Compositions and methods related to tumor activated antibodies targeting EGFR and effector cell antigens

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Apr. 29, 2022, is named 52426-738_601_SL.txt and is 542,564 bytes in size.

SUMMARY

Described herein, in certain embodiments, are polypeptides or polypeptide complexes according to Formula I:

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wherein: A₁comprises a first antigen recognizing molecule that binds to an effector cell antigen; P, comprises a peptide that binds to A₁; L₁comprises a linking moiety that connects A₁to P₁and is a substrate for a tumor specific protease; H₁comprises a half-life extending molecule; and A₂comprises a second antigen recognizing molecule that binds to epidermal growth factor receptor (EGFR). In some embodiments, the first antigen recognizing molecule comprises an antibody or antibody fragment. In some embodiments, first antigen recognizing molecule comprises an antibody or antibody fragment that is human or humanized. In some embodiments, L₁is bound to N-terminus of the first antigen recognizing molecule. In some embodiments, A₂is bound to C-terminus of the first antigen recognizing molecule. In some embodiments, L₁is bound to C-terminus of the first antigen recognizing molecule. In some embodiments, A₂is bound to N-terminus of the first antigen recognizing molecule. In some embodiments, the antibody or antibody fragment comprises a single chain variable fragment, a single domain antibody, or a Fab fragment. In some embodiments, A₁is the single chain variable fragment (scFv). In some embodiments, the scFv comprises a scFv heavy chain polypeptide and a scFv light chain polypeptide. In some embodiments, A₁is the single domain antibody. In some embodiments, the antibody or antibody fragment comprises a single chain variable fragment (scFv), a heavy chain variable domain (VH domain), a light chain variable domain (VL domain), or a variable domain (VHH) of a camelid derived single domain antibody. In some embodiments, A₁comprises an anti-CD3ε single chain variable fragment. In some embodiments, A₁comprises an anti-CD3ε single chain variable fragment that has a K_Dbinding of 1 μM or less to CD3 on CD3 expressing cells. In some embodiments, the effector cell antigen comprises CD3. In some embodiments, A₁comprises a variable light chain and variable heavy chain each of which is capable of specifically binding to human CD3. In some embodiments, A₁comprises complementary determining regions (CDRs) 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. In some embodiments, the isolated polypeptide or polypeptide complex of Formula I binds to an effector cell when L₁is cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex of Formula I binds to an effector cell when L₁is cleaved by the tumor specific protease and A₁binds to the effector cell. In some embodiments, the effector cell is a T cell. In some embodiments, A₁binds to a polypeptide that is part of a TCR-CD3 complex on the effector cell. In some embodiments, the polypeptide that is part of the TCR-CD3 complex is human CD3. In some embodiments, the effector cell antigen comprises CD3, wherein the scFv comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the scFv LC-CDR1: SEQ ID NO: 1, LC-CDR2: SEQ ID NO: 2, and LC-CDR3: SEQ ID NO: 3; and the scFv comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the scFv: HC-CDR1: SEQ ID NO: 4, HC-CDR2: SEQ ID NO: 5, and HC-CDR3: SEQ ID NO: 6. In some embodiments, the effector cell antigen comprises CD3, and the scFv comprises an amino acid sequence according to SEQ ID NO: 13. In some embodiments, the effector cell antigen comprises CD3, wherein the scFv comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the scFv LC-CDR1: SEQ ID NO: 7, LC-CDR2: SEQ ID NO: 8, and LC-CDR3: SEQ ID NO: 9; and the scFv comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the scFv: HC-CDR1: SEQ ID NO: 10, HC-CDR2: SEQ ID NO: 11, and HC-CDR3: SEQ ID NO: 12. In some embodiments, the effector cell antigen comprises CD3, and the scFv comprises an amino acid sequence according to SEQ ID NO: 14. In some embodiments, second antigen recognizing molecule 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, 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), a variable domain (VHH) of a camelid derived single domain antibody. In some embodiments, the antibody or antibody fragment thereof is humanized or human. In some embodiments, A₂is the 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, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the Fab comprise LC-CDR1: SEQ ID NO: 15, LC-CDR2: SEQ ID NO:16, and LC-CDR3: SEQ ID NO: 17; and the Fab comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the Fab comprise: HC-CDR1: SEQ ID NO: 18, HC-CDR2: SEQ ID NO: 19, and HC-CDR3: SEQ ID NO: 20. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 21. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 22. In some embodiments, Fab heavy chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 23. In some embodiments, Fab heavy chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 24. In some embodiments, the Fab light chain polypeptide of A₂is bound to a C-terminus of the single chain variable fragment (scFv) of A₁. In some embodiments, the Fab heavy chain polypeptide of A₂is bound to a C-terminus of the single chain variable fragment (scFv) A₁. In some embodiments, the Fab light chain polypeptide of A₂is bound to a N-terminus of the single chain variable fragment (scFv) of A₁. In some embodiments, the Fab heavy chain polypeptide of A₂is bound to a N-terminus of the single chain variable fragment (scFv) A₁. In some embodiments, the Fab heavy chain polypeptide of A₂is bound to the scFv heavy chain polypeptide of A₁. In some embodiments, the Fab light chain polypeptide of A₂is bound to the scFv heavy chain polypeptide of A₁. In some embodiments, the Fab heavy chain polypeptide of A₂is bound to the scFv light chain polypeptide of A₁. In some embodiments, the Fab light chain polypeptide of A₂is bound to the scFv light chain polypeptide of A₁. In some embodiments, A₂further comprises P₂and L₂, wherein P₂comprises a peptide that binds to A₂; and L₂comprises a linking moiety that connects A₂to P₂and is a substrate for a tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex is according to Formula Ia:

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In some embodiments, the Fab heavy chain polypeptide of A₂is bound to the scFv heavy chain polypeptide of A₁and L₂is bound to the Fab light chain polypeptide of A₂. In some embodiments, the Fab light chain polypeptide of A₂is bound to the scFv heavy chain polypeptide of A₁and L₂is bound to the Fab heavy chain polypeptide of A₂. In some embodiments, the Fab heavy chain polypeptide of A₂is bound to the scFv light chain polypeptide of A₁and L₂is bound to the Fab light chain polypeptide of A₂. In some embodiments, the Fab light chain polypeptide of A₂is bound to the scFv light chain polypeptide of A₁and L₂is bound to the Fab heavy chain polypeptide of A₂. In some embodiments, P₁impairs binding of A₁to the effector cell antigen. In some embodiments, P₁is bound to A₁through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bonding interactions, or a combination thereof. In some embodiments, P₁has less than 70% sequence homology to the effector cell antigen. In some embodiments, P₂impairs binding of A₂to EGFR. In some embodiments, P₂is bound to A₂through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bonding interactions, or a combination thereof. In some embodiments, P₂is bound to A₂at or near an antigen binding site. In some embodiments, P₂has less than 70% sequence homology to EGFR. In some embodiments, P₁or P₂comprises a peptide sequence of at least 10 amino acids in length. In some embodiments, P₁or P₂comprises a peptide sequence of at least 10 amino acids in length and no more than 20 amino acids in length. In some embodiments, P₁or P₂comprises a peptide sequence of at least 16 amino acids in length. In some embodiments, P₁or P₂comprises a peptide sequence of no more than 40 amino acids in length. In some embodiments, P₁or P₂comprises at least two cysteine amino acid residues. In some embodiments, P₁or P₂comprises a cyclic peptide or a linear peptide. In some embodiments, P₁or P₂comprises a cyclic peptide. In some embodiments, P₁or P₂comprises a linear peptide. In some embodiments, P₁comprises at least two cysteine amino acid residues. In some embodiments, P₁comprises an amino acid sequence according to SEQ ID NO: 25. In some embodiments, P₂comprises an amino acid sequence according to SEQ ID NO: 26. In some embodiments, P₂comprises an amino acid sequence according to SEQ ID NO: 115. In some embodiments, P₂comprises an amino acid sequence according to SEQ ID NO: 116. In some embodiments, P₁comprises an amino acid sequence according to SEQ ID NO: 70. In some embodiments, L₁is bound to N-terminus of A₁. In some embodiments, L₁is bound to C-terminus of A₁. In some embodiments, L₂is bound to N-terminus of A₂. In some embodiments, L₂is bound to C-terminus of A₂. In some embodiments, L₁or L₂is a peptide sequence having at least 5 to no more than 50 amino acids. In some embodiments, L₁or L₂is a peptide sequence having at least 10 to no more than 30 amino acids. In some embodiments, L₁or L₂is a peptide sequence having at least 10 amino acids. In some embodiments, L₁or L₂is a peptide sequence having at least 18 amino acids. In some embodiments, L₁or L₂is a peptide sequence having at least 26 amino acids. In some embodiments, L₁or L₂has a formula comprising (G₂S)_n, wherein n is an integer from 1 to 3 (SEQ ID NO: 840). In some embodiments, L₁has a formula selected from the group consisting of (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, P₁becomes unbound from A₁when L₁is cleaved by the tumor specific protease thereby exposing A₁to the effector cell antigen. In some embodiments, P₂becomes unbound from A₂when L₂is cleaved by the tumor specific protease thereby exposing A₂to EGFR. In some embodiments, the tumor specific protease is selected from the group consisting of a matrix metalloprotease (MMP), serine protease, cysteine protease, threonine protease, and aspartic 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, L₁or L₂comprises a urokinase cleavable amino acid sequence, a matriptase cleavable amino acid sequence, matrix metalloprotease cleavable amino acid sequence, or a legumain cleavable amino acid sequence. In some embodiments, L₁or L₂comprises an amino acid sequence according to SEQ ID NO: 30 or 31. In some embodiments, L₁or L₂comprises an amino acid sequence according to any one of SEQ ID NOs: 27-35. In some embodiments, L₁or L₂comprises an 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 that has 1, 2, or 3 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, H₁comprises a polymer. In some embodiments, the polymer is polyethylene glycol (PEG). In some embodiments, H₁comprises albumin. In some embodiments, H₁comprises an Fc domain. In some embodiments, the albumin is serum albumin. In some embodiments, the albumin is human serum albumin. In some embodiments, H₁comprises a polypeptide, a ligand, or a small molecule. In some embodiments, the polypeptide, the ligand or the small molecule binds serum protein or a fragment thereof, a circulating immunoglobulin or a fragment thereof, or CD35/CR1. In some embodiments, the serum protein comprises a thyroxine-binding protein, a transthyretin, a 1-acid glycoprotein, a transferrin, transferrin receptor or a transferrin-binding portion thereof, a fibrinogen, or an 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 or humanized antibody. In some embodiments, the single domain antibody is 645gH1gL1. In some embodiments, the single domain antibody is 645dsgH5gL4. In some embodiments, the single domain antibody is 23-13-A01-sc02. In some embodiments, the single domain antibody is A 10m3 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, the HC-CDR2, and the HC-CDR3 of the single domain antibody comprise: HC-CDR1: SEQ ID NO: 36, HC-CDR2: SEQ ID NO: 37, and HC-CDR3: 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, the HC-CDR2, and the HC-CDR3 of the single domain antibody comprise: HC-CDR1: SEQ ID NO: 39, HC-CDR2: SEQ ID NO: 40, and HC-CDR3: SEQ ID NO: 41. 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, H₁comprises a linking moiety (L₃) that connects H₁to P₁. In some embodiments, L₃is a peptide sequence having at least 5 to no more than 50 amino acids. In some embodiments, L₃is a peptide sequence having at least 10 to no more than 30 amino acids. In some embodiments, L₃is a peptide sequence having at least 10 amino acids. In some embodiments, L₃is a peptide sequence having at least 18 amino acids. In some embodiments, L₃is a peptide sequence having at least 26 amino acids. In some embodiments, L₃has a formula selected from the group consisting of (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, L₃comprises an amino acid sequence according to SEQ ID NO: 29. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence with 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 with at least 95% sequence identity to SEQ ID NO: 50. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 51. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence with 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 with 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 with 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 with 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 with 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 with 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 with 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 with 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 with 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 with 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 with at least 95% sequence identity to SEQ ID NO: 64 and SEQ ID NO: 65.

Described herein, in certain embodiments, are pharmaceutical compositions comprising: (a) the isolated polypeptide or polypeptide complex described herein; and (b) a pharmaceutically acceptable excipient.

Described herein, in certain embodiments, are isolated recombinant nucleic acid molecules encoding the isolated polypeptide or polypeptide complex described herein.

Described herein, in certain embodiments, are polypeptide or polypeptide complexes according to

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wherein: L_1acomprises a tumor specific protease-cleaved linking moiety that when uncleaved connects P_1ato a first antigen recognizing molecule that binds to an effector cell antigen and the first antigen recognizing molecule is connected to a second antigen recognizing molecule that binds to EGFR; P_1acomprises a peptide that binds to the first antigen recognizing molecule when L_1ais uncleaved; and H_1acomprises a half-life extending molecule. In some embodiments, Pa when L_1ais uncleaved impairs binding of the first antigen recognizing molecule to the effector cell antigen. In some embodiments, the first antigen recognizing 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_1ahas less than 70% sequence homology to the effector cell antigen. In some embodiments, P_1acomprises a peptide sequence of at least 10 amino acids in length. In some embodiments, P_1acomprises a peptide sequence of at least 10 amino acids in length and no more than 20 amino acids in length. In some embodiments, P_1acomprises a peptide sequence of at least 16 amino acids in length. In some embodiments, P_1acomprises a peptide sequence of no more than 40 amino acids in length. In some embodiments, P_1acomprises at least two cysteine amino acid residues. In some embodiments, P_1acomprises a cyclic peptide or a linear peptide. In some embodiments, P_1acomprises a cyclic peptide. In some embodiments, P_1acomprises a linear peptide. In some embodiments, P_1acomprises an amino acid sequence selected from the group consisting of SEQ ID NO: 25. In some embodiments, P₁comprises an amino acid sequence according to SEQ ID NO: 70. In some embodiments, H_1acomprises a polymer. In some embodiments, the polymer is polyethylene glycol (PEG). In some embodiments, H_1acomprises albumin. In some embodiments, H_1acomprises an Fc domain. In some embodiments, the albumin is serum albumin. In some embodiments, the albumin is human serum albumin. In some embodiments, H_1acomprises a polypeptide, a ligand, or a small molecule. In some embodiments, the polypeptide, the ligand or the small molecule binds a serum protein or a fragment thereof, a circulating immunoglobulin or a fragment thereof, or CD35/CR1. In some embodiments, the serum protein comprises a thyroxine-binding protein, a transthyretin, a 1-acid glycoprotein, a transferrin, transferrin receptor or a transferrin-binding portion thereof, a fibrinogen, or an 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 antibody comprises a single domain antibody that binds to albumin. In some embodiments, the antibody is a human or humanized antibody. In some embodiments, the single domain antibody is 645gH1gL1. In some embodiments, the single domain antibody is 645dsgH5gL4. In some embodiments, the single domain antibody is 23-13-A01-sc02. In some embodiments, the single domain antibody is A10m3 or a fragment thereof. In some embodiments, the single domain antibody is DOM7r-31. In some embodiments, the single domain antibody is DOM7h-11-15. In some embodiments, the single domain antibody is Alb-1, Alb-8, or Alb-23. In some embodiments, the single domain antibody is 10G. In some embodiments, the single domain antibody comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the single domain antibody comprise: HC-CDR1: SEQ ID NO: 36, HC-CDR2: SEQ ID NO: 37, and HC-CDR3: 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, the HC-CDR2, and the HC-CDR3 of the single domain antibody comprise: HC-CDR1: SEQ ID NO: 39, HC-CDR2: SEQ ID NO: 40, and HC-CDR3: SEQ ID NO: 41. In some embodiments, the single domain antibody is SA21. In some embodiments, H_1acomprises a linking moiety (L_1a) that connects H_1ato P_1a. In some embodiments, L_1ais a peptide sequence having at least 5 to no more than 50 amino acids. In some embodiments, L_1ais a peptide sequence having at least 10 to no more than 30 amino acids. In some embodiments, L_1ais a peptide sequence having at least 10 amino acids. In some embodiments, L_1ais a peptide sequence having at least 18 amino acids. In some embodiments, L_1ais a peptide sequence having at least 26 amino acids. In some embodiments, L_1ahas a formula selected from the group consisting of (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 an amino acid sequence according to SEQ ID NO: 30 or 31. In some embodiments, L₁or L₂comprises an amino acid sequence according to any one of SEQ ID NOs: 27-35. In some embodiments, L₁or L2 comprises an 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 that has 1, 2, or 3 amino acid substitutions, additions, or deletions relative to the amino acid sequence of Linker 4, Linker 5, Linker 6, or Linker 7.

Described herein, in certain embodiments, are polypeptide complexes comprising a structural arrangement according to Configuration 1 (FIG. 1A), 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 P₁is linked to a N-terminus of the heavy chain variable domain of the scFv with a linking moiety (L₁) that is a substrate for a tumor specific protease, and P₁is further linked to a half-life extending molecule; 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, wherein the Fab light chain polypeptide is linked to a C terminus of the light chain variable domain of the scFv, and wherein the Fab is linked to P₂and L₂, wherein P₂comprises a peptide that impairs binding to EGFR; and L₂comprises a linking moiety that connects the Fab heavy chain polypeptide to P₂and is a substrate for a tumor specific protease.

Described herein, in certain embodiments, are polypeptide complexes comprising a structural arrangement according to Configuration 2 (FIG. 1B), 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 (Pt) that impairs binding of the scFv to an effector cell antigen and P₁is linked to a N-terminus of the heavy chain variable domain of the scFv with a linking moiety (L₁) that is a substrate for a tumor specific protease, and P₁is further linked to a half-life extending molecule; 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, wherein the Fab heavy chain polypeptide is linked to a C terminus of the light chain variable domain of the scFv, and wherein the Fab is linked to P₂and L₂, wherein P₂comprises a peptide that impairs binding to EGFR; and L₂comprises a linking moiety that connects the Fab light chain polypeptide to P₂and is a substrate for a tumor specific protease.

INCORPORATION 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 DESCRIPTION OF THE DRAWINGS

The novel features of the 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 of which:

FIGS. 1A-1B illustrate polypeptide complexes of this disclosure.

FIG. 2 illustrates binding to EGFR for several polypeptide complexes of this disclosure measured by ELISA.

FIG. 3 illustrates binding to CD3 for several polypeptide complexes of this disclosure measured by ELISA.

FIG. 4 illustrates polypeptide complexes of this disclosure mediated HCT116 tumor cell killing in the presence of CD8+ T cells.

FIG. 5 illustrates polypeptide complexes of this disclosure mediated A431 tumor cell killing in the presence of CD8+ T cells.

FIG. 6 illustrates kinetic binding and cross reactivity against human and cynomolgus monkey EGFR for polypeptide complexes of this disclosure.

FIG. 7 illustrates binding and cross reactivity against human and cynomolgus EGFR for polypeptide complexes of this disclosure equilibrium.

FIG. 8 illustrates kinetic binding and cross reactivity against human and cynomolgus CD3ε for several polypeptide complexes.

FIG. 9 illustrates equilibrium binding and cross reactivity against human and cynomolgus CD3ε for polypeptide complexes of this disclosure.

FIGS. 10A-10B illustrate pharmacokinetics in cynomolgus monkeys after a single IV bolus injection for polypeptide complexes of this disclosure.

FIGS. 11A-11L illustrate cytokine release in cynomolgus monkeys after single IV bolus for polypeptide complexes of this disclosure.

FIG. 12 illustrates dosing of polypeptide complexes of the disclosure on inflammatory cytokine levels.

FIGS. 13A-13D illustrate serum liver enzymes in cynomolgus monkeys after single IV bolus for polypeptide complexes of this disclosure.

FIG. 14 illustrates binding curves for binding of αEGFR IgG by peptides of the present disclosure as measured by ELISA.

FIG. 15 illustrates inhibition of αEGFR IgG binding to the EGFR antigen by peptides of the present disclosure as measured by ELISA.

FIG. 16 illustrates binding curves for binding of αEGFR Fab by peptides of the present disclosure as measured by ELISA.

FIG. 17 illustrates inhibition of αEGFR Fab binding to the EGFR antigen by peptides of the present disclosure as measured by ELISA.

FIG. 18 illustrates inhibition of αEGFR Fab binding to the EGFR antigen by alanine scanning peptides of peptide-8 as measured by ELISA.

FIG. 19 illustrates inhibition of αEGFR Fab binding to the EGFR antigen by alanine scanning peptides of peptide-8 as measured by ELISA.

FIG. 20 illustrates a consensus sequence of optimized αEGFR Fab peptide-8 generated using WebLogo 3.7.4.

FIG. 21 illustrates αEGFR Fab binding by peptides identified by phage display.

FIG. 22 illustrates αEGFR Fab binding by peptides identified by phage display.

FIG. 23 illustrates inhibition of αEGFR Fab binding to EGFR by peptides identified phage display.

FIG. 24 illustrates inhibition of αEGFR Fab binding to EGFR by peptides identified by phage display.

FIG. 25 shows in vivo tumor growth inhibition with different amounts of PC1 in human PBMC engrafted NCG mice bearing HCT116 xenograft tumors.

FIG. 26 shows in vivo tumor growth inhibition with different amounts of PC5 in human PBMC engrafted NCG mice bearing HCT116 xenograft tumors.

FIG. 27 shows in vivo tumor growth inhibition with different amounts of PC7 in human PBMC engrafted NCG mice bearing HCT116 xenograft tumors.

FIG. 28 shows in vivo tumor growth inhibition with different amounts of PC6 in human PBMC engrafted NCG mice bearing HCT116 xenograft tumors.

FIG. 29 shows PC1 and PC4 binding to human CD3ε as measured by ELISA. PC4 was treated with protease where indicated.

FIG. 30 shows PC1 and PC4 binding to cynomolgus monkey CD3ε as measure by ELISA. PC4 was treated with protease where indicated.

FIG. 31 shows PC1 and PC4 binding to human EGFR as measured by ELISA. PC4 was treated with protease where indicated.

FIG. 32 shows PC1 and PC4 binding to cynomolgus EGFR as measured by ELISA. PC4 was treated with protease where indicated.

FIG. 33 shows PC4 binding to human albumin as measured by ELISA.

FIG. 34 shows PC4 binding to cynomolgus monkey albumin as measured by ELISA.

FIG. 35 shows PC1 and PC4 mediated killing of A549 cells in the presence of peripheral blood mononuclear cells (PBMCs). PC4 was treated with protease where indicated.

FIG. 36 shows IFNγ cytokine levels in EGFR positive HCT116 tumor cells with increasing concentrations of PC4 and PC1. PC4 was protease treated where indicated.

FIG. 37 shows TNF cytokine levels in EGFR positive HCT116 tumor cells with increasing concentrations of PC4 and PC1. PC4 was protease treated where indicated.

FIG. 38 shows IFNγ cytokine levels in EGFR positive A549 tumor cells with increasing concentrations of PC4 and PC1. PC4 was protease treated where indicated.

FIG. 39 shows TNF cytokine levels in EGFR positive A549 tumor cells with increasing concentrations of PC4 and PC1. PC4 was protease treated where indicated.

FIG. 40 shows a schematic representation of a HCT116 mouse tumor model study of the present disclosure.

FIG. 41 shows mean tumor volume versus days of treatment with PC1, PC7, and PC6 in PBMC engrafted mice implanted with HCT116 tumor cells.

FIG. 42 shows tumor volume versus days of treatment with PC5 and vehicle in PBMC engrafted mice implanted with HCT116 tumor cells.

FIG. 43 shows percent survival in PBMC engrafted mice implanted with HCT116 tumor cells with treatment with PC1, PC5, and PC6.

FIG. 44 shows a schematic representation a human colorectal cancer (CRC) organoid study of the present disclosure.

FIG. 45 shows induction of activation markers CD25, CD69, 4-1BB, and Granzyme with PC1 and PC5 in human colorectal cancer tumoroids.

FIG. 46 shows maximum tolerated dose (MTD) for PC1 and no observed adverse effect level (NOAEL) for PC4 in cynomolgus monkeys.

FIG. 47 shows pharmacokinetics in cynomolgus monkeys with different dosing amounts of PC4 after IV bolus injections.

FIG. 48 shows PC10 and PC11 binding to EGFR measured by ELISA.

FIG. 49 shows PC10 and PC11 binding to CD3 measured by ELISA.

FIG. 50 shows a graph of PC10 and PC11 mediated HCT116 tumor cell killing in the presence of CD8+ T cells.

FIGS. 51A-51F illustrate anti-CD3 scFv binding by alanine scanning peptides of anti-CD3 scFv Peptide-A and Peptide-B as measured by ELISA.

FIGS. 52A-52F illustrate 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.

FIGS. 53A-53B illustrate anti-CD3 scFv binding by optimized anti-CD3 scFv Peptide-B sequences as measured by ELISA.

FIGS. 54A-54B illustrate inhibition of anti-CD3 scFv binding to CD3 by optimized anti-CD3 scFv Peptide-B sequences as measured by ELISA.

FIG. 55 illustrates the core sequence motif of optimized anti-CD3 scFv Peptide-B sequences generated using WebLogo 3.7.4.

DETAILED DESCRIPTION

Multispecific antibodies combine the benefits of different binding specificities derived from two or more antibodies into a single composition. Multispecific antibodies for redirecting T cells to cancers have shown promise in both pre-clinical and clinical studies. This approach relies on binding of one antigen interacting portion of the antibody to a tumor-associated antigen or marker, while a second antigen interacting portion can bind to an effector cell antigen on a T cell, such as CD3, which then triggers cytotoxic activity. One such tumor-associated antigen is 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 and is widely expressed throughout the body, resulting in systemic toxicities with EGFR-directed therapies.

T cell engagers (TCEs) therapeutics have several benefits including they are not cell therapies and thus can be offered as off-the-shelf therapies as opposed to chimeric antigen receptor T cell (CAR T cell) therapies. While TCE therapeutics have displayed potent anti-tumor activity in hematological cancers, developing TCEs to treat solid tumors has faced challenges due to the limitations of prior TCE technologies, namely (i) overactivation of the immune system leading to cytokine release syndrome (CRS), (ii) on-target, healthy tissue toxicities and (iii) poor pharmacokinetics (PK) leading to short half-life. CRS arises from the systemic activation of T cells and can result in life-threatening elevations in inflammatory cytokines such as interleukin-6 (IL-6). Severe and acute CRS leading to dose limited toxicities and deaths have been observed upon the dosing of T cell engagers develop using other platforms to treat cancer patients in poor clinical studies. This toxicity restricts the maximum blood levels of T cell engagers that can be safely dosed. T cell engager effectiveness has also been limited because of on-target, healthy tissue toxicity. T cell engagers developed using a platform not designed for tumor-specification activation have resulted in clinicals holds and dose-limiting toxicities resulting from target expression in healthy tissues. T cell engagers have also been limited by short half-lives. T cell engagers quickly reach sub-therapeutic levels after being administered as they are quickly eliminated from the body due to their short exposure half-lives. For this reason, T cell engagers such as blinatumomab are typically administered by a low-dose, continuous infusion pump over a period of weeks to overcome the challenge of a short half-life and to maintain therapeutic levels of drug in the body. A continuous dosing regimen represents a significant burden for patients.

To overcome these challenges associated with the effectiveness of T cell engagers, described herein, are polypeptide or polypeptide complexes that comprise binding domains that selectively bind to an effector cell antigen and EGFR, in which one or more of the binding domains is selectively activated in the tumor microenvironment and the isolated polypeptide or polypeptide complex comprises a half-life extending molecule. Such modifications reduce CRS and on-target healthy tissue toxicity risk, improves stability in the bloodstream and serum half-life prior to activation. The polypeptide or polypeptide complexes described herein have activity at low levels of target expression, and are easily manufactured.

In some embodiments, the polypeptides or polypeptide complexes described herein are used in a method 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 a method 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/rectum 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 a method of treating subjects who are resistant to EGFR inhibitor treatment. In some embodiments, the polypeptides or polypeptide complexes described herein are used in a method of treating subjects who harbor KRAS mutations. In some embodiments, the polypeptides or polypeptide complexes described herein are used in a method of treating subjects who are resistant to EGFR inhibitor treatment and harbor KRAS mutations. In some embodiments, are methods of treating cancer comprising administering to a subject in need thereof an isolated polypeptide or polypeptide complex according to Formula I:

embedded image

wherein: A₁comprises a first antigen recognizing molecule that binds to an effector cell antigen; P₁comprises a peptide that binds to A₁; L₁comprises a linking moiety that connects A₁to P₁and is a substrate for a tumor specific protease; H₁comprises a half-life extending molecule; and A₂comprises a second antigen recognizing molecule that binds to epidermal growth factor receptor (EGFR).

Disclosed herein, in some embodiments, are polypeptides or polypeptide complexes according to Formula I:

embedded image

wherein: A₁is a first antigen recognizing molecule that binds to an effector cell antigen; P₁is a peptide that binds to A₁; L₁is a linking moiety that connects A₁to P₁and is a substrate for a tumor specific protease; H₁is a half-life extending molecule; and A₂is a second antigen recognizing molecule that binds to epidermal growth factor receptor (EGFR).

Disclosed herein, in some embodiments, are polypeptides or polypeptide complexes comprising Formula I:

Disclosed herein, in some embodiments, are polypeptides or polypeptide complexes according to Formula I:

embedded image

wherein: A₁comprises a first antigen recognizing molecule that binds to epidermal growth factor receptor (EGFR); P₁comprises a peptide that binds to A₁; L₁comprises a linking moiety that connects A₁to P₁and is a substrate for a tumor specific protease; H₁comprises a half-life extending molecule; and A₂comprises a second antigen recognizing molecule that binds to an effector cell antigen.

Disclosed herein, in some embodiments, are polypeptides or polypeptide complexes according to Formula I:

embedded image

wherein: A₁is a first antigen recognizing molecule that binds to epidermal growth factor receptor (EGFR); P₁is a peptide that binds to A₁; L₁is a linking moiety that connects A₁to P₁and is a substrate for a tumor specific protease; H₁is a half-life extending molecule; and A₂is a second antigen recognizing molecule that binds to effector cell antigen.

Disclosed herein, in some embodiments, are polypeptides or polypeptide complexes comprising Formula I:

embedded image

wherein: A₁is a first antigen recognizing molecule that binds epidermal growth factor receptor (EGFR); P₁is a peptide that binds to A₁; L₁is a linking moiety that connects A₁to P₁and is a substrate for a tumor specific protease; H₁is a half-life extending molecule; and A₂is a second antigen recognizing molecule that binds to an effector cell antigen.

Disclosed herein, in some embodiments, are polypeptides or polypeptide complexes according to Formula Ia:

embedded image

wherein A₂further comprises P₂and L₂, wherein P₂comprises a peptide that binds to A₂; and L₂comprises a linking moiety that connects A₂to P₂and is a substrate for a tumor specific protease.

Disclosed herein, in some embodiments, are polypeptides or polypeptide complexes according to Formula Ia:

embedded image

wherein A₂further comprises P₂and L₂, wherein P₂is a peptide that binds to A₂; and L₂is a linking moiety that connects A₂to P₂and is a substrate for a tumor specific protease.

Disclosed herein, in some embodiments, are polypeptides or polypeptide complexes comprising Formula Ia:

Disclosed herein, in some embodiments, are polypeptides or polypeptide complexes according to Formula II:

Disclosed herein, in some embodiments, are polypeptides or polypeptide complexes comprising Formula II:

Disclosed herein, in some embodiments, are polypeptides or polypeptide complexes according to Formula II:

embedded image

wherein: L_1ais a tumor specific protease-cleaved linking moiety that when uncleaved connects P_1ato a first antigen recognizing molecule that binds to an effector cell antigen and the first antigen recognizing molecule is connected to a second antigen recognizing molecule that binds to epidermal growth factor receptor (EGFR); P_1ais a peptide that binds to the first antigen recognizing molecule when L_1ais uncleaved; and H_1ais a half-life extending molecule.

Disclosed herein, in some embodiments, are polypeptides or polypeptide complexes comprising Formula II:

Disclosed herein, in some embodiments, are polypeptides or polypeptide complexes, wherein the first antigen recognizing molecule binds to an effector cell antigen and the second antigen recognizing molecule binds to EGFR. In some embodiments, the effector cell antigen comprises CD3. In some embodiments, A₁comprises a first antigen recognizing molecule that binds to an effector cell antigen.

In some embodiments, A₁comprises an antibody or antibody fragment. In some embodiments, A₁comprises an antibody or antibody fragment that is human or humanized. In some embodiments, L₁is bound to N-terminus of the antibody or antibody fragment. In some embodiments, L₁is bound to N-terminus of the antibody or antibody fragment and A₂is bound to the other N-terminus of the antibody or antibody fragment. In some embodiments, A₂is bound to C-terminus of the antibody or antibody fragment. In some embodiments, L₁is bound to C-terminus of the antibody or antibody fragment. In some embodiments, A₂is bound to 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, A₁is the single chain variable fragment (scFv). In some embodiments, the scFv comprises a scFv heavy chain polypeptide and a scFv light chain polypeptide. In some embodiments, A₁is the single domain antibody. In some embodiments, A₁comprises a variable light chain and variable heavy chain each of which is capable of specifically binding to human CD3. In some embodiments, the effector cell antigen comprises CD3. In some embodiments, A₁comprises an anti-CD3ε single chain variable fragment. In some embodiments, A₁comprises an anti-CD3ε single chain variable fragment that has a K_Dbinding of 1 μM or less to CD3 on CD3 expressing cells. In some embodiments, A₁comprises complementary determining regions (CDRs) 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.

In some embodiments, A₁comprises a first antigen recognizing molecule that binds EGFR. In some embodiments, A₁comprises a variable light chain and variable heavy chain each of which is capable of specifically binding to human EGFR.

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

In some embodiments, the scFv heavy chain variable domain comprises at least one, two, or three complementarity determining regions (CDR)s disclosed in Table 1 or a sequence substantially identical thereto (e.g., a sequence that has at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity); and the scFv light chain variable domain comprises at least one, two, or three complementarity determining regions (CDR)s disclosed in Table 1 or a sequence substantially identical thereto (e.g., a sequence that has at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity).

TABLE 1

anti-CD3 amino acid sequences (CDRs as determined by IMGT numbering system)

Amino Acid Sequence
SEQ ID

Construct Description
(N to C)
NO:

SP34.185 CD3: LC: CDR1
TGAVTSGNY
1

SP34.185 CD3: LC: CDR2
GTK
2

SP34.185 CD3: LC: CDR3
VLWYSNRWV
3

SP34.185 CD3: HC: CDR1
GFTFNKYA
4

SP34.185 CD3: HC: CDR2
IRSKYNNYAT
5

SP34.185 CD3: HC: CDR3
VRHGNFGNSYISYWAY
6

SP34.194 CD3: LC: CDR1
TGAVTTSNY
7

SP34.194 CD3: LC: CDR2
GTN
8

SP34.194 CD3: LC: CDR3
ALWYSNLWV
9

SP34.194 CD3: HC: CDR1
GFTFNTYA
10

SP34.194 CD3: HC: CDR2
IRSKYNNYAT
11

SP34.194 CD3: HC: CDR3
VRHGNFGNSYVSWFAY
12

SP34.185 scFv
EVQLVESGGGLVQPGGSLKLS
13

(VH-linker 1-VL)
CAASGFTFNKYAMNWVRQA

PGKGLEWVARIRSKYNNYAT

YYADSVKDRFTISRDDSKNTA

YLQMNNLKTEDTAVYYCVRH

GNFGNSYISYWAY
WGQGTLV

TVSSGGGGSGGGGSGGGGSQT

VVTQEPSLTVSPGGTVTLTCGS

STGAVTSGNYPNWVQQKPGQ

APRGLIGGTKFLAPGTPARFSG

SLLGGKAALTLSGVQPEDEAE

YYCVLWYSNRWVFGGGTKL

TVL

SP34.194 scFv
QTVVTQEPSLTVSPGGTVTLT
14

(VL-linker 1-VH)
CRSSTGAVTTSNYANWVQQK

PGQAPRGLIGGTNKRAPGTPA

RFSGSLLGGKAALTLSGVQPE

DEAEYYCALWYSNLWVFGG

GTKLTVLGGGGSGGGGSGGG

GSEVQLVESGGGLVQPGGSLK

LSCAASGFTFNTYAMNWVRQ

APGKGLEWVARIRSKYNNYA

T
YYADSVKDRFTISRDDSKNT

AYLQMNNLKTEDTAVYYCVR

HGNFGNSYVSWFAY
WGQGT

LVTVSS

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, the LC-CDR2, and the LC-CDR3 of the scFv light chain variable domain comprise: LC-CDR1: SEQ ID NO: 1, LC-CDR2: SEQ ID NO: 2, and LC-CDR3: SEQ ID NO: 3, and wherein the CDRs comprise from 0-2 amino acid modifications in at least one of the LC-CDR1, LC-CDR2, or 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, the LC-CDR2, and the LC-CDR3 of the scFv light chain variable domain comprise: LC-CDR1: SEQ ID NO: 7, LC-CDR2: SEQ ID NO: 8, and LC-CDR3: SEQ ID NO: 9, and wherein the CDRs comprise from 0-2 amino acid modifications in at least one of the LC-CDR1, LC-CDR2, or 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, the HC-CDR2, and the HC-CDR3 of the scFv heavy chain variable domain comprise: HC-CDR1: SEQ ID NO: 4, HC-CDR2: SEQ ID NO: 5, and HC-CDR3: SEQ ID NO: 6, and wherein the CDRs comprise from 0-2 amino acid modifications in at least one of the HC-CDR1, HC-CDR2, or 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, the HC-CDR2, and the HC-CDR3 of the scFv heavy chain variable domain comprise: HC-CDR1: SEQ ID NO: 10, HC-CDR2: SEQ ID NO: 11, and HC-CDR3: SEQ ID NO: 12, and wherein the CDRs comprise from 0-2 amino acid modifications in at least one of the HC-CDR1, HC-CDR2, or HC-CDR3.

In some embodiments, the isolated polypeptide or polypeptide complex of Formula I binds to an effector cell when L₁is cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex of Formula I binds to an effector cell when L₁is cleaved by the tumor specific protease and A₁binds to the effector cell. In some embodiments, the effector cell is a T cell. In some embodiments, A₁binds to a polypeptide that is part of a TCR-CD3 complex on the effector cell. In some embodiments, the polypeptide that is part of the TCR-CD3 complex is human CD3R. In some embodiments, the effector cell antigen comprises CD3, wherein the effector cell antigen comprises CD3, wherein the scFv comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the scFv LC-CDR1: SEQ ID NO: 1, LC-CDR2: SEQ ID NO: 2, and LC-CDR3: SEQ ID NO: 3; and the scFv comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the scFv: HC-CDR1: SEQ ID NO: 4, HC-CDR2: SEQ ID NO: 5, and HC-CDR3: SEQ ID NO: 6. In some embodiments, the effector cell antigen comprises CD3, and the scFv comprises an amino acid sequence according to SEQ ID NO: 13. In some embodiments, the effector cell antigen comprises CD3, wherein the effector cell antigen comprises CD3, wherein the scFv comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the scFv LC-CDR1: SEQ ID NO: 7, LC-CDR2: SEQ ID NO: 8, and LC-CDR3: SEQ ID NO: 9; and the scFv comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the scFv: HC-CDR1: SEQ ID NO: 10, HC-CDR2: SEQ ID NO: 11, and HC-CDR3: SEQ ID NO: 12. In some embodiments, the effector cell antigen comprises CD3, and the scFv comprises an amino acid sequence according to SEQ ID NO: 14.

In some embodiments, the effector cell antigen comprises CD3, and A₁comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of A₁comprise LC-CDR1: SEQ ID NO: 1, LC-CDR2: SEQ ID NO: 2, and LC-CDR3: SEQ ID NO: 3; and A₁comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of A₁comprise: HC-CDR1: SEQ ID NO: 4, HC-CDR2: SEQ ID NO: 5, and HC-CDR3: SEQ ID NO: 6.

In some embodiments, the effector cell antigen comprises CD3, and A₁comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of A₁comprise: LC-CDR1: SEQ ID NO: 7, LC-CDR2: SEQ ID NO: 8, and LC-CDR3: SEQ ID NO: 9; and A₁comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of A₁comprise: HC-CDR1: SEQ ID NO: 10, HC-CDR2: SEQ ID NO: 11, and HC-CDR3: SEQ ID NO: 12.

In some embodiments, A₁comprises an amino acid sequence according to SEQ ID NO: 13. In some embodiments, A₁comprises an amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 13. In some embodiments, A₁comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 13. In some embodiments, A₁comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 13. In some embodiments, A₁comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 13. In some embodiments, A₁comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NO: 13.

In some embodiments, A₁comprises an amino acid sequence according to SEQ ID NO: 14. In some embodiments, A₁comprises an amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 14. In some embodiments, A₁comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 14. In some embodiments, A₁comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 14. In some embodiments, A₁comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 14. In some embodiments, A₁comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NO: 14.

In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen as compared to the binding affinity for the tumor cell antigen of an isolated polypeptide or polypeptide complex that does not have P₁or L₁. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 5× higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex that does not have P₁or L₁. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 8× higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex that does not have P₁or L₁. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 10× higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex that does not have P₁or L₁. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 15× higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex that does not have P₁or L₁. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 20× higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex that does not have P₁or L₁. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 25× higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex that does not have P₁or L₁. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 30× higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex that does not have P₁or L₁. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 35× higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex that does not have P₁or L₁. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 40× higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex that does not have P₁or L₁. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 45× higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex that does not have P₁or L₁. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 50× higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex that does not have P₁or L₁. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 55× higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex that does not have P₁or L₁. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 60× higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex that does not have P₁or L₁. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 65× higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex that does not have P₁or L₁. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 70× higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex that does not have P₁or L₁. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 75× higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex that does not have P₁or L₁. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 80× higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex that does not have P₁or L₁. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 85× higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex that does not have P₁or L₁. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 90× higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex that does not have P₁or L₁. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 95× higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex that does not have P₁or L₁. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 100× higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex that does not have P₁or L₁. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 120× higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex that does not have P₁or L₁. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 1000× higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex that does not have P₁or L₁.

In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen as compared to the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex in which L₁has been cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 5× higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex in which L₁has been cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 8× higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex in which L₁has been cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 10× higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex in which L₁has been cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 15× higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex in which L₁has been cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 20× higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex in which L₁has been cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 25× higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex in which L₁has been cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 30× higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex in which L₁has been cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 35× higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex in which L₁has been cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 40× higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex in which L₁has been cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 45× higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex in which L₁has been cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 50× higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex in which L₁has been cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 55× higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex in which L₁has been cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 60× higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex in which L₁has been cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 65× higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex in which L₁has been cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 70× higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex in which L₁has been cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 75× higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex in which L₁has been cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 80× higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex in which L₁has been cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 85× higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex in which L₁has been cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 90× higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex in which L₁has been cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 95× higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex in which L₁has been cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 100× higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex in which L₁has been cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 120× higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex in which L₁has been cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 1000× higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex in which L₁has been cleaved by the tumor specific protease.

In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC₅₀in an IFNγ release T-cell activation assay as compared to the EC₅₀in an IFNγ release T-cell activation assay of an isolated polypeptide or polypeptide complex that does not have P₁or L₁. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC₅₀in an IFNγ release T-cell activation assay that is at least 10× higher than the EC₅₀in an IFNγ release T-cell activation assay of a form of the isolated polypeptide or polypeptide complex that does not have P₁or L₁. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC₅₀in an IFNγ release T-cell activation assay that is at least 20× higher than the EC₅₀in an IFNγ release T-cell activation assay of a form of the isolated polypeptide or polypeptide complex that does not have P₁or L₁. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC₅₀in an IFNγ release T-cell activation assay that is at least 30× higher than the EC₅₀in an IFNγ release T-cell activation assay of a form of the isolated polypeptide or polypeptide complex that does not have P₁or L₁. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC₅₀in an IFNγ release T-cell activation assay that is at least 40× higher than the EC₅₀in an IFNγ release T-cell activation assay of a form of the isolated polypeptide or polypeptide complex that does not have P₁or L₁. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC₅₀in an IFNγ release T-cell activation assay that is at least 50× higher than the EC₅₀in an IFNγ release T-cell activation assay of a form of the isolated polypeptide or polypeptide complex that does not have P₁or L₁. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC₅₀in an IFNγ release T-cell activation assay that is at least 60× higher than the EC₅₀in an IFNγ release T-cell activation assay of a form of the isolated polypeptide or polypeptide complex that does not have P₁or L₁. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC₅₀in an IFNγ release T-cell activation assay that is at least 70× higher than the EC₅₀in an IFNγ release T-cell activation assay of a form of the isolated polypeptide or polypeptide complex that does not have P₁or L₁. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC₅₀in an IFNγ release T-cell activation assay that is at least 80× higher than the EC₅₀in an IFNγ release T-cell activation assay of a form of the isolated polypeptide or polypeptide complex that does not have P₁or L₁. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC₅₀in an IFNγ release T-cell activation assay that is at least 90× higher than the EC₅₀in an IFNγ release T-cell activation assay of a form of the isolated polypeptide or polypeptide complex that does not have P₁or L₁. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC₅₀in an IFNγ release T-cell activation assay that is at least 100× higher than the EC₅₀in an IFNγ release T-cell activation assay of a form of the isolated polypeptide or polypeptide complex that does not have P₁or L₁. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC₅₀in an IFNγ release T-cell activation assay that is at least 1000× higher than the EC₅₀in an IFNγ release T-cell activation assay of a form of the isolated polypeptide or polypeptide complex that does not have P₁or L₁.

In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC₅₀in an IFNγ release T-cell activation assay as compared to the EC₅₀in an IFNγ release T-cell activation assay of the isolated polypeptide or polypeptide complex in which L₁has been cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC₅₀in an IFNγ release T-cell activation assay that is at least 10× higher than the EC₅₀in an IFNγ release T-cell activation assay of the isolated polypeptide or polypeptide complex in which L₁has been cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC₅₀in an IFNγ release T-cell activation assay that is at least 20× higher than the EC₅₀in an IFNγ release T-cell activation assay of the isolated polypeptide or polypeptide complex in which L₁has been cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC₅₀in an IFNγ release T-cell activation assay that is at least 30× higher than the EC₅₀in an IFNγ release T-cell activation assay of the isolated polypeptide or polypeptide complex in which L₁has been cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC₅₀in an IFNγ release T-cell activation assay that is at least 40× higher than the EC₅₀in an IFNγ release T-cell activation assay of the isolated polypeptide or polypeptide complex in which L₁has been cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC₅₀in an IFNγ release T-cell activation assay that is at least 50× higher than the EC₅₀in an IFNγ release T-cell activation assay of the isolated polypeptide or polypeptide complex in which L₁has been cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC₅₀in an IFNγ release T-cell activation assay that is at least 60× higher than the EC₅₀in an IFNγ release T-cell activation assay of the isolated polypeptide or polypeptide complex in which L₁has been cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC₅₀in an IFNγ release T-cell activation assay that is at least 70× higher than the EC₅₀in an IFNγ release T-cell activation assay of the isolated polypeptide or polypeptide complex in which L₁has been cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC₅₀in an IFNγ release T-cell activation assay that is at least 80× higher than the EC₅₀in an IFNγ release T-cell activation assay of the isolated polypeptide or polypeptide complex in which L₁has been cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC₅₀in an IFNγ release T-cell activation assay that is at least 90× higher than the EC₅₀in an IFNγ release T-cell activation assay of the isolated polypeptide or polypeptide complex in which L₁has been cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC₅₀in an IFNγ release T-cell activation assay that is at least 100× higher than the EC₅₀in an IFNγ release T-cell activation assay of the isolated polypeptide or polypeptide complex in which L₁has been cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC₅₀in an IFNγ release T-cell activation assay that is at least 1000× higher than the EC₅₀in an IFNγ release T-cell activation assay of the isolated polypeptide or polypeptide complex in which L₁has been cleaved by the tumor specific protease.

In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC₅₀in a T-cell cytolysis assay as compared to the EC₅₀in a T-cell cytolysis assay of an isolated polypeptide or polypeptide complex that does not have P₁or L₁. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC₅₀in a T-cell cytolysis assay that is at least 10× higher than the EC₅₀in a T-cell cytolysis assay of a form of the isolated polypeptide or polypeptide complex that does not have P₁or L₁. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC₅₀in a T-cell cytolysis assay that is at least 20× higher than the EC₅₀in a T-cell cytolysis assay of a form of the isolated polypeptide or polypeptide complex that does not have P₁or L₁. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC₅₀in a T-cell cytolysis assay that is at least 30× higher than the EC₅₀in a T-cell cytolysis assay of a form of the isolated polypeptide or polypeptide complex that does not have P₁or L₁. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC₅₀in a T-cell cytolysis assay that is at least 40× higher than the EC₅₀in a T-cell cytolysis assay of a form of the isolated polypeptide or polypeptide complex that does not have P₁or L₁. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC₅₀in a T-cell cytolysis assay that is at least 50× higher than the EC₅₀in a T-cell cytolysis assay of a form of the isolated polypeptide or polypeptide complex that does not have P₁or L₁. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC₅₀in a T-cell cytolysis assay that is at least 60× higher than the EC₅₀in a T-cell cytolysis assay of a form of the isolated polypeptide or polypeptide complex that does not have P₁or L₁. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC₅₀in a T-cell cytolysis assay that is at least 70× higher than the EC₅₀in a T-cell cytolysis assay of a form of the isolated polypeptide or polypeptide complex that does not have P₁or L₁. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC₅₀in a T-cell cytolysis assay that is at least 80× higher than the EC₅₀in a T-cell cytolysis assay of a form of the isolated polypeptide or polypeptide complex that does not have P₁or L₁. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC₅₀in a T-cell cytolysis assay that is at least 90× higher than the EC₅₀in a T-cell cytolysis assay of a form of the isolated polypeptide or polypeptide complex that does not have P₁or L₁. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC₅₀in a T-cell cytolysis assay that is at least 100× higher than the EC₅₀in a T-cell cytolysis assay of an isolated polypeptide or polypeptide complex that does not have P₁or L₁. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC₅₀in a T-cell cytolysis assay that is at least 1000× higher than the EC₅₀in a T-cell cytolysis assay of an isolated polypeptide or polypeptide complex that does not have P₁or L₁.

In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC₅₀in a T-cell cytolysis assay as compared to the EC₅₀in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex in which L₁has been cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC₅₀in a T-cell cytolysis assay that is at least 10× higher than the EC₅₀in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex in which L₁has been cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC₅₀in a T-cell cytolysis assay that is at least 20× higher than the EC₅₀in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex in which L₁has been cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC₅₀in a T-cell cytolysis assay that is at least 30× higher than the EC₅₀in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex in which L₁has been cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC₅₀in a T-cell cytolysis assay that is at least 40× higher than the EC₅₀in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex in which L₁has been cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC₅₀in a T-cell cytolysis assay that is at least 50× higher than the EC₅₀in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex in which L₁has been cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC₅₀in a T-cell cytolysis assay that is at least 60× higher than the EC₅₀in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex in which L₁has been cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC₅₀in a T-cell cytolysis assay that is at least 70× higher than the EC₅₀in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex in which L₁has been cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC₅₀in a T-cell cytolysis assay that is at least 80× higher than the EC₅₀in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex in which L₁has been cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC₅₀in a T-cell cytolysis assay that is at least 90× higher than the EC₅₀in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex in which L₁has been cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC₅₀in a T-cell cytolysis assay that is at least 100× higher than the EC₅₀in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex in which L₁has been cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC₅₀in a T-cell cytolysis assay that is at least 1,000× higher than the EC₅₀in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex in which L₁has been cleaved by the 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 for the tumor cell antigen as compared to the binding affinity for the tumor cell antigen of an isolated polypeptide or polypeptide complex of Formula Ia that does not have 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 for the tumor cell antigen that is at least 10× higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex of Formula Ia that does not have 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 for the tumor cell antigen that is at least 50× higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex of Formula Ia that does not have P₁, L₁, P₂, or L₂. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ta) has weaker binding affinity for the tumor cell antigen that is at least 75× higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex of Formula Ia that does not have 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 for the tumor cell antigen that is at least 100× higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex of Formula Ia that does not have 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 for the tumor cell antigen that is at least 120× higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex of Formula Ia that does not have 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 for the tumor cell antigen that is at least 200× higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex of Formula Ia that does not have 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 for the tumor cell antigen that is at least 300× higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex of Formula Ia that does not have 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 for the tumor cell antigen that is at least 400× higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex of Formula Ia that does not have 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 for the tumor cell antigen that is at least 500× higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex of Formula Ia that does not have 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 for the tumor cell antigen that is at least 600× higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex of Formula Ia that does not have 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 for the tumor cell antigen that is at least 700× higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex of Formula Ia that does not have 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 for the tumor cell antigen that is at least 800× higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex of Formula Ia that does not have 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 for the tumor cell antigen that is at least 900× higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex of Formula Ia that does not have 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 for the tumor cell antigen that is at least 1000× higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex of Formula Ia that does not have P₁, L₁, P₂, or L₂. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ta) has weaker binding affinity for the tumor cell antigen that is at least 10,000× higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex of Formula Ia that does not have 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 for the tumor cell antigen as compared to the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex of Formula Ia in which L₁and L₂have been cleaved by the tumor specific proteases. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H_j(Formula Ia) has weaker binding affinity for the tumor cell antigen that is at least 10× higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex of Formula Ia in which L₁and L₂have been cleaved by the tumor specific proteases. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has weaker binding affinity for the tumor cell antigen that is at least 50× higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex of Formula Ia in which L₁and L₂have been cleaved by the tumor specific proteases. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has weaker binding affinity for the tumor cell antigen that is at least 75× higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex of Formula Ia in which L₁and L₂have been cleaved by the tumor specific proteases. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has weaker binding affinity for the tumor cell antigen that is at least 100× higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex of Formula Ia in which L₁and L₂have been cleaved by the tumor specific proteases. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has weaker binding affinity for the tumor cell antigen that is at least 120× higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex of Formula Ia in which L₁and L₂have been cleaved by the tumor specific proteases. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has weaker binding affinity for the tumor cell antigen that is at least 200× higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex of Formula Ia in which L₁and L₂have been cleaved by the tumor specific proteases. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has weaker binding affinity for the tumor cell antigen that is at least 300× higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex of Formula Ia in which L₁and L₂have been cleaved by the tumor specific proteases. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has weaker binding affinity for the tumor cell antigen that is at least 400× higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex of Formula Ia in which L₁and L₂have been cleaved by the tumor specific proteases. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has weaker binding affinity for the tumor cell antigen that is at least 500× higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex of Formula Ia in which L₁and L₂have been cleaved by the tumor specific proteases. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has weaker binding affinity for the tumor cell antigen that is at least 600× higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex of Formula Ia in which L₁and L₂have been cleaved by the tumor specific proteases. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has weaker binding affinity for the tumor cell antigen that is at least 700× higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex of Formula Ia in which L₁and L₂have been cleaved by the tumor specific proteases. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has weaker binding affinity for the tumor cell antigen that is at least 800× higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex of Formula Ia in which L₁and L₂have been cleaved by the tumor specific proteases. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has weaker binding affinity for the tumor cell antigen that is at least 900× higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex of Formula Ia in which L₁and L₂have been cleaved by the tumor specific proteases. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has weaker binding affinity for the tumor cell antigen that is at least 1000× higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex of Formula Ia in which L₁and L₂have been cleaved by the tumor specific proteases. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has weaker binding affinity for the tumor cell antigen that is at least 10,000× higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex of Formula Ia in which L₁and L₂have been cleaved by the tumor specific proteases.

