and L2 are cleaved by tumor-specific proteases. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weak binding affinity to tumor cell antigens, which is at least 900 times higher than the binding affinity to tumor cell antigens of the polypeptide or polypeptide complex of Formula Ia in which L1 and L2 are cleaved by tumor-specific proteases. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weak binding affinity to tumor cell antigens, which is at least 1000 times higher than the binding affinity to tumor cell antigens of the polypeptide or polypeptide complex of Formula Ia in which L1 and L2 are cleaved by tumor-specific proteases. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weak binding affinity to tumor cell antigens, which is at least 10000 times higher than the binding affinity to tumor cell antigens of the polypeptide or polypeptide complex of Formula Ia in which L1 and L2 are cleaved by tumor-specific proteases.

[0052] In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in IFNγ-releasing T cell activation assays of isolated polypeptides or polypeptide complexes of Formula Ia that do not have P1, L1, P2, or L2. 50 In comparison with EC in IFNγ-releasing T cell activation assays 50 The EC2 is elevated. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in IFNγ-releasing T cell activation assays. 50Elevated EC in IFNγ-releasing T cell activation assays for polypeptides or polypeptide complexes of formula Ia that do not have P1, L1, P2, or L2. 50 It is at least 10 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in EC in IFNγ-releasing T cell activation assays. 50 Elevated EC in IFNγ-releasing T cell activation assays for polypeptides or polypeptide complexes of formula Ia that do not have P1, L1, P2, or L2. 50 It is at least 50 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in EC in IFNγ-releasing T cell activation assays. 50 Elevated EC in IFNγ-releasing T cell activation assays for polypeptides or polypeptide complexes of formula Ia that do not have P1, L1, P2, or L2. 50 This is at least 75 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in IFNγ-releasing T cell activation assays. 50 Elevated EC in IFNγ-releasing T cell activation assays for polypeptides or polypeptide complexes of formula Ia that do not have P1, L1, P2, or L2. 50 It is at least 100 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in IFNγ-releasing T cell activation assays. 50 Elevated EC in IFNγ-releasing T cell activation assays for polypeptides or polypeptide complexes of formula Ia that do not have P1, L1, P2, or L2. 50 It is at least 200 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in EC in IFNγ-releasing T cell activation assays. 50Elevated EC in IFNγ-releasing T cell activation assays for polypeptides or polypeptide complexes of formula Ia that do not have P1, L1, P2, or L2. 50 It is at least 300 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in EC in IFNγ-releasing T cell activation assays. 50 Elevated EC in IFNγ-releasing T cell activation assays for polypeptides or polypeptide complexes of formula Ia that do not have P1, L1, P2, or L2. 50 It is at least 400 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in IFNγ-releasing T cell activation assays. 50 Elevated EC in IFNγ-releasing T cell activation assays for polypeptides or polypeptide complexes of formula Ia that do not have P1, L1, P2, or L2. 50 It is at least 500 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in IFNγ-releasing T cell activation assays. 50 Elevated EC in IFNγ-releasing T cell activation assays for polypeptides or polypeptide complexes of formula Ia that do not have P1, L1, P2, or L2. 50 It is at least 600 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in EC in IFNγ-releasing T cell activation assays. 50 Elevated EC in IFNγ-releasing T cell activation assays for polypeptides or polypeptide complexes of formula Ia that do not have P1, L1, P2, or L2. 50 This is at least 700 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in EC in IFNγ-releasing T cell activation assays. 50Elevated EC in IFNγ-releasing T cell activation assays for polypeptides or polypeptide complexes of formula Ia that do not have P1, L1, P2, or L2. 50 This is at least 800 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in EC in IFNγ-releasing T cell activation assays. 50 Elevated EC in IFNγ-releasing T cell activation assays for polypeptides or polypeptide complexes of formula Ia that do not have P1, L1, P2, or L2. 50 It is at least 900 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in EC in IFNγ-releasing T cell activation assays. 50 Elevated EC in IFNγ-releasing T cell activation assays for polypeptides or polypeptide complexes of formula Ia that do not have P1, L1, P2, or L2. 50 It is at least 1000 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in EC in IFNγ-releasing T cell activation assays. 50 Elevated EC in IFNγ-releasing T cell activation assays for polypeptides or polypeptide complexes of formula Ia that do not have P1, L1, P2, or L2. 50 It is at least 10,000 times higher compared to [the other].

[0053] In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in T cell lysis assays of the polypeptide or polypeptide complex of Formula Ia in which L1 and L2 are cleaved by tumor-specific proteases. 50 In comparison, the binding affinity to tumor cell antigens is increased. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in EC2 lysis assays. 50Elevated EC in T cell lysis assays of polypeptides or polypeptide complexes of formula Ia where L1 and L2 are cleaved by tumor-specific proteases. 50 It is at least 10 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in EC2-12 T cell lysis assays. 50 Elevated EC in T cell lysis assays of polypeptides or polypeptide complexes of formula Ia where L1 and L2 are cleaved by tumor-specific proteases. 50 It is at least 50 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in EC2-12 T cell lysis assays. 50 Elevated EC in T cell lysis assays of polypeptides or polypeptide complexes of formula Ia where L1 and L2 are cleaved by tumor-specific proteases. 50 It is at least 75 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in EC2-12 T cell lysis assays. 50 Elevated EC in T cell lysis assays of polypeptides or polypeptide complexes of formula Ia where L1 and L2 are cleaved by tumor-specific proteases. 50 It is at least 100 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in EC2-12 T cell lysis assays. 50 Elevated EC in T cell lysis assays of polypeptides or polypeptide complexes of formula Ia where L1 and L2 are cleaved by tumor-specific proteases. 50 It is at least 200 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in EC2-12 T cell lysis assays. 50Elevated EC in T cell lysis assays of polypeptides or polypeptide complexes of formula Ia where L1 and L2 are cleaved by tumor-specific proteases. 50 It is at least 300 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in EC2-12 T cell lysis assays. 50 Elevated EC in T cell lysis assays of polypeptides or polypeptide complexes of formula Ia where L1 and L2 are cleaved by tumor-specific proteases. 50 It is at least 400 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in EC2-12 T cell lysis assays. 50 Elevated EC in T cell lysis assays of polypeptides or polypeptide complexes of formula Ia where L1 and L2 are cleaved by tumor-specific proteases. 50 It is at least 500 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in EC2-12 T cell lysis assays. 50 Elevated EC in T cell lysis assays of polypeptides or polypeptide complexes of formula Ia where L1 and L2 are cleaved by tumor-specific proteases. 50 It is at least 600 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in EC2-12 T cell lysis assays. 50 Elevated EC in T cell lysis assays of polypeptides or polypeptide complexes of formula Ia where L1 and L2 are cleaved by tumor-specific proteases. 50 It is at least 700 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in EC2-12 T cell lysis assays. 50Elevated EC in T cell lysis assays of polypeptides or polypeptide complexes of formula Ia where L1 and L2 are cleaved by tumor-specific proteases. 50 It is at least 800 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in EC2-12 T cell lysis assays. 50 Elevated EC in T cell lysis assays of polypeptides or polypeptide complexes of formula Ia where L1 and L2 are cleaved by tumor-specific proteases. 50 It is at least 900 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in EC2-12 T cell lysis assays. 50 Elevated EC in T cell lysis assays of polypeptides or polypeptide complexes of formula Ia where L1 and L2 are cleaved by tumor-specific proteases. 50 It is at least 1000 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in EC2-12 T cell lysis assays. 50 Elevated EC in T cell lysis assays of polypeptides or polypeptide complexes of formula Ia where L1 and L2 are cleaved by tumor-specific proteases. 50 It is at least 10,000 times higher compared to [the other].

[0054] In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is isolated polypeptide or polypeptide complex that does not have P1, L1, P2, or L2 in a T cell lysis assay. 50 In comparison with EC in T cell lysis assays 50 The EC2 is elevated. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in T cell lysis assays. 50Elevated EC in T cell lysis assays of polypeptides or polypeptide complexes of formula Ia that do not have P1, L1, P2, or L2. 50 It is at least 10 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in EC2-12 T cell lysis assays. 50 Elevated EC in T cell lysis assays of polypeptides or polypeptide complexes of formula Ia that do not have P1, L1, P2, or L2. 50 It is at least 50 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in EC2-12 T cell lysis assays. 50 Elevated EC in T cell lysis assays of polypeptides or polypeptide complexes of formula Ia that do not have P1, L1, P2, or L2. 50 It is at least 75 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in EC2-12 T cell lysis assays. 50 Elevated EC in T cell lysis assays of isolated polypeptides or polypeptide complexes lacking P1, L1, P2, or L2 50 It is at least 100 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in EC2-12 T cell lysis assays. 50 Elevated EC in T cell lysis assays of isolated polypeptides or polypeptide complexes lacking P1, L1, P2, or L2 50 It is at least 200 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in EC2-12 T cell lysis assays. 50 Elevated EC in T cell lysis assays of isolated polypeptides or polypeptide complexes lacking P1, L1, P2, or L2 50It is at least 300 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in EC2-12 T cell lysis assays. 50 Elevated EC in T cell lysis assays of isolated polypeptides or polypeptide complexes lacking P1, L1, P2, or L2 50 It is at least 400 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in EC2-12 T cell lysis assays. 50 Elevated EC in T cell lysis assays of isolated polypeptides or polypeptide complexes lacking P1, L1, P2, or L2 50 It is at least 500 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in EC2-12 T cell lysis assays. 50 Elevated EC in T cell lysis assays of isolated polypeptides or polypeptide complexes lacking P1, L1, P2, or L2 50 It is at least 600 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in EC2-12 T cell lysis assays. 50 Elevated EC in T cell lysis assays of isolated polypeptides or polypeptide complexes lacking P1, L1, P2, or L2 50 It is at least 700 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in EC2-12 T cell lysis assays. 50 Elevated EC in T cell lysis assays of isolated polypeptides or polypeptide complexes lacking P1, L1, P2, or L2 50It is at least 800 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in EC2-12 T cell lysis assays. 50 Elevated EC in T cell lysis assays of isolated polypeptides or polypeptide complexes lacking P1, L1, P2, or L2 50 It is at least 900 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in EC2-12 T cell lysis assays. 50 Elevated EC in T cell lysis assays of isolated polypeptides or polypeptide complexes lacking P1, L1, P2, or L2 50 It is at least 1000 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in EC2-12 T cell lysis assays. 50 Elevated EC in T cell lysis assays of isolated polypeptides or polypeptide complexes lacking P1, L1, P2, or L2 50 It is at least 10,000 times higher compared to [the other].

[0055] In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in T cell lysis assays of the polypeptide or polypeptide complex of Formula Ia in which L1 and L2 are cleaved by tumor-specific proteases. 50 Compared to the above, the binding affinity to tumor cell antigens is increased. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in EC2 lysis assays. 50 Elevated EC in T cell lysis assays of polypeptides or polypeptide complexes of formula Ia where L1 and L2 are cleaved by tumor-specific proteases. 50It is at least 10 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in EC2-12 T cell lysis assays. 50 Elevated EC in T cell lysis assays of polypeptides or polypeptide complexes of formula Ia where L1 and L2 are cleaved by tumor-specific proteases. 50 It is at least 50 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in EC2-12 T cell lysis assays. 50 Elevated EC in T cell lysis assays of polypeptides or polypeptide complexes of formula Ia where L1 and L2 are cleaved by tumor-specific proteases. 50 It is at least 75 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in EC2-12 T cell lysis assays. 50 Elevated EC in T cell lysis assays of polypeptides or polypeptide complexes of formula Ia where L1 and L2 are cleaved by tumor-specific proteases. 50 It is at least 100 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in EC2-12 T cell lysis assays. 50 Elevated EC in T cell lysis assays of polypeptides or polypeptide complexes of formula Ia where L1 and L2 are cleaved by tumor-specific proteases. 50 It is at least 200 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in EC2-12 T cell lysis assays. 50 Elevated EC in T cell lysis assays of polypeptides or polypeptide complexes of formula Ia where L1 and L2 are cleaved by tumor-specific proteases. 50It is at least 300 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in EC2-12 T cell lysis assays. 50 Elevated EC in T cell lysis assays of polypeptides or polypeptide complexes of formula Ia where L1 and L2 are cleaved by tumor-specific proteases. 50 It is at least 400 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in EC2-12 T cell lysis assays. 50 Elevated EC in T cell lysis assays of polypeptides or polypeptide complexes of formula Ia where L1 and L2 are cleaved by tumor-specific proteases. 50 It is at least 500 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in EC2-12 T cell lysis assays. 50 Elevated EC in T cell lysis assays of polypeptides or polypeptide complexes of formula Ia where L1 and L2 are cleaved by tumor-specific proteases. 50 It is at least 600 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in EC2-12 T cell lysis assays. 50 Elevated EC in T cell lysis assays of polypeptides or polypeptide complexes of formula Ia where L1 and L2 are cleaved by tumor-specific proteases. 50 It is at least 700 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in EC2-12 T cell lysis assays. 50 Elevated EC in T cell lysis assays of polypeptides or polypeptide complexes of formula Ia where L1 and L2 are cleaved by tumor-specific proteases. 50It is at least 800 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in EC2-12 T cell lysis assays. 50 Elevated EC in T cell lysis assays of polypeptides or polypeptide complexes of formula Ia where L1 and L2 are cleaved by tumor-specific proteases. 50 It is at least 900 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in EC2-12 T cell lysis assays. 50 Elevated EC in T cell lysis assays of polypeptides or polypeptide complexes of formula Ia where L1 and L2 are cleaved by tumor-specific proteases. 50 It is at least 1000 times higher compared to [another factor]. In some embodiments, the polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is used in EC2-12 T cell lysis assays. 50 Elevated EC in T cell lysis assays of polypeptides or polypeptide complexes of formula Ia where L1 and L2 are cleaved by tumor-specific proteases. 50 It is at least 10,000 times higher compared to [the other].

[0056] Second antigen-recognition molecule (A2) In some embodiments, A2 comprises an antibody or antibody fragment. In some embodiments, the antibody or antibody fragment comprises a single-chain variable fragment, a single-domain antibody, Fab, or Fab'. In some embodiments, the antibody or antibody fragment comprises a single-chain variable fragment (scFv), a heavy-chain variable domain (VH domain), a light-chain variable domain (VL domain), or a variable domain (VHH) of a camelid-derived single-domain antibody. In some embodiments, the antibody or antibody fragment is humanized or human. In some embodiments, A2 is Fab or Fab'. In some embodiments, Fab or Fab' comprises (a) a Fab light-chain polypeptide and (b) a Fab heavy-chain polypeptide. In some embodiments, the antibody or antibody fragment comprises a PSMA-binding region.

[0057] In some embodiments, the antigen-binding fragment (Fab) or Fab' that binds to PSMA comprises a Fab light chain polypeptide chain and a Fab heavy chain polypeptide. In some embodiments, the Fab light chain polypeptide comprises a Fab light chain variable domain. In some embodiments, the Fab heavy chain polypeptide comprises a Fab heavy chain variable domain. In some embodiments, the Fab heavy chain variable domain comprises at least one, two, or three complementarity-determining regions (CDRs) disclosed in Table 2, or substantially identical sequences thereto (e.g., sequences having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity). In some embodiments, the Fab light chain variable domain comprises at least one, two, or three complementarity-determining regions (CDRs) disclosed in Table 2, or substantially identical sequences thereto (e.g., sequences having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity).

[0058] In some embodiments, the Fab heavy chain variable domain includes at least one, two, or three complementarity-determining regions (CDRs) disclosed in Table 2, or substantially identical sequences thereto (e.g., sequences having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity), and the Fab light chain variable domain includes at least one, two, or three complementarity-determining regions (CDRs) disclosed in Table 2, or substantially identical sequences thereto (e.g., sequences having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity).

[0059] [Table 2]

[0060] In some embodiments, the Fab includes HC-CDR1, HC-CDR2, and HC-CDR3 as complementarity determination regions (CDRs), where HC-CDR1, HC-CDR2, and HC-CDR3 of the Fab include sequence number 8 for HC-CDR1, sequence number 9 for HC-CDR2, and sequence number 10 for HC-CDR3. The Fab also includes LC-CDR1, LC-CDR2, and LC-CDR3 as CDRs, where LC-CDR1, LC-CDR2, and LC-CDR3 of the Fab include sequence number 11 for LC-CDR1, sequence number 12 for LC-CDR2, and sequence number 13 for LC-CDR3. In some embodiments, Fab includes HC-CDR1, HC-CDR2, and HC-CDR3 as complementarity-determining regions (CDRs), where HC-CDR1 includes SEQ ID NO: 8, HC-CDR2 includes SEQ ID NO: 9, and HC-CDR3 includes SEQ ID NO: 10, and at least one of HC-CDR1, HC-CDR2, or HC-CDR3 contains 0 to 2 amino acid modifications. Alternatively, Fab may include LC-CDR1, LC-CDR2, and LC-CDR3 as CDRs, where LC-CDR1, LC-CDR2, and LC-CDR3 include SEQ ID NO: 11, LC-CDR2 includes SEQ ID NO: 12, and LC-CDR3 includes SEQ ID NO: 13, and at least one of LC-CDR1, LC-CDR2, or LC-CDR3 contains 0 to 2 amino acid modifications.

[0061] In some embodiments, A2 includes HC-CDR1, HC-CDR2, and HC-CDR3 as complementary determination regions (CDRs), where HC-CDR1, HC-CDR2, and HC-CDR3 of A2 include sequence number 8 for HC-CDR1, sequence number 9 for HC-CDR2, and sequence number 10 for HC-CDR3; A2 also includes LC-CDR1, LC-CDR2, and LC-CDR3 as CDRs, where LC-CDR1, LC-CDR2, and LC-CDR3 of A2 include sequence number 11 for LC-CDR1, sequence number 12 for LC-CDR2, and sequence number 13 for LC-CDR3. In some embodiments, A2 includes HC-CDR1, HC-CDR2, and HC-CDR3 as complementarity-determining regions (CDRs), where HC-CDR1 includes SEQ ID NO: 8, HC-CDR2 includes SEQ ID NO: 9, and HC-CDR3 includes SEQ ID NO: 10, and at least one of HC-CDR1, HC-CDR2, or HC-CDR3 contains 0 to 2 amino acid modifications. Alternatively, A2 includes LC-CDR1, LC-CDR2, and LC-CDR3 as CDRs, where LC-CDR1, LC-CDR2, and LC-CDR3 include SEQ ID NO: 11, LC-CDR2 includes SEQ ID NO: 12, and LC-CDR3 includes SEQ ID NO: 13, and at least one of LC-CDR1, LC-CDR2, or LC-CDR3 contains 0 to 2 amino acid modifications.

[0062] In some embodiments, the Fab light chain polypeptide includes the amino acid sequence specified by SEQ ID NO: 14. In some embodiments, the Fab light chain polypeptide includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 14. In some embodiments, the Fab light chain polypeptide includes an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 14. In some embodiments, the Fab light chain polypeptide includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 14. In some embodiments, the Fab light chain polypeptide includes an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 14. In some embodiments, the Fab light chain polypeptide includes an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 14.

[0063] In some embodiments, the Fab heavy chain polypeptide includes the amino acid sequence specified by SEQ ID NO: 15. In some embodiments, the Fab heavy chain polypeptide includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 15. In some embodiments, the Fab heavy chain polypeptide includes an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 15. In some embodiments, the Fab heavy chain polypeptide includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 15. In some embodiments, the Fab heavy chain polypeptide includes an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 15. In some embodiments, the Fab heavy chain polypeptide includes an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 15.

[0064] In some embodiments, the Fab light chain polypeptide of A2 is bound to the C-terminus of the single-chain variable fragment (scFv) of A1. In some embodiments, the Fab heavy chain polypeptide of A2 is bound to the C-terminus of the single-chain variable fragment (scFv) of A1. In some embodiments, the Fab light chain polypeptide of A2 is bound to the N-terminus of the single-chain variable fragment (scFv) of A1. In some embodiments, the Fab heavy chain polypeptide of A2 is bound to the N-terminus of the single-chain variable fragment (scFv) of A1. In some embodiments, the Fab heavy chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of A1. In some embodiments, the Fab light chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of A1. In some embodiments, the Fab heavy chain polypeptide of A2 is bound to the scFv light chain polypeptide of A1. In some embodiments, the Fab light chain polypeptide of A2 is bound to the scFv light chain polypeptide of A1.

[0065] In some embodiments, A2 further comprises P2 and L2, where P2 comprises a peptide that binds to A2, and L2 comprises a linking portion that connects A1 to P1 and is a substrate for a tumor-specific protease. 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.

[0066] 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.

[0067] Peptides (P1, P2, and P 1a ) In some embodiments, P1, P2, or P 1a This includes sequences disclosed in Table 3, or sequences substantially identical thereto (e.g., sequences having 0, 1, or 2 amino acid modifications).

[0068] [Table 3]

[0069] In some embodiments, P1 impairs the binding of A1 to a first target antigen. In some embodiments, P1 impairs the binding of A1 to an effector cell antigen. In some embodiments, P1 binds to A1 by ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-binding interactions, or a combination thereof. In some embodiments, P1 binds to A1 at or near the antigen-binding site. In some embodiments, if L1 is cleaved by a tumor-specific protease, thereby exposing A1 to the effector cell antigen, P1 becomes unbound from A1. In some embodiments, the protease comprises a matrix metalloproteinase (MMP) or a serine protease. In some embodiments, the matrix metalloproteinase comprises MMP2, MMP7, MMP9, MMP13, or MMP14. In some embodiments, the serine protease comprises a matryptase (MTSP1), urokinase, or hepsin. In some embodiments, P1 has less than 70% sequence identity with the effector cell antigen. In some embodiments, P1 has less than 75% sequence identity with the effector cell antigen. In some embodiments, P1 has less than 80% sequence identity with the effector cell antigen. In some embodiments, P1 has less than 85% sequence identity with the effector cell antigen. In some embodiments, P1 has less than 90% sequence identity with the effector cell antigen. In some embodiments, P1 has less than 95% sequence identity with the effector cell antigen. In some embodiments, P1 has less than 98% sequence identity with the effector cell antigen. In some embodiments, P1 has less than 99% sequence identity with the effector cell antigen. In some embodiments, P1 includes a de novo amino acid sequence that shares less than 10% sequence identity with the effector cell antigen. In some embodiments, P1 includes an amino acid sequence from any one of sequence numbers 16-19. In some embodiments, P1 includes the amino acid sequence from sequence number 16.In some embodiments, P1 includes the amino acid sequence of SEQ ID NO: 17. In some embodiments, P1 includes the amino acid sequence of SEQ ID NO: 18. In some embodiments, P1 includes the amino acid sequence of SEQ ID NO: 19. In some embodiments, P1 includes the amino acid sequence of SEQ ID NO: 78.

