COMPOSITIONS AND METHODS RELATED TO TUMOR ACTIVATED ANTIBODIES TARGETING TROP2 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 Nov. 29, 2021, is named 52426-729_601_SL.txt and is 934,933 bytes in size.

SUMMARY

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

A₂-A₁-L₁-P₁-H₁ (Formula I)

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 TROP2. 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-CD3e single chain variable fragment. In some embodiments, A₁comprises an anti-CD3e 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 CD3ε. In some embodiments, the effector cell antigen comprises CD3, wherein 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: 1, HC-CDR2: SEQ ID NO: 2, and HC-CDR3: SEQ ID NO: 3; and 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: 4, LC-CDR2: SEQ ID NO:5, and LC-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 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: 7, HC-CDR2: SEQ ID NO: 8, and HC-CDR3: SEQ ID NO: 9; and 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: 10, LC-CDR2: SEQ ID NO: 11, and LC-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 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: 15, HC-CDR2: SEQ ID NO: 16, and HC-CDR3: SEQ ID NO: 17; and 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: 18, LC-CDR2: SEQ ID NO: 19, and LC-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, Fab heavy chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 22. 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 P₂-L₂-A₂-A₁-L₁-P₁-H₁(Formula Ia) 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 TROP2. 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 TROP2. 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: 26, 27, or 122. In some embodiments, P₂comprises an amino acid sequence according to any one of SEQ ID NOs: 23-25. 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(SEQ ID NO: 243), wherein n is an integer from 1 to 3. In some embodiments, L₁has a formula selected from the group consisting of (G₂S)_n, (GS)_n, (GSGGS)_n(SEQ ID NO: 62), (GGGS)_n(SEQ ID NO: 63), (GGGGS)_n(SEQ ID NO: 64), and (GSSGGS)_n(SEQ ID NO: 65), 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 TROP2. 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: 31 or 32. In some embodiments, L₁or L₂comprises an amino acid sequence according to SEQ ID NO: 58 or 59. In some embodiments, L₁or L₂comprises an amino acid sequence according to any one of SEQ ID NOs: 28-61. In some embodiments, L₁or L₂comprises an amino acid sequence of Linker 25 (ISSGLLSGRSDAG) (SEQ ID NO: 54), Linker 26 (AAGLLAPPGGLSGRSDAG) (SEQ ID NO: 55), Linker 27 (SPLGLSGRSDAG) (SEQ ID NO: 56), or Linker 28 (LSGRSDAGSPLGLAG) (SEQ ID NO: 57), 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 25, Linker 26, Linker 27, or Linker 28. 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 A10m3 or a fragment thereof. In some embodiments, the single domain antibody is DOM7r-31. In some embodiments, the single domain antibody is DOM7h-11-15. In some embodiments, the single domain antibody is Alb-1, Alb-8, or Alb-23. In some embodiments, the single domain antibody is 10E. In some embodiments, the single domain antibody comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the single domain antibody comprise: HC-CDR1: SEQ ID NO: 66, HC-CDR2: SEQ ID NO: 67, and HC-CDR3: SEQ ID NO: 68. 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: 70, HC-CDR2: SEQ ID NO: 71, and HC-CDR3: SEQ ID NO: 72. In some embodiments, the single domain antibody comprises an amino acid sequence according to SEQ ID NO: 73. 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: 62), (GGGS)_n(SEQ ID NO: 63), (GGGGS)_n(SEQ ID NO: 64), and (GSSGGS)_n(SEQ ID NO: 65), wherein n is an integer of at least 1. In some embodiments, L₃comprises an amino acid sequence according to SEQ ID NO: 30. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NOs: 74-121. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 82. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 83. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 74 and SEQ ID NO: 75. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 76 and SEQ ID NO: 77. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 78 and SEQ ID NO: 79. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 80 and SEQ ID NO: 81. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 82 and SEQ ID NO: 83. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 84 and SEQ ID NO: 85. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 86 and SEQ ID NO: 87. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 88 and SEQ ID NO: 89. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 90 and SEQ ID NO: 91. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 92 and SEQ ID NO: 93. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 94 and SEQ ID NO: 95. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 96 and SEQ ID NO: 97. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 98 and SEQ ID NO: 99. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 100 and SEQ ID NO: 101. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 102 and SEQ ID NO: 103. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 104 and SEQ ID NO: 105. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 106 and SEQ ID NO: 107. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 108 and SEQ ID NO: 109. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 110 and SEQ ID NO: 111. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 112 and SEQ ID NO: 113. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 114 and SEQ ID NO: 115. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 116 and SEQ ID NO: 117. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 118 and SEQ ID NO: 119. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 120 and SEQ ID NO: 121.

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

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

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

L_1a-P_1a-H_1a (Formula II)

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 TROP2; 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, P_1awhen 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: 26, 27, or 122. 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 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: 66, HC-CDR2: SEQ ID NO: 67, and HC-CDR3: SEQ ID NO: 68. 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: 70, HC-CDR2: SEQ ID NO: 71, and HC-CDR3: SEQ ID NO: 72. In some embodiments, the single domain antibody comprises an amino acid sequence according to SEQ ID NO: 73. In some embodiments, the single domain antibody is SA21. In some embodiments, H_1acomprises a linking moiety (L_1a) that connects H_1ato Pia. 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: 62), (GGGS)_n(SEQ ID NO: 63), (GGGGS)_n(SEQ ID NO: 64), and (GSSGGS)_n(SEQ ID NO: 65), wherein n is an integer of at least 1. In some embodiments, L_1acomprises an amino acid sequence according to any one of SEQ ID NOs: 28-61. Disclosed herein, in some embodiments, are polypeptide complexes comprising a structural arrangement according to Configuration 1:

wherein the polypeptide complex comprises a single chain variable fragment (scFv) comprising a light chain variable domain and a heavy chain variable domain, wherein the scFv further comprises a peptide that impairs binding of the scFv to an effector cell antigen and the peptide is linked to a N-terminus of the heavy chain variable domain of the scFv with a linking moiety that is a substrate for a tumor specific protease, and the peptide further comprises a half-life extending molecule; and a Fab that binds to TROP2, 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 further comprises P₂and L₂, wherein P₂comprises a peptide that impairs binding to TROP2; and L₂comprises a linking moiety that connects the Fab light chain polypeptide to P₂and is a substrate for a tumor specific protease. Disclosed herein, in some embodiments, are polypeptide complexes comprising a structural arrangement according to Configuration 2:

wherein the polypeptide complex comprises a single chain variable fragment (scFv) comprising a light chain variable domain and a heavy chain variable domain, wherein the scFv further comprises a peptide that impairs binding of the scFv to an effector cell antigen and the peptide is linked to a N-terminus of the heavy chain variable domain of the scFv with a linking moiety that is a substrate for a tumor specific protease, and the peptide further comprises a half-life extending molecule; and a Fab that binds to TROP2, 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 further comprises P₂and L₂, wherein P₂comprises a peptide that impairs binding to TROP2; and L₂comprises a linking moiety that connects the Fab heavy 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 illustrates polypeptide complexes of this disclosure in a normal orientation (FIG. 1A) and flipped orientation (FIG. 1B).

FIG. 2A illustrates titration data for TROP2 binding for several polypeptide complexes of this disclosure.

FIG. 2B illustrates titration data for TROP2 binding for several polypeptide complexes of this disclosure.

FIG. 3A illustrates titration data for CD3ε binding for several polypeptide complexes of this disclosure.

FIG. 3B illustrates titration data for CD3ε binding for several polypeptide complexes of this disclosure.

FIG. 4A illustrates cell viability data for HCT116 cells treated with polypeptide complexes of this disclosure.

FIG. 4B illustrates cell viability data for HCT116 cells treated with polypeptide complexes of this disclosure.

FIG. 4C illustrates cell viability data for MDAMB231 cells treated with polypeptide complexes of this disclosure.

FIG. 4D illustrates cell viability data for MDAMB231 cells treated with polypeptide complexes of this disclosure.

FIG. 5A illustrates titration data for TROP2 binding for several polypeptide complexes of this disclosure.

FIG. 5B illustrates titration data for TROP2 binding for several polypeptide complexes of this disclosure.

FIG. 6A illustrates titration data for CD3ε binding for several polypeptide complexes of this disclosure.

FIG. 6B illustrates titration data for CD3ε binding for several polypeptide complexes of this disclosure.

FIG. 7A illustrates cell viability data for HCT116 cells treated with polypeptide complexes of this disclosure.

FIG. 7B illustrates cell viability data for HCT116 cells treated with polypeptide complexes of this disclosure.

FIGS. 8A-8C illustrate polypeptide complex mediated H292 tumor cell killing in the presence of CD8+ T cells.

FIGS. 9A-9D illustrate polypeptide complex mediated HCT116 tumor cell killing in the presence of CD8+ T cells.

FIGS. 10A-10C illustrate polypeptide complex mediated MDAMB231 tumor cell killing in the presence of CD8+ T cells.

FIG. 11A illustrates polypeptide complex PC1 pharmacokinetics in cynomolgus monkeys after a single IV bolus injection.

FIG. 11B illustrates polypeptide complex PC5 pharmacokinetics in cynomolgus monkeys after a single IV bolus injection.

FIG. 11C illustrates polypeptide complex PC18 pharmacokinetics in cynomolgus monkeys after a single IV bolus injection.

FIG. 11D illustrates polypeptide complex PC21 pharmacokinetics in cynomolgus monkeys after a single IV bolus injection.

FIG. 12A illustrates cytokine release in cynomolgus monkeys after single IV bolus of PC1.

FIG. 12B illustrates cytokine release in cynomolgus monkeys after single IV bolus of polypeptide complex PC5.

FIG. 12C illustrates cytokine release in cynomolgus monkeys after single IV bolus of polypeptide complex PC18.

FIG. 12D illustrates cytokine release in cynomolgus monkeys after single IV bolus of polypeptide complex PC21.

FIG. 12E illustrates plasma cytokine level after administration of polypeptide complexes (unmasked TROP2-TCE and masked TROP2-TRACTr) described herein.

FIG. 13A illustrates serum liver enzymes in cynomolgus monkeys after single IV bolus of PC1.

FIG. 13B illustrates serum liver enzymes in cynomolgus monkeys after single IV bolus of polypeptide complex PC5.

FIG. 13C illustrates serum liver enzymes in cynomolgus monkeys after single IV bolus of polypeptide complex PC18.

FIG. 13D illustrates serum liver enzymes in cynomolgus monkeys after single IV bolus of polypeptide complex PC21.

FIG. 14A and FIG. 14B illustrate TROP2 Fab inhibition by alanine scanning peptides of TROP2 Fab Peptide-1 as measured by ELISA.

FIG. 15A and FIG. 15B illustrate TROP2 Fab binding by alanine scanning peptides of TROP2 Fab Peptide-2 as measured by ELISA.

FIG. 16A and FIG. 16B illustrate TROP2 Fab inhibition by alanine scanning peptides of TROP2 Fab Peptide-2 as measured by ELISA.

FIG. 17A-FIG. 17C illustrate optimized TROP2 Fab Peptide-1 sequences evaluated for peptide inhibition of TROP2 Fab.

FIG. 18 illustrates the core sequence motif of optimized TROP2 Fab Peptide-1 sequences generated using WebLogo 3.7.4.

FIG. 19A-FIG. 19C illustrate optimized TROP2 Fab Peptide-2 sequences evaluated for peptide binding to TROP2 Fab.

FIG. 20A-FIG. 20C illustrate optimized TROP2 Fab Peptide-2 sequences evaluated for peptide inhibition of TROP2 Fab.

FIG. 21 illustrates the core sequence motif of optimized TROP2 Fab Peptide-2 sequences generated using WebLogo 3.7.4.

FIG. 22 illustrates titration data for TROP2 binding for several polypeptide complexes of this disclosure.

FIG. 23 illustrates titration data for CD3ε binding for several polypeptide complexes of this disclosure.