In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has an increased EC₅₀in an IFNγ release T-cell activation assay as compared to the EC₅₀in an IFNγ release T-cell activation assay of an isolated polypeptide or polypeptide complex of Formula Ia that does not have P₁, L₁, P₂, or L₂. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has an increased EC₅₀in an IFNγ release T-cell activation assay that is at least 10× higher than the EC₅₀in an IFNγ release T-cell activation assay of a form of the isolated polypeptide or polypeptide complex of Formula Ia that does not have P₁, L₁, P₂, or L₂. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has an increased EC₅₀in an IFNγ release T-cell activation assay that is at least 50× higher than the EC₅₀in an IFNγ release T-cell activation assay of a form of the isolated polypeptide or polypeptide complex of Formula Ia that does not have P₁, L₁, P₂, or L₂. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has an increased EC₅₀in an IFNγ release T-cell activation assay that is at least 75× higher than the EC₅₀in an IFNγ release T-cell activation assay of a form of the isolated polypeptide or polypeptide complex of Formula Ia that does not have P₁, L₁, P₂, or L₂. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has an increased EC₅₀in an IFNγ release T-cell activation assay that is at least 100× higher than the EC₅₀in an IFNγ release T-cell activation assay of a form of the isolated polypeptide or polypeptide complex of Formula Ia that does not have P₁, L₁, P₂, or L₂. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has an increased EC₅₀in an IFNγ release T-cell activation assay that is at least 200× higher than the EC₅₀in an IFNγ release T-cell activation assay of a form of the isolated polypeptide or polypeptide complex of Formula Ia that does not have P₁, L₁, P₂, or L₂. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has an increased EC₅₀in an IFNγ release T-cell activation assay that is at least 300× higher than the EC₅₀in an IFNγ release T-cell activation assay of a form of the isolated polypeptide or polypeptide complex of Formula Ia that does not have P₁, L₁, P₂, or L₂. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has an increased EC₅₀in an IFNγ release T-cell activation assay that is at least 400× higher than the EC₅₀in an IFNγ release T-cell activation assay of a form of the isolated polypeptide or polypeptide complex of Formula Ia that does not have P₁, L₁, P₂, or L₂. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has an increased EC₅₀in an IFNγ release T-cell activation assay that is at least 500× higher than the EC₅₀in an IFNγ release T-cell activation assay of a form of the isolated polypeptide or polypeptide complex of Formula Ia that does not have P₁, L₁, P₂, or L₂. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has an increased EC₅₀in an IFNγ release T-cell activation assay that is at least 600× higher than the EC₅₀in an IFNγ release T-cell activation assay of a form of the isolated polypeptide or polypeptide complex of Formula Ia that does not have P₁, L₁, P₂, or L₂. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has an increased EC₅₀in an IFNγ release T-cell activation assay that is at least 700× higher than the EC₅₀in an IFNγ release T-cell activation assay of a form of the isolated polypeptide or polypeptide complex of Formula Ia that does not P₁, L₁, P₂, or L₂. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H_j(Formula Ia) has an increased EC₅₀in an IFNγ release T-cell activation assay that is at least 800× higher than the EC₅₀in an IFNγ release T-cell activation assay of a form of the isolated polypeptide or polypeptide complex of Formula Ia that does not have P₁, L₁, P₂, or L₂. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has an increased EC₅₀in an IFNγ release T-cell activation assay that is at least 900× higher than the EC₅₀in an IFNγ release T-cell activation assay of a form of the isolated polypeptide or polypeptide complex of Formula Ia that does not have P₁, L₁, P₂, or L₂. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has an increased EC₅₀in an IFNγ release T-cell activation assay that is at least 1000× higher than the EC₅₀in an IFNγ release T-cell activation assay of a form of the isolated polypeptide or polypeptide complex of Formula Ia that does not have P₁, L₁, P₂, or L₂. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has an increased EC₅₀in an IFNγ release T-cell activation assay that is at least 10,000× higher than the EC₅₀in an IFNγ release T-cell activation assay of a form of the isolated polypeptide or polypeptide complex of Formula Ia that does not have P₁, L₁, P₂, or L₂.

In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has an increased EC₅₀in a T-cell cytolysis assay as compared to the EC₅₀in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula Ia in which L₁and L₂have been cleaved by the tumor specific proteases. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has an increased EC₅₀in a T-cell cytolysis assay that is at least 10× higher than the EC₅₀in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula Ia in which L₁and L₂have been cleaved by the tumor specific proteases, some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has an increased EC₅₀in a T-cell cytolysis assay that is at least 50× higher than the EC₅₀in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula Ia in which L₁and L₂have been cleaved by the tumor specific proteases. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has an increased EC₅₀in a T-cell cytolysis assay that is at least 75× higher than the EC₅₀in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula Ia in which L₁and L₂have been cleaved by the tumor specific proteases. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has an increased EC₅₀in a T-cell cytolysis assay that is at least 100× higher than the EC₅₀in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula Ia in which L₁and L₂have been cleaved by the tumor specific proteases. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has an increased EC₅₀in a T-cell cytolysis assay that is at least 200× higher than the EC₅₀in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula Ia in which L₁and L₂have been cleaved by the tumor specific proteases. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has an increased EC₅₀in a T-cell cytolysis assay that is at least 300× higher than the EC₅₀in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula Ia in which L₁and L₂have been cleaved by the tumor specific proteases. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has an increased EC₅₀in a T-cell cytolysis assay that is at least 400× higher than the EC₅₀in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula Ia in which L₁and L₂have been cleaved by the tumor specific proteases. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H1 (Formula Ia) has an increased EC₅₀in a T-cell cytolysis assay that is at least 500× higher than the EC₅₀in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula Ia in which L₁and L₂have been cleaved by the tumor specific proteases. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has an increased EC₅₀in a T-cell cytolysis assay that is at least 600× higher than the EC₅₀in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula Ia in which L₁and L₂have been cleaved by the tumor specific proteases. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ta) has an increased EC₅₀in a T-cell cytolysis assay that is at least 700× higher than the EC₅₀in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula Ia in which L₁and L₂have been cleaved by the tumor specific proteases. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has an increased EC₅₀in a T-cell cytolysis assay that is at least 800× higher than the EC₅₀in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula Ia in which L₁and L₂have been cleaved by the tumor specific proteases. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has an increased EC₅₀in a T-cell cytolysis assay that is at least 900× higher than the EC₅₀in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula Ia in which L₁and L₂have been cleaved by the tumor specific proteases. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has an increased EC₅₀in a T-cell cytolysis assay that is at least 1000× higher than the EC₅₀in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula Ia in which L₁and L₂have been cleaved by the tumor specific proteases. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has an increased EC₅₀in a T-cell cytolysis assay that is at least 10,000× higher than the EC₅₀in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula Ia in which L₁and L₂have been cleaved by the tumor specific proteases.

In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has an increased EC₅₀in a T-cell cytolysis assay as compared to the EC₅₀in a T-cell cytolysis assay of an isolated polypeptide or polypeptide complex that does not have P₁, L₁, P₂, or L₂. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has an increased EC₅₀in a T-cell cytolysis assay that is at least 10× higher than the EC₅₀in a T-cell cytolysis assay of a form of the isolated polypeptide or polypeptide complex of Formula Ia that does not have P₁, L₁, P₂, or L₂. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has an increased EC₅₀in a T-cell cytolysis assay that is at least 50× higher than the EC₅₀in a T-cell cytolysis assay of a form of the isolated polypeptide or polypeptide complex of Formula Ia that does not have P₁, L₁, P₂, or L₂. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has an increased EC₅₀in a T-cell cytolysis assay that is at least 75× higher than the EC₅₀in a T-cell cytolysis assay of a form of the isolated polypeptide or polypeptide complex of Formula Ia that does not have P₁, L₁, P₂, or L₂. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has an increased EC₅₀in a T-cell cytolysis assay that is at least 100× higher than the EC₅₀in a T-cell cytolysis assay of an isolated polypeptide or polypeptide complex that does not have P₁, L₁, P₂, or L₂. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has an increased EC₅₀in a T-cell cytolysis assay that is at least 200× higher than the EC₅₀in a T-cell cytolysis assay of an isolated polypeptide or polypeptide complex that does not have P₁, L₁, P₂, or L₂. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has an increased EC₅₀in a T-cell cytolysis assay that is at least 300× higher than the EC₅₀in a T-cell cytolysis assay of an isolated polypeptide or polypeptide complex that does not have P₁, L₁, P₂, or L₂. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has an increased EC₅₀in a T-cell cytolysis assay that is at least 400× higher than the EC₅₀in a T-cell cytolysis assay of an isolated polypeptide or polypeptide complex that does not have P₁, L₁, P₂, or L₂. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has an increased EC₅₀in a T-cell cytolysis assay that is at least 500× higher than the EC₅₀in a T-cell cytolysis assay of an isolated polypeptide or polypeptide complex that does not have P₁, L₁, P₂, or L₂. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ta) has an increased EC₀in a T-cell cytolysis assay that is at least 600× higher than the EC₅₀in a T-cell cytolysis assay of an isolated polypeptide or polypeptide complex that does not have P₁, L₁, P₂, or L₂. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has an increased EC₀in a T-cell cytolysis assay that is at least 700× higher than the EC₅₀in a T-cell cytolysis assay of an isolated polypeptide or polypeptide complex that does not have P₁, L₁, P₂, or L₂. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has an increased EC₅₀in a T-cell cytolysis assay that is at least 800× higher than the EC₅₀in a T-cell cytolysis assay of an isolated polypeptide or polypeptide complex that does not have P₁, L₁, P₂, or L₂. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has an increased EC₅₀in a T-cell cytolysis assay that is at least 900× higher than the EC₅₀in a T-cell cytolysis assay of an isolated polypeptide or polypeptide complex that does not have P₁, L₁, P₂, or L₂. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has an increased EC₅₀in a T-cell cytolysis assay that is at least 1000× higher than the EC₅₀in a T-cell cytolysis assay of an isolated polypeptide or polypeptide complex that does not have P₁, L₁, P₂, or L₂. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has an increased EC₅₀in a T-cell cytolysis assay that is at least 10,000× higher than the EC₅₀in a T-cell cytolysis assay of an isolated polypeptide or polypeptide complex that does not have P₁, L₁, P₂, or L₂.

In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has an increased EC₅₀in a T-cell cytolysis assay as compared to the EC₅₀in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula Ia in which L₁and L₂have been cleaved by the tumor specific proteases. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has an increased EC₅₀in a T-cell cytolysis assay that is at least 10× higher than the EC₅₀in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula Ia in which L₁and L₂have been cleaved by the tumor specific proteases. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has an increased EC₅₀in a T-cell cytolysis assay that is at least 50× higher than the EC₅₀in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula Ia in which L₁and L₂have been cleaved by the tumor specific proteases. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has an increased EC₅₀in a T-cell cytolysis assay that is at least 75× higher than the EC₅₀in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula Ia in which L₁and L₂have been cleaved by the tumor specific proteases. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has an increased EC₅₀in a T-cell cytolysis assay that is at least 100× higher than the EC₅₀in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula Ia in which L₁and L₂have been cleaved by the tumor specific proteases. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has an increased EC₅₀in a T-cell cytolysis assay that is at least 200× higher than the EC₅₀in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula Ia in which L₁and L₂have been cleaved by the tumor specific proteases. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H_j(Formula Ia) has an increased EC₅₀in a T-cell cytolysis assay that is at least 300× higher than the EC₅₀in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula Ia in which L₁and L₂have been cleaved by the tumor specific proteases. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has an increased EC₅₀in a T-cell cytolysis assay that is at least 400× higher than the EC₅₀in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula Ia in which L₁and L₂have been cleaved by the tumor specific proteases. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has an increased EC₅₀in a T-cell cytolysis assay that is at least 500× higher than the EC₅₀in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula Ia in which L₁and L₂have been cleaved by the tumor specific proteases. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has an increased EC₅₀in a T-cell cytolysis assay that is at least 600× higher than the EC₅₀in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula Ia in which L₁and L₂have been cleaved by the tumor specific proteases. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has an increased EC₅₀in a T-cell cytolysis assay that is at least 700× higher than the EC₅₀in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula Ia in which L₁and L₂have been cleaved by the tumor specific proteases. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has an increased EC₅₀in a T-cell cytolysis assay that is at least 800× higher than the EC₅₀in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula Ia in which L₁and L₂have been cleaved by the tumor specific proteases. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has an increased EC₅₀in a T-cell cytolysis assay that is at least 900× higher than the EC₅₀in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula Ia in which L₁and L₂have been cleaved by the tumor specific proteases. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has an increased EC₅₀in a T-cell cytolysis assay that is at least 1000× higher than the EC₅₀in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula Ia in which L₁and L₂have been cleaved by the tumor specific proteases. In some embodiments, the isolated polypeptide or polypeptide complex P₂-L₂-A₂-A₁-L₁-P₁—H₁(Formula Ia) has an increased EC₅₀in a T-cell cytolysis assay that is at least 10,000× higher than the EC₅₀in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula Ia in which L₁and L₂have been cleaved by the tumor specific proteases.

Second Antigen Recognizing Molecule (A₂)

In some embodiments, A₂comprises an antibody or antibody fragment. In some embodiments, the antibody or antibody fragment thereof comprises a single chain variable fragment, a single domain antibody, a Fab, 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), a variable domain (VHH) of a camelid derived single domain antibody. In some embodiments, the antibody or antibody fragment thereof is humanized or human. In some embodiments, A₂is the 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 a EGFR binding domain.

In some embodiments, the antigen binding fragment (Fab) or Fab′ that binds to EGFR comprises a Fab light chain polypeptide chain and a Fab heavy chain polypeptide. In some embodiments, the Fab light chain polypeptide comprises a Fab light chain variable domain. In some embodiments, the Fab heavy chain polypeptide comprises a Fab heavy chain variable domain. In some embodiments, the Fab heavy chain variable domain comprises at least one, two, or three complementarity determining regions (CDR)s disclosed in Table 2 or a sequence substantially identical thereto (e.g., a sequence that has 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 (CDR)s disclosed in Table 2 or a sequence substantially identical thereto (e.g., a sequence that has at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity).

In some embodiments, the Fab heavy chain variable domain comprises at least one, two, or three complementarity determining regions (CDR)s disclosed in Table 2 or a sequence substantially identical thereto (e.g., a sequence that has at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity); and the Fab light chain variable domain comprises at least one, two, or three complementarity determining regions (CDR)s disclosed in Table 2 or a sequence substantially identical thereto (e.g., a sequence that has at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity).

TABLE 2

anti-EGFR amino acid sequences (as determined by IMGT numbering system)

Construct
Amino Acid Sequence
SEQ

Description
(N to C)
ID NO:

EGFR: LC:
QSIGTN
15

CDR1

EGFR: LC:
YAS
16

CDR2

EGFR: LC:
QQNNNWPTT
17

CDR3

EGFR: HC:
GFSLTNYG
18

CDR1

EGFR: HC:
IWSGGNT
19

CDR2

EGFR: HC:
ARALTYYDYEFAY
20

CDR3

EGFR Fab LC v1
QILLTQSPVILSVSPGERVSFSCRASQSIGTNIHW
21

YQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTD

FTLSINSVESEDIADYYCQQNNNWPTTFGAGTK

LELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLN

NFYPREAKVQWKVDNALQSGNSQESVTEQDSK

DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS

SPVTKSFNRGEC

EGFR Fab LC v2
DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHW
22

YQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTD

FTLSINSVESEDIADYYCQQNNNWPTTFGAGTK

LELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLN

NFYPREAKVQWKVDNALQSGNSQESVTEQDSK

DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS

SPVTKSFNRGEC

EGFR Fab HC
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYG
23

VHWVRQSPGKGLEWLGVIWSGGNTDYNTPFT

SRLSINKDNSKSQVFFKMNSLQSNDTAIYYCAR

ALTYYDYEFAY
WGQGTLVTVSAASTKGPSVFP

LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS

GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG

TQTYICNVNHKPSNTKVDKKVEPKSC

EGFR Fab HC
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYG
24

(N88Q)
VHWVRQSPGKGLEWLGVIWSGGNTDYNTPFT

SRLSINKDNSKSQVFFKMNSLQSQDTAIYYCAR

ALTYYDYEFAY
WGQGTLVTVSAASTKGPSVFP

LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS

GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG

TQTYICNVNHKPSNTKVDKKVEPKSC

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

In some embodiments, A₂comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of A₂comprise LC-CDR1: SEQ ID NO: 15, LC-CDR2: SEQ ID NO: 16, and LC-CDR3: SEQ ID NO: 17; and A₂comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of A₂comprise: HC-CDR1: SEQ ID NO: 18, HC-CDR2: SEQ ID NO: 19, and HC-CDR3: SEQ ID NO: 20.

In some embodiments, the Fab light chain polypeptide comprises the amino acid sequence according to SEQ ID NO: 21. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 80% sequence identity according to SEQ ID NO: 21. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 85% sequence identity according to SEQ ID NO: 21. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 90% sequence identity according to SEQ ID NO: 21. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 95% sequence identity according to SEQ ID NO: 21. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 99% sequence identity according to SEQ ID NO: 21.

In some embodiments, the Fab light chain polypeptide comprises the amino acid sequence according to SEQ ID NO: 22. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 80% sequence identity according to SEQ ID NO: 22. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 85% sequence identity according to SEQ ID NO: 22. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 90% sequence identity according to SEQ ID NO: 22. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 95% sequence identity according to SEQ ID NO: 22. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 99% sequence identity according to SEQ ID NO: 22.

In some embodiments, the Fab heavy chain polypeptide comprises the amino acid sequence according to SEQ ID NO: 23. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 80% sequence identity according to SEQ ID NO: 23. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 85% sequence identity according to SEQ ID NO: 23. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 90% sequence identity according to SEQ ID NO: 23. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 95% sequence identity according to SEQ ID NO: 23. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 99% sequence identity according to SEQ ID NO: 23.

In some embodiments, the Fab heavy chain polypeptide comprises the amino acid sequence according to SEQ ID NO: 24. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 80% sequence identity according to SEQ ID NO: 24. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 85% sequence identity according to SEQ ID NO: 24. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 90% sequence identity according to SEQ ID NO: 24. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 95% sequence identity according to SEQ ID NO: 24. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 99% sequence identity according to SEQ ID NO: 24.

In some embodiments, the Fab light chain polypeptide of A₂is bound to a C-terminus of the single chain variable fragment (scFv) of A₁. In some embodiments, the Fab heavy chain polypeptide of A₂is bound to a C-terminus of the single chain variable fragment (scFv) A₁. In some embodiments, the Fab light chain polypeptide of A₂is bound to a N-terminus of the single chain variable fragment (scFv) of A₁. In some embodiments, the Fab heavy chain polypeptide of A₂is bound to a N-terminus of the single chain variable fragment (scFv) A₁. In some embodiments, the Fab heavy chain polypeptide of A₂is bound to the scFv heavy chain polypeptide of A₁. In some embodiments, Fab light chain polypeptide of A₂is bound to the scFv heavy chain polypeptide of A₁. In some embodiments, the Fab heavy chain polypeptide of A₂is bound to the scFv light chain polypeptide of A₁. In some embodiments, the Fab light chain polypeptide of A₂is bound to the scFv light chain polypeptide of A₁.

In some embodiments, A₂further comprises P₂and L₂, wherein P₂comprises a peptide that binds to A₂; and L₂comprises a linking moiety that connects A₂to P₂and is a substrate for a tumor specific protease. In some embodiments, the Fab heavy chain polypeptide of A₂is bound to the scFv heavy chain polypeptide of A₁and L₂is bound to the Fab light chain polypeptide of A₂. In some embodiments, the Fab light chain polypeptide of A₂is bound to the scFv heavy chain polypeptide of A₁and L₂is bound to the Fab heavy chain polypeptide of A₂. In some embodiments, the Fab heavy chain polypeptide of A₂is bound to the scFv light chain polypeptide of A₁and L₂is bound to the Fab light chain polypeptide of A₂. In some embodiments, the Fab light chain polypeptide of A₂is bound to the scFv light chain polypeptide of A₁and L₂is bound to the Fab heavy chain polypeptide of A₂.

Peptide (P₁and P₂and P_1a)

In some embodiments, P₁, P₂, or P_1acomprises a sequence as disclosed in Table 3 or a sequence substantially identical thereto (e.g., a sequence that has 0, 1, or 2 amino acid modifications).

TABLE 3

P₁ and P₂ and P_1a Sequences

Amino Acid Sequence
SEQ ID

Peptide
(N to C)
NO:

SP34.185 scFv mask
VYCGPEFDESVGCM
25

EGFR Fab mask
PCRSHIDVAKPICV
26

SP34.194 scFv mask
GYLWGCEWNCAGITT
70

In some embodiments, P₁impairs binding of A₁to a first target antigen. In some embodiments, P₁impairs binding of A₁to the effector cell antigen. In some embodiments, P₁is bound to A₁through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bonding interactions, or a combination thereof. In some embodiments, P₁is bound to A₁at or near an antigen binding site. In some embodiments, P₁becomes unbound from A₁when L₁is cleaved by the tumor specific protease thereby exposing A₁to 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, P₁has less than 70% sequence identity to the effector cell antigen. In some embodiments, P₁has less than 75% sequence identity to the effector cell antigen. In some embodiments, P₁has less than 80% sequence identity to the effector cell antigen. In some embodiments, P₁has less than 85% sequence identity to the effector cell antigen. In some embodiments, P₁has less than 90% sequence identity to the effector cell antigen. In some embodiments, P₁has less than 95% sequence identity to the effector cell antigen. In some embodiments, P₁has less than 98% sequence identity to the effector cell antigen. In some embodiments, P₁has less than 99% sequence identity to the effector cell antigen. In some embodiments, P₁comprises a de novo amino acid sequence that shares less than 10% sequence identity to the effector cell antigen. In some embodiments, P₁comprises an amino acid sequence according to SEQ ID NO: 25.

In some embodiments, A₁comprises a first antigen recognizing molecule that comprises an antibody or antibody binding fragment that binds to CD3. In some embodiments, P₁comprises an amino acid sequence according to any one of SEQ ID NOs: 25, 797-835, or 843-1690 or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 25, 797-835, or 843-1690.

In some embodiments, P₁comprises an amino acid sequence according to Z₁—Z₂—C—Z₄—P—Z₆—Z₇—Z₈—Z₉—Z₁₀—Z₁₁— Z₁₂—C—Z₁₄and Z₁is selected from D, Y, F, I, N, V, H, L, A, T, S, and P; Z₂is selected from D, Y, L, F, I, N, A, V, H, T, and S; Z₄is selected from G and W; Z₆is selected from E, D, V, and P; Z₇is selected from W, L, F, V, G, M, I, and Y; Z₈is selected from E, D, P, and Q; Z₉is selected from E, D, Y, V, F, W, P, L, and Q; Z₁₀is selected from S, D, Y, T, I, F, V, N, A, P, L, and H; Z₁is selected from I, Y, F, V, L, T, N, S, D, A, and H; Z₁₂is selected from F, D, Y, L, I, V, A, N, T, P, S, and H; and Z₁₄is selected from D, Y, N, F, I, P, V, A, T, H, L and S. In some embodiments, Z₁is selected from D, Y, F, I, and N; Z₂is selected from D, Y, L, F, I, and N; Z₄is selected from G and W; Z₆is selected from E and D; Z₇is selected from W, L, F, and V; Z₈is selected from E and D; Z₉is selected from E, D, Y, and V; Z₁₀is selected from S, D, Y, T, and I; Z₁₁is selected from I, Y, F, V, L, and T; Z₁₂is selected from F, D, Y, L, I, V, A, and N; and Z₁₄is selected from D, Y, N, F, I, and P. In some embodiments, Z₁is selected from D, Y, and F; Z₂is selected from D, Y, L, and F; Z₄is selected from G and W; Z₆is selected from E and D; Z₇is selected from W, L, and F; Z₈is selected from E and D; Z₉is selected from E and D; Z₁₀is selected from S, D, and Y; Z₁₁is selected from I, Y, and F; Z₁₂is selected from F, D, Y, and L; and Z₁₄is selected from D, Y, and N. In some embodiments, P₁comprises an amino acid sequence according to U₁—U₂—C—U₄—P—U₆—U₇—U₈—U₉—U₁₀—U₁₁—U₁₂—C—U₁₄and U₁is selected from D, Y, F, I, N, V, H, L, A, T, S, and P; U₂is selected from D, Y, L, F, I, N, A, V, H, T, and S; U₄is selected from G and W; U₆is selected from E, D, V, and P; U₇is selected from W, L, F, V, G, M, I, and Y; U₈is selected from E, D, P, and Q; U₉is selected from E, D, Y, V, F, W, P, L, and Q; U₁₀is selected from S, D, Y, T, I, F, V, N, A, P, L, and H; U₁₁is selected from I, Y, F, V, L, T, N, S, D, A, and H; U₁₂is selected from F, D, Y, L, I, V, A, N, T, P, S, G, and H; and U₁₄is selected from D, Y, N, F, I, P, V, A, T, H, L, M, and S. In some embodiments, U₁is selected from D, Y, F, I, V, and N; U₂is selected from D, Y, L, F, I, and N; U₄is selected from G and W; U₆is selected from E and D; U₇is selected from W, L, F, G, and V; U₈is selected from E and D; U₉is selected from E, D, Y, and V; U₁₀is selected from S, D, Y, T, and I; U₁₁is selected from I, Y, F, V, L, and T; U₁₂is selected from F, D, Y, L, I, V, A, G, and N; and U₁₄is selected from D, Y, N, F, I, M, and P. In some embodiments, U₁is selected from D, Y, V, and F; U₂is selected from D, Y, L, and F; U₄is selected from G and W; U₆is selected from E and D; U₇is selected from W, L, G, and F; U₈is selected from E and D; U₉is selected from E and D; U₁₀is selected from S, D, T, and Y; U₁₁is selected from I, Y, V, L, and F; U₁₂is selected from F, D, Y, G, A, and L; and U₁₄is selected from D, Y, M, and N.

In some embodiments, P₁comprises the amino acid sequences according to any one of SEQ ID NOs: 797-823.

In some embodiments, P₁comprises an amino acid sequences according to any one of SEQ ID NOs: 25, 824-835 and 843-1690.

In some embodiments, P₁comprises the amino acid sequences according to any of SEQ ID NOs: 824-835.

In some embodiments, P₁comprises the amino acid sequence according to SEQ ID NO: 810 or an amino acid sequence that has 1, 2, or 3, amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 810.

In some embodiments, P₁comprises the amino acid sequence according to SEQ ID NO: 811 or an amino acid sequence that has 1, 2, or 3, amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 811.

In some embodiments, P₁comprises the amino acid sequence according to SEQ ID NO: 834 or an amino acid sequence that has 1, 2, or 3, amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 834.

In some embodiments, P₁comprises the amino acid sequence according to SEQ ID NO: 810.

In some embodiments, P₁comprises the amino acid sequence according to SEQ ID NO: 811.

In some embodiments, P₁comprises the amino acid sequence according to SEQ ID NO: 834.

In some embodiments, P₂impairs binding of A₂to the second target antigen. In some embodiments, wherein P₂impairs binding of A₂to EGFR. In some embodiments, P₂is bound to A₂through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bonding interactions, or a combination thereof. In some embodiments, P₂is bound to A₂at or near an antigen binding site. In some embodiments, P₂becomes unbound from A₂when L₂is cleaved by the tumor specific protease thereby exposing A₂to the 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, P₂has less than 70% sequence identity to the EGFR. In some embodiments, P₂has less than 75% sequence identity to the EGFR. In some embodiments, P₂has less than 80% sequence identity to the EGFR. In some embodiments, P₂has less than 85% sequence identity to the EGFR. In some embodiments, P₂has less than 90% sequence identity to the EGFR. In some embodiments, P₂has less than 95% sequence identity to the EGFR. In some embodiments, P₂has less than 98% sequence identity to the EGFR. In some embodiments, P₂has less than 99% sequence identity to the EGFR. In some embodiments, P₂comprises a de novo amino acid sequence that shares less than 10% sequence identity to the EGFR. In some embodiments, P₂comprises an amino acid sequence according to SEQ ID NO: 26. In some embodiments, P₂comprises an amino acid sequence according to SEQ ID NO: 115. In some embodiments, P₂comprises an amino acid sequence according to SEQ ID NO: 116.

In some embodiments, P₂comprises an amino acid sequence according to any one of SEQ ID NOs: 26, 71-96, 98-776, or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 26, 71-96, 98-776. In some embodiments, P₂comprises an an amino acid sequence according to X₁—C—X₂—X₃—X₄—X₅-D-X₆-A-X₇—P—X₈—C—X₉wherein X₁is selected from P and L; X₂is selected from R, L, T, A, N, I, V, S, H, and P; X₃is selected from S, P, F, and Y; X₄is selected from H, L, Q, P, R, F, N; X₅is selected from I, F, Y, H, N, T, S, D, A, L, and V; X₆is selected from T, P, N, L, I, V, S, D, H, A, and Y; X₇is selected from K and Y; X₈is selected from I, P, L, and M; and X₉is selected from A, V, I, T, L, S, D, F, V, and H (SEQ ID NO: 841). In some embodiments, X₁is selected from P and L; X₂is selected from R, L, T, A, and N; X₃is selected from S, P, and F; X₄is selected from H, L, Q, and P; X₅is selected from I, F, Y, H, N, and T; X₆is selected from T, P, N, L, I, and V; X₇is K; X₈is I; and X₉is selected from A, V, I, T, L, and S. In some embodiments, X₁is P; X₂is selected from R, L, and T; X₃is S; X₄is selected from H, L, Q, and P; X₅is selected from I, F, Y, and T; X₆is selected from T, P, N, and V; X₇is K; X₈is I; and X₉is selected from A, V, and I.

In some embodiments, P₂comprises an amino acid sequence according to any one of SEQ ID NOs: 26, 86-96, or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of 26, 86-96. In some embodiments, P₂comprises an amino acid sequence according to any one of SEQ ID NOs: 26, 86-96.

In some embodiments, P₂comprises an amino acid sequence according to any one of SEQ ID NOs: 98-776 or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 98-776. In some embodiments, P₂comprises an amino acid sequence according to any one of SEQ ID NOs: SEQ ID NOs: 98-776.

In some embodiments, P₂comprises an amino acid sequence according to any one of SEQ ID NOs: 99-118 or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of 99-118. In some embodiments, P₂comprises an amino acid sequence according to any one of SEQ ID NOs: 99-118.

In some embodiments, P₂comprises an amino acid sequence according to SEQ ID NO: 26 or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to SEQ ID NO: 26. In some embodiments, P₂comprises the amino acid sequence according to SEQ ID NO: 26.

In some embodiments, P₂comprises an amino acid sequence according to SEQ ID NO: 115 or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to SEQ ID NO: 115. In some embodiments, P₂comprises the amino acid sequence according to SEQ ID NO: 115.

In some embodiments, P₂comprises an amino acid sequence according to SEQ ID NO: 115 or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to SEQ ID NO: 116. In some embodiments, P₂comprises the amino acid sequence according to SEQ ID NO: 116.

In some embodiments, P_1awhen L_1ais uncleaved impairs binding of the antigen recognizing molecule to the target antigen. In some embodiments, the antigen recognizing 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_1ahas less than 70% sequence identity to the target antigen. In some embodiments, P_1ahas less than 75% sequence identity to the target antigen. In some embodiments, P_1ahas less than 80% sequence identity to the target antigen. In some embodiments, P_1ahas less than 85% sequence identity to the target antigen. In some embodiments, P_1ahas less than 90% sequence identity to the target antigen. In some embodiments, P_1ahas less than 95% sequence identity to the target antigen. In some embodiments, P_1ahas less than 98% sequence identity to the target antigen. In some embodiments, P_1ahas less than 99% sequence identity to the target antigen. In some embodiments, P_1acomprises a de novo amino acid sequence that shares less than 10% sequence identity to the second target antigen.

In some embodiments, P_1acomprises an amino acid sequence according to any one of SEQ ID NOs: 25, 797-835, or 843-1690 or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 25, 797-835, or 843-1690.

In some embodiments, P_1acomprises an amino acid sequence according to Z₁—Z₂—C—Z₄—P—Z₆-Z₇—Z₈—Z₉-Z₁₀—Z₁₁—Z₁₂—C—Z₁₄and Z1 is selected from D, Y, F, I, N, V, H, L, A, T, S, and P; Z₂is selected from D, Y, L, F, I, N, A, V, H, T, and S; Z₄is selected from G and W; Z₆is selected from E, D, V, and P; Z₇is selected from W, L, F, V, G, M, I, and Y; Z₈is selected from E, D, P, and Q; Z₉is selected from E, D, Y, V, F, W, P, L, and Q; Z₁₀is selected from S, D, Y, T, I, F, V, N, A, P, L, and H; Z₁₁is selected from I, Y, F, V, L, T, N, S, D, A, and H; Z₁₂is selected from F, D, Y, L, I, V, A, N, T, P, S, and H; and Z₁₄is selected from D, Y, N, F, I, P, V, A, T, H, L and S. In some embodiments, Z, is selected from D, Y, F, I, and N; Z₂is selected from D, Y, L, F, I, and N; Z₄is selected from G and W; Z₆is selected from E and D; Z₇is selected from W, L, F, and V; Z₈is selected from E and D; Z₉is selected from E, D, Y, and V; Z₁₀is selected from S, D, Y, T, and I; Z₁₁is selected from I, Y, F, V, L, and T; Z₁₂is selected from F, D, Y, L, I, V, A, and N; and Z₁₄is selected from D, Y, N, F, I, and P. In some embodiments, Z₁is selected from D, Y, and F; Z₂is selected from D, Y, L, and F; Z₄is selected from G and W; Z₆is selected from E and D; Z₇is selected from W, L, and F; Z₈is selected from E and D; Z₉is selected from E and D; Z₁₀is selected from S, D, and Y; Z₁₁is selected from I, Y, and F; Z₁₂is selected from F, D, Y, and L; and Z₁₄is selected from D, Y, and N. In some embodiments, P₁comprises an amino acid sequence according to U₁—U₂—C—U₄—P—U₆—U₇—U₈—U₉—U₁₀—U₁n-U₁₂—C—U₁₄and U₁is selected from D, Y, F, I, N, V, H, L, A, T, S, and P; U₂is selected from D, Y, L, F, I, N, A, V, H, T, and S; U₄is selected from G and W; U₆is selected from E, D, V, and P; U₇is selected from W, L, F, V, G, M, I, and Y; U₈is selected from E, D, P, and Q; U₉is selected from E, D, Y, V, F, W, P, L, and Q; U₁₀is selected from S, D, Y, T, I, F, V, N, A, P, L, and H; U₁₁is selected from I, Y, F, V, L, T, N, S, D, A, and H; U₁₂is selected from F, D, Y, L, I, V, A, N, T, P, S, G, and H; and U₁₄is selected from D, Y, N, F, I, P, V, A, T, H, L, M, and S. In some embodiments, U₁is selected from D, Y, F, I, V, and N; U₂is selected from D, Y, L, F, I, and N; U₄is selected from G and W; U₆is selected from E and D; U₇is selected from W, L, F, G, and V; U₈is selected from E and D; U₉is selected from E, D, Y, and V; U₁₀is selected from S, D, Y, T, and I; U₁₁is selected from I, Y, F, V, L, and T; U₁₂is selected from F, D, Y, L, I, V, A, G, and N; and U₁₄is selected from D, Y, N, F, I, M, and P. In some embodiments, U₁is selected from D, Y, V, and F; U₂is selected from D, Y, L, and F; U₄is selected from G and W; U₆is selected from E and D; U₇is selected from W, L, G, and F; U₈is selected from E and D; U₉is selected from E and D; U₁₀is selected from S, D, T, and Y; U₁₁is selected from I, Y, V, L, and F; U₁₂is selected from F, D, Y, G, A, and L; and U₁₄is selected from D, Y, M, and N.

In some embodiments, P_1acomprises the amino acid sequences according to any one of SEQ ID NOs: 797-823.

In some embodiments, P_1acomprises an amino acid sequences according to any one of SEQ ID NOs: 25, 824-835 or 843-1690.

In some embodiments, P_1acomprises the amino acid sequences according to any of SEQ ID NOs: 824-835.

In some embodiments, P_1acomprises the amino acid sequence according to SEQ ID NO: 810 or an amino acid sequence that has 1, 2, or 3, amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 810.

In some embodiments, P_1acomprises the amino acid sequence according to SEQ ID NO: 811 or an amino acid sequence that has 1, 2, or 3, amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 811.

In some embodiments, P_1acomprises the amino acid sequence according to SEQ ID NO: 834 or an amino acid sequence that has 1, 2, or 3, amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 834.

In some embodiments, P_1acomprises the amino acid sequence according to SEQ ID NO: 810.

In some embodiments, P_1acomprises the amino acid sequence according to SEQ ID NO: 811.

In some embodiments, P_1acomprises the amino acid sequence according to SEQ ID NO: 834.

In some embodiments, P₁, P₂, or P_1acomprises a peptide sequence of at least 5 amino acids in length. In some embodiments, P₁, P₂, or P_1acomprises a peptide sequence of at least 6 amino acids in length. In some embodiments, P₁, P₂, or P_1acomprises a peptide sequence of at least 10 amino acids in length. In some embodiments, P₁, P₂, or P_1acomprises a peptide sequence of at least 10 amino acids in length and no more than 20 amino acids in length. In some embodiments, P₁, P₂, or P_1acomprises a peptide sequence of at least 16 amino acids in length. In some embodiments, P₁, P₂, or P_1acomprises a peptide sequence of no more than 40 amino acids in length. In some embodiments, P₁, P₂, or P_1acomprises at least two cysteine amino acid residues. In some embodiments, P₁, P₂, or P_1acomprises a cyclic peptide or a linear peptide. In some embodiments, P₁, P₂, or P_1acomprises a cyclic peptide. In some embodiments, P₁, P₂, or P_1acomprises a linear peptide.

In some embodiments, P₁, P₂, or P_1aor P₁, P₂, and P_1acomprise a modified amino acid or non-natural amino acid, or a modified non-natural amino acid, or a combination thereof. In some embodiments, the modified amino acid or a modified non-natural amino acid comprises a post-translational modification. In some embodiments P₁, P₂, or P_1aor P₁, P₂, and P_1acomprise a modification including, but 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 crosslinks, 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 are made anywhere to P₁, P₂, or P_1aor P₁, P₂, and P_1aincluding the peptide backbone, the amino acid side chains, and the terminus.

In some embodiments, P₁, P₂, or P_1adoes not comprise albumin or an albumin fragment. In some embodiments, P₁, P₂, or P_1adoes not comprise an albumin binding domain.

Linking Moiety (L₁, L₂, L₃, and L_1a)

In some embodiments, L₁, L₂, L₃, or L_1ais a peptide sequence having at least 5 to no more than 50 amino acids. In some embodiments L₁, L₂, L₃, or L_1ais a peptide sequence having at least 10 to no more than 30 amino acids. In some embodiments, L₁, L₂, L₃, or L_1ais a peptide sequence having at least 10 amino acids. In some embodiments, L₁, L₂, L₃, or L_1ais a peptide sequence having at least 18 amino acids. In some embodiments, L₁, L₂, L₃, or L_1ais a peptide sequence having at least 26 amino acids. In some embodiments, L₁, L₂, L₃, or L_1ahas a formula comprising (G₂S)_n, wherein n is an integer from 1 to 3 (SEQ ID NO: 840). In some embodiments, L₁, L₂, L₃, or L_1ahas a formula comprising (G₂S)_n, wherein n is an integer of at least 1. In some embodiments, L₁, L₂, L₃, or L_1ahas a formula selected from the group consisting of (G₂S)_n, (GS)₃, (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 metalloprotease, serine protease, cysteine protease, threonine protease, and aspartic protease. In some embodiments L₁, L₂, L₃, or L_1acomprises a urokinase cleavable amino acid sequence, a matriptase (MTSP1) cleavable amino acid sequence, a legumain cleavable amino acid sequence, or a matrix metalloprotease cleavable amino acid sequence. In some instances, the matrix metalloprotease comprises MMP2, MMP7, MMP9, MMP13, or MMP14. In some instances, the serine protease comprises matriptase (MTSP1), urokinase, or hepsin.

In some embodiments, L₁, L₂, L₃, or L_1acomprises a sequence as disclosed in Table 4 or a sequence substantially identical thereto (e.g., a sequence that has 0, 1, or 2 amino acid modifications).

In some embodiments, L₁comprises an 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 that has 1, 2, or 3 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, L₁comprises an amino acid sequence of Linker 4 (ISSGLLSGRSDAG) (SEQ ID NO: 66). In some embodiments, L₁comprises an amino acid sequence of Linker 5 (AAGLLAPPGGLSGRSDAG) (SEQ ID NO: 67). In some embodiments, L₁comprises an amino acid sequence of Linker 6 (SPLGLSGRSDAG) (SEQ ID NO: 68). In some embodiments, L₁comprises an amino acid sequence of Linker 7 (LSGRSDAGSPLGLAG) (SEQ ID NO: 69).

In some embodiments, L₂comprises an 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 that has 1, 2, or 3 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, L₂comprises an amino acid sequence of Linker 4 (ISSGLLSGRSDAG) (SEQ ID NO: 66). In some embodiments, L₁comprises an amino acid sequence of Linker 5 (AAGLLAPPGGLSGRSDAG) (SEQ ID NO: 67). In some embodiments, L₂comprises an amino acid sequence of Linker 6 (SPLGLSGRSDAG) (SEQ ID NO: 68). In some embodiments, L₂comprises an amino acid sequence of Linker 7 (LSGRSDAGSPLGLAG) (SEQ ID NO: 69).

TABLE 4

L₁, L₂, L₃, and L_1a Sequences

Amino Acid Sequence
SEQ ID

Construct Description
(N to C)
NO:

Linker 1
GGGGSGGGGSGGGGS
27

Linker 2
GGGGS
28

Linker 3
GGGGSGGGS
29

Cleavable linker 1
GGGGSGGGLSGRSDAGSPLGLAGSGGGS
30

Cleavable linker 2
GGGGSGGLSGRSDAGSPLGLAGSGGS
31

Cleavable linker 3
GGGGSSGGSAAGLLAPPGGLSGRSDAGG
32

GGS

Cleavable linker 4
GSSGGSAAGLLAPPGGLSGRSDAGGGGS
33

Non-cleavable linker 1
GGGGSGGGGGSGGGGSGGASSGAGGS
34

Non-cleavable linker 2
GGGGSGGGSGGGGSGGASSGAGGSGGG
35

S

Linker 4
ISSGLLSGRSDAG
66

Linker 5
AAGLLAPPGGLSGRSDAG
67

Linker 6
SPLGLSGRSDAG
68

Linker 7
LSGRSDAGSPLGLAG
69

In some embodiments, L₁is bound to N-terminus of A₁. In some embodiments, L₁is bound to C-terminus of A₁. In some embodiments, L₂is bound to N-terminus of A₂. In some embodiments, L₂is bound to C-terminus of A₂. In some embodiments, P₁becomes unbound from A₁when L₁is cleaved by the tumor specific protease thereby exposing A₁to the effector cell antigen. In some embodiments, P₂becomes unbound from A₂when L₂is cleaved by the tumor specific protease thereby exposing A₂to EGFR.

In some embodiments, L₁, L₂, L₃, or L_1acomprise a modified amino acid or non-natural amino acid, or a modified non-natural amino acid, or a combination thereof. In some embodiments, the modified amino acid or a modified non-natural amino acid comprises a post-translational modification. In some embodiments, L₁, L₂, L₃, or L_1acomprise a modification including, but 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 crosslinks, 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 are made anywhere to L₁, L₂, L₃, or L_1aincluding the peptide backbone, or the amino acid side chains.

In some embodiments, the cleavable linker is cleavable by a protease. In some embodiments, the protease is present in higher levels in a disease-state microenvironment relative to levels in healthy tissue or a microenvironment that is not the disease-state 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 a variety of 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.

Half-Life Extending Molecule (H₁and H_1a)

In some embodiments, H₁does not block A, binding to the effector cell antigen. In some embodiments, H₁comprises a linking moiety (L₃) that connects H₁to P₁. In some embodiments, H_1adoes not block the first antigen recognizing molecule binding to the effector cell antigen. In some embodiments, H_1acomprises a linking moiety (L₃) that connects H_1ato P_1a. In some embodiments, half-life extending molecule (H₁or H_1a) does not have binding affinity to antigen recognizing molecule. In some embodiments, half-life extending molecule (H₁or H_1a) does not have binding affinity to the effector cell antigen. In some embodiments, half-life extending molecule (H₁or H_1a) does not shield antigen recognizing molecule from the effector cell antigen. In some embodiments, half-life extending molecule (H₁or H_1a) is not directly linked to antigen recognizing molecule.

In some embodiments, H₁or H_1acomprises a sequence as disclosed in Table 5 or a sequence substantially identical thereto (e.g., a sequence that has at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity).

TABLE 5

H₁ and H_1a Sequences

Amino Acid Sequence
SEQ ID

Construct Description
(N to C)
NO:

Anti-Albumin: CDR-H1
GSTFYTAV
36

Anti-Albumin: CDR-H2
IRWTALTT
37

Anti-Albumin: CDR-H3
AARGTLGLFTTADSYDY
38

10G
GFTFSKFG
39

Anti-Albumin: CDR-H1

10G
ISGSGRDT
40

Anti-Albumin: CDR-H2

10G
TIGGSLSV
41

Anti-Albumin: CDR-H3

Anti-albumin
EVQLVESGGGLVQPGGSLRLS
42

CAASGSTFYTAVMGWVRQAP

GKGLEWVAAIRWTALTTSYA

DSVKGRFTISRDGAKTTLYLQ

MNSLRPEDTAVYYCAARGTL

GLFTTADSYDY
WGQGTLVTV

SS

10G anti-albumin
EVQLVESGGGLVQPGNSLRLS
43

CAASGFTFSKFGMSWVRQAP

GKGLEWVSSISGSGRDTLYAD

SVKGRFTISRDNAKTTLYLQM

NSLRPEDTAVYYCTIGGSLSV

SSQGTLVTVSS

In some embodiments, H₁or H_1acomprise an amino acid sequence that has repetitive sequence motifs. In some embodiments, H₁or H_1acomprises an amino acid sequence that has highly ordered secondary structure. “Highly ordered secondary structure,” as used in this context, means that at least about 50%, or about 70%, or about 80%, or about 90%, of amino acid residues of H₁or H_1acontribute 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 Garnier-Osguthorpe-Robson (“GOR”) algorithm.

In some embodiments, H₁or H_1acomprises a polymer. In some embodiments, the polymer is polyethylene glycol (PEG). In some embodiments, H₁or H_1acomprises albumin. In some embodiments, H₁or H_1acomprises an Fc domain. In some embodiments, the albumin is serum albumin. In some embodiments, the albumin is human serum albumin. In some embodiments, H₁or H_1acomprises a polypeptide, a ligand, or a small molecule. In some embodiments, the polypeptide, the ligand or the small molecule binds serum protein or a fragment thereof, a circulating immunoglobulin or a fragment thereof, or CD35/CR1. In some embodiments, the serum protein comprises a thyroxine-binding protein, a transthyretin, a 1-acid glycoprotein, a transferrin, transferrin receptor or a transferrin-binding portion thereof, a fibrinogen, or an albumin. In some embodiments, the circulating immunoglobulin molecule comprises IgG1, IgG2, IgG3, IgG4, s1gA, 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 or humanized antibody. In some embodiments, the single domain antibody is selected from the group consisting of 645gH1gL1, 645dsgH5gL4, 23-13-A01-sc02, A10m3 or a fragment thereof, DOM7r-31, DOM7h-11-15, Alb-1, Alb-8, Alb-23, 10G, 10E and SA21. In some embodiments, the single domain antibody comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the single domain antibody comprise: HC-CDR1: SEQ ID NO: 36, HC-CDR2: SEQ ID NO: 37, and HC-CDR3: 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, the HC-CDR2, and the HC-CDR3 of the single domain antibody comprise: HC-CDR1: SEQ ID NO: 36, HC-CDR2: SEQ ID NO: 37, and HC-CDR3: SEQ ID NO: 38; and wherein the CDRs comprise from 0-2 amino acid modifications in at least one of the HC-CDR1, HC-CDR2, or 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, the HC-CDR2, and the HC-CDR3 of the single domain antibody comprise: HC-CDR1: SEQ ID NO: 39, HC-CDR2: SEQ ID NO: 40, and HC-CDR3: 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, the HC-CDR2, and the HC-CDR3 of the single domain antibody comprise: HC-CDR1: SEQ ID NO: 39, HC-CDR2: SEQ ID NO: 40, and HC-CDR3: SEQ ID NO: 41; and wherein the CDRs comprise from 0-2 amino acid modifications in at least one of the HC-CDR1, HC-CDR2, or HC-CDR3.

In some embodiments, H₁comprises an amino acid sequence according to SEQ ID NO: 42. In some embodiments, H₁comprises an amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 42. In some embodiments, H₁comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 42. In some embodiments, H₁comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 42. In some embodiments, H₁comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 42. In some embodiments, H₁comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NO: 42.

In some embodiments, H_1acomprises an amino acid sequence according to SEQ ID NO: 42. In some embodiments, H_1acomprises an amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 42. In some embodiments, H_1acomprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 42. In some embodiments, H_1acomprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 42. In some embodiments, H_1acomprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 42. In some embodiments, H_1acomprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NO: 42.

In some embodiments, H₁comprises an amino acid sequence according to SEQ ID NO: 43. In some embodiments, H₁comprises an amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 43. In some embodiments, H₁comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 43. In some embodiments, H₁comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 43. In some embodiments, H₁comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 43. In some embodiments, H₁comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NO: 43.

In some embodiments, H_1acomprises an amino acid sequence according to SEQ ID NO: 43. In some embodiments, H_1acomprises an amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 43. In some embodiments, H_1acomprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 43. In some embodiments, H_1acomprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 43. In some embodiments, H_1acomprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 43. In some embodiments, H_1acomprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NO: 43.

In some embodiments, H₁or H_1aor H₁and H_1acomprise a modified amino acid or non-natural amino acid, or a modified non-natural amino acid, or a combination thereof. In some embodiments, the modified amino acid or a modified non-natural amino acid comprises a post-translational modification. In some embodiments H₁or H_1aor H₁and H_acomprise a modification including, but 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 crosslinks, 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 are made anywhere to H₁or H_1aor H₁and H_1aincluding the peptide backbone, the amino acid side chains, and the terminus.

In some embodiments, H₁comprises a linking moiety (L₃) that connects H₁to P₁. In some embodiments, L₃is a peptide sequence having at least 5 to no more than 50 amino acids. In some embodiments, L₃is a peptide sequence having at least 10 to no more than 30 amino acids. In some embodiments, L₃is a peptide sequence having at least 10 amino acids. In some embodiments, L₃is a peptide sequence having at least 18 amino acids. In some embodiments, L₃is a peptide sequence having at least 26 amino acids. In some embodiments, L₃has a formula selected from the group consisting of (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, L₃comprises an amino acid sequence according to SEQ ID NO: 29.

In some embodiments, H_1acomprises a linking moiety (L_1a) that connects H_1ato P_1a. In some embodiments, L_1ais a peptide sequence having at least 5 to no more than 50 amino acids. In some embodiments, L_1ais a peptide sequence having at least 10 to no more than 30 amino acids. In some embodiments, L_1ais a peptide sequence having at least 10 amino acids. In some embodiments, L_1ais a peptide sequence having at least 18 amino acids. In some embodiments, L_1ais a peptide sequence having at least 26 amino acids. In some embodiments, L_1ahas a formula selected from the group consisting of (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 an amino acid sequence according to SEQ ID NO: 30 or 31.

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 that has 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 with 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 with at least 95% sequence identity to SEQ ID NO: 50. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 51.