[0070] In some embodiments, P1 is Z1-Z2-C-Z4-P-Z6-Z7-Z8-Z9-Z 10 -Z 11 -Z 12 -CZ 14 The amino acid sequence is as follows: Z1 is selected from D, Y, F, I, N, V, H, L, A, T, S, and P; Z2 is selected from D, Y, L, F, I, N, A, V, H, T, and S; Z4 is selected from G and W; Z6 is selected from E, D, V, and P; Z7 is selected from W, L, F, V, G, M, I, and Y; Z8 is selected from E, D, P, and Q; Z9 is selected from E, D, Y, V, F, W, P, L, and Q; Z 10 is selected from S, D, Y, T, I, F, V, N, A, P, L, and H, and Z 11 is selected from I, Y, F, V, L, T, N, S, D, A, and H, and Z 12 is selected from F, D, Y, L, I, V, A, N, T, P, S, and H, and Z 14 Z1 is selected from D, Y, N, F, I, P, V, A, T, H, L, and S. In some embodiments, Z1 is selected from D, Y, F, I, and N, Z2 is selected from D, Y, L, F, I, and N, Z4 is selected from G and W, Z6 is selected from E and D, Z7 is selected from W, L, F, and V, Z8 is selected from E and D, Z9 is selected from E, D, Y, and V, Z 10 is selected from S, D, Y, T, and I, and Z 11 is selected from I, Y, F, V, L, and T, and Z 12 is selected from F, D, Y, L, I, V, A, and N, and Z 14Z1 is selected from D, Y, N, F, I, and P. In some embodiments, Z1 is selected from D, Y, and F, Z2 is selected from D, Y, L, and F, Z4 is selected from G and W, Z6 is selected from E and D, Z7 is selected from W, L, and F, Z8 is selected from E and D, Z9 is selected from E and D, Z 10 The following are selected from S, D, and Y, and Z 11 is selected from I, Y, and F, and Z 12 The following are selected from F, D, Y, and L, and Z 14 This is selected from D, Y, and N.

[0071] In some embodiments, P1 is U1-U2-C-U4-P-U6-U7-U8-U9-U 10 -U 11 -U 12 -CU 14 The amino acid sequence includes, U1 is selected from D, Y, F, I, N, V, H, L, A, T, S, and P, U2 is selected from D, Y, L, F, I, N, A, V, H, T, and S, U4 is selected from G and W, U6 is selected from E, D, V, and P, U7 is selected from W, L, F, V, G, M, I, and Y, U8 is selected from E, D, P, and Q, U9 is selected from E, D, Y, V, F, W, P, L, and Q, and U 10 The following are selected from S, D, Y, T, I, F, V, N, A, P, L, and H, U 11 is selected from I, Y, F, V, L, T, N, S, D, A, and H, U 12 is selected from F, D, Y, L, I, V, A, N, T, P, S, G, and H, U 14 U1 is selected from D, Y, N, F, I, P, V, A, T, H, L, M, and S. In some embodiments, U1 is selected from D, Y, F, I, V, and N, U2 is selected from D, Y, L, F, I, and N, U4 is selected from G and W, U6 is selected from E and D, U7 is selected from W, L, F, G, and V, U8 is selected from E and D, U9 is selected from E, D, Y, and V, U 10It is selected from S, D, Y, T, and I, U 11 It is selected from I, Y, F, V, L, and T, U 12 is selected from F, D, Y, L, I, V, A, G, and N, U 14 U1 is selected from D, Y, N, F, I, M, and P. In some embodiments, U1 is selected from D, Y, V, and F, U2 is selected from D, Y, L, and F, U4 is selected from G and W, U6 is selected from E and D, U7 is selected from W, L, G, and F, U8 is selected from E and D, U9 is selected from E and D, U 10 It is selected from S, D, T, and Y, U 11 is selected from I, Y, V, L, and F, U 12 The following are selected from F, D, Y, G, A, and L, and U 14 This is selected from D, Y, M, and N.

[0072] In some embodiments, P1 comprises an amino acid sequence from any one of sequence numbers 79 to 105. In some embodiments, P1 comprises an amino acid sequence from any one of the sequences in Table 20. In some embodiments, P1 comprises an amino acid sequence from any one of sequence numbers 106 to 117.

[0073] In some embodiments, P1 includes the amino acid sequence according to SEQ ID NO: 18, or a peptide sequence having one, two, or three amino acid substitutions, additions, or deletions compared to SEQ ID NO: 18.

[0074] In some embodiments, P1 includes the amino acid sequence according to SEQ ID NO: 19, or a peptide sequence having one, two, or three amino acid substitutions, additions, or deletions compared to SEQ ID NO: 19.

[0075] In some embodiments, P1 includes the amino acid sequence according to SEQ ID NO: 116, or a peptide sequence having one, two, or three amino acid substitutions, additions, or deletions compared to SEQ ID NO: 116.

[0076] In some embodiments, P1 includes the amino acid sequence given by SEQ ID NO: 18.

[0077] In some embodiments, P1 includes the amino acid sequence given by SEQ ID NO: 19.

[0078] In some embodiments, P1 comprises the amino acid sequence given by SEQ ID NO: 116.

[0079] In some embodiments, P2 impairs the binding of A2 to a second target antigen. In some embodiments, P2 impairs the binding of A2 to PSMA. In some embodiments, P2 binds to A2 by ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bonding interactions, or a combination thereof. In some embodiments, P2 binds to A2 at or near the antigen-binding site. In some embodiments, if L2 is cleaved by a tumor-specific protease, thereby exposing A2 to PSMA, P2 becomes unbound from A2. In some embodiments, the protease comprises a matrix metalloproteinase (MMP) or a serine protease. In some embodiments, the matrix metalloproteinase comprises MMP2, MMP7, MMP9, MMP13, or MMP14. In some embodiments, the serine protease comprises a matryptase (MTSP1), urokinase, or hepsin. In some embodiments, P2 has less than 70% sequence identity with PSMA. In some embodiments, P2 has less than 75% sequence identity with PSMA. In some embodiments, P2 has less than 80% sequence identity with PSMA. In some embodiments, P2 has less than 85% sequence identity with PSMA. In some embodiments, P2 has less than 90% sequence identity with PSMA. In some embodiments, P2 has less than 95% sequence identity with PSMA. In some embodiments, P2 has less than 98% sequence identity with PSMA. In some embodiments, P2 has less than 99% sequence identity with PSMA. In some embodiments, P2 includes a de novo amino acid sequence that shares less than 10% sequence identity with PSMA.

[0080] In some embodiments, P 1a L 1aIf the molecule is not cleaved, it impairs the binding of the first antigen-recognition molecule to the target antigen. In some embodiments, the antigen-recognition molecule includes an antibody or antibody fragment. In some embodiments, the target antigen is an anti-CD3 effector cell antigen. In some embodiments, the target antigen is prostate-specific membrane antigen (PSMA). In some embodiments, P 1a It has less than 70% sequence identity with respect to the target antigen. In some embodiments, P 1a It has less than 75% sequence identity with respect to the target antigen. In some embodiments, P 1a It has less than 80% sequence identity with respect to the target antigen. In some embodiments, P 1a It has less than 85% sequence identity with respect to the target antigen. In some embodiments, P 1a It has less than 90% sequence identity with respect to the target antigen. In some embodiments, P 1a It has less than 95% sequence identity with respect to the target antigen. In some embodiments, P 1a It has less than 98% sequence identity with respect to the target antigen. In some embodiments, P 1a It has less than 99% sequence identity with respect to the target antigen. In some embodiments, P 1a It contains a de novo amino acid sequence that shares less than 10% sequence identity with the second target antigen.

[0081] In some embodiments, P 1a is Z1-Z2-C-Z4-P-Z6-Z7-Z8-Z9-Z 10 -Z 11 -Z 12 -CZ 14The amino acid sequence is as follows: Z1 is selected from D, Y, F, I, N, V, H, L, A, T, S, and P; Z2 is selected from D, Y, L, F, I, N, A, V, H, T, and S; Z4 is selected from G and W; Z6 is selected from E, D, V, and P; Z7 is selected from W, L, F, V, G, M, I, and Y; Z8 is selected from E, D, P, and Q; Z9 is selected from E, D, Y, V, F, W, P, L, and Q; Z 10 is selected from S, D, Y, T, I, F, V, N, A, P, L, and H, and Z 11 is selected from I, Y, F, V, L, T, N, S, D, A, and H, and Z 12 is selected from F, D, Y, L, I, V, A, N, T, P, S, and H, and Z 14 Z1 is selected from D, Y, N, F, I, P, V, A, T, H, L, and S. In some embodiments, Z1 is selected from D, Y, F, I, and N, Z2 is selected from D, Y, L, F, I, and N, Z4 is selected from G and W, Z6 is selected from E and D, Z7 is selected from W, L, F, and V, Z8 is selected from E and D, Z9 is selected from E, D, Y, and V, Z 10 is selected from S, D, Y, T, and I, and Z 11 is selected from I, Y, F, V, L, and T, and Z 12 is selected from F, D, Y, L, I, V, A, and N, and Z 14 Z1 is selected from D, Y, N, F, I, and P. In some embodiments, Z1 is selected from D, Y, and F, Z2 is selected from D, Y, L, and F, Z4 is selected from G and W, Z6 is selected from E and D, Z7 is selected from W, L, and F, Z8 is selected from E and D, Z9 is selected from E and D, Z 10 The following are selected from S, D, and Y, and Z 11 is selected from I, Y, and F, and Z 12 The following are selected from F, D, Y, and L, and Z 14 This is selected from D, Y, and N.

[0082] In some embodiments, P 1a is U1-U2-C-U4-P-U6-U7-U8-U9-U 10 -U 11 -U 12 -CU 14 The amino acid sequence includes, U1 is selected from D, Y, F, I, N, V, H, L, A, T, S, and P, U2 is selected from D, Y, L, F, I, N, A, V, H, T, and S, U4 is selected from G and W, U6 is selected from E, D, V, and P, U7 is selected from W, L, F, V, G, M, I, and Y, U8 is selected from E, D, P, and Q, U9 is selected from E, D, Y, V, F, W, P, L, and Q, and U 10 The following are selected from S, D, Y, T, I, F, V, N, A, P, L, and H, U 11 is selected from I, Y, F, V, L, T, N, S, D, A, and H, U 12 is selected from F, D, Y, L, I, V, A, N, T, P, S, G, and H, U 14 U1 is selected from D, Y, N, F, I, P, V, A, T, H, L, M, and S. In some embodiments, U1 is selected from D, Y, F, I, V, and N, U2 is selected from D, Y, L, F, I, and N, U4 is selected from G and W, U6 is selected from E and D, U7 is selected from W, L, F, G, and V, U8 is selected from E and D, U9 is selected from E, D, Y, and V, U 10 It is selected from S, D, Y, T, and I, U 11 It is selected from I, Y, F, V, L, and T, U 12 is selected from F, D, Y, L, I, V, A, G, and N, U 14 U1 is selected from D, Y, N, F, I, M, and P. In some embodiments, U1 is selected from D, Y, V, and F, U2 is selected from D, Y, L, and F, U4 is selected from G and W, U6 is selected from E and D, U7 is selected from W, L, G, and F, U8 is selected from E and D, U9 is selected from E and D, U 10 It is selected from S, D, T, and Y, U11 is selected from I, Y, V, L, and F, U 12 The following are selected from F, D, Y, G, A, and L, and U 14 This is selected from D, Y, M, and N.

[0083] In some embodiments, P 1a It contains an amino acid sequence corresponding to one of sequence numbers 79-105.

[0084] In some embodiments, P 1a This includes an amino acid sequence consisting of one of the sequences in Table 20.

[0085] In some embodiments, P 1a This contains the amino acid sequence of any one of sequence numbers 106-117.

[0086] In some embodiments, P 1a This includes the amino acid sequence according to SEQ ID NO: 18, or a peptide sequence having one, two, or three amino acid substitutions, additions, or deletions compared to SEQ ID NO: 18.

[0087] In some embodiments, P 1a This includes the amino acid sequence according to SEQ ID NO: 19, or a peptide sequence having one, two, or three amino acid substitutions, additions, or deletions compared to SEQ ID NO: 19.

[0088] In some embodiments, P 1a This includes the amino acid sequence according to SEQ ID NO: 116, or a peptide sequence having one, two, or three amino acid substitutions, additions, or deletions compared to SEQ ID NO: 116.

[0089] In some embodiments, P 1a This includes the amino acid sequence according to SEQ ID NO: 18.

[0090] In some embodiments, P 1a This includes the amino acid sequence according to SEQ ID NO: 19.

[0091] In some embodiments, P 1a This includes the amino acid sequence according to SEQ ID NO: 116.

[0092] In some embodiments, P1, P2, or P 1a It comprises a peptide sequence with a length of at least 5 amino acids. In some embodiments, P1, P2, or P 1a It comprises a peptide sequence with a length of at least 6 amino acids. In some embodiments, P1, P2, or P 1a It comprises a peptide sequence with a length of at least 10 amino acids. In some embodiments, P1, P2, or P 1a It comprises a peptide sequence having a length of at least 10 amino acids and no more than 20 amino acids. In some embodiments, P1, P2, or P 1a It comprises a peptide sequence with a length of at least 16 amino acids. In some embodiments, P1, P2, or P 1a It contains a peptide sequence with a length of 40 amino acids or less. In some embodiments, P1, P2, or P 1a It contains at least two cysteine ​​amino acid residues. In some embodiments, P1, P2, or P 1a This includes cyclic peptides or linear peptides. In some embodiments, P1, P2, or P 1a It contains a cyclic peptide. In some embodiments, P1, P2, or P 1a It contains linear peptides.

[0093] In some embodiments, P1, P2, or P 1a , or P1, P2, and P 1a This includes modified amino acids, unnatural amino acids, unnatural modified amino acids, or combinations thereof. In some embodiments, the modified amino acids or unnatural modified amino acids include post-translational modifications. In some embodiments, P1, P2, or P 1a , or P1, P2, and P 1aThis includes modifications such as acetylation, acylation, ADP-ribosylation, amidation, covalent bonding of flavins, covalent bonding of heme moieties, covalent bonding of nucleotides or nucleotide derivatives, covalent bonding of lipids or lipid derivatives, covalent bonding of phosphatidylinositol, crosslinking, cyclization, disulfide bond formation, demethylation, covalent crosslinking, cystine formation, pyroglutamate formation, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodization, methylation, myristoylation, oxidation, proteolysis, phosphorylation, prenylation, racemization, selenoylation, sulfated, arginylation, and other transfer RNA-mediated addition of amino acids to proteins, as well as ubiquitination. The modifications include P1, P2, or P2, which include the peptide backbone, amino acid side chains, and endpoints. 1a , or P1, P2, and P 1a It will be done everywhere.

[0094] In some embodiments, P1, P2, or P 1a It does not contain albumin or albumin fragments. In some embodiments, P1, P2, or P 1a It does not contain an albumin-binding domain.

[0095] Connecting parts (L1, L2, L3, and L 1a ) In some embodiments, L1, L2, L3, or L 1a This is a peptide sequence having at least 5 to 50 amino acids. In some embodiments, L1, L2, L3, or L 1a This is a peptide sequence having at least 10 to 30 amino acids. In some embodiments, L1, L2, L3, or L 1a This is a peptide sequence having at least 10 amino acids. In some embodiments, L1, L2, L3, or L 1a This is a peptide sequence having at least 18 amino acids. In some embodiments, L1, L2, L3, or L 1aThis is a peptide sequence having at least 26 amino acids. In some embodiments, L1, L2, L3, or L 1a (G2S) n It has an expression that includes, where n is an integer from 1 to 3 (SEQ ID NO: 118). In some embodiments, L1, L2, L3, or L 1a (G2S) n The expression includes a formula in which n is at least an integer of 1. In some embodiments, L1, L2, L3, or L 1a (G2S) n , (GS) n (GSGGS) n (Sequence ID 50), (GGGS) n (Sequence ID 51), (GGGGS) n (Sequence ID 52), and (GSSGGS) n The formula is selected from the group consisting of (SEQ ID NO: 53), where n is at least an integer of 1. In some embodiments, the tumor-specific protease is selected from the group consisting of metalloproteases, serine proteases, cysteine ​​proteases, threonine proteases, and aspartate proteases. In some embodiments, L1, L2, L3, or L 1a This includes a urokinase-cleavable amino acid sequence, a matryptase-cleavable amino acid sequence, a regmine-cleavable amino acid sequence, or a matrix metalloproteinase-cleavable amino acid sequence. In some embodiments, the matrix metalloproteinase includes MMP2, MMP7, MMP9, MMP13, or MMP14. In some embodiments, the serine protease includes matryptase (MTSP1), urokinase, or hepsin.

[0096] In some embodiments, L1, L2, L3, or L 1a This includes sequences disclosed in Table 4, or sequences substantially identical thereto (e.g., sequences having 0, 1, or 2 amino acid modifications).

[0097] In some embodiments, L1 includes an array of linkers 25 (sequence number 45). In some embodiments, L1 includes an array of linkers 26 (sequence number 46). In some embodiments, L1 includes an array of linkers 27 (sequence number 47). In some embodiments, L1 includes an array of linkers 28 (sequence number 48).

[0098] In some embodiments, L2 includes an array of linkers 25 (sequence number 45). In some embodiments, L2 includes an array of linkers 26 (sequence number 46). In some embodiments, L2 includes an array of linkers 27 (sequence number 47). In some embodiments, L2 includes an array of linkers 28 (sequence number 48).

[0099] [Table 4]

[0100] 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, if L1 is cleaved by a tumor-specific protease, thereby exposing A1 to effector cell antigens, P1 is debounded from A1. In some embodiments, if L2 is cleaved by a tumor-specific protease, thereby exposing A2 to PSMA, P2 is debounded from A2.

[0101] In some embodiments, L1, L2, L3, or L 1a This includes modified amino acids, unnatural amino acids, unnatural modified amino acids, or combinations thereof. In some embodiments, the modified amino acids or unnatural modified amino acids include post-translational modifications. In some embodiments, L1, L2, L3, or L 1aThis includes modifications such as acetylation, acylation, ADP-ribosylation, amidation, covalent bonding of flavins, covalent bonding of heme moieties, covalent bonding of nucleotides or nucleotide derivatives, covalent bonding of lipids or lipid derivatives, covalent bonding of phosphatidylinositol, crosslinking, cyclization, disulfide bond formation, demethylation, covalent crosslinking, cystine formation, pyroglutamate formation, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodization, methylation, myristoylation, oxidation, proteolysis, phosphorylation, prenylation, racemization, selenoylation, sulfated, arginylation, and other transfer RNA-mediated addition of amino acids to proteins, as well as ubiquitination. The modifications include L1, L2, L3, or L2, including the peptide backbone or amino acid side chains. 1a It will be done everywhere.

[0102] In some embodiments, the cleavable linker is cleavable by a protease. In some embodiments, the protease is present in the disease state microenvironment at a higher level compared to the level in healthy tissue or the microenvironment that is not the disease state microenvironment. In some embodiments, the protease includes a tumor-specific protease. In some embodiments, the protease includes a matrix metalloproteinase (MMP) or a serine protease. In some embodiments, the matrix metalloproteinase includes MMP2, MMP7, MMP9, MMP13, or MMP14. In some embodiments, the matrix metalloproteinase is selected from the group consisting of MMP2, MMP7, MMP9, MMP13, and MMP14. In some embodiments, the matrix metalloproteinase includes MMP2. In some embodiments, the matrix metalloproteinase includes MMP7. In some embodiments, the matrix metalloproteinase includes MMP9. In some embodiments, the matrix metalloproteinase includes MMP13. In some embodiments, the matrix metalloproteinase comprises MMP14. In some embodiments, the serine protease comprises matryptase (MTSP1), urokinase, or hepsin. In some embodiments, the serine protease is selected from the group consisting of matryptase (MTSP1), urokinase, and hepsin. In some embodiments, the serine protease comprises matryptase (MTSP1). In some embodiments, the serine protease comprises urokinase. In some embodiments, the serine protease comprises hepsin. In some embodiments, the cleavable linker is cleaved by various proteases. In some embodiments, the cleavable linker is cleaved by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more than 20 various proteases.

[0103] Half-life extension molecule (H1 and H 1a ) In some embodiments, H1 does not interfere with A1 binding to effector cell antigens. In some embodiments, H1 includes a ligation portion (L3) that connects H1 to P1. In some embodiments, H 1a This does not interfere with the binding of the first antigen-recognition molecule to the effector cell antigen. In some embodiments, H 1a P 1a H 1a It includes a connecting portion (L3) that connects them. In some embodiments, the half-life extension molecule (H1 or H 1a ) does not have binding affinity to the antigen recognition molecule. In some embodiments, the half-life extension molecule (H1 or H 1a ) does not have binding affinity to effector cell antigens. In some embodiments, the half-life extension molecule (H1 or H 1a ) does not shield the antigen-recognition molecule from the effector cell antigen. In some embodiments, the half-life extension molecule (H1 or H 1a ) is not directly bound to the antigen recognition molecule.

[0104] In some embodiments, H1 or H 1a This includes sequences disclosed in Table 5, or sequences substantially identical thereto (for example, sequences having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity).

[0105] [Table 5]

[0106] In some embodiments, H1 or H 1a This includes an amino acid sequence having a repeating sequence motif. In some embodiments, H1 or H 1a This includes an amino acid sequence having a highly ordered secondary structure. When "highly ordered secondary structure" is used in this context, it refers to H1 or H 1aThis means that at least about 50%, about 70%, about 80%, or about 90% of the amino acid residues contribute to the secondary structure when measured or determined by spectrophotometric methods (e.g., circular dichroism spectroscopy in the "far ultraviolet" spectral region (190–250 nm)) and by means including, but not limited to, computer programs or algorithms such as the Chou-Fasman algorithm or the Garnier-Osguthorpe-Robson ("GOR") algorithm.

[0107] In some embodiments, H1 or H 1a It contains a polymer. In some embodiments, the polymer is polyethylene glycol (PEG). In some embodiments, H1 or H 1a It contains albumin. In some embodiments, H1 or H 1a It contains an Fc domain. In some embodiments, albumin is serum albumin. In some embodiments, albumin is human serum albumin. In some embodiments, H1 or H 1aThis comprises a polypeptide, ligand, or small molecule. In some embodiments, the polypeptide, ligand, or small molecule binds to a serum protein or fragment thereof, a circulating immunoglobulin or fragment thereof, or CD35 / CR1. In some embodiments, the serum protein includes thyroxine-binding protein, transthyretin, α1-acid glycoprotein, transferrin, transferrin receptor or its transferrin-binding moiety, fibrinogen, or albumin. In some embodiments, the circulating immunoglobulin molecule includes 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 includes a single-domain antibody, a single-chain variable fragment, or Fab. In some embodiments, the single-domain antibody includes a single-domain antibody that binds to albumin. In some embodiments, the single-domain antibody is a human antibody or a humanized antibody. In some embodiments, the single-domain antibody is selected from the group consisting of 645gH1gL1, 645dsgH5gL4, 23-13-A01-sc02, A10m3 or its fragment, DOM7r-31, DOM7h-11-15, Alb-1, Alb-8, Alb-23, 10G, 10E, and SA21. In some embodiments, the single-domain antibody includes HC-CDR1, HC-CDR2, and HC-CDR3 as complementarity-determining regions (CDRs), where HC-CDR1 includes SEQ ID NO: 54, HC-CDR2 includes SEQ ID NO: 55, and HC-CDR3 includes SEQ ID NO: 56. In some embodiments, the single-domain antibody comprises HC-CDR1, HC-CDR2, and HC-CDR3 as complementarity-determining regions (CDRs), where HC-CDR1 comprises SEQ ID NO: 54, HC-CDR2 comprises SEQ ID NO: 55, and HC-CDR3 comprises SEQ ID NO: 56, and at least one of HC-CDR1, HC-CDR2, or HC-CDR3 contains 0 to 2 amino acid modifications.In some embodiments, a single-domain antibody comprises HC-CDR1, HC-CDR2, and HC-CDR3 as complementarity-determining regions (CDRs), where HC-CDR1 contains SEQ ID NO: 58, HC-CDR2 contains SEQ ID NO: 59, and HC-CDR3 contains SEQ ID NO: 60. In some embodiments, a single-domain antibody comprises HC-CDR1, HC-CDR2, and HC-CDR3 as complementarity-determining regions (CDRs), where HC-CDR1, HC-CDR2, and HC-CDR3 contain SEQ ID NO: 58, HC-CDR2 contains SEQ ID NO: 59, and HC-CDR3 contains SEQ ID NO: 60, and at least one of HC-CDR1, HC-CDR2, or HC-CDR3 contains 0 to 2 amino acid modifications.