FIG. 24A illustrates PC25 mediated HCT116 tumor cell killing in the presence of CD8+ T cells.

FIG. 24B illustrates PC26 mediated HCT116 tumor cell killing in the presence of CD8+ T cells.

FIG. 24C illustrates PC25 mediated MDAMB231 tumor cell killing in the presence of CD8+ T cells.

FIG. 25 illustrates PC22 pharmacokinetics in cynomolgus monkeys after a single IV bolus injection.

FIGS. 26A-26F illustrate cytokine release in cynomolgus monkeys after single IV bolus of PC22.

FIGS. 27A-27B illustrate serum liver enzyme levels in cynomolgus monkeys after single IV bolus of PC22.

FIGS. 28A-28D illustrate in vivo tumor growth inhibition in human PBMC engrafted NCG mice bearing MDAMB231 xenograft tumors. The anti-tumor activity observed was protease dependent in that the polypeptide complex lacking the protease substrate within the cleavable linker was equivalent to vehicle controls. Shown are PC3 (FIG. 28A), PC17 (FIG. 28B), PC23 (FIG. 28C), PC24 (FIG. 28D).

FIGS. 29A-29F illustrate anti-CD3 scFv binding by alanine scanning peptides of anti-CD3 scFv Peptide-A and Peptide-B as measured by ELISA.

FIGS. 30A-30F 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. 31A-31B illustrate anti-CD3 scFv binding by optimized anti-CD3 scFv Peptide-B sequences as measured by ELISA.

FIGS. 32A-32B illustrate inhibition of anti-CD3 scFv binding to CD3 by optimized anti-CD3 scFv Peptide-B sequences as measured by ELISA.

FIG. 33 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 TROP2. TROP2 (also known as tumor-associated calcium signal transducer 2 or epithelial glycoprotein-1) is the protein product of the TACSTD2 gene, and is a transmembrane glycoprotein that functions in variety of cell signaling pathways, many of which are associated with tumorigenesis. TROP2 has been shown to be overexpressed in multiple human carcinomas, including lung, breast, cervical, ovarian, colorectal, pancreatic, and gastric cancers, and its expression has been correlated with poor patient prognosis. Furthermore, TROP2 functions as an oncogene capable of driving both tumorigenesis and metastasis in epithelial cancers such as colorectal cancer. TROP2 expression in cancer cells has long been correlated with drug resistance, and high levels of TROP2 expression have been shown to correlate with poor prognosis in a variety of cancer types. In a meta-analysis, including data from approximately 2,500 patients, increased TROP2 expression was associated with poor overall survival and disease-free survival outcomes across several solid tumors.

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 isolated polypeptide or polypeptide complexes that comprise binding domains that selectively bind to an effector cell antigen and TROP2, 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 isolated polypeptide or polypeptide complexes described herein have activity at low levels of target expression, and are easily manufactured.

In some embodiments, the isolated polypeptides or polypeptide complexes described herein are used in a method of treating cancer. In some embodiments, the cancer has cells that express TROP2. In some instances, the cancer is a solid tumor cancer. In some embodiments, the cancer is lung, breast (e.g. HER2+; ER/PR+; TNBC), cervical, ovarian, colorectal, pancreatic or gastric. In some embodiments, the polypeptides or polypeptide complexes described herein are used in a method of treating triple-negative breast cancer (TNBC), urothelial cancer (UC), non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), gastric cancer, esophageal cancer, head and neck cancer, prostate cancer, or endometrial cancer. In some embodiments, the polypeptides or polypeptide complexes described herein are used in a method of treating breast cancer, lung cancer, urothelial cancer, endometrial cancer, ovarian cancer, prostate cancer, pancreatic cancer, gastric cancer, colon cancer, head and neck cancer, and glioma. 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:

A₂-A₁-L₁-P₁-H₁ (Formula I)

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

A₂-A₁-L₁-P₁-H₁ (Formula I)

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

A₂-A₁-L₁-P₁-H₁ (Formula I)

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 TROP2.

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

A₂-A₁-L₁-P₁-H₁ (Formula I)

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

A₂-A₁-L₁-P₁-H₁ (Formula I)

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

A₂-A₁-L₁-P₁-H₁ (Formula I)

wherein: A₁comprises a first antigen recognizing molecule that binds to TROP2; 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 isolated polypeptides or polypeptide complexes according to Formula I:

A₂-A₁-L₁-P₁-H₁ (Formula I)

wherein: A₁is a first antigen recognizing molecule that binds to TROP2; 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 isolated polypeptides or polypeptide complexes comprising Formula I:

A₂-A₁-L₁-P₁-H₁ (Formula I)

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

A₂-A₁-L₁-P₁-H₁ (Formula I)

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

P₂-L₂-A₂-A₁-L₁-P₁-H₁ (Formula Ia)

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 isolated polypeptides or polypeptide complexes according to Formula Ia:

P₂-L₂-A₂-A₁-L₁-P₁-H₁ (Formula Ia)

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 isolated polypeptides or polypeptide complexes comprising Formula Ia:

P₂-L₂-A₂-A₁-L₁-P₁-H₁ (Formula Ia)

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

P₂-L₂-A₂-A₁-L₁-P₁-H₁ (Formula Ia)

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 isolated polypeptides or polypeptide complexes according to Formula II:

L_1a-P_1a-H_1a (Formula II)

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

L_1a-P_1a-H_1a (Formula II)

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

L_1a-P_1a-H_1a (Formula II)

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 TROP2; 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 isolated polypeptides or polypeptide complexes comprising Formula II:

L_1a-P_1a-H_1a (Formula II)

First Antigen Recognizing Molecule (A₁)

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 TROP2. 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-CD3e single chain variable fragment. In some embodiments, A₁comprises an anti-CD3e 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 TROP2. In some embodiments, A₁comprises a variable light chain and variable heavy chain each of which is capable of specifically binding to human TROP2.

