Compositions and methods relating to antibody targeting tumor-activated EGFR and effector cell antigens - Patents.com

Abstract

Provided herein are multispecific antibodies for redirecting T cells to cancer that rely on binding of one antigen interacting portion of the antibody to a tumor-associated antigen or marker, such as epidermal growth factor receptor (EGFR), while a second antigen interacting portion can bind to an effector cell antigen on T cells, such as CD3, pharmaceutical compositions thereof, and nucleic acids, and methods for making and discovering the multispecific antibodies.

Description

[Technical Field]

[0001] (cross reference) This application claims the benefit of U.S. Provisional Patent Application No. 63 / 187,690, filed March 12, 2021, and U.S. Provisional Patent Application No. 63 / 327,317, filed April 4, 2022, each of which is incorporated by reference in its entirety.

[0002] (Sequence Listing) This application contains a Sequence Listing, which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on April 29, 2022, is named 52426-738_601_SL.txt and is 542,564 bytes in size. Summary of the Invention

[0003] In one embodiment, a polypeptide or polypeptide complex is provided as set forth in Formula I below:

[0004] [ka] Described herein are polypeptides or polypeptide complexes, wherein A1 comprises a first antigen recognition molecule that binds to an effector cell antigen, P1 comprises a peptide that binds to A1, L1 comprises a linking moiety connecting 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 recognition molecule that binds to epidermal growth factor receptor (EGFR). In some embodiments, the first antigen recognition molecule comprises an antibody or antibody fragment. In some embodiments, the first antigen recognition molecule comprises a human antibody or a humanized antibody or antibody fragment. In some embodiments, L1 is attached to the N-terminus of the first antigen recognition molecule. In some embodiments, A2 is attached to the C-terminus of the first antigen recognition molecule. In some embodiments, L1 is attached to the C-terminus of the first antigen recognition molecule. In some embodiments, A2 is attached to the N-terminus of the first antigen recognition 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 of a camelid-derived single-domain antibody (VHH). In some embodiments, A1 comprises an anti-CD3e single-chain variable fragment. In some embodiments, A1 has a K of 1 μM or less to CD3 on CD3-expressing cells. DIn some embodiments, A1 comprises an anti-CD3e single-chain variable fragment having binding activity. In some embodiments, the effector cell antigen comprises CD3. In some embodiments, A1 comprises a variable light chain and a variable heavy chain, each capable of specifically binding to human CD3. In some embodiments, A1 is selected from the group consisting of muromonab-CD3 (OKT3), otelixizumab (TRX4), teplizumab (MGA031), visilizumab (Nuvion), SP34, X35, VIT3, BMA030 (BW264 / 56), CLB-T3 / 3, CRIS7, YTH12.5, F111-409, CLB-T3.4.2, TR-66, WT32, SP 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, the isolated polypeptide or polypeptide complex of Formula I binds to an effector cell when L1 is cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex of Formula I binds to an effector cell when L1 is cleaved by a tumor-specific protease and A1 binds to the effector cell. In some embodiments, the effector cell is a T cell. In some embodiments, A1 binds to a polypeptide that is part of a TCR-CD3 complex on an effector cell. In some embodiments, the polypeptide that is part of a TCR-CD3 complex is human CD3ε.In some embodiments, the effector cell antigen comprises CD3, and the scFv comprises CDRs LC-CDR1, LC-CDR2, and LC-CDR3, wherein LC-CDR1 is set forth in SEQ ID NO: 1, LC-CDR2 is set forth in SEQ ID NO: 2, and LC-CDR3 is set forth in SEQ ID NO: 3, and the scFv comprises complementarity determining regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3, wherein HC-CDR1 is set forth in SEQ ID NO: 4, HC-CDR2 is set forth in SEQ ID NO: 5, and HC-CDR3 is set forth in SEQ ID NO: 6. In some embodiments, the effector cell antigen comprises CD3, and the scFv comprises the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the effector cell antigen comprises CD3, and the scFv comprises CDRs LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, LC-CDR2, and LC-CDR3 of the scFv comprise LC-CDR1 of SEQ ID NO: 7, LC-CDR2 of SEQ ID NO: 8, and LC-CDR3 of SEQ ID NO: 9, and the scFv comprises complementarity determining regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 of the scFv comprise HC-CDR1 of SEQ ID NO: 10, HC-CDR2 of SEQ ID NO: 11, and HC-CDR3 of SEQ ID NO: 12. In some embodiments, the effector cell antigen comprises CD3, and the scFv comprises the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the second antigen recognition molecule comprises an antibody or an antibody fragment. In some embodiments, the antibody or antibody fragment thereof comprises a single-chain variable fragment, a single-domain antibody, or a Fab. In some embodiments, the antibody or antibody fragment thereof 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 of a camelid-derived single-domain antibody (VHH). In some embodiments, the antibody or antibody fragment thereof is humanized or human. In some embodiments, A2 is a Fab.In some embodiments, the Fab comprises (a) a Fab light chain polypeptide and (b) a Fab heavy chain polypeptide. In some embodiments, the Fab comprises CDRs LC-CDR1, LC-CDR2, and LC-CDR3, where the LC-CDR1, LC-CDR2, and LC-CDR3 of the Fab comprise the LC-CDR1 of SEQ ID NO: 15, the LC-CDR2 of SEQ ID NO: 16, and the LC-CDR3 of SEQ ID NO: 17, and the Fab comprises complementarity-determining regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3 of A1, where the HC-CDR1, HC-CDR2, and HC-CDR3 of A1 comprise the HC-CDR1 of SEQ ID NO: 18, the HC-CDR2 of SEQ ID NO: 19, and the HC-CDR3 of SEQ ID NO: 20. In some embodiments, the Fab light chain polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 21. In some embodiments, the Fab light chain polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 22. In some embodiments, the Fab heavy chain polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 23. In some embodiments, the Fab heavy chain polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 24. In some embodiments, the Fab light chain polypeptide of A2 is attached to the C-terminus of the single-chain variable fragment (scFv) of A1. In some embodiments, the Fab heavy chain polypeptide of A2 is attached to the C-terminus of the single-chain variable fragment of A1. In some embodiments, the Fab light chain polypeptide of A2 is attached to the N-terminus of the single-chain variable fragment of A1. In some embodiments, the Fab heavy chain polypeptide of A2 is attached to the N-terminus of the single-chain variable fragment of A1. In some embodiments, the Fab heavy chain polypeptide of A2 is attached to the scFv heavy chain polypeptide of A1. In some embodiments, the Fab light chain polypeptide of A2 is attached to the scFv heavy chain polypeptide of A1. In some embodiments, the Fab heavy chain polypeptide of A2 is attached to the scFv light chain polypeptide of A1. In some embodiments, the Fab light chain polypeptide of A2 is attached 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 moiety connecting A2 to P2 and is a substrate for a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex is described below in Formula Ia:

[0005] [ka] 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 binding of A1 to an effector cell antigen. In some embodiments, P1 is bound to A1 by ionic interactions, electrostatic interactions, hydrophobic interactions, Pi stacking interactions, and hydrogen 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 binding of A2 to EGFR. In some embodiments, P2 is bound to A2 by ionic interactions, electrostatic interactions, hydrophobic interactions, Pi stacking interactions, and hydrogen bonding interactions, or a combination thereof. In some embodiments, P2 is bound to A2 at or near the antigen binding site. In some embodiments, P2 has less than 70% sequence homology to EGFR. In some embodiments, P1 or P2 comprises a peptide sequence at least 10 amino acids in length. In some embodiments, P1 or P2 comprises a peptide sequence at least 10 amino acids in length and no more than 20 amino acids in length. In some embodiments, P1 or P2 comprises a peptide sequence at least 16 amino acids in length. In some embodiments, P1 or P2 comprises a peptide sequence no more than 40 amino acids in length. In some embodiments, P1 or P2 comprises at least two cysteine ​​amino acid residues. In some embodiments, P1 or P2 comprises a cyclic or linear peptide. In some embodiments, P1 or P2 comprises a cyclic peptide.In some embodiments, P1 or P2 comprises a linear peptide. In some embodiments, P1 comprises at least two cysteine ​​amino acid residues. In some embodiments, P1 comprises the amino acid sequence set forth in SEQ ID NO:25. In some embodiments, P2 comprises the amino acid sequence set forth in SEQ ID NO:26. In some embodiments, P2 comprises the amino acid sequence set forth in SEQ ID NO:115. In some embodiments, P2 comprises the amino acid sequence set forth in SEQ ID NO:116. In some embodiments, P1 comprises the amino acid sequence set forth in SEQ ID NO:70. In some embodiments, L1 is attached to the N-terminus of A1. In some embodiments, L1 is attached to the C-terminus of A1. In some embodiments, L2 is attached to the N-terminus of A2. In some embodiments, L2 is attached to the C-terminus of A2. In some embodiments, L1 or L2 is a peptide sequence having at least 5-50 amino acids or less. In some embodiments, L1 or L2 is a peptide sequence having at least 10-30 amino acids or less. 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 wherein n is an integer from 1 to 3 (SEQ ID NO: 840). In some embodiments, L1 has a formula comprising (G2S) n , (GS) n , (GSGGS) n (SEQ ID NO: 836), (GGGS) n (SEQ ID NO: 837), (GGGGS) n (SEQ ID NO: 838), and (GSSGGS) n(SEQ ID NO: 839), wherein n is an integer of at least 1. In some embodiments, P1 is uncoupled from A1 when L1 is cleaved by a tumor-specific protease, thereby exposing A1 to an effector cell antigen. In some embodiments, P2 is uncoupled from A2 when L2 is cleaved by a tumor-specific protease, thereby exposing A2 to EGFR. In some embodiments, the tumor-specific protease is selected from the group consisting of matrix metalloproteases (MMPs), serine proteases, cysteine ​​proteases, threonine proteases, and aspartic acid proteases. In some embodiments, the matrix metalloproteases include MMP2, MMP7, MMP9, MMP13, or MMP14. In some embodiments, the serine proteases include matriptase (MTSP1), urokinase, or hepsin. In some embodiments, L1 or L2 comprises an amino acid sequence cleavable by urokinase, matriptase, matrix metalloproteinase, or legumain. In some embodiments, L1 or L2 comprises the amino acid sequence set forth in SEQ ID NO: 30 or 31. In some embodiments, L1 or L2 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 27-35. In some embodiments, L1 or L2 comprises the amino acid sequence of linker 4 (ISSGLLSGRSDAG) (SEQ ID NO: 66), linker 5 (AAGLLAPPGGLSGRSDAG) (SEQ ID NO: 67), linker 6 (SPLGLSGRSDAG) (SEQ ID NO: 68), or linker 7 (LSGRSDAGSPLGLAG) (SEQ ID NO: 69), or an amino acid sequence having one, two, or three amino acid substitutions, additions, or deletions relative to the amino acid sequence of linker 4, linker 5, linker 6, or linker 7. In some embodiments, H1 comprises a polymer. In some embodiments, the polymer is polyethylene glycol (PEG). In some embodiments, H1 comprises albumin. In some embodiments, H1 comprises an Fc domain.In some embodiments, the albumin is serum albumin. In some embodiments, the albumin is human serum albumin. In some embodiments, H1 comprises a polypeptide, ligand, or small molecule. In some embodiments, the polypeptide, ligand, or small molecule binds a serum protein or fragment thereof, a circulating immunoglobulin or fragment thereof, or CD35 / CR1. In some embodiments, the serum protein comprises thyroxine-binding protein, transthyretin, 1-acid glycoprotein, transferrin, transferrin receptor or a transferrin-binding portion thereof, fibrinogen, or albumin. In some embodiments, the circulating immunoglobulin molecule comprises IgG1, IgG2, IgG3, IgG4, slgA, IgM, or IgD. In some embodiments, the serum protein is albumin. In some embodiments, the polypeptide is an antibody. In some embodiments, the antibody comprises a single-domain antibody, a single-chain variable fragment, or a Fab. In some embodiments, the single-domain antibody comprises a single-domain antibody that binds to albumin. In some embodiments, the single-domain antibody is a human antibody or a humanized antibody. In some embodiments, the single domain antibody is 645gH1gL1. In some embodiments, the single domain antibody is 645dsgH5gL4. In some embodiments, the single domain antibody is 23-13-A01 -sc02. In some embodiments, the single domain antibody is A10m3 or a fragment thereof. In some embodiments, the single domain antibody is DOM7r-31. In some embodiments, the single domain antibody is DOM7h-11-15. In some embodiments, the single domain antibody is Alb-1, Alb-8, or Alb-23. In some embodiments, the single domain antibody is 10E.In some embodiments, the single domain antibody comprises complementarity determining regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 of the single domain antibody comprise HC-CDR1 of SEQ ID NO: 36, HC-CDR2 of SEQ ID NO: 37, and HC-CDR3 of SEQ ID NO: 38. In some embodiments, the single domain antibody comprises complementarity determining regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3 of the single domain antibody comprise HC-CDR1 of SEQ ID NO: 39, HC-CDR2 of SEQ ID NO: 40, and HC-CDR3 of SEQ ID NO: 41. In some embodiments, the single domain antibody is SA21. In some embodiments, the isolated polypeptide or polypeptide complex comprises modified amino acids, unnatural amino acids, modified unnatural amino acids, or combinations thereof. In some embodiments, the modified amino acid or modified unnatural amino acid comprises a post-translational modification. In some embodiments, H1 comprises a linking moiety (L3) connecting H1 to P1. In some embodiments, L3 is a peptide sequence having at least 5-50 amino acids or less. In some embodiments, L3 is a peptide sequence having at least 10-30 amino acids or less. 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: 836), (GGGS) n (SEQ ID NO: 837), (GGGGS) n (SEQ ID NO: 838), and (GSSGGS) n (SEQ ID NO: 839), wherein n is is an integer of at least 1. In some embodiments, L3 comprises the amino acid sequence set forth in SEQ ID NO:29. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NOs:44-61. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:50. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:51. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:44 and SEQ ID NO:45. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:46 and SEQ ID NO:47. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:48 and SEQ ID NO:49. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:50 and SEQ ID NO:51. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:52 and SEQ ID NO:53. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:54 and SEQ ID NO:55. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:56 and SEQ ID NO:57. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:58 and SEQ ID NO:59.In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 60 and SEQ ID NO: 61. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 62 and SEQ ID NO: 63. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 64 and SEQ ID NO: 65.

[0006] In certain embodiments, described herein are pharmaceutical compositions comprising (a) an isolated polypeptide or polypeptide complex described herein and (b) a pharmaceutically acceptable excipient.

[0007] In certain embodiments, described herein are isolated recombinant nucleic acid molecules that encode an isolated polypeptide or polypeptide complex described herein.

[0008] In one embodiment, a polypeptide or polypeptide complex is provided as set forth in Formula II below:

[0009] [ka] In the formula, L 1a When uncut, P 1a a linking moiety cleaved by a tumor-specific protease connecting P to a first antigen recognition molecule that binds to an effector cell antigen, and the first antigen recognition molecule is bound to a second antigen recognition molecule that binds to EGFR; 1a L 1a comprises a peptide that binds to the first antigen recognition molecule when uncleaved, and 1a In some embodiments, P 1a L 1aWhen uncleaved, P impairs binding of the first antigen recognition molecule to the effector cell antigen. 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 has less than 70% sequence homology to the effector cell antigen. 1a comprises a peptide sequence of at least 10 amino acids in length. 1a comprises a peptide sequence at least 10 amino acids in length and no more than 20 amino acids in length. 1a comprises a peptide sequence of at least 16 amino acids in length. 1a comprises a peptide sequence of 40 amino acids or less in length. 1a contains at least two cysteine ​​amino acid residues. In some embodiments, P 1a comprises a cyclic peptide or a linear peptide. In some embodiments, P 1a In some embodiments, P 1a comprises a linear peptide. In some embodiments, P 1a comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 25. In some embodiments, P 1a comprises the amino acid sequence set forth in SEQ ID NO: 70. In some embodiments, H 1a comprises a polymer. In some embodiments, the polymer is polyethylene glycol (PEG). In some embodiments, H 1a In some embodiments, H 1a In some embodiments, the albumin is serum albumin. In some embodiments, the albumin is human serum albumin. In some embodiments, the H 1acomprises a polypeptide, ligand, or small molecule. In some embodiments, the polypeptide, ligand, or small molecule binds a serum protein or fragment thereof, a circulating immunoglobulin or fragment thereof, or CD35 / CR1. In some embodiments, the serum protein comprises thyroxine-binding protein, transthyretin, 1-acid glycoprotein, transferrin, transferrin receptor or a transferrin-binding portion thereof, fibrinogen, or albumin. In some embodiments, the circulating immunoglobulin molecule comprises IgG1, IgG2, IgG3, IgG4, slgA, IgM, or IgD. In some embodiments, the serum protein is albumin. In some embodiments, the polypeptide is an antibody. In some embodiments, the antibody comprises a single-domain antibody, a single-chain variable fragment, or Fab. In some embodiments, the antibody comprises a single-domain antibody that binds 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 10G. In some embodiments, the single domain antibody comprises complementarity determining regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 of the single domain antibody comprise HC-CDR1 of SEQ ID NO: 36, HC-CDR2 of SEQ ID NO: 37, and HC-CDR3 of SEQ ID NO: 38.In some embodiments, the single domain antibody comprises complementarity determining regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3, and the HC-CDR1, HC-CDR2, and HC-CDR3 of the single domain antibody comprise HC-CDR1 of SEQ ID NO: 39, HC-CDR2 of SEQ ID NO: 40, and HC-CDR3 of SEQ ID NO: 41. In some embodiments, the single domain antibody is SA21. In some embodiments, H. 1a is H 1a P 1a The connecting part (L 1a In some embodiments, L 1a is a peptide sequence having at least 5 to 50 amino acids. 1a is a peptide sequence having at least 10 to 30 amino acids. 1a is a peptide sequence having at least 10 amino acids. In some embodiments, L 1a is a peptide sequence having at least 18 amino acids. In some embodiments, L 1a is a peptide sequence having at least 26 amino acids. 1a (G2S) n , (GS) n , (GSGGS) n (SEQ ID NO: 836), (GGGS) n (SEQ ID NO: 837), (GGGGS) n (SEQ ID NO: 838), and (GSSGGS) n (SEQ ID NO: 839), wherein n is an integer of at least 1. In some embodiments, L 1acomprises the amino acid sequence set forth in SEQ ID NO: 30 or 31. In some embodiments, L1 or L2 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 27-35. In some embodiments, L1 or L2 comprises the amino acid sequence of linker 4 (ISSGLLSGRSDAG) (SEQ ID NO: 66), linker 5 (AAGLLAPPGGLSGRSDAG) (SEQ ID NO: 67), linker 6 (SPLGLSGRSDAG) (SEQ ID NO: 68), or linker 7 (LSGRSDAGSPLGLAG) (SEQ ID NO: 69), or an amino acid sequence having one, two, or three amino acid substitutions, additions, or deletions relative to the amino acid sequence of linker 4, linker 5, linker 6, or linker 7.

[0010] In one embodiment, a polypeptide complex comprising the structural arrangement described in Configuration 1 below,

[0011] [ka] Described herein is an isolated polypeptide or polypeptide complex comprising a single-chain variable fragment (scFv) comprising a light chain variable domain and a heavy chain variable domain, wherein the scFv is linked to a peptide (P1) that impairs binding of the scFv to an effector cell antigen, and P1 is linked to the N-terminus of the heavy chain variable domain of the scFv by a linking moiety (L1) that is a substrate for a tumor-specific protease, and P1 is further linked to a half-life extension and a Fab that binds to epidermal growth factor receptor (EGFR), wherein the Fab comprises a Fab light chain polypeptide and a Fab heavy chain polypeptide, the Fab light chain polypeptide is linked to the C-terminus of the light chain variable domain of the scFv, and the Fab is linked to P2 and L2, wherein P2 comprises the peptide that impairs binding to EGFR, and L2 connects the Fab heavy chain polypeptide to P2 and comprises a linking moiety that is a substrate for a tumor-specific protease.

[0012] In one embodiment, a polypeptide complex comprising the structural arrangement described in Configuration 2 below,

[0013] [ka] Described herein is an isolated polypeptide or polypeptide complex comprising a single-chain variable fragment (scFv) comprising a light chain variable domain and a heavy chain variable domain, wherein the scFv is linked to a peptide (P1) that impairs binding of the scFv to an effector cell antigen, and P1 is linked to the N-terminus of the heavy chain variable domain of the scFv by a linking moiety (L1) that is a substrate for a tumor-specific protease, and P1 is further linked to a half-life extension and a Fab that binds to epidermal growth factor receptor (EGFR), wherein the Fab comprises a Fab light chain polypeptide and a Fab heavy chain polypeptide, and the Fab heavy chain polypeptide is linked to the C-terminus of the light chain variable domain of the scFv, and the Fab is linked to P2 and L2, wherein P2 comprises a peptide that impairs binding to EGFR, and L2 comprises a linking moiety that joins the Fab light chain polypeptide to P2 and is a substrate for a tumor-specific protease.

[0014] (Incorporated by reference) All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. [Brief explanation of the drawings]

[0015] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings. [Figure 1A] 1A-1B illustrate a polypeptide complex of the present disclosure. [Figure 1B]1A-1B illustrate a polypeptide complex of the present disclosure. [Figure 2] FIG. 2 illustrates the binding of several polypeptide complexes of the present disclosure to EGFR as measured by ELISA. [Figure 3] FIG. 3 illustrates the binding of several polypeptide complexes of the present disclosure to CD3 as measured by ELISA. [Figure 4] FIG. 4 illustrates that polypeptide complexes of the present disclosure mediated HCT116 tumor cell killing in the presence of CD8+ T cells. [Figure 5] FIG. 5 illustrates that polypeptide complexes of the present disclosure mediated A431 tumor cell killing in the presence of CD8+ T cells. [Figure 6] FIG. 6 illustrates the kinetic binding and cross-reactivity of polypeptide complexes of the present disclosure to human and cynomolgus monkey EGFR. [Figure 7] FIG. 7 illustrates the equilibrium binding and cross-reactivity of polypeptide complexes of the present disclosure to human and cynomolgus monkey EGFR. [Figure 8] FIG. 8 illustrates the kinetic binding and cross-reactivity of several polypeptide complexes to human and cynomolgus monkey CD3ε. [Figure 9] FIG. 9 illustrates the equilibrium binding and cross-reactivity of polypeptide complexes of the present disclosure to human and cynomolgus monkey CD3ε. [Figure 10A] 10A-10B illustrate the pharmacokinetics in cynomolgus monkeys after a single IV bolus injection of a polypeptide conjugate of the present disclosure. [Figure 10B] 10A-10B illustrate the pharmacokinetics in cynomolgus monkeys after a single IV bolus injection of a polypeptide conjugate of the present disclosure. [Figure 11A] 11A-11L illustrate cytokine release in cynomolgus monkeys after a single IV bolus of a polypeptide conjugate of the present disclosure. [Figure 11B]11A-11L illustrate cytokine release in cynomolgus monkeys after a single IV bolus of a polypeptide conjugate of the present disclosure. [Figure 11C] 11A-11L illustrate cytokine release in cynomolgus monkeys after a single IV bolus of a polypeptide conjugate of the present disclosure. [Figure 11D] 11A-11L illustrate cytokine release in cynomolgus monkeys after a single IV bolus of a polypeptide conjugate of the present disclosure. [Figure 11E] 11A-11L illustrate cytokine release in cynomolgus monkeys after a single IV bolus of a polypeptide conjugate of the present disclosure. [Figure 11F] 11A-11L illustrate cytokine release in cynomolgus monkeys after a single IV bolus of a polypeptide conjugate of the present disclosure. [Figure 11G] 11A-11L illustrate cytokine release in cynomolgus monkeys after a single IV bolus of a polypeptide conjugate of the present disclosure. [Figure 11H] 11A-11L illustrate cytokine release in cynomolgus monkeys after a single IV bolus of a polypeptide conjugate of the present disclosure. [Figure 11I] 11A-11L illustrate cytokine release in cynomolgus monkeys after a single IV bolus of a polypeptide conjugate of the present disclosure. [Figure 11J] 11A-11L illustrate cytokine release in cynomolgus monkeys after a single IV bolus of a polypeptide conjugate of the present disclosure. [Figure 11K] 11A-11L illustrate cytokine release in cynomolgus monkeys after a single IV bolus of a polypeptide conjugate of the present disclosure. [Figure 11L] 11A-11L illustrate cytokine release in cynomolgus monkeys after a single IV bolus of a polypeptide conjugate of the present disclosure. [Figure 12] FIG. 12 illustrates administration of a polypeptide complex of the present disclosure on inflammatory cytokine levels. [Figure 13A]13A-13D illustrate serum liver enzymes in cynomolgus monkeys after a single IV bolus of a polypeptide conjugate of the present disclosure. [Figure 13B] 13A-13D illustrate serum liver enzymes in cynomolgus monkeys after a single IV bolus of a polypeptide conjugate of the present disclosure. [Figure 13C] 13A-13D illustrate serum liver enzymes in cynomolgus monkeys after a single IV bolus of a polypeptide conjugate of the present disclosure. [Figure 13D] 13A-13D illustrate serum liver enzymes in cynomolgus monkeys after a single IV bolus of a polypeptide conjugate of the present disclosure. [Figure 14] FIG. 14 illustrates the binding curves of αEGFR IgG binding by peptides of the disclosure as measured by ELISA. [Figure 15] FIG. 15 illustrates the inhibition of αEGFR IgG binding to the EGFR antigen by peptides of the present disclosure as measured by ELISA. [Figure 16] FIG. 16 illustrates the binding curves of αEGFR Fab binding by peptides of the disclosure as measured by ELISA. [Figure 17] FIG. 17 illustrates the inhibition of αEGFR Fab binding to EGFR antigen by peptides of the present disclosure as measured by ELISA. [Figure 18] FIG. 18 illustrates the inhibition of αEGFR Fab binding to the EGFR antigen by alanine scanning peptides of peptide-8 as measured by ELISA. [Figure 19] FIG. 19 illustrates the inhibition of αEGFR Fab binding to the EGFR antigen by alanine-canned peptides of peptide-8 as measured by ELISA. [Figure 20] FIG. 20 illustrates the optimized αEGFR Fab peptide-8 consensus sequence generated using WebLogo 3.7.4. [Figure 21]FIG. 21 illustrates αEGFR Fab binding by peptides identified by phage display. [Figure 22] FIG. 22 illustrates αEGFR Fab binding by peptides identified by phage display. [Figure 23] FIG. 23 illustrates the inhibition of αEGFR Fab binding to EGFR by peptides identified by phage display. [Figure 24] FIG. 24 illustrates the inhibition of αEGFR Fab binding to EGFR by peptides identified by phage display. [Figure 25] FIG. 25 illustrates in vivo tumor growth inhibition with different amounts of PC1 in human PBMC-engrafted NCG mice resistant to HCT116 xenograft tumors. [Figure 26] FIG. 26 illustrates in vivo tumor growth inhibition with different amounts of PC5 in human PBMC-engrafted NCG mice resistant to HCT116 xenograft tumors. [Figure 27] FIG. 27 illustrates in vivo tumor growth inhibition with different amounts of PC7 in human PBMC-engrafted NCG mice resistant to HCT116 xenograft tumors. [Figure 28] FIG. 28 illustrates in vivo tumor growth inhibition with different amounts of PC6 in human PBMC-engrafted NCG mice resistant to HCT116 xenograft tumors. [Figure 29] FIG. 29 shows PC1 and PC4 binding to human CD3ε measured by ELISA. [Figure 30] Figure 30 shows PC1 and PC4 binding to cynomolgus monkey CD3ε as measured by ELISA. Where indicated, PC4 was treated with protease. [Figure 31] Figure 31 shows PC1 and PC4 binding to human EGFR as measured by ELISA. Where indicated, PC4 was treated with proteases. [Figure 32]Figure 32 shows PC1 and PC4 binding to cynomolgus monkey EGFR as measured by ELISA. Where indicated, PC4 was treated with protease. [Figure 33] FIG. 33 shows PC4 binding to human albumin as measured by ELISA. [Figure 34] FIG. 34 shows PC4 binding to cynomolgus monkey albumin as measured by ELISA. [Figure 35] Figure 35 shows that PC1 and PC4 mediated killing of A549 cells in the presence of human peripheral blood mononuclear cells (PBMCs). Where indicated, PC4 was treated with proteases. [Figure 36] Figure 36 shows IFNγ cytokine levels in EGFR-positive HCT116 tumor cells with increasing levels of PC4 and PC1. Where indicated, PC4 was treated with protease. [Figure 37] Figure 37 shows TNF cytokine levels in EGFR-positive HCT116 tumor cells with increasing levels of PC4 and PC1. Where indicated, PC4 was treated with proteases. [Figure 38] Figure 38 shows IFNγ cytokine levels in EGFR-positive A549 tumor cells with increasing levels of PC4 and PC1. Where indicated, PC4 was treated with protease. [Figure 39] Figure 39 shows TNF cytokine levels in EGFR-positive A549 tumor cells with increasing levels of PC4 and PC1. Where indicated, PC4 was treated with proteases. [Figure 40] FIG. 40 shows a schematic of the HCT116 mouse tumor model study of the present disclosure. [Figure 41] FIG. 41 shows the mean tumor volume versus days of treatment with PC1, PC7, and PC6 in PBMC-engrafted mice implanted with HCT116 tumor cells. [Figure 42]FIG. 42 shows the mean tumor volume versus days of treatment with PC5 and vehicle in PBMC-engrafted mice implanted with HCT116 tumor cells. [Figure 43] FIG. 43 shows survival in PBMC-engrafted mice transplanted with HCT116 tumor cells when treated with PC1, PC5, and PC6. [Figure 44] Figure 44 shows a schematic of the human colon cancer (CRC) organoid study of the present disclosure. [Figure 45] Figure 45 shows the transduction of activation markers CD25, CD69, 4-1BB, and granzymes using PC1 and PC5 in human colon cancer tumor organoids. [Figure 46] FIG. 46 shows the maximum tolerated dose (MTD) of PC1 and the no-observed-toxicity dose of PC4 in cynomolgus monkeys. [Figure 47] FIG. 47 shows the pharmacokinetics in cynomolgus monkeys with different doses of PC4 after IV bolus injection. [Figure 48] FIG. 48 shows PC10 and PC11 binding to EGFR as measured by ELISA. [Figure 49] FIG. 49 shows PC10 and PC11 binding to CD3 as measured by ELISA. [Figure 50] FIG. 50 shows a graph of PC10 and PC11 mediated killing of HCT116 tumor cells in the presence of CD8+ T cells. [Figure 51A] Figures 51A-51F illustrate anti-CD3 scFv binding by alanine scanning peptides of anti-CD3 scFv peptide-A and peptide-B as measured by ELISA. [Figure 51B] Figures 51A-51F illustrate anti-CD3 scFv binding by alanine scanning peptides of anti-CD3 scFv peptide-A and peptide-B as measured by ELISA. [Figure 51C]Figures 51A-51F illustrate anti-CD3 scFv binding by alanine scanning peptides of anti-CD3 scFv peptide-A and peptide-B as measured by ELISA. [Figure 51D] Figures 51A-51F illustrate anti-CD3 scFv binding by alanine scanning peptides of anti-CD3 scFv peptide-A and peptide-B as measured by ELISA. [Figure 51E] Figures 51A-51F illustrate anti-CD3 scFv binding by alanine scanning peptides of anti-CD3 scFv peptide-A and peptide-B as measured by ELISA. [Figure 51F] Figures 51A-51F illustrate anti-CD3 scFv binding by alanine scanning peptides of anti-CD3 scFv peptide-A and peptide-B as measured by ELISA. [Figure 52A] Figures 52A-52F illustrate 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 52B] Figures 52A-52F illustrate 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 52C] Figures 52A-52F illustrate 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 52D] Figures 52A-52F illustrate 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 52E] Figures 52A-52F illustrate 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 52F] Figures 52A-52F illustrate 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 53A] Figures 53A-53B illustrate anti-CD3 scFv binding by optimized anti-CD3 scFv peptide-B sequences measured by ELISA. [Figure 53B] Figures 53A-53B illustrate anti-CD3 scFv binding by optimized anti-CD3 scFv peptide-B sequences measured by ELISA. [Figure 54A] Figures 54A-54B illustrate the inhibition of anti-CD3 scFv binding to CD3 by the optimized anti-CD3 scFv peptide-B sequence as measured by ELISA. [Figure 54B] Figures 54A-54B illustrate the inhibition of anti-CD3 scFv binding to CD3 by the optimized anti-CD3 scFv peptide-B sequence as measured by ELISA. [Figure 55] Figure 55 illustrates the core sequence motif of the optimized anti-CD3 scFv peptide-B sequence using WebLogo 3.7.4.