TABLE 6

Polypeptide complex sequences

Construct
Amino Acid Sequence
SEQ ID

Description
(N to C)
NO:

PC1: LC
QILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRT
44

EGFR Fab LC
NGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESE

DIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFP

PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS

GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC

EVTHQGLSSPVTKSFNRGEC

PC1: HC
EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW
45

SP34.185 scFv
VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTIS

(VH - linker 1
RDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYI

- VL) + Linker

SYWAY
WGQGTLVTVSSGGGGSGGGGSGGGGSQTVVT

2 + EGFR Fab
QEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKP

HC
GQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGV

QPEDEAEYYCVLWYSNRWVFGGGTKLTVLGGGGSQV

QLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQS

PGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQ

VFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGT

LVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY

FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT

VPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

PC2: LC
QILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRT
46

EGFR Fab LC
NGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESE

DIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFP

PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS

GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC

EVTHQGLSSPVTKSFNRGEC

PC2: HC
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANW
47

SP34.194 scFv
VQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAAL

(VL-linker 1-
TLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVLGG

VH) + Linker
GGSGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAA

2 + EGFR Fab
SGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYAT

HC
YYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYY

CVRHGNFGNSYVSWFAYWGQGTLVTVSSGGGGSQV

QLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQS

PGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQ

VFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGT

LVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY

FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT

VPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

PC3: LC
DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRT
48

EGFR Fab LC
NGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESE

DIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFP

PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS

GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC

EVTHQGLSSPVTKSFNRGEC

PC3: HC
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANW
49

SP34.194 scFv
VQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAAL

(VL-linker 1-
TLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVLGG

VH) + Linker
GGSGGGGGGGGSEVQLVESGGGLVQPGGSLKLSCAA

2 + EGFR Fab
SGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYAT

HC
YYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYY

CVRHGNFGNSYVSWFAYWGQGTLVTVSSGGGGSQV

QLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQS

PGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQ

VFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGT

LVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY

FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT

VPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

PC4: LC
GGPCRSHIDVAKPICVGGGGSGGLSGRSDAGSPLGLAG
50

EGFR Fab
SGGSDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWY

mask +
QQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINS

cleavable linker
VESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPS

2 + EGFR Fab
VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN

LC
ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK

VYACEVTHQGLSSPVTKSFNRGEC

PC4: HC
EVQLVESGGGLVQPGGSLRLSCAASGSTFYTAVMGWV
51

Anti-albumin +
RQAPGKGLEWVAAIRWTALTTSYADSVKGRFTISRDG

Linker 3 +
AKTTLYLQMNSLRPEDTAVYYCAARGTLGLFTTADS

SP34.185 scFv

YDY
WGQGTLVTVSSGGGGSGGGSGGVYCGPEFDESVG

mask +
CMGGGGSGGGLSGRSDAGSPLGLAGSGGGSEVQLVES

Cleavable
GGGLVQPGGSLKLSCAASGFTENKYAMNWVRQAPGK

Linker 1 +
GLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNT

SP34.185 scFv
AYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYW

(VH-linker 1-
GQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTV

VL) + Linker
SPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGL

2 + EGFR Fab
IGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAE

HC
YYCVLWYSNRWVFGGGTKLTVLGGGGSQVQLKQSGP

GLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLE

WLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMN

SLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAA

STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS

WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT

QTYICNVNHKPSNTKVDKKVEPKSC

PC5: LC
GGPCRSHIDVAKPICVGGGGSGGLSGRSDAGSPLGLAG
52

EGFR Fab
SGGSDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWY

mask +
QQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINS

cleavable linker
VESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPS

2 + EGFR Fab
VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN

LC v2
ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK

VYACEVTHQGLSSPVTKSFNRGEC

PC5: HC
EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWV
53

10G + Linker 3
RQAPGKGLEWVSSISGSGRDTLYADSVKGRFTISRDNA

+ SP34.185
KTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTV

scFv mask +
SSGGGGSGGGSGGVYCGPEFDESVGCMGGGGSGGGLS

Cleavable
GRSDAGSPLGLAGSGGGSEVQLVESGGGLVQPGGSLKL

Linker 1 +
SCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYN

SP34.185 scFv

NYAT
YYADSVKDRFTISRDDSKNTAYLQMNNLKTEDT

(VH-linker 1-
AVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGG

VL) + Linker
SGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTG

2 + EGFR Fab

AVTSGNY
PNWVQQKPGQAPRGLIGGTKFLAPGTPARF

HC
SGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVF

GGGTKLTVLGGGGSQVQLKQSGPGLVQPSQSLSITCTV

SGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYN

TPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARAL

TYYDYEFAY
WGQGTLVTVSAASTKGPSVFPLAPSSKST

SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA

VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV

DKKVEPKSC

PC6: LC
GGPCRSHIDVAKPICVGGGGSGGGGGSGGGGSGGASSG
54

EGFR Fab
AGGSDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWY

mask + Non-
QQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINS

cleavable linker
VESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPS

1 + EGFR Fab
VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN

LC v2
ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK

VYACEVTHQGLSSPVTKSFNRGEC

PC6: HC
EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWV
55

10G + Linker 3
RQAPGKGLEWVSSISGSGRDTLYADSVKGRFTISRDNA

+ SP34.185
KTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTV

scFv mask +
SSGGGGSGGGSGGVYCGPEFDESVGCMGGGGSGGGSG

Non-cleavable
GGGSGGASSGAGGSGGGSEVQLVESGGGLVQPGGSLK

linker 2 +
LSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKY

SP34.185 scFv

NNYAT
YYADSVKDRFTISRDDSKNTAYLQMNNLKTED

(VH-linker 1-
TAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGG

VL) + Linker
GSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSST

2 + EGFR Fab

GAVTSGNY
PNWVQQKPGQAPRGLIGGTKFLAPGTPAR

HC (N88Q)
FSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVF

GGGTKLTVLGGGGSQVQLKQSGPGLVQPSQSLSITCTV

SGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYN

TPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARAL

TYYDYEFAY
WGQGTLVTVSAASTKGPSVFPLAPSSKST

SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA

VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV

DKKVEPKSC

PC7: LC
GGPCRSHIDVAKPICVGGGGSSGGSAAGLLAPPGGLSG
56

EGFR Fab
RSDAGGGGSDILLTQSPVILSVSPGERVSFSCRASQSIGT

mask +

N
IHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDF

cleavable linker
TLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRT

3 + EGFR Fab
VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ

LC v2
WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD

YEKHKVYACEVTHQGLSSPVTKSFNRGEC

PC7: HC
EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWV
57

10G + Linker 3
RQAPGKGLEWVSSISGSGRDTLYADSVKGRFTISRDNA

+ SP34.185
KTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTV

scFv mask +
SSGGGGSGGGSGGVYCGPEFDESVGCMGSSGGSAAGL

Cleavable
LAPPGGLSGRSDAGGGGSEVQLVESGGGLVQPGGSLKL

Linker 4 +
SCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYN

SP34.185 scFv

NYAT
YYADSVKDRFTISRDDSKNTAYLQMNNLKTEDT

(VH-linker 1-
AVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGG

VL) + Linker
SGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTG

2 + EGFR Fab

AVTSGNY
PNWVQQKPGQAPRGLIGGTKFLAPGTPARF

HC (N88Q)
SGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVF

GGGTKLTVLGGGGSQVQLKQSGPGLVQPSQSLSITCTV

SGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYN

TPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARAL

TYYDYEFAY
WGQGTLVTVSAASTKGPSVFPLAPSSKST

SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA

VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV

DKKVEPKSC

PC8: LC
GGPCRSHIDVAKPICVGGGGSGGLSGRSDAGSPLGLAG
58

EGFR Fab
SGGSDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWY

mask +
QQRINGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINS

cleavable linker
VESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPS

2 + EGFR Fab
VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN

LC v2
ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK

VYACEVTHQGLSSPVTKSFNRGEC

PC8: HC
EVQLVESGGGLVQPGGSLRLSCAASGSTFYTAVMGWV
59

Anti-albumin +
RQAPGKGLEWVAAIRWTALTTSYADSVKGRFTISRDG

Linker 3 +
AKTTLYLQMNSLRPEDTAVYYCAARGTLGLFTTADS

SP34.185 scFv

YDY
WGQGTLVTVSSGGGGSGGGSGGVYCGPEFDESVG

mask +
CMGGGGSGGGLSGRSDAGSPLGLAGSGGGSEVQLVES

Cleavable
GGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGK

Linker 1 +
GLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNT

SP34.185 scFv
AYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYW

(VH-linker 1-
GQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTV

VL) + Linker
SPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGL

2 + EGFR Fab
IGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAE

HC (N88Q)
YYCVLWYSNRWVFGGGTKLTVLGGGGSQVQLKQSGP

GLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLE

WLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMN

SLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSAA

STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS

WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT

QTYICNVNHKPSNTKVDKKVEPKSC

PC9: LC
GGPCRSHIDVAKPICVGGGGSGGLSGRSDAGSPLGLAG
60

EGFR Fab
SGGSDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWY

mask +
QQRINGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINS

cleavable linker
VESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPS

2 + EGFR Fab
VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN

LC v2
ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK

VYACEVTHQGLSSPVTKSFNRGEC

PC9: HC
EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWV
61

10G + Linker 3
RQAPGKGLEWVSSISGSGRDTLYADSVKGRFTISRDNA

+ SP34.185
KTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTV

scFv mask +
SSGGGGSGGGSGGVYCGPEFDESVGCMGGGGSGGGLS

Cleavable
GRSDAGSPLGLAGSGGGSEVQLVESGGGLVQPGGSLKL

Linker 1 +
SCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYN

SP34.185 scFv

NYAT
YYADSVKDRFTISRDDSKNTAYLQMNNLKTEDT

(VH-linker 1-
AVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGG

VL) + Linker
SGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTG

2 + EGFR Fab

AVTSGNY
PNWVQQKPGQAPRGLIGGTKFLAPGTPARF

HC (N88Q)
SGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVF

GGGTKLTVLGGGGSQVQLKQSGPGLVQPSQSLSITCTV

SGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYN

TPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARAL

TYYDYEFAY
WGQGTLVTVSAASTKGPSVFPLAPSSKST

SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA

VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV

DKKVEPKSC

PC10: LC
EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW
62

SP34.185 scFv
VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTIS

(VH-linker 1-
RDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYI

VL) + Linker

SYWAY
WGQGTLVTVSSGGGGSGGGGSGGGGSQTVVT

2 + EGFR Fab
QEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKP

LC
GQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGV

QPEDEAEYYCVLWYSNRWVFGGGTKLTVLGGGGSQI

LLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTN

GSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDI

ADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPP

SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG

NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE

VTHQGLSSPVTKSFNRGEC

PC10: HC
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVR
63

EGFR Fab HC
QSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKS

(N88Q)
QVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQG

TLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKD

YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV

TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

PC11: LC
EVQLVESGGGLVQPGGSLRLSCAASGSTFYTAVMGWV
64

Anti-albumin +
RQAPGKGLEWVAAIRWTALTTSYADSVKGRFTISRDG

Linker 3 +
AKTTLYLQMNSLRPEDTAVYYCAARGTLGLFTTADS

SP34.185 scFv

YDY
WGQGTLVTVSSGGGGSGGGSGGVYCGPEFDESVG

mask +
CMGGGGSGGGLSGRSDAGSPLGLAGSGGGSEVQLVES

Cleavable
GGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGK

Linker 1 +
GLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNT

SP34.185 scFv
AYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYW

(VH-linker 1-
GQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTV

VL) + Linker
SPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGL

2 + EGFR Fab
IGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAE

LC
YYCVLWYSNRWVFGGGTKLTVLGGGGSQILLTQSPVI

LSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIK

YAS
ESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQ

QNNNWPTT
FGAGTKLELKRTVAAPSVFIFPPSDEQLKS

GTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT

EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS

SPVTKSFNRGEC

PC11: HC
GGPCRSHIDVAKPICVGGGGSGGLSGRSDAGSPLGLAG
65

EGFR Fab
SGGSQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGV

mask +
HWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINK

cleavable linker
DNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAY

2 + EGFR Fab
WGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGC

HC (N88Q)
LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences according to SEQ ID NO: 44 and SEQ ID NO: 45. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with 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 amino acid sequences with 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 amino acid sequences with at least 99% sequence identity to SEQ ID NO: 44 and SEQ ID NO: 45.

In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences according to SEQ ID NO: 46 and SEQ ID NO: 47. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with 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 amino acid sequences with 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 amino acid sequences with at least 99% sequence identity to SEQ ID NO: 46 and SEQ ID NO: 47.

In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences according to SEQ ID NO: 48 and SEQ ID NO: 49. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with 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 amino acid sequences with 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 amino acid sequences with at least 99% sequence identity to SEQ ID NO: 48 and SEQ ID NO: 49.

In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences according to SEQ ID NO: 50 and SEQ ID NO: 51. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with 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 amino acid sequences with 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 amino acid sequences with at least 99% sequence identity to SEQ ID NO: 50 and SEQ ID NO: 51.

In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences according to SEQ ID NO: 52 and SEQ ID NO: 53. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with 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 amino acid sequences with 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 amino acid sequences with at least 99% sequence identity to SEQ ID NO: 52 and SEQ ID NO: 53.

In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences according to SEQ ID NO: 54 and SEQ ID NO: 55. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with 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 amino acid sequences with at least 95% sequence identity SEQ ID NO: 54 and SEQ ID NO: 55. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 99% sequence identity to SEQ ID NO: 54 and SEQ ID NO: 55.

In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences according to SEQ ID NO: 56 and SEQ ID NO: 57. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with 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 amino acid sequences with at least 95% sequence identity SEQ ID NO: 56 and SEQ ID NO: 57. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 99% sequence identity to SEQ ID NO: 56 and SEQ ID NO: 57.

In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences according to SEQ ID NO: 58 and SEQ ID NO: 59. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with 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 amino acid sequences with at least 95% sequence identity SEQ ID NO: 58 and SEQ ID NO: 59. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 99% sequence identity to SEQ ID NO: 58 and SEQ ID NO: 59.

In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences according to SEQ ID NO: 60 and SEQ ID NO: 61. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with 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 amino acid sequences with at least 95% sequence identity SEQ ID NO: 60 and SEQ ID NO: 61. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 99% sequence identity to SEQ ID NO: 60 and SEQ ID NO: 61.

In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences according to SEQ ID NO: 62 and SEQ ID NO: 63. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with 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 amino acid sequences with at least 95% sequence identity SEQ ID NO: 62 and SEQ ID NO: 63. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 99% sequence identity to SEQ ID NO: 62 and SEQ ID NO: 63.

In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences according to SEQ ID NO: 64 and SEQ ID NO: 65. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with 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 amino acid sequences with at least 95% sequence identity SEQ ID NO: 64 and SEQ ID NO: 65. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 99% sequence identity to SEQ ID NO: 64 and SEQ ID NO: 65.

Polypeptides or polypeptide complexes, in some embodiments, comprise 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 instances, the sequence comprises at least or about 95% homology to SEQ ID NOs: 44-65. In some instances, the sequence comprises at least or about 97% homology to SEQ ID NOs: 44-65. In some instances, the sequence comprises at least or about 99% homology to SEQ ID NOs: 44-65. In some instances, the sequence comprises at least or about 100% homology to SEQ ID NOs: 44-65. In some instances, the sequence comprises 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: 44, 46, 48, 50, 52, 54, 56, 58, 60, 63, or 65. In some instances, the sequence comprises 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 instances, the sequence comprises 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, 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 of any one of SEQ ID NOs: 61 or 64.

As used herein, the term “percent (%) amino acid sequence identity” with respect to a sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, 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 needed to achieve maximal alignment over the full length of the sequences being compared. In situations where ALIGN-2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X/Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where 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 equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

Disclosed herein, in some embodiments, are polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 1 (FIG. 1A), 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 P₁is linked to a N-terminus of the heavy chain variable domain of the scFv with a linking moiety (L₁) that is a substrate for a tumor specific protease, and P₁is further linked to a half-life extending molecule; 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, wherein the Fab light chain polypeptide is linked to a C terminus of the light chain variable domain of the scFv, and wherein the Fab is linked to P₂and L₂, wherein P₂comprises a peptide that impairs binding to EGFR; and L₂comprises a linking moiety that connects the Fab heavy chain polypeptide to P₂and is a substrate for a tumor specific protease.

Disclosed herein, in some embodiments, are polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 2 (FIG. 1B), 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 P₁is linked to a N-terminus of the heavy chain variable domain of the scFv with a linking moiety (L₁) that is a substrate for a tumor specific protease, and P₁is further linked to a half-life extending molecule; 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, wherein the Fab heavy chain polypeptide is linked to a C terminus of the light chain variable domain of the scFv, and wherein the Fab is linked to P₂and L₂, wherein P₂comprises a peptide that impairs binding to EGFR; and L₂comprises a linking moiety that connects the Fab light chain polypeptide to P₂and is a substrate for a tumor specific protease.

Polynucleotides Encoding Polypeptides or Polypeptide Complexes

Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes as disclosed herein. In some embodiments, the polypeptides or polypeptide complexes comprise an antibody or an antibody fragment. In some embodiments, the polypeptides or polypeptide complexes comprise a Fab and a single chain variable fragment (scFv).

Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes according to Formula I:

Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes comprising Formula I:

Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes according to Formula Ia:

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Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes according to Formula II:

Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes comprising Formula II:

Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes according to Formula II:

Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes comprising Formula II:

Disclosed herein, in some embodiments, are isolated nucleic acid molecules encoding polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 1 (FIG. 1A), 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 P₁is linked to a N-terminus of the heavy chain variable domain of the scFv with a linking moiety (L₁) that is a substrate for a tumor specific protease, and P₁is further linked to a half-life extending molecule; 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, wherein the Fab light chain polypeptide is linked to a C terminus of the light chain variable domain of the scFv, and wherein the Fab is linked to P₂and L₂, wherein P₂comprises a peptide that impairs binding to EGFR; and L₂comprises a linking moiety that connects the Fab heavy chain polypeptide to P₂and is a substrate for a tumor specific protease.

Disclosed herein, in some embodiments, are isolated nucleic acid molecules encoding polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 2 (FIG. 1B), 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 P₁is linked to a N-terminus of the heavy chain variable domain of the scFv with a linking moiety (L₁) that is a substrate for a tumor specific protease, and P₁is further linked to a half-life extending molecule; 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, wherein the Fab heavy chain polypeptide is linked to a C terminus of the light chain variable domain of the scFv, and wherein the Fab is linked to P₂and L₂, wherein P₂comprises a peptide that impairs binding to EGFR; and L₂comprises a linking moiety that connects the Fab light chain polypeptide to P₂and is a substrate for a tumor specific protease.

Pharmaceutical Compositions

Disclosed herein, in some embodiments, are pharmaceutical compositions comprising: (a) the polypeptides or polypeptide complexes as disclosed herein; and (b) a pharmaceutically acceptable excipient.

In some embodiments, the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes according to Formula I:

In some embodiments, the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes comprising Formula I:

In some embodiments, the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes according to Formula Ia:

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and (b) a pharmaceutically acceptable excipient.

In some embodiments, the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes according to Formula II:

In some embodiments, the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes comprising Formula II:

In some embodiments, the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes according to Formula II:

In some embodiments, the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes comprising Formula II:

Disclosed herein, in some embodiments, the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 1 (FIG. 1A), 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 P₁is linked to a N-terminus of the heavy chain variable domain of the scFv with a linking moiety (L₁) that is a substrate for a tumor specific protease, and P₁is further linked to a half-life extending molecule; 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, wherein the Fab light chain polypeptide is linked to a C terminus of the light chain variable domain of the scFv, and wherein the Fab is linked to P₂and L₂, wherein P₂comprises a peptide that impairs binding to EGFR; and L₂comprises a linking moiety that connects the Fab heavy chain polypeptide to P₂and is a substrate for a tumor specific protease; and (b) a pharmaceutically acceptable excipient.

Disclosed herein, in some embodiments, the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 2 (FIG. 1B), 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 P₁is linked to a N-terminus of the heavy chain variable domain of the scFv with a linking moiety (L₁) that is a substrate for a tumor specific protease, and P₁is further linked to a half-life extending molecule; 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, wherein the Fab heavy chain polypeptide is linked to a C terminus of the light chain variable domain of the scFv, and wherein the Fab is linked to P₂and L₂, wherein P₂comprises a peptide that impairs binding to EGFR; and L₂comprises a linking moiety that connects the Fab light chain polypeptide to P₂and is a substrate for a tumor specific protease; and (b) a pharmaceutically acceptable excipient.

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

For administration to a subject, the isolated polypeptide or polypeptide complex as disclosed herein, may be provided in a pharmaceutical composition together with one or more pharmaceutically acceptable carriers or excipients. The term “pharmaceutically acceptable carrier” includes, but is not limited to, any carrier that does not interfere with the effectiveness of the biological activity of the ingredients and that 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 solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Such carriers can be formulated by conventional methods and can be administered to the subject at a suitable dose. Preferably, the compositions are sterile. These compositions may also contain adjuvants such as preservative, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents.

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

The pharmaceutical composition may be adapted for administration by any appropriate route, including a parenteral (e.g., subcutaneous, intramuscular, or intravenous) route. Such compositions may be prepared by any method known in the art of pharmacy, for example by mixing the active ingredient with the carrier(s) or excipient(s) under sterile conditions.

Dosages of the substances of the present disclosure can vary between wide limits, depending upon the disease or disorder to be treated, the age and condition of the individual to be treated, etc. and a physician will ultimately determine appropriate dosages to be used.

Peptides that Impair Binding of Anti-EGFR Binding Domains to EGFR

Disclosed herein are polypeptide or polypeptide complex comprising an anti-EGFR binding domain that are linked to a peptide that impairs binding of the anti-EGFR binding domain to EGFR wherein the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 26, 71-96, 98-776 or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 26, 71-96, 98-776.

In some embodiments, the peptide comprises an amino acid sequence according to any one of 26, 71-96, 98-776.

In some embodiments, the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 26, 86-96 or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 26, 86-96.

In some embodiments, the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 26, 86-96.

In some embodiments, the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 98-776 or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any of SEQ ID NOs: 98-776.

In some embodiments, the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 98-776.

In some embodiments, the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 99-118 or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 99-118.

In some embodiments, the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 99-118.

In some embodiments, the peptide comprises the amino acid sequence according to SEQ ID NO: 71 or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to SEQ ID NO: 71.

In some embodiments, the peptide comprises the amino acid sequence according to SEQ ID NO: 71.

In some embodiments, the peptide comprises the amino acid sequence according to SEQ ID NO: 26 or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to SEQ ID NO: 26.

In some embodiments, the peptide comprises the amino acid sequence according to SEQ ID NO: 26.

In some embodiments, the peptide comprises the amino acid sequence according to SEQ ID NO: 115 or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to SEQ ID NO: 115. In some embodiments, the peptide comprises the amino acid sequence according to SEQ ID NO: 115.

In some embodiments, the peptide comprises the amino acid sequence according to SEQ ID NO: 116 or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to SEQ ID NO: 116. In some embodiments, the peptide comprises the amino acid sequence according to SEQ ID NO: 116.

Disclosed herein are polypeptide or polypeptide complex comprising an anti-EGFR binding domain that are linked to a peptide that impairs binding of the anti-EGFR binding to EGFR wherein the peptide comprises an amino acid sequence according to X₁—C—X₂—X₃—X₄—X₅-D-X₆-A-X₇—P—X₈—C—X₉wherein X₁is selected from P and L; X₂is selected from R, L, T, A, N, I, V, S, H, and P; X₃is selected from S, P, F, and Y; X₄is selected from H, L, Q, P, R, F, N; X₅is selected from I, F, Y, H, N, T, S, D, A, L, and V; X₆is selected from T, P, N, L, I, V, S, D, H, A, and Y; X₇is selected from K and Y; X₈is selected from I, P, L, and M; and X₉is selected from A, V, I, T, L, S, D, F, V, and H (SEQ ID NO: 841). In some embodiments, X₁is selected from P and L; X₂is selected from R, L, T, A, and N; X₃is selected from S, P, and F; X₄is selected from H, L, Q, and P; X₅is selected from I, F, Y, H, N, and T; X₆is selected from T, P, N, L, I, and V, X₇is K; X₈is I; and X₉is selected from A, V, I, T, L, and S. In some embodiments, X₁is P; X₂is selected from R, L, and T; X₃is S; X₄is selected from H, L, Q, and P; X₅is selected from I, F, Y, and T; X₆is selected from T, P, N, and V; X₇is K; X₈is I; and X₉is selected from A, V, and I;

In some embodiments, the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 98-776. In some embodiments, the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 99-118. In some embodiments, the peptide comprises the amino acid sequence according to SEQ ID NO: 26. In some embodiments, the peptide comprises the amino acid sequence according to SEQ ID NO: 115. In some embodiments, the peptide comprises the amino acid sequence according to SEQ ID NO: 116.

In some embodiments, the anti-EGFR binding domain comprises an antibody or an 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, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 comprise: HC-CDR1: SEQ ID NO: 15, HC-CDR2: SEQ ID NO: 16, and HC-CDR3: SEQ ID NO: 17; and the anti-EGFR binding domain comprises light chain complementarity determining regions CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the Fab comprise LC-CDR1: SEQ ID NO: 18, LC-CDR2: SEQ ID NO:19, and LC-CDR3: SEQ ID NO: 20.

In some embodiments, the antibody or antibody fragment comprises the Fab. In some embodiments, the anti-EGFR binding domain comprises an amino acid sequences according to any of SEQ ID NOs: 21-24.

In some embodiments, the anti-EGFR binding domain is linked to the peptide through a linking moiety (L₁). In some embodiments, L₁is a substrate for a tumor specific protease. In some embodiments, L₁is bound to N-terminus of the anti-EGFR binding domain. In some embodiments, L₁is bound to C-terminus of the anti-EGFR binding domain. In some embodiments, L₁is a peptide sequence having at least 5 to no more than 50 amino acids. In some embodiments, L₁is a peptide sequence having at least 10 to no more than 30 amino acids. In some embodiments, L₁is a peptide sequence having at least 10 amino acids. In some embodiments, L₁is a peptide sequence having at least 18 amino acids. In some embodiments, L₁is a peptide sequence having at least 26 amino acids. In some embodiments, L₁has a formula comprising (G₂S)_n, wherein n is an integer from 1 to 3 (SEQ ID NO: 840). In some embodiments, L₁has a formula selected from the group consisting of (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 becomes unbound from the anti-EGFR binding domain when L₁is cleaved by the 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 a matrix metalloprotease (MMP), serine protease, cysteine protease, threonine protease, and aspartic 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, L₁comprises a urokinase cleavable amino acid sequence, a matriptase cleavable amino acid sequence, matrix metalloprotease cleavable amino acid sequence, or a legumain cleavable amino acid sequence. In some embodiments, L₁comprises an amino acid sequence according to any of SEQ ID NO: 30 or 31. In some embodiments, L₁comprises an amino acid sequence according to SEQ ID NO: 27-35. In some embodiments, L₁comprises an amino acid sequence of 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 that has 1, 2, or 3 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, the isolated polypeptide or polypeptide complex further comprises a half-life extending molecule (H₁). In some embodiments, the half-life extending molecule is linked to the peptide. In some embodiments, H₁comprises a polymer. In some embodiments, the polymer is polyethylene glycol (PEG). In some embodiments, H₁comprises albumin. In some embodiments, H₁comprises an Fc domain. In some embodiments, the albumin is serum albumin. In some embodiments, the albumin is human serum albumin. In some embodiments, H₁comprises a polypeptide, a ligand, or a small molecule. In some embodiments, the polypeptide, the ligand or the small molecule binds serum protein or a fragment thereof, a circulating immunoglobulin or a fragment thereof, or CD35/CR1. In some embodiments, the serum protein comprises a thyroxine-binding protein, a transthyretin, a 1-acid glycoprotein, a transferrin, transferrin receptor or a transferrin-binding portion thereof, a fibrinogen, or an 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 or humanized antibody. In some embodiments, the single domain antibody is 645gH1gL1. In some embodiments, the single domain antibody is 645dsgH5gL4. In some embodiments, the single domain antibody is 23-13-A01-sc02. In some embodiments, the single domain antibody is A 10m3 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, the HC-CDR2, and the HC-CDR3 of the single domain antibody comprise: HC-CDR1: SEQ ID NO: 36, HC-CDR2: SEQ ID NO: 37, and HC-CDR3: SEQ ID NO: 38. In some embodiments, the single domain antibody comprises an amino acid sequence according to SEQ ID NO: 69. In some embodiments, the single domain antibody comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the single domain antibody comprise: HC-CDR1: SEQ ID NO: 36, HC-CDR2: SEQ ID NO: 37, and HC-CDR3: SEQ ID NO: 38. In some embodiments, the single domain antibody comprises an amino acid sequence according to 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, H₁comprises a linking moiety (L₃) that connects H₁to the peptide. In some embodiments, L₃is a peptide sequence having at least 5 to no more than 50 amino acids. In some embodiments, L₃is a peptide sequence having at least 10 to no more than 30 amino acids. In some embodiments, L₃is a peptide sequence having at least 10 amino acids. In some embodiments, L₃is a peptide sequence having at least 18 amino acids. In some embodiments, L₃is a peptide sequence having at least 26 amino acids. In some embodiments, L₃has a formula selected from the group consisting of (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, L₃comprises an amino acid sequence according to SEQ ID NO: 29.

Methods of Treatment

Described herein, in some embodiments, are polypeptides or polypeptide complexes, wherein the polypeptides or polypeptide complexes comprise a long half-life. In some instances, the half-life of the polypeptides or polypeptide complexes 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 instances, the half-life of the polypeptides or polypeptide complexes is in a range of about 12 hours to about 300 hours, about 20 hours to about 280 hours, about 40 hours to about 240 hours, about 60 hours to about 200 hours, or about 80 hours to about 140 hours.

Described herein, in some embodiments, are polypeptide or polypeptide complexes administered as once weekly. In some embodiments, the polypeptide or polypeptide complexes are administered once weekly by intravenous, intramuscular, intralesional, topical, subcutaneous, infusion, or oral. In some embodiments, the polypeptide or polypeptide complexes are administered once weekly by bolus injection. In some embodiments, the polypeptide or polypeptide complexes are administered once weekly by continuous infusion. In some embodiments, the isolated polypeptide or polypeptide complex is administered to the subject once a week as a continuous infusion over a period of no more than 60 minutes. In some embodiments, the isolated polypeptide or polypeptide complex is administered to the subject once a week as a continuous intravenous infusion over a period of no more than 30 minutes. In some embodiments, the isolated polypeptide or polypeptide complex is administered to the subject once a week as a continuous intravenous infusion over a period of at least 10 minutes.

In some embodiments, the isolated polypeptide or polypeptide complex is administered to the subject once a week and the isolated polypeptide or polypeptide complex has a half-life of at least 30 hours. In some embodiments, the isolated polypeptide or polypeptide complex is administered to the 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 the 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 the 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 the 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 the 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 the 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 the 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 the subject once a week and the isolated polypeptide or polypeptide complex has a half-life of at least 115 hours.

Production of Antibodies that Bind to EGFR and CD3

In some embodiments, polypeptides described herein (e.g., antibodies and its binding fragments) are produced using any method known in the art to be useful for the synthesis of polypeptides (e.g., antibodies), in particular, by chemical synthesis or by recombinant expression, and are preferably produced by recombinant expression techniques.

In some instances, an antibody or its binding fragment thereof is expressed recombinantly, and the nucleic acid encoding the antibody or its binding fragment is assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., 1994, BioTechniques 17:242), which involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligation of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.

Alternatively, a nucleic acid molecule encoding an antibody is optionally generated from a suitable source (e.g., an antibody cDNA library, or cDNA library generated from any tissue or cells expressing the immunoglobulin) by PCR amplification using synthetic primers hybridizable to the 3′ and 5′ ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence.

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

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

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).

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

In some embodiments, a variety of host-expression vector systems is utilized to express an antibody, or its binding fragment described herein. Such host-expression systems represent vehicles by which the coding sequences of the antibody is produced and subsequently purified, but also represent cells that are, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody or its binding fragment in situ. These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing an antibody or its binding fragment coding sequences; yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing an antibody or its binding fragment coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing an antibody or its binding fragment coding sequences; plant cell systems infected with recombinant virus 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 an antibody or its binding fragment coding sequences; 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. the adenovirus late promoter; the vaccinia virus 7.5K promoter).

For long-term, high-yield production of recombinant proteins, stable expression is preferred. In some instances, cell lines that stably express an antibody are optionally engineered. Rather than using expression vectors that contain 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. Following the introduction of the foreign DNA, engineered cells are then allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci that in turn are cloned and expanded into cell lines. This method can advantageously be used to engineer cell lines which express the antibody or its binding fragments.

In some instances, a number of selection systems are used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 192, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes are employed in tk-, hgprt- or aprt-cells, respectively. Also, antimetabolite resistance are used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Proc. Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 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 TECH11(5):155-215) and hygro, which confers resistance to hygromycin (Santerre et al., 1984, Gene 30:147). Methods commonly known in the art of recombinant DNA technology which 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).

In some instances, the expression levels of an antibody 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)). When a marker in the vector system expressing an antibody is amplifiable, an increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the nucleotide sequence of the antibody, production of the antibody will also increase (Crouse et al., 1983, Mol. Cell Biol. 3:257).

In some instances, any method known in the art for purification of an antibody is used, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.

Expression Vectors

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

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

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

Exemplary algae vectors include pChlamy-4 vector or MCS vector.

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

In some instances, a cell-free system is a mixture of cytoplasmic and/or nuclear components from a cell and is used for in vitro nucleic acid synthesis. In some cases, a cell-free system utilizes either prokaryotic cell components or eukaryotic cell components. Sometimes, a nucleic acid synthesis is obtained in a cell-free system based on for example Drosophila cell, Xenopus egg, or HeLa cells. Exemplary cell-free systems include, but are not limited to, E. coli S30 Extract system, E. coli T7 S30 system, or PURExpress@.

Host Cells

In some embodiments, a host cell includes any suitable cell such as a naturally derived cell or a genetically modified cell. In some instances, a host cell is a production host cell. In some instances, a host cell is a eukaryotic cell. In other instances, a host cell is a prokaryotic cell. In some cases, a eukaryotic cell includes fungi (e.g., yeast cells), animal cell or plant cell. In some cases, a prokaryotic cell is a bacterial cell. Examples of bacterial cell include gram-positive bacteria or gram-negative bacteria. Sometimes the gram-negative bacteria is anaerobic, rod-shaped, or both.

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

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, Stb12™, Stb13™, or Stb14™.

In some instances, animal cells include a cell from a vertebrate or from an invertebrate. In some cases, an animal cell includes a cell from a marine invertebrate, fish, insects, amphibian, reptile, or mammal. In some cases, a fungus cell includes a yeast cell, such as brewer's yeast, baker's yeast, or wine yeast.

Fungi include ascomycetes such as yeast, mold, filamentous fungi, basidiomycetes, or zygomycetes. In some instances, yeast includes Ascomycota or Basidiomycota. In some cases, Ascomycota includes Saccharomycotina (true yeasts, e.g. Saccharomyces cerevisiae (baker's yeast)) or Taphrinomycotina (e.g. Schizosaccharomycetes (fission yeasts)). In some cases, Basidiomycota includes Agaricomycotina (e.g. Tremellomycetes) or Pucciniomycotina (e.g. Microbotryomycetes).

Exemplary yeast or filamentous fungi include, for example, the genus: Saccharomyces, Schizosaccharomyces, Candida, Pichia, Hansenula, Kluyveromyces, Zygosaccharomyces, Yarrowia, Trichosporon, Rhodosporidi, Aspergillus, Fusarium, or Trichoderma. Exemplary yeast 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 lusitaniae, Rhodotorula mucilaginosa, Pichia metanolica, Pichia angusta, Pichia pastoris, Pichia anomala, Hansenula polymorpha, Kluyveromyces lactis, Zygosaccharomyces rouxii, Yarrowia lipolytica, Trichosporon pullulans, Rhodosporidium toru-Aspergillus niger, Aspergillus nidulans, Aspergillus awamori, Aspergillus oryzae, Trichoderma reesei, Yarrowia lipolytica, Brettanomyces bruxellensis, Candida stellata, Schizosaccharomyces pombe, Torulaspora delbrueckii, Zygosaccharomyces bailii, Cryptococcus neoformans, Cryptococcus gattii, or Saccharomyces boulardii.

Exemplary yeast host cells include, but are not limited to, Pichia pastoris yeast strains such as GS115, KM71H1 SMD1168, SMD1168H1 and X-33; and Saccharomyces cerevisiae yeast strain such as INVSc1.

In some instances, additional animal cells include cells obtained from a mollusk, arthropod, annelid or sponge. In some cases, an additional animal cell is a mammalian cell, e.g., from a primate, ape, equine, bovine, porcine, canine, feline or rodent. In some cases, a rodent includes mouse, rat, hamster, gerbil, hamster, chinchilla, fancy rat, or guinea pig.

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 line, GS-CHO cell line, HepaRG™ cells, T-REx™ Jurkat cell line, Per.C6 cells, T-REx™-293 cell line, T-REx™—CHO cell line, and T-REx™-HeLa cell line.

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

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

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

Articles of Manufacture

In another aspect of the invention, an article of manufacture containing materials useful for the treatment, prevention and/or diagnosis of the disorders described above 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, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper that is pierceable by a hypodermic injection needle). At least one active agent in the composition is a bispecific antibody comprising a first antigen-binding site that specifically binds to CD3 and a second antigen-binding site that specifically binds to EGFR as defined herein before.

The label or package insert indicates that the composition is used for treating the condition of choice. Moreover, the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises the bispecific antibody of the invention; and (b) a second container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherwise therapeutic agent. The article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the compositions can be used to treat a particular condition.

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

Certain Definitions

The terminology used herein is for the purpose of describing particular cases only and is not intended to be limiting. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”

The term “antibody” is used in the broadest sense and covers fully assembled antibodies, antibody fragments that can bind antigen, for example, Fab, F(ab′)2, Fv, single chain antibodies (scFv), diabodies, antibody chimeras, hybrid antibodies, bispecific antibodies, and the like.

The term “complementarity determining region” or “CDR” is a segment of the variable region of an antibody that is complementary in structure to the epitope to which the antibody binds and is more variable than the rest of the variable region. Accordingly, a CDR is sometimes referred to as hypervariable region. A variable region comprises three CDRs. CDR peptides can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are 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).

The term “Fab” refers to a protein that contains 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 residue(s) of the constant domains bear a free thiol group. Fab′ fragments are produced by reducing the F(ab′)2 fragment's heavy chain disulfide bridge. Other chemical couplings of antibody fragments are also known.

A “single-chain variable fragment (scFv)” is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of an antibody, connected with a short linker peptide of ten to about 25 amino acids. The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa. This protein retains the specificity of the original antibody, despite removal of the constant regions and the introduction of the linker. scFv antibodies are, e.g. described in Houston, J. S., Methods in Enzymol. 203 (1991) 46-96). In addition, antibody fragments comprise single chain polypeptides having the characteristics of a VH domain, namely being able to assemble together with a VL domain, or of a VL domain, namely being able to assemble together with a VH domain to a functional antigen binding site and thereby providing the antigen binding property of full length antibodies.

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 now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the disclosure. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

EMBODIMENTS

Embodiment 1 comprises an isolated polypeptide or polypeptide complex according to Formula I: A₂-A₁-L₁-P₁—H₁wherein A₁comprises a first antigen recognizing molecule that binds to an effector cell antigen; P₁comprises a peptide that binds to A₁; L₁comprises a linking moiety that connects A₁to P₁and is a substrate for a tumor specific protease; H₁comprises a half-life extending molecule; and A₂comprises a second antigen recognizing molecule that binds to epidermal growth factor receptor (EGFR).

Embodiment 2 comprises the isolated polypeptide or polypeptide complex of embodiment 1, wherein the first antigen recognizing molecule comprises an antibody or antibody fragment.

Embodiment 3 comprises the isolated polypeptide or polypeptide complex of embodiment 1, wherein first antigen recognizing molecule comprises an antibody or antibody fragment that is human or humanized.

Embodiment 4 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-3, wherein L₁is bound to N-terminus of the first antigen recognizing molecule.

Embodiment 5 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-3, wherein A₂is bound to C-terminus of the first antigen recognizing molecule.

Embodiment 6 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-3, wherein L₁is bound to C-terminus of the first antigen recognizing molecule.

Embodiment 7 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-3, wherein A₂is bound to N-terminus of the first antigen recognizing molecule.

Embodiment 8 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 2-7, wherein the antibody or antibody fragment comprises a single chain variable fragment, a single domain antibody, or a Fab fragment.

Embodiment 9 comprises the isolated polypeptide or polypeptide complex of embodiment 8, wherein A₁is the single chain variable fragment (scFv).

Embodiment 10 comprises the isolated polypeptide or polypeptide complex of embodiment 9, wherein the scFv comprises a scFv heavy chain polypeptide and a scFv light chain polypeptide.

Embodiment 11 comprises the isolated polypeptide or polypeptide complex of embodiment 8, wherein A₁is the single domain antibody,

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 (VHH) of a camelid derived single domain antibody.

Embodiment 13 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-12, wherein A₁comprises an anti-CD3ε single chain variable fragment.

Embodiment 14 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-12, wherein A₁comprises an anti-CD3ε single chain variable fragment that has a KD binding of 1 μM or less to CD3 on CD3 expressing cells.

Embodiment 15 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-14, wherein the effector cell antigen comprises CD3.

Embodiment 16 comprises the isolated polypeptide or polypeptide complex of embodiment 1, wherein A₁comprises a variable light chain and variable heavy chain each of which is capable of specifically binding to human CD3.

Embodiment 17 comprises the isolated polypeptide or polypeptide complex of embodiment 1, wherein A₁comprises complementary determining regions (CDRs) 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.

Embodiment 18 comprises the isolated polypeptide or polypeptide complex of embodiment 1, wherein the isolated polypeptide or polypeptide complex of Formula I binds to an effector cell when L₁is cleaved by the tumor specific protease.

Embodiment 19 comprises the isolated polypeptide or polypeptide complex of embodiment 1, wherein the isolated polypeptide or polypeptide complex of Formula I binds to an effector cell when L₁is cleaved by the tumor specific protease and A₁binds to the effector cell.

Embodiment 20 comprises the isolated polypeptide or polypeptide complex of embodiment 19, wherein the effector cell is a T cell.

Embodiment 21 comprises the isolated polypeptide or polypeptide complex of embodiment 1, wherein A₁binds to a polypeptide that is part of a TCR-CD3 complex on the effector cell.

Embodiment 22 comprises the isolated polypeptide or polypeptide complex of embodiment 21, wherein the polypeptide that is part of the TCR-CD3 complex is human CD3R.

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

Embodiment 24 comprises the isolated polypeptide or polypeptide complex of embodiment 1, wherein the effector cell antigen comprises CD3, and A₁comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 ofA₁comprise LC-CDR1: SEQ ID NO: 1, LC-CDR2: SEQ ID NO: 2, and LC-CDR3: SEQ ID NO: 3; and A₁comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of A₁comprise: HC-CDR1: SEQ ID NO: 4, HC-CDR2: SEQ ID NO: 5, and HC-CDR3: SEQ ID NO: 6.

Embodiment 25 comprises the isolated polypeptide or polypeptide complex of embodiment 1, wherein the effector cell antigen comprises CD3, and the scFv comprises an amino acid sequence according to SEQ ID NO: 13.

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

Embodiment 27 comprises the isolated polypeptide or polypeptide complex of embodiment 1, wherein the effector cell antigen comprises CD3, and A₁comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of A₁comprise: LC-CDR1: SEQ ID NO: 7, LC-CDR2: SEQ ID NO: 8, and LC-CDR3: SEQ ID NO: 9; and A₁comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of A₁comprise: HC-CDR1: SEQ ID NO: 10, HC-CDR2: SEQ ID NO: 11, and HC-CDR3: SEQ ID NO: 12.

Embodiment 28 comprises the isolated polypeptide or polypeptide complex of embodiment 1, wherein the effector cell antigen comprises CD3, and the scFv comprises an amino acid sequence according to SEQ ID NO: 14.

Embodiment 29 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-28, wherein second antigen recognizing molecule comprises an antibody or antibody fragment.

Embodiment 30 comprises 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.

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), a variable domain (VHH) of a camelid derived single domain antibody.

Embodiment 32 comprises the isolated polypeptide or polypeptide complex of embodiment 29, wherein the antibody or antibody fragment thereof is humanized or human.

Embodiment 33 comprises the isolated polypeptide or polypeptide complex of embodiment 30, wherein A₂is the Fab.

Embodiment 34 comprises 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.

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

Embodiment 36 comprises the isolated polypeptide or polypeptide complex of embodiment 29, wherein A₂comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of A₂comprise LC-CDR1: SEQ ID NO: 15, LC-CDR2: SEQ ID NO:16, and LC-CDR3: SEQ ID NO: 17; and A₂comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of A₂comprise: HC-CDR1: SEQ ID NO: 18, HC-CDR2: SEQ ID NO: 19, and HC-CDR3: SEQ ID NO: 20.

Embodiment 37 comprises the isolated polypeptide or polypeptide complex of embodiment 34, wherein the Fab light chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 21.

Embodiment 38 comprises the isolated polypeptide or polypeptide complex of embodiment 34, wherein the Fab light chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 22.

Embodiment 39 comprises the isolated polypeptide or polypeptide complex of embodiment 34, wherein Fab heavy chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 23.

Embodiment 40 comprises the isolated polypeptide or polypeptide complex of embodiment 34, wherein Fab heavy chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 24.

Embodiment 41 comprises the isolated polypeptide or polypeptide complex of embodiment 34, wherein the Fab light chain polypeptide of A₂is bound to a C-terminus of the single chain variable fragment (scFv) of A₁.

Embodiment 42 comprises the isolated polypeptide or polypeptide complex of embodiment 34, wherein the Fab heavy chain polypeptide of A₂is bound to a C-terminus of the single chain variable fragment (scFv) A₁.

Embodiment 43 comprises the isolated polypeptide or polypeptide complex of embodiment 34, wherein the Fab light chain polypeptide of A₂is bound to a N-terminus of the single chain variable fragment (scFv) of A₁.

Embodiment 44 comprises the isolated polypeptide or polypeptide complex of embodiment 34, wherein the Fab heavy chain polypeptide of A₂is bound to a N-terminus of the single chain variable fragment (scFv) A₁.

Embodiment 45 comprises the isolated polypeptide or polypeptide complex of embodiment 34, wherein the Fab heavy chain polypeptide of A₂is bound to the scFv heavy chain polypeptide of A₁.

Embodiment 46 comprises the isolated polypeptide or polypeptide complex of embodiment 34, wherein the Fab light chain polypeptide of A₂is bound to the scFv heavy chain polypeptide of A₁.

Embodiment 47 comprises the isolated polypeptide or polypeptide complex of embodiment 34, wherein the Fab heavy chain polypeptide of A₂is bound to the scFv light chain polypeptide of A₁.

Embodiment 48 comprises the isolated polypeptide or polypeptide complex of embodiment 34, wherein the Fab light chain polypeptide of A₂is bound to the scFv light chain polypeptide of A₁.

Embodiment 49 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-48, wherein A₂further comprises P₂and L₂, wherein P₂comprises a peptide that binds to A₂; and L₂comprises a linking moiety that connects A₂to P₂and is a substrate for a tumor specific protease.

Embodiment 50 comprises the isolated polypeptide or polypeptide complex of embodiment 49, wherein the isolated polypeptide or polypeptide complex is according to Formula Ia: P₂-L₂-A₂-A₁-L₁-P₁—H₁.

Embodiment 51 comprises the isolated polypeptide or polypeptide complex of embodiment 50, wherein the Fab heavy chain polypeptide of A₂is bound to the scFv heavy chain polypeptide of A₁and L₂is bound to the Fab light chain polypeptide of A₂.

Embodiment 52 comprises the isolated polypeptide or polypeptide complex of embodiment 50, wherein the Fab light chain polypeptide of A₂is bound to the scFv heavy chain polypeptide of A₁and L₂is bound to the Fab heavy chain polypeptide of A₂.

Embodiment 53 comprises the isolated polypeptide or polypeptide complex of embodiment 50, wherein the Fab heavy chain polypeptide of A₂is bound to the scFv light chain polypeptide of A₁and L₂is bound to the Fab light chain polypeptide of A₂.

Embodiment 54 comprises the isolated polypeptide or polypeptide complex of embodiment 50, wherein the Fab light chain polypeptide of A₂is bound to the scFv light chain polypeptide of A₁and L₂is bound to the Fab heavy chain polypeptide of A₂.

Embodiment 55 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-54, wherein P₁impairs binding of A₁to the effector cell antigen.

Embodiment 56 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-55, wherein P₁is bound to A₁through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bonding interactions, or a combination thereof.

Embodiment 57 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-56, wherein P₁has less than 70% sequence homology to the effector cell antigen.

Embodiment 58 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 49-57, wherein P₂impairs binding of A₂to EGFR.

Embodiment 59 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 49-58, wherein P₂is bound to A₂through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bonding interactions, or a combination thereof.

Embodiment 60 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 49-59, wherein P₂is bound to A₂at or near an antigen binding site.

Embodiment 61 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 49-60, wherein P₂has less than 70% sequence homology to EGFR.

Embodiment 62 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-61, wherein P₁or P₂comprises a peptide sequence of at least 10 amino acids in length.

Embodiment 63 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-62, wherein P₁or P₂comprises a peptide sequence of at least 10 amino acids in length and no more than 20 amino acids in length.

Embodiment 64 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-63, wherein P₁or P₂comprises a peptide sequence of at least 16 amino acids in length.

Embodiment 65 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-61, wherein P₁or P₂comprises a peptide sequence of no more than 40 amino acids in length.

Embodiment 66 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-61, wherein P₁or P₂comprises at least two cysteine amino acid residues.

Embodiment 67 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-61, wherein P₁or P₂comprises a cyclic peptide or a linear peptide.

Embodiment 68 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-61, wherein P₁or P₂comprises a cyclic peptide.

Embodiment 69 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-61, wherein P₁or P₂comprises a linear peptide.

Embodiment 70 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-61, wherein P₁comprises at least two cysteine amino acid residues.

Embodiment 71 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-61, wherein P₁comprises an amino acid sequence according to SEQ ID NO: 25.

Embodiment 72 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-61, wherein P₂comprises an amino acid sequence according to SEQ ID NO: 26.

Embodiment 73 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-61, wherein P₁comprises an amino acid sequence according to SEQ ID NO: 70.

Embodiment 74 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-61, wherein P₂comprises an amino acid sequence according to any one of SEQ ID NOs: 26, 71-96, 98-776, or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 26, 71-96, 98-776.

Embodiment 75 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-61, wherein P₂comprises an an amino acid sequence according to X₁—C—X₂—X₃—X₄—X₅-D-X₆-A-X₇—P—X₈—C—X₉wherein X₁is selected from P and L; X₂is selected from R, L, T, A, N, I, V, S, H, and P; X₃is selected from S, P, F, and Y; X₄is selected from H, L, Q, P, R, F, N; X₅is selected from I, F, Y, H, N, T, S, D, A, L, and V; X₆is selected from T, P, N, L, I, V, S, D, H, A, and Y; X₇is selected from K and Y; X₈is selected from I, P, L, and M; and X₉is selected from A, V, I, T, L, S, D, F, V, and H (SEQ ID NO: 841).

Embodiment 76 comprises the isolated polypeptide or polypeptide complex of embodiment 75, wherein X₁is selected from P and L; X₂is selected from R, L, T, A, and N; X₃is selected from S, P, and F; X₄is selected from H, L, Q, and P; X₅is selected from I, F, Y, H, N, and T; X₆is selected from T, P, N, L, I, and V; X₇is K; X₈is I; and X₉is selected from A, V, I, T, L, and S.

Embodiment 77 comprises the isolated polypeptide or polypeptide complex of embodiment 76, wherein X₁is P; X₂is selected from R, L, and T; X₃is S; X₄is selected from H, L, Q, and P; X₅is selected from I, F, Y, and T; X₆is selected from T, P, N, and V; X₇is K; X₈is I; and X₉is selected from A, V, and I.

Embodiment 78 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-77, wherein P₂comprises an amino acid sequence according to any one of SEQ ID NOs: 26, 86-96, or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 26, 86-96.

Embodiment 79 comprises the isolated polypeptide or polypeptide complex of embodiment 78, wherein P₂comprises an amino acid sequence according to any one of SEQ ID NOs: 26, 86-96.

Embodiment 80 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-77, wherein P₂comprises an amino acid sequence according to any one of SEQ ID NOs: 98-776 or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 98-776.

Embodiment 81 comprises the isolated polypeptide or polypeptide complex of embodiment 80, wherein P₂comprises an amino acid sequence according to any one of SEQ ID NOs: 98-776.

Embodiment 82 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-77, wherein P₂comprises an amino acid sequence according to any one of SEQ ID NOs: 99-118 or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 99-118.

Embodiment 83 comprises the isolated polypeptide or polypeptide complex of embodiment 82, wherein P₂comprises an amino acid sequence according to any one of SEQ ID NOs: 99-118.

Embodiment 84 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-77, wherein P₂comprises an amino acid sequence according to SEQ ID NO: 26 or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to SEQ ID NO: 26.

Embodiment 85 comprises the isolated polypeptide or polypeptide complex of embodiment 84, wherein P₂comprises the amino acid sequence according to SEQ ID NO: 26.

Embodiment 86 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-85, wherein P₁comprises an amino acid sequence according to any one of SEQ ID NOs: 25, 797-835, or 843-1690 or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 25, 797-835, or 843-1690.

Embodiment 87 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-85, wherein P₁comprises an amino acid sequence according to Z₁—Z₂—C—Z₄—P—Z₆-Z₇—Z₈—Z₉-Z₁₀—Z₁₁—Z₁₂—C—Z₁₄and Z₁is selected from D, Y, F, I, N, V, H, L, A, T, S, and P; Z₂is selected from D, Y, L, F, I, N, A, V, H, T, and S; Z₄is selected from G and W; Z₆is selected from E, D, V, and P; Z₇is selected from W, L, F, V, G, M, I, and Y; Z₈is selected from E, D, P, and Q; Z₉is selected from E, D, Y, V, F, W, P, L, and Q; Z₁₀is selected from S, D, Y, T, I, F, V, N, A, P, L, and H; Z₁₁is selected from I, Y, F, V, L, T, N, S, D, A, and H; Z₁₂is selected from F, D, Y, L, I, V, A, N, T, P, S, and H; and Z₁₄is selected from D, Y, N, F, I, P, V, A, T, H, L and S.

Embodiment 88 comprises the isolated polypeptide or polypeptide complex of embodiment 87, wherein Z₁is selected from D, Y, F, I, and N; Z₂is selected from D, Y, L, F, I, and N; Z₄is selected from G and W; Z₆is selected from E and D; Z₇is selected from W, L, F, and V; Z₈is selected from E and D; Z₉is selected from E, D, Y, and V; Z₁₀is selected from S, D, Y, T, and I; Z₁₁is selected from I, Y, F, V, L, and T; Z₁₂is selected from F, D, Y, L, I, V, A, and N; and Z₁₄is selected from D, Y, N, F, I, and P.

Embodiment 89 comprises the isolated polypeptide or polypeptide complex of embodiment 88, wherein Z₁is selected from D, Y, and F; Z₂is selected from D, Y, L, and F; Z₄is selected from G and W; Z₆is selected from E and D; Z₇, is selected from W, L, and F; Z₈is selected from E and D; Z₉is selected from E and D; Z₁₀is selected from S, D, and Y; Z₁₁is selected from I, Y, and F; Z₁₂is selected from F, D, Y, and L; and Z₁₄is selected from D, Y, and N.