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: HC: CDR1
GFTFNKYA
1

SP34.185 CD3: HC: CDR2
IRSKYNNYAT
2

SP34.185 CD3: HC: CDR3
VRHGNFGNSYISYWAY
3

SP34.185 CD3: LC: CDR1
TGAVTSGNY
4

SP34.185 CD3: LC: CDR2
GTK
5

SP34.185 CD3: LC: CDR3
VLWYSNRWV
6

SP34.194 CD3: HC: CDR1
GFTFNTYA
7

SP34.194 CD3: HC: CDR2
IRSKYNNYAT
8

SP34.194 CD3: HC: CDR3
VRHGNFGNSYVSWFAY
9

SP34.194 CD3: LC: CDR1
TGAVTTSNY
10

SP34.194 CD3: LC: CDR2
GT
11

SP34.194 CD3: LC: CDR3
ALWYSNLWV
12

SP34.185 scFv
EVQLVESGGGLVQPGGSLKLSCA
13

(VH-linker 1-VL)
ASGFTFNKYAMNWVRQAPGKG

LEWVARIRSKYNNYATYYADSV

KDRFTISRDDSKNTAYLQMNNLK

TEDTAVYYCVRHGNFGNSYISY

WAY
WGQGTLVTVSSGGGGSGGG

GSGGGGSQTVVTQEPSLTVSPGG

TVTLTCGSSTGAVTSGNYPNWV

QQKPGQAPRGLIGGTKFLAPGTP

ARFSGSLLGGKAALTLSGVQPED

EAEYYCVLWYSNRWVFGGGTKL

TVL

SP34.194 scFv
QTVVTQEPSLTVSPGGTVTLTCRS
14

(VL-linker 1-VH)
STGAVTTSNYANWVQQKPGQAP

RGLIGGTNKRAPGTPARFSGSLLG

GKAALTLSGVQPEDEAEYYCAL

WYSNLWV
FGGGTKLTVLGGGGS

GGGGSGGGGSEVQLVESGGGLV

QPGGSLKLSCAASGFTFNTYAMN

WVRQAPGKGLEWVARIRSKYNN

YAT
YYADSVKDRFTISRDDSKNT

AYLQMNNLKTEDTAVYYCVRHG

NFGNSYVSWFAY
WGQGTLVTVS

S

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: 1; HC-CDR2: SEQ ID NO: 2; HC-CDR3: SEQ ID NO: 3, 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: 7; HC-CDR2: SEQ ID NO: 8; HC-CDR3: SEQ ID NO: 9, 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 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-CDR11: SEQ ID NO: 4; LC-CDR2: SEQ ID NO: 5; and LC-CDR3: SEQ ID NO: 6, 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: 10; LC-CDR2: SEQ ID NO: 11; and LC-CDR3: SEQ ID NO: 12, 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 effector cell antigen comprises CD3, wherein 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: 1, HC-CDR2: SEQ ID NO: 2, and HC-CDR3: SEQ ID NO: 3; 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: 4, LC-CDR2: SEQ ID NO:5, and LC-CDR3: SEQ ID NO: 6.

In some embodiments, the effector cell antigen comprises CD3, wherein 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: 7, HC-CDR2: SEQ ID NO: 8, and HC-CDR3: SEQ ID NO: 9; 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: 10, LC-CDR2: SEQ ID NO: 11, and LC-CDR3: SEQ ID NO: 12.

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 CD3ε. In some embodiments, the effector cell antigen comprises CD3, wherein 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: 1, HC-CDR2: SEQ ID NO: 2, and HC-CDR3: SEQ ID NO: 3; and 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: 4, LC-CDR2: SEQ ID NO:5, and LC-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 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: 7, HC-CDR2: SEQ ID NO: 8, and HC-CDR3: SEQ ID NO: 9; and 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: 10, LC-CDR2: SEQ ID NO: 11, and LC-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, 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 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 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 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 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-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 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-H₁(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. 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-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 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.

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 Pi, 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 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 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 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.

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 TROP2 binding domain.

In some embodiments, the antigen binding fragment (Fab) or Fab′ that binds to TROP2 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-TROP2 amino acid sequences (CDRs as determined by IMGT numbering system)

Amino Acid Sequence
SEQ ID

Construct Description
(N to C)
NO:

TROP2: HC: CDR1
GYTFTNYG
15

TROP2: HC: CDR2
INTYTGEP
16

TROP2: HC: CDR3
ARGGFGSSYWYFDV
17

TROP2: LC: CDR1
QDVSIA
18

TROP2: LC: CDR2
SAS
19

TROP2: LC: CDR3
QQHYITPLT
20

TROP2 Fab LC
DIQLTQSPSSLSASVGDRVSITCKA
21

SQDVSIAVAWYQQKPGKAPKLLI

YSASYRYTGVPDRFSGSGSGTDFT

LTISSLQPEDFAVYYCQQHYITPL

T
FGAGTKVEIKRTVAAPSVFIFPPS

DEQLKSGTASVVCLLNNFYPREA

KVQWKVDNALQSGNSQESVTEQ

DSKDSTYSLSSTLTLSKADYEKHK

VYACEVTHQGLSSPVTKSFNRGE

C

TROP2 Fab HC
QVQLQQSGSELKKPGASVKVSCK
22

ASGYTFTNYGMNWVKQAPGQG

LKWMGWINTYTGEPTYTDDFKG

RFAFSLDTSVSTAYLQISSLKADD

TAVYFCARGGFGSSYWYFDVW

GQGSLVTVSSASTKGPSVFPLAPS

SKSTSGGTAALGCLVKDYFPEPV

TVSWNSGALTSGVHTFPAVLQSS

GLYSLSSVVTVPSSSLGTQTYICN

VNHKPSNTKVDKKVEPKSC

In some embodiments, 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-CDR11: SEQ ID NO: 15, HC-CDR2: SEQ ID NO: 16, and HC-CDR3: SEQ ID NO: 17; and 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: 18, LC-CDR2: SEQ ID NO: 19, and LC-CDR3: SEQ ID NO: 20. In some embodiments, 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: 15, HC-CDR2: SEQ ID NO: 16, and HC-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: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the Fab comprise LC-CDR11: SEQ ID NO: 18, LC-CDR2: SEQ ID NO: 19, and LC-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 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: 15, HC-CDR2: SEQ ID NO: 16, and HC-CDR3: SEQ ID NO: 17; and 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: 18, LC-CDR2: SEQ ID NO: 19, and LC-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 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 heavy chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 22. In some embodiments, the Fab heavy 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 heavy 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 heavy 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 heavy 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 heavy 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 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.