[0108] In some embodiments, H1 includes the amino acid sequence of SEQ ID NO: 57. In some embodiments, H1 includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 57. In some embodiments, H1 includes an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 57. In some embodiments, H1 includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 57. In some embodiments, H1 includes an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 57. In some embodiments, H1 includes an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 57.

[0109] In some embodiments, H 1a This includes the amino acid sequence according to SEQ ID NO: 57. In some embodiments, H 1a This includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 57. In some embodiments, H 1a This includes an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 57. In some embodiments, H 1aThis includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 57. In some embodiments, H 1a This includes an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 57. In some embodiments, H 1a It contains an amino acid sequence that has at least 99% sequence identity with SEQ ID NO: 57.

[0110] In some embodiments, H1 includes the amino acid sequence specified by SEQ ID NO: 61. In some embodiments, H1 includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 61. In some embodiments, H1 includes an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 61. In some embodiments, H1 includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 61. In some embodiments, H1 includes an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 61. In some embodiments, H1 includes an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 61.

[0111] In some embodiments, H 1a This includes the amino acid sequence according to SEQ ID NO: 61. In some embodiments, H 1a This includes an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 61. In some embodiments, H 1a This includes an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 61. In some embodiments, H 1a This includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 61. In some embodiments, H 1a This includes an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 61. In some embodiments, H 1a It contains an amino acid sequence that has at least 99% sequence identity with SEQ ID NO: 61.

[0112] In some embodiments, H1 or H 1a, or H1 and H 1a This includes modified amino acids, unnatural amino acids, unnatural modified amino acids, or combinations thereof. In some embodiments, the modified amino acids or unnatural modified amino acids include post-translational modifications. In some embodiments, H1 or H 1a , or H1 and H 1a This includes modifications such as acetylation, acylation, ADP-ribosylation, amidation, covalent bonding of flavins, covalent bonding of heme moieties, covalent bonding of nucleotides or nucleotide derivatives, covalent bonding of lipids or lipid derivatives, covalent bonding of phosphatidylinositol, crosslinking, cyclization, disulfide bond formation, demethylation, covalent crosslinking, cystine formation, pyroglutamate formation, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodization, methylation, myristoylation, oxidation, proteolysis, phosphorylation, prenylation, racemization, selenoylation, sulfated, arginylation, and other transfer RNA-mediated addition of amino acids to proteins, as well as ubiquitination. The modifications include H1 or H2, including the peptide backbone, amino acid side chains, and terminus. 1a , or H1 and H 1a It will be done everywhere.

[0113] In some embodiments, H1 includes a linking portion (L3) that connects 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 ID 50), (GGGS) n (Sequence ID 51), (GGGGS) n(Sequence ID 52), and (GSSGGS) n The formula is selected from the group consisting of (SEQ ID NO: 53), where n is at least an integer of 1. In some embodiments, L3 includes the amino acid sequence according to SEQ ID NO: 22.

[0114] In some embodiments, H 1a P 1a H 1a The connecting part (L 1a ) includes. In some embodiments, L 1a This is a peptide sequence having at least 5 to 50 amino acids. In some embodiments, L 1a This is a peptide sequence having at least 10 to 30 amino acids. In some embodiments, L 1a This is a peptide sequence having at least 10 amino acids. In some embodiments, L 1a This is a peptide sequence having at least 18 amino acids. In some embodiments, L 1a This is a peptide sequence having at least 26 amino acids. In some embodiments, L 1a (G2S) n , (GS) n (GSGGS) n (Sequence ID 50), (GGGS) n (Sequence ID 51), (GGGGS) n (Sequence ID 52), and (GSSGGS) n The formula has an expression selected from the group consisting of (Sequence ID 53), where n is at least an integer of 1. In some embodiments, L 1a This includes the amino acid sequence according to SEQ ID NO: 22.

[0115] PSMA and CD3-binding antibodies In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence disclosed in Table 6, or a sequence substantially identical thereto (e.g., a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity). In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NOs. 62-77. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NOs. 72. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NOs. 73.

[0116] [Table 6-1]

[0117] [Table 6-2]

[0118] [Table 6-3]

[0119] [Table 6-4]

[0120] In some embodiments, the polypeptide or polypeptide complex includes the amino acid sequences given by SEQ ID NOs. 62 and 63. In some embodiments, the polypeptide or polypeptide complex includes an amino acid sequence having at least 90% sequence identity with respect to SEQ ID NOs. 62 and 63. In some embodiments, the polypeptide or polypeptide complex includes an amino acid sequence having at least 95% sequence identity with respect to SEQ ID NOs. 62 and 63. In some embodiments, the polypeptide or polypeptide complex includes an amino acid sequence having at least 99% sequence identity with respect to SEQ ID NOs. 62 and 63.

[0121] In some embodiments, the polypeptide or polypeptide complex includes the amino acid sequences given by SEQ ID NOs. 64 and 65. In some embodiments, the polypeptide or polypeptide complex includes an amino acid sequence having at least 90% sequence identity with respect to SEQ ID NOs. 64 and 65. In some embodiments, the polypeptide or polypeptide complex includes an amino acid sequence having at least 95% sequence identity with respect to SEQ ID NOs. 64 and 65. In some embodiments, the polypeptide or polypeptide complex includes an amino acid sequence having at least 99% sequence identity with respect to SEQ ID NOs. 64 and 65.

[0122] In some embodiments, the polypeptide or polypeptide complex includes the amino acid sequences given by SEQ ID NOs. 66 and 67. In some embodiments, the polypeptide or polypeptide complex includes an amino acid sequence having at least 90% sequence identity with respect to SEQ ID NOs. 66 and 67. In some embodiments, the polypeptide or polypeptide complex includes an amino acid sequence having at least 95% sequence identity with respect to SEQ ID NOs. 66 and 67. In some embodiments, the polypeptide or polypeptide complex includes an amino acid sequence having at least 99% sequence identity with respect to SEQ ID NOs. 66 and 67.

[0123] In some embodiments, the polypeptide or polypeptide complex includes the amino acid sequences given by SEQ ID NOs. 68 and 69. In some embodiments, the polypeptide or polypeptide complex includes an amino acid sequence having at least 90% sequence identity with respect to SEQ ID NOs. 68 and 69. In some embodiments, the polypeptide or polypeptide complex includes an amino acid sequence having at least 95% sequence identity with respect to SEQ ID NOs. 68 and 69. In some embodiments, the polypeptide or polypeptide complex includes an amino acid sequence having at least 99% sequence identity with respect to SEQ ID NOs. 68 and 69.

[0124] In some embodiments, the polypeptide or polypeptide complex includes the amino acid sequences given by SEQ ID NOs. 70 and 71. In some embodiments, the polypeptide or polypeptide complex includes an amino acid sequence having at least 90% sequence identity with SEQ ID NOs. 70 and 71. In some embodiments, the polypeptide or polypeptide complex includes an amino acid sequence having at least 95% sequence identity with SEQ ID NOs. 70 and 71. In some embodiments, the polypeptide or polypeptide complex includes an amino acid sequence having at least 99% sequence identity with SEQ ID NOs. 70 and 71.

[0125] In some embodiments, the polypeptide or polypeptide complex includes the amino acid sequences given by SEQ ID NOs. 72 and 73. In some embodiments, the polypeptide or polypeptide complex includes an amino acid sequence having at least 90% sequence identity with respect to SEQ ID NOs. 72 and 73. In some embodiments, the polypeptide or polypeptide complex includes an amino acid sequence having at least 95% sequence identity with respect to SEQ ID NOs. 72 and 73. In some embodiments, the polypeptide or polypeptide complex includes an amino acid sequence having at least 99% sequence identity with respect to SEQ ID NOs. 72 and 73.

[0126] In some embodiments, the polypeptide or polypeptide complex includes the amino acid sequences given by SEQ ID NOs. 74 and 75. In some embodiments, the polypeptide or polypeptide complex includes an amino acid sequence having at least 90% sequence identity with respect to SEQ ID NOs. 74 and 75. In some embodiments, the polypeptide or polypeptide complex includes an amino acid sequence having at least 95% sequence identity with respect to SEQ ID NOs. 74 and 75. In some embodiments, the polypeptide or polypeptide complex includes an amino acid sequence having at least 99% sequence identity with respect to SEQ ID NOs. 74 and 75.

[0127] In some embodiments, the polypeptide or polypeptide complex includes the amino acid sequences given by SEQ ID NOs. 76 and 77. In some embodiments, the polypeptide or polypeptide complex includes an amino acid sequence having at least 90% sequence identity with respect to SEQ ID NOs. 76 and 77. In some embodiments, the polypeptide or polypeptide complex includes an amino acid sequence having at least 95% sequence identity with respect to SEQ ID NOs. 76 and 77. In some embodiments, the polypeptide or polypeptide complex includes an amino acid sequence having at least 99% sequence identity with respect to SEQ ID NOs. 76 and 77.

[0128] In some embodiments, the polypeptide or polypeptide complex includes sequences as specified in Table 6. In some embodiments, the sequences include at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs. 62–77. In some embodiments, the sequences include at least or about 95% sequence identity to any one of SEQ ID NOs. 62–77. In some embodiments, the sequences include at least or about 97% sequence identity to any one of SEQ ID NOs. 62–77. In some embodiments, the sequences include at least or about 99% sequence identity to any one of SEQ ID NOs. 62–77. In some embodiments, the sequences include at least or about 100% sequence identity to any one of SEQ ID NOs. 62–77. In some examples, the sequence includes at least a portion of one of sequence numbers 62, 65, 66, 69, 70, 73, 75, or 77, having at least or about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, or more than 210 amino acids. In some examples, the sequence contains at least a portion of either sequence number 63 or 64 having at least or about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, or more than 450 amino acids.In some examples, the array has at least or approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, Contains at least a portion having 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, or more than 640 amino acids.

[0129] As used herein, the term “amino acid sequence identity percentage (%)” with respect to a sequence is defined as the percentage of amino acid residues in a candidate sequence that, after aligning the sequences as necessary and introducing gaps, is identical to an amino acid residue in a given sequence and achieves the maximum sequence identity percentage, without considering conservative substitutions as part of sequence identity. Alignment for the purpose of determining amino acid sequence identity percentage can be achieved in a variety of ways within the scope of what a person skilled in the art can imagine, 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 can determine appropriate parameters for measuring the alignment, including any algorithms necessary to achieve the largest possible sequence that exceeds the full length of the sequences being compared. In situations where ALIGN-2 is used for amino acid sequence comparison, the amino acid sequence of a given amino acid sequence B (to, with, or against) a given amino acid sequence A (or a given amino acid sequence A that has or contains a specific amino acid sequence identity % to a given amino acid sequence B) is calculated as 100 × fraction X / Y, where X is the number of amino acid residues scored as identical 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 is recognized 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 % between A and B will not be equal to the amino acid sequence identity % between B and A. Unless otherwise specified, all amino acid sequence identity % used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

[0130] In some embodiments herein, stereochemical configuration 1:

[0131] [ka] Disclosed is an isolated polypeptide or polypeptide complex comprising a structural configuration such that the polypeptide complex comprises a single-chain variable fragment (scFv) comprising a light-chain variable domain and a heavy-chain variable domain, the scFv further comprising a peptide that impairs the binding of the scFv to an effector cell antigen, the peptide being linked to the heavy-chain variable domain of the scFv by a linking portion which is a substrate of a tumor-specific protease, and the peptide further comprising a half-life-extending molecule; and a prostate-specific membrane antigen (PSMA-binding) Fab or Fab', the Fab or Fab' comprising a Fab light-chain polypeptide chain and a Fab heavy-chain polypeptide chain, the Fab heavy-chain polypeptide chain being linked to the C-terminus of the light-chain variable domain of the scFv.

[0132] In some embodiments herein, stereochemical configuration 2:

[0133] [ka] Disclosed is an isolated polypeptide or polypeptide complex comprising a structural configuration such that the polypeptide complex comprises a single-chain variable fragment (scFv) comprising a light-chain variable domain and a heavy-chain variable domain, the scFv further comprising a peptide that impairs the binding of the scFv to an effector cell antigen, the peptide being ligated to the N-terminus of the heavy-chain variable domain of the scFv by a ligation portion which is a substrate of a tumor-specific protease, and the peptide further comprising a half-life-extending molecule; and a prostate-specific membrane antigen (PSMA-binding Fab or Fab', the Fab or Fab' comprising a Fab light-chain polypeptide chain and a Fab heavy-chain polypeptide chain, the Fab light-chain polypeptide chain being ligated to the C-terminus of the light-chain variable domain of the scFv.

[0134] In some embodiments described herein, stereochemical configuration 3:

[0135] [ka] Disclosed is an isolated polypeptide or polypeptide complex comprising a structural configuration such as, the polypeptide or polypeptide complex comprising a single-chain variable fragment (scFv) comprising a light-chain variable domain and a heavy-chain variable domain, the scFv being linked to a peptide (P1) that impairs the binding of scFv to effector cell antigens, P1 being linked to the N-terminus of the light-chain variable domain of scFv by a linking portion (L1) which is a substrate of a tumor-specific protease, and P1 being further linked to a half-life extension molecule; and a prostate-specific membrane antigen (PSMA-binding Fab, the Fab comprising a Fab light-chain polypeptide and a Fab heavy-chain polypeptide, the Fab heavy-chain polypeptide being linked to the C-terminus of the heavy-chain variable domain of scFv, the Fab being linked to P2 and L2, P2 comprising a peptide that impairs the binding of Fab to PSMA, and L2 comprising a linking portion that connects the Fab light-chain polypeptide to P2 and is a substrate of a tumor-specific protease.

[0136] In some embodiments herein, stereochemical configuration 4:

[0137] [ka] Disclosed is an isolated polypeptide or polypeptide complex comprising a structural configuration such that the polypeptide or polypeptide complex comprises a single-chain variable fragment (scFv) comprising a light-chain variable domain and a heavy-chain variable domain, the scFv being linked to a peptide that impairs the binding of the scFv to an effector cell antigen, the peptide being linked to the light-chain variable domain of the scFv by a linking portion that is a substrate of a tumor-specific protease, and the peptide being further linked to a half-life extension molecule; and a prostate-specific membrane antigen (PSMA-binding Fab, the Fab comprising a Fab light-chain polypeptide chain and a Fab heavy-chain polypeptide chain, the Fab heavy-chain polypeptide chain being linked to the C-terminus of the heavy-chain variable domain of the scFv.

[0138] In some embodiments herein, stereochemical configuration 5:

[0139] [ka] Disclosed is an isolated polypeptide or polypeptide complex comprising a structural configuration such as, the polypeptide or polypeptide complex comprising a single-chain variable fragment (scFv) comprising a light-chain variable domain and a heavy-chain variable domain, the scFv being linked to a peptide (P1) that impairs the binding of scFv to effector cell antigens, P1 being linked to the N-terminus of the light-chain variable domain of scFv by a linking portion that is a substrate of a tumor-specific protease, and P1 being further linked to a half-life extension molecule; and a prostate-specific membrane antigen (PSMA-binding) Fab, the Fab comprising a Fab light-chain polypeptide and a Fab heavy-chain polypeptide, the Fab light-chain polypeptide being linked to the C-terminus of the heavy-chain variable domain of scFv, the Fab being linked to P2 and L2, P2 comprising a peptide that impairs the binding of Fab to PSMA, and L2 comprising a linking portion that connects the Fab heavy-chain polypeptide to P2 and is a substrate of a tumor-specific protease.

[0140] In some embodiments described herein, stereochemical configuration 6:

[0141] [ka] Disclosed is an isolated polypeptide or polypeptide complex comprising a structural configuration such that the polypeptide or polypeptide complex comprises a single-chain variable fragment (scFv) comprising a light-chain variable domain and a heavy-chain variable domain, the scFv being further ligated to a peptide that impairs the binding of the scFv to an effector cell antigen, the peptide being ligated to the N-terminus of the light-chain variable domain of the scFv by a ligation portion that is a substrate of a tumor-specific protease, and the peptide being further ligated to a half-life extension molecule; and a Fab that binds to a prostate-specific membrane antigen (PSMA), the Fab comprising a Fab light-chain polypeptide chain and a Fab heavy-chain polypeptide chain, the Fab light-chain polypeptide chain being ligated to the C-terminus of the heavy-chain variable domain of the scFv.

[0142] In some embodiments herein, stereochemical configuration 7:

[0143] [ka] Disclosed is an isolated polypeptide or polypeptide complex comprising a structural configuration such as, the polypeptide or polypeptide complex comprising a single-chain variable fragment (scFv) comprising a light-chain variable domain and a heavy-chain variable domain, the scFv being linked to a peptide (P1) that impairs the binding of scFv to effector cell antigens, P1 being linked to the N-terminus of the heavy-chain variable domain of scFv by a linking portion (L1) which is a substrate of a tumor-specific protease, and P1 being further linked to a half-life extension molecule; and a prostate-specific membrane antigen (PSMA-binding Fab, the Fab comprising a Fab light-chain polypeptide and a Fab heavy-chain polypeptide, the Fab heavy-chain polypeptide being linked to the C-terminus of the light-chain variable domain of scFv, the Fab being linked to P2 and L2, P2 comprising a peptide that impairs the binding of Fab to PSMA, and L2 comprising a linking portion that connects the Fab light-chain polypeptide to P2 and is a substrate of a tumor-specific protease.

[0144] In some embodiments herein, stereochemical configuration 8:

[0145] [ka] Disclosed is an isolated polypeptide or polypeptide complex comprising a structural configuration such as, the polypeptide or polypeptide complex comprising a single-chain variable fragment (scFv) comprising a light-chain variable domain and a heavy-chain variable domain, the scFv being linked to a peptide (P1) that impairs the binding of scFv to effector cell antigens, P1 being linked to the N-terminus of the heavy-chain variable domain of scFv by a linking portion (L1) which is a substrate of a tumor-specific protease, and P1 being further linked to a half-life extension molecule; and a prostate-specific membrane antigen (PSMA-binding Fab, the Fab comprising a Fab light-chain polypeptide and a Fab heavy-chain polypeptide, the Fab light-chain polypeptide being linked to the C-terminus of the light-chain variable domain of scFv, the Fab being linked to P2 and L2, P2 comprising a peptide that impairs the binding of Fab to PSMA, and L2 comprising a linking portion that connects the Fab heavy-chain polypeptide to P2 and is a substrate of a tumor-specific protease.

[0146] In some embodiments herein, stereochemical configuration 9:

[0147] [ka] An isolated polypeptide or polypeptide complex comprising a structural configuration thereof, wherein the polypeptide or polypeptide complex is Fab that binds to prostate-specific membrane antigen (PSMA), and Fab comprises a Fab light chain polypeptide and a Fab heavy chain polypeptide, Fab is linked to a peptide (P1) that impairs the binding of Fab to PSMA, P1 is linked to the N-terminus of the Fab light chain polypeptide by a linking portion (L1) which is a substrate of a tumor-specific protease, and P1 is further linked to a half-life extension molecule, Fab and effector molecule Disclosed is an isolated polypeptide or polypeptide complex comprising a single-chain variable fragment (scFv) that binds to a cell antigen, the scFv comprising a light-chain variable domain and a heavy-chain variable domain, the heavy-chain variable domain of the scFv being ligated to the N-terminus of a Fab heavy-chain polypeptide, the scFv being ligated to P2 and L2, the P2 comprising a peptide that impairs the binding of the scFv to the effector cell antigen, and the L2 comprising a ligation portion that ligates the light-chain variable domain of the scFv to P2 and is a substrate of a tumor-specific protease.

[0148] In some embodiments of this specification, stereochemical configuration 10:

[0149] [ka] Disclosed is an isolated polypeptide or polypeptide complex comprising a structural configuration such that the polypeptide or polypeptide complex comprises Fab, which binds to prostate-specific membrane antigen (PSMA), wherein Fab comprises a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein Fab is linked to a peptide that impairs the binding of Fab to PSMA, the peptide being linked to the N-terminus of the Fab light chain polypeptide by a linking portion (L1) which is a substrate of a tumor-specific protease, and the peptide being further linked to a half-life extension molecule; and a single-chain variable fragment (scFv) which binds to an effector cell antigen, wherein scFv comprises a light chain variable domain and a heavy chain variable domain, the heavy chain variable domain of scFv being linked to the N-terminus of the Fab heavy chain polypeptide.

[0150] In some embodiments herein, stereochemical configuration 11:

[0151] [ka] An isolated polypeptide or polypeptide complex comprising a structural configuration thereof, wherein the polypeptide or polypeptide complex is Fab that binds to prostate-specific membrane antigen (PSMA), and Fab comprises a Fab light chain polypeptide and a Fab heavy chain polypeptide, Fab is linked to a peptide (P1) that impairs the binding of Fab to PSMA, P1 is linked to the N-terminus of the Fab heavy chain polypeptide by a linking portion (L1) which is a substrate of a tumor-specific protease, and P1 is further linked to a half-life extension molecule, Fab and effector cell anti Disclosed is an isolated polypeptide or polypeptide complex comprising a single-chain variable fragment (scFv) that binds to a progenitor, the scFv comprising a light-chain variable domain and a heavy-chain variable domain, the heavy-chain variable domain of the scFv being ligated to the N-terminus of a Fab light-chain polypeptide, the scFv being further ligated to P2 and L2, the P2 comprising a peptide that impairs the binding of the scFv to an effector cell antigen, and the L2 comprising a ligation portion that ligates the light-chain variable domain of the scFv to P2 and is a substrate of a tumor-specific protease.

[0152] In some embodiments herein, stereochemical configuration 12:

[0153] [ka] Disclosed is an isolated polypeptide or polypeptide complex comprising a structural configuration such that the polypeptide or polypeptide complex comprises Fab, which binds to prostate-specific membrane antigen (PSMA), wherein Fab comprises a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein Fab is linked to a peptide that impairs the binding of Fab to PSMA, the peptide being linked to the N-terminus of the Fab heavy chain polypeptide by a linking portion (L1) which is a substrate of a tumor-specific protease, and the peptide being further linked to a half-life extension molecule; and a single-chain variable fragment (scFv) which binds to an effector cell antigen, wherein scFv comprises a light chain variable domain and a heavy chain variable domain, the heavy chain variable domain of scFv being linked to the N-terminus of the Fab light chain polypeptide.