[0016] (Detailed Description of the Invention) Multispecific antibodies combine the benefits of different binding specificities 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 studies. This approach relies on the binding of one antigen-interacting portion of the antibody to a tumor-associated antigen or marker, while the second antigen-interacting portion can bind to an effector cell antigen on T cells, such as CD3, which then elicits cytotoxic activity. One such tumor-associated antigen is the epidermal growth factor receptor (EGFR). EGFR is a transmembrane protein that is a receptor for members of the epidermal growth factor family of extracellular protein ligands. EGFR is the most commonly overexpressed membrane protein in cancer. However, EGFR expression is not limited to tumors but is widely expressed throughout the body, resulting in systemic toxicity from EGFR-directed therapies.

[0017] T cell engager (TCE) therapy has several advantages, including not being a cell therapy and therefore can be provided as an off-the-shelf therapy, in contrast to chimeric antigen receptor T cell (CAR T cell) therapy. While TCE therapy has demonstrated potent antitumor activity in hematological cancers, the development of TCEs to treat solid tumors faces challenges due to limitations of conventional TCE technology: (i) immune system overactivation resulting in cytokine release syndrome (CRS), (ii) on-target healthy tissue toxicity, and (iii) poor pharmacokinetics (PK) resulting in a short half-life. CRS arises from systemic activation of T cells and can result in life-threatening elevations of inflammatory cytokines such as interleukin-6 (IL-6). Severe and acute CRS resulting in dose-limiting toxicity and death has been observed upon administration of T cell engagers developed using other platforms to treat cancer patients in unsuccessful clinical studies. This toxicity limits the maximum blood concentration of T cell engagers that can be safely administered. The efficacy of T cell engagers is also limited due to on-target healthy tissue toxicity. T cell engagers developed using platforms not designed for tumor-specific activation have resulted in clinical holdup and dose-limiting toxicities resulting from target expression in healthy tissues. T cell engagers are also limited by their short half-lives. Because of their short exposure half-life, T cell engagers are rapidly cleared from the body, rapidly reaching subtherapeutic levels after administration. For this reason, T cell engagers such as blinatumomab are typically administered via low-dose continuous infusion pumps over several weeks to overcome the short half-life challenge and maintain therapeutic drug levels in the body. Continuous administration regimens impose a significant burden on patients.

[0018] To overcome these challenges related to the efficacy of T cell engagers, described herein are polypeptides or polypeptide complexes comprising binding domains that selectively bind to an effector cell antigen and EGFR, wherein one or more binding domains are selectively activated in the tumor microenvironment, and the isolated polypeptides or polypeptide complexes comprise half-life extension molecules. Such modifications reduce the risk of CRS and on-target healthy tissue toxicity, and improve stability in the bloodstream and serum half-life before activation. The polypeptides or polypeptide complexes described herein are active at low levels of target expression and are easily manufactured.

[0019] In some embodiments, the polypeptides or polypeptide complexes described herein are used in methods of treating cancer. In some embodiments, the cancer has cells that express EGFR. In some embodiments, the polypeptides or polypeptide complexes described herein are used in methods of treating colorectal cancer (CRC), squamous cell carcinoma of the head and neck (SCCHN), non-small cell lung cancer (NSCLC), prostate cancer, breast cancer, colon cancer, head and neck cancer, esophagogastric cancer, liver cancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, kidney cancer, or pancreatic cancer. In some embodiments, the polypeptides or polypeptide complexes described herein are used in methods of treating a subject resistant to EGFR inhibitor therapy. In some embodiments, the polypeptides or polypeptide complexes described herein are used in methods of treating a subject with a KRAS mutation. In some embodiments, the polypeptides or polypeptide complexes described herein are used in methods of treating a subject resistant to EGFR inhibitor therapy and with a KRAS mutation. Some embodiments include a method of treating cancer comprising administering to a subject in need thereof an isolated polypeptide or polypeptide complex described in Formula I below,

[0020] [ka] wherein A1 comprises a first antigen recognition molecule that binds to an effector cell antigen, P1 comprises a peptide that binds to A1, L1 comprises a linking moiety connecting 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 recognition molecule that binds to an epidermal growth factor receptor (EGFR).

[0021] In some embodiments, a polypeptide or polypeptide complex is described in Formula I below,

[0022] [ka] Described herein are polypeptides or polypeptide complexes, wherein A1 comprises a first antigen recognition molecule that binds to an effector cell antigen, P1 comprises a peptide that binds to A1, L1 comprises a linking moiety connecting 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 recognition molecule that binds to an epidermal growth factor receptor (EGFR).

[0023] In some embodiments, a polypeptide or polypeptide complex as described in I below,

[0024] [ka] Described herein are polypeptides or polypeptide complexes, wherein A1 is a first antigen-recognition molecule that binds to an effector cell antigen, P1 is a peptide that binds to A1, L1 is a linking moiety that connects A1 to P1 and is a substrate for a tumor-specific protease, H1 is a half-life extending molecule, and A2 is a second antigen-recognition molecule that binds to an epidermal growth factor receptor (EGFR).

[0025] In some embodiments, a polypeptide or polypeptide complex comprising Formula I:

[0026] [ka] Described herein are polypeptides or polypeptide complexes, wherein A1 comprises a first antigen recognition molecule that binds to an effector cell antigen, P1 comprises a peptide that binds to A1, L1 comprises a linking moiety connecting 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 recognition molecule that binds to an epidermal growth factor receptor (EGFR).

[0027] In some embodiments, a polypeptide or polypeptide complex comprising Formula I:

[0028] [ka] Described herein are polypeptides or polypeptide complexes, wherein A1 is a first antigen-recognition molecule that binds to an effector cell antigen, P1 is a peptide that binds to A1, L1 is a linking moiety that connects A1 to P1 and is a substrate for a tumor-specific protease, H1 is a half-life extending molecule, and A2 is a second antigen-recognition molecule that binds to an epidermal growth factor receptor (EGFR).

[0029] In some embodiments, a polypeptide or polypeptide complex is described in Formula I below,

[0030] [ka] Described herein are polypeptides or polypeptide complexes, wherein A1 comprises a first antigen recognition molecule that binds to epidermal growth factor receptor (EGFR), P1 comprises a peptide that binds to A1, L1 comprises a linking moiety 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 recognition molecule that binds to an effector cell antigen.

[0031] In some embodiments, a polypeptide or polypeptide complex is described in Formula I below,

[0032] [ka] Described herein are polypeptides or polypeptide complexes, wherein A1 is a first antigen-recognition molecule that binds to epidermal growth factor receptor (EGFR), P1 is a peptide that binds to A1, L1 is a linking moiety that connects A1 to P1 and is a substrate for a tumor-specific protease, H1 is a half-life extending molecule, and A2 is a second antigen-recognition molecule that binds to an effector cell antigen.

[0033] In some embodiments, a polypeptide or polypeptide complex comprising Formula I:

[0034] [ka] Described herein are polypeptides or polypeptide complexes, wherein A1 comprises a first antigen recognition molecule that binds to epidermal growth factor receptor (EGFR), P1 comprises a peptide that binds to A1, L1 comprises a linking moiety 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 recognition molecule that binds to an effector cell antigen.

[0035] In some embodiments, a polypeptide or polypeptide complex comprising Formula I:

[0036] [ka] Described herein are polypeptides or polypeptide complexes, wherein A1 is a first antigen-recognition molecule that binds to epidermal growth factor receptor (EGFR), P1 is a peptide that binds to A1, L1 is a linking moiety that connects A1 to P1 and is a substrate for a tumor-specific protease, H1 is a half-life extending molecule, and A2 is a second antigen-recognition molecule that binds to an effector cell antigen.

[0037] In some embodiments, a polypeptide or polypeptide complex described in formula Ia,

[0038] [ka] Described herein are polypeptides or polypeptide complexes that are substrates for tumor-specific proteases, wherein A2 further comprises P2 and L2, P2 comprises a peptide that binds to A2, and L2 comprises a linking moiety that connects A2 to P2.

[0039] In some embodiments, a polypeptide or polypeptide complex described in formula Ia,

[0040] [ka] Described herein are polypeptides or polypeptide complexes wherein A2 further comprises P2 and L2, where P2 is a peptide that binds to A2, and L2 is a linking moiety that connects A2 to P2 and is a substrate for a tumor-specific protease.

[0041] In some embodiments, a polypeptide or polypeptide complex comprising Formula Ia:

[0042] [ka] Described herein are polypeptides or polypeptide complexes that are substrates for tumor-specific proteases, wherein A2 further comprises P2 and L2, P2 comprises a peptide that binds to A2, and L2 comprises a linking moiety that connects A2 to P2.

[0043] In some embodiments, a polypeptide or polypeptide complex comprising Formula Ia:

[0044] [ka] Described herein are polypeptides or polypeptide complexes wherein A2 further comprises P2 and L2, where P2 is a peptide that binds to A2, and L2 is a linking moiety that connects A2 to P2 and is a substrate for a tumor-specific protease.

[0045] In some embodiments, a polypeptide or polypeptide complex described in Formula II,

[0046] [ka] In the formula, L 1a When uncut, P 1a a linking moiety cleaved by a tumor-specific protease connecting P to a first antigen recognition molecule that binds to an effector cell antigen, and the first antigen recognition molecule is linked to a second antigen recognition molecule that binds to an epidermal growth factor receptor (EGFR), 1a L 1a comprises a peptide that binds to the first antigen recognition molecule when uncleaved, and 1a Described herein are polypeptides or polypeptide complexes that include a half-life extending molecule.

[0047] In some embodiments, a polypeptide or polypeptide complex comprising Formula II,

[0048] [ka] In the formula, L 1a When uncut, P 1a a linking moiety cleaved by a tumor-specific protease connecting P to a first antigen recognition molecule that binds to an effector cell antigen, and the first antigen recognition molecule is linked to a second antigen recognition molecule that binds to an epidermal growth factor receptor (EGFR), 1a L 1a comprises a peptide that binds to the first antigen recognition molecule when uncleaved, and 1a Described herein are polypeptides or polypeptide complexes that include a half-life extending molecule.

[0049] In some embodiments, a polypeptide or polypeptide complex described in Formula II,

[0050] [ka] In the formula, L 1a When uncut, P 1aa linking moiety cleaved by a tumor-specific protease connecting P to a first antigen recognition molecule that binds to an effector cell antigen, and the first antigen recognition molecule is bound to a second antigen recognition molecule that binds to an epidermal growth factor receptor (EGFR), 1a L 1a is a peptide that binds to the first antigen recognition molecule when uncleaved, and H 1a Described herein are polypeptides or polypeptide complexes that are half-life extending molecules.

[0051] In some embodiments, a polypeptide or polypeptide complex comprising Formula II,

[0052] [ka] In the formula, L 1a When uncut, P 1a a linking moiety cleaved by a tumor-specific protease connecting P to a first antigen recognition molecule that binds to an effector cell antigen, and the first antigen recognition molecule is bound to a second antigen recognition molecule that binds to an epidermal growth factor receptor (EGFR), 1a L 1a is a peptide that binds to the first antigen recognition molecule when uncleaved, and H 1a Described herein are polypeptides or polypeptide complexes that are half-life extending molecules.

[0053] In some embodiments, the present disclosure provides a polypeptide or polypeptide complex, wherein a first antigen recognition molecule binds to an effector cell antigen, and a second antigen recognition molecule binds to EGFR. In some embodiments, the effector cell antigen comprises CD3. In some embodiments, A1 comprises a first antigen recognition molecule that binds to an effector cell antigen.

[0054] In some embodiments, A1 comprises an antibody or antibody fragment. In some embodiments, A1 comprises a human antibody or humanized antibody or antibody fragment. In some embodiments, L1 is attached to the N-terminus of the antibody or antibody fragment. In some embodiments, L1 is attached to the N-terminus of the antibody or antibody fragment and A2 is attached to the other N-terminus of the antibody or antibody fragment. In some embodiments, A2 is attached to the C-terminus of the antibody or antibody fragment. In some embodiments, L1 is attached to the C-terminus of the antibody or antibody fragment. In some embodiments, A2 is attached 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 capable of specifically 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 K of 1 μM or less for CD3 on CD3-expressing cells. DIn some embodiments, A1 comprises an anti-CD3e single-chain variable fragment having binding affinity. In some embodiments, A1 is selected from the group consisting of muromonab-CD3 (OKT3), otelixizumab (TRX4), teplizumab (MGA031), vicibuzumab (Nuvion), SP34, X35, VIT3, BMA030 (BW264 / 56), CLB-T3 / 3, CRIS7, YTH12.5, FI Complementarity determining regions (CDRs) selected from the group consisting of 11-409, CLB-T3.4.2, TR-66, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII-87, 12F6, T3 / RW2-8C8, T3 / RW2-4B6, OKT3D, M-T301, SMC2, F101.01, UCHT-1, WT-31, 15865, 15865vl2, 15865vl6, and 15865vl9.

[0055] In some embodiments, A1 comprises a first antigen recognition molecule that binds to EGFR. In some embodiments, A1 comprises a variable light chain and a variable heavy chain, each of which can specifically bind to human EGFR.

[0056] In some embodiments, the scFv that binds to CD3 comprises an scFv light chain variable domain and an scFv heavy chain variable domain. In some embodiments, the scFv heavy chain variable domain comprises at least one, two, or three complementarity determining regions (CDRs) disclosed in Table 1, or a sequence substantially identical thereto (e.g., a sequence with at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity). In some embodiments, the scFv light chain variable domain comprises at least one, two, or three complementarity determining regions (CDRs) disclosed in Table 1, or a sequence substantially identical thereto (e.g., a sequence with at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity).

[0057] In some embodiments, the scFv heavy chain variable domain comprises at least one, two, or three complementarity determining regions (CDRs) disclosed in Table 1, or a sequence substantially identical thereto (e.g., a sequence with at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity), or the scFv light chain variable domain comprises at least one, two, or three complementarity determining regions (CDRs) disclosed in Table 1, or a sequence substantially identical thereto (e.g., a sequence with at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity).

[0058] [Table 1-1]

[0059] [Table 1-2]

[0060] Table 1-3

[0061] In some embodiments, the scFv light chain variable domain comprises complementarity determining regions (CDRs) LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, LC-CDR2, and LC-CDR3 of the scFv light chain variable domain comprise LC-CDR1 of SEQ ID NO: 1, LC-CDR2 of SEQ ID NO: 2, and LC-CDR3 of SEQ ID NO: 3, and the CDRs comprise 0 to 2 amino acid modifications in at least one of LC-CDR1, LC-CDR2, and LC-CDR3. In some embodiments, the scFv light chain variable domain comprises complementarity determining regions (CDRs) LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, LC-CDR2, and LC-CDR3 of the scFv light chain variable domain comprise LC-CDR1 of SEQ ID NO: 7, LC-CDR2 of SEQ ID NO: 8, and LC-CDR3 of SEQ ID NO: 9, and the CDRs comprise 0 to 2 amino acid modifications in at least one of LC-CDR1, LC-CDR2, and LC-CDR3. In some embodiments, the scFv heavy chain variable domain comprises complementarity determining regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 of the scFv heavy chain variable domain comprise LC-CDR1 of SEQ ID NO: 4, LC-CDR2 of SEQ ID NO: 5, and LC-CDR3 of SEQ ID NO: 6, and the CDRs comprise 0 to 2 amino acid modifications in at least one of HC-CDR1, HC-CDR2, and HC-CDR3. In some embodiments, the scFv heavy chain variable domain comprises complementarity determining regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 of the scFv heavy chain variable domain comprise LC-CDR1 of SEQ ID NO: 10, LC-CDR2 of SEQ ID NO: 11, and LC-CDR3 of SEQ ID NO: 12, and the CDRs comprise 0 to 2 amino acid modifications in at least one of HC-CDR1, HC-CDR2, and HC-CDR3.

[0062] In some embodiments, the isolated polypeptide or polypeptide complex of Formula I binds to an effector cell when L1 is cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex of Formula I binds to an effector cell when L1 is cleaved by a tumor-specific protease and A1 binds to the effector cell. In some embodiments, the effector cell is a T cell. In some embodiments, A1 binds to a polypeptide that is part of a TCR-CD3 complex on the effector cell. In some embodiments, the polypeptide that is part of a TCR-CD3 complex is human CD3ε. In some embodiments, the effector cell antigen comprises CD3, the effector cell antigen comprises CD3, the scFv comprises complementarity determining regions (CDRs) LC-CDR1, LC-CDR2, and LC-CDR3, wherein LC-CDR1, LC-CDR2, and LC-CDR3 of the scFv are SEQ ID NO: 1, LC-CDR2, and LC-CDR3, and wherein LC-CDR1, LC-CDR2, and LC-CDR3 of the scFv are SEQ ID NO: 3, and the scFv comprises complementarity determining regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3 of the scFv are SEQ ID NO: 4, HC-CDR2, and HC-CDR3 of the scFv, wherein HC-CDR1, HC-CDR2, and HC-CDR3 of the scFv are SEQ ID NO: 6. In some embodiments, the effector cell antigen comprises CD3, and the scFv comprises the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the effector cell antigen comprises CD3, the effector cell antigen comprises CD3, the scFv comprises CDRs LC-CDR1, LC-CDR2, and LC-CDR3, wherein in the LC-CDR1, LC-CDR2, and LC-CDR3 of the scFv, LC-CDR1 is SEQ ID NO: 7, LC-CDR2 is SEQ ID NO: 8, and LC-CDR3 is SEQ ID NO: 9, and the scFv comprises complementarity determining regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3, wherein in the HC-CDR1, HC-CDR2, and HC-CDR3 of the scFv, HC-CDR1 is SEQ ID NO: 10, HC-CDR2 is SEQ ID NO: 11, and HC-CDR3 is SEQ ID NO: 12.In some embodiments, the effector cell antigen comprises CD3 and the scFv comprises the amino acid sequence set forth in SEQ ID NO:14.

[0063] In some embodiments, the effector cell antigen comprises CD3, and A1 comprises CDRs LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, LC-CDR2, and LC-CDR3 of A1 comprise LC-CDR1 of SEQ ID NO: 1, LC-CDR2 of SEQ ID NO: 2, and LC-CDR3 of SEQ ID NO: 3, and A1 comprises complementarity determining regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 of A1 comprise HC-CDR1 of SEQ ID NO: 4, HC-CDR2 of SEQ ID NO: 5, and HC-CDR3 of SEQ ID NO: 6.

[0064] In some embodiments, the effector cell antigen comprises CD3, and A1 comprises CDRs LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, LC-CDR2, and LC-CDR3 of A1 comprise LC-CDR1 of SEQ ID NO: 7, LC-CDR2 of SEQ ID NO: 8, and LC-CDR3 of SEQ ID NO: 9, and A1 comprises complementarity determining regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 of A1 comprise HC-CDR1 of SEQ ID NO: 10, HC-CDR2 of SEQ ID NO: 11, and HC-CDR3 of SEQ ID NO: 12.

[0065] In some embodiments, A1 comprises the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, A1 comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 13. In some embodiments, A1 comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 13. In some embodiments, A1 comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 13. In some embodiments, A1 comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 13. In some embodiments, A1 comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 13.

[0066] In some embodiments, A1 comprises the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, A1 comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 14. In some embodiments, A1 comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 14. In some embodiments, A1 comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 14. In some embodiments, A1 comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 14. In some embodiments, A1 comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 14.

[0067] In some embodiments, the isolated polypeptide or polypeptide complex has a weaker binding affinity to a tumor cell antigen compared to the binding affinity of the isolated polypeptide or polypeptide complex without P1 or L1. In some embodiments, the isolated polypeptide or polypeptide complex has a weaker binding affinity to a tumor cell antigen, which is at least 5-fold higher than the binding affinity of the isolated polypeptide or polypeptide complex without P1 or L1. In some embodiments, the isolated polypeptide or polypeptide complex has a weaker binding affinity to a tumor cell antigen, which is at least 8-fold higher than the binding affinity of the isolated polypeptide or polypeptide complex without P1 or L1. In some embodiments, the isolated polypeptide or polypeptide complex has a weaker binding affinity to a tumor cell antigen, which is at least 10-fold higher than the binding affinity of the isolated polypeptide or polypeptide complex without P1 or L1. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity to the tumor cell antigen, which is at least 15-fold higher than the binding affinity of the isolated polypeptide or polypeptide complex to the tumor cell antigen in the form of the isolated polypeptide without P1 or L1. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity to the tumor cell antigen, which is at least 20-fold higher than the binding affinity of the isolated polypeptide or polypeptide complex to the tumor cell antigen in the form of the isolated polypeptide without P1 or L1. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity to the tumor cell antigen, which is at least 25-fold higher than the binding affinity of the isolated polypeptide or polypeptide complex to the tumor cell antigen in the form of the isolated polypeptide without P1 or L1.In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity to the tumor cell antigen, which is at least 30-fold higher than the binding affinity of the isolated polypeptide or polypeptide complex in the form of the isolated polypeptide without P1 or L1. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity to the tumor cell antigen, which is at least 35-fold higher than the binding affinity of the isolated polypeptide or polypeptide complex in the form of the isolated polypeptide without P1 or L1. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity to the tumor cell antigen, which is at least 40-fold higher than the binding affinity of the isolated polypeptide or polypeptide complex in the form of the isolated polypeptide without P1 or L1. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity to the tumor cell antigen, which is at least 45-fold higher than the binding affinity of the isolated polypeptide or polypeptide complex in the form of the isolated polypeptide without P1 or L1. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity to the tumor cell antigen, which is at least 50-fold higher than the binding affinity of the isolated polypeptide or polypeptide complex to the tumor cell antigen in the form of the isolated polypeptide without P1 or L1. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity to the tumor cell antigen, which is at least 55-fold higher than the binding affinity of the isolated polypeptide or polypeptide complex to the tumor cell antigen in the form of the isolated polypeptide without P1 or L1. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity to the tumor cell antigen, which is at least 60-fold higher than the binding affinity of the isolated polypeptide or polypeptide complex to the tumor cell antigen in the form of the isolated polypeptide without P1 or L1.In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity to the tumor cell antigen, which is at least 65-fold higher than the binding affinity of the isolated polypeptide or polypeptide complex to the tumor cell antigen in the form of the isolated polypeptide without P1 or L1. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity to the tumor cell antigen, which is at least 70-fold higher than the binding affinity of the isolated polypeptide or polypeptide complex to the tumor cell antigen in the form of the isolated polypeptide without P1 or L1. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity to the tumor cell antigen, which is at least 75-fold higher than the binding affinity of the isolated polypeptide or polypeptide complex to the tumor cell antigen in the form of the isolated polypeptide without P1 or L1. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity to the tumor cell antigen, which is at least 80-fold higher than the binding affinity of the isolated polypeptide or polypeptide complex to the tumor cell antigen in the form of the isolated polypeptide without P1 or L1. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity to the tumor cell antigen, which is at least 85-fold higher than the binding affinity of the isolated polypeptide or polypeptide complex in the form of the isolated polypeptide without P1 or L1. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity to the tumor cell antigen, which is at least 90-fold higher than the binding affinity of the isolated polypeptide or polypeptide complex in the form of the isolated polypeptide without P1 or L1. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity to the tumor cell antigen, which is at least 95-fold higher than the binding affinity of the isolated polypeptide or polypeptide complex in the form of the isolated polypeptide without P1 or L1.In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity to the tumor cell antigen, which is at least 100-fold higher than the binding affinity of the isolated polypeptide or polypeptide complex to the tumor cell antigen in the form of the isolated polypeptide without P1 or L1. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity to the tumor cell antigen, which is at least 120-fold higher than the binding affinity of the isolated polypeptide or polypeptide complex to the tumor cell antigen in the form of the isolated polypeptide without P1 or L1. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity to the tumor cell antigen, which is at least 1000-fold higher than the binding affinity of the isolated polypeptide or polypeptide complex to the tumor cell antigen in the form of the isolated polypeptide without P1 or L1.

[0068] In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity to a tumor cell antigen than the binding affinity to a tumor cell antigen of an isolated polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity to a tumor cell antigen, which is at least 5-fold higher than the binding affinity to a tumor cell antigen of an isolated polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity to a tumor cell antigen, which is at least 8-fold higher than the binding affinity to a tumor cell antigen of an isolated polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity to a tumor cell antigen, which is at least 10-fold higher than the binding affinity to a tumor cell antigen of an isolated polypeptide or polypeptide complex in which L1 is cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity to a tumor cell antigen, which is at least 15-fold higher than the binding affinity of the isolated polypeptide or polypeptide complex in which L1 has been cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity to a tumor cell antigen, which is at least 20-fold higher than the binding affinity of the isolated polypeptide or polypeptide complex in which L1 has been cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity to a tumor cell antigen, which is at least 25-fold higher than the binding affinity of the isolated polypeptide or polypeptide complex in which L1 has been cleaved by a tumor-specific protease.In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity to a tumor cell antigen, which is at least 30-fold higher than the binding affinity of an isolated polypeptide or polypeptide complex in which L1 has been cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity to a tumor cell antigen, which is at least 35-fold higher than the binding affinity of an isolated polypeptide or polypeptide complex in which L1 has been cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity to a tumor cell antigen, which is at least 40-fold higher than the binding affinity of an isolated polypeptide or polypeptide complex in which L1 has been cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity to a tumor cell antigen, which is at least 45-fold higher than the binding affinity of an isolated polypeptide or polypeptide complex in which L1 has been cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity to a tumor cell antigen, which is at least 50-fold higher than the binding affinity of the isolated polypeptide or polypeptide complex in which L1 has been cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity to a tumor cell antigen, which is at least 55-fold higher than the binding affinity of the isolated polypeptide or polypeptide complex in which L1 has been cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity to a tumor cell antigen, which is at least 60-fold higher than the binding affinity of the isolated polypeptide or polypeptide complex in which L1 has been cleaved by a tumor-specific protease.In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity to a tumor cell antigen, which is at least 65-fold higher than the binding affinity of an isolated polypeptide or polypeptide complex in which L1 has been cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity to a tumor cell antigen, which is at least 70-fold higher than the binding affinity of an isolated polypeptide or polypeptide complex in which L1 has been cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity to a tumor cell antigen, which is at least 75-fold higher than the binding affinity of an isolated polypeptide or polypeptide complex in which L1 has been cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity to a tumor cell antigen, which is at least 80-fold higher than the binding affinity of an isolated polypeptide or polypeptide complex in which L1 has been cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity to a tumor cell antigen, which is at least 85-fold higher than the binding affinity of the isolated polypeptide or polypeptide complex in which L1 has been cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity to a tumor cell antigen, which is at least 90-fold higher than the binding affinity of the isolated polypeptide or polypeptide complex in which L1 has been cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity to a tumor cell antigen, which is at least 95-fold higher than the binding affinity of the isolated polypeptide or polypeptide complex in which L1 has been cleaved by a tumor-specific protease.In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity to a tumor cell antigen, which is at least 100-fold higher than the binding affinity of the isolated polypeptide or polypeptide complex in which L1 has been cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity to a tumor cell antigen, which is at least 120-fold higher than the binding affinity of the isolated polypeptide or polypeptide complex in which L1 has been cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity to a tumor cell antigen, which is at least 1000-fold higher than the binding affinity of the isolated polypeptide or polypeptide complex in which L1 has been cleaved by a tumor-specific protease.