Embodiment 90 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-85, wherein P₁comprises an amino acid sequence according to U₁—U₂—C—U₄—P—U₆—U₇—U₈—U₉—U₁₀—U₁₁—U₁₂—C—U₁₄and U₁is selected from D, Y, F, I, N, V, H, L, A, T, S, and P; U₂is selected from D, Y, L, F, I, N, A, V, H, T, and S; U₄is selected from G and W; U₆is selected from E, D, V, and P; U₇is selected from W, L, F, V, G, M, I, and Y; U₈is selected from E, D, P, and Q; U₉is selected from E, D, Y, V, F, W, P, L, and Q; U₁₀is selected from S, D, Y, T, I, F, V, N, A, P, L, and H; U₁₁is selected from I, Y, F, V, L, T, N, S, D, A, and H; U₁₂is selected from F, D, Y, L, I, V, A, N, T, P, S, G, and H; and U₁₄is selected from D, Y, N, F, I, P, V, A, T, H, L, M, and S.

Embodiment 91 comprises the isolated polypeptide or polypeptide complex of embodiment 90, wherein U₁is selected from D, Y, F, I, V, and N; U₂is selected from D, Y, L, F, I, and N; U₄is selected from G and W; U₆is selected from E and D; U₇is selected from W, L, F, G, and V; U₈is selected from E and D; U₉is selected from E, D, Y, and V; U₁₀is selected from S, D, Y, T, and I; U₁₁is selected from I, Y, F, V, L, and T; U₁₂is selected from F, D, Y, L, I, V, A, G, and N; and U₁₄is selected from D, Y, N, F, I, M, and P.

Embodiment 92 comprises the isolated polypeptide or polypeptide complex of embodiment 91, wherein U₁is selected from D, Y, V, and F; U₂is selected from D, Y, L, and F; U₄is selected from G and W; U₆is selected from E and D; U₇is selected from W, L, G, and F; U₈is selected from E and D; U₉is selected from E and D; U₁₀is selected from S, D, T, and Y; U₁₁is selected from I, Y, V, L, and F; U₁₂is selected from F, D, Y, G, A, and L; and U₁₄is selected from D, Y, M, and N.

Embodiment 93 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-92, wherein P₁comprises the amino acid sequences according to any one of SEQ ID NOs: 797-823.

Embodiment 94 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-92, wherein P₁comprises an amino acid sequences according to any one of SEQ ID NOs: 25, 824-835, or 843-1690.

Embodiment 95 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-92, wherein P₁comprises the amino acid sequences according to any of SEQ ID NOs: 824-835.

Embodiment 96 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-92, wherein P₁comprises the amino acid sequence according to SEQ ID NO: 810 or an amino acid sequence that has 1, 2, or 3, amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 810.

Embodiment 97 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-92, wherein P₁comprises the amino acid sequence according to SEQ ID NO: 811 or an amino acid sequence that has 1, 2, or 3, amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 811.

Embodiment 98 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-92, wherein P₁comprises the amino acid sequence according to SEQ ID NO: 834 or an amino acid sequence that has 1, 2, or 3, amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 834.

Embodiment 99 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-92, wherein P₁comprises the amino acid sequence according to SEQ ID NO: 810.

Embodiment 100 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-92, wherein P₁comprises the amino acid sequence according to SEQ ID NO: 811.

Embodiment 101 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-92, wherein P₁comprises the amino acid sequence according to SEQ ID NO: 834.

Embodiment 102 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-101, wherein L₁is bound to N-terminus of A₁.

Embodiment 103 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-101, wherein L₁is bound to C-terminus of A₁.

Embodiment 104 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 49-101, wherein L₂is bound to N-terminus of A₂.

Embodiment 105 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 49-101, wherein L₂is bound to C-terminus of A₂.

Embodiment 106 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-105, wherein L₁or L₂is a peptide sequence having at least 5 to no more than 50 amino acids.

Embodiment 107 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-105, wherein L₁or L₂is a peptide sequence having at least 10 to no more than 30 amino acids.

Embodiment 108 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-105, wherein L₁or L₂is a peptide sequence having at least 10 amino acids.

Embodiment 109 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-105, wherein L₁or L₂is a peptide sequence having at least 18 amino acids.

Embodiment 110 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-105, wherein L₁or L₂is a peptide sequence having at least 26 amino acids.

Embodiment 111 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-105, wherein L₁or L₂has a formula comprising (G₂S)_n, wherein n is an integer from 1 to 3 (SEQ ID NO: 840).

Embodiment 112 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-105, wherein L₁has a formula selected from the group consisting of (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.

Embodiment 113 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-105, wherein P₁becomes unbound from A₁when L₁is cleaved by the tumor specific protease thereby exposing A₁to the effector cell antigen.

Embodiment 114 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-105, wherein P₂becomes unbound from A₂when L₂is cleaved by the tumor specific protease thereby exposing A₂to EGFR.

Embodiment 115 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-105, wherein the tumor specific protease is selected from the group consisting of a matrix metalloprotease (MMP), serine protease, cysteine protease, threonine protease, and aspartic protease.

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

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

Embodiment 118 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-105, wherein L₁or L₂comprises a urokinase cleavable amino acid sequence, a matriptase cleavable amino acid sequence, matrix metalloprotease cleavable amino acid sequence, or a legumain cleavable amino acid sequence.

Embodiment 119 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-105, wherein L₁or L₂comprises an amino acid sequence according to SEQ ID NO: 30 or 31.

Embodiment 120 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-105, wherein L₁or L₂comprises an amino acid sequence according to any one of SEQ ID NOs: 27-35.

Embodiment 121 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-105, wherein L₁or L₂comprises an 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 that has 1, 2, or 3 amino acid substitutions, additions, or deletions relative to the amino acid sequence of Linker 4, Linker 5, Linker 6, or Linker 7.

Embodiment 122 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-121, wherein H₁comprises a polymer.

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

Embodiment 124 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-121, wherein H₁comprises albumin.

Embodiment 125 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-121, wherein H₁comprises an Fc domain.

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

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

Embodiment 128 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-121, wherein H₁comprises a polypeptide, a ligand, or a small molecule.

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

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

Embodiment 131 comprises the isolated polypeptide or polypeptide complex of embodiment 129, wherein the circulating immunoglobulin molecule comprises IgG1, IgG2, IgG3, IgG4, slgA, IgM or IgD.

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

Embodiment 133 comprises the isolated polypeptide or polypeptide complex of embodiment 128, wherein the polypeptide is an antibody.

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.

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.

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

Embodiment 137 comprises the isolated polypeptide or polypeptide complex of embodiment 134, wherein the single domain antibody is 645gH1 gL1.

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

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

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

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

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

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.

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

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

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

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

Embodiment 148 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-147, 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.

Embodiment 149 comprises the isolated polypeptide or polypeptide complex of embodiment 148, wherein the modified amino acid or modified non-natural amino acid comprises a post-translational modification.

Embodiment 150 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-149, wherein H₁comprises a linking moiety (L₃) that connects H₁to P₁.

Embodiment 151 comprises the isolated polypeptide or polypeptide complex of embodiment 150, wherein L₃is a peptide sequence having at least 5 to no more than 50 amino acids.

Embodiment 152 comprises the isolated polypeptide or polypeptide complex of embodiment 150, wherein L₃is a peptide sequence having at least 10 to no more than 30 amino acids.

Embodiment 153 comprises the isolated polypeptide or polypeptide complex of embodiment 150, wherein L₃is a peptide sequence having at least 10 amino acids.

Embodiment 154 comprises the isolated polypeptide or polypeptide complex of embodiment 150, wherein L₃is a peptide sequence having at least 18 amino acids.

Embodiment 155 comprises the isolated polypeptide or polypeptide complex of embodiment 150, wherein L₃is a peptide sequence having at least 26 amino acids.

Embodiment 156 comprises the isolated polypeptide or polypeptide complex of embodiment 150, wherein L₃has a formula selected from the group consisting of (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.

Embodiment 157 comprises the isolated polypeptide or polypeptide complex of embodiment 150, wherein L₃comprises an amino acid sequence according to SEQ ID NO: 29.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Embodiment 172 comprises a pharmaceutical composition comprising: (a) the isolated polypeptide or polypeptide complex of any one of embodiments 1-171; and (b) a pharmaceutically acceptable excipient.

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

Embodiment 174 an isolated polypeptide or polypeptide complex according to Formula II: L_1a-P_1aH_1awherein: L_1acomprises a tumor specific protease-cleaved linking moiety that when uncleaved connects P_1ato a first antigen recognizing molecule that binds to an effector cell antigen and the first antigen recognizing molecule is connected to a second antigen recognizing molecule that binds to EGFR; P_1acomprises a peptide that binds to the first antigen recognizing molecule when L_1ais uncleaved; and H_1acomprises a half-life extending molecule.

Embodiment 175 comprises the isolated polypeptide or polypeptide complex of embodiment 174, wherein P_1awhen L_1ais uncleaved impairs binding of the first antigen recognizing molecule to the effector cell antigen.

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

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

Embodiment 178 comprises the isolated polypeptide or polypeptide complex of embodiment 174, wherein P_1ahas less than 70% sequence homology to the effector cell antigen.

Embodiment 179 comprises the isolated polypeptide or polypeptide complex of embodiment 174, wherein P_1acomprises a peptide sequence of at least 10 amino acids in length.

Embodiment 180 comprises the isolated polypeptide or polypeptide complex of embodiment 174, wherein P_1acomprises a peptide sequence of at least 10 amino acids in length and no more than 20 amino acids in length.

Embodiment 181 comprises the isolated polypeptide or polypeptide complex of embodiment 174, wherein P_1acomprises a peptide sequence of at least 16 amino acids in length.

Embodiment 182 comprises the isolated polypeptide or polypeptide complex of embodiment 174, wherein P_1acomprises a peptide sequence of no more than 40 amino acids in length.

Embodiment 183 comprises the isolated polypeptide or polypeptide complex of embodiment 174, wherein P_1acomprises at least two cysteine amino acid residues.

Embodiment 184 comprises the isolated polypeptide or polypeptide complex of embodiment 174, wherein P_1acomprises a cyclic peptide or a linear peptide.

Embodiment 185 comprises the isolated polypeptide or polypeptide complex of embodiment 174, wherein P_1acomprises a cyclic peptide.

Embodiment 186 comprises the isolated polypeptide or polypeptide complex of embodiment 174, wherein P_1acomprises a linear peptide.

Embodiment 187 comprises the isolated polypeptide or polypeptide complex of embodiment 174, wherein P_1acomprises an amino acid sequence selected from the group consisting of SEQ ID NO: 25.

Embodiment 188 comprises the isolated polypeptide or polypeptide complex of embodiment 174, wherein P_1acomprises an amino acid sequence according to SEQ ID NO: 70.

Embodiment 189 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 174-187, wherein H_1acomprises a polymer.

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

Embodiment 191 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 174-190, wherein P_1acomprises an amino acid sequence according to any one of SEQ ID NOs: 25, 797-835, or 843-1690, or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOS: 25, 797-835, or 843-1690.

Embodiment 192 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 174-190, wherein P_1acomprises an amino acid sequence according to Z₁—Z₂—C—Z₄—P—Z₆-Z₇—Z₈—Z₉-Z₁₀—Z₁₁—Z₁₂—C—Z₁₄and Z₁is selected from D, Y, F, I, N, V, H, L, A, T, S, and P; Z₂is selected from D, Y, L, F, I, N, A, V, H, T, and S; Z₄is selected from G and W; Z₆is selected from E, D, V, and P; Z₇is selected from W, L, F, V, G, M, I, and Y; Z₈is selected from E, D, P, and Q; Z₉is selected from E, D, Y, V, F, W, P, L, and Q; Z₁₀is selected from S, D, Y, T, I, F, V, N, A, P, L, and H; Z₁₁is selected from I, Y, F, V, L, T, N, S, D, A, and H; Z₁₂is selected from F, D, Y, L, I, V, A, N, T, P, S, and H; and Z₁₄is selected from D, Y, N, F, I, P, V, A, T, H, L and S.

Embodiment 193 comprises the isolated polypeptide or polypeptide complex of embodiment 192, wherein Z₁is selected from D, Y, F, I, and N; Z₂is selected from D, Y, L, F, I, and N; Z₄is selected from G and W; Z₆is selected from E and D; Z₇is selected from W, L, F, and V; Z₈is selected from E and D; Z₉is selected from E, D, Y, and V; Z₁₀is selected from S, D, Y, T, and I; Z₁₁is selected from I, Y, F, V, L, and T; Z₁₂is selected from F, D, Y, L, I, V, A, and N; and Z₁₄is selected from D, Y, N, F, I, and P.

Embodiment 194 comprises the isolated polypeptide or polypeptide complex of embodiment 193, wherein Z₁is selected from D, Y, and F; Z₂is selected from D, Y, L, and F; Z₄is selected from G and W; Z₆is selected from E and D; Z₇is selected from W, L, and F; Z₈is selected from E and D; Z₉is selected from E and D; Z₁₀is selected from S, D, and Y; Z₁₁is selected from I, Y, and F; Z₁₂is selected from F, D, Y, and L; and Z₁₄is selected from D, Y, and N.

Embodiment 195 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 174-190, wherein P_1acomprises an amino acid sequence according to U₁—U₂—C—U₄—P—U₆—U₇—U₈—U₉—U₁₀—U₁₁—U₁₂—C—U₁₄and U₁is selected from D, Y, F, I, N, V, H, L, A, T, S, and P; U₂is selected from D, Y, L, F, I, N, A, V, H, T, and S; U₄is selected from G and W; U₆is selected from E, D, V, and P; U₇is selected from W, L, F, V, G, M, I, and Y; U₈is selected from E, D, P, and Q; U₉is selected from E, D, Y, V, F, W, P, L, and Q; U₁₀is selected from S, D, Y, T, I, F, V, N, A, P, L, and H; U₁₁is selected from I, Y, F, V, L, T, N, S, D, A, and H; U₁₂is selected from F, D, Y, L, I, V, A, N, T, P, S, G, and H; and U₁₄is selected from D, Y, N, F, I, P, V, A, T, H, L, M, and S.

Embodiment 196 comprises the isolated polypeptide or polypeptide complex of embodiment 195, wherein U₁is selected from D, Y, F, 1, V, and N; U₂is selected from D, Y, L, F, I, and N; U₄is selected from G and W; U₆is selected from E and D; U₇is selected from W, L, F, G, and V; U₈is selected from E and D; U₉is selected from E, D, Y, and V; U₁₀is selected from S, D, Y, T, and I; U₁₁is selected from I, Y, F, V, L, and T; U₁₂is selected from F, D, Y, L, I, V, A, G, and N; and U₁₄is selected from D, Y, N, F, I, M, and P.

Embodiment 197 comprises the isolated polypeptide or polypeptide complex of embodiment 196, wherein U₁is selected from D, Y, V, and F; U₂is selected from D, Y, L, and F; U₄is selected from G and W; U₆is selected from E and D; U₇is selected from W, L, G, and F; U₈is selected from E and D; U9 is selected from E and D; U₁₀is selected from S, D, T, and Y; U₁₁is selected from I, Y, V, L, and F; U₁₂is selected from F, D, Y, G, A, and L; and U₁₄is selected from D, Y, M, and N.

Embodiment 198 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 174-197, wherein P_1acomprises an amino acid sequence according to any one of SEQ ID NOs: 797-823.

Embodiment 199 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 174-197, wherein P_1acomprises an amino acid sequence according to any one of SEQ ID NOs: 25, 824-835 or 843-1690.

Embodiment 200 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 174-197, wherein P_1acomprises an amino acid sequence according to any one of SEQ ID NOs: 824-835.

Embodiment 201 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 174-197, wherein P_1acomprises the amino acid sequence according to SEQ ID NO: 810 or an amino acid sequence that has 1, 2, or 3, amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 810.

Embodiment 202 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 174-197, wherein P_1acomprises the amino acid sequence according to SEQ ID NO: 811 or an amino acid sequence that has 1, 2, or 3, amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 811.

Embodiment 203 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 174-197, wherein P_1acomprises the amino acid sequence according to SEQ ID NO: 834 or an amino acid sequence that has 1, 2, or 3, amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 834.

Embodiment 204 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 174-197, wherein P_1acomprises the amino acid sequence according to SEQ ID NO: 810.

Embodiment 205 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 174-197, wherein P_1acomprises the amino acid sequence according to SEQ ID NO: 811.

Embodiment 206 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 174-197, wherein P_1acomprises the amino acid sequence according to SEQ ID NO: 834.

Embodiment 207 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 174-206, wherein H_1acomprises albumin.

Embodiment 208 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 174-206, wherein H_1acomprises an Fc domain.

Embodiment 209 comprises the isolated polypeptide or polypeptide complex of embodiment 207, wherein the albumin is serum albumin.

Embodiment 210 comprises the isolated polypeptide or polypeptide complex of embodiment 209, wherein the albumin is human serum albumin.

Embodiment 211 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 174-206, wherein H_1acomprises a polypeptide, a ligand, or a small molecule.

Embodiment 212 comprises the isolated polypeptide or polypeptide complex of embodiment 211, wherein the polypeptide, the ligand or the small molecule binds a serum protein or a fragment thereof, a circulating immunoglobulin or a fragment thereof, or CD35/CR1.

Embodiment 213 comprises the isolated polypeptide or polypeptide complex of embodiment 212, wherein the serum protein comprises a thyroxine-binding protein, a transthyretin, a 1-acid glycoprotein, a transferrin, transferrin receptor or a transferrin-binding portion thereof, a fibrinogen, or an albumin.

Embodiment 214 comprises the isolated polypeptide or polypeptide complex of embodiment 212, wherein the circulating immunoglobulin molecule comprises IgG1, IgG2, IgG3, IgG4, slgA, IgM or IgD.

Embodiment 215 comprises the isolated polypeptide or polypeptide complex of embodiment 212, wherein the serum protein is albumin.

Embodiment 216 comprises the isolated polypeptide or polypeptide complex of embodiment 211, wherein the polypeptide is an antibody.

Embodiment 217 comprises the isolated polypeptide or polypeptide complex of embodiment 216, wherein the antibody comprises a single domain antibody, a single chain variable fragment or a Fab.

Embodiment 218 comprises the isolated polypeptide or polypeptide complex of embodiment 216, wherein the antibody comprises a single domain antibody that binds to albumin.

Embodiment 219 comprises the isolated polypeptide or polypeptide complex of embodiment 216, wherein the antibody is a human or humanized antibody.

Embodiment 220 comprises the isolated polypeptide or polypeptide complex of embodiment 217, wherein the single domain antibody is 645gH1gL1.

Embodiment 221 comprises the isolated polypeptide or polypeptide complex of embodiment 217, wherein the single domain antibody is 645dsgH5gL4.

Embodiment 222 comprises the isolated polypeptide or polypeptide complex of embodiment 217, wherein the single domain antibody is 23-13-A01-sc02.

Embodiment 223 comprises the isolated polypeptide or polypeptide complex of embodiment 217, wherein the single domain antibody is A 10m3 or a fragment thereof.

Embodiment 224 comprises the isolated polypeptide or polypeptide complex of embodiment 217, wherein the single domain antibody is DOM7r-31.

Embodiment 225 comprises the isolated polypeptide or polypeptide complex of embodiment 217, wherein the single domain antibody is DOM7h-11-15.

Embodiment 226 comprises the isolated polypeptide or polypeptide complex of embodiment 217, wherein the single domain antibody is Alb-1, Alb-8, or Alb-23.

Embodiment 227 comprises the isolated polypeptide or polypeptide complex of embodiment 217, wherein the single domain antibody is 10G.

Embodiment 228 comprises the isolated polypeptide or polypeptide complex of embodiment 217, wherein the single domain antibody comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the single domain antibody comprise: HC-CDR1: SEQ ID NO: 36, HC-CDR2: SEQ ID NO: 37, and HC-CDR3: SEQ ID NO: 38.

Embodiment 229 comprises the isolated polypeptide or polypeptide complex of embodiment 217, wherein the single domain antibody comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the single domain antibody comprise: HC-CDR1: SEQ ID NO: 39, HC-CDR2: SEQ ID NO: 40, and HC-CDR3: SEQ ID NO: 41.

Embodiment 230 comprises the isolated polypeptide or polypeptide complex of embodiment 217, wherein the single domain antibody is SA21.

Embodiment 231 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 174-230, wherein H_1acomprises a linking moiety (L_1a) that connects H_1ato P_1a.

Embodiment 232 comprises the isolated polypeptide or polypeptide complex of embodiment 231, wherein L_1ais a peptide sequence having at least 5 to no more than 50 amino acids.

Embodiment 233 comprises the isolated polypeptide or polypeptide complex of embodiment 231, wherein L_1ais a peptide sequence having at least 10 to no more than 30 amino acids.

Embodiment 234 comprises the isolated polypeptide or polypeptide complex of embodiment 231, wherein L_1ais a peptide sequence having at least 10 amino acids.

Embodiment 235 comprises the isolated polypeptide or polypeptide complex of embodiment 231, wherein L_1ais a peptide sequence having at least 18 amino acids.

Embodiment 236 comprises the isolated polypeptide or polypeptide complex of embodiment 231, wherein L_1ais a peptide sequence having at least 26 amino acids.

Embodiment 237 comprises the isolated polypeptide or polypeptide complex of embodiment 231, wherein L_1ahas a formula selected from the group consisting of (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.

Embodiment 238 comprises the isolated polypeptide or polypeptide complex of embodiment 231, wherein L_1acomprises an amino acid sequence according to SEQ ID NO: 30 or 31.

Embodiment 239 comprises the isolated polypeptide or polypeptide complex of embodiment 231, wherein L₁or L₂comprises an amino acid sequence according to any one of SEQ ID NOs: 27-35.

Embodiment 240 comprises the isolated polypeptide or polypeptide complex of embodiment 231, wherein L₁or L₂comprises an 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 that has 1, 2, or 3 amino acid substitutions, additions, or deletions relative to the amino acid sequence of Linker 4, Linker 5, Linker 6, or Linker 7.

Embodiment 241 comprises an isolated polypeptide complex comprising a structural arrangement according to Configuration 1 (FIG. 1A), 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 P₁is linked to a N-terminus of the heavy chain variable domain of the scFv with a linking moiety (L₁) that is a substrate for a tumor specific protease, and P₁is further linked to a half-life extending molecule; 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, wherein the Fab light chain polypeptide is linked to a C terminus of the light chain variable domain of the scFv, and wherein the Fab is linked to P₂and L₂, wherein P₂comprises a peptide that impairs binding to EGFR; and L₂comprises a linking moiety that connects the Fab heavy chain polypeptide to P₂and is a substrate for a tumor specific protease.

Embodiment 242 comprises an isolated polypeptide complex comprising a structural arrangement according to Configuration 2 (FIG. 1B), 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 P₁is linked to a N-terminus of the heavy chain variable domain of the scFv with a linking moiety (L₁) that is a substrate for a tumor specific protease, and P₁is further linked to a half-life extending molecule; 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, wherein the Fab heavy chain polypeptide is linked to a C terminus of the light chain variable domain of the scFv, and wherein the Fab is linked to P₂and L₂, wherein P₂comprises a peptide that impairs binding to EGFR; and L₂comprises a linking moiety that connects the Fab light chain polypeptide to P₂and is a substrate for a tumor specific protease.

Embodiment 243 comprises an isolated polypeptide or polypeptide complex comprising an anti-EGFR binding domain that is linked to a peptide that impairs binding of the anti-EGFR binding domain to EGFR wherein the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 26, 71-96, 98-776 or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 26, 71-96, 98-776.

Embodiment 244 comprises the isolated polypeptide or polypeptide complex of embodiment 243, wherein the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 26, 71-96, 98-776.

Embodiment 245 comprises the isolated polypeptide or polypeptide complex of embodiment 243, wherein the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 26, 86-96 or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 26, 86-96.

Embodiment 246 comprises the isolated polypeptide or polypeptide complex of embodiment 245, wherein the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 26, 86-96.

Embodiment 247 comprises the isolated polypeptide or polypeptide complex of embodiment 243, wherein the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 98-776 or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any of SEQ ID NOs: 98-776.

Embodiment 248 comprises the isolated polypeptide or polypeptide complex of embodiment 247, wherein the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 98-776.

Embodiment 249 comprises the isolated polypeptide or polypeptide complex of embodiment 243, wherein the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 99-118 or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 99-118.

Embodiment 250 comprises the isolated polypeptide or polypeptide complex of embodiment 249, wherein the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 99-118.

Embodiment 251 comprises the isolated polypeptide or polypeptide complex of embodiment 243, wherein the peptide comprises the amino acid sequence according to SEQ ID NO: 71 or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to SEQ ID NO: 71.

Embodiment 252 comprises the isolated polypeptide or polypeptide complex of embodiment 251, wherein the peptide comprises the amino acid sequence according to SEQ ID NO: 71.

Embodiment 253 comprises the isolated polypeptide or polypeptide complex of embodiment 243, wherein the peptide comprises the amino acid sequence according to SEQ ID NO: 26 or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to SEQ ID NO: 26.

Embodiment 254 comprises the isolated polypeptide or polypeptide complex of embodiment 253, wherein the peptide comprises the amino acid sequence according to SEQ ID NO: 26.

Embodiment 255 comprises an isolated polypeptide or polypeptide complex comprising an anti-EGFR binding domain that is linked to a peptide that impairs binding of the anti-EGFR binding to EGFR wherein the peptide comprises an amino acid sequence according to X₁—C—X₂—X₃—X₄—X₅-D-X₆-A-X₇—P—X₈—C—X₉wherein X₁is selected from P and L; X₂is selected from R, L, T, A, N, I, V, S, H, and P; X₃is selected from S, P, F, and Y; X₄is selected from H, L, Q, P, R, F, N; X₅is selected from I, F, Y, H, N, T, S, D, A, L, and V; X₆is selected from T, P, N, L, I, V, S, D, H, A, and Y; X₇is selected from K and Y; X₈is selected from I, P, L, and M; and X₉is selected from A, V, I, T, L, S, D, F, V, and H (SEQ ID NO: 841).

Embodiment 256 comprises the isolated polypeptide or polypeptide complex of embodiment 255, wherein X₁is selected from P and L; X₂is selected from R, L, T, A, and N; X₃is selected from S, P, and F; X₄is selected from H, L, Q, and P; X₅is selected from I, F, Y, H, N, and T; X₆is selected from T, P, N, L, I, and V; X₇is K; X₈is I; and X₉is selected from A, V, I, T, L, and S.

Embodiment 257 comprises the isolated polypeptide or polypeptide complex of embodiment 256, wherein X₁is P; X₂is selected from R, L, and T; X₃is S; X₄is selected from H, L, Q, and P; X₅is selected from I, F, Y, and T; X₆is selected from T, P, N, and V; X₇is K; X₈is I; and X₉is selected from A, V, and I.

Embodiment 258 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 255-257, wherein the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 98-776.

Embodiment 259 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 255-258, wherein the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 99-118.

Embodiment 260 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 255-257, wherein the peptide comprises the amino acid sequence according to SEQ ID NO: 26.

Embodiment 261 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 243-260, wherein the anti-EGFR binding domain comprises an antibody or an antibody fragment.

Embodiment 262 comprises the isolated polypeptide or polypeptide complex of embodiment 261, wherein the antibody or antibody fragment comprises a single chain variable fragment, a single domain antibody, Fab, or Fab′.

Embodiment 263 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 243-262, 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: SEQ ID NO: 15, HC-CDR2: SEQ ID NO: 16, and HC-CDR3: SEQ ID NO: 17; and the anti-EGFR binding domain comprises light chain complementarity determining regions CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the Fab comprise LC-CDR1: SEQ ID NO: 18, LC-CDR2: SEQ ID NO:19, and LC-CDR3: SEQ ID NO: 20.

Embodiment 264 comprises the isolated polypeptide or polypeptide complex of embodiment 261, wherein the antibody or antibody fragment comprises the Fab.

Embodiment 265 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 243-264, wherein the anti-EGFR binding domain comprises an amino acid sequence according to any of SEQ ID NOs: 21-24.

Embodiment 266 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 243-265, wherein the anti-EGFR binding domain is linked to the peptide through a linking moiety (L₁).

Embodiment 267 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 243-266, wherein L₁is a substrate for a tumor specific protease.

Embodiment 268 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 243-267, wherein L₁is bound to N-terminus of the anti-EGFR binding domain.

Embodiment 269 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 243-267, wherein L₁is bound to C-terminus of the anti-EGFR binding domain.

Embodiment 270 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 243-267, wherein L₁is a peptide sequence having at least 5 to no more than 50 amino acids.

Embodiment 271 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 243-267, wherein L₁is a peptide sequence having at least 10 to no more than 30 amino acids.

Embodiment 272 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 243-267, wherein L₁is a peptide sequence having at least 10 amino acids.

Embodiment 273 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 243-267, wherein L₁is a peptide sequence having at least 18 amino acids.

Embodiment 274 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 243-267, wherein L₁is a peptide sequence having at least 26 amino acids.

Embodiment 275 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 243-267, wherein L₁has a formula comprising (G₂S)_n, wherein n is an integer from 1 to 3 (SEQ ID NO: 840).

Embodiment 276 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 243-267, wherein L₁has a formula selected from the group consisting of (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.

Embodiment 277 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 243-276, wherein the peptide becomes unbound from the anti-EGFR binding domain when L₁is cleaved by the tumor specific protease thereby exposing the anti-EGFR binding domain to EGFR.

Embodiment 278 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 267-277, wherein the tumor specific protease is selected from the group consisting of a matrix metalloprotease (MMP), serine protease, cysteine protease, threonine protease, and aspartic protease.

Embodiment 279 comprises the isolated polypeptide or polypeptide complex of embodiment 278, wherein the matrix metalloprotease comprises MMP2, MMP7, MMP9, MMP13, or MMP14.

Embodiment 280 comprises the isolated polypeptide or polypeptide complex of embodiment 278, wherein the serine protease comprises matriptase (MTSP1), urokinase, or hepsin.

Embodiment 281 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 267-277, wherein L₁comprises a urokinase cleavable amino acid sequence, a matriptase cleavable amino acid sequence, matrix metalloprotease cleavable amino acid sequence, or a legumain cleavable amino acid sequence.

Embodiment 282 comprises the isolated polypeptide or polypeptide complex of embodiment 266, wherein L₁comprises an amino acid sequence according to any of SEQ ID NO: 30 or 31.

Embodiment 283 comprises the isolated polypeptide or polypeptide complex of embodiment 266, wherein L₁comprises an amino acid sequence according to SEQ ID NO: 27-35.

Embodiment 284 comprises the isolated polypeptide or polypeptide complex of embodiment 266, wherein L₁comprises an 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 that has 1, 2, or 3 amino acid substitutions, additions, or deletions relative to the amino acid sequence of Linker 4, Linker 5, Linker 6, or Linker 7.

Embodiment 285 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 243-284, wherein the isolated polypeptide or polypeptide complex further comprises a half-life extending molecule (H₁)

Embodiment 286 comprises the isolated polypeptide or polypeptide complex of embodiment 285, wherein the half-life extending molecule is linked to the peptide.

Embodiment 287 comprises the isolated polypeptide or polypeptide complex of embodiment 285, wherein H₁comprises a polymer.

Embodiment 288 comprises the isolated polypeptide or polypeptide complex of embodiment 287, wherein the polymer is polyethylene glycol (PEG).

Embodiment 289 comprises the isolated polypeptide or polypeptide complex of embodiment 285, wherein H₁comprises albumin.

Embodiment 290 comprises the isolated polypeptide or polypeptide complex of embodiment 285, wherein H₁comprises an Fc domain.

Embodiment 291 comprises the isolated polypeptide or polypeptide complex of embodiment 289, wherein the albumin is serum albumin.

Embodiment 292 comprises the isolated polypeptide or polypeptide complex of embodiment 289, wherein the albumin is human serum albumin.

Embodiment 293 comprises the isolated polypeptide or polypeptide complex of embodiment 285, wherein H₁comprises a polypeptide, a ligand, or a small molecule.

Embodiment 294 comprises the isolated polypeptide or polypeptide complex of embodiment 293, wherein the polypeptide, the ligand or the small molecule binds serum protein or a fragment thereof, a circulating immunoglobulin or a fragment thereof, or CD35/CR1.

Embodiment 295 comprises the isolated polypeptide or polypeptide complex of embodiment 294, wherein the serum protein comprises a thyroxine-binding protein, a transthyretin, a 1-acid glycoprotein, a transferrin, transferrin receptor or a transferrin-binding portion thereof, a fibrinogen, or an albumin.

Embodiment 296 comprises the isolated polypeptide or polypeptide complex of embodiment 294, wherein the circulating immunoglobulin molecule comprises IgG1, IgG2, IgG3, IgG4, slgA, IgM or IgD.

Embodiment 297 comprises the isolated polypeptide or polypeptide complex of embodiment 294, wherein the serum protein is albumin.

Embodiment 298 comprises the isolated polypeptide or polypeptide complex of embodiment 294, wherein the polypeptide is an antibody.

Embodiment 299 comprises the isolated polypeptide or polypeptide complex of embodiment 298, wherein the antibody comprises a single domain antibody, a single chain variable fragment, or a Fab.

Embodiment 300 comprises the isolated polypeptide or polypeptide complex of embodiment 299, wherein the single domain antibody comprises a single domain antibody that binds to albumin.

Embodiment 301 comprises the isolated polypeptide or polypeptide complex of embodiment 300, wherein the single domain antibody is a human or humanized antibody.

Embodiment 302 comprises the isolated polypeptide or polypeptide complex of embodiment 299, wherein the single domain antibody is 645gH1gL1.

Embodiment 303 comprises the isolated polypeptide or polypeptide complex of embodiment 299, wherein the single domain antibody is 645dsgH5gL4.

Embodiment 304 comprises the isolated polypeptide or polypeptide complex of embodiment 299, wherein the single domain antibody is 23-13-A01-sc02.

Embodiment 305 comprises the isolated polypeptide or polypeptide complex of embodiment 299, wherein the single domain antibody is A 10m3 or a fragment thereof.

Embodiment 306 comprises the isolated polypeptide or polypeptide complex of embodiment 299, wherein the single domain antibody is DOM7r-31.

Embodiment 307 comprises the isolated polypeptide or polypeptide complex of embodiment 299, wherein the single domain antibody is DOM7h-11-15.

Embodiment 308 comprises the isolated polypeptide or polypeptide complex of embodiment 299, wherein the single domain antibody is Alb-1, Alb-8, or Alb-23.

Embodiment 309 comprises the isolated polypeptide or polypeptide complex of embodiment 299, wherein the single domain antibody is I0E.

Embodiment 310 comprises the isolated polypeptide or polypeptide complex of embodiment 299, wherein the single domain antibody comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the single domain antibody comprise: HC-CDR1: SEQ ID NO: 36, HC-CDR2: SEQ ID NO: 37, and HC-CDR3: SEQ ID NO: 38.

Embodiment 311 comprises the isolated polypeptide or polypeptide complex of embodiment 299, wherein the single domain antibody comprises an amino acid sequence according to SEQ ID NO: 69.

Embodiment 312 comprises the isolated polypeptide or polypeptide complex of embodiment 299, wherein the single domain antibody comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the single domain antibody comprise: HC-CDR1: SEQ ID NO: 36, HC-CDR2: SEQ ID NO: 37, and HC-CDR3: SEQ ID NO: 38.

Embodiment 313 comprises the isolated polypeptide or polypeptide complex of embodiment 299, wherein the single domain antibody comprises an amino acid sequence according to SEQ ID NO: 42.

Embodiment 314 comprises the isolated polypeptide or polypeptide complex of embodiment 299, wherein the single domain antibody is SA21.

Embodiment 315 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-314, 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.

Embodiment 316 comprises the isolated polypeptide or polypeptide complex of embodiment 315, wherein the modified amino acid or modified non-natural amino acid comprises a post-translational modification.

Embodiment 317 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 285-316, wherein H₁comprises a linking moiety (L₃) that connects H₁to P₁.

Embodiment 318 comprises the isolated polypeptide or polypeptide complex of embodiment 317, wherein L₃is a peptide sequence having at least 5 to no more than 50 amino acids.

Embodiment 319 comprises the isolated polypeptide or polypeptide complex of embodiment 317, wherein L₃is a peptide sequence having at least 10 to no more than 30 amino acids.

Embodiment 320 comprises the isolated polypeptide or polypeptide complex of embodiment 317, wherein L₃is a peptide sequence having at least 10 amino acids.

Embodiment 321 comprises the isolated polypeptide or polypeptide complex of embodiment 317, wherein L₃is a peptide sequence having at least 18 amino acids.

Embodiment 322 comprises the isolated polypeptide or polypeptide complex of embodiment 317, wherein L₃is a peptide sequence having at least 26 amino acids.

Embodiment 323 comprises the isolated polypeptide or polypeptide complex of embodiment 317, wherein L₃has a formula selected from the group consisting of (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.

Embodiment 324 comprises the isolated polypeptide or polypeptide complex of embodiment 317, wherein L₃comprises an amino acid sequence according to SEQ ID NO: 29.

Embodiment 325 comprises a method of treating cancer comprising administering the polypeptides or polypeptide complexes of any of the above embodiments to a subject in need thereof.

Embodiment 326 comprises the method of embodiment 325, wherein the cancer has cells that express EGFR.

Embodiment 327 comprises a method 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/rectum cancer, head and neck cancer, esophagogastric cancer, liver cancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, kidney cancer, or pancreatic cancer comprising administering the polypeptides or polypeptide complexes of any of the above embodiments to a subject in need thereof.

Embodiment 328 comprises the method of embodiment 327, wherein the subject in need thereof is resistant to EGFR inhibitor treatment.

Embodiment 329 comprises the method of embodiment 327, wherein the subject in need thereof harbors KRAS mutations.

Embodiment 330 comprises the method of embodiment 327, wherein the subject in need thereof is resistant to EGFR inhibitor treatment and harbors KRAS mutations.

Embodiment 331 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-77, wherein P₂comprises the amino acid sequence according to SEQ ID NO: 115 or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to SEQ ID NO: 115.

Embodiment 332 comprises the isolated polypeptide or polypeptide complex of embodiment 331, wherein P₂comprises the amino acid sequence according to SEQ ID NOs: 115.

Embodiment 333 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-77, wherein P₂comprises the amino acid sequence according to SEQ ID NO: 116 or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to SEQ ID NO: 116.

Embodiment 334 comprises the isolated polypeptide or polypeptide complex of embodiment 333, wherein P₂comprises the amino acid sequence according to SEQ ID NOs: 116.

Embodiment 335 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 255-257, wherein the peptide comprises the amino acid sequence according to SEQ ID NO: 115.

Embodiment 336 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 255-257, wherein the peptide comprises the amino acid sequence according to SEQ ID NO: 116.

EXAMPLES
Example 1: EGFR Polypeptide Complex Binding

The EGFR-CD3 polypeptide complexes were evaluated for EGFR and CD3s binding.

Briefly, the polypeptide complex molecules were evaluated for their ability to bind EGFR as well as CD3 in a standard enzyme linked immunosorbent assay (ELISA) format. Polypeptide complex binding of EGFR or CD3 were measured before and after protease treatment. Briefly, biotinylated antigen was captured on neutravidin coated plates. Polypeptide complex molecules were treated with active matriptase (MTSP1) where indicated. Polypeptide complex molecules diluted in buffer were then added to the antigen coated plates. Bound polypeptide complex was detected using a standard horse radish peroxidase conjugate secondary antibody. The concentration of polypeptide complex required to achieve 50% maximal signal (EC50) was calculated using Graphpad Prism software.

FIG. 2 shows representative polypeptide molecule binding to EGFR measured by ELISA. FIG. 3 shows representative polypeptide binding to CD3 measured by ELISA.

Example 2: Polypeptide Complex Mediated Tumor Cytotoxicity and T Cell Activation

Polypeptide complexes were evaluated in a functional in vitro tumor cell killing assay using the EGFR positive tumor cell lines HCT116 and A₄₃₁. Tumor cell killing was measured using an xCelligence real time cell analyzer from Agilent that relies on sensor impedance measurements (cell index) that increased as tumor cells adhere, spread, and expand on the surface of the sensor. Likewise, as the tumor cells were killed the impedance decreased. 10,000 tumor cells were added per well and allowed to adhere overnight on a 96 well E-Plate. The following day polypeptide complexes titrated in human serum supplemented medium along with 30,000 CD8+ T cells were added to the wells. Cell index measurements were taken every 10 minutes for an additional 72 hours. The cell index times number of hours (tumor cell growth kinetics) was then plotted versus concentration of polypeptide complex where the concentration required to reduce the tumor growth 50% (IC50) was calculated using Graphpad Prism software.

FIG. 4 shows a graph of polypeptide complex mediated HCT116 tumor cell killing in the presence of CD8+ T cells. FIG. 5 shows a graph of polypeptide complex mediated A₄₃₁tumor cell killing in the presence of CD8+ T cells.

Example 3: Polypeptide Complex Binding

Polypeptide complexes were evaluated in binding studies to EGFR and CD3R.

Kinetic binding of polypeptide molecules to human and cynomolgus monkey EGFR was evaluated by bio-layer interferometry using an Octet RED96 instrument. Briefly, biosensors were loaded with antigen and baselined in buffer. Polypeptide molecules were titrated in solution at 50 nM, 25 nM, 12.5 nM, and 6.25 nM then associated onto the antigen loaded sensors. After a short association period, sensors were transferred into buffer and the dissociation of bound polypeptide molecules was measured. The timing and steps of the experiment are shown in Table 7. Association and dissociation signals were recorded in real time and analyzed using a 1:1 binding model within the instrument software. Analysis using a 1:1 binding model enabled the calculation of the on and off rate constants as well as affinity, KD. Off rate constants were converted to half-life shown in the accompanying figures. Data is seen in FIG. 6.

TABLE 7

Step
Time
pH

Baseline: Octet buffer
60 sec
pH 7.4

Load:
300 sec
pH 7.4

15 nM human EGFR

15 nM cyno EGFR

Baseline: Octet buffer
300 sec
pH 7.4

Association in octet buffer
300 sec
pH 7.4

50 nM PC1

25 nM PC1

12.5 nM PC1

6.25 nM PC1

Dissociation: Octet Buffer
900 sec
pH 7.4

The polypeptide complex molecules were also evaluated for their ability to bind human and cynomolgus monkey antigen EGFR in a standard enzyme linked immunosorbent assay (ELISA) format. Briefly, antigens fused to a human Fc domain were directly coated on high binding ELISA plates. Polypeptide complex molecules diluted in buffer were then added to the antigen coated plates. Bound polypeptide complex was detected using a standard horse radish peroxidase conjugate secondary antibody. The concentration of polypeptide complex required to achieve 50% maximal signal (EC50) was calculated using Graphpad Prism software. Data is seen in FIG. 7.

Kinetic binding of polypeptide molecules to human and cyno CD3 was evaluated by bio-layer interferometry using an Octet RED96 instrument. Briefly, biosensors were loaded with antigen and baselined in buffer. Polypeptide molecules were titrated in solution at 50 nM, 25 nM, 12.5 nM, and 6.25 nM then associated onto the antigen loaded sensors. After a short association period, sensors were transferred into buffer and the dissociation of bound polypeptide molecules was measured. The timing and steps of the experiment are shown in Table 8. Association and dissociation signals were recorded in real time and analyzed using a 1:1 binding model within the instrument software. Analysis using a 1:1 binding model enabled the calculation of the on and off rate constants as well as affinity, KD. Off rate constants were converted to half-life shown in the accompanying figures. Data is seen in FIG. 8.

TABLE 8

Step
Time
pH

Baseline: Octet buffer
60 sec
pH 7.4

Load:
300 sec
pH 7.4

15 nM human CD3e

15 nM cyno CD3e

Baseline: Octet buffer
300 sec
pH 7.4

Association in octet buffer
300 sec
pH 7.4

50 nM PC1

25 nM PC1

12.5 nM PC1

6.25 nM PC1

Dissociation: Octet Buffer
900 sec
pH 7.4

The polypeptide complex molecules were evaluated for their ability to bind human and cynomolgus monkey antigen CD3 in a standard enzyme linked immunosorbent assay (ELISA) format. Briefly, antigens fused to a human Fc domain were directly coated on high binding ELISA plates. Polypeptide complex molecules diluted in buffer were then added to the antigen coated plates. Bound polypeptide complex was detected using a standard horse radish peroxidase conjugate secondary antibody. The concentration of polypeptide complex required to achieve 50% maximal signal (EC50) was calculated using Graphpad Prism software. Data is seen in FIG. 9.

Example 4: Polypeptide Complex Pharmacokinetics in Cynomolgus Monkey

Pharmacokinetics and exploratory safety of polypeptide molecules were evaluated in cynomolgus monkeys. Briefly, cynomolgus monkeys of approximately 3 kg bodyweight were administered polypeptides as an IV bolus and observed daily for signs of adverse events. No in-life adverse events were observed. After dosing, blood was collected in K2 EDTA tubes at specific timepoints and processed to plasma. Plasma was stored frozen until analysis. Concentration of polypeptide molecules in plasma was measured via standard ELISA techniques relative to a reference standard diluted in control cyno plasma. Plasma concentration curves were fit to a standard two phase exponential equation representing distribution and elimination phases. Fitting of phannacokinetics enabled the calculation of Cmax, half-life, volume of distribution, clearance, and 7 day area under the curve (AUC) shown in Tables 9-10. Measured pharmacokinetics in cynomolgus supports once weekly dosing in humans. Data is seen in FIGS. 10A-10B. FIG. 10A demonstrates that the polypeptide complexes described herein comprising a mask was over 8,500 less potent at T cell-mediated killing of EGFR-expressing tumor cells than an equivalent unmasked TCE in an in vitro assay. As seen in FIG. 10B and Table 9 below PC4 (masked) exhibited a half-life of approximately 94 hours, which supports once-weekly dosing in humans, as compared to an unmasked polypeptide complex that exhibited a half-life of about an hour.

TABLE 9

PC4 100 ug/kg
Units

C_MAX
27.05
nM

t_1/2
94.47
hr

Vd
0.12
L

VSS
0.25
L

CL
0.28
mL/hr/kg

BW
3.00
kg

7 day AUC
96.143
nM · min

TABLE 10

Unmasked polypeptide 10 ug/kg
Units

C_MAX
1.66
nM

t_1/2
1.30
hr

Vd
0.24
L

VSS
1.08
L

CL
41.72
mL/hr/kg

BW
3.00
kg

7 day AUC
75
nM · min

Example 5: Polypeptide Complexes in Cynomolgus Cytokine Release

Cytokine release after polypeptide molecule administration by IV bolus was evaluated in cynomolgus monkeys. Briefly, cynomolgus monkeys of approximately 3 kg bodyweight were administered polypeptides as an IV bolus and observed daily for signs of adverse events. No in-life adverse events were observed. After dosing, blood was collected in K2 EDTA tubes at specific timepoints and processed to plasma. Plasma was stored frozen until analysis. Plasma samples were analyzed for cytokines using a non-human primate cytometric Th1/Th2 bead array kit from BD biosciences following the manufacturer's instructions. Interferon gamma, tumor necrosis factor alpha, interleukin 6, interleukin 5, interleukin 4, and interleukin 2 levels in plasma were calculated relative to reference standards provided with the bead array kit. Data is seen in FIGS. 11A-11L and FIG. 12. FIG. 12 shows dosing of polypeptide complexes (EGFR TRACTr) described herein at 100 μg/kg resulted in minimal levels of inflammatory cytokine release, relative to an unmasked EGFR-TCE at 10 μg/kg, which led to a greater than 20-fold expression of IL-6.

Example 6: Polypeptide Complexes in Cynomolgus Toxicity

Systemic liver enzymes after polypeptide molecule administration by IV bolus was evaluated in cynomolgus monkeys. Briefly, cynomolgus monkeys of approximately 3 kg bodyweight were administered polypeptides as an IV bolus and observed daily for signs of adverse events. No in-life adverse events were observed. After dosing, blood was collected in K2 EDTA tubes at specific timepoints and processed to plasma. Plasma was stored frozen until analysis. Plasma samples were analyzed for the presence of liver enzymes aspartate transaminase (AST) and alanine aminotransferase (ALT) as signs of potential liver toxicity. AST and ALT levels were remained within the normal ranges for all timepoints tested after dosing suggesting a lack of liver toxicity. AST and ALT were quantified following the instructions provided in a commercially available kit from Millipore. AST and ALT levels were calculated according to manufacturer's instructions relative to a positive control reference standard. Data is seen in FIGS. 13A-13D.

Example 7: Peptides Bind EGFR IgG and Inhibit EGFR IgG from Binding to the EGFR Antigen

The peptide sequences (peptide-1 to peptide-6) of Table 11 were evaluated for binding to EGFR IgG (see Table 11) in an ELISA format. Briefly, biotinylated peptides were captured on neutravidin coated plates. EGFR IgG diluted in buffer was then added to the peptide coated plates. Bound EGFR IgG was detected using a standard horse radish peroxidase conjugate secondary antibody. The ELISA signal was plotted versus the log-scale antibody concentration. The concentrations of antibody required to observe half maximal binding signal (EC₅₀s) were calculated using Graphpad Prism software. FIG. 14 shows binding curves for peptide binding to EGFR IgG, and Table 12 provides the calculated EC₅₀s for peptide binding to EGFR IgG. Peptide-6 of SEQ ID NO: 76 showed the strongest binding to EGFR IgG (lowest EC₅₀) among the peptides tested in this series.

TABLE 11

Peptide sequences for EGFR IgG binding and EGFR IgG Sequence

Amino Acid Sequence
SEQ ID

Description
(N to C)
NO:

Peptide-1
DWCRSLMSYTDLCP
71

Peptide-2
TSCADAHLIAPSCS
72

Peptide-3
NCQWDRVEHTYACS
73

Peptide-4
WVSCHDGSHMTCFH
74

Peptide-5
MNCLNRLWVEYCLV
75

Peptide-6
YCGQDNTWVREGCF
76

EGFR IgG (Light
DILLTQSPVILSVSPGERVSFSCRASQSIGTN
22

chain)
IHWYQQRINGSPRLLIKYASESISGIPSRFSG

SGSGTDFTLSINSVESEDIADYYCQQNNNW

PTTFGAGTKLELKRTVAAPSVFIFPPSDEQL

KSGTASVVCLLNNFYPREAKVQWKVDNA

LQSGNSQESVTEQDSKDSTYSLSSTLTLSK

ADYEKHKVYACEVTHQGLSSPVTKSFNRG

EC

EGFR IgG (Heavy
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTN
842

chain)
YGVHWVRQSPGKGLEWLGVIWSGGNTDY

NTPFTSRLSINKDNSKSQVFFKMNSLQSND

TAIYYCARALTYYDYEFAYWGQGTLVTVS

AASTKGPSVFPLAPSSKSTSGGTAALGCLV

KDYFPEPVTVSWNSGALTSGVHTFPAVLQ

SSGLYSLSSVVTVPSSSLGTQTYICNVNHKP

SNTKVDKKVEPKSCDKTHTCPPCPAPELLG

GPSVFLFPPKPKDTLMISRTPEVTCVVVDV

SHEDPEVKFNWYVDGVEVHNAKTKPREE

QYNSTYRVVSVLTVLHQDWLNGKEYKCK

VSNKALPAPIEKTISKAKGQPREPQVYTLPP

SRDELTKNQVSLTCLVKGFYPSDIAVEWES

NGQPENNYKTTPPVLDSDGSFFLYSKLTVD

KSRWQQGNVFSCSVMHEALHNHYTQKSL

SLSPGK

TABLE 12

EC₅₀s for peptide binding to EGFR IgG

Pep-
Pep-
Pep-
Pep-
Pep-
Pep-

tide-1
tide-2
tide-3
tide-4
tide-5
tide-6

EC₅₀
52
84
413
>1000
2
0.3

(nM)

For a peptide to function as a mask, it must also inhibit the antibody of interest from binding to its cognate antigen. As such, ELISA-based competitive inhibition studies were also used to test the ability of each peptide to inhibit EGFR IgG from binding to the EGFR antigen. Biotinylated antigen was captured on neutravidin coated plates, quenched using biocytin, and washed. Inhibitory peptides were titrated in a dilution series and pre-incubated with a constant concentration of antibody. Inhibitory peptide and antibody mixtures were then incubated on the antigen captured plates. A horseradish peroxidase conjugate secondary antibody was then used to detect the antibody binding to the plate-bound antigen. The ELISA signal was plotted versus log-scale peptide concentration (see FIG. 15). A dose dependent decrease of signal was indicative of peptides that compete for antibody binding to the cognate antigen. Graphpad Prism software was used to calculate the inhibitory concentrations of peptide required to achieve 50% maximal signal (ICsos). The IC₅₀data is provided in Table 13. Of the peptides tested in this series, peptide-6 was the most effective at inhibiting EGFR IgG binding to the EGFR antigen.

TABLE 13

IC₅₀s for peptide inhibition of EGFR IgG binding to EGFR antigen

Pep-
Pep-
Pep-
Pep-
Pep-
Pep-

tide-1
tide-2
tide-3
tide-4
tide-5
tide-6

IC₅₀
>300
>300
>300
>300
>300
50

(nM)

Example 8: Peptides Bind EGFR Fab and Inhibit EGFR Fab from Binding to the EGFR Antigen

The peptide sequences of Table 14 were evaluated for binding to EGFR Fab in an ELISA format. Binding studies were carried out as described above in Example 7. Binding curves are shown in FIG. 16 and EC₅₀s for binding are provided in Tables 15-16. Peptide-8 and peptide-15 showed the strongest binding affinities (lowest EC₅₀s) for EGFR Fab among the peptides tested in this series.

TABLE 14

Peptide sequences for αEGFR IgG binding

Amino Acid Sequence
SEQ ID

Description
(N to C)
NO:

Peptide-7
WCTVWGDRQEVRCL
77

Peptide-8
PCRSHIDVAKPICV
26

Peptide-9
STCVHYWMQLRSCV
78

Peptide-10
GGCAHWVMRLSWCQ
79

Peptide-11
LGCQHFLMKLATCG
80

Peptide-12
GCGDGLHRMVRMWC
81

Peptide-13
ACTVMGARQEVRCV
82

Peptide-14
PCPALIDVAEVICV
83

Peptide-15
YCNSVWQRGQLYWC
84

Peptide-16
ECSHMEGTRWPLCS
85

TABLE 15

EC₅₀s for peptide binding to EGFR Fab

Pep-
Pep-
Pep-
Pep-
Pep-
Pep-

tide-7
tide-8
tide-9
tide-10
tide-11
tide-12

EC₅₀
>1000
8
>1000
>1000
42
>1000

(nM)

TABLE 16

EC₅₀s for peptide binding to αEGFR Fab

Peptide-13
Peptide-14
Peptide-15
Peptide-16
Peptide-6

EC₅₀(nM)
>1000
>1000
7
34
458

Competitive inhibition studies were also used to test the ability of each peptide to inhibit EGFR Fab from binding to the EGFR antigen in an ELISA format. Competitive inhibition measurements were carried out as described above in Example 7. Dose-dependent peptide inhibition of EGFR Fab binding to the EGFR antigen is shown in FIG. 17, and IC₅₀data is provided in Tables 17-18. Notably, peptide-8 (SEQ ID NO: 26) was the most effective at inhibiting EGFR Fab binding to the EGFR antigen.