[0154] In some embodiments of this specification, stereochemical configuration 13:

[0155] [ka] An isolated polypeptide or polypeptide complex comprising a structural configuration thereof, wherein the polypeptide or polypeptide complex is Fab that binds to prostate-specific membrane antigen (PSMA), wherein Fab comprises a Fab light chain polypeptide and a Fab heavy chain polypeptide, Fab is linked to a peptide (P1) that impairs the binding of Fab to PSMA, P1 is linked to the N-terminus of the Fab light chain polypeptide by a linking portion (L1) which is a substrate of a tumor-specific protease, and P1 is further linked to a half-life extension molecule, Fab and effector cells Disclosed is an isolated polypeptide or polypeptide complex comprising an antigen-binding single-chain variable fragment (scFv), the scFv comprising a light-chain variable domain and a heavy-chain variable domain, the light-chain variable domain of the scFv being ligated to the N-terminus of a Fab heavy-chain polypeptide, the scFv being ligated to P2 and L2, the scFv comprising a peptide that impairs the binding of the scFv to the effector cell antigen, and the L2 comprising a ligation portion that ligates the heavy-chain variable domain of the scFv to P2 and is a substrate of a tumor-specific protease.

[0156] In some embodiments herein, stereochemical configuration 14:

[0157] [ka] Disclosed is an isolated polypeptide or polypeptide complex comprising a structural configuration such that the polypeptide or polypeptide complex comprises Fab, which binds to prostate-specific membrane antigen (PSMA), wherein Fab comprises a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein Fab is linked to a peptide that impairs the binding of Fab to PSMA, the peptide being linked to the N-terminus of the Fab light chain polypeptide by a linking portion that is a substrate of a tumor-specific protease, and the peptide being further linked to a half-life extension molecule; and a single-chain variable fragment (scFv) which binds to an effector cell antigen, wherein scFv comprises a light chain variable domain and a heavy chain variable domain, the light chain variable domain of scFv being linked to the N-terminus of the Fab heavy chain polypeptide.

[0158] In some embodiments of this specification, stereochemical configuration 15:

[0159] [ka] An isolated polypeptide or polypeptide complex comprising a structural configuration thereof, wherein the polypeptide or polypeptide complex is Fab that binds to prostate-specific membrane antigen (PSMA), wherein Fab comprises a Fab light chain polypeptide and a Fab heavy chain polypeptide, Fab is linked to a linkage (P1) that impairs the binding of Fab to PSMA, P1 is linked to the N-terminus of the Fab heavy chain polypeptide by a linkage (L1) which is a substrate of a tumor-specific protease, and P1 is further linked to a half-life extension molecule, Fab and effector cell antigen Disclosed is an isolated polypeptide or polypeptide complex comprising a single-chain variable fragment (scFv) that binds to a Fab light chain polypeptide, the scFv comprising a light chain variable domain and a heavy chain variable domain, the light chain variable domain of the scFv being ligated to the N-terminus of the Fab light chain polypeptide, the scFv being ligated to P2 and L2, the scFv comprising a peptide that impairs the binding of the scFv to the effector cell antigen, and the L2 comprising a ligation portion that connects the heavy chain variable domain of the scFv to P2 and is a substrate of a tumor-specific protease.

[0160] In some embodiments herein, stereochemical configuration 16:

[0161] [ka] Disclosed is an isolated polypeptide or polypeptide complex comprising a structural configuration such that the polypeptide or polypeptide complex comprises Fab, which binds to prostate-specific membrane antigen (PSMA), wherein Fab comprises a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein Fab is linked to a peptide that impairs the binding of Fab to PSMA, the peptide being linked to the N-terminus of the Fab heavy chain polypeptide by a linking portion (L1) which is a substrate of a tumor-specific protease, and the peptide being further linked to a half-life extension molecule; and a single-chain variable fragment (scFv) which binds to an effector cell antigen, wherein scFv comprises a light chain variable domain and a heavy chain variable domain, the light chain variable domain of scFv being linked to the N-terminus of the Fab light chain polypeptide.

[0162] Polynucleotides that encode polypeptides or polypeptide complexes In some embodiments herein, isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes disclosed herein are disclosed. In some embodiments, the polypeptide or polypeptide complex comprises an antibody or antibody fragment. In some embodiments, the polypeptide or polypeptide complex comprises a Fab and a single-chain variable fragment (scFv).

[0163] In some embodiments of this specification, Formula I: A2-A1-L1-P1-H1 (Formula I) Isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes by An isolated recombinant nucleic acid molecule is disclosed, wherein A1 comprises a first antigen-recognizing molecule that binds to an effector cell antigen, P1 comprises a peptide that binds to A1, L1 comprises a ligation portion that connects A1 to P1 and is a substrate for a tumor-specific protease, H1 comprises a half-life extension molecule, and A2 comprises a second antigen-recognizing molecule that binds to prostate-specific membrane antigen (PSMA).

[0164] In some embodiments of this specification, Formula I: A2-A1-L1-P1-H1 (Formula I) Isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes by An isolated recombinant nucleic acid molecule is disclosed, wherein A1 is a first antigen-recognizing molecule that binds to effector cell antigens, P1 is a peptide that binds to A1, L1 is a ligation portion that connects A1 to P1 and is a substrate for tumor-specific proteases, H1 is a half-life extension molecule, and A2 is a second antigen-recognizing molecule that binds to prostate-specific membrane antigen (PSMA).

[0165] In some embodiments of this specification, Formula I: A2-A1-L1-P1-H1 (Formula I) Isolated recombinant nucleic acid molecules encoding a polypeptide or polypeptide complex containing, An isolated recombinant nucleic acid molecule is disclosed, wherein A1 comprises a first antigen-recognizing molecule that binds to an effector cell antigen, P1 comprises a peptide that binds to A1, L1 comprises a ligation portion that connects A1 to P1 and is a substrate for a tumor-specific protease, H1 comprises a half-life extension molecule, and A2 comprises a second antigen-recognizing molecule that binds to prostate-specific membrane antigen (PSMA).

[0166] In some embodiments of this specification, Formula I: A2-A1-L1-P1-H1 (Formula I) Isolated recombinant nucleic acid molecules encoding a polypeptide or polypeptide complex containing, An isolated recombinant nucleic acid molecule is disclosed, wherein A1 is a first antigen-recognizing molecule that binds to effector cell antigens, P1 is a peptide that binds to A1, L1 is a ligation portion that connects A1 to P1 and is a substrate for tumor-specific proteases, H1 is a half-life extension molecule, and A2 is a second antigen-recognizing molecule that binds to prostate-specific membrane antigen (PSMA).

[0167] In some embodiments herein, Formula Ia: P2-L2-A2-A1-L1-P1-H1 (Formula Ia) Isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes are disclosed.

[0168] In some embodiments herein, Formula II: L 1a -P 1a -H 1a (Formula II) Isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes by In the formula, L 1a If not cleaved, P is the first antigen-recognition molecule that binds to the effector cell antigen. 1a The first antigen-recognition molecule is connected to a second antigen-recognition molecule that binds to prostate-specific membrane antigen (PSMA), and the first antigen-recognition molecule is connected to a second antigen-recognition molecule that binds to prostate-specific membrane antigen (PSMA). 1a L 1a If it is not cleaved, it contains a peptide that binds to the first antigen recognition molecule, H 1a This discloses isolated recombinant nucleic acid molecules, including half-life extension molecules.

[0169] In some embodiments herein, Formula II: L 1a -P 1a -H 1a (Formula II) Isolated recombinant nucleic acid molecules encoding a polypeptide or polypeptide complex containing, In the formula, L 1a If not cleaved, P is the first antigen-recognition molecule that binds to the effector cell antigen. 1a The first antigen-recognition molecule is connected to a second antigen-recognition molecule that binds to prostate-specific membrane antigen (PSMA), and the first antigen-recognition molecule is connected to a second antigen-recognition molecule that binds to prostate-specific membrane antigen (PSMA). 1a L 1a If it is not cleaved, it contains a peptide that binds to the first antigen recognition molecule, H 1aThis discloses isolated recombinant nucleic acid molecules, including half-life extension molecules.

[0170] In some embodiments herein, Formula II: L 1a -P 1a -H 1a (Formula II) Isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes by In the formula, L 1a If not cleaved, P is the first antigen-recognition molecule that binds to the effector cell antigen. 1a The linkage is cleaved by a tumor-specific protease, and the first antigen-recognizing molecule is connected to a second antigen-recognizing molecule that binds to prostate-specific membrane antigen (PSMA), P 1a L 1a If it is not cleaved, it is a peptide that binds to the first antigen recognition molecule, H 1a This discloses isolated recombinant nucleic acid molecules that are half-life extension molecules.

[0171] In some embodiments herein, Formula II: L 1a -P 1a -H 1a (Formula II) Isolated recombinant nucleic acid molecules encoding a polypeptide or polypeptide complex containing, In the formula, L 1a If not cleaved, P is the first antigen-recognition molecule that binds to the effector cell antigen. 1a The linkage is cleaved by a tumor-specific protease, and the first antigen-recognizing molecule is connected to a second antigen-recognizing molecule that binds to prostate-specific membrane antigen (PSMA), P 1a L 1a If it is not cleaved, it is a peptide that binds to the first antigen recognition molecule, H 1a This discloses isolated recombinant nucleic acid molecules that are half-life extension molecules.

[0172] In some embodiments herein, stereochemical configuration 1:

[0173] [ka] Disclosed is an isolated nucleic acid molecule encoding a polypeptide or polypeptide complex having a structural configuration thereof, wherein the polypeptide complex is a single-chain variable fragment (scFv) comprising a light-chain variable domain and a heavy-chain variable domain, the scFv further comprising a peptide that impairs the binding of the scFv to an effector cell antigen, the peptide being linked to the heavy-chain variable domain of the scFv by a linking portion which is a substrate of a tumor-specific protease, and the peptide further comprising a half-life-extending molecule, and an isolated nucleic acid molecule comprising a Fab or Fab' that binds to prostate-specific membrane antigen (PSMA), the Fab or Fab' comprising a Fab light-chain polypeptide chain and a Fab heavy-chain polypeptide chain, the Fab heavy-chain polypeptide chain being linked to the C-terminus of the light-chain variable domain of the scFv.

[0174] In some embodiments herein, stereochemical configuration 2:

[0175] [ka] Disclosed is an isolated nucleic acid molecule encoding a polypeptide or polypeptide complex having a structural configuration thereof, wherein the polypeptide complex is a single-chain variable fragment (scFv) comprising a light-chain variable domain and a heavy-chain variable domain, the scFv further comprising a peptide that impairs the binding of scFv to effector cell antigens, the peptide being ligated to the N-terminus of the heavy-chain variable domain of scFv by a ligation portion which is a substrate of a tumor-specific protease, and the peptide further comprising a half-life extension molecule, and an isolated nucleic acid molecule comprising Fab or Fab' that binds to prostate-specific membrane antigen (PSMA), the Fab or Fab' comprising a Fab light-chain polypeptide chain and a Fab heavy-chain polypeptide chain, the Fab light-chain polypeptide chain being ligated to the C-terminus of the light-chain variable domain of scFv.

[0176] Pharmaceutical composition In some embodiments herein, pharmaceutical compositions are disclosed comprising (a) a polypeptide or polypeptide complex disclosed herein and (b) a pharmaceutically acceptable excipient.

[0177] In some embodiments, the pharmaceutical composition is (a) Formula I: A2-A1-L1-P1-H1 (Formula I) A polypeptide or polypeptide complex comprising, (b) a polypeptide or polypeptide complex comprising: (a) a pharmaceutically acceptable excipient, wherein A1 comprises a first antigen-recognizing molecule that binds to an effector cell antigen; P1 comprises a peptide that binds to A1; L1 comprises a linking portion that connects A1 to P1 and is a substrate for a tumor-specific protease; H1 comprises a half-life-extending molecule; and A2 comprises a second antigen-recognizing molecule that binds to prostate-specific membrane antigen (PSMA).

[0178] In some embodiments, the pharmaceutical composition is (a) Formula I: A2-A1-L1-P1-H1 (Formula I) A polypeptide or polypeptide complex comprising, The formula comprises (b) a polypeptide or polypeptide complex in which A1 is a first antigen-recognizing molecule that binds to effector cell antigens, P1 is a peptide that binds to A1, L1 is a ligation portion that connects A1 to P1 and is a substrate for tumor-specific proteases, H1 is a half-life extension molecule, and A2 is a second antigen-recognizing molecule that binds to prostate-specific membrane antigen (PSMA), and (b) a pharmaceutically acceptable excipient.

[0179] In some embodiments, the pharmaceutical composition is (a) Formula I: A2-A1-L1-P1-H1 (Formula I) A polypeptide or polypeptide complex comprising, (b) a polypeptide or polypeptide complex comprising: (a) a pharmaceutically acceptable excipient, wherein A1 comprises a first antigen-recognizing molecule that binds to an effector cell antigen; P1 comprises a peptide that binds to A1; L1 comprises a linking portion that connects A1 to P1 and is a substrate for a tumor-specific protease; H1 comprises a half-life-extending molecule; and A2 comprises a second antigen-recognizing molecule that binds to prostate-specific membrane antigen (PSMA).

[0180] In some embodiments, the pharmaceutical composition is (a) Formula I: A2-A1-L1-P1-H1 (Formula I) A polypeptide or polypeptide complex comprising, The formula comprises (b) a polypeptide or polypeptide complex in which A1 is a first antigen-recognizing molecule that binds to effector cell antigens, P1 is a peptide that binds to A1, L1 is a ligation portion that connects A1 to P1 and is a substrate for tumor-specific proteases, H1 is a half-life extension molecule, and A2 is a second antigen-recognizing molecule that binds to prostate-specific membrane antigen (PSMA), and (b) a pharmaceutically acceptable excipient.

[0181] In some embodiments, the pharmaceutical composition is (a) Formula Ia: P2-L2-A2-A1-L1-P1-H1 (Formula Ia) (b) a polypeptide or polypeptide complex and (b) a pharmaceutically acceptable excipient.

[0182] In some embodiments, the pharmaceutical composition is (a) Formula II: L 1a -P 1a -H 1a (Formula II) A polypeptide or polypeptide complex comprising, In the formula, L 1a If not cleaved, P is the first antigen-recognition molecule that binds to the effector cell antigen. 1a The first antigen-recognition molecule is connected to a second antigen-recognition molecule that binds to prostate-specific membrane antigen (PSMA), and the first antigen-recognition molecule is connected to a second antigen-recognition molecule that binds to prostate-specific membrane antigen (PSMA). 1a L 1a If it is not cleaved, it contains a peptide that binds to the first antigen recognition molecule, H 1a (b) a polypeptide or polypeptide complex containing a half-life extender, and (b) a pharmaceutically acceptable excipient.

[0183] In some embodiments, the pharmaceutical composition is (a) Formula II: L 1a -P 1a -H 1a (Formula II) A polypeptide or polypeptide complex comprising, In the formula, L 1a If not cleaved, P is the first antigen-recognition molecule that binds to the effector cell antigen. 1a The first antigen-recognition molecule is connected to a second antigen-recognition molecule that binds to prostate-specific membrane antigen (PSMA), and the first antigen-recognition molecule is connected to a second antigen-recognition molecule that binds to prostate-specific membrane antigen (PSMA). 1a L 1a If it is not cleaved, it contains a peptide that binds to the first antigen recognition molecule, H 1a (b) a polypeptide or polypeptide complex containing a half-life extender, and (b) a pharmaceutically acceptable excipient.

[0184] In some embodiments, the pharmaceutical composition is (a) Formula II: L 1a -P 1a -H 1a (Formula II) A polypeptide or polypeptide complex comprising, In the formula, L 1a If not cleaved, P is the first antigen-recognition molecule that binds to the effector cell antigen. 1a The linkage is cleaved by a tumor-specific protease, and the first antigen-recognizing molecule is connected to a second antigen-recognizing molecule that binds to prostate-specific membrane antigen (PSMA), P 1a L 1a If it is not cleaved, it is a peptide that binds to the first antigen recognition molecule, H 1a (b) a polypeptide or polypeptide complex that is a half-life extension molecule, and (b) a pharmaceutically acceptable excipient.

[0185] In some embodiments, the pharmaceutical composition is (a) Formula II: L 1a -P 1a -H 1a (Formula II) A polypeptide or polypeptide complex comprising, In the formula, L 1a If not cleaved, P is the first antigen-recognition molecule that binds to the effector cell antigen. 1a The linkage is cleaved by a tumor-specific protease, and the first antigen-recognizing molecule is connected to a second antigen-recognizing molecule that binds to prostate-specific membrane antigen (PSMA), P 1a L 1a If it is not cleaved, it is a peptide that binds to the first antigen recognition molecule, H 1a (b) a polypeptide or polypeptide complex that is a half-life extension molecule, and (b) a pharmaceutically acceptable excipient.

[0186] In some embodiments herein, the pharmaceutical composition is (a) stereostructure 1:

[0187] [ka] A polypeptide or polypeptide complex comprising a structural configuration thereof, wherein the polypeptide complex comprises a single-chain variable fragment (scFv) comprising a light-chain variable domain and a heavy-chain variable domain, the scFv further comprising a peptide that impairs the binding of the scFv to an effector cell antigen, the peptide being linked to the heavy-chain variable domain of the scFv by a linking portion that is a substrate of a tumor-specific protease, and the peptide further comprising a half-life-extending molecule, and (b) a polypeptide or polypeptide complex comprising Fab or Fab' that binds to prostate-specific membrane antigen (PSMA), the Fab or Fab' comprising a Fab light-chain polypeptide chain and a Fab heavy-chain polypeptide chain, the Fab heavy-chain polypeptide chain being linked to the C-terminus of the light-chain variable domain of the scFv, and (b) a pharmaceutically acceptable excipient.

[0188] In some embodiments herein, the pharmaceutical composition is (a) stereoconformation 2:

[0189] [ka] A polypeptide or polypeptide complex comprising a structural configuration thereof, wherein the polypeptide complex comprises a single-chain variable fragment (scFv) comprising a light-chain variable domain and a heavy-chain variable domain, the scFv further comprising a peptide that impairs the binding of scFv to effector cell antigens, the peptide being ligated to the N-terminus of the heavy-chain variable domain of scFv by a ligation portion which is a substrate of a tumor-specific protease, and the peptide further comprising a half-life-extending molecule, wherein each time, a polypeptide or polypeptide complex comprising Fab or Fab' that binds to prostate-specific membrane antigen (PSMA), wherein Fab or Fab' comprises a Fab light-chain polypeptide chain and a Fab heavy-chain polypeptide chain, the Fab light-chain polypeptide chain being ligated to the C-terminus of the light-chain variable domain of scFv, and (b) a pharmaceutically acceptable excipient.

[0190] 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, an radiolabel, an enzyme, a nucleic acid probe, or a contrast agent.

[0191] For administration to a subject, the polypeptides or polypeptide complexes disclosed herein may be provided in a pharmaceutical composition with one or more pharmaceutically acceptable carriers or additives. The term “pharmaceutically acceptable carrier” includes, but is not limited to, any carrier that does not interfere with the efficacy of the bioactivity of the component and is non-toxic to the patient receiving the administration. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate-buffered saline, water, emulsions such as oil / water emulsions, various types of wetting agents, and sterile solutions. Such carriers can be formulated in conventional methods and administered to the subject in a suitable dose. Preferably, the composition is sterile. These compositions may further contain adjuvants such as preservatives, emulsifiers, and dispersants. Prevention of microbial activity may be ensured by inclusion bodies of various antimicrobial and antifungal agents.

[0192] The pharmaceutical composition may be in any preferred form (depending on the desired method of administration). The pharmaceutical composition may be provided in unit dosage forms, in sealed containers, and as part of a kit. Such a kit may include instructions for use. The kit may contain multiple of the unit dosage forms described above.

[0193] Pharmaceutical compositions may be suitable for administration via any appropriate route, including parenteral (e.g., subcutaneous, intramuscular, or intravenous) routes. Such compositions may be prepared by any method known in the pharmaceutical art, for example, by mixing an active ingredient with a carrier or excipient under sterile conditions.

[0194] The dosage of the substances disclosed herein may vary widely depending on the disease or disorder being treated, the age and condition of the individual being treated, and the physician will ultimately determine the appropriate dosage to be used.

[0195] Treatment method Some embodiments are methods for treating a target cancer requiring treatment, comprising the step of administering an isolated polypeptide or polypeptide complex described herein to the target. In some embodiments, the cancer has cells expressing PSMA. In some examples, the cancer is a solid tumor carcinoma. In some embodiments, the cancer is a cancer of the lung, breast (e.g., HER2+, ER / PR+, TNBC), cervix, ovary, colon, pancreas, or stomach.

[0196] Some embodiments are methods for treating prostate cancer in subjects requiring treatment for prostate cancer, comprising the step of administering an isolated polypeptide or polypeptide complex described herein to the subject. Some embodiments are methods for treating metastatic castration-resistant prostate cancer (mCRPC) in subjects requiring treatment for mCRPC, comprising the step of administering an isolated polypeptide or polypeptide complex described herein to the subject.

[0197] In some embodiments herein, isolated polypeptides or polypeptide complexes having long half-lives are described. In some examples, the half-lives of the polypeptides or polypeptide complexes are at least or about 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 84 hours, 96 hours, 100 hours, 108 hours, 119 hours, 120 hours, 140 hours, 160 hours, 180 hours, 200 hours, or more than 200 hours. In some examples, the half-lives of the polypeptides or polypeptide complexes are in the range of about 12 hours to about 300 hours, about 20 hours to about 280 hours, about 40 hours to about 240 hours, about 60 hours to about 200 hours, or about 80 hours to about 140 hours.

[0198] In some embodiments of this specification, polypeptides or polypeptide complexes administered once weekly are described. In some embodiments, the polypeptide or polypeptide complex is administered once weekly by intravenous, intramuscular, intra-injury, topical, subcutaneous, infusion, or orally. In some embodiments, the polypeptide or polypeptide complex is administered once weekly by bolus injection. In some embodiments, the polypeptide or polypeptide complex is administered once weekly by continuous infusion. In some embodiments, the polypeptide or polypeptide complex is administered once weekly to a subject as a continuous infusion for a period of 60 minutes or less. In some embodiments, the polypeptide or polypeptide complex is administered once weekly to a subject as a continuous intravenous infusion for a period of 60 minutes or less. In some embodiments, the polypeptide or polypeptide complex is administered once weekly to a subject as a continuous intravenous infusion for a period of at least 10 minutes.

[0199] In some embodiments, the polypeptide or polypeptide complex is administered to the subject once a week, and the half-life of the polypeptide or polypeptide complex is at least 30 hours. In some embodiments, the polypeptide or polypeptide complex is administered to the subject once a week, and the half-life of the polypeptide or polypeptide complex is at least 50 hours. In some embodiments, the polypeptide or polypeptide complex is administered to the subject once a week, and the half-life of the polypeptide or polypeptide complex is at least 60 hours. In some embodiments, the polypeptide or polypeptide complex is administered to the subject once a week, and the half-life of the polypeptide or polypeptide complex is at least 70 hours. In some embodiments, the polypeptide or polypeptide complex is administered to the subject once a week, and the half-life of the polypeptide or polypeptide complex is at least 80 hours. In some embodiments, the polypeptide or polypeptide complex is administered to the subject once a week, and the half-life of the polypeptide or polypeptide complex is at least 90 hours. In some embodiments, the polypeptide or polypeptide complex is administered to the subject once a week, and the half-life of the polypeptide or polypeptide complex is at least 100 hours. In some embodiments, the polypeptide or polypeptide complex is administered to the subject once a week, and the half-life of the polypeptide or polypeptide complex is at least 110 hours. In some embodiments, the polypeptide or polypeptide complex is administered to the subject once a week, and the half-life of the polypeptide or polypeptide complex is at least 115 hours. In some embodiments, the polypeptide or polypeptide complex is administered to the subject once a week, and the half-life of the polypeptide or polypeptide complex is at least 119 hours.