[0069] In some embodiments, the isolated polypeptide or polypeptide complex exhibits a EC2 activity in an IFNγ release T cell activation assay of an isolated polypeptide or polypeptide complex without P1 or L1. 50 Increased EC in IFNγ-releasing T cell activation assay compared to 50 In some embodiments, the isolated polypeptide or polypeptide complex has increased EC in an IFNγ release T cell activation assay. 50 This EC 50 The IFNγ release of isolated polypeptides or polypeptide complexes without P1 or L1 was observed in ECs in a T cell activation assay. 50 In some embodiments, the isolated polypeptide or polypeptide complex exhibits increased EC in an IFNγ release T cell activation assay. 50 This EC 50 The IFNγ release of isolated polypeptides or polypeptide complexes without P1 or L1 was observed in ECs in a T cell activation assay. 50In some embodiments, the isolated polypeptide or polypeptide complex exhibits increased EC in an IFNγ release T cell activation assay. 50 This EC 50 The IFNγ release of isolated polypeptides or polypeptide complexes without P1 or L1 was observed in ECs in a T cell activation assay. 50 In some embodiments, the isolated polypeptide or polypeptide complex exhibits increased EC20 activity in an IFNγ release T cell activation assay. 50 This EC 50 The IFNγ release of isolated polypeptides or polypeptide complexes without P1 or L1 was observed in ECs in a T cell activation assay. 50 In some embodiments, the isolated polypeptide or polypeptide complex exhibits increased EC2 activity in an IFNγ release T cell activation assay. 50 This EC 50 The IFNγ release of isolated polypeptides or polypeptide complexes without P1 or L1 was observed in ECs in a T cell activation assay. 50 In some embodiments, the isolated polypeptide or polypeptide complex exhibits increased EC20 activity in an IFNγ release T cell activation assay. 50 This EC 50 The IFNγ release of isolated polypeptides or polypeptide complexes without P1 or L1 was observed in ECs in a T cell activation assay. 50 In some embodiments, the isolated polypeptide or polypeptide complex exhibits increased EC2 activity in an IFNγ release T cell activation assay. 50 This EC 50 The IFNγ release of isolated polypeptides or polypeptide complexes without P1 or L1 was observed in ECs in a T cell activation assay. 50 In some embodiments, the isolated polypeptide or polypeptide complex exhibits increased EC2 activity in an IFNγ release T cell activation assay. 50 This EC50 The IFNγ release of isolated polypeptides or polypeptide complexes without P1 or L1 was observed in ECs in a T cell activation assay. 50 In some embodiments, the isolated polypeptide or polypeptide complex exhibits increased EC2 activity in an IFNγ release T cell activation assay. 50 This EC 50 The IFNγ release of isolated polypeptides or polypeptide complexes without P1 or L1 was observed in ECs in a T cell activation assay. 50 In some embodiments, the isolated polypeptide or polypeptide complex exhibits increased EC2 activity in an IFNγ release T cell activation assay. 50 This EC 50 The IFNγ release of isolated polypeptides or polypeptide complexes without P1 or L1 was observed in ECs in a T cell activation assay. 50 In some embodiments, the isolated polypeptide or polypeptide complex exhibits increased EC2 activity in an IFNγ release T cell activation assay. 50 This EC 50 The IFNγ release of isolated polypeptides or polypeptide complexes without P1 or L1 was observed in ECs in a T cell activation assay. 50 In some embodiments, the isolated polypeptide or polypeptide complex exhibits increased EC2 activity in an IFNγ release T cell activation assay. 50 This EC 50 The IFNγ release of isolated polypeptides or polypeptide complexes without P1 or L1 was observed in ECs in a T cell activation assay. 50 1000 times higher than

[0070] In some embodiments, the isolated polypeptide or polypeptide complex is a polypeptide in which L1 is cleaved by a tumor-specific protease, and ... 50Increased EC in IFNγ-releasing T cell activation assay compared to 50 In some embodiments, the isolated polypeptide or polypeptide complex has increased EC in an IFNγ release T cell activation assay. 50 This EC 50 The isolated polypeptides or polypeptide complexes in which L1 has been cleaved by tumor-specific proteases release IFNγ and inhibit EC in T cell activation assays. 50 In some embodiments, the isolated polypeptide or polypeptide complex exhibits increased EC2 activity in an IFNγ release T cell activation assay. 50 This EC 50 The isolated polypeptides or polypeptide complexes in which L1 has been cleaved by tumor-specific proteases release IFNγ and inhibit EC in T cell activation assays. 50 In some embodiments, the isolated polypeptide or polypeptide complex exhibits increased EC20 activity in an IFNγ release T cell activation assay. 50 This EC 50 The isolated polypeptides or polypeptide complexes in which L1 has been cleaved by tumor-specific proteases release IFNγ and inhibit EC in T cell activation assays. 50 In some embodiments, the isolated polypeptide or polypeptide complex exhibits increased EC in an IFNγ release T cell activation assay. 50 This EC 50 The isolated polypeptides or polypeptide complexes in which L1 has been cleaved by tumor-specific proteases release IFNγ and inhibit EC in T cell activation assays. 50 In some embodiments, the isolated polypeptide or polypeptide complex exhibits increased EC in an IFNγ release T cell activation assay. 50 This EC 50The isolated polypeptides or polypeptide complexes in which L1 has been cleaved by tumor-specific proteases release IFNγ and inhibit EC in T cell activation assays. 50 In some embodiments, the isolated polypeptide or polypeptide complex exhibits increased EC2 activity in an IFNγ release T cell activation assay. 50 This EC 50 The isolated polypeptides or polypeptide complexes in which L1 has been cleaved by tumor-specific proteases release IFNγ and inhibit EC in T cell activation assays. 50 In some embodiments, the isolated polypeptide or polypeptide complex exhibits increased EC2 activity in an IFNγ release T cell activation assay. 50 This EC 50 The isolated polypeptides or polypeptide complexes in which L1 has been cleaved by tumor-specific proteases release IFNγ and inhibit EC in T cell activation assays. 50 In some embodiments, the isolated polypeptide or polypeptide complex exhibits increased EC in an IFNγ release T cell activation assay. 50 This EC 50 The isolated polypeptides or polypeptide complexes in which L1 has been cleaved by tumor-specific proteases release IFNγ and inhibit EC in T cell activation assays. 50 In some embodiments, the isolated polypeptide or polypeptide complex exhibits increased EC in an IFNγ release T cell activation assay. 50 This EC 50 The isolated polypeptides or polypeptide complexes in which L1 has been cleaved by tumor-specific proteases release IFNγ and inhibit EC in T cell activation assays. 50 In some embodiments, the isolated polypeptide or polypeptide complex exhibits increased EC in an IFNγ release T cell activation assay. 50 This EC 50The isolated polypeptides or polypeptide complexes in which L1 has been cleaved by tumor-specific proteases release IFNγ and inhibit EC in T cell activation assays. 50 In some embodiments, the isolated polypeptide or polypeptide complex exhibits increased EC in an IFNγ release T cell activation assay. 50 This EC 50 The isolated polypeptides or polypeptide complexes in which L1 has been cleaved by tumor-specific proteases release IFNγ and inhibit EC in T cell activation assays. 50 at least 1000 times higher than

[0071] In some embodiments, the isolated polypeptide or polypeptide complex exhibits an EC2 activity in a T cell lytic assay of a polypeptide or polypeptide complex without P1 or L1. 50 Increased EC in T cell lytic assays compared to 50 In some embodiments, the isolated polypeptide or polypeptide complex has increased EC in a T cell lytic assay. 50 This EC 50 EC in T cell lytic assays in the form of polypeptide or polypeptide complex without P1 or L1 50 In some embodiments, the isolated polypeptide or polypeptide complex exhibits an increased EC in a T cell lytic assay. 50 This EC 50 EC in T cell lytic assays in the form of polypeptide or polypeptide complex without P1 or L1 50 In some embodiments, the isolated polypeptide or polypeptide complex exhibits an increased EC in a T cell lytic assay. 50 This EC 50 EC in T cell lytic assays in the form of polypeptide or polypeptide complex without P1 or L1 50In some embodiments, the isolated polypeptide or polypeptide complex exhibits an increased EC in a T cell lytic assay. 50 This EC 50 EC in T cell lytic assays in the form of polypeptide or polypeptide complex without P1 or L1 50 In some embodiments, the isolated polypeptide or polypeptide complex exhibits an increased EC in a T cell lytic assay. 50 This EC 50 EC in T cell lytic assays in the form of polypeptide or polypeptide complex without P1 or L1 50 In some embodiments, the isolated polypeptide or polypeptide complex exhibits an increased EC in a T cell lytic assay. 50 This EC 50 EC in T cell lytic assays in the form of polypeptide or polypeptide complex without P1 or L1 50 In some embodiments, the isolated polypeptide or polypeptide complex exhibits an increased EC in a T cell lytic assay. 50 This EC 50 EC in T cell lytic assays in the form of polypeptide or polypeptide complex without P1 or L1 50 In some embodiments, the isolated polypeptide or polypeptide complex exhibits an increased EC in a T cell lytic assay. 50 This EC 50 EC in T cell lytic assays in the form of polypeptide or polypeptide complex without P1 or L1 50 In some embodiments, the isolated polypeptide or polypeptide complex exhibits an increased EC in a T cell lytic assay. 50 This EC 50 EC in T cell lytic assays in the form of polypeptide or polypeptide complex without P1 or L1 50In some embodiments, the isolated polypeptide or polypeptide complex exhibits an increased EC in a T cell lytic assay. 50 This EC 50 EC in T cell lytic assays of polypeptides or polypeptide complexes without P1 or L1 50 In some embodiments, the isolated polypeptide or polypeptide complex exhibits an increased EC in a T cell lytic assay. 50 This EC 50 EC in T cell lytic assays of polypeptides or polypeptide complexes without P1 or L1 50 at least 1000 times higher than

[0072] In some embodiments, the isolated polypeptide or polypeptide complex is a polypeptide that inhibits EC2 in a T cell lytic assay. 50 Increased EC in T cell lytic assays compared to 50 In some embodiments, the isolated polypeptide or polypeptide complex has increased EC in a T cell lytic assay. 50 This EC 50 EC in T cell lytic assays of isolated polypeptides or polypeptide complexes in which L1 has been cleaved by tumor-specific proteases 50 In some embodiments, the isolated polypeptide or polypeptide complex exhibits an increased EC in a T cell lytic assay. 50 This EC 50 EC in T cell lytic assays of isolated polypeptides or polypeptide complexes in which L1 has been cleaved by tumor-specific proteases 50 In some embodiments, the isolated polypeptide or polypeptide complex exhibits an increased EC in a T cell lytic assay. 50 This EC 50EC in T cell lytic assays of isolated polypeptides or polypeptide complexes in which L1 has been cleaved by tumor-specific proteases 50 In some embodiments, the isolated polypeptide or polypeptide complex exhibits an increased EC in a T cell lytic assay. 50 This EC 50 EC in T cell lytic assays of isolated polypeptides or polypeptide complexes in which L1 has been cleaved by tumor-specific proteases 50 In some embodiments, the isolated polypeptide or polypeptide complex exhibits an increased EC in a T cell lytic assay. 50 This EC 50 EC in T cell lytic assays of isolated polypeptides or polypeptide complexes in which L1 has been cleaved by tumor-specific proteases 50 In some embodiments, the isolated polypeptide or polypeptide complex exhibits an increased EC in a T cell lytic assay. 50 This EC 50 EC in T cell lytic assays of isolated polypeptides or polypeptide complexes in which L1 has been cleaved by tumor-specific proteases 50 In some embodiments, the isolated polypeptide or polypeptide complex exhibits an increased EC in a T cell lytic assay. 50 This EC 50 EC in T cell lytic assays of isolated polypeptides or polypeptide complexes in which L1 has been cleaved by tumor-specific proteases 50 In some embodiments, the isolated polypeptide or polypeptide complex exhibits an increased EC in a T cell lytic assay. 50 This EC 50 EC in T cell lytic assays of isolated polypeptides or polypeptide complexes in which L1 has been cleaved by tumor-specific proteases 50In some embodiments, the isolated polypeptide or polypeptide complex exhibits an increased EC in a T cell lytic assay. 50 This EC 50 EC in T cell lytic assays of isolated polypeptides or polypeptide complexes in which L1 has been cleaved by tumor-specific proteases 50 In some embodiments, the isolated polypeptide or polypeptide complex exhibits an increased EC in a T cell lytic assay. 50 This EC 50 EC in T cell lytic assays of isolated polypeptides or polypeptide complexes in which L1 has been cleaved by tumor-specific proteases 50 In some embodiments, the isolated polypeptide or polypeptide complex exhibits an increased EC in a T cell lytic assay. 50 This EC 50 EC in T cell lytic assays of isolated polypeptides or polypeptide complexes in which L1 has been cleaved by tumor-specific proteases 50 at least 1000 times higher than

[0073] In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity to a tumor cell antigen compared to the binding affinity to a tumor cell antigen of the isolated polypeptide or polypeptide complex of Formula Ia without P1, L1, P2, or L2. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity to a tumor cell antigen that is at least 10-fold higher than the binding affinity to a tumor cell antigen of the isolated polypeptide or polypeptide complex of Formula Ia without P1, L1, P2, or L2. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has weaker binding affinity to a tumor cell antigen that is at least 50-fold higher than the binding affinity of the isolated polypeptide or polypeptide complex of Formula Ia without P1, L1, P2, or L2 to the tumor cell antigen. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has weaker binding affinity to a tumor cell antigen that is at least 75-fold higher than the binding affinity of the isolated polypeptide or polypeptide complex of Formula Ia without P1, L1, P2, or L2 to the tumor cell antigen. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity to a tumor cell antigen that is at least 100 times greater than the binding affinity to a tumor cell antigen of the isolated polypeptide or polypeptide complex form of Formula Ia without P1, L1, P2, or L2.In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has weaker binding affinity to a tumor cell antigen that is at least 120-fold higher than the binding affinity of the isolated polypeptide or polypeptide complex of Formula Ia without P1, L1, P2, or L2. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has weaker binding affinity to a tumor cell antigen that is at least 200-fold higher than the binding affinity of the isolated polypeptide or polypeptide complex of Formula Ia without P1, L1, P2, or L2. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has weaker binding affinity to a tumor cell antigen that is at least 300-fold higher than the binding affinity of the isolated polypeptide or polypeptide complex of Formula Ia without P1, L1, P2, or L2 to the tumor cell antigen. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has weaker binding affinity to a tumor cell antigen that is at least 400-fold higher than the binding affinity of the isolated polypeptide or polypeptide complex of Formula Ia without P1, L1, P2, or L2 to the tumor cell antigen. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity to a tumor cell antigen that is at least 500 times greater than the binding affinity to a tumor cell antigen of the isolated polypeptide or polypeptide complex form of Formula Ia without P1, L1, P2, or L2.In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has weaker binding affinity to a tumor cell antigen that is at least 600-fold higher than the binding affinity of the isolated polypeptide or polypeptide complex of Formula Ia without P1, L1, P2, or L2 to the tumor cell antigen. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has weaker binding affinity to a tumor cell antigen that is at least 700-fold higher than the binding affinity of the isolated polypeptide or polypeptide complex of Formula Ia without P1, L1, P2, or L2 to the tumor cell antigen. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has weaker binding affinity to a tumor cell antigen that is at least 800-fold higher than the binding affinity of the isolated polypeptide or polypeptide complex of Formula Ia without P1, L1, P2, or L2 to the tumor cell antigen. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has weaker binding affinity to a tumor cell antigen that is at least 900-fold higher than the binding affinity of the isolated polypeptide or polypeptide complex of Formula Ia without P1, L1, P2, or L2 to the tumor cell antigen. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity to a tumor cell antigen that is at least 1000 times greater than the binding affinity to a tumor cell antigen of the isolated polypeptide or polypeptide complex form of Formula Ia without P1, L1, P2, or L2.In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity to a tumor cell antigen that is at least 10,000 times greater than the binding affinity to a tumor cell antigen of the isolated polypeptide or polypeptide complex form of Formula Ia without P1, L1, P2, or L2.

[0074] In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity to a tumor cell antigen compared to the binding affinity to a tumor cell antigen of an isolated polypeptide or polypeptide complex of Formula Ia in which L1 and L2 have been cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity to a tumor cell antigen that is at least 10-fold higher than the binding affinity to a tumor cell antigen of an isolated polypeptide or polypeptide complex of Formula Ia in which L1 and L2 have been cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has weaker binding affinity to a tumor cell antigen that is at least 50-fold higher than the binding affinity of an isolated polypeptide or polypeptide complex of Formula Ia in which L1 and L2 have been cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has weaker binding affinity to a tumor cell antigen that is at least 75-fold higher than the binding affinity of an isolated polypeptide or polypeptide complex of Formula Ia in which L1 and L2 have been cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity to a tumor cell antigen that is at least 100-fold greater than the binding affinity to a tumor cell antigen of an isolated polypeptide or polypeptide complex of Formula Ia in which L1 and L2 have been cleaved by a tumor-specific protease.In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has weaker binding affinity to a tumor cell antigen that is at least 120-fold higher than the binding affinity of an isolated polypeptide or polypeptide complex of Formula Ia in which L1 and L2 have been cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has weaker binding affinity to a tumor cell antigen that is at least 200-fold higher than the binding affinity of an isolated polypeptide or polypeptide complex of Formula Ia in which L1 and L2 have been cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has weaker binding affinity to a tumor cell antigen that is at least 300-fold higher than the binding affinity of an isolated polypeptide or polypeptide complex of Formula Ia in which L1 and L2 have been cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has weaker binding affinity to a tumor cell antigen that is at least 400-fold higher than the binding affinity of an isolated polypeptide or polypeptide complex of Formula Ia in which L1 and L2 have been cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity to a tumor cell antigen that is at least 500 times greater than the binding affinity to a tumor cell antigen of an isolated polypeptide or polypeptide complex of Formula Ia in which L1 and L2 have been cleaved by a tumor-specific protease.In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has weaker binding affinity to a tumor cell antigen that is at least 600-fold higher than the binding affinity of an isolated polypeptide or polypeptide complex of Formula Ia in which L1 and L2 have been cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has weaker binding affinity to a tumor cell antigen that is at least 700-fold higher than the binding affinity of an isolated polypeptide or polypeptide complex of Formula Ia in which L1 and L2 have been cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has weaker binding affinity to a tumor cell antigen that is at least 800-fold higher than the binding affinity of an isolated polypeptide or polypeptide complex of Formula Ia in which L1 and L2 have been cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has weaker binding affinity to a tumor cell antigen that is at least 900-fold higher than the binding affinity of an isolated polypeptide or polypeptide complex of Formula Ia in which L1 and L2 have been cleaved by a tumor-specific protease. In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity to a tumor cell antigen that is at least 1000 times greater than the binding affinity to a tumor cell antigen of an isolated polypeptide or polypeptide complex of Formula Ia in which L1 and L2 have been cleaved by a tumor-specific protease.In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has a weaker binding affinity to a tumor cell antigen that is at least 10,000 times greater than the binding affinity to a tumor cell antigen of an isolated polypeptide or polypeptide complex of Formula Ia in which L1 and L2 have been cleaved by a tumor-specific protease.

[0075] In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has an EC20 activity greater than or equal to 1000 fold in an IFNγ release T cell activation assay compared to an isolated polypeptide or polypeptide complex of Formula Ia without P1, L1, P2, or L2. 50 Increased EC in IFNγ-releasing T cell activation assay compared to 50 In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits increased EC in an IFNγ release T cell activation assay. 50 This EC 50 The present invention relates to an EC200 / 2000 IFNγ release T cell activation assay in which an isolated polypeptide or polypeptide complex of Formula Ia in the form of an isolated polypeptide or polypeptide complex without P1, L1, P2, or L2 is inhibited. 50 In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits increased EC2 activity in an IFNγ release T cell activation assay. 50 This EC 50 The present invention relates to an EC200 / 2000 IFNγ release T cell activation assay in which an isolated polypeptide or polypeptide complex of Formula Ia in the form of an isolated polypeptide or polypeptide complex without P1, L1, P2, or L2 is inhibited. 50 In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits increased EC2 activity in an IFNγ release T cell activation assay. 50 This EC 50The present invention relates to an EC200 / 2000 IFNγ release T cell activation assay in which an isolated polypeptide or polypeptide complex of Formula Ia in the form of an isolated polypeptide or polypeptide complex without P1, L1, P2, or L2 is inhibited. 50 In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits increased EC2 activity in an IFNγ release T cell activation assay. 50 This EC 50 The present invention relates to an EC200 / 2000 IFNγ release T cell activation assay in which an isolated polypeptide or polypeptide complex of Formula Ia in the form of an isolated polypeptide or polypeptide complex without P1, L1, P2, or L2 is inhibited. 50 In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits increased EC2 activity in an IFNγ release T cell activation assay. 50 This EC 50 The present invention relates to an EC200 / 2000 IFNγ release T cell activation assay in which an isolated polypeptide or polypeptide complex of Formula Ia in the form of an isolated polypeptide or polypeptide complex without P1, L1, P2, or L2 is inhibited. 50 In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits increased EC20 activity in an IFNγ release T cell activation assay. 50 This EC 50 The present invention relates to an EC200 / 2000 IFNγ release T cell activation assay in which an isolated polypeptide or polypeptide complex of Formula Ia in the form of an isolated polypeptide or polypeptide complex without P1, L1, P2, or L2 is inhibited. 50 In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits increased EC2 activity in an IFNγ release T cell activation assay. 50 This EC 50 The present invention relates to an EC200 / 2000 IFNγ release T cell activation assay in which an isolated polypeptide or polypeptide complex of Formula Ia in the form of an isolated polypeptide or polypeptide complex without P1, L1, P2, or L2 is inhibited. 50In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits increased EC20 activity in an IFNγ release T cell activation assay. 50 This EC 50 The present invention relates to an EC200 / 2000 IFNγ release T cell activation assay in which an isolated polypeptide or polypeptide complex of Formula Ia in the form of an isolated polypeptide or polypeptide complex without P1, L1, P2, or L2 is inhibited. 50 In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits increased EC2 activity in an IFNγ release T cell activation assay. 50 This EC 50 The present invention relates to an EC200 / 2000 IFNγ release T cell activation assay in which an isolated polypeptide or polypeptide complex of Formula Ia in the form of an isolated polypeptide or polypeptide complex without P1, L1, P2, or L2 is inhibited. 50 In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits increased EC2 activity in an IFNγ release T cell activation assay. 50 This EC 50 The present invention relates to an EC200 / 2000 IFNγ release T cell activation assay in which an isolated polypeptide or polypeptide complex of Formula Ia in the form of an isolated polypeptide or polypeptide complex without P1, L1, P2, or L2 is inhibited. 50 In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits increased EC20 activity in an IFNγ release T cell activation assay. 50 This EC 50 The present invention relates to an EC200 / 2000 IFNγ release T cell activation assay in which an isolated polypeptide or polypeptide complex of Formula Ia in the form of an isolated polypeptide or polypeptide complex without P1, L1, P2, or L2 is inhibited. 50In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits increased EC20 activity in an IFNγ release T cell activation assay. 50 This EC 50 The present invention relates to an EC200 / 2000 IFNγ release T cell activation assay in which an isolated polypeptide or polypeptide complex of Formula Ia in the form of an isolated polypeptide or polypeptide complex without P1, L1, P2, or L2 is inhibited. 50 In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits increased EC20 activity in an IFNγ release T cell activation assay. 50 This EC 50 The present invention relates to an EC200 / 2000 IFNγ release T cell activation assay in which an isolated polypeptide or polypeptide complex of Formula Ia in the form of an isolated polypeptide or polypeptide complex without P1, L1, P2, or L2 is inhibited. 50 In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits increased EC2 activity in an IFNγ release T cell activation assay. 50 This EC 50 The present invention relates to an EC200 / 2000 IFNγ release T cell activation assay in which an isolated polypeptide or polypeptide complex of Formula Ia in the form of an isolated polypeptide or polypeptide complex without P1, L1, P2, or L2 is inhibited. 50 At least 10,000 times higher than

[0076] In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is a polypeptide having an EC2 activity in a T cell lysis assay of an isolated polypeptide or polypeptide complex of Formula Ia in which L1 and L2 have been cleaved by a tumor-specific protease. 50 Increased EC in T cell lytic assays compared to 50In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has an increased EC 50 This EC 50 The EC in a T cell lytic assay of an isolated polypeptide or polypeptide complex of formula Ia in which L1 and L2 have been cleaved by a tumor-specific protease 50 In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits increased EC2 in a T cell lytic assay. 50 This EC 50 The EC in a T cell lytic assay of an isolated polypeptide or polypeptide complex of formula Ia in which L1 and L2 have been cleaved by a tumor-specific protease 50 In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits increased EC2 in a T cell lytic assay. 50 This EC 50 The EC in a T cell lytic assay of an isolated polypeptide or polypeptide complex of formula Ia in which L1 and L2 have been cleaved by a tumor-specific protease 50 In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits increased EC2 in a T cell lytic assay. 50 This EC 50 The EC in a T cell lytic assay of an isolated polypeptide or polypeptide complex of formula Ia in which L1 and L2 have been cleaved by a tumor-specific protease 50 In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits increased EC2 in a T cell lytic assay. 50 This EC50 The EC in a T cell lytic assay of an isolated polypeptide or polypeptide complex of formula Ia in which L1 and L2 have been cleaved by a tumor-specific protease 50 In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits increased EC20 activity in a T cell lytic assay. 50 This EC 50 The EC in a T cell lytic assay of an isolated polypeptide or polypeptide complex of formula Ia in which L1 and L2 have been cleaved by a tumor-specific protease 50 In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits increased EC2 in a T cell lytic assay. 50 This EC 50 The EC in a T cell lytic assay of an isolated polypeptide or polypeptide complex of formula Ia in which L1 and L2 have been cleaved by a tumor-specific protease 50 In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits increased EC20 activity in a T cell lytic assay. 50 This EC 50 The EC in a T cell lytic assay of an isolated polypeptide or polypeptide complex of formula Ia in which L1 and L2 have been cleaved by a tumor-specific protease 50 In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits increased EC2 in a T cell lytic assay. 50 This EC 50 The EC in a T cell lytic assay of an isolated polypeptide or polypeptide complex of formula Ia in which L1 and L2 have been cleaved by a tumor-specific protease 50In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits increased EC2 in a T cell lytic assay. 50 This EC 50 The EC in a T cell lytic assay of an isolated polypeptide or polypeptide complex of formula Ia in which L1 and L2 have been cleaved by a tumor-specific protease 50 In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits increased EC20 activity in a T cell lytic assay. 50 This EC 50 The EC in a T cell lytic assay of an isolated polypeptide or polypeptide complex of formula Ia in which L1 and L2 have been cleaved by a tumor-specific protease 50 In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits increased EC2 in a T cell lytic assay. 50 This EC 50 The EC in a T cell lytic assay of an isolated polypeptide or polypeptide complex of formula Ia in which L1 and L2 have been cleaved by a tumor-specific protease 50 In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits increased EC2 in a T cell lytic assay. 50 This EC 50 The EC in a T cell lytic assay of an isolated polypeptide or polypeptide complex of formula Ia in which L1 and L2 have been cleaved by a tumor-specific protease 50 In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits increased EC2 in a T cell lytic assay. 50This EC 50 The EC in a T cell lytic assay of an isolated polypeptide or polypeptide complex of formula Ia in which L1 and L2 have been cleaved by a tumor-specific protease 50 At least 10,000 times higher than