TABLE 17

IC₅₀s for peptide inhibition of

αEGFR Fab binding to EGFR antigen

Pep-
Pep-
Pep-
Pep-
Pep-
Pep-

tide-7
tide-8
tide-9
tide-10
tide-11
tide-12

IC₅₀
>300
0.3
>300
>300
>300
>300

(nM)

TABLE 18

IC₅₀s for peptide inhibition of

αEGFR Fab binding to EGFR antigen

Peptide-13
Peptide-14
Peptide-15
Peptide-16
Peptide-6

IC₅₀(nM)
>300
>300
>300
>300
13

Example 9: Sequence Activity Relationships—EGFR Fab Peptide

Sequence activity relationships (SAR) were established for peptide-8 which was found to exhibit strong binding to EGFR Fab and inhibition of EGFR Fab binding to the EFGR antigen. The sequence activity relationships were established by mutating individual residues within the peptide to alanine and measuring the influence of each mutation on the ability of the peptide to inhibit EGFR Fab binding to the EGFR antigen. The alanine scanning sequences are provided in Table 19.

TABLE 19

Alanine scan peptide sequences

Amino Acid Sequence
SEQ ID

Description
(N to C)
NO:

Peptide-8 (wild type)
PCRSHIDVAKPICV
26

Peptide-17

A
CRSHIDVAKPICV
86

Peptide-18
PCASHIDVAKPICV
87

Peptide-19
PCRAHIDVAKPICV
88

Peptide-20
PCRSAIDVAKPICV
89

Peptide-21
PCRSHADVAKPICV
90

Peptide-22
PCRSHIAVAKPICV
91

Peptide-23
PCRSHIDAAKPICV
92

Peptide-24
PCRSHIDVAAPICV
93

Peptide-25
PCRSHIDVAKAICV
94

Peptide-26
PCRSHIDVAKPACV
95

Peptide-27
PCRSHIDVAKPICA
96

Inhibition measurements were carried out in an ELISA format as described above in Example 7. The competitive inhibition plots for the alanine scanning peptides are shown in FIGS. 18-19, and the calculated IC₅₀s are provided in Tables 20-21. Peptide residues whose alanine mutations significantly diminished the inhibitory activity (or significantly raised the IC₅₀) of the peptide were considered key residues where mutations were not tolerated (classified as not tolerated, or “NT”). Peptide residues whose alanine mutations only somewhat diminished the inhibitory activity of the peptide were considered somewhat tolerated (classified as somewhat tolerated, or “ST”). Peptide residues whose alanine mutations performed similarly to the non-mutated peptide-8 sequence were considered non-critical sites where mutations were indeed tolerated (classified as tolerated, or “T”)

TABLE 20

Competitive inhibition and SAR data for

peptide-8 alanine scanning mutants

Fold shift in

IC₅₀relative

to Peptide-8

(IC₅₀Ala

Peptide
SEQ

mutant/IC₅₀
Ala scan
Mutation

description
ID NO:
IC₅₀(μM)
Peptide-8)
position
tolerated?

Peptide-8
26
3.9
—
—
—

(wild type)

Peptide-17
86
16
4.1
Pro-1
NT

Peptide-18
87
5.6
1.4
Arg-3
T

Peptide-19
88
93
24
Ser-4
NT

Peptide-20
89
11
2.8
His-5
ST

Peptide-21
90
1.95
0.5
Ile-6
T

Peptide-22
91
>100
ND
Asp-7
NT

TABLE 21

Competitive inhibition and SAR data for

peptide-8 alanine scanning mutants

Fold shift in

IC₅₀relative

to Peptide-8

(IC₅₀Ala

Peptide
SEQ

mutant/IC₅₀
Ala scan
Mutation

description
ID NO:
IC₅₀(μM)
Peptide-8)
position
tolerated?

Peptide-8
26
1.4
—
—
—

(wild type)

Peptide-23
92
3.8
2.7
Val-8
T

Peptide-24
93
>100
ND
Lys-10
NT

Peptide-25
94
>100
ND
Pro-11
NT

Peptide-26
95
>100
ND
Ile-12
NT

Peptide-27
96
3.1
2.2
Val-14
T

As can be seen from Tables 20-21, alanine mutations at Asp-7, Lys-10, Pro-11, and Ile-12 of peptide-8 were the most poorly tolerated, suggesting that these residues are key residues for inhibiting EGFR Fab binding to the EGFR antigen. The mutation studies indicate that Pro-1 and Ser-4 may also be important for binding inhibition. Additionally, the results indicate that Arg-3, Ile-6, Val-8, and Val-14 are non-critical residues for inhibition of EGFR binding to the EGFR antigen.

Example 10: Optimized Phage Library Construction of Peptide-8

Using the peptide sequence activity relationships of Example 9, DNA oligo libraries were constructed where codons encoding critical residues within peptide-8 were minimally mutated, and codons encoding non-critical residues that were tolerated in the alanine scanning mutation studies (e.g., Arg-3, Ile-6, Val-8, and Val-14) were heavily mutated. The resulting oligos were cloned into bacteriophage vectors used to display the SAR guided peptides via fusion to the pIII filament of the bacteriophage. The relevant vectors were then used to produce the phage optimization libraries via amplification in bacteria using standard techniques in the field.

Example 11: Panning of the Optimized Phage Library

Once the phage optimization libraries were completed, phage libraries were bio-panned using EGFR Fab loaded beads. Multiple rounds of panning were performed where bacteriophage was allowed to bind to EGFR Fab loaded beads, washed, eluted, and amplified. Additional selective pressure was included during each round of panning using a fixed concentration of EGFR antigen or peptide-8. After panning, phage infected bacteria were plated out and colonies picked into 96 well blocks. Clonal phage was then amplified and separated from bacterial cells via centrifugation. Phage containing supernatants were tested in binding ELISAs against EGFR Fab coated plates in the presence or absence of saturating concentrations of EGFR antigen. Phage able to bind EGFR Fab were selected for sequence analysis if the binding signal was reduced in the presence of pEGFR.

Example 12: Panning ELISAs—EGFR Fab Peptides

Clonal phage were harvested as crude supernatants and screened via standard ELISAs. Briefly, biotinylated EGFR Fab was captured on neutravidin coated plates. Prior to the addition of clonal phage, wells were incubated with blocking buffer and EGFR antigen or blocking buffer alone. Without washing or aspirating, clonal phage supernatants were then added to the wells and incubated for a short time. Wells were then washed followed by detection of bound phage using a horse radish conjugated anti-M13 antibody. Clonal phage of interest were then sent for sequence analysis.

Phage panning results of peptide-8 library sequences are shown in Table 22. 679 clonal phage sequences were identified. A consensus sequence was calculated from all the sequences of Table 22 using WebLogo 3.7.4. The consensus sequence is shown in FIG. 20.

TABLE 22

Phage panning results of αEGFR Fab peptide-8 library sequences. (—)

indicates the same amino acid as in aEGFR Fab peptide-8 corresponding position.

Phage binding ELISA

EGFR Fab

EGFR
signal in
SEQ

Phage
Amino acid position sequence
Backgroud
Fab
presence of
ID

number
1
2
3
4
5
6
7
8
9
10
11
12
13
14
signal
signal
EGFR
NO:

Phage-1
P
C
R
S
H
I
D
V
A
K
P
I
C
V
0.071
2.596
0.084
98

Phage-2
—
—
L
—
P
F
—
T
—
—
—
—
—
A
0.067
2.525
0.078
99

Phage-3
—
—
T
—
—
F
—
T
—
—
—
—
—
T
0.068
2.592
0.082
100

Phage-4
—
—
T
—
—
F
—
T
—
—
—
—
—
I
0.070
2.595
0.093
101

Phage-5
—
—
L
—
—
F
—
T
—
—
—
—
—
L
0.078
2.582
0.077
102

Phage-6
—
—
L
—
—
T
—
T
—
—
—
—
—
A
0.071
2.523
0.070
103

Phage-7
—
—
L
—
—
H
—
T
—
—
—
—
—
A
0.069
2.599
0.076
104

Phage-8
—
—
L
—
—
F
—
T
—
—
—
—
—
A
0.070
2.543
0.077
105

Phage-9
—
—
L
—
—
F
—
T
—
—
—
—
—
—
0.073
2.513
0.083
106

Phage-10
—
—
L
—
—
N
—
P
—
—
—
—
—
S
0.064
2.564
0.078
107

Phage-11
—
—
T
—
Q
H
—
T
—
—
—
—
—
I
0.072
2.590
0.090
108

Phage-12
—
—
T
—
—
F
—
T
—
—
—
—
—
A
0.073
2.503
0.091
109

Phage-13
—
—
L
—
—
T
—
T
—
—
—
—
—
—
0.068
2.517
0.076
110

Phage-14
—
—
L
—
—
N
—
P
—
—
—
—
—
A
0.066
2.589
0.068
111

Phage-15
—
—
L
—
—
Y
—
T
—
—
—
—
—
A
0.074
2.584
0.086
112

Phage-16
—
—
N
—
—
Y
—
L
—
—
—
—
—
T
0.082
2.545
0.084
113

Phage-17
—
—
N
—
—
F
—
I
—
—
—
—
—
L
0.067
2.606
0.090
114

Phage-18
—
—
A
Y
—
N
—
P
—
—
—
—
—
S
0.075
2.577
0.089
115

Phage-19
—
—
L
F
—
F
—
P
—
—
—
—
—
S
0.077
2.590
0.126
116

Phage-20
—
—
T
—
—
F
—
P
—
—
—
—
—
A
0.071
2.625
0.376
117

Phage-21
—
—
T
—
—
N
—
N
—
—
—
—
—
A
0.087
2.679
0.175
118

Phage-22
—
—
L
—
—
F
—
T
—
—
—
—
—
I
0.074
2.588
0.070
119

Phage-23
—
—
T
—
—
T
—
L
—
—
—
—
—
—
0.075
2.488
0.072
120

Phage-24
—
—
L
—
Q
F
—
T
—
—
—
—
—
S
0.128
2.485
0.113
121

Phage-25
—
—
L
—
L
F
—
T
—
—
—
—
—
I
0.096
2.475
0.085
122

Phage-26
—
—
L
—
—
F
—
N
—
—
—
—
—
—
0.123
2.463
0.119
123

Phage-27
—
—
I
—
—
F
—
T
—
—
—
—
—
A
0.097
2.461
0.085
124

Phage-28
—
—
I
—
—
Y
—
P
—
—
—
—
—
S
0.078
2.501
0.078
125

Phage-29
—
—
L
—
—
H
—
P
—
—
—
—
—
A
0.076
2.597
0.072
126

Phage-30
—
—
L
—
—
F
—
D
—
—
—
—
—
A
0.078
2.596
0.073
127

Phage-31
—
—
A
—
—
F
—
T
—
—
—
—
—
I
0.083
2.595
0.079
128

Phage-32
—
—
L
—
—
H
—
T
—
—
—
—
—
—
0.071
2.595
0.071
129

Phage-33
—
—
T
—
R
F
—
T
—
—
—
—
—
A
0.083
2.593
0.081
130

Phage-34
—
—
L
—
—
Y
—
L
—
—
—
—
—
I
0.089
2.576
0.078
131

Phage-35
—
—
T
—
—
H
—
L
—
—
—
—
—
—
0.083
2.575
0.079
132

Phage-36
—
—
L
—
—
F
—
I
—
—
—
—
—
A
0.068
2.567
0.066
133

Phage-37
—
—
V
—
—
Y
—
T
—
—
—
—
—
A
0.070
2.533
0.068
134

Phage-38
—
—
L
—
—
H
—
A
—
—
—
—
—
L
0.069
2.513
0.068
135

Phage-39
—
—
—
—
L
N
—
T
—
—
—
—
—
—
0.071
2.505
0.071
136

Phage-40
—
—
L
—
Q
F
—
T
—
—
—
—
—
A
0.076
2.503
0.072
137

Phage-41
—
—
L
—
L
F
—
T
—
Q
—
—
—
—
0.092
2.503
0.089
138

Phage-42
—
—
T
—
—
H
—
T
—
—
—
—
—
—
0.079
2.500
0.079
139

Phage-43
—
—
N
—
—
Y
—
L
—
—
—
—
—
I
0.071
2.496
0.067
140

Phage-44
—
—
L
—
L
Y
—
N
—
—
—
—
—
L
0.071
2.494
0.070
141

Phage-45
—
—
L
—
—
D
—
T
—
—
—
—
—
T
0.106
2.494
0.083
142

Phage-46
—
—
A
—
—
F
—
—
—
—
—
—
—
A
0.075
2.493
0.068
143

Phage-47
—
—
P
F
—
F
—
P
—
—
—
—
—
—
0.086
2.492
0.080
144

Phage-48
—
—
A
—
—
N
—
L
—
—
—
—
—
A
0.073
2.487
0.071
145

Phage-49
—
—
I
—
Q
F
—
T
—
—
—
—
—
A
0.109
2.481
0.080
146

Phage-50
—
—
I
—
L
Y
—
T
—
—
—
—
—
—
0.077
2.476
0.076
147

Phage-51
—
—
L
—
L
T
—
P
—
—
—
—
—
A
0.085
2.474
0.073
148

Phage-52
—
—
T
—
—
H
—
T
—
—
—
—
—
A
0.104
2.473
0.088
149

Phage-53
—
—
L
Y
—
H
—
P
—
—
—
—
—
A
0.099
2.472
0.092
150

Phage-54
—
—
H
—
—
Y
—
T
—
—
—
—
—
A
0.074
2.470
0.072
151

Phage-55
—
—
L
—
—
S
—
P
—
—
—
—
—
S
0.112
2.470
0.108
152

Phage-56
—
—
L
—
—
T
—
I
—
—
—
—
—
L
0.120
2.468
0.117
153

Phage-57
—
—
L
F
—
F
—
P
—
—
—
—
—
A
0.112
2.464
0.097
154

Phage-58
—
—
L
—
—
F
—
P
—
—
—
—
—
A
0.110
2.463
0.105
155

Phage-59
—
—
L
—
P
H
—
T
—
—
—
—
—
L
0.076
2.462
0.073
156

Phage-60
—
—
T
—
—
F
—
L
—
—
—
—
—
—
0.106
2.461
0.084
157

Phage-61
—
—
V
—
L
Y
—
T
—
—
—
—
—
L
0.103
2.461
0.095
158

Phage-62
—
—
T
—
—
L
—
T
—
—
—
—
—
A
0.123
2.459
0.114
159

Phage-63
—
—
L
—
L
F
—
D
—
—
—
—
—
A
0.081
2.456
0.079
160

Phage-64
—
—
N
—
—
H
—
L
—
—
—
—
—
—
0.074
2.448
0.074
161

Phage-65
—
—
I
—
—
Y
—
T
—
—
—
—
—
I
0.142
2.446
0.132
162

Phage-66
—
—
A
—
—
F
—
—
—
—
—
—
—
L
0.103
2.445
0.097
163

Phage-67
—
—
T
—
—
F
—
S
—
—
—
—
—
I
0.069
2.442
0.066
164

Phage-68
—
—
T
—
—
F
—
S
—
—
—
—
—
S
0.132
2.433
0.124
165

Phage-69
—
—
T
—
L
V
—
T
—
—
—
—
—
A
0.132
2.424
0.120
166

Phage-70
—
—
L
—
L
N
—
P
—
—
—
—
—
A
0.153
2.423
0.136
167

Phage-71
—
—
I
—
—
N
—
T
—
—
—
—
—
A
0.114
2.419
0.113
168

Phage-72
—
—
V
—
—
F
—
T
—
—
—
—
—
A
0.122
2.415
0.102
169

Phage-73
—
—
N
—
P
F
—
T
—
—
—
—
—
—
0.109
2.369
0.087
170

Phage-74
—
—
V
—
—
N
—
—
—
—
—
—
—
A
0.108
2.358
0.093
171

Phage-75
—
—
L
—
—
V
—
P
—
—
—
—
—
—
0.092
2.349
0.089
172

Phage-76
—
—
L
—
—
S
—
D
—
—
—
—
—
A
0.074
2.331
0.071
173

Phage-77
—
—
T
—
—
H
—
—
—
—
—
—
—
—
0.109
2.316
0.085
174

Phage-78
—
—
L
—
F
Y
—
L
—
—
—
—
—
S
0.127
2.263
0.127
175

Phage-79
—
—
S
—
—
Y
—
T
—
—
—
—
—
F
0.074
2.199
0.073
176

Phage-80
—
—
A
—
—
V
—
T
—
—
—
—
—
—
0.069
2.170
0.068
177

Phage-81
—
—
L
—
L
D
—
P
—
—
—
—
—
A
0.073
2.166
0.072
178

Phage-82
—
—
L
—
—
—
—
D
—
—
—
—
—
—
0.154
2.150
0.139
179

Phage-83
—
—
T
—
—
A
—
T
—
N
—
—
—
A
0.074
2.132
0.071
180

Phage-84
—
—
L
—
L
T
—
H
—
—
—
—
—
I
0.088
2.125
0.075
181

Phage-85
—
—
F
—
—
H
—
P
—
—
—
—
—
—
0.100
2.121
0.088
182

Phage-86
—
—
S
—
—
F
—
N
—
—
—
—
—
A
0.081
2.086
0.078
183

Phage-87
—
—
A
—
L
T
—
T
—
—
—
—
—
A
0.128
1.993
0.123
184

Phage-88
—
—
S
—
—
H
—
S
—
—
—
—
—
T
0.069
1.993
0.067
185

Phage-89
—
—
T
—
P
Y
—
—
—
—
—
—
—
I
0.077
1.983
0.075
186

Phage-90
—
—
L
—
P
L
—
S
—
—
—
—
—
A
0.114
1.980
0.096
187

Phage-91
—
—
L
—
—
T
—
T
—
—
—
—
—
I
0.072
1.972
0.071
188

Phage-92
—
—
L
—
L
F
—
T
—
—
—
—
—
H
0.083
1.969
0.079
189

Phage-93
—
—
A
—
—
Y
—
D
—
—
—
—
—
S
0.091
1.804
0.073
190

Phage-94
—
—
S
—
—
Y
—
—
—
—
—
—
—
A
0.091
1.785
0.083
191

Phage-95
—
—
A
—
—
F
—
N
—
—
—
—
—
A
0.092
1.688
0.076
192

Phage-96
—
—
T
—
Q
Y
—
T
—
—
—
—
—
T
0.074
1.676
0.074
193

Phage-97
—
—
A
—
—
H
—
L
—
—
—
—
—
I
0.080
1.670
0.080
194

Phage-98
—
—
I
—
Q
Y
—
T
—
—
—
—
—
T
0.075
1.469
0.073
195

Phage-99
—
—
A
—
L
Y
—
—
—
—
—
—
—
A
0.093
1.462
0.082
196

Phage-100
—
—
L
—
—
S
—
A
—
—
—
—
—
—
0.123
1.428
0.087
197

Phage-101
—
—
A
—
—
H
—
T
—
—
—
—
—
L
0.078
1.428
0.076
198

Phage-102
—
—
L
—
—
N
—
S
—
—
—
—
—
H
0.081
1.394
0.078
199

Phage-103
—
—
—
—
—
L
—
P
—
—
—
—
—
S
0.078
1.164
0.074
200

Phage-104
—
—
L
—
Q
T
—
D
—
—
—
—
—
—
0.080
1.138
0.075
201

Phage-105
—
—
S
—
P
Y
—
S
—
—
—
—
—
A
0.106
1.008
0.090
202

Phage-106
—
—
V
—
—
F
Y
T
—
Y
—
—
—
—
0.098
0.968
0.083
203

Phage-107
—
—
T
—
L
H
—
N
—
—
—
—
—
T
0.077
0.635
0.075
204

Phage-108
—
—
V
—
—
A
—
P
—
—
—
—
—
A
0.072
0.459
0.072
205

Phage-109
—
—
A
—
L
S
—
D
—
—
—
—
—
I
0.075
0.329
0.067
206

Phage-110
W
—
H
P
Q
S
—
P
—
Y
—
—
—
D
0.094
0.265
0.070
207

Phage-111
—
—
A
—
Q
H
—
L
—
—
—
—
—
I
0.085
0.213
0.081
208

Phage-112
L
—
H
P
Q
N
—
A
—
Y
—
N
—
F
0.081
0.071
0.090
209

Phage-113
F
—
H
P
Q
A
—
L
—
Y
—
M
—
—
0.076
0.069
0.081
210

Phage-114
W
—
H
P
Q
N
—
F
—
Y
—
M
—
D
0.078
0.067
0.086
211

Phage-115
—
—
L
—
—
T
—
L
—
—
—
—
—
L
0.074
2.491
0.074
212

Phage-116
—
—
A
—
—
Y
—
T
—
—
—
—
—
I
0.074
2.485
0.074
213

Phage-117
—
—
V
—
—
Y
—
T
—
—
—
—
—
T
0.091
2.455
0.091
214

Phage-118
—
—
V
—
—
T
—
T
—
—
—
—
—
S
0.070
2.362
0.070
215

Phage-119
—
—
T
G
P
Y
—
T
—
—
—
—
—
A
0.090
2.129
0.090
216

Phage-120
—
—
N
—
M
Y
—
T
—
—
—
—
—
A
0.068
2.496
0.068
217

Phage-121
—
—
L
—
L
H
—
D
—
—
—
—
—
L
0.072
2.430
0.073
218

Phage-122
—
—
T
—
—
—
—
T
—
—
—
—
—
I
0.070
2.169
0.070
219

Phage-123
—
—
N
—
—
Y
—
L
—
—
—
—
—
—
0.069
2.479
0.069
220

Phage-124
—
—
T
—
—
Y
—
L
—
—
—
—
—
T
0.097
2.440
0.098
221

Phage-125
—
—
I
G
P
Y
—
T
—
—
—
—
—
A
0.072
2.576
0.073
222

Phage-126
—
—
T
—
—
F
—
P
—
—
—
—
—
S
0.072
2.562
0.073
223

Phage-127
—
—
L
—
—
H
—
N
—
—
—
—
—
A
0.070
2.528
0.071
224

Phage-128
—
—
P
—
—
F
—
T
—
—
—
—
—
S
0.081
2.509
0.082
225

Phage-129
—
—
S
—
L
F
—
T
—
—
—
—
—
I
0.067
2.479
0.068
226

Phage-130
—
—
V
—
—
Y
—
T
—
—
—
—
—
—
0.110
2.445
0.111
227

Phage-131
—
—
T
—
—
H
—
T
—
—
—
—
—
L
0.088
2.485
0.090
228

Phage-132
—
—
T
—
P
Y
—
T
—
—
—
—
—
T
0.071
2.472
0.073
229

Phage-133
—
—
T
—
—
F
—
—
—
—
—
—
—
A
0.107
2.395
0.108
230

Phage-134
—
—
T
—
—
N
—
T
—
—
—
—
—
A
0.080
2.037
0.082
231

Phage-135
—
—
T
—
—
F
—
I
—
—
—
—
—
A
0.079
2.649
0.081
232

Phage-136
—
—
L
—
—
A
—
T
—
—
—
—
—
I
0.072
2.589
0.074
233

Phage-137
—
—
T
—
—
V
—
T
—
—
—
—
—
A
0.070
2.517
0.072
234

Phage-138
—
—
L
—
—
N
—
T
—
—
—
—
—
—
0.071
2.482
0.072
235

Phage-139
—
—
L
—
—
T
—
T
—
—
—
—
—
T
0.082
2.161
0.083
236

Phage-140
—
—
L
Y
P
S
—
P
—
—
—
—
—
A
0.073
2.483
0.075
237

Phage-141
—
—
P
—
—
H
—
S
—
—
—
—
—
—
0.072
1.781
0.073
238

Phage-142
—
—
N
—
—
T
—
N
—
—
—
—
—
T
0.077
2.601
0.079
239

Phage-143
—
—
N
—
—
F
—
S
—
—
—
—
—
A
0.077
2.006
0.079
240

Phage-144
—
—
A
—
L
H
—
T
—
—
—
—
—
S
0.076
2.481
0.078
241

Phage-145
—
—
S
—
L
Y
—
A
—
—
—
—
—
—
0.068
2.581
0.070
242

Phage-146
—
—
T
—
—
T
—
T
—
—
—
—
—
I
0.074
2.395
0.076
243

Phage-147
—
—
T
—
—
Y
—
H
—
—
—
—
—
A
0.118
2.404
0.120
244

Phage-148
—
—
N
—
L
H
—
T
—
—
—
—
—
—
0.070
2.506
0.072
245

Phage-149
—
—
L
—
L
T
—
T
—
—
—
—
—
T
0.067
2.600
0.070
246

Phage-150
—
—
A
—
—
F
—
P
—
—
—
—
—
F
0.107
2.340
0.110
247

Phage-151
—
—
T
—
—
F
—
I
—
—
—
—
—
L
0.073
2.628
0.076
248

Phage-152
—
—
L
—
—
V
—
T
—
—
—
—
—
A
0.127
2.419
0.130
249

Phage-153
—
—
T
—
—
H
—
P
—
—
—
—
—
I
0.076
2.473
0.079
250

Phage-154
—
—
L
F
—
H
—
T
—
—
—
—
—
L
0.093
2.485
0.096
251

Phage-155
—
—
T
—
—
Y
—
T
—
—
—
—
—
A
0.068
2.546
0.072
252

Phage-156
—
—
L
—
L
F
—
—
—
—
—
—
—
L
0.073
2.449
0.076
253

Phage-157
—
—
I
—
L
V
—
T
—
—
—
—
—
—
0.070
2.234
0.073
254

Phage-158
—
—
L
—
—
Y
—
N
—
—
—
—
—
I
0.069
2.491
0.072
255

Phage-159
—
—
N
—
—
F
—
T
—
—
—
—
—
T
0.073
1.550
0.075
256

Phage-160
—
—
L
—
L
T
—
—
—
—
—
—
—
S
0.072
2.152
0.075
257

Phage-161
—
—
L
—
—
D
—
D
—
—
—
—
—
A
0.084
2.459
0.088
258

Phage-162
—
—
T
—
—
—
—
T
—
—
—
—
—
A
0.073
2.498
0.077
259

Phage-163
—
—
L
—
—
H
—
N
—
—
—
—
—
S
0.075
2.483
0.079
260

Phage-164
—
—
S
—
—
N
—
P
—
—
—
—
—
S
0.072
2.575
0.076
261

Phage-165
—
—
T
G
P
F
—
T
—
—
—
—
—
A
0.073
2.512
0.078
262

Phage-166
—
—
N
—
L
F
—
D
—
—
—
—
—
—
0.068
2.396
0.072
263

Phage-167
—
—
V
—
—
H
—
P
—
—
—
—
—
A
0.072
2.021
0.076
264

Phage-168
—
—
T
—
—
F
—
—
—
—
—
—
—
T
0.070
2.581
0.074
265

Phage-169
—
—
T
—
P
Y
—
T
—
—
—
—
—
—
0.130
2.419
0.134
266

Phage-170
—
—
T
—
P
Y
—
T
—
—
—
—
—
S
0.074
2.478
0.078
267

Phage-171
—
—
T
—
—
T
—
T
—
—
—
—
—
—
0.073
2.525
0.078
268

Phage-172
—
—
T
—
—
H
—
L
—
—
—
—
—
A
0.071
2.440
0.076
269

Phage-173
—
—
T
—
L
D
—
T
—
—
—
—
—
T
0.073
2.238
0.078
270

Phage-174
—
—
A
—
L
F
—
T
—
—
—
—
—
A
0.070
2.403
0.075
271

Phage-175
—
—
N
—
—
Y
—
T
—
—
—
—
—
S
0.072
2.496
0.078
272

Phage-176
—
—
T
G
P
H
—
—
—
—
—
—
—
A
0.074
2.465
0.080
273

Phage-177
—
—
P
F
—
N
—
P
—
—
—
—
—
A
0.073
2.593
0.078
274

Phage-178
—
—
L
—
—
Y
—
T
—
—
—
—
—
—
0.073
2.504
0.078
275

Phage-179
—
—
A
Y
—
N
—
P
—
—
—
—
—
A
0.073
2.508
0.079
276

Phage-180
—
—
L
—
—
H
—
T
—
—
—
—
—
I
0.102
2.526
0.108
277

Phage-181
—
—
S
—
—
T
—
P
—
—
—
—
—
A
0.071
1.659
0.075
278

Phage-182
—
—
L
—
—
T
—
T—
—
Q
—
—
—
—
0.109
2.458
0.115
279

Phage-183
—
—
A
—
N
Y
—
L
—
—
—
—
—
T
0.077
2.344
0.082
280

Phage-184
—
—
T
—
—
T
—
D
—
—
—
—
—
—
0.073
1.572
0.076
281

Phage-185
—
—
A
F
G
N
—
P
—
—
—
—
—
S
0.065
2.489
0.072
282

Phage-186
—
—
L
—
—
A
—
T
—
—
—
—
—
T
0.070
2.504
0.077
283

Phage-187
—
—
L
—
—
N
—
T
—
—
—
—
—
I
0.071
2.449
0.078
284

Phage-188
—
—
L
—
N
T
—
—
—
—
—
—
—
I
0.090
0.618
0.092
285

Phage-189
—
—
T
—
—
F
—
T
—
—
—
—
—
L
0.074
2.574
0.080
286

Phage-190
—
—
L
—
—
A
—
P
—
—
—
—
—
A
0.108
2.494
0.115
287

Phage-191
—
—
L
—
—
H
—
S
—
—
—
—
—
S
0.077
2.473
0.084
288

Phage-192
—
—
L
F
P
H
—
P
—
—
—
—
—
A
0.074
2.477
0.081
289

Phage-193
—
—
T
—
—
S
—
—
—
—
—
—
—
A
0.077
1.708
0.081
290

Phage-194
—
—
L
—
L
H
—
T
—
—
—
—
—
T
0.085
2.485
0.092
291

Phage-195
—
—
A
—
—
T
—
T
—
—
—
—
—
—
0.082
2.466
0.090
292

Phage-196
—
—
V
—
—
F
—
L
—
—
—
—
—
L
0.067
2.482
0.074
293

Phage-197
—
—
T
—
—
H
—
—
—
—
—
—
—
T
0.070
2.576
0.077
294

Phage-198
—
—
I
—
—
Y
—
T
—
—
—
—
—
L
0.071
2.481
0.079
295

Phage-199
—
—
I
—
—
F
—
T
—
—
—
—
—
—
0.070
2.442
0.078
296

Phage-200
—
—
L
—
—
A
—
T
—
—
—
—
—
A
0.069
2.456
0.077
297

Phage-201
—
—
T
—
—
H
—
—
—
—
—
—
—
T
0.076
2.616
0.084
298

Phage-202
—
—
T
—
—
F
—
L
—
—
—
—
—
A
0.069
2.505
0.076
299

Phage-203
—
—
T
—
—
A
—
T
—
—
—
—
—
T
0.071
2.537
0.079
300

Phage-204
—
—
A
—
P
Y
—
D
—
—
—
—
—
T
0.068
1.894
0.074
301

Phage-205
—
—
L
—
P
V
—
T
—
—
—
—
—
A
0.071
2.410
0.079
302

Phage-206
—
—
A
—
L
N
—
T
—
—
—
—
—
—
0.070
2.553
0.079
303

Phage-207
—
—
L
—
—
N
—
N
—
—
—
—
—
A
0.072
2.564
0.081
304

Phage-208
—
—
L
—
M
T
—
T
—
—
—
—
—
T
0.070
2.480
0.078
305

Phage-209
—
—
L
—
L
F
—
I
—
—
—
—
—
I
0.072
2.505
0.081
306

Phage-210
—
—
T
—
—
F
—
L
—
—
—
—
—
—
0.072
2.498
0.080
307

Phage-211
—
—
—
—
Y
F
H
S
P
—
—
—
—
A
0.083
2.504
0.092
308

Phage-212
—
—
T
—
P
F
—
T
—
—
—
—
—
I
0.074
2.354
0.082
309

Phage-213
—
—
S
—
L
F
—
L
—
—
—
—
—
S
0.080
2.502
0.089
310

Phage-214
—
—
V
—
—
S
—
T
—
—
—
—
—
A
0.068
1.836
0.075
311

Phage-215
—
—
N
—
—
F
—
T
—
—
—
—
—
A
0.068
2.519
0.077
312

Phage-216
—
—
A
—
—
F
—
N
—
—
—
—
—
S
0.082
2.570
0.092
313

Phage-217
—
—
—
—
R
Y
—
T
—
—
—
—
—
—
0.067
2.077
0.075
314

Phage-218
—
—
S
—
—
T
—
L
—
—
—
—
—
—
0.076
0.724
0.078
315

Phage-219
—
—
T
—
—
L
—
T
—
—
—
—
—
F
0.067
2.602
0.077
316

Phage-220
—
—
L
—
—
S
—
N
—
—
—
—
—
F
0.070
2.376
0.080
317

Phage-221
—
—
A
—
—
H
—
T
—
—
—
—
—
A
0.070
2.594
0.081
318

Phage-222
—
—
V
—
—
F
—
P
—
—
—
—
—
F
0.074
2.491
0.084
319

Phage-223
—
—
L
—
—
Y
—
H
—
—
—
—
—
S
0.074
2.463
0.084
320

Phage-224
—
—
T
—
—
N
—
L
—
—
—
—
—
—
0.071
2.477
0.081
321

Phage-225
—
—
T
—
—
F
—
A
—
—
—
—
—
S
0.073
2.380
0.083
322

Phage-226
—
—
L
—
—
V
—
—
—
—
—
—
—
T
0.075
2.445
0.085
323

Phage-227
—
—
L
—
Q
Y
—
D
—
—
—
—
—
A
0.079
2.412
0.090
324

Phage-228
—
—
H
—
—
T
—
T
—
—
—
—
—
L
0.072
2.269
0.082
325

Phage-229
—
—
T
—
Q
Y
—
T
—
—
—
—
—
—
0.070
2.450
0.081
326

Phage-230
—
—
N
—
—
T
—
T
—
—
—
—
—
—
0.085
2.472
0.096
327

Phage-231
—
—
L
—
—
L
—
T
—
—
—
—
—
T
0.073
2.495
0.084
328

Phage-232
—
—
S
—
L
Y
—
T
—
—
—
—
—
Y
0.065
1.908
0.074
329

Phage-233
—
—
A
—
L
F
—
N
—
—
—
—
—
I
0.070
2.119
0.080
330

Phage-234
—
—
T
—
—
LL
—
L
—
—
—
—
—
T
0.081
2.483
0.092
331

Phage-235
—
—
L
—
Q
H
—
D
—
—
—
—
—
—
0.069
0.848
0.072
332

Phage-236
—
—
V
—
P
F
—
T
—
—
—
—
—
L
0.074
2.641
0.087
333

Phage-237
—
—
L
—
—
T
—
S
—
—
—
—
—
—
0.124
2.441
0.136
334

Phage-238
R
—
S
—
—
H
—
T
—
—
I
—
—
G
0.071
2.472
0.083
335

Phage-239
—
—
L
—
—
H
—
Y
—
—
—
—
—
L
0.074
2.572
0.087
336

Phage-240
—
—
N
—
—
Y
—
T
—
—
—
—
—
A
0.093
2.501
0.106
337

Phage-241
—
—
L
F
—
Y
—
P
—
—
—
—
—
S
0.075
2.502
0.088
338

Phage-242
—
—
L
F
—
N
—
P
—
—
—
—
—
Y
0.074
2.535
0.087
339

Phage-243
—
—
T
—
—
F
—
A
—
—
—
—
—
A
0.074
2.289
0.086
340

Phage-244
—
—
A
—
—
H
—
N
—
—
—
—
—
L
0.071
2.348
0.083
341

Phage-245
—
—
N
—
—
F
—
T
—
—
—
—
—
L
0.068
2.588
0.081
342

Phage-246
—
—
L
—
—
F
—
Y
—
—
—
—
—
T
0.066
2.449
0.079
343

Phage-247
—
V
C
L
S
F
R
Y
R
Q
A
D
L
C
0.130
2.417
0.142
344

Phage-248
—
—
I
—
P
F
—
T
—
—
—
—
—
A
0.069
2.617
0.082
345

Phage-249
—
—
L
—
P
F
—
N
—
—
—
—
—
T
0.100
2.362
0.113
346

Phage-250
—
—
T
—
—
F
—
N
—
—
—
—
—
L
0.072
2.507
0.085
347

Phage-251
—
—
L
—
—
S
—
T
—
—
—
—
—
I
0.068
1.565
0.077
348

Phage-252
—
—
L
—
—
S
—
T
—
—
—
—
—
L
0.067
2.496
0.080
349

Phage-253
—
—
T
—
P
F
—
L
—
—
—
—
—
T
0.084
0.883
0.088
350

Phage-254
—
—
T
—
P
Y
—
—
—
—
—
—
—
A
0.073
1.856
0.083
351

Phage-255
—
—
A
—
—
Y
—
T
—
—
—
—
—
S
0.071
2.369
0.084
352

Phage-256
—
—
P
—
—
T
—
T
—
—
—
—
—
—
0.069
2.503
0.084
353

Phage-257
—
—
L
—
—
S
—
S
—
—
—
—
—
—
0.080
2.231
0.093
354

Phage-258
—
—
T
—
L
F
—
T
—
—
—
—
—
T
0.069
2.597
0.084
355

Phage-259
—
—
L
—
—
V
—
N
—
—
—
—
—
—
0.070
1.452
0.079
356

Phage-260
—
—
L
—
Q
Y
—
N
—
—
—
—
—
A
0.070
1.384
0.078
357

Phage-261
—
—
L
G
P
F
—
T
—
—
—
—
—
S
0.076
2.322
0.090
358

Phage-262
—
—
L
—
—
T
—
P
—
—
—
—
—
I
0.076
2.462
0.091
359

Phage-263
—
—
T
—
—
Y
—
S
—
—
—
—
—
A
0.067
2.375
0.082
360

Phage-264
—
—
L
—
F
T
—
—
—
—
—
—
—
S
0.093
2.466
0.108
361

Phage-265
—
—
L
—
—
S
—
N
—
—
—
—
—
T
0.082
1.920
0.094
362

Phage-266
—
—
T
—
—
F
—
N
—
—
—
—
—
A
0.081
2.458
0.096
363

Phage-267
—
—
T
—
—
N
—
T
—
—
—
—
—
S
0.070
2.476
0.086
364

Phage-268
—
—
V
—
L
Y
—
T
—
—
—
—
—
T
0.080
2.494
0.096
365

Phage-269
—
—
L
—
L
T
—
S
—
—
—
—
—
A
0.072
2.320
0.087
366

Phage-270
—
—
T
—
—
Y
—
—
—
—
—
—
—
L
0.076
2.572
0.093
367

Phage-271
—
—
L
—
—
F
—
P
—
—
—
—
—
I
0.115
2.428
0.131
368

Phage-272
—
—
T
—
—
S
—
N
—
—
—
—
—
A
0.073
2.492
0.090
369

Phage-273
—
—
A
Y
—
N
—
P
—
—
T
—
—
S
0.072
2.468
0.089
370

Phage-274
—
—
L
—
P
H
—
T
—
—
—
—
—
A
0.070
2.473
0.087
371

Phage-275
—
—
I
—
L
T
—
N
—
—
—
—
—
S
0.075
1.899
0.089
372

Phage-276
—
—
A
—
Q
F
—
N
—
—
—
—
—
A
0.090
1.979
0.104
373

Phage-277
—
—
T
—
L
T
—
T
—
—
—
—
—
—
0.077
2.492
0.095
374

Phage-278
—
—
L
—
—
Y
—
L
—
—
—
—
—
A
0.075
2.489
0.094
375

Phage-279
—
—
I
—
P
Y
—
N
—
—
—
—
—
I
0.084
0.989
0.091
376

Phage-280
—
—
I
—
Q
T
—
T
—
—
—
—
—
A
0.074
1.579
0.086
377

Phage-281
—
—
T
—
L
H
—
T
—
—
—
—
—
T
0.070
2.573
0.091
378

Phage-282
—
—
S
—
F
D
N
P
T
Q
—
—
—
A
0.080
2.480
0.100
379

Phage-283
—
—
N
—
—
H
—
T
—
—
—
—
—
A
0.077
2.485
0.097
380

Phage-284
—
—
P
—
—
Y
—
A
—
—
—
—
—
—
0.068
2.552
0.090
381

Phage-285
—
—
I
—
—
Y
—
T
—
—
—
—
—
A
0.073
2.412
0.093
382

Phage-286
—
—
L
—
—
Y
—
P
—
—
—
—
—
A
0.075
2.512
0.098
383

Phage-287
—
—
V
—
L
T
—
T
—
—
—
—
—
A
0.072
2.466
0.096
384

Phage-288
—
—
T
—
L
A
—
P
—
—
—
—
—
I
0.074
1.189
0.085
385

Phage-289
—
—
T
—
L
LL
—
D
—
—
—
—
—
T
0.071
1.094
0.082
386

Phage-290
—
—
A
—
P
Y
—
T
—
—
—
—
—
T
0.106
2.374
0.130
387

Phage-291
—
—
A
—
P
Y
—
L
—
—
—
—
—
T
0.080
2.529
0.106
388

Phage-292
—
—
L
—
—
N
—
S
—
—
—
—
—
A
0.112
2.417
0.136
389

Phage-293
—
—
T
—
L
T
—
T
—
—
—
—
—
T
0.071
2.478
0.097
390

Phage-294
—
—
L
—
—
N
—
H
S
—
—
—
—
S
0.075
2.502
0.101
391

Phage-295
—
—
T
—
—
F
—
—
—
—
—
—
—
—
0.071
1.560
0.088
392

Phage-296
—
—
L
Y
L
N
—
P
—
—
—
—
—
L
0.079
2.295
0.103
393

Phage-297
—
—
L
—
—
F
—
T
—
—
—
—
—
S
0.066
2.624
0.095
394

Phage-298
—
—
N
Y
—
N
—
P
—
—
—
—
—
A
0.070
2.418
0.097
395

Phage-299
—
—
A
—
Q
Y
—
T
—
—
—
—
—
T
0.089
1.848
0.110
396

Phage-300
—
—
T
—
L
H
—
D
—
—
—
—
—
L
0.077
1.446
0.095
397

Phage-301
—
—
T
—
—
N
N
N
—
—
—
—
—
A
0.074
2.486
0.108
398

Phage-302
—
—
L
F
—
N
—
P
—
Q
—
—
—
A
0.076
2.597
0.112
399

Phage-303
—
—
L
—
—
D
—
A
—
—
—
—
—
P
0.105
2.443
0.139
400

Phage-304
—
—
L
—
—
Y
—
N
—
—
—
—
—
T
0.072
2.525
0.110
401

Phage-305
—
—
A
—
—
A
—
T
—
—
—
—
—
A
0.098
2.443
0.140
402

Phage-306
—
—
T
—
—
H
—
P
S
—
—
—
—
—
0.126
2.443
0.168
403

Phage-307
—
—
T
—
L
Y
—
T
—
—
—
—
—
T
0.068
2.484
0.121
404

Phage-308
—
—
A
—
—
F
—
N
—
—
—
—
—
—
0.075
0.831
0.093
405

Phage-309
—
—
S
—
P
F
—
T
—
—
—
—
—
A
0.072
2.633
0.169
406

Phage-310
—
—
S
—
P
F
—
P
—
—
—
—
—
A
0.063
2.617
0.264
407

Phage-311
—
—
A
—
—
H
—
—
—
—
—
—
—
L
0.073
0.121
0.079
408

Phage-312
L
—
F
L
—
P
E
D
—
S
—
F
—
F
0.070
0.074
0.071
409

Phage-313
—
—
H
P
Q
N
—
T
—
Y
—
F
—
S
0.089
0.088
0.071
410

Phage-314
H
—
H
P
Q
F
H
S
—
Y
—
F
—
A
0.081
0.077
0.075
411

Phage-315
—
—
T
—
—
T
—
I
—
—
—
—
—
L
0.079
2.573
0.079
412

Phage-316
—
—
A
—
—
Y
—
T
—
—
—
—
—
A
0.073
2.558
0.066
413

Phage-317
—
—
T
—
—
T
—
T
—
—
—
—
—
A
0.069
2.537
0.067
414

Phage-318
—
—
L
—
—
D
—
T
—
—
—
—
—
A
0.092
2.531
0.091
415

Phage-319
—
—
T
—
—
H
—
N
—
—
—
—
—
H
0.086
2.510
0.076
416

Phage-320
—
—
T
—
—
F
—
T
—
—
—
—
—
—
0.072
2.655
0.073
417

Phage-321
—
—
A
—
—
H
—
T
—
—
—
—
—
I
0.074
2.497
0.075
418

Phage-322
—
—
L
—
L
N
—
N
—
—
—
—
—
S
0.076
2.624
0.079
419

Phage-323
—
—
L
—
—
F
—
T
—
—
—
—
—
T
0.069
2.518
0.074
420

Phage-324
—
—
L
—
—
S
—
T
—
—
—
—
—
A
0.074
2.576
0.080
421

Phage-325
—
—
N
—
L
Y
—
T
—
—
—
—
—
L
0.064
2.590
0.070
422

Phage-326
—
—
A
—
—
D
—
T
—
—
—
—
—
A
0.069
2.434
0.076
423

Phage-327
—
—
T
—
—
H
—
L
—
—
—
—
—
T
0.070
2.471
0.077
424

Phage-328
—
—
L
F
L
N
—
P
—
—
—
—
—
A
0.106
2.519
0.114
425

Phage-329
—
—
L
—
—
F
—
—
—
—
—
—
—
A
0.066
2.492
0.074
426

Phage-330
—
—
A
—
—
Y
—
N
—
—
—
—
—
—
0.084
2.342
0.092
427

Phage-331
—
—
L
—
L
F
—
T
—
—
—
—
—
A
0.070
2.586
0.079
428

Phage-332
—
—
L
—
—
N
—
T
—
—
—
—
—
A
0.068
2.611
0.078
429

Phage-333
—
—
I
—
—
F
—
T
—
—
—
—
—
L
0.070
2.509
0.080
430

Phage-334
—
—
L
—
P
F
—
T
—
—
—
—
—
I
0.104
2.468
0.115
431

Phage-335
—
—
T
—
—
Y
—
T
—
—
—
—
—
S
0.070
2.494
0.081
432

Phage-336
—
—
L
—
L
Y
—
T
—
—
—
—
—
I
0.067
2.607
0.079
433

Phage-337
—
—
A
—
—
T
—
T
—
—
—
—
—
T
0.067
2.461
0.083
434

Phage-338
—
—
L
—
—
Y
—
T
—
—
—
—
—
I
0.068
2.541
0.084
435

Phage-339
—
—
T
—
—
Y
—
T
—
—
—
—
—
—
0.085
2.513
0.107
436

Phage-340
—
—
N
—
L
Y
—
T
—
—
—
—
—
I
0.073
2.502
0.095
437

Phage-341
—
—
T
—
—
H
—
T
—
—
—
—
—
—
0.074
2.592
0.069
438

Phage-342
—
—
L
—
—
Y
—
T
—
—
—
—
—
—
0.085
2.587
0.076
439

Phage-343
—
—
A
—
—
T
—
T
—
—
—
—
—
A
0.067
2.509
0.066
440

Phage-344
—
—
L
—
—
Y
—
T
—
—
—
—
—
T
0.088
2.497
0.075
441

Phage-345
—
—
L
—
—
H
—
T
—
—
—
—
—
L
0.068
2.603
0.070
442

Phage-346
—
—
L
—
—
T
—
T
—
—
—
—
—
L
0.072
2.600
0.075
443

Phage-347
—
—
N
—
—
Y
—
T
—
—
—
—
—
L
0.075
2.499
0.083
444

Phage-348
—
—
L
Y
—
N
—
P
—
—
—
—
—
A
0.071
2.519
0.097
445

Phage-349
—
—
L
F
—
N
—
P
—
—
—
—
—
A
0.074
2.678
0.124
446

Phage-350
—
—
T
—
—
Y
—
T
—
—
—
—
—
L
0.073
2.586
0.072
447

Phage-351
—
—
L
—
—
Y
—
T
—
—
—
—
—
L
0.072
2.584
0.069
448

Phage-352
—
—
T
—
—
Y
—
T
—
—
—
—
—
T
0.093
2.552
0.089
449

Phage-353
—
—
N
—
—
Y
—
L
—
—
—
—
—
L
0.070
2.612
0.101
450

Phage-354
—
—
L
—
—
F
—
P
—
—
—
—
—
S
0.082
2.592
0.075
451

Phage-355
—
—
L
—
—
Y
—
I
—
—
—
—
—
A
0.093
2.583
0.077
452

Phage-356
—
—
A
Y
—
H
—
P
—
—
—
—
—
A
0.084
2.573
0.083
453

Phage-357
—
—
N
—
L
H
—
T
—
—
—
—
—
L
0.090
2.564
0.074
454

Phage-358
—
—
T
—
—
Y
—
—
—
—
—
—
—
A
0.073
2.558
0.064
455

Phage-359
—
—
A
—
—
F
—
l
—
—
—
—
—
—
0.085
2.550
0.070
456

Phage-360
—
—
A
—
—
Y
—
T
—
—
—
—
—
L
0.072
2.532
0.069
457

Phage-361
—
—
V
—
—
T
—
T
—
—
—
—
—
L
0.082
2.523
0.082
458

Phage-362
—
—
N
—
—
F
—
N
—
—
—
—
—
A
0.071
2.483
0.069
459

Phage-363
—
—
A
—
L
F
—
N
—
—
—
—
—
T
0.104
2.479
0.093
460

Phage-364
—
—
L
—
—
F
—
—
—
—
—
—
—
i
0.104
2.474
0.092
461

Phage-365
—
—
V
—
—
N
—
T
—
—
—
—
—
L
0.089
2.471
0.075
462

Phage-366
—
—
L
—
—
D
—
P
—
—
—
—
—
L
0.092
2.465
0.089
463

Phage-367
—
—
I
—
—
Y
—
T
—
—
—
—
—
—
0.080
2.457
0.073
464

Phage-368
—
—
N
—
—
N
—
P
—
—
—
—
—
A
0.071
2.456
0.067
465

Phage-369
—
—
T
—
—
H
—
T
—
—
—
—
—
S
0.101
2.455
0.101
466

Phage-370
—
—
L
—
—
F
—
N
—
—
—
—
—
A
0.077
2.455
0.071
467

Phage-371
—
—
L
—
—
S
—
P
—
—
—
—
—
A
0.075
2.454
0.070
468

Phage-372
—
—
T
—
L
H
—
T
—
—
—
—
—
L
0.092
2.446
0.086
469

Phage-373
—
—
N
—
L
H
—
T
—
—
—
—
—
I
0.084
2.445
0.076
470

Phage-374
—
—
L
—
Q
Y
—
T
—
—
—
—
—
A
0.126
2.435
0.111
471

Phage-375
—
—
L
—
—
H
—
T
—
—
—
—
—
S
0.107
2.432
0.091
472

Phage-376
—
—
L
—
P
F
—
D
—
—
—
—
—
A
0.127
2.430
0.123
473

Phage-377
—
—
A
—
—
A
—
T
—
—
—
—
—
T
0.073
2.428
0.073
474

Phage-378
—
—
T
—
—
N
—
P
—
—
—
—
—
I
0.089
2.425
0.075
475

Phage-379
—
—
A
—
P
Y
—
T
—
—
—
—
—
A
0.083
2.423
0.071
476

Phage-380
—
—
L
—
—
H
—
I
—
—
—
—
—
S
0.140
2.422
0.121
477

Phage-381
—
—
A
—
—
H
—
T
—
—
—
—
—
—
0.092
2.419
0.090
478

Phage-382
—
—
T
—
—
Y
—
P
—
—
—
—
—
S
0.066
2.419
0.065
479

Phage-383
—
—
P
—
—
Y
—
T
—
—
—
—
—
A
0.146
2.413
0.126
480

Phage-384
—
—
S
—
P
Y
—
T
—
—
—
—
—
L
0.082
2.412
0.077
481

Phage-385
—
—
T
—
—
Y
—
N
—
—
—
—
—
S
0.075
2.406
0.073
482

Phage-386
—
—
V
—
—
T
—
S
—
—
—
—
—
—
0.086
2.398
0.078
483

Phage-387
—
—
V
—
—
F
—
—
—
—
—
—
—
A
0.096
2.372
0.077
484

Phage-388
—
—
L
—
—
—
—
P
—
—
—
—
—
A
0.070
2.367
0.069
485

Phage-389
—
—
L
—
—
F
—
L
—
—
—
—
—
L
0.107
2.367
0.081
486

Phage-390
L
—
Y
—
—
H
W
I
—
—
L
—
—
H
2.341
2.360
2.220
487

Phage-391
—
—
T
—
—
A
—
—
—
—
—
—
—
A
0.105
2.357
0.090
488

Phage-392
—
—
T
—
L
N
—
T
—
—
—
—
—
T
0.149
2.343
0.139
489

Phage-393
—
—
N
—
P
Y
—
T
—
—
—
—
—
A
0.072
2.341
0.069
490

Phage-394
—
—
T
—
—
T
—
N
—
—
—
—
—
T
0.142
2.334
0.128
491

Phage-395
V
—
L
A
Y
R
S
R
Q
A
D
L
—
R
0.082
2.309
0.079
492

Phage-396
—
—
N
—
L
Y
—
T
—
—
—
—
—
A
0.079
2.253
0.072
493

Phage-397
—
—
I
—
—
N
—
N
—
—
—
—
—
A
0.071
2.246
0.069
494

Phage-398
—
—
A
—
—
D
—
T
—
—
—
—
—
T
0.072
2.238
0.069
495

Phage-399
—
—
A
—
P
F
—
T
—
—
—
—
—
Y
0.104
2.230
0.085
496

Phage-400
—
—
A
—
—
N
—
T
—
—
—
—
—
I
0.090
2.208
0.085
497

Phage-401
—
—
T
—
Q
T
—
I
—
—
—
—
—
A
0.078
2.192
0.074
498

Phage-402
—
—
L
—
—
S
—
N
—
—
—
—
—
L
0.074
2.185
0.072
499

Phage-403
—
—
L
—
—
N
—
S
—
—
—
—
—
T
0.145
2.170
0.129
500

Phage-404
—
—
L
—
—
F
—
D
—
—
—
—
—
—
0.115
2.159
0.103
501

Phage-405
—
—
T
—
L
T
—
N
—
—
—
—
—
T
0.076
2.128
0.070
502

Phage-406
—
—
A
—
P
H
—
T
—
—
—
—
—
L
0.073
2.112
0.066
503

Phage-407
—
—
N
—
Q
Y
—
T
—
—
—
—
—
A
0.116
2.094
0.116
504

Phage-408
—
—
A
—
—
F
—
S
—
—
—
—
—
A
0.083
2.093
0.076
505

Phage-409
—
—
A
—
—
F
—
A
—
—
—
—
—
A
0.073
2.082
0.073
506

Phage-410
—
—
S
—
Q
Y
—
I
—
—
—
—
—
A
0.104
2.053
0.084
507

Phage-411
—
—
L
—
—
Y
—
S
—
—
—
—
—
A
0.071
1.973
0.070
508

Phage-412
—
—
L
—
Q
Y
—
T
—
—
—
—
—
I
0.092
1.967
0.090
509

Phage-413
—
—
V
—
—
Y
—
—
—
—
—
—
—
A
0.086
1.927
0.077
510

Phage-414
—
—
T
—
—
—
—
I
—
—
—
—
—
A
0.152
1.856
0.120
511

Phage-415
—
—
V
—
—
N
—
T
—
—
—
—
—
T
0.071
1.812
0.068
512

Phage-416
—
—
V
—
L
T
—
N
—
—
—
—
—
T
0.084
1.715
0.072
513

Phage-417
—
—
V
—
L
T
—
N
—
—
—
—
—
L
0.070
1.621
0.068
514

Phage-418
V
—
L
—
V
R
S
R
Q
A
D
L
—
R
0.130
1.558
0.126
515

Phage-419
—
—
H
—
—
H
—
T
—
—
—
—
—
T
0.095
1.527
0.088
516

Phage-420
—
—
L
—
P
Y
—
—
—
—
—
—
—
—
0.086
1.518
0.069
517

Phage-421
—
—
N
—
—
D
—
T
—
—
—
—
—
S
0.092
1.421
0.089
518

Phage-422
—
—
A
Y
Q
N
—
P
—
—
—
—
—
S
0.106
1.313
0.105
519

Phage-423
—
—
S
—
—
F
—
L
—
—
—
—
—
—
0.106
1.297
0.105
520

Phage-424
—
—
I
—
—
H
—
A
—
—
—
—
—
A
0.112
1.252
0.097
521

Phage-425
—
—
H
—
R
H
—
T
—
—
—
—
—
A
0.119
1.244
0.076
522

Phage-426
—
—
T
—
—
F
—
H
—
—
—
—
—
T
0.084
1.196
0.080
523

Phage-427
—
—
I
—
L
Y
—
H
—
—
—
—
—
—
0.097
1.055
0.092
524

Phage-428
—
—
L
—
—
T
—
T
—
—
—
—
—
S
0.097
0.952
0.089
525

Phage-429
—
—
I
A
—
N
—
P
—
—
—
—
—
A
0.076
0.927
0.071
526

Phage-430
—
—
L
—
—
N
—
T
—
—
—
—
—
T
0.111
0.918
0.094
527

Phage-431
—
—
A
—
Q
Y
—
N
—
—
—
—
—
A
0.103
0.879
0.083
528

Phage-432
—
—
L
—
L
S
—
P
—
—
—
—
—
T
0.082
0.848
0.078
529

Phage-433
—
—
A
—
—
F
—
L
—
—
—
—
—
T
0.154
0.759
0.123
530

Phage-434
—
—
L
—
—
N
—
L
—
—
—
—
—
—
0.088
0.746
0.085
531

Phage-435
—
—
S
—
—
N
—
N
—
—
—
—
—
L
0.080
0.712
0.075
532

Phage-436
—
—
A
—
N
H
—
D
—
—
—
—
—
L
0.079
0.686
0.075
533

Phage-437
—
—
N
—
P
Y
—
T
—
—
—
—
—
F
0.105
0.613
0.092
534

Phage-438
—
—
A
—
—
F
—
F
—
—
—
—
—
A
0.070
0.600
0.069
535

Phage-439
—
—
I
—
—
N
—
H
—
—
—
—
—
I
0.069
0.580
0.069
536

Phage-440
—
—
V
—
N
F
—
T
—
—
—
—
—
T
0.072
0.472
0.070
537

Phage-441
—
—
A
—
—
H
—
T
—
—
—
—
—
Y
0.093
0.426
0.076
538

Phage-442
T
—
T
—
—
F
—
T
—
—
—
—
—
—
0.065
0.422
0.065
539

Phage-443
—
—
L
—
—
T
—
T
—
—
—
—
—
Y
0.084
0.397
0.079
540

Phage-444
—
—
N
—
—
T
—
—
—
—
—
—
—
Y
0.078
0.254
0.077
541

Phage-445
—
—
L
—
Q
F
—
T
—
—
—
—
—
L
0.120
0.218
0.086
542

Phage-446
—
—
F
—
—
H
—
P
—
—
—
—
—
A
0.077
0.189
0.077
543

Phage-447
—
—
T
—
—
T
—
—
—
—
—
—
—
—
0.094
0.165
0.090
544

Phage-448
—
—
A
—
M
Y
—
D
—
—
—
—
—
H
0.078
0.137
0.074
545

Phage-449
—
—
V
—
P
F
—
T
—
—
—
—
—
I
0.102
0.135
0.093
546

Phage-450
—
—
N
—
L
Y
—
N
—
—
—
—
—
—
0.066
0.133
0.065
547

Phage-451
—
—
L
—
L
N
—
P
—
—
—
—
—
T
0.076
0.126
0.074
548

Phage-452
—
—
L
—
L
H
—
I
—
—
—
—
—
A
0.070
0.117
0.070
549

Phage-453
T
—
S
L
Q
H
Q
Y
—
Y
—
M
—
I
0.114
0.105
0.146
550

Phage-454
—
—
A
—
—
H
—
Y
—
—
—
—
—
T
0.081
0.087
0.069
551

Phage-455
—
—
V
—
—
N
—
I
—
—
—
—
—
L
0.076
0.080
0.075
552

Phage-456
M
—
H
P
Q
N
—
A
—
Y
—
F
—
D
0.091
0.075
0.107
553

Phage-457
—
—
N
—
—
A
—
—
—
—
—
—
—
I
0.073
0.074
0.067
554

Phage-458
—
—
N
—
—
T
—
S
—
—
—
—
—
A
0.072
0.072
0.069
555

Phage-459
L
—
H
P
Q
N
—
A
—
Y
—
M
—
D
0.076
0.072
0.088
556

Phage-460
G
—
F
—
—
P
Q
N
—
H
—
S
—
F
0.073
0.069
0.077
557

Phage-461
L
—
H
P
Q
F
—
F
—
Y
—
M
—
D
0.070
0.067
0.076
558

Phage-462
W
—
H
P
Q
H
—
A
—
Y
—
F
—
S
0.068
0.066
0.073
559

Phage-463
W
—
H
P
Q
F
—
Y
—
Y
—
M
—
D
0.071
0.065
0.078
560

Phage-464
F
—
H
P
Q
N
—
—
—
Y
—
—
—
A
0.067
0.064
0.073
561

Phage-465
—
—
V
—
—
T
—
T
—
—
—
—
—
—
0.082
1.564
0.082
562

Phage-466
—
—
N
—
F
T
—
T
—
—
—
—
—
A
0.074
2.351
0.074
563

Phage-467
L
—
L
A
L
R
—
R
Q
A
D
L
—
R
0.072
2.482
0.072
564

Phage-468
—
—
L
—
—
N
—
I
—
—
—
—
—
A
0.089
2.287
0.090
565

Phage-469
—
—
L
—
—
H
—
—
—
—
—
—
—
—
0.067
2.426
0.068
566

Phage-470
—
—
I
—
—
H
—
T
—
—
—
—
—
L
0.068
2.419
0.069
567

Phage-471
—
—
L
Y
—
D
—
P
—
—
—
—
—
S
0.131
2.417
0.132
568

Phage-472
—
—
L
—
N
Y
—
T
—
—
—
—
—
S
0.067
2.347
0.068
569

Phage-473
—
—
S
—
—
H
—
T
—
—
—
—
—
L
0.078
2.569
0.079
570

Phage-474
—
—
A
—
P
F
—
T
—
—
—
—
—
—
0.073
1.500
0.073
571

Phage-475
—
—
V
—
—
N
—
I
—
—
—
—
—
A
0.093
1.127
0.093
572

Phage-476
—
—
T
—
—
D
—
N
—
—
—
—
—
—
0.071
0.993
0.071
573

Phage-477
—
—
T
—
L
V
—
—
—
—
—
—
—
L
0.067
2.455
0.069
574

Phage-478
—
—
A
—
Q
F
—
L
—
—
—
—
—
T
0.065
2.367
0.067
575

Phage-479
—
—
S
—
—
Y
—
T
—
—
—
—
—
L
0.066
2.428
0.068
576

Phage-480
—
—
T
—
L
H
—
P
—
—
—
—
—
A
0.076
2.589
0.078
577

Phage-481
—
—
A
Y
—
G
—
N
—
—
—
—
—
L
0.071
2.556
0.073
578

Phage-482
—
—
I
—
—
N
—
H
—
—
—
—
—
S
0.078
2.542
0.080
579

Phage-483
—
—
L
Y
—
Y
—
P
—
—
—
—
—
S
0.072
0.531
0.073
580

Phage-484
—
—
S
—
—
Y
—
T
—
—
—
—
—
—
0.069
2.426
0.071
581

Phage-485
—
—
T
—
R
F
—
I
—
—
—
—
—
A
0.071
1.940
0.073
582

Phage-486
—
—
L
—
P
N
—
I
—
—
—
—
—
A
0.091
1.291
0.092
583

Phage-487
—
—
T
—
L
Y
—
T
—
—
—
—
—
I
0.071
2.445
0.073
584

Phage-488
—
—
S
—
—
Y
—
T
—
—
—
—
—
A
0.073
2.388
0.075
585

Phage-489
—
—
T
—
N
F
—
T
—
—
—
—
—
A
0.106
2.064
0.107
586

Phage-490
—
—
S
—
—
F
—
L
—
—
—
—
—
T
0.105
2.314
0.107
587

Phage-491
—
—
T
—
—
L
—
P
—
—
—
—
—
A
0.083
2.377
0.086
588

Phage-492
—
—
A
—
—
T
—
T
—
—
—
—
—
I
0.066
2.513
0.068
589

Phage-493
—
—
I
—
—
N
—
P
—
—
—
—
—
S
0.085
2.563
0.087
590

Phage-494
—
—
L
—
—
F
—
P
—
—
—
—
—
L
0.074
2.534
0.077
591

Phage-495
—
—
V
—
L
Y
—
T
—
—
—
—
—
—
0.072
2.466
0.075
592

Phage-496
—
—
L
—
—
F
—
A
—
—
—
—
—
I
0.110
2.253
0.113
593

Phage-497
—
—
T
—
L
Y
—
T
—
—
—
—
—
—
0.084
2.605
0.087
594

Phage-498
—
—
V
—
L
T
—
T
—
—
—
—
—
I
0.115
2.249
0.117
595

Phage-499
—
—
—
—
—
L
—
I
—
—
—
—
—
A
0.067
2.420
0.071
596

Phage-500
—
—
A
—
—
F
—
H
—
—
—
—
—
L
0.074
2.332
0.077
597

Phage-501
—
—
T
—
L
F
—
N
—
—
—
—
—
A
0.074
2.278
0.077
598

Phage-502
—
—
T
—
—
H
—
P
—
—
—
—
—
A
0.071
2.487
0.075
599

Phage-503
—
—
A
—
—
T
—
N
—
—
—
—
—
A
0.076
1.934
0.079
600

Phage-504
—
—
T
—
L
S
—
T
—
—
—
—
—
T
0.073
2.406
0.077
601

Phage-505
—
—
I
—
—
S
—
T
—
—
—
—
—
I
0.103
1.750
0.106
602

Phage-506
—
—
V
—
Q
Y
—
L
—
—
—
—
—
—
0.073
2.406
0.077
603

Phage-507
—
—
A
—
—
F
—
L
—
—
—
—
—
I
0.106
2.411
0.110
604

Phage-508
—
—
L
—
R
F
—
—
—
—
—
—
—
T
0.068
2.030
0.071
605

Phage-509
—
—
S
—
—
F
—
P
—
—
—
—
—
—
0.067
2.255
0.072
606

Phage-510
—
—
N
—
—
Y
—
T
—
—
—
—
—
I
0.081
2.466
0.085
607

Phage-511
—
—
P
—
—
F
—
T
—
—
—
—
—
I
0.068
2.413
0.072
608

Phage-512
—
—
L
—
—
T
—
I
—
—
—
—
—
A
0.069
2.453
0.074
609

Phage-513
—
—
A
—
—
T
—
L
—
—
—
—
—
T
0.070
2.442
0.075
610

Phage-514
—
—
I
—
P
H
—
L
—
—
—
—
—
S
0.068
0.963
0.070
611

Phage-515
—
—
T
—
—
Y
—
F
—
—
—
—
—
I
0.066
2.224
0.070
612

Phage-516
—
—
—
—
—
Y
—
N
—
—
—
—
—
L
0.072
2.240
0.076
613

Phage-517
—
—
S
—
—
F
—
T
—
—
—
—
—
A
0.094
2.477
0.099
614

Phage-518
—
—
N
—
L
S
E
T
—
—
—
—
—
—
0.082
2.453
0.088
615

Phage-519
—
—
L
—
—
D
—
—
—
—
—
—
—
T
0.073
2.229
0.078
616

Phage-520
—
—
T
—
—
Y
—
—
—
—
—
—
—
—
0.065
2.170
0.070
617

Phage-521
—
—
A
—
—
F
—
T
—
—
—
—
—
A
0.081
2.451
0.086
618

Phage-522
—
—
T
—
L
F
—
T
—
—
—
—
—
S
0.066
2.509
0.072
619

Phage-523
—
—
N
—
R
Y
—
T
—
—
—
—
—
A
0.063
1.794
0.067
620

Phage-524
—
—
T
—
—
A
—
I
—
—
—
—
—
T
0.070
1.982
0.075
621

Phage-525
—
—
L
—
R
F
—
T
—
—
—
—
—
I
0.072
2.517
0.079
622

Phage-526
—
—
L
G
P
T
—
N
—
—
—
—
—
A
0.071
2.571
0.077
623

Phage-527
—
—
T
—
Q
Y
—
—
—
—
—
—
—
A
0.071
2.441
0.077
624

Phage-528
—
—
L
—
Q
F
—
—
—
—
—
—
—
A
0.068
1.852
0.073
625

Phage-529
—
—
V
—
L
F
—
T
—
—
—
—
—
L
0.070
2.465
0.076
626

Phage-530
—
—
S
—
—
T
—
T
—
—
—
—
—
I
0.071
2.421
0.078
627

Phage-531
—
—
T
—
N
F
—
T
—
—
—
—
—
S
0.080
0.848
0.082
628

Phage-532
—
—
L
—
—
T
—
N
—
—
—
—
—
I
0.063
2.493
0.069
629

Phage-533
—
—
I
—
—
S
—
P
—
—
—
—
—
I
0.070
2.320
0.077
630

Phage-534
—
—
L
—
—
L
—
T
—
—
—
—
—
A
0.069
2.432
0.076
631

Phage-535
—
—
L
—
F
N
—
T
—
—
—
—
—
A
0.069
2.324
0.075
632

Phage-536
—
—
N
—
—
T
—
T
—
—
—
—
—
I
0.067
1.804
0.072
633

Phage-537
—
—
T
—
—
L
—
N
—
—
—
—
—
A
0.068
2.491
0.075
634

Phage-538
—
—
A
—
—
Y
—
N
—
—
—
—
—
A
0.066
2.253
0.073
635

Phage-539
—
—
T
—
L
F
—
T
—
—
—
—
—
—
0.064
2.207
0.070
636

Phage-540
—
—
T
—
—
F
—
—
—
—
—
—
—
S
0.067
2.536
0.075
637

Phage-541
—
—
L
—
L
V
—
T
—
—
—
—
—
I
0.068
2.505
0.076
638

Phage-542
—
—
L
—
L
F
—
P
—
—
—
—
—
A
0.073
2.596
0.081
639

Phage-543
—
—
I
—
—
S
—
T
—
—
—
—
—
A
0.071
1.410
0.075
640

Phage-544
—
—
I
—
—
F
—
T
—
—
—
—
—
S
0.068
1.927
0.075
641

Phage-545
—
—
N
—
L
Y
—
I
—
—
—
—
—
A
0.073
2.447
0.082
642

Phage-546
—
—
L
F
—
H
—
P
—
—
—
—
—
N
0.070
2.269
0.078
643

Phage-547
—
—
S
—
—
T
—
P
—
—
—
—
—
I
0.070
1.596
0.076
644

Phage-548
—
—
N
—
—
H
—
T
—
—
—
—
—
I
0.066
2.530
0.075
645

Phage-549
—
—
V
—
—
S
—
T
—
—
—
—
—
—
0.074
2.290
0.081
646

Phage-550
—
—
N
—
L
F
—
T
—
—
—
—
—
L
0.071
2.585
0.080
647

Phage-551
—
—
A
—
—
F
—
—
—
—
—
—
—
I
0.083
2.221
0.091
648

Phage-552
—
—
L
—
L
N
—
N
—
—
—
—
—
—
0.070
2.273
0.078
649

Phage-553
—
—
I
—
A
T
—
T
—
—
—
—
—
—
0.070
0.950
0.073
650

Phage-554
—
—
A
F
L
N
—
P
—
—
—
—
—
S
0.068
2.580
0.078
651

Phage-555
—
—
L
—
P
T
—
T
—
—
—
—
—
A
0.068
0.956
0.071
652

Phage-556
—
—
T
—
—
H
—
Y
—
—
—
—
—
L
0.070
2.403
0.079
653

Phage-557
—
—
A
—
—
Y
—
L
—
—
—
—
—
I
0.073
2.576
0.083
654

Phage-558
—
—
T
—
P
Y
—
T
—
—
—
—
—
A
0.068
2.333
0.077
655

Phage-559
—
—
L
—
—
V
—
T
—
—
—
—
—
S
0.073
2.465
0.082
656

Phage-560
—
—
V
—
—
Y
—
L
—
—
—
—
—
L
0.076
2.392
0.085
657

Phage-561
—
—
A
—
—
Y
—
P
—
—
—
—
—
S
0.066
2.137
0.074
658

Phage-562
—
—
S
—
Q
F
—
T
—
—
—
—
—
A
0.069
2.455
0.079
659

Phage-563
—
—
L
—
—
N
—
N
—
—
—
—
—
L
0.069
2.460
0.079
660

Phage-564
—
—
L
—
L
L
—
T
—
—
—
—
—
I
0.082
2.213
0.091
661

Phage-565
—
—
T
—
L
F
—
I
—
—
—
—
—
—
0.068
2.597
0.079
662

Phage-566
—
—
A
—
—
Y
—
S
—
—
—
—
—
T
0.085
1.380
0.091
663

Phage-567
—
—
V
—
L
L
—
T
—
—
—
—
—
I
0.069
2.333
0.079
664

Phage-568
—
—
T
—
L
F
—
D
—
—
—
—
—
A
0.069
2.414
0.080
665

Phage-569
—
—
A
—
—
F
—
T
—
—
—
—
—
L
0.071
2.498
0.082
666

Phage-570
—
—
L
—
L
F
—
T
—
—
—
—
—
T
0.065
2.565
0.076
667

Phage-571
—
—
L
—
Q
Y
—
I
—
—
—
—
—
L
0.118
2.285
0.128
668

Phage-572
—
—
L
—
—
F
—
—
—
—
—
—
—
L
0.071
2.496
0.082
669

Phage-573
—
—
L
—
—
F
—
D
—
—
—
—
—
S
0.119
2.388
0.130
670

Phage-574
—
—
A
—
L
T
—
T
—
—
—
—
—
T
0.069
2.582
0.081
671

Phage-575
—
—
L
—
F
N
—
P
—
—
—
—
—
A
0.071
2.478
0.083
672

Phage-576
—
—
T
—
Q
F
—
T
—
—
—
—
—
A
0.065
2.577
0.078
673

Phage-577
—
—
L
—
—
Y
—
Y
—
—
—
—
—
S
0.089
1.403
0.096
674

Phage-578
—
—
P
F
L
N
—
P
—
—
—
—
—
A
0.071
2.465
0.083
675

Phage-579
—
—
N
—
—
F
—
—
—
—
—
—
—
—
0.081
2.294
0.092
676

Phage-580
—
—
N
—
—
F
—
N
—
—
—
—
—
L
0.073
2.412
0.085
677

Phage-581
—
—
N
—
—
F
—
H
—
—
—
—
—
I
0.072
2.289
0.084
678

Phage-582
—
—
L
—
Q
N
—
P
—
—
—
—
—
A
0.074
2.498
0.087
679

Phage-583
—
—
A
—
—
T
—
T
—
—
—
—
—
S
0.075
1.392
0.082
680

Phage-584
—
—
S
—
Q
F
—
—
—
—
—
—
—
A
0.081
0.877
0.085
681

Phage-585
—
—
H
—
L
F
—
L
—
—
—
—
—
A
0.068
0.178
0.069
682

Phage-586
—
—
L
—
—
T
—
—
—
—
—
—
—
A
0.066
2.605
0.080
683

Phage-587
—
—
L
—
L
H
—
P
—
—
—
—
—
—
0.070
1.851
0.080
684

Phage-588
—
—
I
—
L
D
—
T
—
—
—
—
—
L
0.072
1.357
0.079
685

Phage-589
—
—
V
—
—
L
—
T
—
—
—
—
—
A
0.074
2.463
0.088
686

Phage-590
—
—
N
—
L
F
—
T
—
—
—
—
—
A
0.073
2.367
0.086
687

Phage-591
—
—
N
—
L
T
—
T
—
—
—
—
—
S
0.066
2.419
0.079
688

Phage-592
—
—
L
—
—
L
—
P
—
—
—
—
—
A
0.129
2.446
0.143
689

Phage-593
—
—
A
—
—
H
—
S
—
—
—
—
—
—
0.071
2.599
0.086
690

Phage-594
—
—
T
—
—
F
—
H
—
—
—
—
—
L
0.073
2.488
0.087
691

Phage-595
—
—
L
—
L
H
—
T
—
—
—
—
—
A
0.068
2.596
0.083
692

Phage-596
—
—
L
—
—
N
—
H
—
—
—
—
—
T
0.069
2.505
0.084
693

Phage-597
—
—
S
—
—
F
—
A
—
—
—
—
—
Y
0.075
2.447
0.089
694

Phage-598
—
—
T
—
—
T
—
P
—
—
—
—
—
A
0.071
2.576
0.087
695

Phage-599
—
—
V
—
—
Y
—
T
—
—
—
—
—
I
0.072
2.064
0.084
696

Phage-600
—
—
L
—
L
V
—
N
—
—
—
—
—
A
0.072
1.402
0.080
697

Phage-601
—
—
A
—
P
F
—
L
—
—
—
—
—
S
0.080
1.785
0.092
698

Phage-602
—
—
I
—
—
F
—
T
—
—
—
—
—
N
0.071
1.154
0.078
699

Phage-603
—
—
L
—
F
T
—
P
—
—
—
—
—
A
0.075
2.448
0.091
700

Phage-604
—
—
N
—
Q
Y
—
T
—
—
—
—
—
L
0.068
2.259
0.083
701

Phage-605
—
—
I
—
—
Y
—
L
—
—
—
—
—
L
0.068
2.443
0.085
702

Phage-606
—
—
T
—
—
H
—
H
—
—
—
—
—
L
0.067
2.506
0.085
703

Phage-607
—
—
A
—
—
Y
—
L
—
—
—
—
—
S
0.067
2.453
0.085
704

Phage-608
—
—
T
—
—
H
—
D
—
—
—
—
—
A
0.072
1.237
0.080
705

Phage-609
—
—
A
—
R
Y
—
I
—
—
—
—
—
—
0.072
1.337
0.081
706

Phage-610
—
—
N
—
—
F
—
L
—
—
—
—
—
A
0.075
2.358
0.093
707

Phage-611
—
—
A
—
—
H
—
L
—
—
—
—
—
—
0.069
1.799
0.083
708

Phage-612
—
—
V
—
F
Y
—
T
—
—
—
—
—
A
0.068
2.489
0.088
709

Phage-613
—
—
T
—
—
N
—
T
—
—
—
—
—
—
0.070
2.289
0.088
710

Phage-614
—
—
I
—
Q
Y
—
T
—
—
—
—
—
I
0.094
2.107
0.11:
711

Phage-615
—
—
L
—
—
H
—
P
—
—
—
—
—
H
0.089
2.055
0.105
712

Phage-616
—
—
T
—
—
A
—
T
—
—
—
—
—
A
0.070
1.392
0.080
713

Phage-617
—
—
L
—
—
D
—
—
—
—
—
—
—
—
0.076
2.166
0.094
714

Phage-618
—
—
L
—
P
H
—
I
—
—
—
—
—
A
0.077
2.354
0.096
715

Phage-619
—
—
T
—
—
Y
—
T
—
—
—
—
—
I
0.067
2.586
0.089
716

Phage-620
—
—
T
—
—
N
—
T
—
—
—
—
—
T
0.074
0.454
0.078
717

Phage-621
—
—
L
—
P
Y
—
N
—
—
—
—
—
A
0.070
1.177
0.080
718

Phage-622
—
—
I
—
L
S
—
P
—
—
—
—
—
—
0.072
2.281
0.092
719

Phage-623
—
—
L
—
—
Y
—
I
—
—
—
—
—
I
0.079
2.302
0.099
720

Phage-624
—
—
V
—
—
Y
—
H
—
—
—
—
—
L
0.093
2.448
0.115
721

Phage-625
—
—
T
—
—
H
—
N
—
—
—
—
—
A
0.077
2.600
0.101
722

Phage-626
—
—
T
—
—
Y
—
N
—
—
—
—
—
T
0.070
2.248
0.091
723

Phage-627
—
—
I
—
P
F
—
T
—
—
—
—
—
Y
0.074
2.357
0.097
724

Phage-628
—
—
T
—
P
F
—
—
—
—
—
—
—
L
0.119
2.272
0.141
725

Phage-629
—
—
L
—
—
T
—
L
—
—
—
—
—
I
0.077
2.071
0.097
726

Phage-630
—
—
V
—
Q
H
—
T
—
—
—
—
—
A
0.070
0.213
0.072
727

Phage-631
—
—
A
—
—
N
—
T
—
—
—
—
—
A
0.074
2.132
0.096
728

Phage-632
—
—
V
—
—
F
—
H
—
—
—
—
—
—
0.083
0.839
0.091
729

Phage-633
—
—
L
—
N
F
—
P
—
—
—
—
—
I
0.078
1.955
0.099
730

Phage-634
L
—
L
A
S
R
Y
R
Q
A
D
L
—
R
0.071
0.641
0.078
731

Phage-635
—
—
H
—
P
F
—
D
—
—
—
—
—
A
0.066
0.345
0.070
732

Phage-636
—
—
I
—
—
N
—
N
—
—
—
—
—
T
0.068
1.430
0.084
733

Phage-637
—
—
V
—
—
Y
—
T
—
—
—
—
—
L
0.072
0.496
0.077
734

Phage-638
—
—
L
—
L
D
—
P
—
—
—
—
—
—
0.077
1.281
0.092
735

Phage-639
—
—
H
—
—
F
—
S
—
—
—
—
—
S
0.070
1.420
0.087
736

Phage-640
—
—
L
—
Q
T
—
—
—
—
—
—
—
A
0.074
1.644
0.094
737

Phage-641
—
—
T
—
P
Y
—
P
—
—
—
—
—
A
0.113
1.701
0.133
738

Phage-642
—
—
H
—
—
H
—
N
—
—
—
—
—
A
0.067
1.062
0.080
739

Phage-643
—
—
V
—
—
F
—
N
—
—
—
—
—
A
0.065
1.098
0.080
740

Phage-644
—
—
V
—
—
H
—
T
—
—
—
—
—
L
0.083
0.335
0.087
741

Phage-645
—
—
S
—
—
F
—
—
—
—
—
—
—
L
0.067
0.696
0.076
742

Phage-646
—
—
I
—
—
F
—
T
—
—
—
—
—
T
0.070
0.882
0.082
743

Phage-647
—
—
I
—
—
D
—
P
—
—
—
—
—
T
0.090
0.642
0.100
744

Phage-648
—
—
L
—
—
D
—
—
—
—
—
—
—
—
0.081
0.847
0.096
745

Phage-649
—
—
L
—
—
N
—
L
—
—
—
—
—
A
0.069
0.713
0.082
746

Phage-650
—
—
T
—
Q
T
—
T
—
—
—
—
—
I
0.064
1.454
0.108
747

Phage-651
—
—
N
—
—
F
—
L
—
—
—
—
—
I
0.111
0.134
0.112
748

Phage-652
—
—
A
F
L
N
—
P
—
—
—
—
—
A
0.067
0.469
0.081
749

Phage-653
—
—
H
—
—
S
—
T
—
—
—
—
—
—
0.084
0.153
0.086
750

Phage-654
—
—
L
—
Q
Y
—
I
—
—
—
—
—
I
0.082
0.411
0.096
751

Phage-655
—
—
T
—
Q
Y
—
—
—
—
—
—
—
T
0.075
0.309
0.085
752

Phage-656
—
—
L
—
—
N
—
T
—
—
—
—
—
T
0.079
0.299
0.091
753

Phage-657
—
—
T
—
—
S
—
T
—
—
—
—
—
T
0.069
0.287
0.084
754

Phage-658
—
—
L
—
—
F
—
N
—
—
—
—
—
L
0.076
0.167
0.087
755

Phage-659
—
—
T
G
P
S
E
N
—
—
—
—
—
A
0.076
0.206
0.092
756

Phage-660
—
—
—
—
Q
Y
—
T
—
—
—
—
—
A
0.065
0.113
0.071
757

Phage-661
F
—
H
P
Q
F
—
H
—
Y
—
N
—
Y
0.107
0.081
0.103
758

Phage-662
—
—
T
—
L
Y
—
S
—
—
—
—
—
—
0.069
0.126
0.083
759

Phage-663
—
—
T
—
—
L
—
T
—
—
—
—
—
T
0.089
0.110
0.094
760

Phage-664
—
—
H
P
Q
F
H
A
—
Y
—
—
—
D
0.111
0.079
0.103
761

Phage-665
—
—
T
—
L
Y
—
T
—
—
—
—
—
H
0.066
0.088
0.074
762

Phage-666
L
—
H
P
Q
N
—
T
—
Y
—
—
—
D
0.118
0.097
0.110
763

Phage-667
—
—
L
—
R
F
—
H
—
—
—
—
—
T
0.094
0.073
0.085
764

Phage-668
—
—
T
—
M
V
—
S
—
—
—
—
—
T
0.071
0.106
0.089
765

Phage-669
F
—
H
P
Q
A
—
I
—
Y
—
L
—
N
0.098
0.077
0.086
766

Phage-670
—
—
H
P
Q
F
H
P
—
Y
—
L
—
D
0.097
0.079
0.086
767

Phage-671
—
—
L
F
—
N
—
I
—
—
—
—
—
A
0.114
0.150
0.144
768

Phage-672
S
—
H
P
Q
A
—
I
—
Y
—
Y
—
D
0.083
0.070
0.071
769

Phage-673
—
—
A
—
—
V
—
L
—
—
—
—
—
T
0.123
0.110
0.111
770

Phage-674
—
—
V
—
—
N
—
T
—
—
—
—
—
S
0.130
0.116
0.114
771

Phage-675
—
—
A
—
—
L
—
P
—
—
—
—
—
L
0.122
0.113
0.086
772

Phage-676
—
—
I
—
L
T
—
T
—
—
—
—
—
I
0.070
0.091
0.092
773

Phage-677
L
—
H
P
Q
A
—
I
—
F
—
F
—
N
0.069
0.073
0.075
774

Phage-678
F
—
H
P
Q
N
—
S
—
Y
—
—
—
D
0.074
0.072
0.068
775

Phage-679
L
—
H
P
Q
S
—
I
—
Y
—
F
—
D
0.066
0.071
0.072
776

Of the 679 clonal phage, 20 were selected for peptide synthesis and evaluated for binding and inhibition of EGFR Fab. The sequences of the peptides that were selected for synthesis are shown in Table 23. Binding curves for binding of the peptides to EGFR Fab as measured by ELISA are shown in FIGS. 21-22, and EC₅₀s relative to peptide-8 and EGFR-Bio are provided in Tables 24-25. FIGS. 23-24 show dose-dependent inhibition of EGFR Fab binding to the EGFR antigen by the peptides as measured by ELISA. IC₅₀data for binding inhibition by the peptides relative to peptide-8 is provided in Tables 26-27. The binding and inhibition data demonstrate that the phage library identified more potent peptide masks such as peptide-45 and peptide-46.

TABLE 23

Peptide sequences selected for synthesis (EGFR

Fab Peptide-8 Optimization)

Amino Acid Sequence

Description
(N to C)
SEQ ID NO:

Peptide-29
PCLSPFDTAKPICA
99

Peptide-30
PCTSHFDTAKPICT
100

Peptide-31
PCTSHFDTAKPICI
101

Peptide-32
PCLSHFDTAKPICL
102

Peptide-33
PCLSHTDTAKPICA
103

Peptide-34
PCLSHHDTAKPICA
104

Peptide-35
PCLSHFDTAKPICA
105

Peptide-36
PCLSHFDTAKPICV
106

Peptide-37
PCLSHNDPAKPICS
107

Peptide-38
PCTSQHDTAKPICI
108

Peptide-39
PCTSHFDTAKPICA
109

Peptide-40
PCLSHTDTAKPICV
110

Peptide-41
PCLSHNDPAKPICA
111

Peptide-42
PCLSHYDTAKPICA
112

Peptide-43
PCNSHYDLAKPICT
113

Peptide-44
PCNSHFDIAKPICL
114

Peptide-45
PCAYHNDPAKPICS
115

Peptide-46
PCLFHFDPAKPICS
116

Peptide-47
PCTSHFDPAKPICA
117

Peptide-48
PCTSHNDNAKPICA
118

TABLE 24

EC₅₀s for peptide binding to αEGFR Fab

Peptide
EC₅₀(nM)

EGFR-Bio (3 nM)
0.17

Peptide-8
699.6

Peptide-29
150.6

Peptide-30
194.0

Peptide-31
171.8

Peptide-32
66.55

Peptide-33
340.2

Peptide-34
377.5

Peptide-35
149.2

Peptide-36
154.2

Peptide-37
358.0

Peptide-38
1294

TABLE 25

EC₅₀s for peptide binding to αEGFR Fab

Peptide
EC₅₀(nM)

EGFR-Bio (3 nM)
0.17

Peptide-8
340.3

Peptide-39
206.2

Peptide-40
413.7

Peptide-41
292.2

Peptide-42
63.58

Peptide-43
186.4

Peptide-44
340.9

Peptide-45
10.3

Peptide-46
4.9

Peptide-47
379.9

Peptide-48
536

TABLE 26

IC₅₀s for peptide inhibition of

αEGFR Fab binding to EGFR antigen

Peptide
IC₅₀(μM)

Peptide-8
7.76

Peptide-29
2.08

Peptide-30
4.55

Peptide-31
3.72

Peptide-32
2.08

Peptide-33
2.30

Peptide-34
4.91

Peptide-35
2.22

Peptide-36
1.97

Peptide-37
4.16

Peptide-38
29.9

TABLE 27

IC₅₀s for peptide inhibition of

αEGFR Fab binding to EGFR antigen

Peptide
IC₅₀(μM)

Peptide-8
6.29

Peptide-39
2.79

Peptide-40
2.66

Peptide-41
4.25

Peptide-42
2.22

Peptide-43
4.23

Peptide-44
6.57

Peptide-45
0.543

Peptide-46
0.300

Peptide-47
6.25

Peptide-48
8.03

Example 13: Anti-Tumor Efficacy in a Mouse Model of Colorectal Cancer

Female mice were subcutaneously implanted with 5 million HCT116 colorectal tumor cells in 50% matrigel. 20 million human PBMCs were engrafted via the tail vein the same day as HCT116 inoculation. When tumors reached 250 mm³, mice were randomized into groups and compounds were dosed intravenously every day for 10 days. Tumor volume was measured every two to three days and plotted overtime. The tumor volume growth kinetics indicate anti-tumor activity of masked EGFR targeted bispecific compounds. The anti-tumor activity observed was protease dependent in that the compound lacking the protease substrate within the cleavable linker was equivalent to vehicle controls. FIGS. 25-28 show the in vivo tumor growth inhibition data with different dosing amounts of PC1 (FIG. 25), PC5 (FIG. 26), PC7 (FIG. 27), and PC6 (FIG. 28).

Example 14: Binding of PC1 and PC4 to Human and Cynomolgus CD3R, EGFR, and Albumin

Polypeptide complexes PC1 and PC4 were evaluated for binding to human and cynomolgus monkey CD3ε, EGFR, and albumin by ELISA. Briefly, biotinylated antigen was captured on neutravidin coated plates. Polypeptide complex molecules were treated with active protease where indicated. Polypeptide complex molecules diluted in buffer were then added to the antigen coated plates. Bound polypeptide complex was detected using a standard horse radish peroxidase conjugate secondary antibody. Binding curves were fit using Graphpad Prism software. Binding curves for PC1 and PC4 binding to human and cynomolgus monkey CD3ε are shown in FIGS. 29 and 30, respectively. Binding curves for PC1 and PC4 binding to human and cynomolgus monkey EGFR are shown in FIGS. 31 and 32, respectively. As can be seen from the curves, target engagement is cleavage dependent as masking reduces CD3ε and EGFR binding by more than 1000-fold and more than 300-fold, respectively. Treatment of PC4 with protease enzyme enables potent antigen binding comparable to non-masked PC1. Binding curves for PC4 binding to human and cynomolgus monkey albumin are shown in FIGS. 33 and 34, respectively. As can be seen, PC4 exhibits potent binding to both human and monkey albumin.

Example 15: Polypeptide Complex Mediated Tumor Cytotoxicity and T Cell Activation (A549 Cells)

Polypeptide complexes were evaluated in a functional in vitro tumor cell killing assay using the EGFR positive tumor cell line A549. Tumor cell killing was measured using an xCelligence real time cell analyzer from Agilent that relies on sensor impedance measurements (cell index) that increased as tumor cells adhere, spread, and expand on the surface of the sensor. Likewise, as the tumor cells were killed the impedance decreased. Tumor cells were added per well and allowed to adhere overnight on a 96 well E-Plate. The following day polypeptide complexes titrated in human serum supplemented medium along with human PBMCs were added to the wells. Cell index measurements were taken every 10 minutes for an additional 72 hours. The cell index times number of hours (tumor cell growth kinetics) was then plotted versus logarithmic concentration of polypeptide complex where the concentration required to reduce the tumor growth 50% (IC₅₀) was calculated using Graphpad Prism software. FIG. 35 shows a graph of PC1 and PC4 mediated A549 tumor cell killing in the presence of PBMCs. PC4 was treated with protease where indicated. Protease treatment increased the activity of PC4 by about 43000-fold relative to untreated PC4. The activity level of protease treated PC4 was comparable to that of non-masked PC1 (see FIG. 35).

Example 16: Evaluation of Polypeptide Complex in an In Vitro Cytokine Induction Assay

Polypeptide complexes PC1 and PC4 were evaluated in a functional in vitro cytokine induction assay using the EGFR positive tumor cell lines HCT116 (colorectal cancer (CRC) cells—KRAS and PIK3CA mutant) and A549 (non-small cell lung cancer (NSCLC) cells—KRAS mutant). T cell cytokine induction was measured using an Meso Scale Discovery (MSD) electrochemiluminescence instrument. Tumor cells were added to wells and allowed to adhere overnight on a 96 well. The following day polypeptide complexes titrated in human serum supplemented medium along with human PBMCs. Supernatants were harvested 72 hours later and measured for cytokine levels (interferon gamma (IFNγ) and tumor necrosis factor (TNF)). Cytokine levels were then plotted versus logarithmic concentration of polypeptide complex where the concentration required to induce 50% (EC₅₀) of the maximum was calculated using Graphpad Prism software. FIGS. 36-39 show cytokine levels with increasing concentrations of PC1 and PC4 in HCT116 cells (FIGS. 36-37) and in A549 cells (FIGS. 38-39). PC4 was treated with protease where indicated. Masking of PC4 EGFR and CD3 binding domains reduces the capacity to induce cytokine release. Additionally, the functional activity against KRAS mutant anti-EGFR resistant CRC and NSCLC cell lines in T cell co-culture assays was found to be cleavage dependent. Protease-treated PC4 exhibits similar potency relative to non-masked PC1.

Example 17: Activity in HCT116 Mouse Tumor Model

Polypeptide complexes were evaluated in a mouse tumor model study. Female mice were subcutaneously implanted with 5 million HCT116 colorectal tumor cells in 50% matrigel. 20 million human PBMCs were engrafted via the tail vein the same day as HCT116 inoculation. When tumors reached 250 mm³, mice were randomized into groups and compounds were dosed intravenously every day for 10 days (see FIG. 40). Tumor volume was measured every two to three days and plotted overtime. The tumor volume growth kinetics indicate that anti-tumor activity of masked EGFR targeted bispecific compounds is similar to that of the non-masked compound. The anti-tumor activity observed was protease dependent in that the compound lacking the protease substrate within the cleavable linker (non-cleavable) was equivalent to vehicle controls. Survival plots were generated based on number of mice alive in the study versus time. FIG. 41 shows a plot of mean tumor volume versus days of treatment for PC1 (0.5 mg/kg), PC7 (0.5 mg/kg), non-cleavable PC6 (0.5 mg/kg), and vehicle treated mice. FIG. 42 shows a plot of tumor volume versus days of treatment for vehicle and PC5 treated mice. Percent survival of mice versus days of treatment with PC1 (0.5 mg/kg), PC5 (0.5 mg/kg), non-cleavable PC6 (0.5 mg/kg), and vehicle is shown in FIG. 43. Cleavable PC7 and PC1 at equivalent dose levels were found to induce complete tumor eradication in human PBMC engrafted mice implanted with anti-EGFR resistant KRAS- and PIK3CA-mutant HCT116 human tumor cells (see FIG. 41).

Example 18: Cleavage-Dependent Activity in Human Colorectal Cancer (CRC) Organoids

Fresh tumor tissue was procured from colorectal cancer patients, separated into small three-dimensional tumoroids without disruption of the underlying immune compartment or tumor microenvironment, and distributed to 96 well plates. Fresh tumoroids were treated with polypeptide complexes and maintained in culture for 24 to 72 hours (see FIG. 44). At the end of the study, tumoroids were digested and stained using fluorescently labeled antibodies that bind cell markers CD25, CD69, 4-1BB, and Granzyme-B (GzB). Stained cells were run via flow cytometry and fluorescent signals were gated on live cells. Percent of positively stained cells for a given marker were plotted for each polypeptide treatment condition. Referring to FIG. 45, upregulation of CD25, CD69, 4-1BB, and Granzyme was protease cleavage dependent where the polypeptide complex PC6 lacking cleavable substrate (non-cleavable) was inactive and equivalent to vehicle control. The cleavable and non-masked polypeptide complexes were active and able to induce activation markers within the tumor resident immune compartment in these primary human colorectal cancer tumoroids (see FIG. 45). Furthermore, concentration-dependent activity of PC5 was observed (see FIG. 45).

Example 19: Non-Human Primate (NHP) Toxicity Studies

Pharmacokinetics and exploratory safety of polypeptide molecules PC1 and PC4 were evaluated in cynomolgus monkeys. Briefly, cynomolgus monkeys of approximately 3 kg bodyweight were administered polypeptides as an IV bolus once weekly for three weeks or via continuous IV infusion. Animals were observed daily for signs of adverse events. PC1 was dosed at 5 μg/kg/day, 15 μg/kg/day, and 30 μg/kg/day via continuous IV (cIV) infusion. Dosing amounts of 0.1 mg/kg/dose, 0.3 mg/kg/dose, and 0.6 mg/kg/dose was used for PC4 via IV bolus injections. After dosing, blood was collected in K2 EDTA tubes at specific timepoints and processed to plasma. Plasma was stored frozen until analysis. Concentration of polypeptide molecules in plasma was measured via standard ELISA techniques relative to a reference standard diluted in control cynomolgus monkey plasma. Maximum plasma concentrations achieved in animals for masked polypeptide complex PC4 were compared to those achieved using the non-masked polypeptide complex PC1. Comparison of the maximum plasma concentration achieved with the masked polypeptide complex compared to the maximum tolerated plasma concentration for the non-masked polypeptide complex revealed a >3,000× multiple (see FIGS. 46-47). The masked polypeptide complex had no adverse events in the animals at the highest dose tested of 0.6 mg/kg/dose (see FIG. 46). The maximum tolerated dose (MTD) of the non-masked polypeptide complex PC1 was 15 μg/kg/day by continuous IV infusion. Animals dosed with 30 μg/kg/day continuous IV infusion of the non-masked polypeptide complex was not tolerated due to severe adverse events (see FIG. 46). The PC4 toxicity studies support its enhances pharmacokinetics, safety, and design. Notably, PC4 administered once weekly for three weeks was well tolerated without clinical observations or notable changes in clinical chemistry, hematology, or pathology. In contrast, PC1 dosed by continuous IV infusion at 15 μg/kg/day had increased body temperature and increased liver enzymes. Treatment with PC1 at 30 μg/kg/day by continuous IV infusion had to be terminated 24 hrs after dosing due to severe adverse events, including fever, skin rash, GI toxicities, and lack of responsiveness in the animals (see FIG. 46). Clinical observations, body temperature, and clinical pathology measurements support the large safety multiple (>3,000×) for PC4 relative to non-masked PC1. PC4 was found to reduce cytokine release and healthy tissue toxicities (GI, Liver, skin) at high exposures in cynomolgus monkeys despite broad tissue expression of EGFR. The NOAEL (no observed adverse effect level) for PC4 is ≥0.6 mg/kg/dose weekly IV bolus for three weeks (see FIG. 46).

Example 20: EGFR Polypeptide Complex Binding of PC10 and PC11

The EGFR-CD3 polypeptide complexes were evaluated for EGFR and CD3ε binding.

FIG. 48 shows PC10 and PC11 binding to EGFR measured by ELISA.

FIG. 49 shows PC10 and PC11 binding to CD3 measured by ELISA.

Example 21: PC10 and PC11 Mediated Tumor Cytotoxicity and T Cell Activation

Polypeptide complexes PC10 and PC11 were evaluated in a functional in vitro tumor cell killing assay using the EGFR positive tumor cell line HCT116. Tumor cell killing was measured using an xCelligence real time cell analyzer from Agilent that relies on sensor impedance measurements (cell index) that increased as tumor cells adhere, spread, and expand on the surface of the sensor. Likewise, as the tumor cells were killed the impedance decreased. 10,000 tumor cells were added per well and allowed to adhere overnight on a 96 well E-Plate. The following day polypeptide complexes titrated in human serum supplemented medium along with 30,000 CD8+ T cells were added to the wells. Cell index measurements were taken every 10 minutes for an additional 72 hours. The cell index times number of hours (tumor cell growth kinetics) was then plotted versus concentration of polypeptide complex where the concentration required to reduce the tumor growth 50% (IC₅₀) was calculated using Graphpad Prism software.

FIG. 50 shows a graph of PC10 and PC11 mediated HCT116 tumor cell killing in the presence of CD8+ T cells.

Example 22: Optimized Phase Library Construction—CD3 scFv Peptides

Sequence activity relationships (SAR) were established for Peptide-A and Peptide-B by mutating each individual residue within the peptide to alanine and measuring binding and inhibition against SP34.185 scFv. Peptide residues whose alanine mutations significantly weakened binding and inhibition can be considered critical residues where mutations were not tolerated. Peptide residues whose alanine mutations performed similarly to the non-mutated sequence can be considered non-critical sites where mutations were indeed tolerated. Using the peptide SAR, DNA oligo libraries were constructed where codons encoding critical residues within each peptide sequence were minimally mutated and codons encoding non-critical residues were heavily mutated. The resulting oligos were cloned into bacteriophage vectors used to display the SAR guided peptides via fusion to the pIII filament of the bacteriophage. The relevant vectors were then used to produce the phage optimization libraries via amplification in bacteria using standard techniques in the field.

Peptides were evaluated for their ability to bind SP34.185 scFv by standard enzyme linked immunosorbent assays (ELISAs). Briefly, biotinylated peptides were captured on neutravidin coated plates, quenched with biocytin followed by a washing step. SP34.185 scFv was then titrated onto the peptide captured plates. Plates were then washed and bound SP34.185 scFv was detected using a secondary horse radish peroxidase antibody conjugate. After washing again, plates were developed using standard ELISA techniques and stopped using acid. The concentration of SP34.185 scFv required to achieve 50% maximal signal or EC₅₀was calculated using Graphpad prism. Data is shown in FIGS. 51A-51F and summarized in Tables 28A-28D. Peptide Sequences of CD3 Ala Scan Peptides for Peptide A and Peptide-B are shown in Table 30.

TABLE 28A

Summary of FIG. 51B

Pep-
Pep-
Pep-
Pep-
Pep-
Pep-
Pep-

ELISA
tide-A
tide-C
tide-D
tide-E
tide-F
tide-G
tide-H

EC50
1.013
0.9429
1.018
0.9738
1.27
47.5
346.2

nM

TABLE 28B

Summary of FIG. 51C

Pep-
Pep-
Pep-
Pep-
Pep-
Pep-
Pep-

ELISA
tide-A
tide-I
tide-J
tide-K
tide-L
tide-M
tide-N

EC₅₀
0.986
310.8
3.134
1,960
4.363
2.76
1.546

nM

TABLE 28C

Summary of FIG. 51E

Pep-
Pep-
Pep-
Pep-
Pep-
Pep-

ELISA
tide-O
tide-P
tide-Q
tide-R
tide-S
tide-T

EC₅₀
1.356
2.359
30.04
47.50
457.1
4.762

nM

TABLE 28D

Summary of FIG. 51F

Pep-
Pep-
Pep-
Pep-
Pep-
Pep-

ELISA
tide-U
tide-V
tide-W
tide-X
tide-Y
tide-Z

EC₅₀
39.90
2168
1.916
1.948
2.012
1.833

nM

Peptides were evaluated for their ability to inhibit SP34.185 scFv from binding CD3ε by standard enzyme linked immunosorbent assays (ELISAs). Briefly, a fixed concentration of SP34.185 scFv was incubated with varying concentrations of peptides in solution. SP34.185scFv and peptide solutions were incubated for 1 hr prior to addition to CD3 coated plates. Binding was allowed to proceed for 30 min prior to washing. After washing, bound SP34.185 scFv using a secondary horse radish peroxidase antibody conjugate. After washing again, plates were developed using standard ELISA techniques and stopped using acid. The concentration of peptide required to inhibit 50% of the SP34.185 scFv CD3 binding signal (IC₅₀) was calculated using Graphpad prism. Data is shown in FIGS. 52A-52F and summarized in Tables 29A-29D.

TABLE 29A

Summary of FIG. 52B

Pep-
Pep-
Pep-
Pep-
Pep-
Pep-
Pep-

ELISA
tide-A
tide-C
tide-D
tide-E
tide-F
tide-G
tide-H

IC₅₀
0.1926
0.1025
0.2318
0.1905
5.484
>100
>100

uM

TABLE 29B

Summary of FIG. 52C

Pep-
Pep-
Pep-
Pep-
Pep-
Pep-
Pep-

ELISA
tide-A
tide-I
tide-10
tide-K
tide-L
tide-M
tide-N

IC₅₀
0.1138
>100
63.18
>100
86.78
36.66
3.009

uM

TABLE 29C

Summary of FIG. 52E

Pep-
Pep-
Pep-
Pep-
Pep-
Pep-

ELISA
tide-O
tide-P
tide-Q
tide-R
tide-S
tide-T

IC₅₀
0.1473
3.333
>100
>100
>100
41.46

uM

TABLE 29D

Summary of FIG. 52F

Pep-
Pep-
Pep-
Pep-
Pep-
Pep-

ELISA
tide-U
tide-V
tide-W
tide-X
tide-Y
tide-Z

IC₅₀
>100
>100
1.912
0.6992
1.456
0.1180

uM

TABLE 30

CD3 Ala Scan Sequences-Peptide-A and Peptide-B

anti-CD3

SEQ

Peptide-ID
Panned target
Sequence
ID NO:

Peptide-A
SP34.185
GSQCLGPEWEVCPY
797

Peptide-C
SP34.185
ASQCLGPEWEVCPY
798

Peptide-D
SP34.185
GAQCLGPEWEVCPY
799

Peptide-E
SP34.185
GSACLGPEWEVCPY
800

Peptide-F
SP34.185
GSQCAGPEWEVCPY
801

Peptide-G
SP34.185
GSQCLAPEWEVCPY
802

Peptide-H
SP34.185
GSQCLGAEWEVCPY
803

Peptide-I
SP34.185
GSQCLGPAWEVCPY
804

Peptide-J
SP34.185
GSQCLGPEAEVCPY
805

Peptide-K
SP34.185
GSQCLGPEWAVCPY
806

Peptide-L
SP34.185
GSQCLGPEWEACPY
807

Peptide-M
SP34.185
GSQCLGPEWEVCAY
808

Peptide-N
SP34.185
GSQCLGPEWEVCPA
809

Peptide-A
SP34.185
GSQCLGPEWEVCPY
810

Peptide-B
SP34.185
VYCGPEFDESVGCM
811

Peptide-O
SP34.185
AYCGPEFDESVGCM
812

Peptide-P
SP34.185
VACGPEFDESVGCM
813

Peptide-Q
SP34.185
VYCAPEFDESVGCM
814

Peptide-R
SP34.185
VYCGAEFDESVGCM
815

Peptide-S
SP34.185
VYCGPAFDESVGCM
816

Peptide-T
SP34.185
VYCGPEADESVGCM
817

Peptide-U
SP34.185
VYCGPEFAESVGCM
818

Peptide-V
SP34.185
VYCGPEFDASVGCM
819

Peptide-W
SP34.185
VYCGPEFDEAVGCM
820

Peptide-X
SP34.185
VYCGPEFDESAGCM
821

Peptide-Y
SP34.185
VYCGPEFDESVACM
822

Peptide-Z
SP34.185
VYCGPEFDESVGCA
823

Example 23: Panning of the Optimized Phage Library Construction—CD3 scFv Peptides

Once the phage optimization libraries were completed, phage libraries were bio-panned using SP34.185 scFv loaded beads. Multiple rounds of panning were performed where bacteriophage was allowed to bind to SP34.185 scFv loaded beads, washed, eluted, and amplified. Additional selective pressure was included during each round of panning using a fixed concentration of CD3, Peptide-A, or Peptide-B. After panning, phage infected bacteria were plated out and colonies picked into 96 well blocks. Clonal phage was then amplified and separated from bacterial cells via centrifugation. Phage containing supernatants were tested in binding ELISAs against SP34.185 scFv coated plates in the presence or absence of saturating concentration of CD3. Phage able to bind SP34.185 scFv were selected for sequence analysis if the binding signal was reduced in the presence of CD3.