[0200] Production of antibodies that bind to PSMA and CD3 In some embodiments, the polypeptides described herein (e.g., antibodies and their conjugated fragments) are produced by any method known in the art as useful for the synthesis of polypeptides (e.g., antibodies), specifically by chemical synthesis or recombinant expression, and preferably by recombinant expression techniques.

[0201] In some cases, the antibody or its binding fragment is expressed by recombination, and the nucleic acid encoding the antibody or its binding fragment is assembled from chemically synthesized oligonucleotides (e.g., those described by Kutmeier et al. in "BioTechniques 17:242" in 1994), requiring the synthesis of duplicate oligonucleotides containing multiple portions of the antibody-encoding sequence, annealing and ligation of these oligonucleotides, and subsequent PCR amplification of the ligated oligonucleotides.

[0202] Alternatively, nucleic acid molecules encoding antibodies can be optionally generated from a suitable source (e.g., an antibody cDNA library, or a cDNA library 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 particular gene sequence.

[0203] In some cases, the antibody or its binding fragment is optionally generated by immunizing an animal such as a mouse to produce a polyclonal antibody, or more preferably by generating a monoclonal antibody as described, for example, by Kohler and Milstein (1975, Nature 256:495-497), by Kozbor et al. (1983, Immunology Today 4:72), or by 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 can be optionally obtained by screening a Fab expression library for clones of Fab fragments that bind to specific antigens (e.g., as described by Huse et al. in Science 246:1275-1281, 1989) or by screening an antibody library (see, for example, Clackson et al. in Nature 352:624, 1991, and Hane et al. in Proc.Natl.Acad.Sci.USA 94:4937, 1997).

[0204] In some embodiments, techniques developed to produce "chimeric antibodies" by splicing genes from appropriate antigen-specific mouse antibody molecules together with genes from appropriately bioactive human antibody molecules are used (Morrison et al., 1984, Proc.Natl.Acad.Sci.81:851-855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454). Chimeric antibodies are molecules in which various parts originate from different animal species, such as those having a variable region derived from a mouse monoclonal antibody and a constant region from a human immunoglobulin.

[0205] In some embodiments, techniques described for the production of single-chain antibodies (U.S. Patent No. 4,694,778, Science 242:423-42 by Bird, Proc.Natl.Acad.Sci.USA 85:5879-5883 by Huston et al., and Nature 334:544-54 by Ward et al., 1989) are suitable for producing single-chain antibodies. Single-chain antibodies are formed by linking heavy and light chain fragments of the Fv site via amino acid crosslinking, resulting in single-chain polypeptides. Techniques for assembling functional Fv fragments in E. coli are also used optionally (Science 242:1038-1041 by Skerra et al., 1988).

[0206] In some embodiments, an expression vector containing the antibody nucleotide sequence, or the antibody nucleotide sequence itself, is transferred to host cells by conventional techniques (e.g., electroporation, liposome transfection, and calcium phosphate precipitation), and the transfected cells are then cultured by conventional techniques for antibody production. In certain embodiments, antibody expression is regulated by a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

[0207] In some embodiments, various host expression vector systems are used to express the antibodies or their conjugated fragments described herein. Such host expression systems represent not only vehicles that produce and subsequently purify the antibody coding sequence, but also cells that express the antibody or its conjugated fragment in situ when transformed or transfected with a suitable nucleotide coding sequence. These include microorganisms such as bacteria (e.g., Escherichia coli and Bacillus subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the coding sequence of the antibody or its conjugated fragment, yeast (e.g., Saccharomyces picia) transformed with recombinant yeast expression vectors containing the coding sequence of the antibody or its conjugated fragment, insect cell lines infected with recombinant virus expression vectors (e.g., baculovirus) containing the coding sequence of the antibody or its conjugated fragment, and recombinant virus expression vectors (e.g., cauliflower mosaic virus). Examples include, but are not limited to, plant cell lines infected with (CaMV and tobacco mosaic virus (TMV)) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmids) containing the coding sequence of antibodies or their binding fragments, or mammalian cell lines (e.g., COS, CHO, BH, 293, 293T, 3T3 cells) containing recombinant expression constructs derived from mammalian cell genomes (e.g., metallothionein promoter) or mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter).

[0208] For long-term, high-yield production of recombinant proteins, stable expression is desirable. In some cases, cell lines that stably express antibodies are manipulated by arbitrary selection. Rather than using expression vectors that contain viral replication origins, host cells are transformed with DNA controlled by appropriate expression regulators (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and selectable markers. After introduction of exogenous DNA, the manipulated cells are grown in enriched medium for 1-2 days, then switched to selective medium. The selectable markers in the recombinant plasmid provide resistance to selection, allowing the cells to grow to form a foci that stably integrates the plasmid into their chromosomes, is cloned, and expands into a cell line. This method can be advantageously used to manipulate cell lines that express antibodies or their binding fragments.

[0209] In some examples, numerous selection systems have been used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine guanine phosphoribosyltransferase (Szybalska and Szybalski, 192, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes, which are used for tk-, hgprt-, or aprt- cells, respectively. Furthermore, antimetabolite resistance is also associated with the following genes: dhfr, which imparts resistance to methotrexate (Proc.Natl.Acad.Sci.USA 77:357 by Wigler et al., 1980; Proc.Natl.Acad.Sci.USA 78:1527 by O'Hare et al., 1981); gpt, which imparts resistance to mycophenolate (Proc.Natl.Acad.Sci.USA 78:2072 by Mulligan and Berg, 1981); and neo, which imparts resistance to aminoglycoside G-418 (Clinical Pharmacy 12:488-505; Biotherapy by Wu and Wu, 1991). It is used as a selection criterion for hygromycin-resistant hygro (Santerre et al., Gene 30:147, 1984). (3:87-95, Ann. Rev. Pharmacol. Toxicol. 32:573-596 by Tolstoshev, Science 260:926-932, Ann. Rev. Biochem. 62:191-217, 1993; May 1993, TIB TECH 11(5):155-215) by Morgan and Anderson.Well-known methods in the field of recombinant DNA techniques that are available are described in Ausubel et al. (eds.), *Current Protocols in Molecular Biology*, John Wiley & Sons, NY, 1993; Kriegler, *Gene Transfer and Expression*, A Laboratory Manual, Stockton Press, NY, 1990; Chapters 12 and 13 of *Current Protocols in Human Genetics*, John Wiley & Sons, NY, 1994, edited by Dracopoli et al.; and Colberre-Garapin et al., *J.Mol.Biol.150:1*, 1981.

[0210] In some cases, antibody expression levels increase with vector amplification (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) for commentary). If the marker in the antibody-expressing vector system is amplified, the number of copies of the marker gene increases due to the increased level of the inhibitor present in the host cell culture. Since the amplified site is related to the antibody's nucleotide sequence, antibody production also increases (Crouse et al., 1983, Mol. Cell Biol. 3:257).

[0211] In some cases, any method known in the field of antibody purification is used, for example, by chromatography (e.g., by ion exchange, affinity, specifically by affinity of protein A to specific antigens, and sizing column chromatography), centrifugation, differential solubility, or other standard techniques for protein purification.

[0212] Expression vector In some embodiments, the vectors also include any suitable vectors derived from either eukaryotic or prokaryotic sources. In some cases, the vectors are obtained from bacterial (e.g., Escherichia coli), insect, yeast (e.g., Pichia pastris), algae, or mammalian sources. Exemplary bacterial vectors include pACYC177, pASK75, the pBAD vector series, the pBADM vector series, the pET vector series, the pETM vector series, the pGEX vector series, pHAT, pHAT2, pMal-c2, pMal-p2, the pQE vector series, pRSET A, pRSET B, pRSET C, the pTrcHis2 series, pZA31-Luc, pZE21-MCS-1, pFLAG ATS, pFLAG CTS, pFLAG MAC, pFLAG Shift-12c, pTAC-MAT-1, pFLAG CTC, or pTAC-MAT-2.

[0213] Examples of insect vectors include 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, FLAG vectors such as pPolh-FLAG1 or pPolh-MAT2, or MAT vectors such as pPolh-MAT1 or pPolh-MAT2.

[0214] Depending on the case, yeast vectors may 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 pastris vector, pGAPZA,B,&C pichia pastris 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.

[0215] Examples of algal vectors include the pChlamy-4 vector or the MCS vector.

[0216] Examples of mammalian vectors include transient expression vectors and 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 hpBICEP-CMV 4. Stable expression vectors for mammals 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.

[0217] In some cases, cell-free systems are mixtures of cytoplasmic and / or nuclear components from cells used for nucleic acid synthesis in vitro. Depending on the case, cell-free systems utilize either prokaryotic or eukaryotic components. Sometimes, nucleic acid synthesis is obtained using cell-free systems based on, for example, Drosophila cells, Xenopus eggs, or HeLa cells. Exemplary cell-free systems include, but are not limited to, the E. coli S30 extract system, the E. coli T7 S30 system, or PURExpress®.

[0218] host cell In some embodiments, the host cell may be any suitable cell, such as naturally occurring cells or genetically modified cells. In some examples, the host cell is a production host cell. In some examples, the host cell is a eukaryotic cell. In other examples, the host cell is a prokaryotic cell. Sometimes, eukaryotic cells include fungi (e.g., yeast), animal cells, or plant cells. Sometimes, prokaryotic cells are bacterial cells. Examples of bacterial cells include Gram-positive or Gram-negative bacteria. Sometimes, Gram-negative bacteria are anaerobic, rod-shaped, or both.

[0219] In some cases, Gram-positive bacteria include Actinobacteria, Firmicutes, or Tenericutes. In some cases, Gram-negative bacteria include Aquifexus, Deinococcus thermus, Fibrobacter-Chlorobium / Bacteroidetes (FCB group), Fusobacterium, Gemmatimonas, Nitrospira, Planctomyces-Vercomicrobium / Chlamydia (PVC group), Proteobacteria, Spirochetes, or Synergistes. Other bacteria may include Acidobacterium, Chloroflexus, Chrysiogenes, Cyanobacteria, Deferibacter, Dictyoglomi, Thermodesulfobacteria, or Thermotoga. Bacterial cells may also be Escherichia coli, Clostridium botulinum, or Coli bacilli.

[0220] Examples of prokaryotic host cells include, but are not limited to, BL21, Mach1(trademark), DH10B(trademark), TOP10, DH5α, DH10Bac(trademark), OmniMax(trademark), MegaX(trademark), DH12S(trademark), INV110, TOP10F', INVαF, TOP10 / P3, ccdB Survival, PIR1, PIR2, Stbl2(trademark), Stbl3(trademark), or Stbl4(trademark).

[0221] In some cases, animal cells include cells from vertebrates or invertebrates. In some cases, animal cells include cells from marine invertebrates, fish, insects, amphibians, reptiles, or mammals. In some cases, fungal cells include yeast cells such as brewer's yeast, baker's yeast, or wine yeast.

[0222] Fungi include yeasts, molds, filamentous fungi, basidiomycetes, or ascomycetes such as zygomycetes. In some cases, yeasts may belong to the Ascomycota or Basidiomycota phyla. In some cases, Ascomycota may include the Saccharomyces subphylum (true yeasts, e.g., Saccharomyces cerevisiae (baker's yeast)) or the Taflynamita subphylum (e.g., Schizosaccharomyces (fission yeast)). In some cases, Basidiomycota may include the Agaricus subphylum (e.g., Tremella class) or the Rust-forming subphylum (e.g., Microbotryomycetes class).

[0223] Exemplary yeasts or filamentous fungi include, for example, the genera: Saccharomyces, Schizosaccharomyces, Candida, Pichia, Hansenula, Kruywelomyces, Tygosaccharomyces, Yarrowia, Trichosporon, Rhodosporidi, Aspergillus, Fusarium, or Trichoderma. Examples of yeasts or filamentous fungi include, for example, the following species: Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida yutilis, Candida boidini, Candida albicans, Candida tropicalis, Candida stellatoidea, Candida glabrata, Candida crusei, Candida parasylosis, Candida gilliermondi, Candida biswanati, Candida lusitani, Rhodotorula musiraginosa, Pichia metanolica, Pichia angusta Examples include *angusta*, *Pichia pastris*, *Pichia anomala*, *Hansenula polymorpha*, *Cluiveromyces lactis*, *Tygosaccharomyces lucii*, *Yarrowia liporitica*, *Trichosporon pullulan*, *Rhodosporidium toru-Aspergillus niger*, *Aspergillus nidurans*, *Aspergillus awamori*, *Aspergillus oryzae*, *Trichoderma risei*, *Yarrowia liporitica*, *Brettanomyces bruccellensis*, *Candida stellata*, *Schizosaccharomyces pombe*, *Torraspora delbruccii*, *Tygosaccharomyces baili*, *Cryptococcus neoformans*, *Cryptococcus gattii*, or *Saccharomyces braudii*.

[0224] Exemplary yeast host cells include, but are not limited to, Pichia pastris yeast strains such as GS115, KM71H, SMD1168, SMD1168H, and X-33, as well as Saccharomyces cerevisiae yeast strains such as INVSc1.

[0225] In some cases, additional animal cells may be derived from mollusks, arthropods, annelids, or sponges. In some cases, additional animal cells may be mammalian cells from primates, apes, horses, cats, pigs, dogs, cats, or rodents. In some cases, rodents may include mice, rats, hamsters, gerbils, chinchillas, fancy rats, or guinea pigs.

[0226] Exemplary mammalian host cells include 293A cell line, 293FT cell line, 293F cells, 293H cells, CHO DG44 cells, CHO-S cells, CHO-K1 cells, FUT8 KO CHOK1, Expi293F(TM) cells, Flp-In(TM) T-REx(TM) 293 cell line, Flp-In(TM)-293 cell line, Flp-In(TM)-3T3 cell line, Flp-In(TM)-BHK cell line, Flp-In(TM) )-CHO cell line, Flp-In(TM)-CV-1 cell line, Flp-In(TM)-Jurkat cell line, FreeStyle(TM) 293-F cells, FreeStyle(TM) CHO-S cells, GripTite(TM) 293 Examples include, but are not limited to, MSR cell lines, GS-CHO cell lines, HepaRG® cells, T-REx® Jurkat cell lines, Per.C6 cells, T-REx®-293 cell lines, T-REx®-CHO cell lines, and T-REx®-HeLa cell lines.

[0227] In some cases, the mammalian host cell is a stable cell line, i.e., a cell line that has incorporated the target genetic material into its own genome and is capable of expressing the genetic material's products after multiple generations of cell division. In other cases, the mammalian host cell is a transient cell line, i.e., a cell line that has not incorporated the target genetic material into its own genome and is not capable of expressing the genetic material's products after multiple generations of cell division.

[0228] Exemplary insect host cells include, but are not limited to, Drosophila S2 cells, Sf9 cells, Sf21 cells, High Five® cells, and expressSF+® cells.

[0229] In some examples, plant cells include algal cells. Exemplary insect cell lines include Chlamydomonas reinhardtii 137c or Synechococcus elongatus PC7942.

[0230] manufactured goods In another aspect of the present invention, a product is provided containing materials useful for treating, preventing, and / or diagnosing the aforementioned disorders. The product comprises a container and a label or accompanying documentation located on or attached to the container. Suitable containers include, for example, bottles, vials, syringes, and IV solution bags. The container may be formed from a variety of materials, such as glass or plastic. The container may hold one composition, either by itself or in combination with another composition effective for treating, preventing, and / or diagnosing a disease, and may have a sterile access port (for example, the container may be an intravenous injection bag or vial with a stopper through which a subcutaneous needle can be inserted). At least one active agent in the composition is a bispecific antibody comprising a first antigen-binding site that specifically binds to CD3 and a second antigen-binding site that specifically binds to PSMA as defined herein.

[0231] The label or accompanying leaflet indicates that the composition is used to treat a selected disease. Furthermore, the product 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 a cytotoxic or other therapeutic agent. The product in this embodiment of the present invention may further comprise an accompanying leaflet indicating that the composition is available for use in treating a particular disease.

[0232] Alternatively or additionally, the product may further comprise a second (or third) container containing a pharmaceutically acceptable buffer such as bacteriostatic distilled water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. Furthermore, it may comprise other commercially and user-desirable materials, including other buffers, excipients, filters, needles, and syringes.

[0233] Specific definition The terms used herein are intended to describe, and not limit, specific cases. Where used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless otherwise specified in the context. Furthermore, terms such as “including,” “includes,” “having,” “has,” “with,” or variations thereof, are intended to be encompassed to the same extent as “comprising” when used in any form for carrying out the invention and / or in the claims.

[0234] The term "antibody" is used in its broadest sense and includes fully assembled antibodies capable of binding to antigens, antibody fragments such as Fab, F(ab')2, Fv, single-chain antibodies (scFv), diabodies, antibody chimeras, hybrid antibodies, and bispecific antibodies.

[0235] The term "complementarity-determining region" or "CDR" refers to a segment of the variable region of an antibody that is structurally complementary to an epitope to which the antibody binds and which is more variable than the remainder of the variable region. Therefore, CDRs are sometimes referred to as hypervariable regions. A variable region contains three CDRs. CDR peptides can be obtained by constructing a gene that encodes the CDR of the antibody of interest. Such genes are prepared, for example, by using polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. For example, see Larrick et al., Methods: A Companion to Methods in Enzymology 2:106 (1991); Courtenay-Luck, "Genetic Manipulation of Monoclonal Antibodies," in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al. (eds.), pp. 166-179 (Cambridge University Press 1995); and Ward et al., "Genetic Manipulation and Expression of Antibodies," in Monoclonal Antibodies: Principles and Applications, Birch et al. (eds.), pp. 137-185 (Wiley-Liss, Inc. 1995).

[0236] The term "Fab" refers to a protein containing a constant region of the light chain and a first constant region (CH1) of the heavy chain. Fab fragments differ from Fab' fragments by adding a few residues from the antibody hinge region to the carboxyl terminus of the heavy chain CH1 domain, which contains one or more cysteines. Fab'-SH is a designation used herein for Fab' fragments in which the cysteine ​​residues in the constant region carry a free thiol group. Fab' fragments are produced by reducing the heavy chain disulfide crosslinks of the F(ab')2 fragment. Other chemical couplings of antibody fragments are also known.

[0237] A "single-chain variable fragment (scFv)" is a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of an antibody, linked to a short linker peptide of 10 to approximately 25 amino acids. The linker is usually rich in glycine for flexibility and also rich in serine or threonine 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 regardless of the removal of the constant region and the introduction of the linker. scFv antibodies are described, for example, in Houston, JS, Methods in Enzymol. 203 (1991) 46-96. In addition, antibody fragments contain a single-chain polypeptide that has the characteristics of a VH domain that can be assembled together with the VL domain to a functional antigen-binding site, or a VL domain that can be assembled together with the VH domain, thereby providing the antigen-binding properties of a full-length antibody.

[0238] While preferred embodiments of the Disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided only as examples. Numerous variations, modifications, and substitutions will be conceived by those skilled in the art without departing from the Disclosure. It should be understood that various alternatives to the embodiments of the Disclosure described herein may be used in the practice of the Disclosure. The following claims define the scope of the Disclosure, and methods and structures within the scope of these claims and their equivalents are intended to be encompassed thereby.

[0239] Embodiment Embodiment 1 comprises an isolated polypeptide or polypeptide complex according to formula I:A2-A1-L1-P1-H1, wherein A1 comprises a first antigen-recognizing molecule that binds to an effector cell antigen, P1 comprises a peptide that binds to A1, L1 comprises a linking portion that connects A1 to P1 and is a substrate for a tumor-specific protease, H1 comprises a half-life-extending molecule, and A2 comprises a second antigen-recognizing molecule that binds to prostate-specific membrane antigen (PSMA).

[0240] Embodiment 2 comprises the isolated polypeptide or polypeptide complex described in Embodiment 1, wherein the first antigen recognition molecule comprises an antibody or antibody fragment.

[0241] Embodiment 3 comprises the isolated polypeptide or polypeptide complex described in Embodiment 1, wherein the first antigen-recognizing molecule comprises a human or humanized antibody or antibody fragment.

[0242] Embodiment 4 comprises an isolated polypeptide or polypeptide complex according to any one of Embodiments 1 to 3, wherein L1 is bound to the N-terminus of a first antigen recognition molecule.

[0243] Embodiment 5 comprises an isolated polypeptide or polypeptide complex according to any one of Embodiments 1 to 3, wherein A2 is bound to the C-terminus of the first antigen recognition molecule.

[0244] Embodiment 6 comprises an isolated polypeptide or polypeptide complex according to any one of Embodiments 1 to 3, wherein L1 is bound to the C-terminus of a first antigen recognition molecule.

[0245] Embodiment 7 comprises an isolated polypeptide or polypeptide complex according to any one of Embodiments 1 to 3, wherein A2 is bound to the N-terminus of a first antigen recognition molecule.

[0246] Embodiment 8 comprises an isolated polypeptide or polypeptide complex according to any one of Embodiments 2 to 7, wherein the antibody or antibody fragment comprises a single-chain variable fragment, a single-domain antibody, or a Fab fragment.

[0247] Embodiment 9 comprises the isolated polypeptide or polypeptide complex described in Embodiment 8, wherein A1 is a single-chain variable fragment (scFv).

[0248] Embodiment 10 comprises the isolated polypeptide or polypeptide complex described in Embodiment 9, wherein scFv comprises an scFv heavy chain polypeptide and an scFv light chain polypeptide.

[0249] Embodiment 11 comprises the isolated polypeptide or polypeptide complex described in Embodiment 8, wherein A1 is a single-domain antibody.

[0250] Embodiment 12 comprises an isolated polypeptide or polypeptide complex as described in Embodiment 8, wherein the antibody or antibody fragment comprises a single-chain variable fragment (scFv), a heavy-chain variable domain (VH domain), a light-chain variable domain (VL domain), or a variable domain (VHH) of a single-domain antibody derived from a camelid family.

[0251] Embodiment 13 comprises an isolated polypeptide or polypeptide complex according to any one of Embodiments 1 to 12, wherein A1 comprises an anti-CD3e single-chain variable fragment.

[0252] Embodiment 14 is a state in which A1 has a K content of 1 μM or less relative to CD3 on CD3-expressing cells. D The present invention comprises an isolated polypeptide or polypeptide complex according to any one of Embodiments 1 to 12, comprising an anti-CD3e single-chain variable fragment having a binding.

[0253] Embodiment 15 comprises an isolated polypeptide or polypeptide complex according to any one of Embodiments 1 to 14, wherein the effector cell antigen comprises CD3.