[0077] In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has an EC2 activity greater than or equal to that of the polypeptide or polypeptide complex without P1, L1, P2, or L2 in a T cell lytic assay. 50 Increased EC in T cell lytic assays compared to 50 In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has an increased EC 50 This EC 50 EC in a T cell lytic assay of a polypeptide or polypeptide complex of formula Ia in the form of P1, L1, P2, or without L2. 50 In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits increased EC2 in a T cell lytic assay. 50 This EC 50 EC in a T cell lytic assay of a polypeptide or polypeptide complex of formula Ia in the form of P1, L1, P2, or without L2. 50 In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits increased EC2 in a T cell lytic assay. 50 This EC 50 EC in a T cell lytic assay of a polypeptide or polypeptide complex of formula Ia in the form of P1, L1, P2, or without L2. 50In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits increased EC2 in a T cell lytic assay. 50 This EC 50 EC in T cell lytic assays in the form of a polypeptide or polypeptide complex without P1, L1, P2, or L2 50 In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits increased EC2 in a T cell lytic assay. 50 This EC 50 EC in T cell lytic assays in the form of a polypeptide or polypeptide complex without P1, L1, P2, or L2 50 In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits increased EC20 activity in a T cell lytic assay. 50 This EC 50 EC in T cell lytic assays in the form of a polypeptide or polypeptide complex without P1, L1, P2, or L2 50 In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits increased EC2 in a T cell lytic assay. 50 This EC 50 EC in T cell lytic assays in the form of a polypeptide or polypeptide complex without P1, L1, P2, or L2 50 In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits increased EC20 activity in a T cell lytic assay. 50 This EC 50EC in T cell lytic assays in the form of a polypeptide or polypeptide complex without P1, L1, P2, or L2 50 In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits increased EC2 in a T cell lytic assay. 50 This EC 50 EC in T cell lytic assays in the form of a polypeptide or polypeptide complex without P1, L1, P2, or L2 50 In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits increased EC2 in a T cell lytic assay. 50 This EC 50 EC in T cell lytic assays in the form of a polypeptide or polypeptide complex without P1, L1, P2, or L2 50 In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits increased EC20 activity in a T cell lytic assay. 50 This EC 50 EC in T cell lytic assays in the form of a polypeptide or polypeptide complex without P1, L1, P2, or L2 50 In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits increased EC2 in a T cell lytic assay. 50 This EC 50 EC in T cell lytic assays in the form of a polypeptide or polypeptide complex without P1, L1, P2, or L2 50 In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits increased EC2 in a T cell lytic assay. 50This EC 50 EC in T cell lytic assays in the form of a polypeptide or polypeptide complex without P1, L1, P2, or L2 50 In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits increased EC2 in a T cell lytic assay. 50 This EC 50 EC in T cell lytic assays in the form of a polypeptide or polypeptide complex without P1, L1, P2, or L2 50 At least 10,000 times higher than

[0078] In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) is a polypeptide having an EC2 activity in a T cell lysis assay of an isolated polypeptide or polypeptide complex of Formula Ia in which L1 and L2 have been cleaved by a tumor-specific protease. 50 Increased EC in T cell lytic assays compared to 50 In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) has an increased EC 50 This EC 50 The EC in a T cell lytic assay of an isolated polypeptide or polypeptide complex of formula Ia in which L1 and L2 have been cleaved by a tumor-specific protease 50 In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits increased EC2 in a T cell lytic assay. 50 This EC 50 The EC in a T cell lytic assay of an isolated polypeptide or polypeptide complex of formula Ia in which L1 and L2 have been cleaved by a tumor-specific protease 50In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits increased EC2 in a T cell lytic assay. 50 This EC 50 The EC in a T cell lytic assay of an isolated polypeptide or polypeptide complex of formula Ia in which L1 and L2 have been cleaved by a tumor-specific protease 50 In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits increased EC2 in a T cell lytic assay. 50 This EC 50 The EC in a T cell lytic assay of an isolated polypeptide or polypeptide complex of formula Ia in which L1 and L2 have been cleaved by a tumor-specific protease 50 In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits increased EC2 in a T cell lytic assay. 50 This EC 50 The EC in a T cell lytic assay of an isolated polypeptide or polypeptide complex of formula Ia in which L1 and L2 have been cleaved by a tumor-specific protease 50 In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits increased EC20 activity in a T cell lytic assay. 50 This EC 50 The EC in a T cell lytic assay of an isolated polypeptide or polypeptide complex of formula Ia in which L1 and L2 have been cleaved by a tumor-specific protease 50 In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits increased EC2 in a T cell lytic assay. 50 This EC50 The EC in a T cell lytic assay of an isolated polypeptide or polypeptide complex of formula Ia in which L1 and L2 have been cleaved by a tumor-specific protease 50 In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits increased EC20 activity in a T cell lytic assay. 50 This EC 50 The EC in a T cell lytic assay of an isolated polypeptide or polypeptide complex of formula Ia in which L1 and L2 have been cleaved by a tumor-specific protease 50 In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits increased EC2 in a T cell lytic assay. 50 This EC 50 The EC in a T cell lytic assay of an isolated polypeptide or polypeptide complex of formula Ia in which L1 and L2 have been cleaved by a tumor-specific protease 50 In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits increased EC2 in a T cell lytic assay. 50 This EC 50 The EC in a T cell lytic assay of an isolated polypeptide or polypeptide complex of formula Ia in which L1 and L2 have been cleaved by a tumor-specific protease 50 In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits increased EC20 activity in a T cell lytic assay. 50 This EC 50 The EC in a T cell lytic assay of an isolated polypeptide or polypeptide complex of formula Ia in which L1 and L2 have been cleaved by a tumor-specific protease 50In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits increased EC2 in a T cell lytic assay. 50 This EC 50 The EC in a T cell lytic assay of an isolated polypeptide or polypeptide complex of formula Ia in which L1 and L2 have been cleaved by a tumor-specific protease 50 In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits increased EC2 in a T cell lytic assay. 50 This EC 50 The EC in a T cell lytic assay of an isolated polypeptide or polypeptide complex of formula Ia in which L1 and L2 have been cleaved by a tumor-specific protease 50 In some embodiments, the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 (Formula Ia) exhibits increased EC2 in a T cell lytic assay. 50 This EC 50 The EC in a T cell lytic assay of an isolated polypeptide or polypeptide complex of formula Ia in which L1 and L2 have been cleaved by a tumor-specific protease 50 At least 10,000 times higher than

[0079] Second antigen recognition molecule (A2) In some embodiments, A2 comprises an antibody or antibody fragment. In some embodiments, the antibody or antibody fragment thereof comprises a single-chain variable fragment, a single-domain antibody, Fab, or Fab'. In some embodiments, the antibody or antibody fragment thereof 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 of a camelid-derived single-domain antibody (VHH). In some embodiments, the antibody or antibody fragment thereof is humanized or human. In some embodiments, A2 is a Fab or Fab'. In some embodiments, the 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 thereof comprises an EGFR-binding domain.

[0080] In some embodiments, an antigen-binding fragment (Fab) or Fab' that binds to EGFR comprises a Fab light chain polypeptide chain and a Fab heavy chain polypeptide chain. 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 a sequence substantially identical thereto (e.g., a sequence with 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 a sequence substantially identical thereto (e.g., a sequence with at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity).

[0081] 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 a sequence substantially identical thereto (e.g., a sequence with at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity), or the Fab light chain variable domain comprises at least one, two, or three complementarity determining regions (CDRs) disclosed in Table 2, or a sequence substantially identical thereto (e.g., a sequence with at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity).

[0082] [Table 2]

[0083] Table 3-1

[0084] Table 3-2

[0085] In some embodiments, the Fab comprises complementarity determining regions (CDRs) LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, LC-CDR2, and LC-CDR3 of the Fab comprise LC-CDR1 of SEQ ID NO: 15, LC-CDR2 of SEQ ID NO: 16, and LC-CDR3 of SEQ ID NO: 17, and the Fab comprises CDRs HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 of the Fab comprise HC-CDR1 of SEQ ID NO: 18, HC-CDR2 of SEQ ID NO: 19, and HC-CDR3 of SEQ ID NO: 20. In some embodiments, the Fab comprises complementarity determining regions (CDRs) LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, LC-CDR2, and LC-CDR3 of the Fab comprise LC-CDR1 of SEQ ID NO: 15, LC-CDR2 of SEQ ID NO: 16, and LC-CDR3 of SEQ ID NO: 17, and the CDRs comprise 0-2 amino acid modifications in at least one of HC-CDR1, HC-CDR2, or HC-CDR3; and the Fab comprises CDRs HC-CDR1, HC-CDR2, and HC-CDR3 of the Fab comprise HC-CDR1 of SEQ ID NO: 18, HC-CDR2 of SEQ ID NO: 19, and HC-CDR3 of SEQ ID NO: 20, and the CDRs comprise 0-2 amino acid modifications in at least one of LC-CDR1, LC-CDR2, or LC-CDR3.

[0086] In some embodiments, A2 comprises CDRs LC-CDR1, LC-CDR2, and LC-CDR3, and the LC-CDR1, LC-CDR2, and LC-CDR3 of A2 comprise LC-CDR1 of SEQ ID NO: 15, LC-CDR2 of SEQ ID NO: 16, and LC-CDR3 of SEQ ID NO: 17, and A2 comprises complementarity determining regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3, and the HC-CDR1, HC-CDR2, and HC-CDR3 of A2 comprise HC-CDR1 of SEQ ID NO: 18, HC-CDR2 of SEQ ID NO: 19, and HC-CDR3 of SEQ ID NO: 20.

[0087] In some embodiments, the Fab light chain polypeptide comprises the amino acid sequence set forth in SEQ ID NO:21. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence having at least 80% sequence identity to that set forth in SEQ ID NO:21. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence having at least 85% sequence identity to that set forth in SEQ ID NO:21. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence having at least 90% sequence identity to that set forth in SEQ ID NO:21. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence having at least 95% sequence identity to that set forth in SEQ ID NO:21. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence having at least 99% sequence identity to that set forth in SEQ ID NO:21.

[0088] In some embodiments, the Fab light chain polypeptide comprises the amino acid sequence set forth in SEQ ID NO:22. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence having at least 80% sequence identity to that set forth in SEQ ID NO:22. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence having at least 85% sequence identity to that set forth in SEQ ID NO:22. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence having at least 90% sequence identity to that set forth in SEQ ID NO:22. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence having at least 95% sequence identity to that set forth in SEQ ID NO:22. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence having at least 99% sequence identity to that set forth in SEQ ID NO:22.

[0089] In some embodiments, the Fab heavy chain polypeptide comprises the amino acid sequence set forth in SEQ ID NO:23. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence having at least 80% sequence identity to that set forth in SEQ ID NO:23. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence having at least 85% sequence identity to that set forth in SEQ ID NO:23. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence having at least 90% sequence identity to that set forth in SEQ ID NO:23. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence having at least 95% sequence identity to that set forth in SEQ ID NO:23. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence having at least 99% sequence identity to that set forth in SEQ ID NO:23.

[0090] In some embodiments, the Fab heavy chain polypeptide comprises the amino acid sequence set forth in SEQ ID NO:24. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence having at least 80% sequence identity to that set forth in SEQ ID NO:24. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence having at least 85% sequence identity to that set forth in SEQ ID NO:24. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence having at least 90% sequence identity to that set forth in SEQ ID NO:24. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence having at least 95% sequence identity to that set forth in SEQ ID NO:24. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence having at least 99% sequence identity to that set forth in SEQ ID NO:24.

[0091] In some embodiments, the Fab light chain polypeptide of A2 is conjugated to the C-terminus of the single chain variable fragment (scFv) of A1. In some embodiments, the Fab heavy chain polypeptide of A2 is conjugated to the C-terminus of the single chain variable fragment (scFv) of A1. In some embodiments, the Fab light chain polypeptide of A2 is conjugated to the N-terminus of the single chain variable fragment (scFv) of A1. In some embodiments, the Fab heavy chain polypeptide of A2 is conjugated to the N-terminus of the single chain variable fragment (scFv) of A1. In some embodiments, the Fab heavy chain polypeptide of A2 is conjugated to the scFv heavy chain polypeptide of A1. In some embodiments, the Fab light chain polypeptide of A2 is conjugated to the scFv heavy chain polypeptide of A1. In some embodiments, the Fab heavy chain polypeptide of A2 is conjugated to the scFv light chain polypeptide of A1. In some embodiments, the Fab light chain polypeptide of A2 is conjugated to the scFv light chain polypeptide of A1.

[0092] In some embodiments, A2 further comprises P2 and L2, where P2 comprises a peptide that binds to A2, and L2 connects A2 to P2 and comprises a linking moiety that 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.

[0093] In some embodiments, the Fab heavy chain polypeptide of A2 is coupled to the scFv heavy chain polypeptide of A1 and L2 is coupled to the Fab light chain polypeptide of A2. In some embodiments, the Fab light chain polypeptide of A2 is coupled to the scFv heavy chain polypeptide of A1 and L2 is coupled to the Fab heavy chain polypeptide of A2. In some embodiments, the Fab heavy chain polypeptide of A2 is coupled to the scFv light chain polypeptide of A1 and L2 is coupled to the Fab light chain polypeptide of A2. In some embodiments, the Fab light chain polypeptide of A2 is coupled to the scFv light chain polypeptide of A1 and L2 is coupled to the Fab heavy chain polypeptide of A2.

[0094] Peptides (P1 and P2 and P 1a ) In some embodiments, P1, P2, or P 1a includes the sequences disclosed in Table 3, or sequences substantially identical thereto (eg, sequences having 0, 1, or 2 amino acid modifications).

[0095] [Table 4]

[0096] In some embodiments, P1 impairs binding of A1 to the first target antigen. In some embodiments, P1 impairs binding of A1 to an effector cell antigen. In some embodiments, P1 is bound to A1 by ionic interactions, electrostatic interactions, hydrophobic interactions, Pi stacking interactions, and hydrogen bonding interactions, or a combination thereof. In some embodiments, P1 is bound to A1 at or near the antigen-binding site. In some embodiments, P1 is unbound from A1 when L1 is cleaved by a tumor-specific protease, thereby exposing A1 to the effector cell antigen. In some embodiments, the protease comprises a tumor-specific protease. In some embodiments, the protease comprises a matrix metalloprotease (MMP) or a serine protease. In some embodiments, the matrix metalloprotease comprises MMP2, MMP7, MMP9, MMP13, or MMP14. In some embodiments, the serine protease comprises matriptase (MTSP1), urokinase, or hepsin. In some embodiments, P1 has less than 70% sequence identity to the effector cell antigen. In some embodiments, P1 has less than 75% sequence identity to the effector cell antigen. In some embodiments, P1 has less than 80% sequence identity to the effector cell antigen. In some embodiments, P1 has less than 85% sequence identity to the effector cell antigen. In some embodiments, P1 has less than 90% sequence identity to the effector cell antigen. In some embodiments, P1 has less than 95% sequence identity to the effector cell antigen. In some embodiments, P1 has less than 98% sequence identity to the effector cell antigen. In some embodiments, P1 has less than 99% sequence identity to the effector cell antigen. In some embodiments, P1 comprises a de novo amino acid sequence that shares less than 10% sequence identity to the effector cell antigen. In some embodiments, P1 comprises the amino acid sequence set forth in SEQ ID NO:25.

[0097] In some embodiments, A1 comprises a first antigen recognition molecule comprising an antibody or antibody-binding fragment that binds to CD3. In some embodiments, P1 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 25, 797-835, or 843-1690, or an amino acid sequence having one, two, or three amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 25, 797-835, or 843-1690.

[0098] In some embodiments, P1 is Z1-Z2-C-Z4-P-Z6-Z7-Z8-Z9-Z 10 -Z 11 -Z 12 -CZ 14 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; and 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 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, and 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 Z14 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, and Z 10 is selected from S, D, and Y, and Z 11 is selected from I, Y, and F, and Z 12 is selected from F, D, Y, and L, and Z 14 is selected from D, Y, and N. In some embodiments, P1 is U1-U2-C-U4-P-U6-U7-U8-U9-U 10 -U 11 -U 12 -CU 14 wherein 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; and U9 is selected from E, D, Y, V, F, W, P, L, and Q; 10 is selected from S, D, Y, T, I, F, V, N, A, P, L, and H, and U 11 is selected from I, Y, F, V, L, T, N, S, D, A, and H, and U 12 is selected from F, D, Y, L, I, V, A, N, T, P, S, G, and H, and U 14 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, and U 10 is selected from S, D, Y, T, and I, and U 11is selected from I, Y, F, V, L, and T, and U 12 is selected from F, D, Y, L, I, V, A, G, and N, and U 14 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, and U 10 is selected from S, D, T, and Y, and U 11 is selected from I, Y, V, L, and F, and U 12 is selected from F, D, Y, G, A, and L, and U 14 is selected from D, Y, M, and N.

[0099] In some embodiments, P1 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 797-823.

[0100] In some embodiments, P1 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 25, 824-835, and 843-1690.

[0101] In some embodiments, P1 comprises the amino acid sequence set forth in any one of SEQ ID NOs:824-835.

[0102] In some embodiments, P1 comprises the amino acid sequence set forth in SEQ ID NO:810, or an amino acid sequence having one, two, or three amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO:810.

[0103] In some embodiments, P1 comprises the amino acid sequence set forth in SEQ ID NO:811, or an amino acid sequence having one, two, or three amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO:811.

[0104] In some embodiments, P1 comprises the amino acid sequence set forth in SEQ ID NO:834, or an amino acid sequence having one, two, or three amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO:834.

[0105] In some embodiments, P1 comprises the amino acid sequence set forth in SEQ ID NO:810.

[0106] In some embodiments, P1 comprises the amino acid sequence set forth in SEQ ID NO:811.

[0107] In some embodiments, P1 comprises the amino acid sequence set forth in SEQ ID NO:834.

[0108] In some embodiments, P2 impairs binding of A2 to a second target antigen. In some embodiments, P2 impairs binding of A2 to EGFR. In some embodiments, P2 is bound to A2 by ionic interactions, electrostatic interactions, hydrophobic interactions, Pi stacking interactions, and hydrogen bonding interactions, or a combination thereof. In some embodiments, P2 is bound to A2 at or near the antigen-binding site. In some embodiments, P2 is unbound from A2 when L2 is cleaved by a tumor-specific protease, thereby exposing A2 to EGFR. In some embodiments, the protease comprises a tumor-specific protease. In some embodiments, the protease comprises a matrix metalloprotease (MMP) or a serine protease. In some embodiments, the matrix metalloprotease comprises MMP2, MMP7, MMP9, MMP13, or MMP14. In some embodiments, the serine protease comprises matriptase (MTSP1), urokinase, or hepsin. In some embodiments, P2 has less than 70% sequence identity to EGFR. In some embodiments, P2 has less than 75% sequence identity to EGFR. In some embodiments, P2 has less than 80% sequence identity to EGFR. In some embodiments, P2 has less than 85% sequence identity to EGFR. In some embodiments, P2 has less than 90% sequence identity to EGFR. In some embodiments, P2 has less than 95% sequence identity to EGFR. In some embodiments, P2 has less than 98% sequence identity to EGFR. In some embodiments, P2 has less than 99% sequence identity to EGFR. In some embodiments, P2 comprises a de novo amino acid sequence that shares less than 10% sequence identity to EGFR. In some embodiments, P2 comprises the amino acid sequence set forth in SEQ ID NO:26. In some embodiments, P2 comprises the amino acid sequence set forth in SEQ ID NO:115. In some embodiments, P2 comprises the amino acid sequence set forth in SEQ ID NO:116.

[0109] In some embodiments, P2 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 26, 71-96, 98-776, or an amino acid sequence having one, two, or three amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 26, 71-96, 98-776. In some embodiments, P2 comprises the amino acid sequence set forth in X1-C-X2-X3-X4-X5-D-X6-A-X7-P-X8-C-X9, where Xi is selected from P and L, X2 is selected from R, L, T, A, N, I, V, S, H, and P, X3 is selected from S, P, F, and Y, X4 is selected from H, L, Q, P, R, F, N, X5 is selected from I, F, Y, H, N, T, S, D, A, L, and V, X6 is selected from T, P, N, L, I, V, S, D, H, A, and Y, X7 is selected from K and Y, X8 is selected from I, P, L, and M, and X9 is selected from A, V, I, T, L, S, D, F, V, and H (SEQ ID NO: 841). In some embodiments, X1 is selected from P and L, X2 is selected from R, L, T, A, and N, X3 is selected from S, P, and F, X4 is selected from H, L, Q, and P, X5 is selected from I, F, Y, H, N, and T, X6 is selected from T, P, N, L, I, and V, X7 is K, X8 is I, and X9 is selected from A, V, I, T, L, and S. In some embodiments, X1 is P, X2 is selected from R, L, and T, X3 is S, X4 is selected from H, L, Q, and P, X5 is selected from I, F, Y, and T, X6 is selected from T, P, N, and V, X7 is K, and X8 is I, and X9 is selected from A, V, and I.

[0110] In some embodiments, P2 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 26, 86-96, or an amino acid sequence having one, two, or three amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 26, 86-96. In some embodiments, P2 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 26, 86-96.

[0111] In some embodiments, P2 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 98-776, or an amino acid sequence having one, two, or three amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 98-776. In some embodiments, P2 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 98-776.

[0112] In some embodiments, P2 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 99-118, or an amino acid sequence having one, two, or three amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 99-118. In some embodiments, P2 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 99-118.

[0113] In some embodiments, P2 comprises the amino acid sequence set forth in SEQ ID NO: 26, or an amino acid sequence having one, two, or three amino acid mutations, substitutions, or deletions relative to SEQ ID NO: 26. In some embodiments, P2 comprises the amino acid sequence set forth in SEQ ID NO: 26.

[0114] In some embodiments, P2 comprises the amino acid sequence set forth in SEQ ID NO: 115, or an amino acid sequence having one, two, or three amino acid mutations, substitutions, or deletions relative to SEQ ID NO: 115. In some embodiments, P2 comprises the amino acid sequence set forth in SEQ ID NO: 115.

[0115] In some embodiments, P2 comprises the amino acid sequence set forth in SEQ ID NO: 116, or an amino acid sequence having one, two, or three amino acid mutations, substitutions, or deletions relative to SEQ ID NO: 116. In some embodiments, P2 comprises the amino acid sequence set forth in SEQ ID NO: 116.

[0116] In some embodiments, P 1a L 1a When uncleaved, P impairs binding of the antigen recognition molecule to the target antigen. In some embodiments, the antigen recognition molecule comprises 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 epidermal growth factor receptor (EGFR). In some embodiments, P 1a has less than 70% sequence identity to the target antigen. 1a has less than 75% sequence identity to the target antigen. 1a has less than 80% sequence identity to the target antigen. 1a has less than 85% sequence identity to the target antigen. 1a has less than 90% sequence identity to the target antigen. 1a has less than 95% sequence identity to the target antigen. 1a has less than 98% sequence identity to the target antigen. 1a has less than 99% sequence identity to the target antigen. 1a comprises a de novo amino acid sequence that shares less than 10% sequence identity to the second target antigen.

[0117] In some embodiments, P 1acomprises an amino acid sequence set forth in any one of SEQ ID NOs: 25, 797 to 835, or 843 to 1690, or an amino acid sequence having one, two, or three amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 25, 797 to 835, or 843 to 1690.

[0118] In some embodiments, P 1a is Z1-Z2-C-Z4-P-Z6-Z7-Z8-Z9-Z 10 -Z 11 -Z 12 -CZ 14 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; and 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 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, and 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 14is 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, and Z 10 is selected from S, D, and Y, and Z 11 is selected from I, Y, and F, and Z 12 is selected from F, D, Y, and L, and Z 14 is selected from D, Y, and N. In some embodiments, P1 is U1-U2-C-U4-P-U6-U7-U8-U9-U 10 -U 11 -U 12 -CU 14 wherein 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; and U9 is selected from E, D, Y, V, F, W, P, L, and Q; 10 is selected from S, D, Y, T, I, F, V, N, A, P, L, and H, and U 11 is selected from I, Y, F, V, L, T, N, S, D, A, and H, and U 12 is selected from F, D, Y, L, I, V, A, N, T, P, S, G, and H, and U 14 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, and U 10 is selected from S, D, Y, T, and I, and U 11 is selected from I, Y, F, V, L, and T, and U12 is selected from F, D, Y, L, I, V, A, G, and N, and U 14 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, and U 10 is selected from S, D, T, and Y, and U 11 is selected from I, Y, V, L, and F, and U 12 is selected from F, D, Y, G, A, and L, and U 14 is selected from D, Y, M, and N.

[0119] In some embodiments, P 1a comprises the amino acid sequence set forth in any one of SEQ ID NOs: 797 to 823.

[0120] In some embodiments, P 1a comprises the amino acid sequence set forth in any one of SEQ ID NOs: 25, 824 to 835, or 843 to 1690.

[0121] In some embodiments, P 1a comprises the amino acid sequence set forth in any one of SEQ ID NOs: 824 to 835.

[0122] In some embodiments, P 1a comprises the amino acid sequence set forth in SEQ ID NO:810 or an amino acid sequence having one, two, or three amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO:810.

[0123] In some embodiments, P 1a comprises the amino acid sequence set forth in SEQ ID NO:811 or an amino acid sequence having one, two, or three amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO:811.

[0124] In some embodiments, P 1a comprises the amino acid sequence set forth in SEQ ID NO:834 or an amino acid sequence having one, two, or three amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO:834.

[0125] In some embodiments, P 1a comprises the amino acid sequence set forth in SEQ ID NO:810.

[0126] In some embodiments, P 1a comprises the amino acid sequence set forth in SEQ ID NO:811.

[0127] In some embodiments, P 1a comprises the amino acid sequence set forth in SEQ ID NO:834.

[0128] In some embodiments, P1, P2, or P 1a comprises a peptide sequence at least 5 amino acids in length. In some embodiments, P1, P2, or P 1a comprises a peptide sequence of at least 6 amino acids in length. In some embodiments, P1, P2, or P 1a comprises a peptide sequence of at least 10 amino acids in length. In some embodiments, P1, P2, or P 1a comprises a peptide sequence at least 10 amino acids in length and no more than 20 amino acids in length. 1a comprises a peptide sequence of at least 16 amino acids in length. In some embodiments, P1, P2, or P 1a comprises a peptide sequence of 40 amino acids or less. In some embodiments, P1, P2, or P 1a contains at least two cysteine ​​amino acid residues. In some embodiments, P1, P2, or P 1a comprises a cyclic or linear peptide. In some embodiments, P1, P2, or P 1a In some embodiments, P1, P2, or P 1acomprises a linear peptide.

[0129] In some embodiments, P1, P2, or P 1a , or P1, P2, and P 1a In some embodiments, P1, P2, or P3 comprises a modified amino acid or a non-natural amino acid, or a modified non-natural amino acid, or a combination thereof. In some embodiments, the modified amino acid or the modified non-natural amino acid comprises a post-translational modification. In some embodiments, P1, P2, or P4 comprises a modified amino acid or a non-natural amino acid, or a combination thereof. 1a , or P1, P2, and P 1a These modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA-mediated addition of amino acids to proteins such as arginylation, and ubiquitination. Modifications can occur anywhere, including the peptide backbone, amino acid side chains, and termini, at P1, P2, or P3. 1a , or P1, P2, and P 1a It is done.

[0130] In some embodiments, P1, P2, or P 1a does not comprise albumin or an albumin fragment. In some embodiments, P1, P2, or P 1a does not contain an albumin binding domain.

[0131] Linking parts (L1, L2, L3, and L 1a ) In some embodiments, L1, L2, L3, or L 1ais a peptide sequence having at least 5 to 50 amino acids. In some embodiments, L1, L2, L3, or L 1a is a peptide sequence having at least 10 to no more than 30 amino acids. In some embodiments, L1, L2, L3, or L 1a is a peptide sequence having at least 10 amino acids. In some embodiments, L1, L2, L3, or L 1a is a peptide sequence having at least 18 amino acids. In some embodiments, L1, L2, L3, or L 1a is a peptide sequence having at least 26 amino acids. In some embodiments, L1, L2, L3, or L 1a (G2S) n wherein n is an integer from 1 to 3 (SEQ ID NO: 840). In some embodiments, L1, L2, L3, or L 1a (G2S) n wherein n is an integer of at least 1. In some embodiments, L1, L2, L3, or L 1a (G2S) n , (GS) n , (GSGGS) n (SEQ ID NO: 836), (GGGS) n (SEQ ID NO: 837), (GGGGS) n (SEQ ID NO: 838), and (GSSGGS) n (SEQ ID NO: 839), wherein n is an integer of at least 1. In some embodiments, the tumor-specific protease is selected from the group consisting of matrix metalloproteases, serine proteases, cysteine ​​proteases, threonine proteases, and aspartic acid proteases. In some embodiments, L1, L2, L3, or L4 is selected from the group consisting of: 1acomprises an amino acid sequence cleavable by urokinase, an amino acid sequence cleavable by matriptase (MTSP1), an amino acid sequence cleavable by legumain, or an amino acid sequence cleavable by a matrix metalloprotease. In some examples, the matrix metalloprotease comprises MMP2, MMP7, MMP9, MMP13, or MMP14. In some examples, the serine protease comprises matriptase (MTSP1), urokinase, or hepsin.

[0132] In some embodiments, L1, L2, L3, or L 1a includes the sequences disclosed in Table 4, or sequences substantially identical thereto (eg, sequences having 0, 1, or 2 amino acid modifications).

[0133] In some embodiments, L1 comprises the amino acid sequence of linker 4 (ISSGLLSGRSDAG) (SEQ ID NO: 66), linker 5 (AAGLLAPPGGLSGRSDAG) (SEQ ID NO: 67), linker 6 (SPLGLSGRSDAG) (SEQ ID NO: 68), or linker 7 (LSGRSDAGSPLGLAG) (SEQ ID NO: 69), or an amino acid sequence having one, two, or three amino acid substitutions, additions, or deletions relative to the amino acid sequence of linker 4, linker 5, linker 6, or linker 7. In some embodiments, L1 comprises the amino acid sequence of linker 4 (ISSGLLSGRSDAG) (SEQ ID NO: 66). In some embodiments, L1 comprises the amino acid sequence of linker 5 (AAGLLAPPGGLSGRSDAG) (SEQ ID NO: 67). In some embodiments, L1 comprises the amino acid sequence of linker 6 (SPLGLSGRSDAG) (SEQ ID NO: 68). In some embodiments, L1 comprises the amino acid sequence of linker 7 (LSGRSDAGSPLGLAG) (SEQ ID NO: 69).