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

SEQ

Amino Acid
ID

Construct Description
Sequence (N to C)
NO:

TROP2 Fab Peptide-1
SVLFCVKNLYCWVT
23

TROP2 Fab Peptide-2
VDFCKIYSWPVCHQ
24

TROP2 Fab Peptide-3
IDFCMLYNWPICAG
25

SP34.185 scFv Peptide-A
GSQCLGPEWEVCPY
26

SP34.185 scFv Peptide-B
VYCGPEFDESVGCM
27

SP34.194 scFv Peptide-AM
GYLWGCEWNCAGITT
122

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 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: 26 or 27. In some embodiments, P₁comprises the amino acid sequence of SEQ ID NO: 26. In some embodiments, P₁comprises the amino acid sequence of SEQ ID NO: 27. In some embodiments, P₁comprises the amino acid sequence of SEQ ID NO: 122.

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; 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, 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; Z₁is 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; Z₁is 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 SEQ ID NOs: 202-228.

In some embodiments, P₁comprises an amino acid sequences according to any of the sequences of Table 35.

In some embodiments, P₁comprises the amino acid sequences according to SEQ ID NOs: 229-240.

In some embodiments, P₁comprises the amino acid sequence according to SEQ ID NO: 239 or a peptide sequence that has 1, 2, or 3, amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 239.

In some embodiments, P₁comprises the amino acid sequence according to SEQ ID NO: 27 or a peptide sequence that has 1, 2, or 3, amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 27.

In some embodiments, P₁comprises the amino acid sequence according to SEQ ID NO: 26 or a peptide sequence that has 1, 2, or 3, amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 26.

In some embodiments, P₁comprises the amino acid sequence according to SEQ ID NO: 239.

In some embodiments, P₁comprises the amino acid sequence according to SEQ ID NO: 27.

In some embodiments, P₁comprises the amino acid sequence according to SEQ ID NO: 26.

In some embodiments, P₂impairs binding of A₂to a second target antigen. In some embodiments, wherein P₂impairs binding of A₂to TROP2. 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 TROP2. 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 TROP2. In some embodiments, P₂has less than 75% sequence identity to TROP2. In some embodiments, P₂has less than 80% sequence identity to TROP2. In some embodiments, P₂has less than 85% sequence identity to the TROP2. In some embodiments, P₂has less than 90% sequence identity to TROP2. In some embodiments, P₂has less than 95% sequence identity to TROP2. In some embodiments, P₂has less than 98% sequence identity to TROP2. In some embodiments, P₂has less than 99% sequence identity to the TROP2. In some embodiments, P₂comprises a de novo amino acid sequence that shares less than 10% sequence identity to TROP2. In some embodiments, P₂comprises an amino acid sequence according to any one of SEQ ID NOs: 23-25. In some embodiments, P₂comprises the amino acid sequence of SEQ ID NO: 23. In some embodiments, P₂comprises the amino acid sequence of SEQ ID NO: 24. In some embodiments, P₂comprising the amino acid sequence of SEQ ID NO: 25.

In some embodiments, P₂comprises an amino acid sequence according to X₁-X₂-X₃-X₄-C-X₆-X₇-X₈-X₉-X₁₀-C-X₁₂-X₁₃-X₁₄and X₁is selected from N, D, S, Y, A, F, H, T, L, and V; X₂is selected from S, T, D, A, H, V, Y, N, F, I, and L; X₃is selected from L, I, and V; X₄is selected from F, L, V, M, W, I, Y, and H; X₆is selected from V, F, L, I, and W; X₇is selected from K, R, Q, N, H, and M; X₈is selected from N and K; X₉is selected from L, V, and I; X₁₀is selected from Y, W, F, Q, and L; X₁₂is selected from W and V; X₁₃is selected from I, N, H, T, V, Y, and D; X₁₄is selected from D, V, A, S, I, T, N, Y, H, and P. In some embodiments, X₁is selected from N, D, S, Y, A, F, and T; X₂is selected from S, T, D, A, H, V, Y, and N; X₃is L; X₄is selected from F, L, V, M, and W; X₆is selected from V, F, and L; X₇is selected from K, R, Q, and N; X₈is selected from N and K; X₉is selected from L, V, and I; X₁₀is selected from Y, W, and F; X₁₂is W; X₁₃is selected from I, N, H, and T; and X₁₄is selected from D, V, A, S, I, T, and N. In some embodiments, X₁is selected from N, D, S, and Y; X₂is selected from S, T, and D; X₃is L; X₄is selected from F, L, and V; X₆is selected from V and F; X₇is selected from K, R, and Q; X₈is N; X₉is selected from L and V; X₁₀is selected from Y and W; X₁₂is W; X₁₃is selected from I, N, and H; X₁₄is selected from D, V, A, and S.

In some embodiments, P₂comprises an amino acid sequence according to an amino acid sequence according to J₁-J₂-J₃-C-J₅-J₆-J₇-J₈-W-J₁₀-J₁₁-C-J₁₃-J₁₄and J₁is selected V, I, L, P, E, F, and M; J₂is selected from D and N; J₃is selected from F and W; J₅is selected from A, E, S, R, K, Y, L, Q, G, M, F, T, W, and D; J₆is selected from L, M, I, V, F, T, R, and S; J₇is selected from Y, F, and N; J₈is selected from N, R, D, H, K, Q, S, G, A, E, and M; J₁₀is selected from P and R; J₁₁is selected from V and I; J₁₃is selected from D, G, N, R, S, Q, Y, T, A, E, L, V, K, M, I, H, F, and W; and J₁₄is selected from T, S, Q, L, D, N, A, E, K, M, V, R, I, H, P, V, and W. In some embodiments, J₁is selected from V, I, and L; J₂is D; J₃is F; J₅is selected from A, E, S, R, K, Y, and L; J₆is selected from L, M, and I; J₇is Y; J₈is selected from N, R, D, H, K, Q, S, and G; J₁₀is P; J₁is selected from V and I; J₁₃is selected from D, G, N, R, S, Q, Y, T, and A; and J₁₄is selected from T, S, Q, L, D, N, A, and E. In some embodiments, J₁is selected from V, I, and L; J₂is D; J₃is F; J₅is selected from A, E, S, and R; J₆is selected from L, M, and I; J₇is Y; J₈is selected from N, R, and D; J₁₀is P; J₁₁is selected from V and I; J₁₃is selected from D, G, N, R, S, and Q; and J₁₄is selected from T, S, Q, and L.