[0254] Embodiment 16 comprises the isolated polypeptide or polypeptide complex described in Embodiment 1, wherein A1 comprises a variable light chain and a variable heavy chain, each specifically capable of binding to human CD3.

[0255] Embodiment 17 is a combination of A1, muromonab-CD3 (OKT3), otelixizumab (TRX4), teplizumab (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, The isolated polypeptide or polypeptide complex described in Embodiment 1 includes a complementarity-determining region (CDR) selected from the group consisting of XIII-46, XIII-87, 12F6, T3 / RW2-8C8, T3 / RW2-4B6, OKT3D, M-T301, SMC2, F101.01, UCHT-1, WT-31, 15865, 15865v12, 15865v16, and 15865v19.

[0256] Embodiment 18 comprises the isolated polypeptide or polypeptide complex described in Embodiment 1, wherein the polypeptide or polypeptide complex of formula I binds to effector cells when L1 is cleaved by a tumor-specific protease.

[0257] Embodiment 19 comprises the isolated polypeptide or polypeptide complex described in Embodiment 1, wherein the polypeptide or polypeptide complex of formula I binds to the effector cell when L1 is cleaved by a tumor-specific protease and A1 binds to the effector cell.

[0258] Embodiment 20 comprises the isolated polypeptide or polypeptide complex described in Embodiment 19, wherein the effector cell is a T cell.

[0259] Embodiment 21 comprises the isolated polypeptide or polypeptide complex described in Embodiment 1, wherein A1 binds to a polypeptide that is part of the TCR-CD3 complex on an effector cell.

[0260] Embodiment 22 comprises the isolated polypeptide or polypeptide complex described in Embodiment 21, wherein the polypeptide that is part of the TCR-CD3 complex is human CD3ε.

[0261] Embodiment 23 comprises the isolated polypeptide or polypeptide complex described in Embodiment 1, wherein the effector cell antigen comprises CD3, the scFV comprises HC-CDR1, HC-CDR2, and HC-CDR3 as complementarity-determining regions (CDRs), where HC-CDR1, HC-CDR2, and HC-CDR3 of the scFV comprises SEQ ID NO: 1 for HC-CDR1, SEQ ID NO: 2 for HC-CDR2, and SEQ ID NO: 3 for HC-CDR3, the scFV comprises LC-CDR1, LC-CDR2, and LC-CDR3 as CDRs, where LC-CDR1, LC-CDR2, and LC-CDR3 of the scFV comprises SEQ ID NO: 4 for LC-CDR1, SEQ ID NO: 5 for LC-CDR2, and SEQ ID NO: 6 for LC-CDR3.

[0262] Embodiment 24 comprises the isolated polypeptide or polypeptide complex described in Embodiment 1, wherein the effector cell antigen comprises CD3 and the scFv comprises the amino acid sequence according to SEQ ID NO: 7.

[0263] Embodiment 25 comprises an isolated polypeptide or polypeptide complex according to any one of Embodiments 1 to 24, wherein the second antigen recognition molecule comprises an antibody or antibody fragment.

[0264] Embodiment 26 comprises an isolated polypeptide or polypeptide complex as described in Embodiment 25, wherein the antibody or antibody fragment comprises a single-chain variable fragment, a single-domain antibody, or a Fab.

[0265] Embodiment 27 comprises an isolated polypeptide or polypeptide complex according to Embodiment 27, wherein the antibody or antibody fragment comprises a single-chain variable fragment (scFv), a heavy-chain variable domain (VH domain), a light-chain variable domain (VL domain), or a variable domain (VHH) of a single-domain antibody derived from a camelid family.

[0266] Embodiment 28 comprises the isolated polypeptide or polypeptide complex described in Embodiment 25, wherein the antibody or antibody fragment is human or humanized.

[0267] Embodiment 29 comprises the isolated polypeptide or polypeptide complex described in Embodiment 26, wherein A2 is Fab.

[0268] Embodiment 30 comprises the isolated polypeptide or polypeptide complex described in Embodiment 29, wherein Fab comprises (a) Fab light chain polypeptide and (b) Fab heavy chain polypeptide.

[0269] Embodiment 31 comprises the isolated polypeptide or polypeptide complex described in Embodiment 29, wherein the Fab includes HC-CDR1, HC-CDR2, and HC-CDR3 as complementarity-determining regions (CDRs), where HC-CDR1, HC-CDR2, and HC-CDR3 of the Fab include SEQ ID NO: 8 for HC-CDR1, SEQ ID NO: 9 for HC-CDR2, and SEQ ID NO: 10 for HC-CDR3, and the Fab includes LC-CDR1, LC-CDR2, and LC-CDR3 as CDRs, where LC-CDR1, LC-CDR2, and LC-CDR3 of the Fab include SEQ ID NO: 11 for LC-CDR1, SEQ ID NO: 12 for LC-CDR2, and SEQ ID NO: 13 for LC-CDR3.

[0270] Embodiment 32 comprises the isolated polypeptide or polypeptide complex described in Embodiment 30, wherein the Fab light chain polypeptide comprises the amino acid sequence according to SEQ ID NO: 14.

[0271] Embodiment 33 comprises the isolated polypeptide or polypeptide complex described in Embodiment 30, wherein the Fab heavy chain polypeptide comprises the amino acid sequence according to SEQ ID NO: 15.

[0272] Embodiment 34 comprises the isolated polypeptide or polypeptide complex described in Embodiment 30, wherein the Fab light chain polypeptide of A2 is bound to the C-terminus of the single-chain variable fragment (scFv) of A1.

[0273] Embodiment 35 comprises the isolated polypeptide or polypeptide complex described in Embodiment 30, wherein the Fab heavy chain polypeptide of A2 is bound to the C-terminus of the single-chain variable fragment (scFv) of A1.

[0274] Embodiment 36 comprises the isolated polypeptide or polypeptide complex described in Embodiment 30, wherein the Fab light chain polypeptide of A2 is bound to the N-terminus of the single-chain variable fragment (scFv) of A1.

[0275] Embodiment 37 comprises the isolated polypeptide or polypeptide complex described in Embodiment 30, wherein the Fab heavy chain polypeptide of A2 is bound to the N-terminus of the single-chain variable fragment (scFv) of A1.

[0276] Embodiment 38 includes the isolated polypeptide or polypeptide complex described in Embodiment 30, wherein the Fab heavy chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of A1.

[0277] Embodiment 39 includes the isolated polypeptide or polypeptide complex described in Embodiment 30, wherein the Fab light chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of A1.

[0278] Embodiment 40 includes the isolated polypeptide or polypeptide complex described in Embodiment 30, wherein the Fab heavy chain polypeptide of A2 is bound to the scFv light chain polypeptide of A1.

[0279] Embodiment 41 includes the isolated polypeptide or polypeptide complex described in Embodiment 30, wherein the Fab light chain polypeptide of A2 is bound to the scFv light chain polypeptide of A1.

[0280] Embodiment 42 comprises an isolated polypeptide or polypeptide complex according to any one of Embodiments 1 to 41, wherein A2 further comprises P2 and L2, where P2 comprises a peptide that binds to A2, and L2 comprises a linking portion that connects A1 to P1 and is a substrate of a tumor-specific protease.

[0281] Embodiment 43 comprises the isolated polypeptide or polypeptide complex described in Embodiment 42, wherein the polypeptide or polypeptide complex is according to formula Ia:P2-L2-A2-A1-L1-P1-H1.

[0282] Embodiment 44 includes the isolated polypeptide or polypeptide complex described in Embodiment 43, wherein 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.

[0283] Embodiment 45 includes the isolated polypeptide or polypeptide complex described in Embodiment 43, wherein 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.

[0284] Embodiment 46 includes the isolated polypeptide or polypeptide complex described in Embodiment 43, wherein 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.

[0285] Embodiment 47 includes the isolated polypeptide or polypeptide complex described in Embodiment 43, wherein 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.

[0286] Embodiment 48 comprises an isolated polypeptide or polypeptide complex according to any one of Embodiments 1 to 47, wherein P1 impairs the binding of A1 to the effector cell antigen.

[0287] Embodiment 49 comprises an isolated polypeptide or polypeptide complex according to any one of Embodiments 1 to 48, wherein P1 is bound to A1 by ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, or H-bond interactions, or a combination thereof.

[0288] Embodiment 50 comprises an isolated polypeptide or polypeptide complex according to any one of Embodiments 1 to 48, wherein P1 has less than 70% sequence homology to the effector cell antigen.

[0289] Embodiment 51 comprises an isolated polypeptide or polypeptide complex according to any one of Embodiments 1 to 50, wherein P2 impairs the binding of A2 to PSMA.

[0290] Embodiment 52 comprises an isolated polypeptide or polypeptide complex according to any one of Embodiments 1 to 50, wherein P2 is bound to A2 by ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, or H-bond interactions, or a combination thereof.

[0291] Embodiment 53 comprises an isolated polypeptide or polypeptide complex according to any one of Embodiments 1 to 50, wherein P2 is bound to A2 at or near an antigen-binding site.

[0292] Embodiment 54 comprises an isolated polypeptide or polypeptide complex according to any one of Embodiments 1 to 50, wherein P2 has less than 70% sequence homology to PSMA.

[0293] Embodiment 55 comprises an isolated polypeptide or polypeptide complex according to any one of Embodiments 1 to 54, wherein P1 or P2 comprises a peptide sequence having a length of at least 10 amino acids.

[0294] Embodiment 56 comprises an isolated polypeptide or polypeptide complex according to any one of Embodiments 1 to 54, wherein P1 or P2 comprises a peptide sequence having a length of at least 10 amino acids and a length of 20 amino acids or less.

[0295] Embodiment 57 comprises an isolated polypeptide or polypeptide complex according to any one of Embodiments 1 to 54, wherein P1 or P2 comprises a peptide sequence having a length of at least 16 amino acids.

[0296] Embodiment 58 comprises an isolated polypeptide or polypeptide complex according to any one of Embodiments 1 to 54, wherein P1 or P2 comprises a peptide sequence having a length of 40 amino acids or less.

[0297] Embodiment 59 comprises an isolated polypeptide or polypeptide complex according to any one of Embodiments 1 to 54, wherein P1 or P2 comprises at least two cysteine ​​amino acid residues.

[0298] Embodiment 60 comprises an isolated polypeptide or polypeptide complex according to any one of Embodiments 1 to 54, wherein P1 or P2 comprises a cyclic peptide or a linear peptide.

[0299] Embodiment 61 comprises an isolated polypeptide or polypeptide complex according to any one of Embodiments 1 to 54, wherein P1 or P2 comprises a cyclic peptide.

[0300] Embodiment 62 comprises an isolated polypeptide or polypeptide complex according to any one of Embodiments 1 to 54, wherein P1 or P2 comprises a linear peptide.

[0301] Embodiment 63 comprises an isolated polypeptide or polypeptide complex according to any one of Embodiments 1 to 54, wherein P1 comprises at least two cysteine ​​amino acid residues.

[0302] Embodiment 64 comprises an isolated polypeptide or polypeptide complex according to any one of Embodiments 1 to 54, wherein P1 comprises an amino acid sequence according to any one of SEQ ID NOs: 16-19 or 78.

[0303] Embodiment 65 comprises an isolated polypeptide or polypeptide complex according to any one of Embodiments 1 to 64, wherein L1 is bonded to the N-terminus of A1.

[0304] Embodiment 66 comprises an isolated polypeptide or polypeptide complex according to any one of Embodiments 1 to 64, wherein L1 is bonded to the C-terminus of A1.

[0305] Embodiment 67 comprises an isolated polypeptide or polypeptide complex according to any one of Embodiments 1 to 66, wherein L2 is bonded to the N-terminus of A2.

[0306] Embodiment 68 comprises an isolated polypeptide or polypeptide complex according to any one of Embodiments 1 to 66, wherein L2 is bonded to the C-terminus of A2.

[0307] Embodiment 69 comprises an isolated polypeptide or polypeptide complex according to any one of Embodiments 1 to 68, wherein L1 or L2 is a peptide sequence having at least 5 to 50 amino acids.

[0308] Embodiment 70 comprises an isolated polypeptide or polypeptide complex according to any one of Embodiments 1 to 68, wherein L1 or L2 is a peptide sequence having at least 10 to 30 amino acids.

[0309] Embodiment 71 comprises an isolated polypeptide or polypeptide complex according to any one of Embodiments 1 to 68, wherein L1 or L2 is a peptide sequence having at least 10 amino acids.

[0310] Embodiment 72 comprises an isolated polypeptide or polypeptide complex according to any one of Embodiments 1 to 68, wherein L1 or L2 is a peptide sequence having at least 18 amino acids.

[0311] Embodiment 73 comprises an isolated polypeptide or polypeptide complex according to any one of Embodiments 1 to 68, wherein L1 or L2 is a peptide sequence having at least 26 amino acids.

[0312] Embodiment 74 is such that L1 or L2 is (G2S) n The isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 68 comprises a formula including n, where n is an integer from 1 to 3 (SEQ ID NO: 118).

[0313] Embodiment 75 is (G2S) n , (GS) n (GSGGS) n (Sequence ID 50), (GGGS) n (Sequence ID 51), (GGGGS) n (Sequence ID 52), and (GSSGGS) n The isolated polypeptide or polypeptide complex according to any one of Embodiments 1 to 68 comprises a formula selected from the group consisting of (Sequence ID 53), wherein n is at least an integer of 1.

[0314] Embodiment 76 comprises an isolated polypeptide or polypeptide complex according to any one of Embodiments 1 to 68, wherein when L1 is cleaved by a tumor-specific protease, thereby exposing A1 to an effector cell antigen, P1 is decoupled from A1.

[0315] Embodiment 77 includes an isolated polypeptide or polypeptide complex according to any one of Embodiments 1 to 68, wherein when L2 is cleaved by a tumor-specific protease, thereby exposing A2 to PSMA, P2 is decoupled from A2.

[0316] Embodiment 78 comprises an isolated polypeptide or polypeptide complex according to any one of Embodiments 1 to 68, wherein the tumor-specific protease is selected from the group consisting of matrix metalloproteinases (MMPs), serine proteases, cysteine ​​proteases, threonine proteases, and aspartate proteases.

[0317] Embodiment 79 comprises the isolated polypeptide or polypeptide complex described in Embodiment 78, wherein the matrix metalloproteinase comprises MMP2, MMP7, MMP9, MMP13, or MMP14.

[0318] Embodiment 80 comprises an isolated polypeptide or polypeptide complex as described in Embodiment 78, wherein the serine protease comprises a matryptase (MTSP1), urokinase, or hepsin.

[0319] Embodiment 81 comprises an isolated polypeptide or polypeptide complex according to any one of Embodiments 1 to 68, wherein L1 or L2 comprises a urokinase-cleavable amino acid sequence, a matryptase-cleavable amino acid sequence, a matrix metalloproteinase-cleavable amino acid sequence, or a regmine-cleavable amino acid sequence.

[0320] Embodiment 82 comprises an isolated polypeptide or polypeptide complex according to any one of Embodiments 1 to 68, wherein L1 or L2 comprises the amino acid sequence according to SEQ ID NO: 23.

[0321] Embodiment 83 comprises an isolated polypeptide or polypeptide complex according to any one of Embodiments 1 to 68, wherein L1 or L2 comprises an amino acid sequence according to any one of SEQ ID NOs. 20-49 or 78.

[0322] Embodiment 84 comprises an isolated polypeptide or polypeptide complex according to any one of Embodiments 1 to 68, wherein L1 or L2 comprises the amino acid sequence of linker 25 (ISSGLLSGRSDAG) (SEQ ID NO: 45), linker 26 (AAGLLAPPGGLSGRSDAG) (SEQ ID NO: 46), linker 27 (SPLGLSGRSDAG) (SEQ ID NO: 47), or linker 28 (LSGRSDAGSPLGLAG) (SEQ ID NO: 48), or an amino acid sequence having one, two, or three amino acid substitutions, additions, or deletions to the amino acid sequence of linker 25, linker 26, linker 27, or linker 28.

[0323] Embodiment 85 comprises an isolated polypeptide or polypeptide complex according to any one of Embodiments 1 to 83, wherein H1 comprises a cyclic peptide.

[0324] Embodiment 86 comprises an isolated polypeptide or polypeptide complex according to any one of Embodiments 1 to 83, wherein the polymer comprises polyethylene glycol (PEG).

[0325] Embodiment 87 comprises an isolated polypeptide or polypeptide complex according to any one of Embodiments 1 to 83, wherein H1 contains albumin.

[0326] Embodiment 88 comprises an isolated polypeptide or polypeptide complex according to any one of Embodiments 1 to 83, wherein H1 comprises an Fc domain.

[0327] Embodiment 89 comprises the isolated polypeptide or polypeptide complex described in Embodiment 87, wherein the albumin is serum albumin.

[0328] Embodiment 90 comprises the isolated polypeptide or polypeptide complex described in Embodiment 87, wherein the albumin is human serum albumin.

[0329] Embodiment 91 comprises an isolated polypeptide or polypeptide complex according to any one of Embodiments 1 to 83, wherein H1 comprises a polypeptide, ligand, or small molecule.

[0330] Embodiment 92 comprises an isolated polypeptide or polypeptide complex as described in Embodiment 91, wherein the polypeptide, ligand, or small molecule is bound to a serum protein or fragment thereof, a circulating immunoglobulin or fragment thereof, or CD35 / CR1.

[0331] Embodiment 93 comprises an isolated polypeptide or polypeptide complex according to Embodiment 88, wherein the serum protein comprises a thyroxine-binding protein, transthyretin, α1-acid glycoprotein, transferrin, a transferrin receptor or its transferrin-binding moiety, fibrinogen, or albumin.

[0332] Embodiment 94 comprises the isolated polypeptide or polypeptide complex described in Embodiment 88, wherein the circulating immunoglobulin molecule comprises IgG1, IgG2, IgG3, IgG4, slgA, IgM, or IgD.

[0333] Embodiment 95 comprises the isolated polypeptide or polypeptide complex described in Embodiment 92, wherein the serum protein is albumin.

[0334] Embodiment 96 comprises the isolated polypeptide or polypeptide complex described in Embodiment 91, wherein the polypeptide is an antibody.

[0335] Embodiment 97 comprises an isolated polypeptide or polypeptide complex as described in Embodiment 96, wherein the antibody comprises a single-domain antibody, a single-chain variable fragment, or a Fab.

[0336] Embodiment 98 comprises an isolated polypeptide or polypeptide complex as described in Embodiment 97, wherein the single-domain antibody includes a single-domain antibody that binds to albumin.

[0337] Embodiment 99 comprises the isolated polypeptide or polypeptide complex described in Embodiment 97, wherein the single-domain antibody is a human antibody or a humanized antibody.

[0338] Embodiment 100 comprises the isolated polypeptide or polypeptide complex described in Embodiment 97, wherein the single-domain antibody is 645gH1gL1.

[0339] Embodiment 101 comprises the isolated polypeptide or polypeptide complex described in Embodiment 97, wherein the single-domain antibody is 645dsgH5gL4.

[0340] Embodiment 102 comprises the isolated polypeptide or polypeptide complex described in Embodiment 97, wherein the single-domain antibody is 23-13-A01-sc02.

[0341] Embodiment 103 comprises an isolated polypeptide or polypeptide complex as described in Embodiment 97, wherein the single-domain antibody is A10m3 or a fragment thereof.

[0342] Embodiment 104 comprises the isolated polypeptide or polypeptide complex described in Embodiment 97, wherein the single-domain antibody is DOM7r-31.

[0343] Embodiment 105 comprises the isolated polypeptide or polypeptide complex described in Embodiment 97, wherein the single-domain antibody is DOM7h-11-15.

[0344] Embodiment 106 comprises the isolated polypeptide or polypeptide complex described in Embodiment 97, wherein the single-domain antibody is Alb-1, Alb-8, or Alb-23.

[0345] Embodiment 107 comprises the isolated polypeptide or polypeptide complex described in Embodiment 97, wherein the single-domain antibody is 10E.

[0346] Embodiment 108 comprises an isolated polypeptide or polypeptide complex as described in Embodiment 97, wherein the single-domain antibody comprises HC-CDR1, HC-CDR2, and HC-CDR3 as complementarity-determining regions (CDRs), and the single-domain antibody HC-CDR1, HC-CDR2, and HC-CDR3 comprises SEQ ID NO: 54 for HC-CDR1, SEQ ID NO: 55 for HC-CDR2, and SEQ ID NO: 56 for HC-CDR3.

[0347] Embodiment 109 comprises an isolated polypeptide or polypeptide complex as described in Embodiment 97, wherein the single-domain antibody comprises HC-CDR1, HC-CDR2, and HC-CDR3 as complementarity-determining regions (CDRs), and the HC-CDR1, HC-CDR2, and HC-CDR3 of the single-domain antibody comprises SEQ ID NO: 58 for HC-CDR1, SEQ ID NO: 59 for HC-CDR2, and SEQ ID NO: 60 for HC-CDR3.

[0348] Embodiment 110 comprises the isolated polypeptide or polypeptide complex described in Embodiment 97, wherein the single-domain antibody is SA21.

[0349] Embodiment 111 comprises an isolated polypeptide or polypeptide complex according to any one of Embodiments 1 to 110, wherein the polypeptide or polypeptide complex comprises a modified amino acid, a non-natural amino acid, a non-natural modified amino acid, or a combination thereof.

[0350] Embodiment 112 comprises an isolated polypeptide or polypeptide complex as described in Embodiment 111, wherein the modified amino acid or unnaturally modified amino acid includes post-translational modifications.

[0351] Embodiment 113 comprises an isolated polypeptide or polypeptide complex according to any one of Embodiments 1 to 112, wherein H1 includes a connecting portion (L3) that connects H1 to P1.

[0352] Embodiment 114 comprises the isolated polypeptide or polypeptide complex described in Embodiment 113, wherein L3 is a peptide sequence having at least 5 to 50 amino acids.

[0353] Embodiment 115 comprises the isolated polypeptide or polypeptide complex described in Embodiment 113, wherein L3 is a peptide sequence having at least 10 to 30 amino acids.

[0354] Embodiment 116 comprises the isolated polypeptide or polypeptide complex described in Embodiment 113, wherein L3 is a peptide sequence having at least 10 amino acids.

[0355] Embodiment 117 comprises the isolated polypeptide or polypeptide complex described in Embodiment 113, wherein L3 is a peptide sequence having at least 18 amino acids.

[0356] Embodiment 118 comprises the isolated polypeptide or polypeptide complex described in Embodiment 113, wherein L3 is a peptide sequence having at least 26 amino acids.

[0357] Embodiment 119 is such that L3 is (G2S) n , (GS) n (GSGGS) n (Sequence ID 50), (GGGS) n (Sequence ID 51), (GGGGS) n (Sequence ID 52), and (GSSGGS) n The isolated polypeptide or polypeptide complex described in Embodiment 113 has a formula selected from the group consisting of (Sequence ID 53), wherein n is at least an integer of 1.

[0358] Embodiment 120 comprises an isolated polypeptide or polypeptide complex as described in Embodiment 113, wherein L3 comprises the amino acid sequence according to SEQ ID NO: 22.

[0359] Embodiment 121 comprises the isolated polypeptide or polypeptide complex described in Embodiment 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NOs. 62-77.

[0360] Embodiment 122 comprises the isolated polypeptide or polypeptide complex described in Embodiment 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 72.