[0134] In some embodiments, L2 comprises the amino acid sequence of linker 4 (ISSGLLSGRSDAG) (SEQ ID NO: 66), linker 5 (AAGLLAPPGGLSGRSDAG) (SEQ ID NO: 67), linker 6 (SPLGLSGRSDAG) (SEQ ID NO: 68), or linker 7 (LSGRSDAGSPLGLAG) (SEQ ID NO: 69), or an amino acid sequence having one, two, or three amino acid substitutions, additions, or deletions relative to the amino acid sequence of linker 4, linker 5, linker 6, or linker 7. In some embodiments, L2 comprises the amino acid sequence of linker 4 (ISSGLLSGRSDAG) (SEQ ID NO: 66). In some embodiments, L2 comprises the amino acid sequence of linker 5 (AAGLLAPPGGLSGRSDAG) (SEQ ID NO: 67). In some embodiments, L2 comprises the amino acid sequence of linker 6 (SPLGLSGRSDAG) (SEQ ID NO: 68). In some embodiments, L2 comprises the amino acid sequence of linker 7 (LSGRSDAGSPLGLAG) (SEQ ID NO: 69).

[0135] [Table 5-1]

[0136] [Table 5-2]

[0137] In some embodiments, L1 is attached to the N-terminus of A1. In some embodiments, L1 is attached to the C-terminus of A1. In some embodiments, L2 is attached to the N-terminus of A2. In some embodiments, L2 is attached to the C-terminus of A2. In some embodiments, P1 is uncoupled from A1 when L1 is cleaved by a tumor-specific protease, thereby exposing A1 to effector cell antigens. In some embodiments, P2 is uncoupled from A2 when L2 is cleaved by a tumor-specific protease, thereby exposing A2 to effector cell antigens.

[0138] In some embodiments, L1, L2, L3, or L 1a In some embodiments, L1, L2, L3, or L4 comprises a modified amino acid or a non-natural amino acid, or a modified non-natural amino acid, or a combination thereof. In some embodiments, the modified amino acid or the modified non-natural amino acid comprises a post-translational modification. In some embodiments, L1, L2, L3, or L4 comprises a modified amino acid or a non-natural amino acid, or a combination thereof. 1aThese modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA-mediated addition of amino acids to proteins such as arginylation, and ubiquitination. Modifications can occur anywhere including the peptide backbone or at the amino acid side chains, at L1, L2, L3, or L4. 1a It is done.

[0139] In some embodiments, the cleavable linker is cleavable by a protease. In some embodiments, the protease is present at a higher concentration in a diseased microenvironment compared to the concentration in a healthy tissue or a non-diseased microenvironment. In some embodiments, the protease comprises a tumor-specific protease. In some embodiments, the protease comprises a matrix metalloprotease (MMP) or a serine protease. In some embodiments, the matrix metalloprotease comprises MMP2, MMP7, MMP9, MMP13, or MMP14. In some embodiments, the matrix metalloprotease is selected from the group consisting of MMP2, MMP7, MMP9, MMP13, and MMP14. In some embodiments, the matrix metalloprotease comprises MMP2. In some embodiments, the matrix metalloprotease comprises MMP7. In some embodiments, the matrix metalloprotease comprises MMP9. In some embodiments, the matrix metalloprotease comprises MMP13. In some embodiments, the matrix metalloprotease comprises MMP14. In some embodiments, the serine protease comprises matriptase (MTSP1), urokinase, or hepsin. In some embodiments, the serine protease is selected from the group consisting of matriptase (MTSP1), urokinase, and hepsin. In some embodiments, the serine protease comprises matriptase (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 different 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 different proteases.

[0140] Half-life extension molecules (H1 and H 1a ) In some embodiments, H1 does not inhibit binding of A1 to an effector cell antigen. In some embodiments, H1 comprises a linking moiety (L3) that connects H1 to P1. In some embodiments, H 1a does not inhibit binding of the first antigen recognition molecule to an effector cell antigen. 1a is H 1a P 1a In some embodiments, the half-life extension (H1 or H2) comprises a linking moiety (L3) that connects the half-life extension (H1 or H2) to the 1a ) has no binding affinity for the antigen recognition molecule. In some embodiments, the half-life extension molecule (H1 or H 1a ) has no binding affinity for 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 linked to the antigen recognition molecule.

[0141] In some embodiments, H or H 1a includes a sequence disclosed in Table 5, or a sequence substantially identical thereto (e.g., a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity).

[0142] [Table 6]

[0143] [Table 7-1]

[0144] In some embodiments, H or H 1a comprises an amino acid sequence with a repetitive sequence motif. In some embodiments, H1 or H 1a contains an amino acid sequence with a highly ordered secondary structure. As used in this context, "highly ordered secondary structure" refers to an amino acid sequence that is highly ordered, such as an H1 or H2 1a This means that at least about 50%, or about 70%, or about 80%, or about 90% of the amino acid residues of the protein contribute to secondary structure as measured or determined by means including, but not limited to, spectrophotometry (e.g., by circular dichroism spectroscopy in the "far UV" spectral region (190-250 nm)) and computer programs or algorithms such as the Chou-Fasman algorithm and the Gamier-Osguthorpe-Robson ("GOR") algorithm.

[0145] In some embodiments, H or H 1a In some embodiments, H1 comprises a polymer. In some embodiments, the polymer is polyethylene glycol (PEG). In some embodiments, H1 comprises albumin. In some embodiments, H1 or H 1aIn some embodiments, the albumin is serum albumin. In some embodiments, the albumin is human serum albumin. In some embodiments, the albumin is H1 or H 1acomprises a polypeptide, ligand, or small molecule. In some embodiments, the polypeptide, ligand, or small molecule binds a serum protein or fragment thereof, a circulating immunoglobulin or fragment thereof, or CD35 / CR1. In some embodiments, the serum protein comprises thyroxine-binding protein, transthyretin, 1-acid glycoprotein, transferrin, transferrin receptor or a transferrin-binding portion thereof, fibrinogen, or albumin. In some embodiments, the circulating immunoglobulin molecule comprises IgG1, IgG2, IgG3, IgG4, slgA, IgM, or IgD. In some embodiments, the serum protein is albumin. In some embodiments, the polypeptide is an antibody. In some embodiments, the antibody comprises a single-domain antibody, a single-chain variable fragment, or a Fab. In some embodiments, the single-domain antibody comprises a single-domain antibody that binds to albumin. In some embodiments, the single-domain antibody is a human antibody or a humanized antibody. In some embodiments, the single domain antibody is selected from the group consisting of 645gH1gL1, 645dsgH5gL4, 23-13-A01 -sc02, A10m3 or fragments thereof, DOM7r-31, DOM7h-11-15, Alb-1, Alb-8, Alb-23, 10G, 10E, and SA21. In some embodiments, the single domain antibody comprises complementarity determining regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 of the single domain antibody comprise HC-CDR1 of SEQ ID NO: 36, HC-CDR2 of SEQ ID NO: 37, and HC-CDR3 of SEQ ID NO: 38.In some embodiments, the single domain antibody comprises complementarity determining regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 of the single domain antibody comprise HC-CDR1 of SEQ ID NO: 36, HC-CDR2 of SEQ ID NO: 37, and HC-CDR3 of SEQ ID NO: 38, and the CDRs comprise 0 to 2 amino acid modifications in at least one of HC-CDR1, HC-CDR2, and HC-CDR3. In some embodiments, the single domain antibody comprises complementarity determining regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 of the single domain antibody comprise HC-CDR1 of SEQ ID NO: 39, HC-CDR2 of SEQ ID NO: 40, and HC-CDR3 of SEQ ID NO: 41. In some embodiments, the single domain antibody comprises complementarity determining regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 of the single domain antibody comprise HC-CDR1 of SEQ ID NO: 39, HC-CDR2 of SEQ ID NO: 40, and HC-CDR3 of SEQ ID NO: 41, and the CDRs comprise 0 to 2 amino acid modifications in at least one of HC-CDR1, HC-CDR2, and HC-CDR3.

[0146] In some embodiments, H1 comprises the amino acid sequence set forth in SEQ ID NO: 42. In some embodiments, H1 comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 42. H1 comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 42. H1 comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 42. H1 comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 42. H1 comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 42.

[0147] In some embodiments, H 1acomprises the amino acid sequence set forth in SEQ ID NO: 42. In some embodiments, H 1a comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 42. 1a comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 42. 1a comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 42. 1a comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 42. 1a comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:42.

[0148] In some embodiments, H1 comprises the amino acid sequence set forth in SEQ ID NO: 43. In some embodiments, H1 comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 43. H1 comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 43. H1 comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 43. H1 comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 43. H1 comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 43.

[0149] In some embodiments, H 1a comprises the amino acid sequence set forth in SEQ ID NO: 43. In some embodiments, H 1a comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 43. 1a comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 43. 1a comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 43. 1a comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 43. 1a comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:43.

[0150] In some embodiments, H or H 1a or H1 and H 1a comprises a modified amino acid or a non-natural amino acid, or a modified non-natural amino acid, or a combination thereof. In some embodiments, the modified amino acid or the modified non-natural amino acid comprises a post-translational modification. In some embodiments, H1 or H 1a or H1 and H 1a These modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA-mediated addition of amino acids to proteins such as arginylation, and ubiquitination. Modifications can occur anywhere, including the peptide backbone, amino acid side chains, and termini, by addition of H1 or H 1a or H1 and H 1a It is done.

[0151] In some embodiments, H1 comprises a linking moiety (L3) connecting H1 to P1. In some embodiments, L3 is a peptide sequence having at least 5-50 amino acids or less. In some embodiments, L3 is a peptide sequence having at least 10-30 amino acids or less. 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 ... n , (GS) n , (GSGGS) n(SEQ ID NO: 836), (GGGS) n (SEQ ID NO: 837), (GGGGS) n (SEQ ID NO: 838), and (GSSGGS) n (SEQ ID NO: 839), wherein n is an integer of at least 1. In some embodiments, L3 comprises the amino acid sequence set forth in SEQ ID NO: 29.

[0152] In some embodiments, H 1a is H 1a P 1a The connecting part (L 1a In some embodiments, L 1a is a peptide sequence having at least 5 to 50 amino acids. 1a is a peptide sequence having at least 10 to 30 amino acids. 1a is a peptide sequence having at least 10 amino acids. In some embodiments, L 1a is a peptide sequence having at least 18 amino acids. In some embodiments, L 1a is a peptide sequence having at least 26 amino acids. 1a (G2S) n , (GS) n , (GSGGS) n (SEQ ID NO: 836), (GGGS) n (SEQ ID NO: 837), (GGGGS) n (SEQ ID NO: 838), and (GSSGGS) n (SEQ ID NO: 839), wherein n is an integer of at least 1. In some embodiments, L 1a comprises the amino acid sequence set forth in SEQ ID NO: 30 or 31.

[0153] Antibodies that bind to EGFR and CD3 In some embodiments, the isolated 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 isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 44-61. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 50. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 51.

[0154] Table 8-1

[0155] Table 8-2

[0156] Table 8-3

[0157] Table 8-4

[0158] Table 8-5

[0159] [Table 8-6]

[0160] [Table 8-7]

[0161] In some embodiments, the isolated polypeptide or polypeptide complex comprises the amino acid sequence set forth in SEQ ID NO: 44 and SEQ ID NO: 45. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 44 and SEQ ID NO: 45. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 44 and SEQ ID NO: 45. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 44 and SEQ ID NO: 45.

[0162] In some embodiments, the isolated polypeptide or polypeptide complex comprises the amino acid sequence set forth in SEQ ID NO: 46 and SEQ ID NO: 47. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 46 and SEQ ID NO: 47. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 46 and SEQ ID NO: 47. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 46 and SEQ ID NO: 47.

[0163] In some embodiments, the isolated polypeptide or polypeptide complex comprises the amino acid sequence set forth in SEQ ID NO: 48 and SEQ ID NO: 49. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 48 and SEQ ID NO: 49. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 48 and SEQ ID NO: 49. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 48 and SEQ ID NO: 49.

[0164] In some embodiments, the isolated polypeptide or polypeptide complex comprises the amino acid sequence set forth in SEQ ID NO:50 and SEQ ID NO:51. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:50 and SEQ ID NO:51. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:50 and SEQ ID NO:51. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:50 and SEQ ID NO:51.

[0165] In some embodiments, the isolated polypeptide or polypeptide complex comprises the amino acid sequence set forth in SEQ ID NO:52 and SEQ ID NO:53. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:52 and SEQ ID NO:53. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:52 and SEQ ID NO:53. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:52 and SEQ ID NO:53.

[0166] In some embodiments, the isolated polypeptide or polypeptide complex comprises the amino acid sequence set forth in SEQ ID NO:54 and SEQ ID NO:55. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:54 and SEQ ID NO:55. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:54 and SEQ ID NO:55. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:54 and SEQ ID NO:55.

[0167] In some embodiments, the isolated polypeptide or polypeptide complex comprises the amino acid sequence set forth in SEQ ID NO:56 and SEQ ID NO:57. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:56 and SEQ ID NO:57. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:56 and SEQ ID NO:57. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:56 and SEQ ID NO:57.

[0168] In some embodiments, the isolated polypeptide or polypeptide complex comprises the amino acid sequence set forth in SEQ ID NO:58 and SEQ ID NO:59. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:58 and SEQ ID NO:59. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:58 and SEQ ID NO:59. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:58 and SEQ ID NO:59.

[0169] In some embodiments, the isolated polypeptide or polypeptide complex comprises the amino acid sequence set forth in SEQ ID NO:60 and SEQ ID NO:61. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:60 and SEQ ID NO:61. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:60 and SEQ ID NO:61. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:60 and SEQ ID NO:61.

[0170] In some embodiments, the isolated polypeptide or polypeptide complex comprises the amino acid sequence set forth in SEQ ID NO:62 and SEQ ID NO:63. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:62 and SEQ ID NO:63. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:62 and SEQ ID NO:63. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:62 and SEQ ID NO:63.

[0171] In some embodiments, the isolated polypeptide or polypeptide complex comprises the amino acid sequence set forth in SEQ ID NO:64 and SEQ ID NO:65. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:64 and SEQ ID NO:65. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:64 and SEQ ID NO:65. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:64 and SEQ ID NO:65.

[0172] The polypeptide or polypeptide complex, in some embodiments, comprises a sequence set forth in Table 6. In some embodiments, the sequence comprises 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: 44-65. In some examples, the sequence comprises at least or about 95% homology to SEQ ID NOs: 44-65. In some examples, the sequence comprises at least or about 97% homology to SEQ ID NOs: 44-65. In some examples, the sequence comprises at least or about 99% homology to SEQ ID NOs: 44-65. In some examples, the sequence comprises at least or about 100% homology to SEQ ID NOs: 44-65. In some examples, the sequence includes at least a portion 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 of any one of SEQ ID NOs: 4, 46, 48, 50, 52, 54, 56, 58, 60, 63, or 65. In some examples, the sequence includes at least a portion 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 of any one of SEQ ID NOs: 45, 47, 49, 51, 53, 55, 57, 59, or 62.In some examples, the sequence is 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, 460, 470, 480, 490, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, , 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.

[0173] As used herein, the term "percent (%) amino acid sequence identity" for a sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a specific sequence, after aligning the sequences and introducing gaps as necessary to achieve the maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in a variety of ways within the art, for example, using publicly available computer software such as EMBOSS MATCHER, EMBOSS WATER, EMBOSS STRETCHER, EMBOSS NEEDLE, EMBOSS LALIGN, BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared. In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to a given amino acid sequence B (which may alternatively be expressed as a given amino acid sequence A having or containing % amino acid sequence identity to given amino acid sequence B) is calculated as follows: multiply the fraction X / Y by 100, where X is the number of amino acid residues scored by the sequence alignment program ALIGN-2 as identical matches in the sequences A and B in that program, and where Y is the total number of amino acid residues in B. It should be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

[0174] In some embodiments, a polypeptide or polypeptide complex comprising the structural arrangement set forth in Configuration 1 below,

[0175] [ka] Disclosed herein is an isolated polypeptide or polypeptide complex comprising a single-chain variable fragment (scFv) comprising a light chain variable domain and a heavy chain variable domain, wherein the scFv is linked to a peptide (P1) that impairs binding of the scFv to an effector cell antigen, and P1 is linked to the N-terminus of the heavy chain variable domain of the scFv by a linking moiety (L1) that is a substrate for a tumor-specific protease, and P1 is further linked to a half-life extension and a Fab that binds to epidermal growth factor receptor (EGFR), wherein the Fab comprises a Fab light chain polypeptide and a Fab heavy chain polypeptide, the Fab light chain polypeptide is linked to the C-terminus of the light chain variable domain of the scFv, and the Fab is linked to P2 and L2, wherein P2 comprises the peptide that impairs binding to EGFR, and L2 connects the Fab heavy chain polypeptide to P2 and comprises a linking moiety that is a substrate for a tumor-specific protease.

[0176] In some embodiments, a polypeptide or polypeptide complex comprising the structural arrangement set forth in Configuration 2 below,

[0177] [ka] Disclosed herein is an isolated polypeptide or polypeptide complex comprising a single-chain variable fragment (scFv) comprising a light chain variable domain and a heavy chain variable domain, wherein the scFv is linked to a peptide (P1) that impairs binding of the scFv to an effector cell antigen, and P1 is linked to the N-terminus of the heavy chain variable domain of the scFv by a linking moiety (L1) that is a substrate for a tumor-specific protease, and P1 is further linked to a half-life extension and a Fab that binds to epidermal growth factor receptor (EGFR), wherein the Fab comprises a Fab light chain polypeptide and a Fab heavy chain polypeptide, and the Fab heavy chain polypeptide is linked to the C-terminus of the light chain variable domain of the scFv, and the Fab is linked to P2 and L2, wherein P2 comprises a peptide that impairs binding to EGFR, and L2 comprises a linking moiety that joins the Fab light chain polypeptide to P2 and is a substrate for a tumor-specific protease.

[0178] Polynucleotides encoding polypeptides or polypeptide complexes In some embodiments, disclosed herein are isolated recombinant nucleic acid molecules encoding the polypeptides or polypeptide complexes disclosed herein. In some embodiments, the polypeptides or polypeptide complexes comprise antibodies or antibody fragments. In some embodiments, the polypeptides or polypeptide complexes comprise Fab and single-chain variable fragments (scFv).

[0179] In some embodiments, an isolated recombinant nucleic acid molecule encoding a polypeptide or polypeptide complex set forth in Formula I below,

[0180] [ka] wherein A1 comprises a first antigen recognition molecule that binds to an effector cell antigen, P1 comprises a peptide that binds to A1, L1 comprises a linking moiety connecting 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 recognition molecule that binds to an epidermal growth factor receptor (EGFR).

[0181] In some embodiments, an isolated recombinant nucleic acid molecule encoding a polypeptide or polypeptide complex set forth in Formula I below,

[0182] [ka] Disclosed herein is an isolated recombinant nucleic acid molecule, wherein A1 is a first antigen recognition molecule that binds to an effector cell antigen, P1 is a peptide that binds to A1, L1 is a linking moiety that connects A1 to P1 and is a substrate for a tumor-specific protease, H1 is a half-life extending molecule, and A2 is a second antigen recognition molecule that binds to an epidermal growth factor receptor (EGFR).

[0183] In some embodiments, an isolated recombinant nucleic acid molecule encoding a polypeptide or polypeptide complex comprising Formula I:

[0184] [ka] Disclosed herein is an isolated recombinant nucleic acid molecule, wherein A1 comprises a first antigen recognition molecule that binds to an effector cell antigen, P1 comprises a peptide that binds to A1, L1 comprises a linking moiety connecting 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 recognition molecule that binds to an epidermal growth factor receptor (EGFR).

[0185] In some embodiments, an isolated recombinant nucleic acid molecule encoding a polypeptide or polypeptide complex comprising Formula I:

[0186] [ka] Disclosed herein is an isolated recombinant nucleic acid molecule, wherein A1 is a first antigen recognition molecule that binds to an effector cell antigen, P1 is a peptide that binds to A1, L1 is a linking moiety that connects A1 to P1 and is a substrate for a tumor-specific protease, H1 is a half-life extending molecule, and A2 is a second antigen recognition molecule that binds to an epidermal growth factor receptor (EGFR).

[0187] In some embodiments, disclosed herein are isolated and recombinant nucleic acid molecules that encode a polypeptide or polypeptide complex set forth in Formula Ia below.

[0188] [ka]

[0189] In some embodiments, an isolated and recombinant nucleic acid molecule encoding a polypeptide or polypeptide complex set forth in Formula II below:

[0190] [ka] In the formula, L 1a When uncut, P1a a linking moiety cleaved by a tumor-specific protease connecting the P to a first antigen recognition molecule that binds to an effector cell antigen, and the first antigen recognition molecule is bound to a second antigen recognition molecule that binds to an epidermal growth factor receptor (EGFR), 1a L 1a comprises a peptide that binds to the first antigen recognition molecule when uncleaved, and 1a Disclosed herein are isolated recombinant nucleic acid molecules, including half-life extending molecules.

[0191] In some embodiments, an isolated recombinant nucleic acid molecule encoding a polypeptide or polypeptide complex comprising Formula II:

[0192] [ka] In the formula, L 1a When uncut, P 1a a linking moiety cleaved by a tumor-specific protease connecting the P to a first antigen recognition molecule that binds to an effector cell antigen, and the first antigen recognition molecule is bound to a second antigen recognition molecule that binds to an epidermal growth factor receptor (EGFR), 1a L 1a comprises a peptide that binds to the first antigen recognition molecule when uncleaved, and 1a Disclosed herein are isolated recombinant nucleic acid molecules, including half-life extending molecules.

[0193] In some embodiments, an isolated and recombinant nucleic acid molecule encoding a polypeptide or polypeptide complex set forth in Formula II below:

[0194] [ka] In the formula, L 1a When uncut, P 1aa linking moiety cleaved by a tumor-specific protease that connects P to a first antigen recognition molecule that binds to an effector cell antigen, and the first antigen recognition molecule is bound to a second antigen recognition molecule that binds to epidermal growth factor receptor (EGFR), 1a L 1a is a peptide that binds to the first antigen recognition molecule when uncleaved, and H 1a Disclosed herein are isolated recombinant nucleic acid molecules that are half-life extending molecules.

[0195] In some embodiments, an isolated recombinant nucleic acid molecule encoding a polypeptide or polypeptide complex comprising Formula II:

[0196] [ka] In the formula, L 1a When uncut, P 1a a linking moiety cleaved by a tumor-specific protease that connects P to a first antigen recognition molecule that binds to an effector cell antigen, and the first antigen recognition molecule is bound to a second antigen recognition molecule that binds to epidermal growth factor receptor (EGFR), 1a L 1a is a peptide that binds to the first antigen recognition molecule when uncleaved, and H 1a Disclosed herein are isolated recombinant nucleic acid molecules that are half-life extending molecules.

[0197] In some embodiments, an isolated nucleic acid molecule encoding a polypeptide or polypeptide complex comprising the structural arrangement set forth in Configuration 1 below,

[0198] [ka] Disclosed herein is an isolated nucleic acid molecule, wherein the isolated polypeptide or polypeptide complex comprises a single-chain variable fragment (scFv) comprising a light chain variable domain and a heavy chain variable domain, wherein the scFv is linked to a peptide (P1) that impairs binding of the scFv to an effector cell antigen, and P1 is linked to the N-terminus of the heavy chain variable domain of the scFv by a linking moiety (L1) that is a substrate for a tumor-specific protease, and P1 is further linked to a half-life extension and to a Fab that binds to epidermal growth factor receptor (EGFR), wherein the Fab comprises a Fab light chain polypeptide and a Fab heavy chain polypeptide, and the Fab light chain polypeptide is linked to the C-terminus of the light chain variable domain of the scFv, and the Fab is linked to P2 and L2, wherein P2 comprises the peptide that impairs binding to EGFR, and L2 joins the Fab heavy chain polypeptide to P2 and comprises a linking moiety that is a substrate for a tumor-specific protease.

[0199] In some embodiments, an isolated nucleic acid molecule encoding a polypeptide or polypeptide complex comprising the structural arrangement set forth in Configuration 2 below,

[0200] [ka] Disclosed herein is an isolated nucleic acid molecule, wherein the isolated polypeptide or polypeptide complex comprises a single-chain variable fragment (scFv) comprising a light chain variable domain and a heavy chain variable domain, wherein the scFv is linked to a peptide (P1) that impairs binding of the scFv to an effector cell antigen, and P1 is linked to the N-terminus of the heavy chain variable domain of the scFv by a linking moiety (L1) that is a substrate for a tumor-specific protease, and P1 is further linked to a half-life extension and to a Fab that binds to epidermal growth factor receptor (EGFR), wherein the Fab comprises a Fab light chain polypeptide and a Fab heavy chain polypeptide, and the Fab heavy chain polypeptide is linked to the C-terminus of the light chain variable domain of the scFv, and the Fab is linked to P2 and L2, wherein P2 comprises the peptide that impairs binding to EGFR, and L2 joins the Fab light chain polypeptide to P2 and comprises a linking moiety that is a substrate for a tumor-specific protease.

[0201] Pharmaceutical Composition Disclosed herein, in some embodiments, is a pharmaceutical composition comprising (a) a polypeptide or polypeptide complex disclosed herein and (b) a pharmaceutically acceptable excipient.

[0202] In some embodiments, the pharmaceutical composition comprises (a) a polypeptide or polypeptide complex according to Formula I,

[0203] [ka] wherein A1 comprises a first antigen recognition molecule that binds to an effector cell antigen, P1 comprises a peptide that binds to A1, L1 comprises a linking moiety that joins 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 recognition molecule that binds to epidermal growth factor receptor (EGFR), and (b) a pharmaceutically acceptable excipient.

[0204] In some embodiments, the pharmaceutical composition comprises (a) a polypeptide or polypeptide complex according to Formula I,

[0205] [ka] wherein A1 is a first antigen-recognition molecule that binds to an effector cell antigen, P1 is a peptide that binds to A1, L1 is a linking moiety that connects A1 to P1 and is a substrate for a tumor-specific protease, H1 is a half-life extending molecule, and A2 is a second antigen-recognition molecule that binds to epidermal growth factor receptor (EGFR), and (b) a pharmaceutically acceptable excipient.

[0206] In some embodiments, the pharmaceutical composition comprises: (a) a polypeptide or polypeptide complex comprising Formula I:

[0207] [ka] wherein A1 comprises a first antigen recognition molecule that binds to an effector cell antigen, P1 comprises a peptide that binds to A1, L1 comprises a linking moiety that joins 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 recognition molecule that binds to epidermal growth factor receptor (EGFR), and (b) a pharmaceutically acceptable excipient.

[0208] In some embodiments, the pharmaceutical composition comprises: (a) a polypeptide or polypeptide complex comprising Formula I:

[0209] [ka] wherein A1 is a first antigen-recognition molecule that binds to an effector cell antigen, P1 is a peptide that binds to A1, L1 is a linking moiety that connects A1 to P1 and is a substrate for a tumor-specific protease, H1 is a half-life extending molecule, and A2 is a second antigen-recognition molecule that binds to epidermal growth factor receptor (EGFR), and (b) a pharmaceutically acceptable excipient.

[0210] In some embodiments, the pharmaceutical composition comprises: (a) a polypeptide or polypeptide complex described in Formula Ia below:

[0211] [ka] and (b) a pharmaceutically acceptable excipient.

[0212] In some embodiments, the pharmaceutical composition comprises (a) a polypeptide or polypeptide complex according to Formula II below:

[0213] [ka] In the formula, L 1a When uncut, P 1a a linking moiety cleaved by a tumor-specific protease connecting the P to a first antigen recognition molecule that binds to an effector cell antigen, and the first antigen recognition molecule is bound to a second antigen recognition molecule that binds to an epidermal growth factor receptor (EGFR), 1a L 1a comprises a peptide that binds to the first antigen recognition molecule when uncleaved, and 1a comprises (a) a polypeptide or polypeptide complex comprising a half-life extending molecule, and (b) a pharmaceutically acceptable excipient.

[0214] In some embodiments, the pharmaceutical composition comprises: (a) a polypeptide or polypeptide complex comprising Formula II:

[0215] [ka] In the formula, L 1a When uncut, P 1a a linking moiety cleaved by a tumor-specific protease connecting the P to a first antigen recognition molecule that binds to an effector cell antigen, and the first antigen recognition molecule is bound to a second antigen recognition molecule that binds to an epidermal growth factor receptor (EGFR), 1a L 1a comprises a peptide that binds to the first antigen recognition molecule when uncleaved, and 1a comprises (a) a polypeptide or polypeptide complex comprising a half-life extending molecule, and (b) a pharmaceutically acceptable excipient.

[0216] In some embodiments, the pharmaceutical composition comprises (a) a polypeptide or polypeptide complex according to Formula II below:

[0217] [ka] In the formula, L 1a When uncut, P 1a a linking moiety cleaved by a tumor-specific protease that connects P to a first antigen recognition molecule that binds to an effector cell antigen, and the first antigen recognition molecule is bound to a second antigen recognition molecule that binds to epidermal growth factor receptor (EGFR), 1a L 1a is a peptide that binds to the first antigen recognition molecule when uncleaved, and H 1a comprises a polypeptide or polypeptide conjugate that is a half-life extending molecule, and (b) a pharmaceutically acceptable excipient.

[0218] In some embodiments, the pharmaceutical composition comprises: (a) a polypeptide or polypeptide complex comprising Formula II:

[0219] [ka] In the formula, L 1a When uncut, P 1a a linking moiety cleaved by a tumor-specific protease that connects the P to a first antigen recognition molecule that binds to an effector cell antigen, and the first antigen recognition molecule is bound to a second antigen recognition molecule that binds to an epidermal growth factor receptor (EGFR), 1a L 1a is a peptide that binds to the first antigen recognition molecule when uncleaved, and H 1a comprises a polypeptide or polypeptide conjugate that is a half-life extending molecule, and (b) a pharmaceutically acceptable excipient.