In some embodiments, P₂comprises an amino acid sequence according to an amino acid sequence according to B₁-B₂-B₃-C-B₅-B₆-B₇-B₈-W-B₁₀-B₁₁-C-B₁₃-B₁₄and B₁is selected from V, I, L, P, E, F, and M; B₂is selected from D and N; B₃is selected from F and W; B₅is selected from A, E, S, R, K, Y, L, Q, G, M, F, T, W, and D; B₆is selected from L, M, I, V, F, T, R, and S; B₇is selected from Y, F, and N; B₈is selected from N, R, D, H, K, Q, S, G, A, E, and M; B₁₀is selected from P and R; B₁₁is selected from V and I; B₁₃is selected from D, G, N, R, S, Q, Y, T, A, E, L, V, K, M, I, H, F, and W; and B₁₄is selected from T, S, Q, L, D, N, A, E, K, M, V, R, I, H, P, V, G, and W. In some embodiments, B₁is selected from V, I, and L; B₂is D; B₃is F; B₅is selected from A, E, S, R, K, Y, M, G, and L; B₆is selected from L, M, and I; B₇is Y; B₈is selected from N, R, D, H, K, Q, S, and G; B₁₀is P; B₁₁is selected from V and I; B₁₃is selected from D, G, N, R, S, Q, Y, T, H, and A; and B₁₄is selected from T, S, Q, L, D, N, A, G, and E. In some embodiments, B₁is selected from V, I, and L; B₂is D; B₃is F; B₈is selected from A, E, S, K, M, G, and R; B₆is selected from L, M, and I; B₇is Y; B₈is selected from N, R, S, H, and D; B₁₀is P; B₁₁is selected from V and I; B₁₃is selected from D, G, N, R, S, H, A, Y, and Q; B₁₄is selected from T, S, Q, G, and L.

In some embodiments, P₂comprises the amino acid sequences according to SEQ ID NOs: 133-145.

In some embodiments, P₂comprises the amino acid sequences according to SEQ ID NOs: 146-158.

In some embodiments, P₂comprises an amino acid sequence according to any of the sequences of Table 27.

In some embodiments, P₂comprises the amino acid sequences according to SEQ ID NOs: 159-178.

In some embodiments, P₂comprises an amino acid sequence according to any of the sequences of Table 29.

In some embodiments, P₂comprises the amino acid sequences according to SEQ ID NOs: 179-201.

In some embodiments, P₂comprises the amino acid sequence according to SEQ ID NO: 24 or a peptide sequence that has 1, 2, or 3, amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 24.

In some embodiments, P₂comprises the amino acid sequence according to SEQ ID NO: 181 or a peptide sequence that has 1, 2, or 3, amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 181.

In some embodiments, P₂comprises the amino acid sequence according to SEQ ID NO: 186 or a peptide sequence that has 1, 2, or 3, amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 186.

In some embodiments, P₂comprises the amino acid sequence according to SEQ ID NO: 24.

In some embodiments, P₂comprises the amino acid sequence according to SEQ ID NO: 181.

In some embodiments, P₂comprises the amino acid sequence according to SEQ ID NO: 186.

In some embodiments, P_1awhen 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 identity to the effector cell antigen. In some embodiments, P_1ahas less than 75% sequence identity to the effector cell antigen. In some embodiments, P_1ahas less than 80% sequence identity to the effector cell antigen. In some embodiments, P_1ahas less than 85% sequence identity to the effector cell antigen. In some embodiments, P_1ahas less than 90% sequence identity to the effector cell antigen. In some embodiments, P_1ahas less than 95% sequence identity to the effector cell antigen. In some embodiments, P_1ahas less than 98% sequence identity to the effector cell antigen. In some embodiments, P_1ahas less than 99% sequence identity to the effector cell antigen. In some embodiments, P_1acomprises a de novo amino acid sequence that shares less than 10% sequence identity to the second effector cell antigen. In some embodiments, P_1acomprises an amino acid sequence according to SEQ ID NO: 26 or 27 or 122.

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 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. 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; Z₁is 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; Z₁is 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, 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. 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 SEQ ID NOs: 202-228.

In some embodiments, P_1acomprises an amino acid sequence according to any of the sequences of Table 35.

In some embodiments, P_1acomprises the amino acid sequences according to SEQ ID NOs: 229-240.

In some embodiments, P_1acomprises the amino acid sequence according to SEQ ID NO: 239 or a peptide sequence that has 1, 2, or 3, amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 239.

In some embodiments, P_1acomprises the amino acid sequence according to SEQ ID NO: 27 or a peptide sequence that has 1, 2, or 3, amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 27.

In some embodiments, P_1acomprises the amino acid sequence according to SEQ ID NO: 26 or a peptide sequence that has 1, 2, or 3, amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 26.

In some embodiments, P_1acomprises the amino acid sequence according to SEQ ID NO: 239.

In some embodiments, P_1acomprises the amino acid sequence according to SEQ ID NO: 27.

In some embodiments, P_1acomprises the amino acid sequence according to SEQ ID NO: 26.