[0361] Embodiment 123 comprises the isolated polypeptide or polypeptide complex described in Embodiment 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 73.

[0362] Embodiment 124 comprises the isolated polypeptide or polypeptide complex described in Embodiment 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 62 and SEQ ID NO: 63.

[0363] Embodiment 125 comprises the isolated polypeptide or polypeptide complex described in Embodiment 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 64 and SEQ ID NO: 65.

[0364] Embodiment 126 comprises the isolated polypeptide or polypeptide complex described in Embodiment 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 66 and SEQ ID NO: 67.

[0365] Embodiment 127 comprises the isolated polypeptide or polypeptide complex described in Embodiment 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 68 and SEQ ID NO: 69.

[0366] Embodiment 128 comprises the isolated polypeptide or polypeptide complex described in Embodiment 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 70 and SEQ ID NO: 71.

[0367] Embodiment 129 comprises the isolated polypeptide or polypeptide complex described in Embodiment 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NOs. 72 and SEQ ID NOs. 73.

[0368] Embodiment 130 comprises the isolated polypeptide or polypeptide complex described in Embodiment 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 74 and SEQ ID NO: 75.

[0369] Embodiment 131 comprises the isolated polypeptide or polypeptide complex described in Embodiment 1, wherein the polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 76 and SEQ ID NO: 77.

[0370] Embodiment 132 comprises a pharmaceutical composition comprising (a) a polypeptide or polypeptide complex described in any one of Embodiments 1 to 131, and (b) a pharmaceutically acceptable excipient.

[0371] Embodiment 133 comprises an isolated recombinant nucleic acid molecule encoding a polypeptide or polypeptide complex described in any one of Embodiments 1 to 131.

[0372] Embodiment 134 is based on formula II:L 1a -P 1a -H 1a A polypeptide or polypeptide complex isolated by the formula, wherein L 1aIf not cleaved, P is the first antigen-recognition molecule that binds to the effector cell antigen. 1a It includes a linkage portion cleaved by a tumor-specific protease, and the first antigen-recognition molecule is linked to a second antigen-recognition molecule that binds to PSMA, P 1a L 1a If it is not cleaved, it contains a peptide that binds to the first antigen recognition molecule, H 1a This includes isolated polypeptides or polypeptide complexes containing half-life extension molecules.

[0373] Embodiment 135 is L 1a If it is not disconnected, P 1a However, it includes the isolated polypeptide or polypeptide complex described in Embodiment 134, which impairs the binding of the first antigen-recognizing molecule to the effector cell antigen.

[0374] Embodiment 136 comprises an isolated polypeptide or polypeptide complex as described in Embodiment 134, wherein the first antigen recognition molecule comprises an antibody or antibody fragment.

[0375] Embodiment 137 comprises the isolated polypeptide or polypeptide complex described in Embodiment 134, wherein the effector cell antigen is an anti-CD3 effector cell antigen.

[0376] Embodiment 138 is P 1a This comprises an isolated polypeptide or polypeptide complex according to Embodiment 134, which has less than 70% sequence homology to the effector cell antigen.

[0377] Embodiment 139 is P 1a However, it includes an isolated polypeptide or polypeptide complex as described in Embodiment 134, which comprises a peptide sequence having a length of at least 10 amino acids.

[0378] Embodiment 140 is P 1aHowever, it includes an isolated polypeptide or polypeptide complex as described in Embodiment 134, which contains a peptide sequence having a length of at least 10 amino acids and a length of 20 amino acids or less.

[0379] Embodiment 141 is P 1a However, it includes an isolated polypeptide or polypeptide complex as described in Embodiment 134, which comprises a peptide sequence having a length of at least 16 amino acids.

[0380] Embodiment 142 is P 1a However, it includes an isolated polypeptide or polypeptide complex as described in Embodiment 134, which contains a peptide sequence of 40 amino acids or less in length.

[0381] Embodiment 143 is P 1a However, it includes the isolated polypeptide or polypeptide complex described in Embodiment 134, which contains at least two cysteine ​​amino acid residues.

[0382] Embodiment 144 is P 1a However, it includes an isolated polypeptide or polypeptide complex as described in Embodiment 134, which comprises a cyclic peptide or a linear peptide.

[0383] Embodiment 145 comprises the isolated polypeptide or polypeptide complex described in Embodiment 134, wherein the albumin is serum albumin.

[0384] Embodiment 146 is P 1a This comprises an isolated polypeptide or polypeptide complex as described in Embodiment 134, wherein the polypeptide is a linear peptide.

[0385] Embodiment 147 is P 1a However, it includes an isolated polypeptide or polypeptide complex according to Embodiment 134, which contains an amino acid sequence selected from the group consisting of any one of SEQ ID NOs. 16 to 19.

[0386] Embodiment 148 is H 1aThe polymer comprises an isolated polypeptide or polypeptide complex as described in any one of Embodiments 132 to 145.

[0387] Embodiment 149 comprises the isolated polypeptide or polypeptide complex described in Embodiment 148, wherein the polymer contains polyethylene glycol (PEG).

[0388] Embodiment 150 is H 1a The comprising an isolated polypeptide or polypeptide complex according to any one of embodiments 134 to 147, wherein the polypeptide contains albumin.

[0389] Embodiment 151 is H 1a The isolated polypeptide or polypeptide complex described in any one of embodiments 134 to 147 comprises an Fc domain.

[0390] Embodiment 152 comprises the isolated polypeptide or polypeptide complex described in Embodiment 150, wherein the albumin is serum albumin.

[0391] Embodiment 153 comprises the isolated polypeptide or polypeptide complex described in Embodiment 152, wherein the albumin is human serum albumin.

[0392] Embodiment 154 is H 1a However, it includes an isolated polypeptide or polypeptide complex as described in any one of Embodiments 134 to 147, which comprises a polypeptide, ligand, or small molecule.

[0393] Embodiment 155 comprises an isolated polypeptide or polypeptide complex as described in Embodiment 154, wherein the polypeptide, ligand, or small molecule is bound to a serum protein or fragment thereof, a circulating immunoglobulin or fragment thereof, or CD35 / CR1.

[0394] Embodiment 156 comprises an isolated polypeptide or polypeptide complex according to Embodiment 155, wherein the serum protein comprises a thyroxine-binding protein, transthyretin, α1-acid glycoprotein, transferrin, a transferrin receptor or its transferrin-binding moiety, fibrinogen, or albumin.

[0395] Embodiment 157 comprises the isolated polypeptide or polypeptide complex described in Embodiment 155, wherein the circulating immunoglobulin molecule comprises IgG1, IgG2, IgG3, IgG4, slgA, IgM, or IgD.

[0396] Embodiment 158 ​​comprises the isolated polypeptide or polypeptide complex described in Embodiment 153, wherein the serum protein is albumin.

[0397] Embodiment 159 comprises the isolated polypeptide or polypeptide complex described in Embodiment 154, wherein the polypeptide is an antibody.

[0398] Embodiment 160 comprises an isolated polypeptide or polypeptide complex as described in Embodiment 159, wherein the antibody comprises a single-domain antibody, a single-chain variable fragment, or a Fab.

[0399] Embodiment 161 comprises an isolated polypeptide or polypeptide complex as described in Embodiment 159, wherein the single-domain antibody comprises a single-domain antibody that binds to albumin.

[0400] Embodiment 162 comprises the isolated polypeptide or polypeptide complex described in Embodiment 159, wherein the single-domain antibody is a human antibody or a humanized antibody.

[0401] Embodiment 163 comprises the isolated polypeptide or polypeptide complex described in Embodiment 160, wherein the single-domain antibody is 645gH1gL1.

[0402] Embodiment 164 comprises the isolated polypeptide or polypeptide complex described in Embodiment 160, wherein the single-domain antibody is 645dsgH5gL4.

[0403] Embodiment 165 comprises the isolated polypeptide or polypeptide complex described in Embodiment 160, wherein the single-domain antibody is 23-13-A01-sc02.

[0404] Embodiment 166 comprises the isolated polypeptide or polypeptide complex described in Embodiment 160, wherein the single-domain antibody is A10m3 or a fragment thereof.

[0405] Embodiment 167 comprises the isolated polypeptide or polypeptide complex described in Embodiment 160, wherein the single-domain antibody is DOM7r-31.

[0406] Embodiment 168 comprises the isolated polypeptide or polypeptide complex described in Embodiment 160, wherein the single-domain antibody is DOM7h-11-15.

[0407] Embodiment 169 comprises the isolated polypeptide or polypeptide complex described in Embodiment 160, wherein the single-domain antibody is Alb-1, Alb-8, or Alb-23.

[0408] Embodiment 170 comprises the isolated polypeptide or polypeptide complex described in Embodiment 160, wherein the single-domain antibody is 10E.

[0409] Embodiment 171 comprises an isolated polypeptide or polypeptide complex as described in Embodiment 160, wherein the single-domain antibody comprises HC-CDR1, HC-CDR2, and HC-CDR3 as complementarity-determining regions (CDRs), and the single-domain antibody HC-CDR1, HC-CDR2, and HC-CDR3 comprises SEQ ID NO: 54 for HC-CDR1, SEQ ID NO: 55 for HC-CDR2, and SEQ ID NO: 56 for HC-CDR3.

[0410] Embodiment 172 comprises an isolated polypeptide or polypeptide complex as described in Embodiment 158, wherein the single-domain antibody comprises HC-CDR1, HC-CDR2, and HC-CDR3 as complementarity-determining regions (CDRs), and the single-domain antibody HC-CDR1, HC-CDR2, and HC-CDR3 comprises SEQ ID NO: 58 for HC-CDR1, SEQ ID NO: 59 for HC-CDR2, and SEQ ID NO: 60 for HC-CDR3.

[0411] Embodiment 173 comprises the isolated polypeptide or polypeptide complex described in Embodiment 160, wherein the single-domain antibody is SA21.

[0412] Embodiment 174 is H 1a However, P 1a H 1a The connecting part (L 1a The isolated polypeptide or polypeptide complex described in any one of embodiments 134 to 173, including ).

[0413] Embodiment 175 is L 1a However, it includes an isolated polypeptide or polypeptide complex as described in Embodiment 174, which is a peptide sequence having at least 5 to 50 amino acids.

[0414] Embodiment 176 is L 1a However, it includes an isolated polypeptide or polypeptide complex as described in Embodiment 174, which is a peptide sequence having at least 10 to 30 amino acids.

[0415] Embodiment 177 is L 1a However, it includes an isolated polypeptide or polypeptide complex as described in Embodiment 174, which is a peptide sequence having at least 10 amino acids.

[0416] Embodiment 178 is L 1a However, it includes an isolated polypeptide or polypeptide complex as described in Embodiment 174, which is a peptide sequence having at least 18 amino acids.

[0417] Embodiment 179 is L 1a However, it includes the isolated polypeptide or polypeptide complex described in Embodiment 174, which is a peptide sequence having at least 26 amino acids.

[0418] Embodiment 180 is L 1a However, (G2S) n , (GS) n (GSGGS) n (Sequence ID 50), (GGGS) n (Sequence ID 51), (GGGGS) n (Sequence ID 52), and (GSSGGS) n The isolated polypeptide or polypeptide complex according to Embodiment 174 has a formula selected from the group consisting of (Sequence ID 53), wherein n is at least an integer of 1.

[0419] Embodiment 181 is L 1a However, it includes an isolated polypeptide or polypeptide complex as described in Embodiment 174, which contains the amino acid sequence according to Sequence ID No. 23.

[0420] Embodiment 182 is P 1a But Z1-Z2-C-Z4-P-Z6-Z7-Z8-Z9-Z 10 -Z 11 -Z 12 -CZ 14 The amino acid sequence includes, where Z1 is selected from D, Y, F, I, N, V, H, L, A, T, S, and P, Z2 is selected from D, Y, L, F, I, N, A, V, H, T, and S, Z4 is selected from G and W, Z6 is selected from E, D, V, and P, Z7 is selected from W, L, F, V, G, M, I, and Y, Z8 is selected from E, D, P, and Q, Z9 is selected from E, D, Y, V, F, W, P, L, and Q, Z 10 However, it is selected from S, D, Y, T, I, F, V, N, A, P, L, and H, Z 11 However, selected from I, Y, F, V, L, T, N, S, D, A, and H, Z12 However, it is selected from F, D, Y, L, I, V, A, N, T, P, S, and H, Z 14 The comprising an isolated polypeptide or polypeptide complex as described in any one of Embodiments 134 to 181, selected from D, Y, N, F, I, P, V, A, T, H, L, and S.

[0421] Embodiment 183 is characterized in that Z1 is selected from D, Y, F, I, and N, Z2 is selected from D, Y, L, F, I, and N, Z4 is selected from G and W, Z6 is selected from E and D, Z7 is selected from W, L, F, and V, Z8 is selected from E and D, Z9 is selected from E, D, Y, and V, Z 10 However, selected from S, D, Y, T, and I, Z 11 However, it is selected from I, Y, F, V, L, and T, and Z 12 However, it is selected from F, D, Y, L, I, V, A, and N, Z 14 The solution comprises an isolated polypeptide or polypeptide complex as described in Embodiment 182, selected from D, Y, N, F, I, and P.

[0422] Embodiment 184 is characterized in that Z1 is selected from D, Y, and F, Z2 is selected from D, Y, L, and F, Z4 is selected from G and W, Z6 is selected from E and D, Z7 is selected from W, L, and F, Z8 is selected from E and D, Z9 is selected from E and D, Z 10 However, it is selected from S, D, and Y, and Z 11 However, selected from I, Y, and F, Z 12 However, it is selected from F, D, Y, and L, and Z 14 The solution comprises an isolated polypeptide or polypeptide complex as described in any one of embodiments 182 to 183, selected from D, Y, and N.

[0423] Embodiment 185 is P 1a But U1-U2-C-U4-P-U6-U7-U8-U9-U 10 -U 11 -U 12-CU 14 The amino acid sequence includes U1 selected from D, Y, F, I, N, V, H, L, A, T, S, and P, U2 selected from D, Y, L, F, I, N, A, V, H, T, and S, U4 selected from G and W, U6 selected from E, D, V, and P, U7 selected from W, L, F, V, G, M, I, and Y, U8 selected from E, D, P, and Q, U9 selected from E, D, Y, V, F, W, P, L, and Q, and U 10 However, it is selected from S, D, Y, T, I, F, V, N, A, P, L, and H, U 11 However, it is selected from I, Y, F, V, L, T, N, S, D, A, and H, U 12 However, it is selected from F, D, Y, L, I, V, A, N, T, P, S, G, and H, U 14 The comprising an isolated polypeptide or polypeptide complex as described in any one of Embodiments 134 to 181, selected from D, Y, N, F, I, P, V, A, T, H, L, M, and S.

[0424] Embodiment 186 is characterized in that U1 is selected from D, Y, F, I, V, and N, U2 is selected from D, Y, L, F, I, and N, U4 is selected from G and W, U6 is selected from E and D, U7 is selected from W, L, F, G, and V, U8 is selected from E and D, U9 is selected from E, D, Y, and V, U 10 However, it is selected from S, D, Y, T, and I, U 11 However, it is selected from I, Y, F, V, L, and T, U 12 However, it is selected from F, D, Y, L, I, V, A, G, and N, U 14 The solution comprises an isolated polypeptide or polypeptide complex as described in Embodiment 185, selected from D, Y, N, F, I, M, and P.

[0425] Embodiment 187 is characterized in that U1 is selected from D, Y, V, and F, U2 is selected from D, Y, L, and F, U4 is selected from G and W, U6 is selected from E and D, U7 is selected from W, L, G, and F, U8 is selected from E and D, U9 is selected from E and D, and U 10 However, it is selected from S, D, T, and Y, U 11 However, it is selected from I, Y, V, L, and F, U 12 However, it is selected from F, D, Y, G, A, and L, U 14 The solution comprises an isolated polypeptide or polypeptide complex as described in Embodiment 186, selected from D, Y, M, and N.

[0426] Embodiment 188 is P 1a However, it includes an isolated polypeptide or polypeptide complex as described in any one of embodiments 134 to 181 and 70 to 88, comprising the amino acid sequence according to SEQ ID NOs. 79 to 105.

[0427] Embodiment 189 is P 1a However, it includes an isolated polypeptide or polypeptide complex as described in any one of embodiments 134 to 181 and 70 to 88, which contains an amino acid sequence according to one of the sequences in Table 20.

[0428] Embodiment 190 is P 1a However, it includes an isolated polypeptide or polypeptide complex as described in any one of Embodiments 134 to 181 or 189, which contains the amino acid sequence of any one of Sequence IDs 106 to 117.

[0429] Embodiment 191 is P 1a The present invention includes an isolated polypeptide or polypeptide complex according to any one of Embodiments 134 to 181, comprising the amino acid sequence of SEQ ID NO: 18, or a peptide sequence having one, two, or three amino acid substitutions, additions, or deletions relative to SEQ ID NO: 18.

[0430] Embodiment 192 is P1a The present invention includes an isolated polypeptide or polypeptide complex described in any one of Embodiments 134 to 187, which comprises the amino acid sequence according to SEQ ID NO: 19, or a peptide sequence having one, two, or three amino acid substitutions, additions, or deletions relative to SEQ ID NO: 19.

[0431] Embodiment 193 is P 1a The present invention includes an isolated polypeptide or polypeptide complex according to any one of Embodiments 134 to 187, comprising the amino acid sequence of SEQ ID NO: 116, or a peptide sequence having one, two, or three amino acid substitutions, additions, or deletions relative to SEQ ID NO: 116.

[0432] Embodiment 194 is P 1a However, it includes an isolated polypeptide or polypeptide complex as described in Embodiment 191, which contains the amino acid sequence according to Sequence ID No. 18.

[0433] Embodiment 195 is P 1a However, it includes an isolated polypeptide or polypeptide complex as described in Embodiment 192, which contains the amino acid sequence according to Sequence ID No. 19.

[0434] Embodiment 196 is P 1a However, it includes an isolated polypeptide or polypeptide complex as described in Embodiment 193, which contains the amino acid sequence according to SEQ ID NO: 116.

[0435] Embodiment 197 has a three-dimensional configuration 1:

[0436] [ka] A polypeptide complex comprising a structural configuration thereof, wherein the polypeptide complex comprises a single-chain variable fragment (scFv) comprising a light-chain variable domain and a heavy-chain variable domain, the scFv further comprising a peptide that impairs the binding of scFv to effector cell antigens, the peptide being linked to the heavy-chain variable domain of scFv by a linking portion which is a substrate of a tumor-specific protease, and the peptide further comprising a half-life-extending molecule, and a polypeptide complex comprising Fab or Fab' that binds to prostate-specific membrane antigen (PSMA), the Fab or Fab' comprising a Fab light-chain polypeptide chain and a Fab heavy-chain polypeptide chain, the Fab heavy-chain polypeptide chain being linked to the C-terminus of the light-chain variable domain of scFv, and the polypeptide complex comprising Fab or Fab'.

[0437] Embodiment 198 has a three-dimensional configuration 2:

[0438] [ka] A polypeptide complex comprising a structural configuration thereof, wherein the polypeptide complex comprises a single-chain variable fragment (scFv) comprising a light-chain variable domain and a heavy-chain variable domain, the scFv further comprising a peptide that impairs the binding of scFv to effector cell antigens, the peptide being ligated to the N-terminus of the heavy-chain variable domain of scFv by a ligation portion that is a substrate of a tumor-specific protease, and the peptide further comprising a half-life-extending molecule, and a polypeptide complex comprising Fab or Fab' that binds to prostate-specific membrane antigen (PSMA), the Fab or Fab' comprising a Fab light-chain polypeptide chain and a Fab heavy-chain polypeptide chain, the Fab light-chain polypeptide chain being ligated to the C-terminus of the light-chain variable domain of scFv, and the Fab or Fab'.

[0439] Embodiment 199 is such that P1 is Z1-Z2-C-Z4-P-Z6-Z7-Z8-Z9-Z 10 -Z 11 -Z 12 -CZ 14The amino acid sequence includes, where Z1 is selected from D, Y, F, I, N, V, H, L, A, T, S, and P, Z2 is selected from D, Y, L, F, I, N, A, V, H, T, and S, Z4 is selected from G and W, Z6 is selected from E, D, V, and P, Z7 is selected from W, L, F, V, G, M, I, and Y, Z8 is selected from E, D, P, and Q, Z9 is selected from E, D, Y, V, F, W, P, L, and Q, Z 10 However, it is selected from S, D, Y, T, I, F, V, N, A, P, L, and H, Z 11 However, selected from I, Y, F, V, L, T, N, S, D, A, and H, Z 12 However, it is selected from F, D, Y, L, I, V, A, N, T, P, S, and H, Z 14 The comprising an isolated polypeptide or polypeptide complex as described in any one of Embodiments 1 to 133, selected from D, Y, N, F, I, P, V, A, T, H, L, and S.

[0440] Embodiment 200 is characterized in that Z1 is selected from D, Y, F, I, and N, Z2 is selected from D, Y, L, F, I, and N, Z4 is selected from G and W, Z6 is selected from E and D, Z7 is selected from W, L, F, and V, Z8 is selected from E and D, Z9 is selected from E, D, Y, and V, Z 10 However, selected from S, D, Y, T, and I, Z 11 However, it is selected from I, Y, F, V, L, and T, and Z 12 However, it is selected from F, D, Y, L, I, V, A, and N, Z 14 The solution comprises an isolated polypeptide or polypeptide complex as described in Embodiment 199, selected from D, Y, N, F, I, and P.

[0441] Embodiment 201 is characterized in that Z1 is selected from D, Y, and F, Z2 is selected from D, Y, L, and F, Z4 is selected from G and W, Z6 is selected from E and D, Z7 is selected from W, L, and F, Z8 is selected from E and D, Z9 is selected from E and D, Z10 However, it is selected from S, D, and Y, and Z 11 However, selected from I, Y, and F, Z 12 However, it is selected from F, D, Y, and L, and Z 14 The comprising an isolated polypeptide or polypeptide complex as described in any one of Embodiments 199 to 200, selected from D, Y, and N.

[0442] Embodiment 202 is such that P1 is U1-U2-C-U4-P-U6-U7-U8-U9-U 10 -U 11 -U 12 -CU 14 The amino acid sequence includes U1 selected from D, Y, F, I, N, V, H, L, A, T, S, and P, U2 selected from D, Y, L, F, I, N, A, V, H, T, and S, U4 selected from G and W, U6 selected from E, D, V, and P, U7 selected from W, L, F, V, G, M, I, and Y, U8 selected from E, D, P, and Q, U9 selected from E, D, Y, V, F, W, P, L, and Q, and U 10 However, it is selected from S, D, Y, T, I, F, V, N, A, P, L, and H, U 11 However, it is selected from I, Y, F, V, L, T, N, S, D, A, and H, U 12 However, it is selected from F, D, Y, L, I, V, A, N, T, P, S, G, and H, U 14 The comprising an isolated polypeptide or polypeptide complex as described in any one of Embodiments 1 to 133, selected from D, Y, N, F, I, P, V, A, T, H, L, M, and S.