[0220] In some embodiments, there is provided (a) a polynucleotide or polypeptide complex comprising the structural arrangement set forth in Configuration 1 below,

[0221] [ka] In the configuration, the isolated polypeptide or polypeptide complex comprises a single-chain variable fragment (scFv) comprising a light chain variable domain and a heavy chain variable domain, wherein the scFv is linked to a peptide (P1) that impairs binding of the scFv to an effector cell antigen, and P1 is linked to the N-terminus of the heavy chain variable domain of the scFv by a linking moiety (L1) that is a substrate for a tumor-specific protease, and P1 is further linked to a half-life extension molecule and a Fab that binds to the epidermal growth factor receptor (EGFR), and the Fa Disclosed herein is a pharmaceutical composition comprising: a polynucleotide or polypeptide complex, wherein b comprises a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab light chain polypeptide is linked to the C-terminus of the light chain variable domain of the scFv, and wherein the Fab is linked to P2 and L2, wherein P2 comprises a peptide that impairs binding to EGFR, and L2 links the Fab heavy chain polypeptide to P2 and comprises a linking moiety that is a substrate for a tumor-specific protease; and (b) a pharmaceutically acceptable excipient.

[0222] In some embodiments, there is provided (a) a polynucleotide or polypeptide complex comprising the structural arrangement set forth in Configuration 2 below,

[0223] [ka] In the configuration, the isolated polypeptide or polypeptide complex comprises a single-chain variable fragment (scFv) comprising a light chain variable domain and a heavy chain variable domain, wherein the scFv is linked to a peptide (P1) that impairs binding of the scFv to an effector cell antigen, and P1 is linked to the N-terminus of the heavy chain variable domain of the scFv by a linking moiety (L1) that is a substrate for a tumor-specific protease, and P1 is further linked to a half-life extension molecule and a Fab that binds to the epidermal growth factor receptor (EGFR), and the Fa Disclosed herein is a pharmaceutical composition comprising: a polynucleotide or polypeptide complex, wherein b comprises a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab heavy chain polypeptide is linked to the C-terminus of the light chain variable domain of the scFv, and wherein the Fab is linked to P2 and L2, wherein P2 comprises a peptide that impairs binding to EGFR, and L2 comprises a linking moiety that joins the Fab light chain polypeptide to P2 and is a substrate for a tumor-specific protease; and (b) a pharmaceutically acceptable excipient.

[0224] In some embodiments, the isolated polypeptide or polypeptide complex further comprises a detectable label, a therapeutic agent, or a pharmacokinetic modifying moiety, hi some embodiments, the detectable label comprises a fluorescent label, a radioactive label, an enzyme, a nucleic acid probe, or an imaging agent.

[0225] For administration to a subject, an isolated polypeptide or polypeptide complex as disclosed herein can be provided in a pharmaceutical composition together with one or more pharmaceutically acceptable carriers or excipients. The term "pharmaceutically acceptable carrier" includes, but is not limited to, a carrier that does not interfere with the effectiveness of the biological activity of the component and is not toxic to the patient to whom it is administered. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate-buffered saline, water, emulsions such as oil / water emulsions, various types of wetting agents, sterile solutions, etc. Such carriers can be formulated by conventional methods and administered to a subject in appropriate dosages. Preferably, the compositions are sterilized. These compositions can also contain adjuvants such as preservatives, emulsifiers, and dispersing agents. Prevention of microbial attack can be ensured by including various antibacterial and antifungal agents.

[0226] The pharmaceutical composition may be in any suitable form (depending on the desired method of administration). The pharmaceutical composition may be provided in a unit dosage form, in a sealed container, or as part of a kit. Such a kit may include instructions for use. It may include a plurality of unit dosage forms.

[0227] The pharmaceutical compositions can be adapted for administration by any suitable route, including parenteral (e.g., subcutaneous, intramuscular, or intravenous) routes. Such compositions can be prepared by any method known in the art of pharmacy, for example, by mixing the active ingredient with the carrier or excipient under sterile conditions.

[0228] Doses of the substances of the present disclosure may vary between wide limits, depending on the disease or disorder being treated, the age and condition of the individual being treated, etc., and the physician will ultimately determine the appropriate dose to be used.

[0229] Peptides that impair the binding of anti-EGFR binding domains to EGFR Disclosed herein is a polypeptide or polypeptide complex comprising an anti-EGFR binding domain linked to a peptide that impairs binding of the anti-EGFR binding domain to EGFR, wherein the peptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 26, 71-96, 98-776, or an amino acid sequence having one, two, or three amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 26, 71-96, 98-776.

[0230] In some embodiments, the peptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 26, 71-96, 98-776.

[0231] In some embodiments, the peptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 26, 86-96, or an amino acid sequence having one, two, or three amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 26, 86-96.

[0232] In some embodiments, the peptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 26, 86-96.

[0233] In some embodiments, the peptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 98-776, or an amino acid sequence having one, two, or three amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 98-776.

[0234] In some embodiments, the peptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 98-776.

[0235] In some embodiments, the peptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 99-118, or an amino acid sequence having one, two, or three amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 99-118.

[0236] In some embodiments, the peptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 99-118.

[0237] In some embodiments, the peptide comprises the amino acid sequence set forth in SEQ ID NO:71, or an amino acid sequence having one, two, or three amino acid mutations, substitutions, or deletions relative to SEQ ID NO:71.

[0238] In some embodiments, the peptide comprises the amino acid sequence set forth in SEQ ID NO:71.

[0239] In some embodiments, the peptide comprises the amino acid sequence set forth in SEQ ID NO:26, or an amino acid sequence having one, two, or three amino acid mutations, substitutions, or deletions relative to SEQ ID NO:26.

[0240] In some embodiments, the peptide comprises the amino acid sequence set forth in SEQ ID NO:26.

[0241] In some embodiments, the peptide comprises the amino acid sequence set forth in SEQ ID NO: 115, or an amino acid sequence having one, two, or three amino acid mutations, substitutions, or deletions relative to SEQ ID NO: 115. In some embodiments, the peptide comprises the amino acid sequence set forth in SEQ ID NO: 115.

[0242] In some embodiments, the peptide comprises the amino acid sequence set forth in SEQ ID NO: 116, or an amino acid sequence having one, two, or three amino acid mutations, substitutions, or deletions relative to SEQ ID NO: 116. In some embodiments, the peptide comprises the amino acid sequence set forth in SEQ ID NO: 116.

[0243] Disclosed herein is a polypeptide or polypeptide complex comprising an anti-EGFR binding domain linked to a peptide that impairs binding of the anti-EGFR binding domain to EGFR, wherein the peptide comprises an amino acid sequence set forth as X1-C-X2-X3-X4-X5-D-X6-A-X7-P-X8-C-X9, wherein X1 is selected from P and L, X2 is selected from R, L, T, A, N, I, V, S, H, and P, and X3 is S, P, or C. , F, and Y; X4 is selected from H, L, Q, P, R, F, N; X5 is selected from I, F, Y, H, N, T, S, D, A, L, and V; X6 is selected from T, P, N, L, I, V, S, D, H, A, and Y; X7 is selected from K and Y; X8 is selected from I, P, L, and M; and X9 is selected from A, V, I, T, L, S, D, F, V, and H (SEQ ID NO: 841). In some embodiments, X1 is selected from P and L, X2 is selected from R, L, T, A, and N, X3 is selected from S, P, and F, X4 is selected from H, L, Q, and P, X5 is selected from I, F, Y, H, N, and T, X6 is selected from T, P, N, L, I, and V, X7 is K, X8 is I, and X9 is selected from A, V, I, T, L, and S. In some embodiments, X1 is P, X2 is selected from R, L, and T, X3 is S, X4 is selected from H, L, Q, and P, X5 is selected from I, F, Y, and T, X6 is selected from T, P, N, and V, X7 is K, and X8 is I, and X9 is selected from A, V, and I.

[0244] In some embodiments, the peptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 98-776. In some embodiments, the peptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 99-118. In some embodiments, the peptide comprises the amino acid sequence set forth in SEQ ID NO: 26. In some embodiments, the peptide comprises the amino acid sequence set forth in SEQ ID NO: 115. In some embodiments, the peptide comprises the amino acid sequence set forth in SEQ ID NO: 116.

[0245] In some embodiments, the anti-EGFR binding domain 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 anti-EGFR binding domain comprises heavy chain complementarity determining regions HC-CDR1, HC-CDR2, and HC-CDR3, where HC-CDR1, HC-CDR2, and HC-CDR3 comprise HC-CDR1 of SEQ ID NO: 15, HC-CDR2 of SEQ ID NO: 16, and HC-CDR3 of SEQ ID NO: 17, and the anti-EGFR binding domain comprises light chain complementarity determining regions LC-CDR1, LC-CDR2, and LC-CDR3, where LC-CDR1, LC-CDR2, and LC-CDR3 of the Fab comprise LC-CDR1 of SEQ ID NO: 18, LC-CDR2 of SEQ ID NO: 19, and LC-CDR3 of SEQ ID NO: 20.

[0246] In some embodiments, the antibody or antibody fragment comprises a Fab. In some embodiments, the anti-EGFR binding domain comprises the amino acid sequence set forth in any one of SEQ ID NOs: 21-24.

[0247] In some embodiments, the anti-EGFR binding domain is linked to the peptide by a linking moiety (L1). In some embodiments, L1 is a substrate for a tumor-specific protease. In some embodiments, L1 is attached to the N-terminus of the anti-EGFR binding domain. In some embodiments, L1 is attached to the C-terminus of the anti-EGFR binding domain. In some embodiments, L1 is a peptide sequence having at least 5 to 50 amino acids or less. In some embodiments, L1 is a peptide sequence having at least 10 to 30 amino acids or less. In some embodiments, L1 is a peptide sequence having at least 10 amino acids. In some embodiments, L1 is a peptide sequence having at least 18 amino acids. In some embodiments, L1 is a peptide sequence having at least 26 amino acids. In some embodiments, L1 is a (G2S) nwherein n is an integer from 1 to 3 (SEQ ID NO: 840). In some embodiments, L1 has a formula comprising (G2S) n , (GS) n , (GSGGS) n (SEQ ID NO: 836), (GGGS) n (SEQ ID NO: 837), (GGGGS) n (SEQ ID NO: 838), and (GSSGGS) n (SEQ ID NO: 839), wherein n is an integer of at least 1. In some embodiments, the peptide is uncoupled from the anti-EGFR binding domain when L1 is cleaved by a tumor-specific protease, thereby exposing the anti-EGFR binding domain to EGFR. In some embodiments, the tumor-specific protease is selected from the group consisting of matrix metalloproteases (MMPs), serine proteases, cysteine ​​proteases, threonine proteases, and aspartic acid proteases. In some embodiments, the matrix metalloproteases include MMP2, MMP7, MMP9, MMP13, or MMP14. In some embodiments, the serine proteases include matriptase (MTSP1), urokinase, or hepsin. In some embodiments, L1 comprises an amino acid sequence cleavable by urokinase, matriptase, matrix metalloproteinase, or legumain. In some embodiments, L1 comprises the amino acid sequence set forth in any of SEQ ID NOs: 30 or 31. In some embodiments, L1 comprises the amino acid sequence set forth in SEQ ID NOs: 27-35. In some embodiments, L1 comprises the amino acid sequence of linker 4 (ISSGLLSGRSDAG) (SEQ ID NO: 66), linker 5 (AAGLLAPPGGLSGRSDAG) (SEQ ID NO: 67), linker 6 (SPLGLSGRSDAG) (SEQ ID NO: 68), or linker 7 (LSGRSDAGSPLGLAG) (SEQ ID NO: 69), or an amino acid sequence having one, two, or three amino acid substitutions, additions, or deletions relative to the amino acid sequence of linker 4, linker 5, linker 6, or linker 7.

[0248] In some embodiments, the isolated polypeptide or polypeptide complex further comprises a half-life extender (H1). In some embodiments, the half-life extender is linked to the peptide. In some embodiments, H1 comprises a polymer. In some embodiments, the polymer is polyethylene glycol (PEG). In some embodiments, H1 comprises albumin. In some embodiments, H1 comprises an Fc domain. In some embodiments, the albumin is serum albumin. In some embodiments, the albumin is human serum albumin. In some embodiments, H1 comprises a polypeptide, ligand, or small molecule. In some embodiments, the polypeptide, ligand, or small molecule binds a serum protein or fragment thereof, a circulating immunoglobulin or fragment thereof, or CD35 / CR1. In some embodiments, the serum protein comprises thyroxine-binding protein, transthyretin, 1-acid glycoprotein, transferrin, transferrin receptor or a transferrin-binding portion thereof, fibrinogen, or albumin. In some embodiments, the circulating immunoglobulin molecule comprises IgG1, IgG2, IgG3, IgG4, slgA, IgM, or IgD. In some embodiments, the serum protein is albumin. In some embodiments, the polypeptide is an antibody. In some embodiments, the antibody comprises a single domain antibody, a single chain variable fragment, or a Fab. In some embodiments, the single domain antibody comprises a single domain antibody that binds to albumin. In some embodiments, the single domain antibody is a human antibody or a humanized antibody. In some embodiments, the single domain antibody is 645gH1gL1. In some embodiments, the single domain antibody is 645dsgH5gL4. In some embodiments, the single domain antibody is 23-13-A01-sc02. In some embodiments, the single domain antibody is A10m3 or a fragment thereof. In some embodiments, the single domain antibody is DOM7r-31. In some embodiments, the single domain antibody is DOM7h-11-15.In some embodiments, the single domain antibody is Alb-1, Alb-8, or Alb-23. In some embodiments, the single domain antibody is 10E. In some embodiments, the single domain antibody comprises complementarity determining regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 of the single domain antibody comprise the HC-CDR1 of SEQ ID NO: 36, the HC-CDR2 of SEQ ID NO: 37, and the HC-CDR3 of SEQ ID NO: 38. In some embodiments, the single domain antibody comprises the amino acid sequence set forth in SEQ ID NO: 69. In some embodiments, the single domain antibody comprises complementarity determining regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3 of the single domain antibody comprise the HC-CDR1 of SEQ ID NO: 36, the HC-CDR2 of SEQ ID NO: 37, and the HC-CDR3 of SEQ ID NO: 38. In some embodiments, the single domain antibody comprises the amino acid sequence set forth in SEQ ID NO: 42. In some embodiments, the single domain antibody is SA21. In some embodiments, the isolated polypeptide or polypeptide complex comprises a modified amino acid, a non-natural amino acid, a modified non-natural amino acid, or a combination thereof. In some embodiments, the modified amino acid or modified non-natural amino acid comprises a post-translational modification. In some embodiments, H1 comprises a linking moiety (L3) connecting H1 to the peptide. In some embodiments, L3 is a peptide sequence having at least 5-50 amino acids or less. In some embodiments, L3 is a peptide sequence having at least 10-30 amino acids or less. 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: 836), (GGGS)n (SEQ ID NO: 837), (GGGGS) n (SEQ ID NO: 838), and (GSSGGS) n (SEQ ID NO: 839), wherein n is an integer of at least 1. In some embodiments, L3 comprises the amino acid sequence set forth in SEQ ID NO: 29.

[0249] Treatment method In some embodiments, the polypeptides or polypeptide complexes described herein are used in methods for treating cancer. In some embodiments, the cancer has cells that express EGFR. In some embodiments, the polypeptides or polypeptide complexes described herein are used in methods for treating colorectal cancer (CRC), squamous cell carcinoma of the head and neck (SCCHN), non-small cell lung cancer (NSCLC), prostate cancer, breast cancer, colon cancer, head and neck cancer, esophagogastric cancer, liver cancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, renal cancer, or pancreatic cancer. In some embodiments, the polypeptides or polypeptide complexes described herein are used in methods for treating a subject who is resistant to EGFR inhibitor therapy. In some embodiments, the polypeptides or polypeptide complexes described herein are used in methods for treating a subject who has a KRAS mutation. In some embodiments, the polypeptides or polypeptide complexes described herein are used in methods for treating a subject who is resistant to EGFR inhibitor therapy and has a KRAS mutation.

[0250] In some embodiments, the present disclosure provides a polypeptide or polypeptide complex that comprises a long half-life.In some examples, the half-life of polypeptide or polypeptide complex is at least or about 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 84 hours, 96 hours, 100 hours, 108 hours, 120 hours, 140 hours, 160 hours, 180 hours, 200 hours, or more than 200 hours.In some examples, the half-life of polypeptide or polypeptide complex is at least or about 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 84 hours, 96 hours, 100 hours, 108 hours, 120 hours, 140 hours, 160 hours, 180 hours, 200 hours, or more than 200 hours. In some examples, the half-life of the polypeptide or polypeptide complex ranges from about 12 hours to about 300 hours, from about 20 hours to about 280 hours, from about 40 hours to about 240 hours, from about 60 hours to about 200 hours, or from about 80 hours to about 140 hours.

[0251] In some embodiments, polypeptides or polypeptide complexes are described herein that are administered weekly. In some embodiments, the polypeptides or polypeptide complexes are administered intravenously, intramuscularly, intralesionally, topically, subcutaneously, by infusion, or orally once a week. In some embodiments, the polypeptides or polypeptide complexes are administered weekly by bolus injection. In some embodiments, the polypeptides or polypeptide complexes are administered weekly by continuous infusion. In some embodiments, the isolated polypeptides or polypeptide complexes are administered to a subject once a week as a continuous infusion over a period of 60 minutes or less. In some embodiments, the isolated polypeptides or polypeptide complexes are administered to a subject once a week as a continuous intravenous infusion over a period of 30 minutes or less. In some embodiments, the isolated polypeptides or polypeptide complexes are administered to a subject once a week as a continuous intravenous infusion over a period of at least 10 minutes. In some embodiments, the isolated polypeptides or polypeptide complexes are administered to a subject once a week, and the isolated polypeptides or polypeptide complexes have a half-life of at least 30 hours. In some embodiments, the isolated polypeptide or polypeptide complex is administered to a subject once a week, and the isolated polypeptide or polypeptide complex has a half-life of at least 50 hours. In some embodiments, the isolated polypeptide or polypeptide complex is administered to a subject once a week, and the isolated polypeptide or polypeptide complex has a half-life of at least 60 hours. In some embodiments, the isolated polypeptide or polypeptide complex is administered to a subject once a week, and the isolated polypeptide or polypeptide complex has a half-life of at least 70 hours. In some embodiments, the isolated polypeptide or polypeptide complex is administered to a subject once a week, and the isolated polypeptide or polypeptide complex has a half-life of at least 80 hours. In some embodiments, the isolated polypeptide or polypeptide complex is administered to a subject once a week, and the isolated polypeptide or polypeptide complex has a half-life of at least 90 hours.In some embodiments, the isolated polypeptide or polypeptide complex is administered to a subject once a week, and the isolated polypeptide or polypeptide complex has a half-life of at least 100 hours. In some embodiments, the isolated polypeptide or polypeptide complex is administered to a subject once a week, and the isolated polypeptide or polypeptide complex has a half-life of at least 110 hours. In some embodiments, the isolated polypeptide or polypeptide complex is administered to a subject once a week, and the isolated polypeptide or polypeptide complex has a half-life of at least 115 hours.

[0252] Production of antibodies that bind to EGFR and CD3 In some embodiments, the polypeptides (e.g., antibodies and binding fragments thereof) described herein are produced using any method known in the art to aid in the synthesis of polypeptides (e.g., antibodies), inter alia, by chemical synthesis or by recombinant expression, and are preferably produced by recombinant expression techniques.

[0253] In some examples, antibodies or binding fragments thereof are recombinantly expressed, and nucleic acids encoding the antibodies or binding fragments thereof are assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., 1994, BioTechniques 17:242), which involves synthesizing overlapping oligonucleotides containing portions of the antibody-encoding sequence, annealing and ligating those oligonucleotides, and then amplifying the ligated oligonucleotides by PCR.

[0254] Alternatively, nucleic acid molecules encoding antibodies are optionally generated from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from any tissue or cell that expresses immunoglobulins) by PCR amplification using synthetic primers capable of hybridizing to the 3' and 5' ends of the sequence, or by cloning using oligonucleotide probes specific for the particular gene sequence.

[0255] In some examples, the antibody or binding fragment thereof is optionally produced by immunizing an animal such as a mouse to produce polyclonal antibodies, or more preferably, by producing monoclonal antibodies, e.g., as described by Kohler and Milstein (1975, Nature 256:495-497), or 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 the antibody are optionally obtained by screening Fab expression libraries for clones of FAb fragments that bind to a specific antigen (e.g., as described in Huse et al., 1989, Science 246:1275-1281) or by screening antibody libraries (see Clackson et al., 1991, Nature 352:624; Hane et al., 1997 Proc. Natl. Acad. Sci. USA 94:4937).

[0256] In some embodiments, techniques developed for producing "chimeric antibodies" by splicing genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a mouse antibody molecule of appropriate biological activity (Morrison et al., 1984, Proc. Natl. Acad. Sci. 81:851-855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454) are used. Chimeric antibodies are molecules in which different portions are derived from different animal species, such as those having a variable region derived from a mouse monoclonal antibody and a human immunoglobulin constant region.

[0257] In some embodiments, techniques described for the production of single-chain antibodies (U.S. Pat. No. 4,694,778; Bird, 1988, Science 242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989, Nature 334:544-54) are adapted to produce single-chain antibodies. Single-chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single-chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli are also optionally used (Skerra et al., 1988, Science 242:1038-1041).

[0258] In some embodiments, an expression vector containing an antibody nucleotide sequence or the antibody nucleotide sequence is introduced into host cells by conventional techniques (e.g., electroporation, liposome transfection, and calcium phosphate precipitation), and the transfected cells are then cultured by conventional techniques to produce the antibody. In certain embodiments, antibody expression is regulated by a constitutive, inducible, or tissue-specific promoter.

[0259] In some embodiments, various host-expression vector systems are utilized to express the antibodies or binding fragments thereof described herein. Such host-expression systems represent not only the vehicle in which the antibody coding sequence is produced and subsequently purified, but also cells that, when transformed or transfected with the appropriate nucleotide coding sequence, express the antibody or binding fragment thereof in situ. These include, but are not limited to, microorganisms such as bacteria (e.g., Escherichia coli and Bacillus subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the antibody or its binding fragment coding sequence; yeast (e.g., Saccharomyces pichia) transformed with recombinant yeast expression vectors containing the antibody or its binding fragment coding sequence; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing the antibody or its binding fragment coding sequence; plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV)) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the antibody or its binding fragment coding sequence; or mammalian cell systems (e.g., COS, CHO, BH, 293, 293T, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter).

[0260] For long-term, high-yield production of recombinant proteins, stable expression is preferred. In some instances, cell lines that stably express antibodies are optionally engineered. Rather than using expression vectors containing viral origins of replication, host cells are transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.) and a selectable marker. After introduction of the exogenous DNA, engineered cells are grown in rich medium for 1-2 days and then switched to selective medium. The selectable marker on the recombinant plasmid confers resistance to selection, allowing cells to stably integrate the plasmid into their chromosomes and grow to form foci, which are cloned and expanded into cell lines. This method can be advantageously used to engineer cell lines that express antibodies or their binding fragments.

[0261] In some examples, a number of selection systems are used, including, but not limited to, herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 192, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes utilized in tk cells, hgprt cells, or aprt cells, respectively. Similarly, antimetabolite resistance has been used as a selection criterion for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Proc. Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); and the aminoglycoside G-418 (Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May 1993, TIB TECH 11(5):155-215), and neo, which confers resistance to hygromycin (Santerre et al., 1984, Gene 30:147).Methods generally known in the art of recombinant DNA technology that can be used are described in Ausubel et al. (eds., 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY; and in Chapters 12 and 13, Dracopoli et al. (eds), 1994, Current Protocols in Human Genetics, John Wiley & Sons, NY.; Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1).

[0262] In some instances, antibody expression levels are increased by vector amplification (for a review, see Bebbington and Hentschel, *The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning*, Vol. 3 (Academic Press, New York, 1987)). If the marker in the antibody expression vector system is amplifiable, increasing the level of inhibitor present in the host cell culture will increase the number of copies of the marker gene. Because the amplified region is related to the antibody nucleotide sequence, antibody production will also increase (Crouse et al., 1983, Mol. Cell Biol. 3:257).

[0263] In some examples, any method for purification of antibodies known in the art is used, for example, by chromatography (e.g., ion exchange, affinity, especially affinity to a specific antigen followed by Protein A, and sizing column chromatography), centrifugation, differential solubility, or other standard techniques for purification of proteins.

[0264] Expression vector In some embodiments, the vector comprises any suitable vector derived from a eukaryotic or prokaryotic source. In some cases, the vector is obtained from a bacterial (e.g., E. coli), insect, yeast (e.g., Pichia pastoris), algae, or mammalian source. Exemplary bacterial vectors include pACYC177, pASK75, pBAD vector system, pBADM vector system, pET vector system, pETM vector system, pGEX vector system, pHAT, pHAT2, pMal-c2, pMal-p2, pQE vector system, pRSET A, pRSET B, pRSET C, pTrcHis2 system, pZA31-Luc, pZE21-MCS-1, pFLAG ATS, pFLAG CTS, pFLAG MAC, pFLAG Shift-12c, pTAC-MAT-1, pFLAG CTC, or pTAC-MAT-2.

[0265] Exemplary insect vectors include pFastBac1, pFastBac DUAL, pFastBac ET, pFastBac HTa, pFastBac HTb, pFastBac HTc, pFastBac M30a, pFastBact M30b, pFastBac, M30c, pVL1392, pVL1393, pVL1393 M10, pVL1393 M11, pVL1393 M12, a FLAG vector such as pPolh-FLAG1 or pPolh-MAT 2, or a MAT vector such as pPolh-MAT1, or pPolh-MAT2.

[0266] In some cases, the yeast vector comprises a Gateway® pDEST™ 14 vector, a Gateway® pDEST™ 15 vector, a Gateway® pDEST™ 17 vector, a Gateway® pDEST™ 24 vector, a Gateway® pYES-DEST52 vector, a pBAD-DEST49 Gateway® destination vector, a pAO815 Pichia vector, a pFLD1 Pichi pastoris vector, a pGAPZA, B, and C Pichia pastoris vector, a pPIC3.5K Pichia vector, a pPIC6 A, B, and C Pichia vector, a pPIC9K Pichia vector, pTEF1 / Zeo, a pYES2 yeast vector, a pYES2 / CT yeast vector, a pYES2 / NT A, B, and C yeast vector, or a pYES3 / CT yeast vector.

[0267] Exemplary algal vectors include the pChlamy-4 vector or the MCS vector.

[0268] Examples of mammalian vectors include transient expression vectors or stable expression vectors. Mammalian transient expression vectors can include pRK5, p3xFLAG-CMV 8, pFLAG-Myc-CMV 19, pFLAG-Myc-CMV 23, pFLAG-CMV 2, pFLAG-CMV 6a,b,c, pFLAG-CMV 5.1, pFLAG-CMV 5a,b,c, p3xFLAG-CMV 7.1, pFLAG-CMV 20, p3xFLAG-Myc-CMV 24, pCMV-FLAG-MAT1, pCMV-FLAG-MAT2, pBICEP-CMV 3, or pBICEP-CMV 4. Mammalian stable expression vectors can include pFLAG-CMV 3, p3xFLAG-CMV 9, p3xFLAG-CMV 13, pFLAG-Myc-CMV 21, p3xFLAG-Myc-CMV 25, pFLAG-CMV 4, p3xFLAG-CMV 10, p3xFLAG-CMV 14, pFLAG-Myc-CMV 22, p3xFLAG-Myc-CMV 26, pBICEP-CMV 1, or pBICEP-CMV 2.

[0269] In some instances, cell-free systems are mixtures of cytoplasmic and / or nuclear components from cells and are used for in vitro nucleic acid synthesis. In some cases, cell-free systems utilize either prokaryotic or eukaryotic components. Often, nucleic acid synthesis is achieved in cell-free systems based on, for example, Drosophila cells, Xenopus eggs, or HeLa cells. Exemplary cell-free systems include, but are not limited to, the E. coli S30 extract system, the E. coli T7 S30 system, or PURExpress®.

[0270] host cell In some embodiments, the host cell includes any suitable cell, for example, a naturally occurring cell or a genetically modified cell. 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. In some cases, the eukaryotic cell includes a fungus (e.g., yeast), an animal cell, or a plant cell. In some cases, the prokaryotic cell is a bacterial cell. Examples of bacterial cells include gram-positive or gram-negative bacteria. Often, gram-negative bacteria are anaerobic, rod-shaped, or both.

[0271] In some examples, the Gram-positive bacteria include Actinobacteria, Firmicutes, or Tenericutes. In some cases, the Gram-negative bacteria include Aquificae, Deinococcus-Thermus, Fibrobacteres-Chlorobi / Bacteroidetes (FCB group), Fusobacteria, Gemmatimonadetes, Nitrospirae, Planctomycetes-Verrucomicrobia / Chlamydiae (PVC group), Proteobacteria, Spirochaetes, or Synergistetes. The other bacteria can be from the phylum Acidobacteria, Chloroflexi, Chrysiogenetes, Cyanobacteria, Deferribacteres, Dictyoglomi, Thermodesulfobacteria, or Thermotogae. The bacterial cell can be Escherichia coli, Clostridium botulinum, or E. coli.