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(SEQ ID NO: 243), wherein n is an integer from 1 to 3. 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)_n, (GSGGS)_n(SEQ ID NO: 62), (GGGS)_n(SEQ ID NO: 63), (GGGGS)_n(SEQ ID NO: 64), and (GSSGGS)_n(SEQ ID NO: 65), 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 cleavable amino acid sequence, a legumain cleavable amino acid sequence, or a matrix metalloprotease cleavable amino acid sequence. 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, 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, L₁, comprises the sequence of Linker-25 (SEQ ID NO: 54). In some embodiments, L₁, comprises the sequence of Linker-26 (SEQ ID NO: 55). In some embodiments, L₁, comprises the sequence of Linker-27 (SEQ ID NO: 56). In some embodiments, L₁, comprises the sequence of Linker-28 (SEQ ID NO: 57).

In some embodiments, L₂, comprises the sequence of Linker-25 (SEQ ID NO: 54). In some embodiments, L₂, comprises the sequence of Linker-26 (SEQ ID NO: 55). In some embodiments, L₂, comprises the sequence of Linker-27 (SEQ ID NO: 56). In some embodiments, L₂, comprises the sequence of Linker-28 (SEQ ID NO: 57).

TABLE 4

L₁, L₂, L₃, and L_1a Sequences

SEQ

Construct
Amino Acid Sequence
ID

Description
(N to C)
NO:

Linker 1
GGGGSGGGGSGGGGS
28

Linker 2
GGGGS
29

Linker 3
GGGGSGGGS
30

Cleavable
GGGGSGGGLSGRSDAGSPLGL
31

linker 1
AGSGGGS

Cleavable
GGGGSGGLSGRSDAGSPLGLA
32

linker 2
GSGGS

Linker 4
GGGGSLSGRSDNHGSSGT
33

Linker 5
GGGGSSGGSGGSGLSGRSDNH
34

GSSGT

Linker 6
ASGRSDNH
35

Linker 7
LAGRSDNH
36

Linker 8
ISSGLASGRSDNH
37

Linker 9
ISSGLLAGRSDNH
38

Linker 10
LSGRSDNH
39

Linker 11
ISSGLLSGRSDNP
40

Linker 12
ISSGLLSGRSDNH
41

Linker 13
LSGRSDNHSPLGLAGS
42

Linker 14
SPLGLAGSLSGRSDNH
43

Linker 15
SPLGLSGRSDNH
44

Linker 16
LAGRSDNHSPLGLAGS
45

Linker 17
LSGRSDNHVPLSLKMG
46

Linker 18
LSGRSDNHVPLSLSMG
47

Linker 19
GSSGGSGGSGGSGISSGLLSGR
48

SDNHGSSGT

Linker 20
GSSGGSGGSGGISSGLLSGRSD
49

NHGGGS

Linker 21
ASGRSDNH
50

Linker 22
LAGRSDNH
51

Linker 23
ISSGLASGRSDNH
52

Linker 24
LSGRSDAG
53

Linker 25
ISSGLLSGRSDAG
54

Linker 26
AAGLLAPPGGLSGRSDAG
55

Linker 27
SPLGLSGRSDAG
56

Linker 28
LSGRSDAGSPLGLAG
57

Cleavable
GGGGSSGGSAAGLLAPPGGLS
58

linker 3
GRSDAGGGGS

Cleavable
GSSGGSAAGLLAPPGGLSGRS
59

linker 4
DAGGGGS

Non-cleavable
GGGGSGGGGGSGGGGSGGAS
60

linker 1
SGAGGSGGS

Non-cleavable
GGGGSGGGSGGGGSGGASSG
61

linker 2
AGGSGGGS

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 TROP2.

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 A₁binding 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, the half-life extending molecule (H₁or H_1a) does not have binding affinity to the first antigen recognizing molecule. In some embodiments, the half-life extending molecule (H₁or H_1a) does not have binding affinity to the effector cell antigen. In some embodiments, the half-life extending molecule (H₁or H_1a) does not shield the first antigen recognizing molecule from the effector cell antigen. In some embodiments, the half-life extending molecule (H₁or H_1a) is not directly linked to the first 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

Construct Description
(N to C)
SEQ ID NO:

Anti-Albumin: CDR-H1
GSTFYTAV
66

Anti-Albumin: CDR-H2
IRWTALTT
67

Anti-Albumin: CDR-H3
AARGTLGLFTTADSYDY
68

Anti-albumin
EVQLVESGGGLVQPGGSLRLSCAASGSTF
69

YTAV
MGWVRQAPGKGLEWVAAIRWTA

LTT
SYADSVKGRFTISRDGAKTTLYLQM

NSLRPEDTAVYYCAARGTLGLFTTADSY

DY
WGQGTLVTVSS

10G Anti-Albumin: CDR-H1
GFTFSKFG
70

10G Anti-Albumin: CDR-H2
ISGSGRDT
71

10G Anti-Albumin: CDR-H3
TIGGSLSV
72

10G Anti-albumin
EVQLVESGGGLVQPGNSLRLSCAASGFT
73

FSKFG
MSWVRQAPGKGLEWVSSISGSGR

DT
LYADSVKGRFTISRDNAKTTLYLQMN

SLRPEDTAVYYCTIGGSLSVSSQGTLVTV

SS

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 Gamier-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, 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 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: 66, HC-CDR2: SEQ ID NO: 67, and HC-CDR3: SEQ ID NO: 68. 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: 66, HC-CDR2: SEQ ID NO: 67, and HC-CDR3: SEQ ID NO: 68; 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: 70, HC-CDR2: SEQ ID NO: 71, and HC-CDR3: SEQ ID NO: 72. 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: 70, HC-CDR2: SEQ ID NO: 71, and HC-CDR3: SEQ ID NO: 72; 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: 69. In some embodiments, H₁comprises an amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 69. In some embodiments, H₁comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 69. In some embodiments, H₁comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 69. In some embodiments, H₁comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 69. In some embodiments, H₁comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NO: 69.

In some embodiments, H₁comprises an amino acid sequence according to SEQ ID NO: 73. In some embodiments, H₁comprises an amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 73. In some embodiments, H₁comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 73. In some embodiments, H₁comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 73. In some embodiments, H₁comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 73. In some embodiments, H₁comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NO: 73.