[0443] Embodiment 203 is characterized in that U1 is selected from D, Y, F, I, V, and N, U2 is selected from D, Y, L, F, I, and N, U4 is selected from G and W, U6 is selected from E and D, U7 is selected from W, L, F, G, and V, U8 is selected from E and D, U9 is selected from E, D, Y, and V, U 10 However, it is selected from S, D, Y, T, and I, U 11However, it is selected from I, Y, F, V, L, and T, U 12 However, it is selected from F, D, Y, L, I, V, A, G, and N, U 14 The solution comprises an isolated polypeptide or polypeptide complex as described in Embodiment 202, selected from D, Y, N, F, I, M, and P.

[0444] Embodiment 204 is characterized in that U1 is selected from D, Y, V, and F, U2 is selected from D, Y, L, and F, U4 is selected from G and W, U6 is selected from E and D, U7 is selected from W, L, G, and F, U8 is selected from E and D, U9 is selected from E and D, and U 10 However, it is selected from S, D, T, and Y, U 11 However, it is selected from I, Y, V, L, and F, U 12 However, it is selected from F, D, Y, G, A, and L, U 14 The solution comprises an isolated polypeptide or polypeptide complex as described in Embodiment 203, selected from D, Y, M, and N.

[0445] Embodiment 205 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 133 and 200 to 204, wherein P1 comprises an amino acid sequence according to any one of Sequence IDs 79 to 105.

[0446] Embodiment 206 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 133 and 200 to 204, wherein P1 comprises an amino acid sequence according to one of the sequences in Table 20.

[0447] Embodiment 207 comprises an isolated polypeptide or polypeptide complex described in any one of Embodiments 1 to 133 or 206, wherein P1 comprises the amino acid sequence according to any one of Sequence IDs 106 to 117.

[0448] Embodiment 208 comprises an isolated polypeptide or polypeptide complex according to any one of Embodiments 1 to 133, wherein P1 comprises the amino acid sequence according to SEQ ID NO: 18, or a peptide sequence having one, two, or three amino acid substitutions, additions, or deletions relative to SEQ ID NO: 18.

[0449] Embodiment 209 comprises an isolated polypeptide or polypeptide complex according to any one of Embodiments 1 to 133, 200 to 204, wherein P1 comprises the amino acid sequence according to SEQ ID NO: 19, or a peptide sequence having one, two, or three amino acid substitutions, additions, or deletions relative to SEQ ID NO: 19.

[0450] Embodiment 210 comprises an isolated polypeptide or polypeptide complex according to any one of Embodiments 1 to 133, 200 to 204, wherein P1 comprises the amino acid sequence according to SEQ ID NO: 116, or a peptide sequence having one, two, or three amino acid substitutions, additions, or deletions relative to SEQ ID NO: 116.

[0451] Embodiment 211 comprises an isolated polypeptide or polypeptide complex as described in Embodiment 208, wherein P1 comprises the amino acid sequence according to SEQ ID NO: 18.

[0452] Embodiment 212 comprises an isolated polypeptide or polypeptide complex as described in Embodiment 209, wherein P1 comprises the amino acid sequence according to SEQ ID NO: 19.

[0453] Embodiment 213 comprises the isolated polypeptide or polypeptide complex described in Embodiment 210, wherein P1 comprises the amino acid sequence according to SEQ ID NO: 116.

[0454] Embodiment 214 comprises a pharmaceutical composition comprising (a) a polypeptide or polypeptide complex described in any one of Embodiments 1 to 213, and (b) a pharmaceutically acceptable excipient.

[0455] Embodiment 215 comprises an isolated recombinant nucleic acid molecule encoding a polypeptide or polypeptide complex described in any one of Embodiments 1 to 213.

[0456] Embodiment 216 is a method for treating lung cancer, comprising the step of administering an isolated polypeptide or polypeptide complex described in Embodiments 1 to 213 to a subject requiring treatment for lung cancer.

[0457] Embodiment 217 is a method for treating breast cancer, comprising the step of administering an isolated polypeptide or polypeptide complex described in Embodiments 1 to 213 to a subject in need of treatment for breast cancer.

[0458] Embodiment 218 is a method for treating cervical cancer, comprising the step of administering an isolated polypeptide or polypeptide complex described in Embodiments 1 to 213 to a subject requiring treatment for cervical cancer.

[0459] Embodiment 219 is a method for treating ovarian cancer, comprising the step of administering an isolated polypeptide or polypeptide complex described in Embodiments 1 to 213 to a subject in need of treatment for ovarian cancer.

[0460] Embodiment 220 is a method for treating pancreatic cancer, comprising the step of administering an isolated polypeptide or polypeptide complex described in Embodiments 1 to 213 to a subject requiring treatment for pancreatic cancer.

[0461] Embodiment 221 is a method for treating colorectal cancer, comprising the step of administering an isolated polypeptide or polypeptide complex described in Embodiments 1 to 213 to a subject requiring treatment for colorectal cancer.

[0462] Embodiment 222 is a method for treating gastric cancer, comprising the step of administering an isolated polypeptide or polypeptide complex described in Embodiments 1 to 213 to a subject requiring treatment for gastric cancer.

[0463] Embodiment 223 is a method for treating pancreatic cancer, comprising the step of administering an isolated polypeptide or polypeptide complex described in Embodiments 1 to 213 to a subject requiring treatment for pancreatic cancer.

[0464] Embodiment 224 is a method for treating metastatic castration-resistant prostate cancer, comprising the step of administering an isolated polypeptide or polypeptide complex described in Embodiments 1 to 213 to a subject requiring treatment for metastatic castration-resistant prostate cancer. [Examples]

[0465] Example 1: Binding of PSMA polypeptide complex The PSMA-CD3 polypeptide complexes shown in Table 7 were evaluated for their binding to PSMA and CD3ε.

[0466] [Table 7]

[0467] The polypeptide complex molecules listed in Table 7 were evaluated for their binding ability to PSMA and CD3 using standard enzyme-linked immunosorbent assay (ELISA). Polypeptide complex binding to PSMA or CD3 was measured before and after protease treatment. Briefly, biotinylated antigens were captured on plates coated with neutraavidin. Where indicated, polypeptide complex molecules were treated with active matryptase (MTSP1). Subsequently, the polypeptide complex molecules, diluted in buffer, were added to the antigen-coated plates. The bound polypeptide complexes were detected using a standard horseradish peroxidase-conjugated secondary antibody. The polypeptide complex concentration required to achieve a 50% maximum signal (EC50) was calculated using Graphpad Prism.

[0468] Figures 2A, 2B, and 2C show representative PSMA-bound ELISAs. This data is summarized in Table 8. Figures 3A, 3B, and 3C show representative CD3-bound ELISAs. This data is summarized in Table 9. EC of masked polypeptide complexes of PC3. 50 The EC of the masked polypeptide complex of PC5 was approximately 8 times higher than that of PC3 treated with protease. 50 The EC of the masked polypeptide complex of PC4 and PC6 was approximately 95 times higher than that of PC3 treated with protease. 50 These values ​​were approximately 100 times and 230 times higher, respectively, than those of polypeptide complexes treated with protease.

[0469] [Table 8]

[0470] [Table 9]

[0471] Example 2: Polypeptide-mediated tumor cytotoxicity and T cell activation Polypeptide complexes were evaluated in a functional in vitro tumor cell death assay using PSMA-positive tumor cell lines 22Rv1 and LNCaP. Tumor cell death was measured using Acea Biosciences' real-time cell analyzer, which depended on increased sensor impedance measurements (cell index) as tumor cells adhered to, spread, and expanded on the sensor surface. Similarly, impedance decreased as tumor cells died. 25,000 tumor cells were added to each well and allowed to adhere overnight. The following day, polypeptide complexes titrated with 75,000 CD8+ T cells in human serum supplement medium were added to the wells. Cell index measurements were acquired every 10 minutes for a further 96 hours. The cell index × time (dynamics of tumor cell proliferation) was then plotted against the concentration of the polypeptide complex, and the concentration required to reduce tumor growth by 50% (IC50) was calculated using Graphpad Prism.

[0472] The 22Rv1 tumor cell line has a PSMA density of approximately 3000 copies per cell. Figure 4 shows representative viability data for 22Rv1 treated with PC1 or PC2. This data is summarized in Table 10, showing that PC2 is approximately 1000 times more potent than PC1. Figures 5A and 5B, as well as Tables 11 and 12, show viability data for 22Rv1 cells treated with masked or cleaved polypeptide complexes. The IC50 of the masked polypeptide complex of PC5 was more than 50 times that of the unmasked polypeptide complex of PC1, but protease treatment reduced the IC50 to less than that of PC1. Similarly, the IC50s of the masked polypeptide complexes of PC4 and PC6 were approximately 150 and 200 times that of PC2, respectively, and both were rescued to approximately 1.7 and 2.5 times that of PC2 by protease treatment.

[0473] The LNCaP tumor cell line has a PSMA density of approximately 350,000 copies per cell. Figure 6 shows representative viability data for LNCaP. This data is summarized in Table 13, showing that PC2 is approximately 100 times more viable than PC1. Figure 7 and Table 14 show viability data for LNCaP cells treated with masked or cleaved polypeptide complexes. The IC50 of the masked polypeptide complex of PC4 was approximately 30 times that of the unmasked polypeptide complex of PC2, but protease treatment rescued the IC50 to approximately 2.5 times that of the unmasked polypeptide complex.

[0474] [Table 10]

[0475] [Table 11]

[0476] [Table 12]

[0477] [Table 13]

[0478] [Table 14]

[0479] Example 3: Tumor cell death mediated by polypeptide complex The polypeptide complex was evaluated in a functional in vitro tumor cell death assay using the PSMA-positive tumor cell line 22Rv1. Tumor cell death was measured using Agilent's xCelligence real-time cell analyzer, which is dependent on increased sensor impedance measurements (cell index) as tumor cells adhere to, spread, and expand on the sensor surface. Similarly, impedance decreased as tumor cells died. 10,000 tumor cells were added to each well and allowed to adhere overnight on a 96-well E-plate. The following day, the polypeptide complex, titrated with 30,000 CD8+ T cells in human serum supplement medium, was added to the wells. Cell index measurements were acquired every 10 minutes for a further 72 hours. The cell index × time (dynamics of tumor cell proliferation) was then plotted against the concentration of the polypeptide complex, and the concentration required to reduce tumor growth by 50% (IC50) was calculated using Graphpad Prism software. The data are shown in Figures 8A and 8B.

[0480] Example 4: Pharmacokinetics of polypeptide complexes in cynomolgus monkeys The pharmacokinetics and exploratory safety of polypeptide molecules were evaluated in cynomolgus monkeys. Briefly, cynomolgus monkeys weighing approximately 3 kg were administered the polypeptide as an IV bolus, and signs of adverse events were observed daily. No adverse events were observed during survival. Blood was collected in K2 EDTA tubes at specific time points after administration and processed into plasma. The plasma was cryopreserved until analysis. The concentration of polypeptide molecules in the plasma was measured using standard ELISA techniques and compared to a reference standard diluted in control cynomolgus monkey plasma. The plasma concentration curves were fitted to the standard two-stage exponential equation representing the stages of distribution and excretion. Pharmacokinetic fitting allowed for the calculation of Cmax, half-life, volume of distribution, clearance, and area under the curve (AUC) over 7 days, as shown in Table 15 for the PSMA TCE polypeptide complex and Table 16 for the PSMA TRACTr polypeptide complex. The data are shown in Figures 9A-9B. The pharmacokinetics measured in cynomolgus monkeys support once-weekly administration in humans.

[0481] [Table 15]

[0482] [Table 16]

[0483] Example 5: Cytokine release of polypeptide complexes in cynomolgus monkeys Cytokine release after IV bolus administration of polypeptide molecules to cynomolgus monkeys was evaluated. Briefly, cynomolgus monkeys weighing approximately 3 kg were administered polypeptides as IV boluses, and signs of adverse events were observed daily. No adverse events were observed during survival. After administration, blood was collected in K2 EDTA tubes at specific time points and processed into plasma. The plasma was frozen and stored until analysis. Plasma samples were analyzed for cytokines using BD Biosciences' non-human primate cytometric Th1 / Th2 bead array kit, following the manufacturer's instructions. Levels of interferon-γ, tumor necrosis factor-α, interleukin-6, interleukin-5, interleukin-4, and interleukin-2 in the plasma were calculated by comparing them to the reference standards provided in the bead array kit. The data are shown in Figures 10A to 10C.

[0484] Example 6: Toxicity of polypeptide complexes in cynomolgus monkeys Systemic liver enzymes were evaluated in cynomolgus monkeys after IV bolus administration of polypeptide molecules. Briefly, approximately 3 kg cynomolgus monkeys were administered polypeptides as an IV bolus, and signs of adverse events were observed daily. No adverse events were observed during survival. Blood was collected in K2 EDTA tubes at specific time points after administration and processed into plasma. Plasma was frozen and stored until analysis. Plasma samples were analyzed for the presence of liver enzymes aspartate transaminase (AST) and alanine aminotransferase (ALT) as signs of possible hepatotoxicity. AST and ALT levels remained within the normal range at all time points tested after administration, suggesting the absence of hepatotoxicity. AST and ALT were quantified according to the instructions provided in the commercially available Millipore kit. AST and ALT levels were calculated by comparing them to a positive control reference standard according to the manufacturer's instructions. Data are shown in Figures 11A and 11B.

[0485] Example 7: Construction of an optimized phage library - CD3 scFv peptide Sequence activity relationships (SARs) were established for peptides A and B by mutating individual residues within the peptides to alanine and measuring their binding and inhibition against SP34.185 scFv. Peptide residues where binding and inhibition were significantly weakened by the alanine mutation could be identified as critical residues where mutation was unacceptable. Peptide residues where the performance of the alanine mutation was similar to that of the non-mutated sequence could be identified as uncritical sites where mutation was unacceptable. A DNA oligo library was constructed using peptide SARs, where mutations in codons encoding critical residues within each peptide sequence were minimal, while mutations in codons encoding uncritical residues were large. The resulting oligos were cloned into bacteriophage vectors used to represent SAR-guided peptides via fusion to bacteriophage pIII filaments. Subsequently, phage-optimized libraries were produced using the relevant vectors via amplification in bacteria using techniques standard in the art.

[0486] The binding ability of peptides to SP34.185scFv was evaluated using standard ELISA. Briefly, biotinylated peptides were captured on a neutraavidin-coated plate and quenched by biocitin followed by a washing step. SP34.185scFv was then titrated onto the plate containing the captured peptides. The plate was then washed, and binding of SP34.185scFv was detected using a second horseradish peroxidase antibody conjugate. After another washing, the plate was developed using standard ELISA techniques and stopped with acid. The concentration of SP34.185scFv required to achieve a 50% maximum signal or EC50 was calculated using Graphpad prism. The data are shown in Figures 12A to 12F and summarized in Tables 17A to 17D. Table 19 shows the peptide sequences of the CD3 alanine scan (Ala Scan) peptides in peptide A and peptide B.

[0487] [Table 17A]

[0488] [Table 17B]

[0489] [Table 17C]

[0490] [Table 17D]

[0491] The inhibitory activity of peptides on the binding of SP34.185scFv to CD3e was evaluated using standard ELISA. Briefly, fi...

Claims

1. Formula I: A ２ -A １ -L １ -P １ -H １ A polypeptide complex by, During the ceremony, A １ comprises a first antigen recognition molecule that binds to an effector cell antigen, and A １ comprises an anti-CD3 antibody or an antigen-binding fragment thereof, and comprises HC-CDR1, HC-CDR2, and HC-CDR3 as complementarity-determining regions (CDRs), and A １ said HC-CDR1, said HC-CDR2, and said HC-CDR3 of A comprise SEQ ID NO: 1 for HC-CDR1, SEQ ID NO: 2 for HC-CDR2, and SEQ ID NO: 3 for HC-CDR3, and A １ comprises LC-CDR1, LC-CDR2, and LC-CDR3 as CDRs, and A １ said LC-CDR1, said LC-CDR2, and said LC-CDR3 of A comprise SEQ ID NO: 4 for LC-CDR1, SEQ ID NO: 5 for LC-CDR2, and SEQ ID NO: 6 for LC-CDR3, P １ is, A １ It contains a peptide that binds to P １ U １ -U ２ -C-U ４ -P-U ６ -U ７ -U ８ -U ９ -U １０ -U １１ -U １２ -C-U １４ Includes an amino acid sequence by U １ However, it is selected from D, Y, F, I, N, V, H, L, A, T, S, and P, U ２ However, it is selected from D, Y, L, F, I, N, A, V, H, T, and S, U ４ However, it is selected from G and W, U ６ However, selected from E, D, V, and P, U ７ However, it is selected from W, L, F, V, G, M, I, and Y, U ８ However, selected from E, D, P, and Q, U ９ However, it is selected from E, D, Y, V, F, W, P, L, and Q, U １０ However, it is selected from S, D, Y, T, I, F, V, N, A, P, L, and H, U １１ However, it is selected from I, Y, F, V, L, T, N, S, D, A, and H, U １２ However, it is selected from F, D, Y, L, I, V, A, N, T, P, S, G, and H, U １４ However, it is selected from D, Y, N, F, I, P, V, A, T, H, L, M, and S. L １ P １ A １ It connects and includes a ligation portion that is a substrate for tumor-specific proteases. H １ It contains half-life extension molecules, A ２ This is a polypeptide complex comprising a second antigen-recognizing molecule containing an antibody that binds to prostate-specific membrane antigen (PSMA) or an antigen-binding fragment thereof.

2. A ２ However, the complementarity determination region (CDR) includes HC-CDR1, HC-CDR2, and HC-CDR3, and HC-CDR1, HC-CDR2, and HC-CDR3 include sequence number 8 for HC-CDR1, sequence number 9 for HC-CDR2, and sequence number 10 for HC-CDR3, A ２ However, the CD-R includes LC-CDR1, LC-CDR2, and LC-CDR3, A ２ The polypeptide complex according to claim 1, wherein the LC-CDR1, LC-CDR2, and LC-CDR3 each include SEQ ID NO: 11 for LC-CDR1, SEQ ID NO: 12 for LC-CDR2, and SEQ ID NO: 13 for LC-CDR3.

3. The polypeptide complex according to claim 1, wherein the effector cell antigen comprises differentiation antigen group 3 (CD3).

4. A １ The polypeptide complex according to claim 1, wherein the complex comprises a single-chain variable fragment and an antibody form selected from the Fab or Fab' fragment.

5. A ２ The polypeptide complex according to claim 1, wherein the complex comprises a single-chain variable fragment, a single-domain antibody, and an antibody format selected from Fab or Fab' fragment.

6. A １ However, it includes an antibody form of a single-chain variable fragment (scFv), A ２ The polypeptide complex according to claim 1, wherein the antibody form is Fab or Fab'.

7. L １ It is cleaved by tumor-specific proteases, thereby A １ When exposed to the effector cell antigen, P １ A １ The polypeptide complex according to claim 1, which becomes debondable.

8. The polypeptide complex according to claim 1, wherein the tumor-specific protease is selected from the group consisting of matrix metalloproteinase (MMP), serine protease, cysteine ​​protease, threonine protease, and aspartate protease.

9. The polypeptide complex according to claim 8, wherein the matrix metalloproteinase comprises MMP2, MMP7, MMP9, MMP13, or MMP14.

10. The polypeptide complex according to claim 8, wherein the serine protease comprises matryptase (MTSP1), urokinase, or hepsin.

11. L １ The polypeptide complex according to claim 1, wherein it comprises a urokinase-cleavable amino acid sequence, a matryptase-cleavable amino acid sequence, a matrix metalloproteinase-cleavable amino acid sequence, or a regmine-cleavable amino acid sequence.

12. L １ The polypeptide complex according to claim 1, wherein the complex comprises the amino acid sequence specified by SEQ ID NO:

23.

13. L １ The polypeptide complex according to claim 1, wherein the complex comprises an amino acid sequence according to any one of sequence numbers 20 to 49.

14. L １ The polypeptide complex according to claim 1, wherein the linker 25 (ISSGLLSGRSDAG) (SEQ ID NO: 45), linker 26 (AAGLLAAPPGGLSGRSDAG) (SEQ ID NO: 46), linker 27 (SPLGLLSGRSDAG) (SEQ ID NO: 47), or linker 28 (LSGRSDAGSPLGLAG) (SEQ ID NO: 48) comprises the amino acid sequence of linker 25 (ISSGLLSGRSDAG) (SEQ ID NO: 45), linker 26 (AAGLLAAPPGGLSGRSDAG) (SEQ ID NO: 46), linker 27 (SPLGLLSGRSDAG) (SEQ ID NO: 47), or linker 28 (LSGRSDAGSPLGLAG) (SEQ ID NO: 48).

15. H １ The polypeptide complex according to claim 1, wherein the polypeptide complex comprises serum albumin.

16. The polypeptide complex according to claim 15, wherein the serum albumin is human serum albumin.

17. H １ The polypeptide complex according to claim 1, wherein it comprises an anti-albumin single-domain antibody.

18. H １ However, the complementarity determination region (CDR) includes HC-CDR1, HC-CDR2, and HC-CDR3, H １ The polypeptide complex according to claim 17, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 each include SEQ ID NO: 54 for HC-CDR1, SEQ ID NO: 55 for HC-CDR2, and SEQ ID NO: 56 for HC-CDR3.

19. H １ However, the complementarity determination region (CDR) includes HC-CDR1, HC-CDR2, and HC-CDR3, H １ The polypeptide complex according to claim 17, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 each include SEQ ID NO: 58 for HC-CDR1, SEQ ID NO: 59 for HC-CDR2, and SEQ ID NO: 60 for HC-CDR3.

20. U １ However, it is selected from D, Y, F, I, V, and N, U ２ However, it is selected from D, Y, L, F, I, and N, U ４ However, it is selected from G and W, U ６ However, it is selected from E and D, U ７ However, it is selected from W, L, F, G, and V, U ８ However, it is selected from E and D, U ９ However, it is selected from E, D, Y, and V, U １０ However, it is selected from S, D, Y, T, and I, U １１ However, it is selected from I, Y, F, V, L, and T, U １２ However, it is selected from F, D, Y, L, I, V, A, G, and N, U １４ The polypeptide complex according to claim 1, wherein the molecule is selected from D, Y, N, F, I, M, and P.

21. U １ However, it is selected from D, Y, V, and F, U ２ However, it is selected from D, Y, L, and F, U ４ However, it is selected from G and W, U ６ However, it is selected from E and D, U ７ However, it is selected from W, L, G, and F, U ８ However, it is selected from E and D, U ９ However, it is selected from E and D, U １０ However, it is selected from S, D, T, and Y, U １１ However, selected from I, Y, V, L, and F, U １２ However, it is selected from F, D, Y, G, A, and L, U １４ The polypeptide complex according to claim 20, wherein selected from D, Y, M, and N.

22. P １ However, the amino acid sequence of any one of sequence numbers 93-95 and 102-105 The polypeptide complex according to claim 1, comprising:

23. P １ The polypeptide complex according to claim 1, wherein the complex comprises an amino acid sequence according to any one of sequence numbers 106 and 108-117.

24. P １ The polypeptide complex according to claim 1, wherein the complex comprises the amino acid sequence specified by SEQ ID NO:

19.

25. P １ The polypeptide complex according to claim 1, wherein the complex comprises the amino acid sequence specified by SEQ ID NO:

116.

26. The polypeptide complex according to claim 1, comprising the amino acid sequences of SEQ ID NOs: 72 and 73.

Images