[0272] Exemplary prokaryotic host cells include, but are not limited to, BL21, Mach1™, DH10B™, TOP10, DH5α, DH10Bac™, OmniMax™, MegaX™, DH12S™, INV110, TOP10F′, INVαF, TOP10 / P3, ccdB Survival, PIR1, PIR2, Stbl2™, Stbl3™, or Stbl4™.

[0273] In some instances, animal cells include cells derived from vertebrates or invertebrates. In some instances, animal cells include cells derived from marine invertebrates, fish, insects, amphibians, reptiles, or mammals. In some instances, fungal cells include yeasts such as brewer's yeast, baker's yeast, or wine yeast.

[0274] Fungi include ascomycetes, such as yeasts, molds, filamentous fungi, basidiomycetes, or zygomycetes. In some instances, yeasts include ascomycetes or basidiomycetes. In some instances, ascomycetes include the subphylum Saccharomycotina (true yeasts, e.g., Saccharomyces cerevisiae (baker's yeast)) or the subphylum Taphrinicotina (e.g., Schizosaccharomycetes (fission yeast)). In some instances, basidiomycetes include the subphylum Agaricomycotina (e.g., Tremellomycetes) or the subphylum Pucciniomycotina (e.g., Microbotryomycetes).

[0275] Exemplary yeasts or filamentous fungi include, for example, the genera: Saccharomyces, Schizosaccharomyces, Candida, Pichia, Hansenula, Kluyveromyces, Zygosaccharomyces, Yarrowia, Trichosporon, Rhodosporidi, Aspergillus, Fusarium, or Trichoderma. Exemplary yeasts or filamentous fungi include, for example, the species: Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida utilis, Candida boidini, Candida albicans, Candida tropicalis, Candida stellatoidea, Candida glabrata, Candida krusei, Candida parapsilosis, Candida guilliermondii, Candida viswanathii, Candida viswanathii, Candida lusitaniae, Rhodotorula mucilaginosa, Pichia methanolica, Pichia angusta, Pichia pastoris, Pichia anomala, Hansenula polymorpha, Kluyveromyces lactislactis, Zygosaccharomyces rouxii, Yarrowia lipolytica, Trichosporon pullulans, Rhodosporidium toru-Aspergillus niger, Aspergillus nidulans, Aspergillus awamori, Aspergillus oryzae, Trichoderma reesei, Yarrowia lipolytica, Brettanomyces bruxellensis, Candida stellata stellata, fission yeast, Torulaspora delbrueckii, Zygosaccharomyces bailii, Cryptococcus neoformans, Cryptococcus gattii, or Saccharomyces boulardii.

[0276] Exemplary yeast host cells include, but are not limited to, Pichia pastoris yeast strains such as GS115, KM71H, SMD1168, SMD1168H, and X-33; and Saccharomyces cerevisiae yeast strains such as INVSc1.

[0277] In some embodiments, the additional animal cells include cells obtained from a mollusk, arthropod, annelid, or sponge. In some embodiments, the additional animal cells are mammalian cells, e.g., from a primate, ape, horse, cow, pig, dog, cat, or rodent. In some cases, the rodent includes a mouse, rat, hamster, gerbil, hamster, chinchilla, fancy rat, or guinea pig.

[0278] Exemplary mammalian host cells include, but are not limited to, 293A cell line, 293FT cell line, 293F cells, 293 H cells, CHO DG44 cells, CHO-S cells, CHO-K1 cells, FUT8 KO CHOK1, Expi293F™ cells, Flp-In™ T-REx™ 293 cell line, Flp-In™-293 cell line, Flp-In™-3T3 cell line, Flp-In™-BHK cell line, Flp-In™-CHO cell line, Flp-In™-CV-1 cell line, Flp-In™-Jurkat cell line, FreeStyle™ 293-F cells, FreeStyle™ CHO-S cells, GripTite™ 293 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.

[0279] In some instances, the mammalian host cell is a stable cell line or a cell line that has integrated the genetic material of interest into its genome and has the ability to express the product of the genetic material after many generations of cell division. In some instances, the mammalian host cell is a transient cell line or a cell line that has not integrated the genetic material of interest into its genome and does not have the ability to express the product of the genetic material after many generations of cell division.

[0280] Exemplary insect host cells include, but are not limited to, Drosophila S2 cells, Sf9 cells, Sf21 cells, High Five™ cells, and expresSF+® cells.

[0281] In some examples, the plant cells include cells from algae. Exemplary insect cell lines include, but are not limited to, strains from Chlamydomonas reinhardtii 137c or Synechococcus elongatus PPC 7942.

[0282] product In another aspect of the present invention, an article of manufacture containing materials useful for the treatment, prevention, and / or diagnosis of the above-mentioned disorders is provided. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container may be formed from a variety of materials, such as glass or plastic. The container holds a composition effective for treating, preventing, and / or diagnosing a disease, alone or in combination with another composition, and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic needle). At least one active agent in the composition is a bispecific antibody comprising a first antigen-binding site that specifically binds CD3 and a second antigen-binding site that specifically binds EGFR, as defined herein above.

[0283] The label or package insert indicates that the composition is used to treat a selected condition. Additionally, the article of manufacture may include (a) a first container having a composition disposed therein, the composition comprising a bispecific antibody of the invention; and (b) a second container having a composition disposed therein, the composition further comprising a cytotoxic or otherwise therapeutic agent. The article of manufacture in this embodiment of the invention may further include a package insert indicating that the composition can be used to treat a particular condition.

[0284] Alternatively, or additionally, the article of manufacture may further comprise a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution, and may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0285] Specific Definitions The terms used herein are for the purpose of describing particular instances only and are not intended to limit the invention. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms "including," "includes," "having," "has," "with," or variations thereof are used in either the detailed description and / or claims, such terms are intended to be included in a manner similar to the term "comprising."

[0286] The term "antibody" is used in the broadest sense and includes fully assembled antibodies, antibody fragments capable of binding antigen, such as Fab, F(ab'), Fv, single-chain antibodies (scFv), diabodies, antibody chimeras, hybrid antibodies, bispecific antibodies, and the like.

[0287] The term "complementarity-determining region" or "CDR" refers to a segment of an antibody variable region that is structurally complementary to the epitope to which the antibody binds and is more variable than the rest of the variable region. Accordingly, CDRs are often called hypervariable regions. A variable region contains three CDRs. CDR peptides can be obtained by constructing a gene encoding the CDR of an antibody of interest. Such a gene is prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick et al., Methods: A Companion to Methods in Enzymology 2:106 (1991); Courtenay-Luck, "Genetic Manipulation of Monoclonal Antibodies," in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al. (eds.), pages 166-179 (Cambridge University Press 1995); and Ward et al., "Genetic Manipulation and Expression of Antibodies," in Monoclonal Antibodies: Principles and Applications, Birch et al., (eds.), pages 137-185 (Wiley-Liss, Inc. 1995).

[0288] The term "Fab" refers to a protein containing the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab fragments differ from Fab' fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residues of the constant domains bear a free thiol group. Fab' fragments are generated by reduction of the heavy chain disulfide bridges of the F(ab')2 fragment. Other chemical couplings of antibody fragments are also known.

[0289] A "single-chain variable fragment (scFv)" is a fusion protein consisting of the variable regions of an antibody's heavy chain (VH) and light chain (VL) connected by a short linker peptide of 10 to approximately 25 amino acids. The linker is typically rich in glycine for flexibility and serine or threonine for solubility, and can connect the N-terminus of the VH to the C-terminus of the VL, or vice versa. This protein retains the specificity of the original antibody despite the removal of the constant region and the introduction of the linker. scFv antibodies are described, for example, in Houston, J.S., Methods in Enzymol. 203 (1991) 46-96. Additionally, antibody fragments include single-chain polypeptides that have the characteristics of a VH domain, i.e., can assemble with a VL domain, or have the characteristics of a VL domain, i.e., can assemble with a VH domain into a functional antigen-binding site, thereby providing the antigen-binding properties of a full-length antibody.

[0290] While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the present disclosure. It should be understood that various alternatives to the embodiments of the present disclosure described herein may be utilized in practicing the present disclosure. The following claims define the scope of the disclosure, and it is intended that methods and structures within the scope of the claims and their equivalents be covered thereby. DETAILED DESCRIPTION OF THE INVENTION

[0291] Embodiment 1 includes an isolated polypeptide or polypeptide complex set forth in Formula I, A2-A1-L1-P1-H1, where A1 comprises a first antigen recognition molecule that binds to an effector cell antigen, P1 comprises a peptide that binds to A1, L1 comprises a linking moiety 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 recognition molecule that binds to epidermal growth factor receptor (EGFR).

[0292] Embodiment 2 includes the isolated polypeptide or polypeptide complex of embodiment 1, wherein the first antigen recognition molecule comprises an antibody or antibody fragment.

[0293] Embodiment 3 includes the isolated polypeptide or polypeptide complex of embodiment 1, wherein the first antigen recognition molecule comprises a human antibody or a humanized antibody or antibody fragment.

[0294] Embodiment 4 includes the isolated polypeptide or polypeptide complex of any one of Embodiments 1 to 3, wherein L1 is attached to the N-terminus of the first antigen recognition molecule.

[0295] Embodiment 5 includes the isolated polypeptide or polypeptide complex of any one of Embodiments 1 to 3, wherein A2 is attached to the C-terminus of the first antigen recognition molecule.

[0296] Embodiment 6 includes the isolated polypeptide or polypeptide complex of any one of Embodiments 1 to 3, wherein L1 is attached to the C-terminus of the first antigen recognition molecule.

[0297] Embodiment 7 includes the isolated polypeptide or polypeptide complex of any one of Embodiments 1 to 3, wherein A2 is attached to the N-terminus of the first antigen recognition molecule.

[0298] Embodiment 8 includes the isolated polypeptide or polypeptide complex of 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.

[0299] Embodiment 9 includes the isolated polypeptide or polypeptide complex of embodiment 8, wherein A1 is a single chain variable fragment (scFv).

[0300] Embodiment 10 includes the isolated polypeptide or polypeptide complex of embodiment 9, wherein the scFv comprises an scFv heavy chain polypeptide and an scFv light chain polypeptide.

[0301] Embodiment 11 includes the isolated polypeptide or polypeptide complex of embodiment 8, wherein A1 is a single domain antibody.

[0302] Embodiment 12 comprises the isolated polypeptide or polypeptide complex of 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 of a camelid-derived single-domain antibody (VHH).

[0303] Embodiment 13 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1 to 12, wherein A1 comprises an anti-CD3e single chain variable fragment.

[0304] Embodiment 14 comprises the isolated polypeptide or polypeptide complex of any one of Embodiments 1 to 12, wherein A1 comprises an anti-CD3e single chain variable fragment having a KD binding to CD3 on CD3-expressing cells of 1 μM or less.

[0305] Embodiment 15 comprises the isolated polypeptide or polypeptide complex of any one of Embodiments 1 to 14, wherein the effector cell antigen comprises CD3.

[0306] Embodiment 16 includes the isolated polypeptide or polypeptide complex of embodiment 1, wherein A1 comprises a variable light chain and a variable heavy chain, each capable of specifically binding to human CD3.

[0307] Embodiment 17 comprises the isolated polypeptide or polypeptide complex of embodiment 1, wherein A1 is selected from the group consisting of muromonab-CD3 (OKT3), otelixizumab (TRX4), teplizumab (MGA031), visilizumab (Nuvion), SP34, X35, VIT3, BMA030 (BW264 / 56), CLB-T3 / 3, CRIS7, YTH12.5, F111-409, CLB-T3.4.2, TR-66, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII-87, 12F6, T3 / RW2-8C8, T3 / RW2-4B6, OKT3D, M-T301, SMC2, F101.01, UCHT-1, WT-31, 15865, 15865v12, 15865v16, and 15865v19.

[0308] Embodiment 18 includes the isolated polypeptide or polypeptide complex of embodiment 1, wherein the isolated polypeptide or polypeptide complex according to Formula I binds to an effector cell antigen when L1 is cleaved by a tumor-specific protease.

[0309] Embodiment 19 includes the isolated polypeptide or polypeptide complex of embodiment 1, wherein the isolated polypeptide or polypeptide complex according to Formula I binds to an effector cell when L1 is cleaved by a tumor-specific protease and A1 binds to the effector cell.

[0310] Embodiment 20 includes the isolated polypeptide or polypeptide complex of embodiment 19, wherein the effector cell is a T cell.

[0311] Embodiment 21 includes the isolated polypeptide or polypeptide complex of embodiment 1, wherein A1 binds to a polypeptide that is part of a TCR-CD3 complex on an effector cell.

[0312] Embodiment 22 includes the isolated polypeptide or polypeptide complex of embodiment 21, wherein the polypeptide that is part of a TCR-CD3 complex is human CD3ε.

[0313] Embodiment 23 includes the isolated polypeptide or polypeptide complex of embodiment 1, wherein the effector cell antigen comprises CD3, and the scFv comprises CDRs LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, LC-CDR2, and LC-CDR3 of the scFv comprise LC-CDR1 of SEQ ID NO: 1, LC-CDR2 of SEQ ID NO: 2, and LC-CDR3 of SEQ ID NO: 3, and the scFv comprises complementarity determining regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 of the scFv comprise HC-CDR1 of SEQ ID NO: 4, HC-CDR2 of SEQ ID NO: 5, and HC-CDR3 of SEQ ID NO: 6.

[0314] Embodiment 24 includes the isolated polypeptide or polypeptide complex of embodiment 1, wherein the effector cell antigen comprises CD3, A1 comprises CDRs LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, LC-CDR2, and LC-CDR3 of A1 comprise LC-CDR1 of SEQ ID NO: 1, LC-CDR2 of SEQ ID NO: 2, and LC-CDR3 of SEQ ID NO: 3, and A1 comprises complementarity determining regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 of A1 comprise HC-CDR1 of SEQ ID NO: 4, HC-CDR2 of SEQ ID NO: 5, and HC-CDR3 of SEQ ID NO: 6.

[0315] Embodiment 25 includes the isolated polypeptide or polypeptide complex of embodiment 1, wherein the effector cell antigen comprises CD3 and the scFv comprises the amino acid sequence set forth in SEQ ID NO:13.

[0316] Embodiment 26 includes the isolated polypeptide or polypeptide complex of embodiment 1, wherein the effector cell antigen comprises CD3, and the scFv comprises CDRs LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, LC-CDR2, and LC-CDR3 of the scFv comprise LC-CDR1 of SEQ ID NO: 7, LC-CDR2 of SEQ ID NO: 8, and LC-CDR3 of SEQ ID NO: 9, and the scFv comprises complementarity determining regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, HC-CDR2, and HC-CDR3 of the scFv comprise HC-CDR1 of SEQ ID NO: 10, HC-CDR2 of SEQ ID NO: 11, and HC-CDR3 of SEQ ID NO: 12.

[0317] Embodiment 27 includes the isolated polypeptide or polypeptide complex of embodiment 1, wherein the effector cell antigen comprises CD3, A1 comprises CDRs LC-CDR1, LC-CDR2, and LC-CDR3, and the LC-CDR1, LC-CDR2, and LC-CDR3 of the scFv comprise LC-CDR1 of SEQ ID NO: 7, LC-CDR2 of SEQ ID NO: 8, and LC-CDR3 of SEQ ID NO: 9, and A1 comprises complementarity-determining regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3, and the HC-CDR1, HC-CDR2, and HC-CDR3 of A1 comprise HC-CDR1 of SEQ ID NO: 10, HC-CDR2 of SEQ ID NO: 11, and HC-CDR3 of SEQ ID NO: 12.

[0318] Embodiment 28 includes the isolated polypeptide or polypeptide complex of embodiment 1, wherein the effector cell antigen comprises CD3 and the scFv comprises the amino acid sequence set forth in SEQ ID NO:14.

[0319] Embodiment 29 includes the isolated polypeptide or polypeptide complex of any one of Embodiments 1 to 28, wherein the second antigen recognition molecule comprises an antibody or antibody fragment.

[0320] Embodiment 30 includes the isolated polypeptide or polypeptide complex of embodiment 29, wherein the antibody or antibody fragment thereof comprises a single chain variable fragment, a single domain antibody, or a Fab.

[0321] Embodiment 31 comprises the isolated polypeptide or polypeptide complex of embodiment 29, wherein the antibody or antibody fragment thereof 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 of a camelid-derived single-domain antibody (VHH).

[0322] Embodiment 32 includes the isolated polypeptide or polypeptide complex of embodiment 29, wherein the antibody or antibody fragment thereof is humanized or human.

[0323] Embodiment 33 includes the isolated polypeptide or polypeptide complex of embodiment 30, wherein A2 is a Fab.

[0324] Embodiment 34 includes the isolated polypeptide or polypeptide complex of embodiment 33, wherein the Fab comprises (a) a Fab light chain polypeptide and (b) a Fab heavy chain polypeptide.

[0325] Embodiment 35 includes an isolated polypeptide or polypeptide complex according to embodiment 33, wherein the Fab comprises CDRs LC-CDR1, LC-CDR2, and LC-CDR3, and the LC-CDR1, LC-CDR2, and LC-CDR3 of the Fab comprise LC-CDR1 of SEQ ID NO: 15, LC-CDR2 of SEQ ID NO: 16, and LC-CDR3 of SEQ ID NO: 17; and the Fab comprises complementarity-determining regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3, and the HC-CDR1, HC-CDR2, and HC-CDR3 of the Fab comprise HC-CDR1 of SEQ ID NO: 18, HC-CDR2 of SEQ ID NO: 19, and HC-CDR3 of SEQ ID NO: 20.

[0326] Embodiment 36 includes an isolated polypeptide or polypeptide complex according to embodiment 29, wherein A2 comprises CDRs LC-CDR1, LC-CDR2, and LC-CDR3, and the LC-CDR1, LC-CDR2, and LC-CDR3 of A2 comprise LC-CDR1 of SEQ ID NO: 15, LC-CDR2 of SEQ ID NO: 16, and LC-CDR3 of SEQ ID NO: 17, and A2 comprises complementarity determining regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3, and the HC-CDR1, HC-CDR2, and HC-CDR3 of A2 comprise HC-CDR1 of SEQ ID NO: 18, HC-CDR2 of SEQ ID NO: 19, and HC-CDR3 of SEQ ID NO: 20.

[0327] Embodiment 37 comprises the isolated polypeptide or polypeptide complex of embodiment 34, wherein the Fab light chain polypeptide comprises the amino acid sequence set forth in SEQ ID NO:21.

[0328] Embodiment 38 includes the isolated polypeptide or polypeptide complex of embodiment 34, wherein the Fab light chain polypeptide comprises the amino acid sequence set forth in SEQ ID NO:22.

[0329] Embodiment 39 includes the isolated polypeptide or polypeptide complex of embodiment 34, wherein the Fab heavy chain polypeptide comprises the amino acid sequence set forth in SEQ ID NO:23.

[0330] Embodiment 40 comprises the isolated polypeptide or polypeptide complex of embodiment 34, wherein the Fab heavy chain polypeptide comprises the amino acid sequence set forth in SEQ ID NO:24.

[0331] Embodiment 41 comprises the isolated polypeptide or polypeptide complex of embodiment 34, wherein the Fab light chain polypeptide of A2 is linked to the C-terminus of the single chain variable fragment (scFv) of A1.

[0332] Embodiment 42 comprises the isolated polypeptide or polypeptide complex of embodiment 34, wherein the Fab heavy chain polypeptide of A2 is linked to the C-terminus of the single chain variable fragment (scFv) of A1.

[0333] Embodiment 43 comprises the isolated polypeptide or polypeptide complex of embodiment 34, wherein the Fab light chain polypeptide of A2 is linked to the N-terminus of the single chain variable fragment (scFv) of A1.

[0334] Embodiment 44 comprises the isolated polypeptide or polypeptide complex of embodiment 34, wherein the Fab heavy chain polypeptide of A2 is linked to the N-terminus of the single chain variable fragment (scFv) of A1.

[0335] Embodiment 45 comprises the isolated polypeptide or polypeptide complex of embodiment 34, wherein the Fab heavy chain polypeptide of A2 is conjugated to the scFv heavy chain polypeptide of A1.

[0336] Embodiment 46 comprises the isolated polypeptide or polypeptide complex of embodiment 34, wherein the Fab light chain polypeptide of A2 is conjugated to the scFv heavy chain polypeptide of A1.

[0337] Embodiment 47 comprises the isolated polypeptide or polypeptide complex of embodiment 34, wherein the Fab heavy chain polypeptide of A2 is conjugated to the scFv light chain polypeptide of A1.

[0338] Embodiment 48 comprises the isolated polypeptide or polypeptide complex of embodiment 34, wherein the Fab light chain polypeptide of A2 is conjugated to the scFv light chain polypeptide of A1.

[0339] Embodiment 49 includes the isolated polypeptide or polypeptide complex of any one of embodiments 1 to 48, wherein A2 further comprises P2 and L2, wherein P2 comprises a peptide that binds to A2, and L2 comprises a linking moiety that connects A2 to P2 and is a substrate for a tumor-specific protease.

[0340] Embodiment 50 includes the isolated polypeptide or polypeptide complex of embodiment 49, wherein the isolated polypeptide or polypeptide complex is described by formula Ia, P2-L2-A2-A1-L1-P1-H1.

[0341] Embodiment 51 includes the isolated polypeptide or polypeptide complex of embodiment 50, wherein the Fab heavy chain polypeptide of A2 is conjugated to the scFv heavy chain polypeptide of A1, and L2 is conjugated to the Fab light chain polypeptide of A2.

[0342] Embodiment 52 includes the isolated polypeptide or polypeptide complex of embodiment 50, wherein the Fab light chain polypeptide of A2 is conjugated to the scFv heavy chain polypeptide of A1, and L2 is conjugated to the Fab heavy chain polypeptide of A2.

[0343] Embodiment 53 includes the isolated polypeptide or polypeptide complex of embodiment 50, wherein the Fab heavy chain polypeptide of A2 is conjugated to the scFv light chain polypeptide of A1, and L2 is conjugated to the Fab light chain polypeptide of A2.

[0344] Embodiment 54 includes the isolated polypeptide or polypeptide complex of embodiment 50, wherein the Fab light chain polypeptide of A2 is conjugated to the scFv light chain polypeptide of A1, and L2 is conjugated to the Fab heavy chain polypeptide of A2.

[0345] Embodiment 55 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1 to 54, wherein P1 impairs binding of A1 to an effector cell antigen.

[0346] Embodiment 56 includes an isolated polypeptide or polypeptide complex of any one of embodiments 1 to 55, wherein P1 is bound to A1 by ionic interactions, electrostatic interactions, hydrophobic interactions, Pi stacking interactions, and hydrogen bonding interactions, or a combination thereof.

[0347] Embodiment 57 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1 to 56, wherein P1 has less than 70% sequence homology to the effector cell antigen.

[0348] Embodiment 58 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 49 to 57, wherein P2 impairs the binding of A2 to EGFR.

[0349] Embodiment 59 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 49 to 58, wherein P2 is bound to A2 by ionic interactions, electrostatic interactions, hydrophobic interactions, Pi stacking interactions, and hydrogen bonding interactions, or a combination thereof.

[0350] Embodiment 60 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 49 to 59, wherein P2 is bound to A2 at or near the antigen-binding site.

[0351] Embodiment 61 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 49 to 60, wherein P2 has less than 70% sequence homology to EGFR.

[0352] Embodiment 62 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1 to 61, wherein P1 or P2 comprises a peptide sequence at least 10 amino acids in length.

[0353] Embodiment 63 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1 to 62, wherein P1 or P2 comprises a peptide sequence at least 10 amino acids in length and no more than 20 amino acids in length.

[0354] Embodiment 64 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1 to 63, wherein P1 or P2 comprises a peptide sequence at least 16 amino acids in length.

[0355] Embodiment 65 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1 to 61, wherein P1 or P2 comprises a peptide sequence of 40 amino acids or less in length.

[0356] Embodiment 66 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1 to 61, wherein P1 or P2 comprises at least two cysteine ​​amino acid residues.

[0357] Embodiment 67 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1 to 61, wherein P1 or P2 comprises a cyclic or linear peptide.

[0358] Embodiment 68 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1 to 61, wherein P1 or P2 comprises a cyclic peptide.

[0359] Embodiment 69 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1 to 61, wherein P1 or P2 comprises a linear peptide.

[0360] Embodiment 70 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1 to 61, wherein P1 comprises at least two cysteine ​​amino acid residues.

[0361] Embodiment 71 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1 to 61, wherein P1 comprises the amino acid sequence set forth in SEQ ID NO:25.

[0362] Embodiment 72 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1 to 61, wherein P2 comprises the amino acid sequence set forth in SEQ ID NO:26.

[0363] Embodiment 73 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1 to 61, wherein P1 comprises the amino acid sequence set forth in SEQ ID NO:70.

[0364] Embodiment 74 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1 to 61, wherein P2 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 26, 71 to 96, 98 to 776, or an amino acid sequence having one, two, or three amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 26, 71 to 96, 98 to 776.

[0365] Embodiment 75 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1 to 61, wherein P2 comprises the amino acid sequence set forth as X1-C-X2-X3-X4-X5-D-X6-A-X7-P-X8-C-X9, wherein X1 is selected from P and L, X2 is selected from R, L, T, A, N, I, V, S, H, and P, and X3 is selected from S, P, F, and Y. X4 is selected from H, L, Q, P, R, F, N, X5 is selected from I, F, Y, H, N, T, S, D, A, L, and V, X6 is selected from T, P, N, L, I, V, S, D, H, A, and Y, X7 is selected from K and Y, X8 is selected from I, P, L, and M, and X9 is selected from A, V, I, T, L, S, D, F, V, and H (SEQ ID NO: 841).

[0366] Embodiment 76 includes the isolated polypeptide or polypeptide complex of embodiment 75, wherein Xi is selected from P and L, X2 is selected from R, L, T, A, and N, X3 is selected from S, P, and F, X4 is selected from H, L, Q, and P, X5 is selected from I, F, Y, H, N, and T, X6 is selected from T, P, N, L, I, and V, X7 is K, X8 is I, and X9 is selected from A, V, I, T, L, and S.

[0367] Embodiment 77 includes the isolated polypeptide or polypeptide complex of embodiment 76, wherein Xi is P, X2 is selected from R, L, and T, X3 is S, X4 is selected from H, L, Q, and P, X5 is selected from I, F, Y, and T, X6 is selected from T, P, N, and V, X7 is K, and X8 is I, and X9 is selected from A, V, and I.

[0368] Embodiment 78 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1 to 77, wherein P2 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 26, 86-96, or an amino acid sequence having one, two, or three amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 26, 86-96.

[0369] Embodiment 79 includes the isolated polypeptide or polypeptide complex of embodiment 78, wherein P2 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 26, 86-96.

[0370] Embodiment 80 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1 to 77, wherein P2 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 98 to 776, or an amino acid sequence having one, two, or three amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 98 to 776.

[0371] Embodiment 81 comprises the isolated polypeptide or polypeptide complex of embodiment 80, wherein P2 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 98-776.

[0372] Embodiment 82 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1 to 77, wherein P2 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 99 to 118, or an amino acid sequence having one, two, or three amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 99 to 118.

[0373] Embodiment 83 comprises the isolated polypeptide or polypeptide complex of embodiment 82, wherein P2 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 99-118.

[0374] Embodiment 84 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1 to 77, wherein P2 comprises the amino acid sequence set forth in SEQ ID NO:26, or an amino acid sequence having one, two, or three amino acid mutations, substitutions, or deletions relative to SEQ ID NO:26.

[0375] Embodiment 85 comprises the isolated polypeptide or polypeptide complex of embodiment 84, wherein P2 comprises the amino acid sequence set forth in SEQ ID NO:26.

[0376] Embodiment 86 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1 to 85, wherein P1 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 25, 797 to 835, or 843 to 1690, or an amino acid sequence having one, two, or three amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 25, 797 to 835, or 843 to 1690.

[0377] Embodiment 87 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1 to 85, wherein P1 is Z1-Z2-C-Z4-P-Z6-Z7-Z8-Z9-Z 10 -Z 11 -Z 12 -CZ 14 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; and 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 12is selected from F, D, Y, L, I, V, A, N, T, P, S, and H, and Z 14 is selected from D, Y, N, F, I, P, V, A, T, H, L, and S.

[0378] Embodiment 88 includes the isolated polypeptide or polypeptide complex of embodiment 87, wherein 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; and 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 is selected from D, Y, N, F, I, and P.

[0379] Embodiment 89 includes the isolated polypeptide or polypeptide complex of embodiment 88, wherein 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, and Z 10 is selected from S, D, and Y, and Z 11 is selected from I, Y, and F, and Z 12 is selected from F, D, Y, and L, and Z 14 is selected from D, Y, and N.

[0380] Embodiment 90 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1 to 85, wherein P1 is U1-U2-C-U4-P-U6-U7-U8-U9-U 10 -U 11 -U 12 -CU 14wherein 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; and U9 is selected from E, D, Y, V, F, W, P, L, and Q; 10 is selected from S, D, Y, T, I, F, V, N, A, P, L, and H, and U 11 is selected from I, Y, F, V, L, T, N, S, D, A, and H, and U 12 is selected from F, D, Y, L, I, V, A, N, T, P, S, G, and H, and U 14 is selected from D, Y, N, F, I, P, V, A, T, H, L, M, and S.

[0381] Embodiment 91 comprises the isolated polypeptide or polypeptide complex of embodiment 90, wherein 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; and U 10 is selected from S, D, Y, T, and I, and U 11 is selected from I, Y, F, V, L, and T, and U 12 is selected from F, D, Y, L, I, V, A, G, and N, and U 14 is selected from D, Y, N, F, I, M, and P.