Example 24: Panning ELISAs—CD3 scFv Peptides

Clonal phages were harvested as crude supernatants and screened via standard enzyme linked immunosorbent assays (ELISAs). Briefly, biotinylated SP34.185 scFv was captured on neutravidin coated plates. Prior to the addition of clonal phage, wells were incubated with blocking buffer and CD3 or blocking buffer alone. Without washing or aspirating, clonal phage supernatants were then added to the wells and incubated for a short time. Wells were then washed followed by detection of bound phage using a horse radish peroxidase conjugated anti-M13 antibody. Clonal phage of interest was then sent for sequence analysis.

Phage panning results of CD3 scFv Peptide-B library sequences are shown in Table 31. The sequences of those peptides selected for synthesis are shown in Table 32, and further evaluated for binding to anti-CD3 scFv (FIGS. 53A-53B) and inhibition of anti-CD3 scFv binding to CD3 (FIGS. 54A-54B). The consensus sequence shown in FIG. 55 was calculated from all the sequences shown in Table 31 and was generated using WebLogo 3.7.4.

TABLE 31

Clonal Phage Peptide Sequences from the Peptide-B Optimization Library Panning (—) indicates

same amino acid as in CD3 scFv Peptide-B corresponding position (e.g. Phage-1 position).

Phage binding ELISA

SP34.185

SP34.185
scFv signal
SEQ

Phage
Amino acid position sequence
Backgroud
scFv
in presence
ID

ID
1
2
3
4
5
6
7
8
9
10
11
12
13
14
signal
signal
of CD3
NO:

Phage-1/
V
Y
C
G
P
E
F
D
E
S
V
G
C
M
0.06
2.79
0.09
25

Peptide B

Phage-2
D
D
—
W
—
D
W
E
F
D
F
A
—
A
0.08
2.75
0.09
824

Phage-3
Y
I
—
—
L
D
—
P
D
F
L
Y
—
D
0.08
2.88
0.10
825

Phage-4
F
D
—
W
—
D
W
E
—
Y
F
V
—
D
0.08
2.79
0.09
826

Phage-5
Y
I
—
W
—
D
W
E
—
Y
F
D
—
D
0.08
2.74
0.09
827

Phage-6
N
I
—
W
—
D
W
E
D
D
Y
F
—
F
0.09
2.54
0.09
828

Phage-7
N
F
—
W
—
D
W
E
Y
I
Y
P
—
I
0.07
2.77
0.09
829

Phage-8
—
D
—
W
—
D
W
E
—
D
F
L
—
I
0.08
2.54
0.08
830

Phage-9
H
A
—
W
—
D
W
E
—
Y
F
P
—
N
0.08
2.85
0.09
831

Phage-10
Y
D
—
—
—
D
V
—
—
—
Y
V
—
V
0.09
2.63
0.10
832

Phage-11
I
D
—
W
—
D
W
E
D
D
T
F
—
Y
0.09
2.73
0.08
833

Phage-12
Y
L
—
—
—
D
G
—
—
T
L
A
—
Y
0.08
2.66
0.15
834

Phage-13
—
D
—
—
—
D
G
—
—
—
I
L
—
Y
0.11
2.13
0.08
835

Phage-14
F
I
—
W
—
D
W
E
—
D
Y
F
—
A
0.07
2.44
0.09
843

Phage-15
G
D
—
W
—
D
W
E
W
D
F
Y
—
D
0.07
2.71
0.07
844

Phage-16
Y
L
—
W
—
D
W
E
Y
I
D
L
—
D
0.12
2.67
0.08
845

Phage-17
S
F
—
W
—
D
W
E
—
Y
F
D
—
D
0.10
2.60
0.07
846

Phage-18
D
D
—
W
—
D
W
E
—
Y
A
S
—
D
0.09
2.57
0.07
847

Phage-19
N
L
—
W
—
D
W
E
Y
P
F
F
—
D
0.09
2.52
0.09
848

Phage-20
F
D
—
W
—
D
W
E
—
—
F
V
—
D
0.08
2.34
0.09
849

Phage-21
D
I
—
—
—
D
G
—
—
T
I
I
—
D
0.13
2.30
0.10
850

Phage-22
D
D
—
W
—
D
W
E
Y
Y
A
V
—
D
0.09
2.28
0.09
851

Phage-23
Y
D
—
W
—
D
W
E
—
Y
S
N
—
D
0.10
2.17
0.08
852

Phage-24
I
N
—
W
—
D
W
E
D
Y
F
F
—
D
0.07
2.16
0.07
853

Phage-25
N
I
—
W
—
D
W
E
D
D
T
F
—
F
0.06
2.87
0.07
854

Phage-26
N
I
—
W
—
D
W
E
P
N
S
F
—
F
0.09
2.87
0.08
855

Phage-27
Y
D
—
—
—
—
M
—
—
—
I
D
—
F
0.09
2.39
0.08
856

Phage-28
D
F
—
W
—
D
W
E
F
P
F
I
—
H
0.11
2.73
0.12
857

Phage-29
D
F
—
—
—
—
M
—
—
—
I
T
—
I
0.07
2.36
0.08
858

Phage-30
Y
D
—
—
—
—
—
—
—
—
T
V
—
I
0.10
2.32
0.08
859

Phage-31
H
D
—
W
—
D
W
E
W
D
I
F
—
I
0.07
2.26
0.08
860

Phage-32
H
A
—
W
—
D
W
E
—
Y
N
P
—
N
0.11
2.71
0.11
861

Phage-33
D
V
—
W
—
D
W
E
W
D
F
F
—
N
0.08
2.65
0.08
862

Phage-34
N
—
—
W
—
D
W
E
Y
Y
I
P
—
N
0.10
2.57
0.08
863

Phage-35
I
I
—
W
—
D
W
E
F
I
D
Y
—
N
0.08
2.10
0.07
864

Phage-36
S
L
—
W
—
D
W
E
Y
D
I
A
—
P
0.07
2.53
0.08
865

Phage-37
D
L
—
—
—
—
L
—
—
—
I
F
—
P
0.08
2.49
0.09
866

Phage-38
T
N
—
W
—
D
W
E
W
V
L
P
—
P
0.14
2.47
0.10
867

Phage-39
I
E
—
W
—
D
W
E
P
N
Y
F
—
P
0.13
2.29
0.09
868

Phage-40
I
F
—
W
—
D
W
E
D
Y
—
D
—
P
0.07
2.28
0.07
869

Phage-41
I
D
—
W
—
D
W
E
Y
D
F
F
—
P
0.07
2.26
0.08
870

Phage-42
L
F
—
W
—
D
W
E
D
—
F
F
—
P
0.18
2.11
0.13
871

Phage-43
—
D
—
W
—
D
W
E
D
Y
A
D
—
T
0.11
2.20
0.10
872

Phage-44
—
I
—
W
—
D
W
E
Q
Y
F
P
—
V
0.11
2.34
0.09
873

Phage-45
I
E
—
W
—
D
W
E
P
I
Y
P
—
Y
0.09
2.85
0.09
874

Phage-46
I
T
—
W
—
D
W
E
V
Y
F
P
—
Y
0.07
2.55
0.08
875

Phage-47
I
D
—
W
—
D
W
E
Y
I
H
P
—
Y
0.06
2.51
0.09
876

Phage-48
I
D
—
W
—
D
W
E
Y
I
N
P
—
Y
0.12
2.50
0.12
877

Phage-49
A
D
—
W
—
D
W
E
—
A
F
P
—
Y
0.09
2.44
0.09
878

Phage-50
I
D
—
W
—
D
W
E
Y
I
Y
P
—
Y
0.09
2.31
0.07
879

Phage-51
N
I
—
W
—
D
W
E
D
D
N
F
—
F
0.09
2.08
0.09
880

Phage-52
Y
D
—
W
—
D
W
E
Y
V
D
A
—
Y
0.09
2.06
0.09
881

Phage-53
F
—
—
—
—
D
G
—
—
—
Y
V
—
D
0.09
2.03
0.11
882

Phage-54
D
I
—
W
—
D
W
E
Y
I
N
I
—
S
0.11
2.02
0.11
883

Phage-55
F
V
—
W
—
D
W
E
D
F
N
F
—
D
0.07
2.01
0.08
884

Phage-56
F
A
—
W
—
D
W
E
D
Y
—
A
—
D
0.07
2.01
0.09
885

Phage-57
D
N
—
W
—
D
W
E
Y
D
F
F
—
V
0.08
1.99
0.09
886

Phage-58
Y
D
—
W
—
D
W
E
—
Y
N
D
—
A
0.09
1.96
0.11
887

Phage-59
D
D
—
—
—
D
G
—
—
T
I
I
—
V
0.07
1.91
0.09
888

Phage-60
F
P
—
W
—
D
W
E
—
Y
A
I
—
D
0.10
1.89
0.10
889

Phage-61
P
D
—
—
—
D
G
—
—
—
L
F
—
T
0.12
1.86
0.07
890

Phage-62
D
N
—
W
—
D
W
E
Y
D
Y
F
—
V
0.07
1.83
0.07
891

Phage-63
I
F
—
W
—
D
W
E
—
F
Y
D
—
Y
0.12
1.82
0.08
892

Phage-64
A
D
—
W
—
D
W
E
—
Y
F
P
—
N
0.08
1.82
0.08
893

Phage-65
H
T
—
W
—
D
W
E
D
D
I
F
—
N
0.12
1.81
0.10
894

Phage-66
F
A
—
W
—
D
W
E
—
A
F
L
—
L
0.09
1.80
0.09
895

Phage-67
Y
D
—
—
—
—
L
—
—
—
I
A
—
D
0.08
1.77
0.08
896

Phage-68
N
S
—
W
—
D
W
E
Y
D
I
I
—
D
0.08
1.77
0.10
897

Phage-69
F
A
—
W
—
D
W
E
—
V
A
P
—
Y
0.07
1.75
0.07
898

Phage-70
L
D
—
—
—
D
G
—
—
T
L
T
—
Y
0.10
1.75
0.12
899

Phage-71
—
L
—
W
—
D
W
E
—
F
Y
D
—
P
0.07
1.74
0.09
900

Phage-72
H
A
—
W
—
V
W
E
—
Y
F
P
—
N
0.07
1.72
0.08
901

Phage-73
N
E
—
W
—
N
G
E
P
T
F
P
—
T
0.08
1.71
0.07
902

Phage-74
L
T
—
—
—
D
G
—
—
T
L
Y
—
D
0.08
1.70
0.07
903

Phage-75
Y
D
—
—
—
—
Y
—
—
—
—
P
—
I
0.13
1.67
0.09
904

Phage-76
I
E
—
W
—
D
W
E
P
N
S
F
—
D
0.09
1.66
0.08
905

Phage-77
Y
D
—
—
—
—
L
—
—
—
I
H
—
Y
0.12
1.66
0.09
906

Phage-78
I
—
—
—
—
—
—
—
—
—
T
I
—
N
0.08
1.63
0.08
907

Phage-79
I
—
—
—
—
—
V
E
—
A
Y
L
—
Y
0.09
1.62
0.10
908

Phage-80
F
D
—
—
—
D
G
—
—
T
—
Y
—
D
0.09
1.61
0.08
909

Phage-81
I
D
—
—
—
D
G
—
—
T
I
S
—
Y
0.08
1.57
0.11
910

Phage-82
N
—
—
—
—
—
—
—
—
I
S
T
—
L
0.10
1.55
0.11
911

Phage-83
Y
D
—
—
—
D
G
—
—
—
Y
F
—
D
0.08
1.53
0.08
912

Phage-84
N
F
—
W
—
D
W
E
Y
F
N
D
—
N
0.09
1.53
0.09
913

Phage-85
—
L
—
W
—
D
W
E
A
F
F
D
—
D
0.07
1.47
0.07
914

Phage-86
I
—
—
—
—
—
W
E
W
P
—
A
—
N
0.16
1.47
0.10
915

Phage-87
—
F
—
W
—
D
W
E
D
N
F
F
—
N
0.08
1.46
0.10
916

Phage-88
—
V
—
W
—
D
W
E
T
F
F
P
—
D
0.08
1.46
0.08
917

Phage-89
D
N
—
—
—
D
G
—
—
T
Y
I
—
N
0.10
1.45
0.09
918

Phage-90
D
N
—
W
—
D
W
E
Y
N
F
F
—
V
0.07
1.45
0.08
919

Phage-91
F
—
—
—
—
—
V
E
—
D
Y
L
—
I
0.10
1.43
0.10
920

Phage-92
D
N
—
W
—
D
W
E
Y
D
I
F
—
V
0.07
1.43
0.07
921

Phage-93
I
D
—
—
—
—
—
—
—
—
I
A
—
P
0.08
1.42
0.08
922

Phage-94
Y
F
—
—
—
—
V
E
—
Y
T
L
—
F
0.10
1.42
0.10
923

Phage-95
F

—
—
—
—
—
—
—
—
A
P
—
N
0.06
1.37
0.08
924

Phage-96
F
D
—
—
—
—
V
E
—
Y
F
Y
—
A
0.11
1.36
0.08
925

Phage-97
D
F
—
W
—
D
W
E
D
F
F
F
—
A
0.18
1.35
0.12
926

Phage-98
F
F
—
—
—
D
G
—
—
T
L
S
—
N
0.08
1.35
0.09
927

Phage-99
F
I
—
—
—
—
—
—
—
—
—
A
—
L
0.14
1.35
0.09
928

Phage-100
Y
D
—
—
—
—
—
—
—
A
I
—
—
Y
0.09
1.32
0.10
929

Phage-101
Y
T
—
W
—
D
W
E
—
Y
L
Y
—
P
0.10
1.32
0.15
930

Phage-102
F
D
—
W
—
D
W
E
—
P
T
T
—
H
0.08
1.31
0.08
931

Phage-103
Y
D
—
W
—
D
W
E
D
F
P
I
—
D
0.14
1.31
0.10
932

Phage-104
—
V
—
W
—
D
W
E
Y
I
D
D
—
S
0.08
1.30
0.07
933

Phage-105
I
N
—
W
—
D
W
E
V
I
S
F
—
D
0.12
1.30
0.08
934

Phage-106
L
S
—
W
—
D
W
E
—
V
T
P
—
L
0.10
1.29
0.10
935

Phage-107
F
A
—
W
—
D
W
E
—
V
D
I
—
Y
0.09
1.28
0.08
936

Phage-108
Y
D
—
—
—
—
M
—
—
—
I
V
—
D
0.10
1.25
0.08
937

Phage-109
Y
D
—
W
—
D
W
E
V
F
I
V
—
D
0.06
1.25
0.07
938

Phage-110
D
N
—
W
—
D
W
E
H
N
F
F
—
V
0.10
1.25
0.08
939

Phage-111
Y
D
—
—
—
D
G
—
—
—
I
Y
—
P
0.07
1.23
0.08
940

Phage-112
Y
D
—
—
—
—
—
E
F
P
Y
Y
—
F
0.12
1.23
0.12
941

Phage-113
A
D
—
—
—
—
Y
—
—
—
I
P
—
V
0.11
1.22
0.09
942

Phage-114
F
L
—

—
—
V
E
—
V
H
Y
—
S
0.08
1.22
0.10
943

Phage-115
T
D
—
W
—
D
W
E
Y
I
T
S
—
S
0.08
1.22
0.08
944

Phage-116
A
F
—
—
—
—
L
—
—
—
I
T
—
D
0.09
1.21
0.09
945

Phage-117
N
D
—
W
—
D
W
E
—
Y
F
S
—
Y
0.09
1.19
0.09
946

Phage-118
F
D
—
—
—
—
W
E
I
V
T
D
—
Y
0.08
1.19
0.09
947

Phage-119
N
L
—
—
—
—
M
—
—
—
I
I
—
P
0.13
1.19
0.11
948

Phage-120
D
L
—
—
—
—
M
—
—
—
I
Y
—
D
0.10
1.19
0.14
949

Phage-121
F
D
—
—
—
D
G
V
—
D
Y
I
—
D
0.09
1.18
0.09
950

Phage-122
Y
A
—
W
—
D
W
E
—
D
F
A
—
Y
0.11
1.18
0.08
951

Phage-123
H
D
—
—
—
—
M
—
—
—
I
V
—
V
0.10
1.17
0.10
952

Phage-124
—
F
—
—
—
—
—
E
F
I
F
L
—
A
0.07
1.17
0.08
953

Phage-125
Y
D
—

—
—
L
—
—
—
I
L
—
D
0.08
1.16
0.09
954

Phage-126
S
V
—
W
—
D
W
E
—
F
Y
S
—
D
0.11
1.16
0.10
955

Phage-127
P
—
—
—
—
D
G
—
—
T
A
I
—
T
0.13
1.16
0.10
956

Phage-128
D
D
—
—
—
—
L
E
W
Y
Y
P
—
Y
0.09
1.16
0.08
957

Phage-129
F
I
—
—
—
—
—
—
—
—
L
P
—
N
0.08
1.14
0.09
958

Phage-130
I
D
—
—
—
—
—
—
—
I
L
P
—
D
0.11
1.14
0.33
959

Phage-131
F
L
—
—
—
—
—
E
—
D
A
P
—
Y
0.08
1.13
0.08
960

Phage-132
I
F
—
—
—
D
G
—
—
T
H
I
—
H
0.10
1.13
0.07
961

Phage-133
—
F
—
W
—
D
W
E
Y
I
D
F
—
N
0.10
1.11
0.21
962

Phage-134
I
F
—
—
—
—
Y
—
—
—
L
H
—
I
0.12
1.11
0.11
963

Phage-135
H
L
—
W
—
D
W
E
W
Y
—
D
—
P
0.08
1.11
0.10
964

Phage-136
F
I
—
—
—
—
M
—
—
—
I
A
—
N
0.08
1.11
0.09
965

Phage-137
I
F
—
—
—
—
V
E
M
I
F
L
—
N
0.09
1.10
0.08
966

Phage-138
Y
D
—
—
—
—
W
E
F
P
—
D
—
I
0.11
1.09
0.11
967

Phage-139
N
L
—
—
—
—
L
—
—
—
I
T
—
F
0.10
1.09
0.08
968

Phage-140
F
—
—
—
—
—
V
E
D
F
Y
F
—
Y
0.08
1.09
0.08
969

Phage-141
D
—
—
—
—
—
—
—
—
—
L
I
—
N
0.11
1.07
0.11
970

Phage-142
D
—
—
—
—
—
—
—
—
—
L
P
—
D
0.08
1.07
0.08
971

Phage-143
A
I
—
—
—
—
L
—
—
—
I
A
—
P
0.09
1.07
0.09
972

Phage-144
—
I
—
—
—
—
V
E
D
Y
N
L
—
Y
0.08
1.07
0.09
973

Phage-145
H
T
—
W
—
D
W
E
D
Y
T
V
—
P
0.10
1.06
0.09
974

Phage-146
S
D
—
W
—
D
W
E
Y
F
Y
D
—
N
0.10
1.06
0.08
975

Phage-147
—
F
—

—
D
G
—
—
T
—
H
—
D
0.09
1.05
0.08
976

Phage-148
D
—
—
—
—
—
Y
—
—
—
—
H
—
I
0.09
1.05
0.08
977

Phage-149
A
D
—
—
—
D
G
—
—
—
T
T
—
H
0.07
1.05
0.08
978

Phage-150
F
—
—

—
—
L
—
—
—
L
T
—
V
0.10
1.05
0.08
979

Phage-151
I
L
—
—
—
—
V
E
—
D
Y
Y
—
Y
0.11
1.04
0.09
980

Phage-152
H
L
—
W
—
D
W
E
I
Y
H
S
—
D
0.09
1.04
0.09
981

Phage-153
I
F
—
W
—
D
W
E
D
Y
N
F
—
T
0.08
1.04
0.11
982

Phage-154
I
V
—
—
—
D
G
—
—
T
L
I
—
H
0.12
1.04
0.11
983

Phage-155
A
D
—
W
—
D
W
E
W
D
Y
T
—
D
0.12
1.03
0.11
984

Phage-156
I
T
—

—
—
—
—
—
—
T
T
—
N
0.20
1.02
0.21
985

Phage-157
Y
H
—
W
—
D
W
E
—
Y
T
S
—
D
0.20
1.02
0.09
986

Phage-158
N
—
—
—
—
—
V
E
—
Y
A
L
—
T
0.11
1.01
0.10
987

Phage-159
F
I
—
—
—
—
M
—
—
—
I
H
—
D
0.15
1.00
0.19
988

Phage-160
D
N
—
W
—
D
W
E

F
A
V
—
P
0.14
1.00
0.10
989

Phage-161
Y
D
—
—
—
—
L
—
—
T
—
V
—
D
0.10
1.00
0.09
990

Phage-162
Y
D
—
—
—
—
—
—
—
—
I
A
—
Y
0.08
0.99
0.08
991

Phage-163
I
D
—
W
—
D
W
E
Y
T
—
H
—
D
0.07
0.97
0.09
992

Phage-164
D
D
—
—
—
—
L
—
—
—
I
I
—
I
0.09
0.96
0.09
993

Phage-165
—
—
—
—
—
—
Y
—
—
—
S
F
—
F
0.09
0.91
0.08
994

Phage-166
F
N
—
W
—
D
W
E
D
P
Y
F
—
V
0.09
0.86
0.07
995

Phage-167
Y
D
—
—
—
—
Y
—
—
—
S
Y
—
S
0.08
0.82
0.07
996

Phage-168
—
A
—
W
—
D
W
E
Y
T
D
S
—
F
0.13
0.79
0.09
997

Phage-169
T
D
—
—
—
—
—
—
—
—
—
A
—
Y
0.10
0.77
0.09
998

Phage-170
T
D
—
W
—
D
W
E
F
Y
A
D
—
D
0.07
0.75
0.08
999

Phage-171
Y
D
—
—
—
—
L
—
—
—
—
I
—
H
0.09
0.69
0.09
1000

Phage-172
S
D
—
—
—
D
G
—
—
—
I
I
—
T
0.07
0.69
0.07
1001

Phage-173
Y
—
—
—
—
—
—
—
—
—
I
D
—
D
0.08
0.67
0.09
1002

Phage-174
F
F
—
—
—
—
I
—
—
—
I
A
—
V
0.08
0.62
0.09
1003

Phage-175
D
—
—
—
—
—
—
—
—
—
T
F
—
D
0.16
0.60
0.10
1004

Phage-176
Y
D
—
—
—
—
W
E
W
P
I
D
—
V
0.10
0.59
0.10
1005

Phage-177
F
—
—
—
—
—
I
E
L
F
S
F
—
Y
0.13
0.59
0.11
1006

Phage-178
Y
—
—
—
—
—
V
—
—
—
I
T
—
P
0.15
0.42
0.11
1007

Phage-179
I
L
—
—
—
—
—
—
—
—
I
N
—
N
0.09
0.37
0.25
1008

Phage-180
—
V
—
—
—
A
M
G
Q
H
Y
L
—
D
0.08
0.09
0.08
1009

Phage-181
—
V
—
—
T
K
M
G
I
H
Y
L
—
S
0.08
0.08
0.08
1010

Phage-182
Y
D
—
W
—
D
W
E
Y
V
Y
A
—
Y
0.08
0.98
0.08
1011

Phage-183
D
L
—
—
—
—
L
—
—
—
—
N
—
D
0.09
0.98
0.08
1012

Phage-184
Y
—
—
—
—
—
—
—
—
—
T
V
—
Y
0.14
0.97
0.16
1013

Phage-185
L
D
—
W
—
D
W
E
W
P
Y
S
—
N
0.08
0.96
0.09
1014

Phage-186
F
I
—
W
—
D
W
E
D
D
F
F
—
Y
0.08
0.96
0.09
1015

Phage-187
D
L
—
—
—
—
V
E
W
Y
F
F
—
N
0.11
0.95
0.10
1016

Phage-188
Y
D
—
—
—
—
L
—
—
—
I
V
—
F
0.07
0.94
0.08
1017

Phage-189
L
N
—
W
—
V
W
E
D
D
—
F
—
Y
0.09
0.92
0.09
1018

Phage-190
F
N
—
W
—
D
W
E
D
P
N
F
—
V
0.09
0.91
0.09
1019

Phage-191
—
I
—
W
—
D
W
E
D
D
Y
F
—
P
0.10
0.91
0.13
1020

Phage-192
F
L
—
—
—
—
—
—
—
—
S
V
—
Y
0.10
0.91
0.08
1021

Phage-193
Y
D
—
—
—
—
L
—
—
—
I
F
—
Y
0.10
0.91
0.09
1022

Phage-194
H
L
—

—
D
G
—
—
—
F
T
—
F
0.11
0.90
0.10
1023

Phage-195
Y
F
—
—
—
—
M
—
—
—
L
Y
—
I
0.08
0.90
0.08
1024

Phage-196
Y
—
—
—
—
—
V
E
—
Y
A
N
—
Y
0.07
0.90
0.07
1025

Phage-197
N
H
—
—
—
—
—
—
—
I
T
A
—
Y
0.16
0.90
0.58
1026

Phage-198
I
D
—
W
—
D
W
E
—
A
F
N
—
Y
0.09
0.90
0.08
1027

Phage-199
A
—
—
—
—
—
L
E
—
F
F
L
—
T
0.09
0.89
0.08
1028

Phage-200
I
—
—
—
—
—
V
E
—
V
H
H
—
Y
0.08
0.89
0.08
1029

Phage-201
F
F
—
—
—
—
—
—
—
—
—
A
—
D
0.10
0.89
0.11
1030

Phage-202
Y
D
—
—
—
—
L
—
—
T
I
I
—
A
0.08
0.89
0.08
1031

Phage-203
I
L
—
—
—
—
W
E
Y
P
L
D
—
S
0.09
0.89
0.10
1032

Phage-204
F
I
—
—
—
—
—
—
—
—
T
T
—
N
0.09
0.88
0.10
1033

Phage-205
F
—
—
—
—
—
L

—
—
S
—
D
0.17
0.88
0.15
1034

Phage-206
H
L
—
—
—
—
L
—
—
—
—
T
—
F
0.10
0.87
0.10
1035

Phage-207
L
I
—
—
—
—
V
E
D
Y
S
L
—
H
0.09
0.87
0.09
1036

Phage-208
Y
F
—
—
—
—
M
—
—
—
—
Y
—
D
0.08
0.87
0.08
1037

Phage-209
H
I
—
—
—
—
M
—
—
—
I
Y
—
I
0.13
0.87
0.09
1038

Phage-210
F
D
—
—
—
—
L
—
—
—
I
N
—
D
0.08
0.87
0.09
1039

Phage-211
Y
—
—
—
—
—
V
F
I
Y
I
Y
—
T
0.07
0.87
0.08
1040

Phage-212
L
A
—
W
—
V
R
E
—
I
N
A
—
I
0.08
0.85
0.07
1041

Phage-213
I
D
—
W
—
D
W
E
D
I
T
F
—
D
0.08
0.85
0.08
1042

Phage-214
I
V
—
—
—
—
L
—
—
—
I
T
—
P
0.11
0.85
0.15
1043

Phage-215
F
—
—
—
—
—
—
E
L
P
A
D
—
D
0.08
0.85
0.09
1044

Phage-216
F
D
—
—
—
—
—
—
—
—
N
P
—
F
0.10
0.85
0.09
1045

Phage-217
D
A
—
W
—
D
W
E
—
Y
S
S
—
D
0.10
0.83
0.10
1046

Phage-218
D
H
—
W
—
D
W
E
P
N
Y
F
—
V
0.08
0.83
0.09
1047

Phage-219
D
—
—
W
—
D
W
E
I
N
Y
I
—
F
0.09
0.83
0.10
1048

Phage-220
I
—
—
W
—
D
W
E
Y
V
Y
A
—
N
0.10
0.82
0.09
1049

Phage-221
D
F
—
—
—
—
V
E
—
D
Y
L
—
D
0.07
0.82
0.08
1050

Phage-222
H
D
—
—
—
D
G
R
—
D
Y
D
—
A
0.11
0.82
0.09
1051

Phage-223
L
A
—
W
—
D
W
E
D
D
Y
F
—
V
0.08
0.82
0.09
1052

Phage-224
D
I
—
W
—
D
W
E
D
Y
L
P
—
V
0.10
0.82
0.10
1053

Phage-225
I
L
—
—
—
—
T
E
V
Y
A
L
—
P
0.08
0.81
0.10
1054

Phage-226
I
F
—
—
—
—
W
E
F
—
—
L
—
N
0.10
0.81
0.11
1055

Phage-227
T
—
—
—
—
—
V
E
D
F
S
L
—
V
0.07
0.80
0.08
1056

Phage-228
F
I
—
I
—
—
W
E
F
V
D
A
—
F
0.11
0.80
0.09
1057

Phage-229
F
A
—
W
—
D
W
E
—
D
S
P
—
D
0.06
0.80
0.07
1058

Phage-230
I
L
—
—
—
—
V
E
—
L
I
F
—
P
0.12
0.80
0.08
1059

Phage-231
F
—
—
—
—
—
V
E
—
Y
I
Y
—
Y
0.08
0.80
0.08
1060

Phage-232
D
S
—
—
—
—
L
—
—
—
I
I
—
D
0.10
0.79
0.09
1061

Phage-233
F
L
—
—
—
D
G
—
—
T
S
V
—
D
0.11
0.79
0.08
1062

Phage-234
F
N
—
W
—
N
G
E
P
T
Y
F
—
V
0.11
0.79
0.08
1063

Phage-235
L
A
—
W
—
V
W
E
Y
P
—
T
—
I
0.09
0.78
0.09
1064

Phage-236
D
—
—
—
—
—
V
E
—
D
—
Y
—
Y
0.09
0.78
0.09
1065

Phage-237
I
T
—
W
—
D
W
E
—
Y
A
N
—
T
0.08
0.77
0.07
1066

Phage-238
F
F
—
—
—
D
G
—
—
T
Y
S
—
I
0.15
0.77
0.13
1067

Phage-239
T
D
—
W
—
D
W
E
Y
A
T
S
—
D
0.09
0.76
0.09
1068

Phage-240
F
N
—
—
—
D
G
Y
—
D
Y
L
—
D
0.10
0.76
0.11
1069

Phage-241
Y
D
—
W
—
D
W
E
V
D
F
H
—
P
0.11
0.76
0.08
1070

Phage-242
N
I
—
W
—
D
W
E
D
D
S
F
—
F
0.08
0.76
0.08
1071

Phage-243
A
T
—
—
—
—
—
—
—
—
I
—
—
S
0.13
0.75
0.09
1072

Phage-244
S
—
—
—
—
—
—
—
—
—
T
F
—
D
0.10
0.74
0.09
1073

Phage-245
P
I
—
—
—
—
Y
—
—
—
D
V
—
A
0.08
0.74
0.08
1074

Phage-246
Y
—
—
—
—
D
G
—
—
Y
N
S
—
I
0.11
0.74
0.11
1075

Phage-247
—
D
—
W
—
D
W
E
V
F
I
A
—
D
0.11
0.74
0.10
1076

Phage-248
D
L
—
—
—
—
V
E
—
V
N
L
—
L
0.12
0.74
0.10
1077

Phage-249
F
D
—
—
—
—
M
—
—
—
T
T
—
F
0.09
0.74
0.09
1078

Phage-250
N
F
—
W
—
D
W
E
P
I
Y
F
—
T
0.13
0.74
0.14
1079

Phage-251
—
D
—
—
—
D
G
—
—
—
F
F
—
L
0.08
0.73
0.08
1080

Phage-252
—
D
—
—
—
D
G
—
—
T
A
F
—
I
0.10
0.73
0.09
1081

Phage-253
N
I
—
—
—
—
M
—
—
—
L
V
—
I
0.10
0.73
0.09
1082

Phage-254
I
I
—
—
—
—
—
—
—
—
F
F
—
F
0.08
0.73
0.09
1083

Phage-255
N
F
—
—
—
—
Y
—
—
—
I
S
—
I
0.10
0.73
0.38
1084

Phage-256
H
L
—
—
—
—
T
E
—
A
D
T
—
N
0.11
0.73
0.51
1085

Phage-257
D
—
—
—
—
—
V
E
—
D
Y
L
—
D
0.08
0.72
0.09
1086

Phage-258
D
—
—
—
—
—
L
—
—
—
I
N
—
D
0.11
0.72
0.10
1087

Phage-259
F
—
—
—
—
—
L
—
—
—
L
F
—
V
0.09
0.72
0.08
1088

Phage-260
P
D
—
—
—
—
W
E
F
Y
—
T
—
N
0.12
0.72
0.08
1089

Phage-261
F
D
—
—
—
—
—
E
Y
I
Y
A
—
T
0.09
0.72
0.08
1090

Phage-262
D
—
—
—
—
—
I
—
—
—
S
I
—
N
0.12
0.72
0.11
1091

Phage-263
D
F
—
—
—
I
V
E
—
Y
I
F
—
F
0.08
0.72
0.07
1092

Phage-264
P
V
—
W
—
D
W
E
Y
V
S
S
—
D
0.08
0.71
0.08
1093

Phage-265
Y
I
—
—
—
—
R
—
—
—
N
L
—
L
0.09
0.71
0.09
1094

Phage-266
Y
D
—
—
—
—
L
—
—
—
I
V
—
D
0.11
0.71
0.11
1095

Phage-267
H
D
—
W
—
D
W
E
D
F
Y
F
—
V
0.09
0.71
0.08
1096

Phage-268
H
—
—
—
—
—
Y
—
—
—
I
D
—
Y
0.12
0.71
0.10
1097

Phage-269
L
F
—
—
—
—
M
P
—
D
I
F
—
N
0.08
0.71
0.08
1098

Phage-270
H
D
—
—
—
—
L
E
F
H
Y
A
—
Y
0.10
0.71
0.12
1099

Phage-271
D
F
—
—
—
—
L
—
—
—
I
N
—
F
0.08
0.70
0.08
1100

Phage-272
Y
F
—
—
—
—
L
—
—
—
I
A
—
N
0.10
0.70
0.10
1101

Phage-273
T
D
—
W
—
D
W
E
A
D
I
I
—
D
0.10
0.70
0.08
1102

Phage-274
Y
D
—
—
—
—
L
—
—
—
I
Y
—
F
0.09
0.70
0.08
1103

Phage-275
Y
—
—
W
—
D
W
W
—
Y
—
T
—
D
0.10
0.70
0.11
1104

Phage-276
P
I
—
—
—
—
L
E
—
—
Y
L
—
N
0.13
0.69
0.52
1105

Phage-277
F
D
—
—
—
—
I
—
—
—
I
V
—
Y
0.12
0.69
0.11
1106

Phage-278
I
L
—
—
—
D
G
I
—
F
F
D
—
P
0.09
0.68
0.07
1107

Phage-279
A
—
—
—
—
—
Y
—
—
—
L
T
—
V
0.07
0.68
0.07
1108

Phage-280
D
F
—
—
—
—
L
—
—
—
I
I
—
A
0.14
0.68
0.14
1109

Phage-281
Y
D
—
—
—
—
—
—
—
—
L
D
—
N
0.08
0.68
0.08
1110

Phage-282
A
T
—
—
—
—
—
—
—
—
—
A
—
D
0.14
0.67
0.08
1111

Phage-283
L
L
—
—
—
D
G
V
—
D
F
F
—
D
0.10
0.67
0.10
1112

Phage-284
N
F
—
—
—
—
L
P
—
D
I
F
—
F
0.12
0.66
0.13
1113

Phage-285
F
I
—
—
—
—
V
E
—
V
S
L
—
N
0.08
0.66
0.08
1114

Phage-286
Y
D
—
—
—
D
G
Y
—
A
F
Y
—
H
0.12
0.66
0.10
1115

Phage-287
N
F
—
—
—
—
I
E
E
D
Y
L
—
D
0.08
0.65
0.08
1116

Phage-288
D
—
—
—
—
D
G
V
—
D
F
I
—
N
0.06
0.65
0.08
1117

Phage-289
T
D
—
W
—
D
W
E
Y
I
Y
S
—
S
0.08
0.65
0.07
1118

Phage-290
F
—
—
—
—
—
—
E
—
I
T
N
—
I
0.14
0.65
0.11
1119

Phage-291
F
D
—
W
—
D
W
E
—
—
F
F
—
H
0.07
0.64
0.08
1120

Phage-292
D
F
—
—
—
D
G
—
—
—
—
F
—
P
0.08
0.63
0.08
1121

Phage-293
H
N
—
—
—
—
L
—
—
—
L
V
—
D
0.13
0.63
0.09
1122

Phage-294
I
—
—
—
—
D
G
A
—
D
Y
T
—
D
0.07
0.63
0.07
1123

Phage-295
F
D
—
—
—
—
—
E
F
P
—
I
—
F
0.08
0.62
0.08
1124

Phage-296
Y
N
—
—
—
—
L
—
—
—
—
T
—
D
0.09
0.62
0.08
1125

Phage-297
F
D
—
—
—
—
L
—
—
—
I
H
—
A
0.07
0.62
0.08
1126

Phage-298
D
I
—
—
—
—
V
E
—
Y
F
L
—
F
0.15
0.61
0.10
1127

Phage-299
F
D
—
—
—
—
V
—
—
—
L
T
—
F
0.10
0.61
0.09
1128

Phage-300
F
D
—
—
—
—
I
E
—
F
H
L
—
F
0.08
0.61
0.08
1129

Phage-301
A
—
—
—
—
—
L
—
—
—
I
I
—
D
0.12
0.61
0.10
1130

Phage-302
Y
N
—
—
—
—
L
—
—
—
I
T
—
N
0.09
0.61
0.10
1131

Phage-303
F
D
—
W
—
D
W
E
—
P
—
D
—
L
0.08
0.61
0.07
1132

Phage-304
D
V
—
—
—
—
L
—
—
—
—
L
—
P
0.08
0.60
0.08
1133

Phage-305
N
—
—
—
—
—
L
—
—
—
L
P
—
P
0.09
0.60
0.08
1134

Phage-306
Y
—
—
W
—
D
W
E
Y
D
I
F
—
S
0.08
0.60
0.10
1135

Phage-307
D
D
—
—
—
—
—
—
—
—
T
Y
—
N
0.08
0.60
0.09
1136

Phage-308
F
—
—
—
—
—
—
E
—
V
F
H
—
Y
0.09
0.60
0.09
1137

Phage-309
T
D
—
W
—
D
W
E
—
Y
F
L
—
D
0.07
0.60
0.09
1138

Phage-310
—
—
—
—
—
—
W
E
—
—
Y
L
—
P
0.09
0.60
0.09
1139

Phage-311
D
D
—
—
—
N
G
Y
A
T
F
I
—
Y
0.06
0.59
0.08
1140

Phage-312
F
L
—
—
—
—
I
E
D
D
T
H
—
Y
0.16
0.59
0.38
1141

Phage-313
F
A
—
W
—
D
W
E
—
T
I
P
—
H
0.08
0.59
0.08
1142

Phage-314
—
L
—
—
—
—
—
—
—
—
Y
N
—
Y
0.10
0.58
0.08
1143

Phage-315
Y
D
—
—
—
—
—
—
—
—
I
S
—
I
0.09
0.58
0.09
1144

Phage-316
Y
D
—
—
—
—
—
—
—
—
—
N
—
Y
0.12
0.57
0.10
1145

Phage-317
A
I
—
W
—
D
W
E
—
F
—
D
—
Y
0.10
0.57
0.09
1146

Phage-318
L
T
—
W
—
V
R
E
—
I
F
A
—
D
0.07
0.57
0.08
1147

Phage-319
—
L
—
—
—
—
—
—
—
—
Y
Y
—
N
0.09
0.57
0.09
1148

Phage-320
N
V
—
—
—
—
Y
—
—
—
A
P
—
N
0.07
0.56
0.08
1149

Phage-321
H
D
—
—
—
—
—
—
—
—
I
S
—
V
0.11
0.55
0.09
1150

Phage-322
F
D
—
—
—
—
L
—
—
T
—
D
—
N
0.12
0.55
0.41
1151

Phage-323
Y
F
—
—
—
—
V
E
—
H
F
Y
—
Y
0.09
0.55
0.08
1152

Phage-324
D
—
—
—
—
—
L
—
—
—
I
I
—
H
0.09
0.54
0.09
1153

Phage-325
D
D
—
—
—
—
V
P
—
D
I
T
—
Y
0.11
0.54
0.08
1154

Phage-326
D
N
—
—
—
—
L
—
—
—
—
V
—
D
0.10
0.54
0.08
1155

Phage-327
—
H
—
W
—
D
W
E
P
N
Y
V
—
D
0.10
0.54
0.08
1156

Phage-328
D
—
—
—
—
—
L
—
—
—
L
F
—
L
0.09
0.53
0.09
1157

Phage-329
D
D
—
—
—
—
L
—
—
—
—
V
—
A
0.08
0.53
0.11
1158

Phage-330
A
A
—
—
—
—
L
—
—
—
I
V
—
D
0.13
0.53
0.09
1159

Phage-331
D
F
—
—
—
—
—
E
—
I
N
N
—
F
0.14
0.52
0.48
1160

Phage-332
Y
—
—
—
—
—
—
—
—
—
N
A
—
Y
0.08
0.52
0.07
1161

Phage-333
—
L
—
—
—
—
—
—
—
—
N
S
—
Y
0.10
0.52
0.11
1162

Phage-334
Y
D
—
—
—
—
—
—
—
—
I
D
—
D
0.09
0.52
0.09
1163

Phage-335
D
S
—
—
—
—
—
E
F
Y
Y
V
—
F
0.11
0.52
0.14
1164

Phage-336
Y
I
—
—
—
—
L
—
—
—
L
I
—
H
0.10
0.52
0.08
1165

Phage-337
F
D
—
—
—
—
V
E
—
D
Y
F
—
Y
0.11
0.52
0.10
1166

Phage-338
F
D
—
—
—
—
Y
—
—
—
L
Y
—
F
0.08
0.52
0.07
1167

Phage-339
—
L
—
—
—
D
G
—
—
Y
S
F
—
H
0.10
0.51
0.10
1168

Phage-340
I
P
—
—
—
—
M
—
—
—
—
V
—
N
0.12
0.51
0.09
1169

Phage-341
I
I
—
—
—
D
G
Y
—
D
F
T
—
D
0.12
0.51
0.09
1170

Phage-342
I
F
—
—
—
—
L
—
—
—
I
I
—
Y
0.11
0.51
0.08
1171

Phage-343
—
D
—
—
—
—
—
—
—
—
Y
D
—
Y
0.18
0.51
0.18
1172

Phage-344
L
S
—
—
—
—
M
—
—
—
L
Y
—
D
0.12
0.51
0.08
1173

Phage-345
Y
D
—
W
—
D
W
E
Y
N
I
D
—
T
0.08
0.51
0.09
1174

Phage-346
N
H
—
—
—
D
G
—
—
T
T
V
—
F
0.09
0.51
0.08
1175

Phage-347
N
F
—
—
—
—
L
—
—
—
I
P
—
H
0.11
0.50
0.12
1176

Phage-348
F
H
—
—
—
—
I
E
—
Y
A
L
—
D
0.08
0.50
0.09
1177

Phage-349
—
—
—
—
—
—
V
E
D
Y
N
L
—
Y
0.07
0.50
0.08
1178

Phage-350
—
—
—
—
—
D
G
—
—
L
A
N
—
Y
0.09
0.50
0.08
1179

Phage-351
L
I
—
—
—
V
I
A
—
D
L
P
—
N
0.17
0.50
0.26
1180

Phage-352
D
I
—
—
—
—
I
P
—
D
—
S
—
D
0.10
0.50
0.08
1181

Phage-353
I
—
—
—
—
—
W
E
—
A
D
Y
—
D
0.11
0.50
0.45
1182

Phage-354
I
D
—
W
—
D
W
E
D
D
S
I
—
Y
0.10
0.50
0.10
1183

Phage-355
—
L
—
—
—
—
V
E
D
F
T
L
—
D
0.09
0.50
0.11
1184

Phage-356
L
—
—
—
—
V
I
E
—
I
Y
Y
—
Y
0.09
0.49
0.09
1185

Phage-357
F
F
—
—
—
—
—
E
V
H
S
D
—
N
0.14
0.49
0.38
1186

Phage-358
N
D
—
—
—
—
V
E
L
V
S
D
—
N
0.10
0.49
0.08
1187

Phage-359
D
L
—
—
—
—
L
—
—
—
T
V
—
D
0.09
0.49
0.08
1188

Phage-360
I
P
—
—
—
—
V
E
D
Y
N
L
—
N
0.08
0.49
0.08
1189

Phage-361
Y
—
—
—
—
—
L
E
W
P
—
V
—
N
0.10
0.49
0.10
1190

Phage-362
Y
D
—
—
—
—
L
—
—
—
—
I
—
N
0.08
0.49
0.10
1191

Phage-363
—
—
—
—
—
D
G
—
—
—
F
D
—
A
0.08
0.49
0.08
1192

Phage-364
N
D
—
—
—
—
W
E
D
T
Y
F
—
L
0.08
0.49
0.10
1193

Phage-365
P
—
—
—
—
—
M
E
—
L
S
N
—
S
0.13
0.48
0.16
1194

Phage-366
D
D
—
—
—
—
—
E
V
I
S
D
—
Y
0.15
0.48
0.10
1195

Phage-367
D
L
—
—
—
—
—
P
—
D
—
P
—
D
0.08
0.48
0.08
1196

Phage-368
I
—
—
—
—
—
—
—
—
—
F
V
—
Y
0.11
0.48
0.10
1197

Phage-369
A
—
—
—
—
—
Y
E
V
F
A
D
—
N
0.10
0.48
0.11
1198

Phage-370
I
D
—
—
—
—
Y
—
—
—
—
D
—
L
0.09
0.48
0.09
1199

Phage-371
H
I
—
W
—
D
W
E
—
F
H
D
—
N
0.07
0.48
0.08
1200

Phage-372
Y
D
—
—
—
—
L
—
—
T
I
T
—
L
0.08
0.48
0.08
1201

Phage-373
Y
L
—
—
—
—
L
—
—
T
I
L
—
N
0.09
0.48
0.08
1202

Phage-374
F
F
—
—
—
—
—
E
—
A
F
L
—
F
0.10
0.48
0.14
1203

Phage-375
I
L
—
—
—
—
L
—
—
—
F
T
—
A
0.07
0.47
0.08
1204

Phage-376
F
H
—
—
—
—
V
E
L
Y
T
D
—
N
0.09
0.47
0.08
1205

Phage-377
N
L
—
—
—
—
V
E
—
Y
N
F
—
Y
0.08
0.47
0.08
1206

Phage-378
F
D
—
—
—
—
V
E
—
T
Y
Y
—
F
0.21
0.47
0.09
1207

Phage-379
—
F
—
—
—
—
—
E
—
D
H
Y
—
Y
0.12
0.47
0.37
1208

Phage-380
A
I
—
—
—
—
W
E
V
V
A
D
—
N
0.11
0.47
0.11
1209

Phage-381
F
I
—
W
—
D
W
E
—
D
N
Y
—
N
0.12
0.47
0.25
1210

Phage-382
I
—
—
—
—
—
—
—
—
—
F
I
—
D
0.08
0.47
0.10
1211

Phage-383
N
L
—
—
—
—
V
E
D
V
Y
D
—
H
0.12
0.47
0.43
1212

Phage-384
H
—
—
—
—
—
V
E
—
Y
H
N
—
N
0.09
0.47
0.09
1213

Phage-385
D
I
—
—
—
—
Y
—
—
—
Y
S
—
T
0.09
0.47
0.09
1214

Phage-386
D
—
—
—
—
—
L
—
—
T
L
I
—
A
0.13
0.46
0.10
1215

Phage-387
I
A
—
—
—
—
M
P
—
D
I
D
—
Y
0.12
0.46
0.09
1216

Phage-388
I
D
—
—
—
—
L
—
—
—
I
F
—
D
0.10
0.46
0.10
1217

Phage-389
Y
F
—
—
—
D
V
E
—
D
F
A
—
D
0.11
0.46
0.11
1218

Phage-390
Y
N
—
—
—
—
W
E
Y
A
I
L
—
D
0.12
0.46
0.34
1219

Phage-391
I
—
—
—
—
—
V
E
D
Y
I
V
—
N
0.14
0.46
0.22
1220

Phage-392
Y
D
—
—
—
—
I
—
—
—
T
P
—
A
0.07
0.46
0.07
1221

Phage-393
D
T
—
W
—
D
W
E
H
I
Y
A
—
D
0.09
0.46
0.09
1222

Phage-394
D
I
—
—
—
—
M
—
—
—
—
T
—
N
0.13
0.45
0.12
1223

Phage-395
Y
D
—
W
—
D
W
E
R
Y
F
P
—
I
0.10
0.45
0.09
1224

Phage-396
H
L
—
—
—
—
L
—
—
—
—
A
—
S
0.13
0.45
0.11
1225

Phage-397
Y
D
—
—
—
D
G
—
—
T
T
I
—
A
0.09
0.45
0.09
1226

Phage-398
Y
—
—
—
—
—
Y
E
D
V
L
D
—
F
0.07
0.45
0.08
1227

Phage-399
D
F
—
—
—
—
M
—
—
T
I
S
—
D
0.14
0.45
0.10
1228

Phage-400
I
L
—
—
—
—
L
—
—
—
L
V
—
D
0.08
0.44
0.07
1229

Phage-401
L
I
—
—
—
—
W
E
V
I
T
N
—
D
0.12
0.44
0.40
1230

Phage-402
Y
D
—
—
—
—
—
—
—
Y
F
—
—
P
0.09
0.44
0.09
1231

Phage-403
A
L
—
—
—
—
V
E
V
Y
D
V
—
V
0.08
0.44
0.08
1232

Phage-404
Y
H
—
W
—
D
W
E
D
V
N
F
—
Y
0.10
0.44
0.09
1233

Phage-405
F
L
—
—
—
M
G
G
L
T
F
Y
—
Y
0.09
0.44
0.08
1234

Phage-406
I
I
—
—
—
—
—
—
—
Y
—
—
—
F
0.09
0.43
0.19
1235

Phage-407
F
F
—
—
—
—
M
—
—
—
—
H
—
F
0.11
0.43
0.09
1236

Phage-408
A
F
—
—
—
—
—
—
—
—
L
F
—
A
0.09
0.43
0.09
1237

Phage-409
N
—
—
—
—
D
G
—
—
T
N
I
—
D
0.08
0.43
0.08
1238

Phage-410
Y
L
—
—
—
—
W
E
W
V
H
N
—
L
0.13
0.43
0.09
1239

Phage-411
A
T
—
—
—
D
G
—
—
—
H
I
—
A
0.08
0.43
0.09
1240

Phage-412
—
—
—
—
—
—
V
E
V
L
D
Y
—
D
0.07
0.42
0.08
1241

Phage-413
I
H
—
—
—
—
W
E
F
Y
T
D
—
D
0.08
0.42
0.08
1242

Phage-414
D
D
—
—
—
—
L
—
—
T
—
A
—
D
0.13
0.42
0.32
1243

Phage-415
Y
L
—
—
—
—
—
—
—
—
I
D
—
N
0.11
0.42
0.17
1244

Phage-416
L
L
—
—
—
—
V
E
D
V
F
A
—
Y
0.09
0.42
0.09
1245

Phage-417
Y
D
—
—
—
—
L
—
—
—
L
T
—
D
0.08
0.42
0.08
1246

Phage-418
F
D
—
—
—
—
L
—
—
T
—
N
—
Y
0.09
0.41
0.09
1247

Phage-419
F
A
—
W
—
D
W
E
—
I
N
D
—
H
0.08
0.41
0.09
1248

Phage-420
Y
—
—
—
—
—
Y
E
—
D
I
Y
—
N
0.09
0.41
0.09
1249

Phage-421
N
V
—
—
—
—
V
E
D
Y
T
F
—
Y
0.09
0.40
0.08
1250

Phage-422
A
—
—
—
—
—
L
E
—
Y
D
F
—
T
0.12
0.40
0.08
1251

Phage-423
F
D
—
—
—
—
I
—
—
—
T
I
—
T
0.08
0.40
0.09
1252

Phage-424
N
L
—
—
—
—
L
—
—
T
L
V
—
A
0.10
0.40
0.09
1253

Phage-425
Y
S
—
W
—
D
W
E
—
Y
L
A
—
N
0.08
0.40
0.08
1254

Phage-426
G
I
—
—
—
—
V
E
D
Y
N
Y
—
D
0.09
0.40
0.10
1255

Phage-427
F
F
—
—
—
—
L
—
—
—
—
N
—
H
0.07
0.40
0.07
1256

Phage-428
Y
—
—
—
—
—
Y
E
—
D
F
Y
—
F
0.11
0.40
0.09
1257

Phage-429
L
—
—
—
—
—
Y
—
—
—
T
D
—
Y
0.11
0.40
0.10
1258

Phage-430
D
I
—
—
—
—
V
E
—
D
F
L
—
Y
0.10
0.40
0.08
1259

Phage-431
T
L
—
—
—
—
V
E
L
Y
I
F
—
D
0.08
0.40
0.09
1260

Phage-432
F
—
—
—
—
—
—
E
Q
I
A
D
—
Y
0.11
0.40
0.09
1261

Phage-433
D
D
—
—
—
—
V
E
—
Y
H
L
—
D
0.11
0.40
0.18
1262

Phage-434
D
—
—
—
—
—
L
E
D
V
T
L
—
H
0.13
0.39
0.09
1263

Phage-435
—
L
—
—
—
—
V
E
D
V
N
L
—
Y
0.09
0.39
0.08
1264

Phage-436
D
I
—
—
—
—
L
—
—
T
I
D
—
Y
0.09
0.39
0.09
1265

Phage-437
T
—
—
—
—
—
V
E
—
D
I
N
—
Y
0.08
0.39
0.08
1266

Phage-438
I
V
—
W
—
D
W
E
—
Y
P
N
—
D
0.08
0.39
0.08
1267

Phage-439
S
D
—
—
—
—
L
—
—
—
I
I
—
T
0.11
0.39
0.10
1268

Phage-440
Y
D
—
—
—
—
L
P
—
D
Y
D
—
N
0.15
0.39
0.10
1269

Phage-441
N
L
—
W
—
D
W
E
—
Y
Y
A
—
D
0.12
0.39
0.17
1270

Phage-442
D
D
—
—
—
—
L
—
—
—
L
P
—
H
0.10
0.39
0.08
1271

Phage-443
S
L
—
—
—
D
G
Q
—
D
Y
T
—
F
0.08
0.39
0.08
1272

Phage-444
L
I
—
W
—
D
W
E
—
Y
N
F
—
T
0.13
0.39
0.11
1273

Phage-445
F
H
—
—
—
D
G
—
—
T
—
P
—
I
0.08
0.39
0.08
1274

Phage-446
F
D
—
—
—
—
W
E
W
I
Y
D
—
F
0.08
0.38
0.08
1275

Phage-447
I
—
—
—
—
—
W
—
—
—
L
D
—
D
0.08
0.38
0.09
1276

Phage-448
L
I
—
—
—
—
I
—
—
—
A
S
—
N
0.10
0.38
0.11
1277

Phage-449
T
S
—
W
V
D
W
E
—
F
S
D
—
I
0.11
0.38
0.34
1278

Phage-450
Y
—
—
—
—
—
V
E
—
D
Y
V
—
D
0.10
0.38
0.08
1279

Phage-451
D
D
—
—
—
—
Q
E
F
I
Y
A
—
I
0.09
0.38
0.08
1280

Phage-452
A
D
—
W
—
D
W
E
—
Y
A
D
—
Y
0.11
0.38
0.10
1281

Phage-453
Y
—
—
—
—
—
—
—
—
—
I
H
—
I
0.08
0.38
0.07
1282

Phage-454
S
E
—
W
—
D
W
E
P
F
F
D
—
N
0.08
0.37
0.09
1283

Phage-455
Y
H
—
—
—
—
M
—
—
—
L
I
—
T
0.07
0.37
0.07
1284

Phage-456
S
D
—
W
—
D
W
E
D
A
Y
F
—
I
0.09
0.37
0.07
1285

Phage-457
—
D
—
—
—
—
V
E
—
Y
Y
H
—
D
0.08
0.37
0.08
1286

Phage-458
D
N
—
—
—
—
Y
—
—
—
T
A
—
N
0.10
0.37
0.10
1287

Phage-459
L
—
—
—
—
V
—
E
F
Y
D
Y
—
Y
0.08
0.37
0.10
1288

Phage-460
Y
T
—
—
—
—
M
—
—
—
—
T
—
I
0.09
0.37
0.08
1289

Phage-461
N
F
—
W
—
D
W
E
V
N
S
F
—
D
0.09
0.37
0.08
1290

Phage-462
N
A
—
W
—
D
W
E
Y
I
D
F
—
N
0.12
0.36
0.09
1291

Phage-463
Y
N
—
—
—
—
M
—
—
—
I
F
—
S
0.09
0.36
0.07
1292

Phage-464
L
D
—
—
—
—
L
—
—
—
I
T
—
Y
0.08
0.36
0.09
1293

Phage-465
H
—
—
—
—
—
—
—
—
—
I
N
—
D
0.11
0.36
0.08
1294

Phage-466
I
I
—
—
—
—
L
P
—
D
Y
V
—
T
0.08
0.36
0.08
1295

Phage-467
D
I
—
—
—
—
—
—
—
—
I
D
—
S
0.08
0.36
0.08
1296

Phage-468
P
—
—
—
—
—
—
—
—
—
—
L
—
F
0.10
0.36
0.09
1297

Phage-469
—
F
—
—
—
—
—
—
—
—
—
D
—
Y
0.07
0.36
0.08
1298

Phage-470
Y
D
—
W
—
D
W
E
—
A
L
P
—
A
0.08
0.36
0.08
1299

Phage-471
D
D
—
W
—
D
W
E
D
Y
—
F
—
F
0.10
0.36
0.10
1300

Phage-472
H
F
—
—
—
—
W
E
L
F
S
D
—
Y
0.11
0.36
0.11
1301

Phage-473
I
T
—
W
—
D
W
E
V
N
F
P
—
Y
0.07
0.35
0.07
1302

Phage-474
P
D
—
—
—
—
L
—
—
—
I
T
—
N
0.20
0.35
0.16
1303

Phage-475
N
L
—
W
—
D
W
E
A
F
F
P
—
Y
0.08
0.35
0.07
1304

Phage-476
F
—
—
—
—
—
—
E
Y
I
R
D
—
Y
0.08
0.35
0.07
1305

Phage-477
F
F
—
—
—
—
—
—
—
—
I
I
—
D
0.09
0.35
0.10
1306

Phage-478
—
L
—
—
—
K
G
G
P
T
Y
N
—
S
0.08
0.35
0.10
1307

Phage-479
L
A
—
W
—
V
W
E
—
P
G
H
—
D
0.11
0.35
0.10
1308

Phage-480
D
—
—
—
—
—
V
E
D
V
N
D
—
Y
0.07
0.35
0.08
1309

Phage-481
D
—
—
—
—
—
—
E
—
A
H
Y
—
N
0.08
0.35
0.07
1310

Phage-482
—
L
—
—
—
—
L
—
—
T
L
T
—
I
0.08
0.35
0.07
1311

Phage-483
Y
—
—
—
—
—
I
E
D
Y
N
L
—
N
0.10
0.34
0.09
1312

Phage-484
Y
I
—
—
—
—
V
E
—
Y
Y
N
—
F
0.13
0.34
0.14
1313

Phage-485
D
I
—
—
—
—
L
—
—
—
I
F
—
F
0.08
0.34
0.09
1314

Phage-486
D
I
—
—
—
—
V
E
—
D
Y
L
—
Y
0.07
0.34
0.08
1315

Phage-487
T
L
—
—
—
—
—
E
—
D
A
P
—
I
0.10
0.34
0.08
1316

Phage-488
N
—
—
W
—
D
W
E
Y
I
N
S
—
V
0.14
0.34
0.09
1317

Phage-489
N
D
—
—
—
—
V
E
—
Y
Y
Y
—
T
0.07
0.34
0.09
1318

Phage-490
I
T
—
—
—
—
M
—
—
—
I
D
—
N
0.08
0.34
0.08
1319

Phage-491
Y
—
—
—
—
—
M
A
—
D
L
I
—
D
0.10
0.34
0.29
1320

Phage-492
I
D
—
—
—
—
L
—
—
—
I
V
—
T
0.11
0.33
0.09
1321

Phage-493
H
T
—
W
—
D
W
E
W
D
—
Y
—
D
0.08
0.33
0.07
1322

Phage-494
I
H
—
—
—
—
W
E
L
I
D
D
—
L
0.08
0.33
0.10
1323

Phage-495
Y
T
—
—
—
—
L
—
—
—
I
T
—
T
0.08
0.33
0.08
1324

Phage-496
F
H
—
—
—
—
V
E
—
T
—
Y
—
F
0.11
0.33
0.09
1325

Phage-497
D
—
—
—
—
—
L
—
—
—
L
I
—
N
0.07
0.33
0.08
1326

Phage-498
I
—
—
—
—
—
—
—
—
—
D
Y
—
I
0.08
0.33
0.10
1327

Phage-499
D
L
—
—
—
—
I
E
—
D
L
V
—
T
0.09
0.33
0.09
1328

Phage-500
N
I
—
—
—
—
L
Q
—
D
I
V
—
P
0.09
0.33
0.09
1329

Phage-501
N
N
—
—
—
—
M
—
—
—
I
T
—
Y
0.08
0.33
0.08
1330

Phage-502
H
T
—
—
—
—
L
—
—
—
I
V
—
V
0.08
0.33
0.08
1331

Phage-503
Y
—
—
—
—
—
I
E
D
I
L
V
—
T
0.12
0.33
0.23
1332

Phage-504
N
T
—
—
—
—
—
E
F
V
H
L
—
P
0.07
0.33
0.11
1333

Phage-505
D
I
—
—
—
—
M
—
—
—
T
V
—
D
0.10
0.33
0.09
1334

Phage-506
A
I
—
—
—
—
V
E
I
V
N
Y
—
Y
0.09
0.32
0.07
1335

Phage-507
H
L
—
—
—
—
V
E
D
P
T
A
—
V
0.10
0.32
0.28
1336

Phage-508
A
D
—
—
—
—
L
—
—
—
I
S
—
T
0.10
0.32
0.09
1337

Phage-509
F
D
—
—
—
—
L
—
—
—
—
I
—
D
0.07
0.32
0.09
1338

Phage-510
D
V
—
—
—
—
—
—
—
—
I
D
—
N
0.10
0.32
0.08
1339

Phage-511
Y
L
—
—
—
—
V
E
—
I
S
I
—
F
0.08
0.32
0.07
1340

Phage-512
S
A
—
—
—
—
—
—
—
—
L
H
—
V
0.10
0.31
0.30
1341

Phage-513
H
L
—
W
—
D
W
E
—
D
S
A
—
N
0.08
0.31
0.08
1342

Phage-514
H
T
—
W
—
D
W
E
Y
D
Y
D
—
F
0.10
0.31
0.08
1343

Phage-515
Y
D
—
—
—
—
W
E
—
V
A
L
—
N
0.10
0.31
0.10
1344

Phage-516
T
L
—
—
—
—
I
E
—
Y
I
V
—
Y
0.09
0.31
0.23
1345

Phage-517
S
—
—
—
—
—
—
—
—
—
I
F
—
T
0.09
0.31
0.09
1346

Phage-518
D
I
—
—
—
—
—
—
—
—
L
H
—
Y
0.08
0.31
0.08
1347

Phage-519
L
F
—
—
—
—
—
E
—
A
Y
L
—
I
0.08
0.31
0.08
1348

Phage-520
I
F
—
—
—
—
I
E
—
D
F
V
—
T
0.10
0.31
0.10
1349

Phage-521
D
D
—
—
—
—
W
E
Y
Y
—
A
—
V
0.08
0.31
0.08
1350

Phage-522
D
D
—
—
—
—
L
—
—
T
T
I
—
Y
0.10
0.31
0.09
1351

Phage-523
A
S
—
—
—
—
L
—
—
—
—
A
—
D
0.10
0.31
0.09
1352

Phage-524
Y
L
—
—
—
—
V
E
D
Y
D
Y
—
Y
0.08
0.31
0.09
1353

Phage-525
D
L
—
—
—
—
W
E
—
T
I
F
—
A
0.12
0.30
0.09
1354

Phage-526
D
F
—
—
—
D
G
E
—
F
Y
I
—
P
0.12
0.30
0.11
1355

Phage-527
—
—
—
—
—
—
V
E
—
N
I
L
—
H
0.15
0.30
0.17
1356

Phage-528
D
—
—
—
—
—
V
E
—
N
Y
F
—
F
0.12
0.30
0.21
1357

Phage-529
N
D
—
W
—
D
W
Y
—
F
L
S
—
D
0.10
0.30
0.10
1358

Phage-530
R
D
—
W
—
D
W
E
V
P
Y
F
—
D
0.08
0.30
0.09
1359

Phage-531
N
L
—
—
—
—
V
E
—
A
—
Y
—
Y
0.10
0.30
0.13
1360

Phage-532
F
D
—
W
—
D
G
E
L
N
Y
L
—
T
0.23
0.30
0.08
1361

Phage-533
Y
—
—
—
—
—
V
E
D
V
N
L
—
I
0.18
0.30
0.10
1362

Phage-534
D
N
—
—
—
—
Y
—
—
—
I
T
—
L
0.11
0.29
0.09
1363

Phage-535
L
N
—
W
—
D
W
E
—
D
Y
S
—
N
0.09
0.29
0.09
1364

Phage-536
P
T
—
—
—
—
V
E
—
L
L
S
—
N
0.12
0.29
0.26
1365

Phage-537
D
H
—
—
—
—
V
E
L
I
F
Y
—
H
0.11
0.29
0.08
1366

Phage-538
F
H
—
—
—
—
L
—
—
—
I
F
—
Y
0.08
0.29
0.08
1367

Phage-539
F
D
—
—
—
—
L
E
—
T
—
V
—
P
0.16
0.29
0.16
1368

Phage-540
D
F
—
—
—
—
L
—
—
—
L
P
—
A
0.08
0.29
0.08
1369

Phage-541
D
S
—
—
—
—
L
—
—
—
I
Y
—
D
0.09
0.29
0.09
1370

Phage-542
A
D
—
W
—
D
W
E
—
F
L
L
—
F
0.12
0.29
0.10
1371

Phage-543
I
L
—
—
—
—
V
E
—
L
D
F
—
N
0.10
0.28
0.08
1372

Phage-544
F
F
—
—
—
—
—
E
—
I
F
L
—
Y
0.09
0.28
0.07
1373

Phage-545
Y
N
—
—
—
D
G
—
—
—
Y
D
—
H
0.07
0.28
0.07
1374

Phage-546
D
N
—
—
—
—
L
—
—
T
I
T
—
F
0.11
0.28
0.11
1375

Phage-547
H
N
—
—
—
D
G
—
—
A
F
I
—
N
0.09
0.28
0.23
1376

Phage-548
D
—
—
—
—
—
V
E
—
D
L
V
—
P
0.10
0.28
0.23
1377

Phage-549
D
—
—
—
—
—
L
—
—
—
L
N
—
F
0.08
0.28
0.07
1378

Phage-550
D
—
—
—
—
—
—
Q
—
D
F
H
—
H
0.11
0.28
0.27
1379

Phage-551
Y
D
—
—
—
—
W
E
F
T
D
D
—
I
0.10
0.28
0.18
1380

Phage-552
D
I
—
—
—
—
Y
E
—
D
I
I
—
Y
0.14
0.28
0.19
1381

Phage-553
F
D
—
—
—
—
L
—
—
T
—
P
—
P
0.11
0.28
0.08
1382

Phage-554
N
—
—
—
—
—
L
—
—
T
S
V
—
D
0.09
0.28
0.26
1383

Phage-555
Y
—
—
—
—
—
W
E
F
—
F
D
—
D
0.17
0.28
0.11
1384

Phage-556
Y
A
—
—
—
—
L
—
—
—
—
T
—
D
0.09
0.28
0.08
1385

Phage-557
F
L
—
—
—
—
V
E
Q
D
Y
F
—
V
0.08
0.28
0.10
1386

Phage-558
D
N
—
—
—
—
—
—
—
—
—
R
—
D
0.09
0.27
0.26
1387

Phage-559
N
D
—
—
—
D
G
I
—
T
—
D
—
Y
0.10
0.27
0.24
1388

Phage-560
Y
F
—
—
—
—
V
E
D
Y
N
D
—
F
0.08
0.27
0.09
1389

Phage-561
N
L
—
—
—
—
—
—
—
—
I
F
—
Y
0.10
0.27
0.07
1390

Phage-562
I
D
—
W
—
D
W
E
—
Y
I
P
—
T
0.10
0.27
0.08
1391

Phage-563
I
—
—
—
—
D
G
—
—
—
F
I
—
A
0.07
0.27
0.08
1392

Phage-564
D
—
—
—
—
—
—
—
—
—
—
V
—
Y
0.08
0.27
0.07
1393

Phage-565
—
F
—
—
—
—
W
E
D
I
T
D
—
D
0.13
0.27
0.09
1394

Phage-566
L
D
—
—
—
—
V
—
—
T
F
T
—
H
0.08
0.26
0.08
1395

Phage-567
D
D
—
—
—
—
Y
—
—
—
F
A
—
H
0.13
0.26
0.10
1396

Phage-568
I
—
—
—
—
—
Y
Q
—
D
L
P
—
N
0.12
0.26
0.11
1397

Phage-569
L
D
—
—
—
—
V
E
—
Y
N
Y
—
V
0.09
0.26
0.08
1398

Phage-570
Y
V
—
—
—
—
—
—
—
—
S
A
—
N
0.14
0.26
0.08
1399

Phage-571
T
P
—
—
—
—
L
E
—
A
I
—
—
Y
0.10
0.26
0.10
1400

Phage-572
Y
F
—
—
—
—
—
—
—
—
A
D
—
N
0.08
0.26
0.08
1401

Phage-573
D
D
—
—
—
—
—
E
—
D
I
I
—
D
0.12
0.26
0.25
1402

Phage-574
L
—
—
—
—
V
V
E
—
L
N
H
—
N
0.08
0.26
0.09
1403

Phage-575
—
I
—
—
—
D
G
E
—
L
I
A
—
A
0.09
0.26
0.27
1404

Phage-576
F
A
—
W
—
D
W
Q
—
T
Y
V
—
N
0.09
0.25
0.08
1405

Phage-577
Y
I
—
—
—
—
V
E
F
L
F
F
—
N
0.08
0.25
0.08
1406

Phage-578
T
Q
—
—
—
K
G
E
P
T
Y
H
—
Y
0.12
0.25
0.12
1407

Phage-579
N
—
—
—
—
—
V
E
—
Y
H
N
—
D
0.10
0.25
0.17
1408

Phage-580
—
—
—
—
—
—
W
E
F
F
S
D
—
A
0.08
0.25
0.07
1409

Phage-581
F
—
—
—
—
—
L
E
—
—
F
F
—
Y
0.10
0.25
0.22
1410

Phage-582
D
—
—
—
—
—
I
E
—
N
F
Y
—
Y
0.13
0.25
0.09
1411

Phage-583
Y
A
—
—
—
—
V
E
—
Y
—
Y
—
A
0.08
0.25
0.09
1412

Phage-584
—
D
—
—
—
—
L
—
—
—
I
I
—
D
0.08
0.25
0.07
1413

Phage-585
N
D
—
—
—
—
M
—
—
—
I
A
—
Y
0.07
0.25
0.07
1414

Phage-586
—
—
—
—
—
—
—
—
—
Y
L
A
—
A
0.09
0.25
0.21
1415

Phage-587
—
I
—
—
—
—
—
—
—
—
A
N
—
D
0.08
0.25
0.09
1416

Phage-588
L
T
—
W
—
D
W
E
—
D
F
F
—
N
0.07
0.24
0.07
1417

Phage-589
F
—
—
—
—
—
Q
E
—
I
N
Y
—
Y
0.10
0.24
0.23
1418

Phage-590
T
—
—
—
—
—
L
E
—
F
F
L
—
Y
0.13
0.24
0.08
1419

Phage-591
P
D
—
—
—
—
L
—
—
—
—
A
—
H
0.12
0.24
0.09
1420

Phage-592
N
D
—
—
—
—
—
E
—
I
I
F
—
V
0.09
0.24
0.24
1421

Phage-593
Y
I
—
—
—
—
—
—
—
—
F
Y
—
N
0.25
0.24
0.08
1422

Phage-594
H
A
—
—
—
—
L
—
—
—
L
L
—
N
0.20
0.24
0.07
1423

Phage-595
Y
—
—
—
—
—
W
E
—
A
—
L
—
A
0.09
0.24
0.21
1424

Phage-596
A
F
—
—
—
—
V
E
—
Y
D
L
—
N
0.10
0.24
0.16
1425

Phage-597
Y
N
—
—
—
—
—
—
—
—
—
A
—
S
0.15
0.23
0.09
1426

Phage-598
Y
L
—
—
—
—
V
E
D
D
T
L
—
A
0.08
0.23
0.09
1427

Phage-599
A
V
—
—
—
—
—
—
—
—
—
N
—
D
0.08
0.23
0.08
1428

Phage-600
N
—
—
—
—
—
W
E
V
Y
S
L
—
P
0.13
0.23
0.08
1429

Phage-601
D
F
—
—
—
—
V
E
—
D
T
Y
—
H
0.07
0.23
0.07
1430

Phage-602
I
S
—
—
—
—
Y
E
W
D
Y
A
—
N
0.08
0.23
0.10
1431

Phage-603
—
N
—
—
—
—
L
—
—
—
T
I
—
Y
0.08
0.23
0.08
1432

Phage-604
Y
D
—
—
—
—
—
—
—
T
A
P
—
Y
0.07
0.23
0.08
1433

Phage-605
Y
L
—
—
—
—
—
E
—
N
F
L
—
T
0.09
0.23
0.22
1434

Phage-606
F
D
—
—
—
—
V
—
—
—
—
D
—
A
0.08
0.22
0.09
1435

Phage-607
Y
D
—
—
—
—
Q
E
—
I
S
F
—
N
0.09
0.22
0.09
1436

Phage-608
T
D
—
—
—
—
I
E
L
Y
D
D
—
F
0.09
0.22
0.09
1437

Phage-609
T
F
—
—
—
—
L
—
—
—
—
Y
—
Y
0.08
0.22
0.07
1438

Phage-610
F
F
—
—
—
—
I
—
—
—
N
A
—
V
0.09
0.22
0.07
1439

Phage-611
Y
H
—
W
—
D
W
E
P
I
Y
I
—
I
0.12
0.22
0.10
1440

Phage-612
A
I
—
—
—
—
Y
E
—
D
H
Y
—
Y
0.08
0.22
0.08
1441

Phage-613
P
L
—
—
—
D
G
F
—
N
Y
N
—
F
0.12
0.22
0.08
1442

Phage-614
F
P
—
W
—
D
W
E
W
D
N
N
—
H
0.09
0.22
0.09
1443

Phage-615
—
D
—
—
—
D
G
—
—
L
A
A
—
H
0.10
0.22
0.11
1444

Phage-616
—
D
—
W
—
D
W
E
—
Y
Y
S
—
D
0.08
0.22
0.07
1445

Phage-617
—
—
—
—
—
—
Y
—
—
—
Y
D
—
T
0.07
0.21
0.10
1446

Phage-618
N
L
—
—
—
—
W
E
N
F
A
D
—
F
0.08
0.21
0.08
1447

Phage-619
Y
I
—
—
—
—
L
E
V
F
F
V
—
D
0.12
0.21
0.10
1448

Phage-620
I
F
—
—
—
—
L
E
D
Y
S
I
—
F
0.09
0.21
0.08
1449

Phage-621
D
—
—
—
—
—
L
E
Q
Y
D
L
—
F
0.09
0.21
0.08
1450

Phage-622
L
L
—
—
—
V
N
E
D
P
L
D
—
Y
0.11
0.21
0.13
1451

Phage-623
I
D
—
—
—
—
—
—
—
—
—
F
—
Y
0.08
0.21
0.08
1452

Phage-624
I
I
—
—
—
—
V
E
—
I
D
I
—
S
0.08
0.21
0.08
1453

Phage-625
I
A
—
W
—
D
W
E
D
Y
S
S
—
P
0.08
0.21
0.11
1454

Phage-626
Y
—
—
—
—
—
V
E
D
I
N
D
—
I
0.09
0.21
0.07
1455

Phage-627
N
I
—
—
—
—
M
—
—
—
I
D
—
I
0.08
0.21
0.07
1456

Phage-628
F
D
—
W
—
D
W
E
—
L
—
S
—
Y
0.07
0.21
0.08
1457

Phage-629
Y
F
—
—
—
—
W
E
D
H
F
F
—
D
0.09
0.21
0.19
1458

Phage-630
T
—
—
—
—
—
—
E
—
D
S
Y
—
D
0.12
0.20
0.09
1459

Phage-631
N
L
—
—
—
—
V
E
L
I
D
I
—
S
0.11
0.20
0.09
1460

Phage-632
D
N
—
—
—
—
W
E
—
V
Y
L
—
N
0.08
0.20
0.08
1461

Phage-633
F
L
—
—
—
—
—
—
—
—
D
L
—
F
0.08
0.20
0.09
1462

Phage-634
H
I
—
—
—
—
Q
—
—
—
I
—
—
T
0.09
0.20
0.19
1463

Phage-635
F
D
—
W
—
D
W
E
D
N
S
Y
—
D
0.10
0.20
0.09
1464

Phage-636
T
A
—
—
—
—
W
E
F
D
F
N
—
D
0.08
0.20
0.07
1465

Phage-637
H
H
—
W
—
D
W
E
D
Y
S
T
—
P
0.10
0.20
0.11
1466

Phage-638
Y
—
—
—
—
—
—
—
—
—
—
N
—
F
0.07
0.20
0.08
1467

Phage-639
L
H
—
W
—
D
W
E
—
I
D
I
—
D
0.08
0.20
0.09
1468

Phage-640
D
I
—
—
—
D
G
Q
—
D
F
V
—
S
0.08
0.20
0.09
1469

Phage-641
D
V
—
W
—
D
W
E
V
N
Y
F
—
D
0.09
0.20
0.07
1470

Phage-642
—
N
—
—
—
—
M
—
—
—
I
D
—
A
0.12
0.20
0.10
1471

Phage-643
D
N
—
—
—
—
—
—
—
A
T
V
—
N
0.11
0.19
0.19
1472

Phage-644
D
L
—
—
—
—
—
E
—
V
H
N
—
N
0.08
0.19
0.08
1473

Phage-645
—
N
—
—
—
—
—
—
—
—
S
Y
—
F
0.13
0.19
0.09
1474

Phage-646
N
I
—
W
—
D
W
E
—
D
N
F
—
S
0.08
0.19
0.08
1475

Phage-647
F
V
—
—
—
—
W
E
V
Y
D
D
—
D
0.08
0.19
0.08
1476

Phage-648
A
—
—
—
—
—
L
E
V
V
H
L
—
V
0.10
0.19
0.17
1477

Phage-649
P
F
—
—
—
—
M
—
—
T
I
D
—
Y
0.07
0.19
0.09
1478

Phage-650
L
L
—
—
—
V
M
E
D
V
F
A
—
Y
0.08
0.19
0.08
1479

Phage-651
D
L
—
—
—
—
—
—
—
—
T
N
—
Y
0.07
0.19
0.08
1480

Phage-652
H
D
—
—
—
—
M
E
—
Y
Y
L
—
P
0.10
0.18
0.10
1481

Phage-653
T
D
—
—
—
—
Y
—
—
—
I
I
—
P
0.08
0.18
0.09
1482

Phage-654
—
L
—
W
—
D
W
E
D
Y
A
D
—
N
0.09
0.18
0.08
1483

Phage-655
N
D
—
—
—
—
L
—
—
—
L
T
—
D
0.07
0.18
0.09
1484

Phage-656
I
—
—
—
—
—
L
—
—
—
I
A
—
Y
0.11
0.18
0.08
1485

Phage-657
N
—
—
—
—
—
V
E
—
F
N
F
—
H
0.11
0.18
0.14
1486

Phage-658
D
V
—
—
—
—
I
E
—
Y
S
F
—
I
0.08
0.18
0.09
1487

Phage-659
D
L
—
—
—
—
V
E
—
I
T
D
—
A
0.10
0.18
0.12
1488

Phage-660
H
D
—
—
—
—
—
—
—
—
—
F
—
I
0.12
0.18
0.12
1489

Phage-661
P
L
—
—
—
V
L
E
—
D
I
Y
—
Y
0.10
0.18
0.13
1490

Phage-662
D
L
—
—
—
—
—
E
D
I
I
D
—
N
0.10
0.18
0.11
1491

Phage-663
D
—
—
—
—
—
V
E
V
P
S
N
—
N
0.10
0.18
0.18
1492

Phage-664
I
I
—
—
—
—
L
—
—
—
T
A
—
D
0.10
0.18
0.09
1493

Phage-665
D
H
—
—
—
—
—
—
—
—
—
N
—
D
0.10
0.18
0.14
1494

Phage-666
F
D
—
—
—
—
—
—
—
—
L
Y
—
S
0.07
0.18
0.07
1495

Phage-667
F
A
—
W
—
D
W
E
—
V
Y
I
—
Y
0.08
0.18
0.08
1496

Phage-668
L
—
—
—
—
—
—
—
—
—
L
D
—
S
0.08
0.18
0.09
1497

Phage-669
D
L
—
—
—
—
L
E
—
A
F
L
—
A
0.09
0.18
0.08
1498

Phage-670
F
A
—
—
—
—
L
—
—
T
L
T
—
L
0.10
0.18
0.08
1499

Phage-671
F
D
—
—
—
—
V
E
—
I
S
N
—
D
0.17
0.18
0.10
1500

Phage-672
—
H
—
—
—
—
L
E
Y
P
F
D
—
N
0.09
0.17
0.16
1501

Phage-673
A
—
—
—
—
—
—
E
—
H
T
T
—
N
0.10
0.17
0.15
1502

Phage-674
L
—
—
—
—
V
S
E
Q
F
T
F
—
I
0.08
0.17
0.08
1503

Phage-675
D
—
—
—
—
—
L
—
—
—
Y
D
—
N
0.10
0.17
0.09
1504

Phage-676
F
—
—
—
—
—
W
E
—
F
D
V
—
I
0.13
0.17
0.15
1505

Phage-677
F
T
—
—
—
—
V
E
—
Y
D
H
—
I
0.08
0.17
0.09
1506

Phage-678
Y
N
—
—
—
—
—
—
—
—
—
T
—
F
0.12
0.17
0.11
1507

Phage-679
A
V
—
—
—
—
N
—
—
—
N
S
—
A
0.08
0.17
0.08
1508

Phage-680
I
—
—
W
—
D
W
E
V
P
N
D
—
A
0.10
0.17
0.09
1509

Phage-681
Y
F
—
—
—
—
—
E
—
F
F
H
—
Y
0.12
0.17
0.12
1510

Phage-682
Y
V
—
—
—
D
G
—
—
—
S
F
—
D
0.12
0.17
0.12
1511

Phage-683
I
S
—
—
—
—
V
E
—
F
F
Y
—
Y
0.10
0.17
0.08
1512

Phage-684
L
I
—
—
—
V
—
E
—
D
—
Y
—
D
0.17
0.17
0.15
1513

Phage-685
—
—
—
—
—
—
V
E
D
H
N
Y
—
A
0.14
0.17
0.16
1514

Phage-686
L
D
—
—
—
—
—
E
F
V
Y
I
—
A
0.08
0.17
0.10
1515

Phage-687
Y
D
—
—
—
—
—
E
—
D
L
P
—
I
0.17
0.17
0.11
1516

Phage-688
D
V
—
—
—
—
V
E
—
D
Y
Y
—
D
0.10
0.17
0.14
1517

Phage-689
N
D
—
W
—
D
W
E
Y
D
N
V
—
V
0.08
0.17
0.10
1518

Phage-690
D
L
—
—
—
—
—
E
V
A
N
D
—
N
0.10
0.16
0.16
1519

Phage-691
H
D
—
—
—
—
L
—
—
—
I
S
—
N
0.09
0.16
0.07
1520

Phage-692
L
D
—
W
—
D
W
E
—
T
T
H
—
D
0.08
0.16
0.08
1521

Phage-693
I
I
—
—
—
—
V
E
—
D
D
Y
—
L
0.09
0.16
0.09
1522

Phage-694
Y
—
—
W
—
D
W
E
—
V
I
I
—
D
0.09
0.16
0.09
1523

Phage-695
F
D
—
—
—
—
I
—
—
Y
T
N
—
N
0.12
0.16
0.09
1524

Phage-696
I
T
—
—
—
—
L
—
—
T
I
N
—
D
0.08
0.16
0.11
1525

Phage-697
D
S
—
—
—
—
V
E
—
D
I
Y
—
I
0.07
0.16
0.08
1526

Phage-698
Y
L
—
—
—
—
—
—
—
—
G
N
—
H
0.07
0.16
0.08
1527

Phage-699
D
N
—
—
—
—
L
P
—
D
Y
F
—
D
0.08
0.16
0.10
1528

Phage-700
—
L
—
—
—
—
—
E
—
V
S
N
—
N
0.11
0.16
0.08
1529

Phage-701
—
D
—
W
—
D
W
E
—
D
I
V
—
D
0.10
0.16
0.09
1530

Phage-702
—
—
—
W
—
D
W
E
D
N
F
P
—
Y
0.07
0.16
0.07
1531

Phage-703
D
—
—
—
—
—
V
E
—
H
F
N
—
H
0.08
0.16
0.08
1532

Phage-704
A
D
—
—
—
—
I
E
—
D
A
Y
—
Y
0.12
0.16
0.09
1533

Phage-705
I
L
—
W
—
D
W
E
D
A
T
F
—
Y
0.09
0.16
0.07
1534

Phage-706
I
H
—
W
—
D
W
E
D
F
N
I
—
P
0.09
0.16
0.08
1535

Phage-707
T
I
—
—
—
—
V
E
D
Y
N
D
—
I
0.07
0.16
0.07
1536

Phage-708
D
D
—
—
—
—
L
—
—
—
—
A
—
I
0.08
0.16
0.08
1537

Phage-709
D
D
—
W
—
D
W
E
D
H
I
F
—
F
0.13
0.16
0.08
1538

Phage-710
—
N
—
—
—
—
V
E
—
I
I
F
—
D
0.12
0.15
0.12
1539

Phage-711
I
F
—
W
—
D
W
E
D
D
T
V
—
I
0.08
0.15
0.09
1540

Phage-712
I
I
—
—
—
—
—
E
—
I
S
D
—
L
0.12
0.15
0.15
1541

Phage-713
F
D
—
—
—
—
V
E
—
Y
N
D
—
D
0.11
0.15
0.09
1542

Phage-714
N
D
—
—
—
—
L
—
—
T
L
Y
—
I
0.08
0.15
0.10
1543

Phage-715
A
I
—
—
—
—
L
E
—
D
I
S
—
N
0.12
0.15
0.17
1544

Phage-716
H
L
—
—
—
—
—
—
—
—
T
N
—
Y
0.07
0.15
0.07
1545

Phage-717
S
—
—
—
—
—
L
—
—
—
—
A
—
I
0.10
0.15
0.11
1546

Phage-718
L
—
—
—
—
—
—
E
Q
L
A
D
—
T
0.08
0.15
0.08
1547

Phage-719
I
D
—
—
—
—
L
—
—
—
I
A
—
N
0.07
0.15
0.08
1548

Phage-720
F
D
—
—
—
D
G
Q
—
D
L
V
—
N
0.10
0.15
0.08
1549

Phage-721
N
L
—
—
—
—
—
E
—
F
F
D
—
Y
0.09
0.15
0.15
1550

Phage-722
S
I
—
—
—
—
L
Q
—
D
I
V
—
P
0.09
0.14
0.14
1551

Phage-723
N
P
—
—
—
—
Y
—
—
—
A
H
—
D
0.08
0.14
0.08
1552

Phage-724
Y
D
—
—
—
—
L
—
—
Y
Y
N
—
N
0.12
0.14
0.11
1553

Phage-725
—
D
—
—
—
—
L
—
—
T
I
F
—
D
0.07
0.14
0.08
1554

Phage-726
D
N
—
—
—
—
L
—
—
—
—
T
—
T
0.09
0.14
0.10
1555

Phage-727
—
D
—
—
—
—
L
—
—
—
S
Y
—
D
0.10
0.14
0.13
1556

Phage-728
Y
—
—
—
—
—
—
E
F
I
D
F
—
F
0.07
0.14
0.07
1557

Phage-729
Y
D
—
W
—
D
W
E
V
I
T
Y
—
N
0.08
0.14
0.09
1558

Phage-730
F
D
—
—
—
—
I
E
—
D
F
F
—
V
0.06
0.14
0.07
1559

Phage-731
I
F
—
W
—
D
W
—
D
I
N
F
—
D
0.10
0.14
0.14
1560

Phage-732
N
F
—
—
—
—
L
P
—
D
I
T
—
Y
0.37
0.14
0.09
1561

Phage-733
S
L
—
—
—
—
—
E
—
Y
Y
H
—
L
0.09
0.14
0.07
1562

Phage-734
A
S
—
—
—
—
L
—
—
—
L
D
—
L
0.12
0.14
0.13
1563

Phage-735
S
F
—
—
—
—
R
E
W
D
L
A
—
Y
0.09
0.14
0.08
1564

Phage-736
H
L
—
—
—
—
—
E
D
V
L
D
—
I
0.08
0.14
0.12
1565

Phage-737
L
D
—
—
—
D
G
—
—
F
Y
Y
—
L
0.20
0.14
0.09
1566

Phage-738
D
N
—
W
—
D
W
E
—
D
I
A
—
T
0.16
0.14
0.11
1567

Phage-739
S
D
—
—
—
—
L
—
—
T
I
H
—
I
0.09
0.14
0.08
1568

Phage-740
N
S
—
—
—
D
G
—
—
—
—
D
—
L
0.08
0.14
0.08
1569

Phage-741
D
L
—
—
—
—
L
—
—
—
T
L
—
I
0.07
0.14
0.08
1570

Phage-742
F
D
—
—
—
—
S
—
—
—
F
N
—
Y
0.09
0.14
0.11
1571

Phage-743
D
L
—
—
—
—
—
E
—
D
D
I
—
Y
0.12
0.14
0.13
1572

Phage-744
H
A
—
—
—
—
—
E
—
D
T
Y
—
F
0.10
0.14
0.14
1573

Phage-745
S
D
—
—
—
—
L
—
—
—
—
A
—
I
0.11
0.14
0.08
1574

Phage-746
I
V
—
—
—
—
L
P
—
D
Y
N
—
Y
0.09
0.13
0.11
1575

Phage-747
D
L
—
—
—
—
—
—
—
—
F
I
—
F
0.09
0.13
0.08
1576

Phage-748
Y
D
—
—
—
—
—
—
—
T
L
T
—
N
0.13
0.13
0.08
1577

Phage-749
Y
D
—
—
—
—
V
E
—
I
—
N
—
D
0.11
0.13
0.12
1578

Phage-750
—
F
—
—
—
—
I
E
D
D
H
V
—
I
0.07
0.13
0.07
1579

Phage-751
F
T
—
—
—
—
W
E
D
D
Y
A
—
S
0.12
0.13
0.12
1580

Phage-752
N
F
—
—
—
—
—
—
—
—
—
—
—
N
0.10
0.13
0.09
1581

Phage-753
N
L
—
—
—
—
V
E
—
I
L
I
—
D
0.07
0.13
0.07
1582

Phage-754
D
S
—
—
—
—
V
E
—
Y
D
L
—
N
0.11
0.13
0.08
1583

Phage-755
T
L
—
—
—
—
—
E
—
I
T
D
—
N
0.09
0.13
0.08
1584

Phage-756
T
V
—
—
—
K
M
E
M
N
S
T
—
D
0.09
0.13
0.09
1585

Phage-757
—
H
—
W
—
D
W
E
D
A
—
S
—
N
0.10
0.12
0.11
1586

Phage-758
D
L
—
—
—
D
G
N
—
L
D
F
—
F
0.08
0.12
0.08
1587

Phage-759
D
L
—
—
—
D
G
E
—
H
Y
Y
—
D
0.09
0.12
0.07
1588

Phage-760
F
N
—
—
—
—
V
E
—
I
L
L
—
T
0.11
0.12
0.12
1589

Phage-761
H
—
—
—
—
—
V
E
N
I
N
D
—
I
0.09
0.12
0.07
1590

Phage-762
I
D
—
—
—
—
L
—
—
—
—
T
—
D
0.09
0.12
0.08
1591

Phage-763
I
F
—
—
—
—
I
E
Q
P
A
L
—
Y
0.08
0.12
0.09
1592

Phage-764
Y
D
—
—
—
—
—
Q
—
D
L
V
—
P
0.10
0.12
0.08
1593

Phage-765
H
A
—
W
—
D
W
E
—
p
N
Y
—
D
0.13
0.12
0.09
1594

Phage-766
N
V
—
W
—
D
W
E
—
D
Y
N
—
Y
0.11
0.12
0.10
1595

Phage-767
S
F
—
—
Q
—
L
G
D
N
Y
D
—
I
0.09
0.12
0.12
1596

Phage-768
D
D
—
—
—
—
L
—
—
T
T
V
—
Y
0.08
0.12
0.09
1597

Phage-769
N
F
—
—
—
—
W
E
V
A
T
L
—
L
0.09
0.12
0.14
1598

Phage-770
D
L
—
—
—
—
V
E
—
D
T
Y
—
N
0.09
0.11
0.07
1599

Phage-771
A
L
—
—
—
—
V
E
Q
V
D
L
—
T
0.08
0.11
0.08
1600

Phage-772
D
D
—
—
—
—
L
—
—
—
—
N
—
N
0.08
0.11
0.08
1601

Phage-773
I
F
—
—
—
—
—
E
Q
I
I
Y
—
D
0.09
0.11
0.11
1602

Phage-774
S
D
—
W
—
D
W
E
—
V
Y
Y
—
S
0.09
0.11
0.10
1603

Phage-775
F
F
—
—
—
D
G
—
—
V
A
I
—
D
0.09
0.11
0.07
1604

Phage-776
D
D
—
W
—
D
W
E
D
D
—
Y
—
Y
0.11
0.11
0.07
1605

Phage-777
Y
D
—
—
—
—
—
—
—
T
—
V
—
P
0.08
0.11
0.08
1606

Phage-778
S
—
—
—
—
—
L
—
—
—
—
N
—
V
0.10
0.11
0.08
1607

Phage-779
A
F
—
V
S
F
Q
Q
S
L
P
H
—
D
0.09
0.11
0.10
1608

Phage-780
—
N
—
—
—
—
V
E
—
Y
F
V
—
F
0.09
0.11
0.09
1609

Phage-781
T
N
—
W
—
D
W
E
—
D
F
A
—
V
0.07
0.11
0.08
1610

Phage-782
D
D
—
—
—
—
—
E
—
I
I
L
—
F
0.08
0.10
0.08
1611

Phage-783
N
S
—
—
—
—
L
E
D
Y
H
L
—
P
0.08
0.10
0.10
1612

Phage-784
I
H
—
—
—
D
S
—
G
F
D
F
—
D
0.09
0.10
0.08
1613

Phage-785
F
D
—
—
—
—
L
E
—
D
H
L
—
F
0.08
0.10
0.08
1614

Phage-786
T
V
—
W
—
D
—
E
—
Y
A
D
—
D
0.10
0.10
0.08
1615

Phage-787
Y
F
—
W
—
D
W
E
—
A
A
D
—
L
0.09
0.10
0.09
1616

Phage-788
—
S
—
—
—
—
—
—
—
—
—
D
—
I
0.08
0.10
0.07
1617

Phage-789
A
V
—
—
—
—
L
P
—
D
I
V
—
Y
0.08
0.10
0.08
1618

Phage-790
—
L
—
—
—
—
V
E
—
Y
H
L
—
A
0.08
0.10
0.30
1619

Phage-791
S
L
—
W
—
D
W
E
—
V
D
N
—
F
0.12
0.10
0.11
1620

Phage-792
T
I
—
—
—
D
G
Q
—
D
Y
N
—
H
0.07
0.10
0.07
1621

Phage-793
F
L
—
—
—
—
G
E
P
T
Y
L
—
T
0.12
0.10
0.09
1622

Phage-794
D
D
—
W
L
—
Q
H
D
I
Y
V
—
A
0.10
0.10
0.08
1623

Phage-795
I
F
—
—
—
—
V
E
—
V
A
F
—
F
0.07
0.10
0.08
1624

Phage-796
A
L
—
—
—
—
V
E
D
D
Y
D
—
L
0.09
0.10
0.09
1625

Phage-797
D
D
—
—
—
—
I
E
L
Y
L
T
—
A
0.08
0.10
0.08
1626

Phage-798
T
D
—
W
—
D
W
E
D
D
S
I
—
D
0.07
0.10
0.07
1627

Phage-799
S
I
—
—
—
—
L
E
—
I
F
L
—
N
0.08
0.10
0.08
1628

Phage-800
N
D
—
—
—
—
L
E
—
D
I
L
—
F
0.07
0.10
0.09
1629

Phage-801
D
D
—
—
—
—
L
—
—
T
Y
S
—
Y
0.09
0.09
0.08
1630

Phage-802
F
V
—
—
—
—
W
E
—
I
D
L
—
I
0.10
0.09
0.09
1631

Phage-803
Y
D
—
—
—
—
L
—
—
—
L
S
—
P
0.07
0.09
0.07
1632

Phage-804
N
L
—
—
—
—
L
—
—
—
I
I
—
P
0.08
0.09
0.08
1633

Phage-805
I
D
—
W
—
D
W
E
—
F
N
N
—
F
0.08
0.09
0.09
1634

Phage-806
A
—
—
—
—
—
L
E
—
H
D
Y
—
Y
0.08
0.09
0.09
1635

Phage-807
D
D
—
S
—
Q
—
—
Q
I
D
L
—
D
0.14
0.09
0.09
1636

Phage-808
S
—
—
—
—
—
S
—
—
—
I
Y
—
Y
0.08
0.09
0.07
1637

Phage-809
S
L
—
—
—
—
—
Q
—
D
A
P
—
N
0.07
0.09
0.07
1638

Phage-810
D
A
—
—
—
—
L
—
—
T
T
H
—
D
0.09
0.09
0.08
1639

Phage-811
N
D
—
—
—
—
V
E
—
V
A
D
—
F
0.07
0.09
0.08
1640

Phage-812
Y
I
—
—
—
—
—
E
Q
D
Y
F
—
F
0.08
0.09
0.08
1641

Phage-813
T
D
—
—
—
D
G
—
—
T
N
Y
—
F
0.11
0.09
0.08
1642

Phage-814
Y
D
—
—
—
—
V
—
—
—
—
L
—
P
0.08
0.09
0.08
1643

Phage-815
I
D
—
—
—
—
—
—
—
—
A
Y
—
T
0.08
0.09
0.08
1644

Phage-816
F
D
—
—
—
—
—
E
—
F
F
H
—
Y
0.09
0.09
0.09
1645

Phage-817
F
P
—
—
—
—
I
E
—
Y
D
Y
—
V
0.09
0.09
0.08
1646

Phage-818
A
D
—
—
—
I
—
E
S
I
D
I
—
V
0.09
0.09
0.07
1647

Phage-819
I
S
—
—
—
D
L
W
P
T
D
I
—
T
0.10
0.09
0.10
1648

Phage-820
D
D
—
—
—
D
G
—
—
V
H
T
—
N
0.09
0.09
0.07
1649

Phage-821
I
D
—
W
—
D
W
E
G
—
F
A
—
N
0.09
0.09
0.08
1650

Phage-822
L
N
—
—
—
D
G
—
—
T
F
Y
—
D
0.08
0.08
0.07
1651

Phage-823
I
H
—
—
—
—
L
G
A
Y
I
S
—
S
0.08
0.08
0.09
1652

Phage-824
T
I
—
W
—
D
W
E
—
D
Y
F
—
Y
0.08
0.08
0.08
1653

Phage-825
H
S
—
L
A
Q
—
—
Q
D
L
V
—
I
0.07
0.08
0.07
1654

Phage-826
N
N
—
A
S
D
L
S
—
D
N
S
—
I
0.07
0.08
0.08
1655

Phage-827
—
N
—
W
—
D
W
E
—
D
—
A
—
N
0.09
0.08
0.07
1656

Phage-828
—
—
—
—
—
—
L
—
—
—
F
Y
—
F
0.08
0.08
0.07
1657

Phage-829
L
F
—
—
T
V
L
—
—
F
F
D
—
D
0.07
0.08
0.07
1658

Phage-830
F
D
—
—
—
—
L
—
—
—
—
S
—
A
0.08
0.08
0.07
1659

Phage-831
Y
—
—
—
—
—
V
E
—
N
—
Y
—
I
0.07
0.08
0.08
1660

Phage-832
D
—
—
—
—
—
L
—
D
—
T
I
—
H
0.12
0.08
0.10
1661

Phage-833
—
F
—
—
—
Q
W
A
—
A
N
A
—
F
0.09
0.08
0.07
1662

Phage-834
F
T
—
—
—
—
Y
—
—
—
I
T
—
P
0.09
0.08
0.09
1663

Phage-835
L
N
—
—
—
V
N
—
—
—
S
V
—
I
0.07
0.08
0.08
1664

Phage-836
A
I
—
W
—
D
W
E
—
F
S
D
—
H
0.07
0.08
0.07
1665

Phage-837
Y
—
—
—
V
D
L
G
A
N
—
Y
—
Y
0.10
0.08
0.09
1666

Phage-838
H
—
—
—
—
—
V
E
—
D
Y
H
—
D
0.07
0.08
0.07
1667

Phage-839
N
D
—
—
S
L
Q
Y
D
I
P
T
—
V
0.08
0.08
0.07
1668

Phage-840
Y
V
—
R
—
Q
L
—
V
Y
H
Y
—
N
0.15
0.08
0.07
1669

Phage-841
H
D
—
—
—
D
G
—
—
—
I
I
—
S
0.08
0.08
0.07
1670

Phage-842
F
D
—
—
—
—
L
—
—
T
I
I
—
P
0.08
0.08
0.08
1671

Phage-843
—
D
—
—
S
D
R
G
—
N
A
A
—
H
0.07
0.08
0.07
1672

Phage-844
N
I
—
L
A
Q
—
N
—
D
P
T
—
N
0.07
0.08
0.08
1673

Phage-845
T
N
—
—
S
K
S
Q
V
—
D
H
—
I
0.10
0.08
0.09
1674

Phage-846
N
H
—
H
—
Q
—
W
—
L
T
N
—
N
0.09
0.07
0.07
1675

Phage-847
L
L
—
H
—
Q
G
—
L
Y
H
L
—
H
0.09
0.07
0.08
1676

Phage-848
T
N
—
D
S
K
L
E
G
D
D
N
—
F
0.09
0.07
0.07
1677

Phage-849
N
D
—
—
—
—
M
—
—
—
L
L
—
D
0.08
0.07
0.07
1678

Phage-850
F
H
—
—
—
—
V
—
—
—
I
N
—
N
0.07
0.07
0.07
1679

Phage-851
D
F
—
—
—
D
G
—
—
T
Y
V
—
S
0.07
0.07
0.08
1680

Phage-852
—
S
—
—
—
Q
—
—
—
N
N
T
—
N
0.07
0.07
0.08
1681

Phage-853
—
—
—
H
L
—
S
E
Q
F
D
I
—
I
0.08
0.07
0.08
1682

Phage-854
D
D
—
—
—
—
W
E
F
V
F
F
—
D
0.08
0.07
0.08
1683

Phage-855
Y
N
—
E
Q
Q
Q
—
—
D
P
S
—
I
0.07
0.07
0.07
1684

Phage-856
N
T
—
—
T
—
Q
H
—
F
N
—
—
L
0.08
0.07
0.08
1685

Phage-857
H
P
—
Q
—
G
I
E
—
V
D
Y
—
V
0.08
0.07
0.08
1686

Phage-858
—
A
—
S
R
Q
L
G
—
D
A
Y
—
N
0.07
0.07
0.07
1687

Phage-859
D
I
—
—
A
Q
E
V
H
V
Y
T
—
P
0.07
0.07
0.07
1688

Phage-860
F
F
—
E
G
N
L
—
A
Y
L
L
—
L
0.08
0.07
0.08
1689

Phage-861
Y
—
—
—
—
D
G
E
—
N
I
V
—
D
0.07
0.07
0.07
1690

TABLE 32

Sequences of those peptides selected for

synthesis (CD3 scFv Peptide-B Optimization)

SEQ ID

Peptide-ID
Sequence
NO:

Peptide-AA
DDCWPDWEFDFACA
824

Peptide-AB
YICGLDFPDFLYCD
825

Peptide-AC
FDCWPDWEEYFVCD
826

Peptide-AD
YICWPDWEEYFDCD
827

Peptide-AE
NICWPDWEDDYFCF
828

Peptide-AF
NFCWPDWEYIYPCI
829

Peptide-AG
VDCWPDWEEDFLCI
830

Peptide-AH
HACWPDWEEYFPCN
831

Peptide-AI
YDCGPDVDESYVCV
832

Peptide-AJ
IDCWPDWEDDTFCY
833

Peptide-AK
YLCGPDGDETLACY
834

Peptide-AL
VDCGPDGDESILCY
835

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.

Number	Name	Date	Kind
6217866	Schlessinger et al.	Apr 2001	B1
7060808	Goldstein et al.	Jun 2006	B1
8557961	Silverman et al.	Oct 2013	B2
8658175	Dubridge et al.	Feb 2014	B2
9249211	Schellenberger et al.	Feb 2016	B2
9493563	Blein et al.	Nov 2016	B2
9540440	Lowman et al.	Jan 2017	B2
9708412	Baeuerle et al.	Jul 2017	B2
9916166	Aizawa	Mar 2018	B2
9976166	Schellenberger et al.	May 2018	B2
10118966	Qian	Nov 2018	B2
10533053	Lowman et al.	Jan 2020	B2
10870874	Schellenberger et al.	Dec 2020	B2
11180562	Lowman et al.	Nov 2021	B2
11512113	Campbell et al.	Nov 2022	B2
11649291	Griswold et al.	May 2023	B2
11773166	Ollier et al.	Oct 2023	B2
11851502	Blein et al.	Dec 2023	B2
11891454	Blein et al.	Feb 2024	B2
20040229310	Simmons	Nov 2004	A1
20150232557	Tan et al.	Aug 2015	A1
20160193332	Lowman et al.	Jul 2016	A1
20160289341	Wu	Oct 2016	A1
20170145115	Blein et al.	May 2017	A1
20180112011	Ollier	Apr 2018	A1
20180193477	Ng et al.	Jul 2018	A1
20180333507	Lowman et al.	Nov 2018	A1
20200102403	Ollier	Apr 2020	A1
20200354452	Qin et al.	Nov 2020	A1
20210047406	Irving et al.	Feb 2021	A1
20210164011	Schellenberger et al.	Jun 2021	A1
20220213227	Ollier	Jul 2022	A1
20220289853	Lowman et al.	Sep 2022	A1
20230061715	Schellenberger et al.	Mar 2023	A1
20230062624	Ollier et al.	Mar 2023	A1
20230220109	Campbell et al.	Jul 2023	A1

Number	Date	Country
0359282	Mar 1990	EP
WO-2012025525	Mar 2012	WO
WO-2020247871	Dec 2020	WO
WO-2022094299	May 2022	WO
WO-2022125562	Jun 2022	WO
WO-2022240637	Nov 2022	WO
WO-2022240865	Nov 2022	WO
WO-2024100451	May 2024	WO
WO-2024102723	May 2024	WO

	Number	Date	Country
	63327317	Apr 2022	US
	63187690	May 2021	US

Compositions and methods related to tumor activated antibodies targeting EGFR and effector cell antigens

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Date Filed

Date Issued

Inventors

Original Assignees

Examiners

Agents

CPC

Field of Search

CPC

International Classifications

Term Extension

Abstract

Description

Claims

CROSS-REFERENCE

PCT Information

US Referenced Citations (36)

Foreign Referenced Citations (9)

Non-Patent Literature Citations (55)

Related Publications (1)

Provisional Applications (2)