[0382] Embodiment 92 comprises the isolated polypeptide or polypeptide complex of embodiment 91, wherein 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 10is selected from S, D, T, and Y, and U 11 is selected from I, Y, V, L, and F, and U 12 is selected from F, D, Y, G, A, and L, and U 14 is selected from D, Y, M, and N.

[0383] Embodiment 93 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1 to 92, wherein P1 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 797 to 823.

[0384] Embodiment 94 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1 to 92, wherein P1 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 25, 824-835, or 843-1690.

[0385] Embodiment 95 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1 to 92, wherein P1 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 824 to 835.

[0386] Embodiment 96 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1 to 92, wherein P1 comprises the amino acid sequence set forth in SEQ ID NO: 810, or an amino acid sequence having one, two, or three amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 810.

[0387] Embodiment 97 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1 to 92, wherein P1 comprises the amino acid sequence set forth in SEQ ID NO: 811, or an amino acid sequence having one, two, or three amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 811.

[0388] Embodiment 98 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1 to 92, wherein P1 comprises the amino acid sequence set forth in SEQ ID NO: 834, or an amino acid sequence having one, two, or three amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 834.

[0389] Embodiment 99 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1 to 92, wherein P1 comprises the amino acid sequence set forth in SEQ ID NO:810.

[0390] Embodiment 100 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1 to 92, wherein P1 comprises the amino acid sequence set forth in SEQ ID NO:811.

[0391] Embodiment 101 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1 to 92, wherein P1 comprises the amino acid sequence set forth in SEQ ID NO:834.

[0392] Embodiment 102 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1 to 101, wherein L1 is attached to the N-terminus of A1.

[0393] Embodiment 103 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1 to 101, wherein L1 is attached to the C-terminus of A1.

[0394] Embodiment 104 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 49 to 101, wherein L2 is attached to the N-terminus of A2.

[0395] Embodiment 105 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 49 to 101, wherein L2 is attached to the C-terminus of A2.

[0396] Embodiment 106 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1 to 105, wherein L1 or L2 is a peptide sequence having at least 5 and no more than 50 amino acids.

[0397] Embodiment 107 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1 to 105, wherein L1 or L2 is a peptide sequence having at least 10 and no more than 30 amino acids.

[0398] Embodiment 108 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1 to 105, wherein L1 or L2 is a peptide sequence having at least 10 amino acids.

[0399] Embodiment 109 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1 to 105, wherein L1 or L2 is a peptide sequence having at least 18 amino acids.

[0400] Embodiment 110 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1 to 105, wherein L1 or L2 is a peptide sequence having at least 26 amino acids.

[0401] Embodiment 111 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1 to 105, wherein L1 or L2 is (G2S) n wherein n is an integer from 1 to 3 (SEQ ID NO: 840).

[0402] Embodiment 112 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1 to 105, wherein L1 is (G2S) n , (GS) n , (GSGGS) n (SEQ ID NO: 836), (GGGS) n (SEQ ID NO: 837), (GGGGS) n(SEQ ID NO: 838), and (GSSGGS) n (SEQ ID NO: 839), wherein n is an integer of at least 1.

[0403] Embodiment 113 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1 to 105, wherein P1 is uncoupled from A1 upon cleavage of L1 by a tumor-specific protease, thereby exposing A1 to an effector cell antigen.

[0404] Embodiment 114 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1 to 105, wherein P2 is uncoupled from A2 when L2 is cleaved by a tumor-specific protease, thereby exposing A2 to EGFR.

[0405] Embodiment 115 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1 to 105, wherein the tumor-specific protease is selected from the group consisting of matrix metalloproteinases (MMPs), serine proteases, cysteine ​​proteases, threonine proteases, and aspartic acid proteases.

[0406] Embodiment 116 comprises the isolated polypeptide or polypeptide complex of embodiment 115, wherein the matrix metalloprotease comprises MMP2, MMP7, MMP9, MMP13, or MMP14.

[0407] Embodiment 117 comprises the isolated polypeptide or polypeptide complex of embodiment 115, wherein the serine protease comprises matriptase (MTSP1), urokinase, or hepsin.

[0408] Embodiment 118 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 1 to 105, wherein L1 or L2 comprises an amino acid sequence cleavable by urokinase, an amino acid sequence cleavable by matriptase, an amino acid sequence cleavable by matrix metalloproteinase, or an amino acid sequence cleavable by legumain.

[0409] Embodiment 119 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1 to 105, wherein L1 or L2 comprises the amino acid sequence set forth in SEQ ID NO: 30 or 31.

[0410] Embodiment 120 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1 to 105, wherein L1 or L2 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 27 to 35.

[0411] Embodiment 121 includes an isolated polypeptide or polypeptide complex of any one of embodiments 1 to 105, wherein L1 or L2 comprises the amino acid sequence of linker 4 (ISSGLLSGRSDAG) (SEQ ID NO: 66), linker 5 (AAGLLAPPGGLSGRSDAG) (SEQ ID NO: 67), linker 6 (SPLGLSGRSDAG) (SEQ ID NO: 68), or linker 7 (LSGRSDAGSPLGLAG) (SEQ ID NO: 69), or an amino acid sequence having one, two, or three amino acid substitutions, additions, or deletions relative to the amino acid sequence of linker 4, linker 5, linker 6, or linker 7.

[0412] Embodiment 122 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1 to 121, wherein H1 comprises a polymer.

[0413] Embodiment 123 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1 to 121, wherein the polymer is polyethylene glycol (PEG).

[0414] Embodiment 124 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1 to 121, wherein H1 comprises albumin.

[0415] Embodiment 125 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1 to 121, wherein H1 comprises an Fc domain.

[0416] Embodiment 126 comprises the isolated polypeptide or polypeptide complex of embodiment 124, wherein the albumin is serum albumin.

[0417] Embodiment 127 comprises the isolated polypeptide or polypeptide complex of embodiment 124, wherein the albumin is human serum albumin.

[0418] Embodiment 128 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1 to 121, wherein H1 comprises a polypeptide, a ligand, or a small molecule.

[0419] Embodiment 129 includes the isolated polypeptide or polypeptide complex of embodiment 128, wherein the polypeptide, ligand, or small molecule binds a serum protein or fragment thereof, a circulating immunoglobulin or fragment thereof, or CD35 / CR1.

[0420] Embodiment 130 includes the isolated polypeptide or polypeptide complex of embodiment 129, wherein the serum protein comprises thyroxine-binding protein, transthyretin, 1-acid glycoprotein, transferrin, transferrin receptor or a transferrin-binding portion thereof, fibrinogen, or albumin.

[0421] Embodiment 131 comprises the isolated polypeptide or polypeptide complex of embodiment 129, wherein the circulating immunoglobulin molecules comprise IgG1, IgG2, IgG3, IgG4, slgA, IgM, or IgD.

[0422] Embodiment 132 comprises the isolated polypeptide or polypeptide complex of embodiment 129, wherein the serum protein is albumin.

[0423] Embodiment 133 includes the isolated polypeptide or polypeptide complex of embodiment 128, wherein the polypeptide is an antibody.

[0424] Embodiment 134 comprises the isolated polypeptide or polypeptide complex of embodiment 133, wherein the antibody comprises a single domain antibody, a single chain variable fragment, or a Fab.

[0425] Embodiment 135 comprises the isolated polypeptide or polypeptide complex of embodiment 134, wherein the single domain antibody comprises a single domain antibody that binds to albumin.

[0426] Embodiment 136 comprises the isolated polypeptide or polypeptide complex of embodiment 134, wherein the single domain antibody is a human antibody or a humanized antibody.

[0427] Embodiment 137 comprises the isolated polypeptide or polypeptide complex of embodiment 134, wherein the single domain antibody is 645gH1gL1.

[0428] Embodiment 138 comprises the isolated polypeptide or polypeptide complex of embodiment 134, wherein the single domain antibody is 645dsgH5gL4.

[0429] Embodiment 139 comprises the isolated polypeptide or polypeptide complex of embodiment 134, wherein the single domain antibody is 23-13-A01-sc02.

[0430] Embodiment 140 comprises the isolated polypeptide or polypeptide complex of embodiment 134, wherein the single domain antibody is A10m3 or a fragment thereof.

[0431] Embodiment 141 comprises the isolated polypeptide or polypeptide complex of embodiment 134, wherein the single domain antibody is DOM7r-31.

[0432] Embodiment 142 comprises the isolated polypeptide or polypeptide complex of embodiment 134, wherein the single domain antibody is DOM7h-11-15.

[0433] Embodiment 143 comprises the isolated polypeptide or polypeptide complex of embodiment 134, wherein the single domain antibody is Alb-1, Alb-8, or Alb-23.

[0434] Embodiment 144 comprises the isolated polypeptide or polypeptide complex of embodiment 134, wherein the single domain antibody is 10E.

[0435] Embodiment 145 includes an isolated polypeptide or polypeptide complex described in embodiment 134, wherein the single domain antibody comprises complementarity determining regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3, and the HC-CDR1, HC-CDR2, and HC-CDR3 of the single domain antibody comprise HC-CDR1 of SEQ ID NO: 36, HC-CDR2 of SEQ ID NO: 37, and HC-CDR3 of SEQ ID NO: 38.

[0436] Embodiment 146 includes an isolated polypeptide or polypeptide complex described in embodiment 134, wherein the single domain antibody comprises complementarity determining regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3, and the HC-CDR1, HC-CDR2, and HC-CDR3 of the single domain antibody comprise HC-CDR1 of SEQ ID NO: 39, HC-CDR2 of SEQ ID NO: 40, and HC-CDR3 of SEQ ID NO: 41.

[0437] Embodiment 147 comprises the isolated polypeptide or polypeptide complex of embodiment 134, wherein the single domain antibody is SA21.

[0438] Embodiment 148 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1 to 147, wherein the isolated polypeptide or polypeptide complex comprises a modified amino acid, a non-natural amino acid, a modified non-natural amino acid, or a combination thereof.

[0439] Embodiment 149 includes the isolated polypeptide or polypeptide complex of embodiment 148, wherein the modified amino acid or modified unnatural amino acid comprises a post-translational modification.

[0440] Embodiment 150 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1 to 149, wherein H1 comprises a linking moiety (L3) connecting H1 to P1.

[0441] Embodiment 151 comprises the isolated polypeptide or polypeptide complex of embodiment 150, wherein L3 is a peptide sequence having at least 5 and no more than 50 amino acids.

[0442] Embodiment 152 comprises the isolated polypeptide or polypeptide complex of embodiment 150, wherein L3 is a peptide sequence having at least 10 and no more than 30 amino acids.

[0443] Embodiment 153 comprises the isolated polypeptide or polypeptide complex of embodiment 150, wherein L3 is a peptide sequence having at least 10 amino acids.

[0444] Embodiment 154 comprises the isolated polypeptide or polypeptide complex of embodiment 150, wherein L3 is a peptide sequence having at least 18 amino acids.

[0445] Embodiment 155 comprises the isolated polypeptide or polypeptide complex of embodiment 150, wherein L3 is a peptide sequence having at least 26 amino acids.

[0446] Embodiment 156 comprises the isolated polypeptide or polypeptide complex of embodiment 150, wherein L3 is (G2S) n , (GS) n , (GSGGS) n (SEQ ID NO: 836), (GGGS) n (SEQ ID NO: 837), (GGGGS) n (SEQ ID NO: 838), and (GSSGGS) n (SEQ ID NO: 839), wherein n is an integer of at least 1.

[0447] Embodiment 157 comprises the isolated polypeptide or polypeptide complex of embodiment 150, wherein L3 comprises the amino acid sequence set forth in SEQ ID NO:29.

[0448] Embodiment 158 ​​comprises the isolated polypeptide or polypeptide complex of embodiment 1, wherein the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NOs: 44-61.

[0449] Embodiment 159 comprises the isolated polypeptide or polypeptide complex of embodiment 1, wherein the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:50.

[0450] Embodiment 160 comprises the isolated polypeptide or polypeptide complex of embodiment 1, wherein the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:51.

[0451] Embodiment 161 comprises the isolated polypeptide or polypeptide complex of embodiment 1, wherein the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:44 and SEQ ID NO:45.

[0452] Embodiment 162 comprises the isolated polypeptide or polypeptide complex of embodiment 1, wherein the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:46 and SEQ ID NO:47.

[0453] Embodiment 163 comprises the isolated polypeptide or polypeptide complex of embodiment 1, wherein the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:48 and SEQ ID NO:49.

[0454] Embodiment 164 comprises the isolated polypeptide or polypeptide complex of embodiment 1, wherein the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:50 and SEQ ID NO:51.

[0455] Embodiment 165 comprises the isolated polypeptide or polypeptide complex of embodiment 1, wherein the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:52 and SEQ ID NO:53.

[0456] Embodiment 166 comprises the isolated polypeptide or polypeptide complex of embodiment 1, wherein the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:54 and SEQ ID NO:55.

[0457] Embodiment 167 comprises the isolated polypeptide or polypeptide complex of embodiment 1, wherein the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:56 and SEQ ID NO:57.

[0458] Embodiment 168 comprises the isolated polypeptide or polypeptide complex of embodiment 1, wherein the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:58 and SEQ ID NO:59.

[0459] Embodiment 169 comprises the isolated polypeptide or polypeptide complex of embodiment 1, wherein the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:60 and SEQ ID NO:61.

[0460] Embodiment 170 includes the isolated polypeptide or polypeptide complex of embodiment 1, wherein the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:62 and SEQ ID NO:63.

[0461] Embodiment 171 comprises the isolated polypeptide or polypeptide complex of embodiment 1, wherein the isolated polypeptide or polypeptide complex comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:64 and SEQ ID NO:65.

[0462] Embodiment 172 includes a pharmaceutical composition comprising (a) an isolated polypeptide or polypeptide complex according to any one of embodiments 1 to 171 and (b) a pharmaceutically acceptable excipient.

[0463] Embodiment 173 comprises an isolated recombinant nucleic acid molecule encoding the isolated polypeptide or polypeptide complex of any one of embodiments 1 to 171.

[0464] Embodiment 174 is a compound of formula II, wherein L 1a -P 1a -H 1a wherein L 1a When uncut, P 1a a linking moiety cleaved by a tumor-specific protease connecting the first antigen recognition molecule to a first antigen recognition molecule that binds to an effector cell antigen, and the first antigen recognition molecule is bound to a second antigen recognition molecule that binds to EGFR, 1a L 1a comprises a peptide that binds to the first antigen recognition molecule when uncleaved, and 1a includes half-life extending molecules.

[0465] Embodiment 175 comprises the isolated polypeptide or polypeptide complex of embodiment 174, 1a L 1a When uncleaved, it impairs binding of the first antigen recognition molecule to the effector cell antigen.

[0466] Embodiment 176 comprises the isolated polypeptide or polypeptide complex of embodiment 174, wherein the first antigen recognition molecule comprises an antibody or antibody fragment.

[0467] Embodiment 177 comprises the isolated polypeptide or polypeptide complex of embodiment 174, wherein the effector cell antigen is an anti-CD3 effector cell antigen.

[0468] Embodiment 178 comprises an isolated polypeptide or polypeptide complex according to embodiment 174, comprising P 1a has less than 70% sequence homology to the effector cell antigen.

[0469] Embodiment 179 comprises an isolated polypeptide or polypeptide complex according to embodiment 174, comprising P 1a comprises a peptide sequence at least 10 amino acids in length.

[0470] Embodiment 180 comprises the isolated polypeptide or polypeptide complex of embodiment 174, 1a comprises a peptide sequence at least 10 amino acids in length and no more than 20 amino acids in length.

[0471] Embodiment 181 comprises the isolated polypeptide or polypeptide complex of embodiment 174, wherein P 1a comprises a peptide sequence at least 16 amino acids in length.

[0472] Embodiment 182 comprises the isolated polypeptide or polypeptide complex of embodiment 174, wherein P 1a comprises a peptide sequence of 40 amino acids or less in length.

[0473] Embodiment 183 comprises an isolated polypeptide or polypeptide complex according to embodiment 174, comprising P 1a contains at least two cysteine ​​amino acid residues.

[0474] Embodiment 184 comprises the isolated polypeptide or polypeptide complex of embodiment 174, wherein P 1a includes cyclic or linear peptides.

[0475] Embodiment 185 comprises the isolated polypeptide or polypeptide complex of embodiment 174, 1a includes cyclic peptides.

[0476] Embodiment 186 comprises the isolated polypeptide or polypeptide complex of embodiment 174, wherein P 1a comprises a linear peptide.

[0477] Embodiment 187 comprises the isolated polypeptide or polypeptide complex of embodiment 174, wherein P 1a comprises an amino acid sequence selected from the group consisting of SEQ ID NO:25.

[0478] Embodiment 188 comprises the isolated polypeptide or polypeptide complex of embodiment 174, wherein P 1a comprises the amino acid sequence set forth in SEQ ID NO:70.

[0479] Embodiment 189 comprises an isolated polypeptide or polypeptide complex according to any one of embodiments 174 to 187, 1a includes polymers.

[0480] Embodiment 190 comprises the isolated polypeptide or polypeptide complex of embodiment 189, wherein the polymer is polyethylene glycol (PEG).

[0481] Embodiment 191 comprises an isolated polypeptide or polypeptide complex according to any one of embodiments 174 to 190, wherein P 1a comprises an amino acid sequence set forth in any one of SEQ ID NOs: 25, 797 to 835, or 843 to 1690, or an amino acid sequence having one, two, or three amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 25, 797 to 835, or 843 to 1690.

[0482] Embodiment 192 comprises an isolated polypeptide or polypeptide complex according to any one of embodiments 174 to 190, wherein P 1a is Z1-Z2-C-Z4-P-Z6-Z7-Z8-Z9-Z 10 -Z 11 -Z 12 -CZ 14 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; and 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 is selected from D, Y, N, F, I, P, V, A, T, H, L, and S.

[0483] Embodiment 193 comprises the isolated polypeptide or polypeptide complex of embodiment 192, wherein 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; and 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 is selected from D, Y, N, F, I, and P.

[0484] Embodiment 194 comprises the isolated polypeptide or polypeptide complex of embodiment 193, wherein 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, and Z 10 is selected from S, D, and Y, and Z 11 is selected from I, Y, and F, and Z 12 is selected from F, D, Y, and L, and Z 14 is selected from D, Y, and N.

[0485] Embodiment 195 comprises an isolated polypeptide or polypeptide complex according to any one of embodiments 174 to 190, wherein P 1a is U1-U2-C-U4-P-U6-U7-U8-U9-U 10 -U 11 -U 12 -CU 14 wherein 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; and U9 is selected from E, D, Y, V, F, W, P, L, and Q; 10 is selected from S, D, Y, T, I, F, V, N, A, P, L, and H, and U 11 is selected from I, Y, F, V, L, T, N, S, D, A, and H, and U 12 is selected from F, D, Y, L, I, V, A, N, T, P, S, G, and H, and U 14 is selected from D, Y, N, F, I, P, V, A, T, H, L, M, and S.

[0486] Embodiment 196 comprises the isolated polypeptide or polypeptide complex of embodiment 195, wherein 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 ...

Claims

1. A polypeptide complex comprising an anti-epidermal growth factor receptor (EGFR) binding domain linked to a peptide that impairs binding of the anti-EGFR binding domain to EGFR, wherein the peptide comprises an amino acid sequence set forth in the formula: X1-C-X2-X3-X4-X5-D-X6-A-X7-P-X8-C-X9 (SEQ ID NO: 841) During the ceremony, X 1 is selected from the group consisting of P and L; X2 is selected from the group consisting of R, L, T, A, N, I, V, S, H, and P; X 3 is selected from the group consisting of S, P, F, and Y; X 4 is selected from the group consisting of H, L, Q, P, R, F, and N; X5 is selected from the group consisting of I, F, Y, H, N, T, S, D, A, L, and V; X6 is selected from the group consisting of T, P, N, L, I, V, S, D, H, A, and Y; X 7 is selected from the group consisting of K and Y; X 8 is selected from the group consisting of I, P, L, and M; X 9 is selected from the group consisting of A, V, I, T, L, S, D, F, V, and H; a polypeptide complex, wherein the anti-EGFR binding domain comprises heavy chain complementarity determining regions HC-CDR1, HC-CDR2 and HC-CDR3, wherein the HC-CDR1, the HC-CDR2 and the HC-CDR3 comprise HC-CDR1 of SEQ ID NO: 18, HC-CDR2 of SEQ ID NO: 19 and HC-CDR3 of SEQ ID NO: 20; and the anti-EGFR binding domain comprises light chain complementarity determining regions LC-CDR1, LC-CDR2 and LC-CDR3, wherein the LC-CDR1, the LC-CDR2 and the LC-CDR3 comprise LC-CDR1 of SEQ ID NO: 15, LC-CDR2 of SEQ ID NO: 16 and LC-CDR3 of SEQ ID NO:

17.

2. X 1 is selected from the group consisting of P and L; X 2 is selected from the group consisting of R, L, T, A, and N; X 3 is selected from the group consisting of S, P, and F; X 4 is selected from the group consisting of H, L, Q, and P; X 5 is selected from the group consisting of I, F, Y, H, N, and T; X 6 is selected from the group consisting of T, P, N, L, I, and V; X 7 is selected from the group consisting of K; X 8 is selected from the group consisting of I; X 9 is selected from the group consisting of A, V, I, T, L, and S; The polypeptide complex of claim 1.

3. X 1 is selected from the group consisting of P; X 2 is selected from the group consisting of R, L, and T; X 3 is selected from the group consisting of S; X 4 is selected from the group consisting of H, L, Q, and P; X 5 is selected from the group consisting of I, F, Y, and T; X 6 is selected from the group consisting of T, P, N, and V; X 7 is selected from the group consisting of K; X 8 is selected from the group consisting of I; X 9 is selected from the group consisting of A, V, and I; The polypeptide complex of claim 2.

4. The polypeptide complex described in claim 1, wherein the peptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 99 to 118.

5. The polypeptide complex described in claim 1, wherein the peptide comprises the amino acid sequence set forth in SEQ ID NO:

115.

6. The polypeptide complex described in claim 1, wherein the peptide comprises the amino acid sequence set forth in SEQ ID NO:

116.

7. The polypeptide complex described in claim 1, wherein the peptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 26, 87, 90, 92, or 96.

8. The polypeptide complex of claim 1, wherein the peptide comprises the amino acid sequence set forth in SEQ ID NO:

26.

9. The polypeptide complex of claim 1, wherein the anti-EGFR binding domain comprises an antibody or an antibody fragment.

10. The polypeptide complex of claim 9, wherein the antibody or antibody fragment comprises a single chain variable fragment, a single domain antibody, Fab, or Fab'.

11. The polypeptide complex of claim 10, wherein the antibody or antibody fragment comprises a Fab.

12. The polypeptide complex of claim 11, wherein the Fab comprises a light chain polypeptide having the amino acid sequence of SEQ ID NO:21, and a heavy chain polypeptide having the amino acid sequence of SEQ ID NO:

24.

13. The polypeptide complex of claim 11, wherein the Fab comprises a light chain polypeptide having the amino acid sequence of SEQ ID NO:21, and a heavy chain polypeptide having the amino acid sequence of SEQ ID NO:

23.

14. The polypeptide complex of claim 11, wherein the Fab comprises a light chain polypeptide having the amino acid sequence of SEQ ID NO:22 and a heavy chain polypeptide having the amino acid sequence of SEQ ID NO:

23.

15. The polypeptide complex of claim 11, wherein the Fab comprises a light chain polypeptide having the amino acid sequence of SEQ ID NO:22 and a heavy chain polypeptide having the amino acid sequence of SEQ ID NO:

24.

16. The polypeptide complex of claim 1, wherein the anti-EGFR binding domain is linked to the peptide via a linking moiety.

17. The polypeptide complex of claim 16, wherein the linking moiety is a substrate for a tumor-specific protease.

18. The polypeptide complex according to claim 1, wherein: P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 (Formula Ia) During the ceremony, A 1 comprises a first antigen recognition molecule that binds to an effector cell antigen comprising differentiation cluster 3 (CD3), A 1 comprises an anti-CD3 binding molecule comprising complementarity determining regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3, said HC-CDR1, said HC-CDR2, and said HC-CDR3 of A 1 comprising HC-CDR1 of SEQ ID NO:4, HC-CDR2 of SEQ ID NO:5, and HC-CDR3 of SEQ ID NO:6, A 1 comprises CDRs LC-CDR1, LC-CDR2, and LC-CDR3 of A 1 comprising LC-CDR1 of SEQ ID NO:1, LC-CDR2 of GTK, and LC-CDR3 of SEQ ID NO:3; P 1 comprises a peptide that binds to A 1 ; L 1 connects A 1 to P 1 and contains a linking moiety that is a substrate for a tumor-specific protease; H 1 comprises a half-life extender; A 2 comprises a second antigen recognition molecule comprising said anti-EGFR binding domain; L 2 connects A 2 to P 2 and contains a linking moiety that is a substrate for a tumor-specific protease; 18. The polypeptide complex of any one of claims 1 to 17, wherein P 2 comprises a peptide that binds to A 2 and impairs binding of the anti-EGFR binding domain to EGFR.

19. The polypeptide complex of claim 18, wherein A 1 comprises an antibody or antibody fragment, including a single chain variable fragment, a single domain antibody, or a Fab fragment.

20. The polypeptide complex of claim 18, wherein A 1 is a single chain variable fragment and A 2 is Fab.

21. The polypeptide complex of claim 20, wherein the single-chain variable fragment comprises the amino acid sequence set forth in SEQ ID NO:

13.

22. The polypeptide complex of claim 20, wherein the single-chain variable fragment comprises the amino acid sequence set forth in SEQ ID NO:

14.

23. The polypeptide complex of claim 18, wherein P 1 is decoupled from A 1 when L 1 is cleaved by said tumor-specific protease, thereby exposing A 1 to said effector cell antigen.

24. P 1 comprises an amino acid sequence described in the following formula: U 1 -U 2 -CU 4 -PU 6 -U 7 -U 8 -U 9 -U 10 -U 11 -U 12 -CU 14 During the ceremony, U 1 is selected from the group consisting of D, Y, F, I, N, V, H, L, A, T, S, and P; U2 is selected from the group consisting of D, Y, L, F, I, N, A, V, H, T, and S; U 4 is selected from the group consisting of G and W; U 6 is selected from the group consisting of E, D, V, and P; U 7 is selected from the group consisting of W, L, F, V, G, M, I, and Y; U 8 is selected from the group consisting of E, D, P, and Q; U 9 ​​is selected from the group consisting of E, D, Y, V, F, W, P, L, and Q; U 10 is selected from the group consisting of S, D, Y, T, I, F, V, N, A, P, L, and H; U 11 is selected from the group consisting of I, Y, F, V, L, T, N, S, D, A, and H; U 12 is selected from the group consisting of F, D, Y, L, I, V, A, N, T, P, S, G, and H; 20. The polypeptide complex of claim 18, wherein U14 is selected from the group consisting of D, Y, N, F, I, P, V, A, T, H, L, M, and S.

25. The polypeptide complex of claim 18, wherein P 1 comprises an amino acid sequence set forth in any one of SEQ ID NOs:25, 811-813, 820-823, 824, and 826-835.

26. The polypeptide complex of claim 18, wherein P 1 comprises the amino acid sequence set forth in SEQ ID NO:

25.

27. The polypeptide complex of claim 18, wherein P 1 comprises the amino acid sequence set forth in SEQ ID NO:

834.

28. The polypeptide complex of claim 18, wherein P 2 is decoupled from A 2 when L 2 is cleaved by the tumor-specific protease, thereby exposing A 2 to EGFR.

29. The polypeptide complex of claim 18, wherein H 1 comprises serum albumin.

30. The polypeptide complex of claim 29, wherein H 1 comprises human serum albumin.

31. The polypeptide complex of claim 18, wherein H 1 comprises a single domain antibody.

32. The polypeptide complex of claim 18, wherein H 1 comprises complementarity determining regions (CDRs) HC-CDR1, HC-CDR2, and HC-CDR3, and the HC-CDR1, HC-CDR2, and HC-CDR3 of H 1 comprise HC-CDR1 of SEQ ID NO:36, HC-CDR2 of SEQ ID NO:37, and HC-CDR3 of SEQ ID NO:

38.

33. The polypeptide complex of claim 18, wherein H 1 comprises the amino acid sequence set forth in SEQ ID NO:

42.

34. The polypeptide complex of claim 18, wherein the tumor-specific protease is selected from the group consisting of matrix metalloproteases (MMPs), serine proteases, cysteine ​​proteases, threonine proteases, and aspartic acid proteases.

35. The polypeptide complex of claim 18, wherein L 1 or L 2 comprises an amino acid sequence set forth in any one of SEQ ID NOs: 27-35, and 66-69.

36. The polypeptide complex of claim 18, wherein L 1 or L 2 comprises the amino acid sequence set forth in SEQ ID NO:30 or SEQ ID NO:

31.

37. The polypeptide complex of claim 18, wherein L 1 or L 2 comprises the amino acid sequence set forth in SEQ ID NO:

69.

38. The polypeptide complex of claim 18, comprising the amino acid sequences set forth in SEQ ID NO:44 and SEQ ID NO:

45.

39. The polypeptide complex of claim 18, comprising the amino acid sequences set forth in SEQ ID NO:50 and SEQ ID NO:

51.

40. The polypeptide complex of claim 18, comprising the amino acid sequences set forth in SEQ ID NO:52 and SEQ ID NO:

53.

41. The polypeptide complex of claim 18, comprising the amino acid sequences set forth in SEQ ID NO:54 and SEQ ID NO:

55.

42. The polypeptide complex of claim 18, comprising the amino acid sequences set forth in SEQ ID NO:56 and SEQ ID NO:57.

Images