In some embodiments, H_1acomprises an amino acid sequence according to SEQ ID NO: 69. In some embodiments, H_1acomprises an amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 69. In some embodiments, H_1acomprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 69. In some embodiments, H_1acomprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 69. In some embodiments, H_1acomprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 69. In some embodiments, H_1acomprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NO: 69.

In some embodiments, H_1acomprises an amino acid sequence according to SEQ ID NO: 73. In some embodiments, H_1acomprises an amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 73. In some embodiments, H_1acomprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 73. In some embodiments, H_1acomprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 73. In some embodiments, H_1acomprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 73. In some embodiments, H_1acomprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NO: 73.

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_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 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: 62), (GGGS)_n(SEQ ID NO: 63), (GGGGS)_n(SEQ ID NO: 64), and (GSSGGS)_n(SEQ ID NO: 65), wherein n is an integer of at least 1. In some embodiments, L₃comprises an amino acid sequence according to SEQ ID NO: 30.

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: 62), (GGGS)_n(SEQ ID NO: 63), (GGGGS)_n(SEQ ID NO: 64), and (GSSGGS)_n(SEQ ID NO: 65), wherein n is an integer of at least 1. In some embodiments, L_1acomprises an amino acid sequence according to any one of SEQ ID NOs: 28-61.

Antibodies that Bind to TROP2 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: 74-132, and 241-242. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 82. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 83.

TABLE 6

Polypeptide complex sequences

Amino Acid Sequence

Construct Description
(N to C)
SEQ ID NO:

PC1: LC
DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVA
74

TROP2 Fab LC
WYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGS

GTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAG

TKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL

NNFYPREAKVQWKVDNALQSGNSQESVTEQDS

KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL

SSPVTKSFNRGEC

PC1: HC
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNY
75

SP34.194 scFv (VH-
ANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGS

linker 1-VL) + Linker
LLGGKAALTLSGVQPEDEAEYYCALWYSNLWV

2 + TROP2 Fab HC
FGGGTKLTVLGGGGSGGGGSGGGGSEVQLVES

GGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQ

APGKGLEWVARIRSKYNNYATYYADSVKDRFT

ISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGN

FGNSYVSWFAY
WGQGTLVTVSSGGGGSQVQLQ

QSGSELKKPGASVKVSCKASGYTFTNYGMNWV

KQAPGQGLKWMGWINTYTGEPTYTDDFKGRF

AFSLDTSVSTAYLQISSLKADDTAVYFCARGGF

GSSYWYFDV
WGQGSLVTVSSASTKGPSVFPLAP

SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL

TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT

YICNVNHKPSNTKVDKKVEPKSC

PC2: LC
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNY
76

SP34.194 scFv (VH-
ANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGS

linker 1-VL) + Linker
LLGGKAALTLSGVQPEDEAEYYCALWYSNLWV

2 + TROP2 Fab LC
FGGGTKLTVLGGGGSGGGGSGGGGSEVQLVES

GGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQ

APGKGLEWVARIRSKYNNYATYYADSVKDRFT

ISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGN

FGNSYVSWFAY
WGQGTLVTVSSGGGGSDIQLT

QSPSSLSASVGDRVSITCKASQDVSIAVAWYQQ

KPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFT

LTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEI

KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY

PREAKVQWKVDNALQSGNSQESVTEQDSKDST

YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV

TKSFNRGEC

PC2: HC
QVQLQQSGSELKKPGASVKVSCKASGYTFTNY
77

TROP2 Fab HC

G
MNWVKQAPGQGLKWMGWINTYTGEPTYTD

DFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFC

ARGGFGSSYWYFDV
WGQGSLVTVSSASTKGPS

VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW

NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS

LGTQTYICNVNHKPSNTKVDKKVEPKSC

PC3: LC
DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVA
78

TROP2 Fab LC
WYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGS

GTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAG

TKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL

NNFYPREAKVQWKVDNALQSGNSQESVTEQDS

KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL

SSPVTKSFNRGEC

PC3: HC
EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYA
79

SP34.185 scFv (VH-
MNWVRQAPGKGLEWVARIRSKYNNYATYYAD

linker 1-VL) + Linker
SVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYY

2 + TROP2 Fab HC
CVRHGNFGNSYISYWAYWGQGTLVTVSSGGG

GSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLT

CGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGT

K
FLAPGTPARFSGSLLGGKAALTLSGVQPEDEAE

YYCVLWYSNRWVFGGGTKLTVLGGGGSQVQL

QQSGSELKKPGASVKVSCKASGYTFTNYGMNW

VKQAPGQGLKWMGWINTYTGEPTYTDDFKGR

FAFSLDTSVSTAYLQISSLKADDTAVYFCARGGF

GSSYWYFDV
WGQGSLVTVSSASTKGPSVFPLAP

SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL

TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT

YICNVNHKPSNTKVDKKVEPKSC

PC4: LC
EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYA
80

SP34.185 scFv (VH-
MNWVRQAPGKGLEWVARIRSKYNNYATYYAD

linker 1-VL) + Linker
SVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYY

2 + TROP2 Fab LC
CVRHGNFGNSYISYWAYWGQGTLVTVSSGGG

GSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLT

CGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGT

K
FLAPGTPARFSGSLLGGKAALTLSGVQPEDEAE

YYCVLWYSNRWVFGGGTKLTVLGGGGSDIQLT

QSPSSLSASVGDRVSITCKASQDVSIAVAWYQQ

KPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFT

LTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEI

KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY

PREAKVQWKVDNALQSGNSQESVTEQDSKDST

YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV

TKSFNRGEC

PC4: HC
QVQLQQSGSELKKPGASVKVSCKASGYTFTNY
81

TROP2 Fab HC

G
MNWVKQAPGQGLKWMGWINTYTGEPTYTD

DFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFC

ARGGFGSSYWYFDV
WGQGSLVTVSSASTKGPS

VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW

NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS

LGTQTYICNVNHKPSNTKVDKKVEPKSC

PC5: LC
GGVDFCKIYSWPVCHQGGGGSGGLSGRSDAGSP
82

TROP2 Fab mask
LGLAGSGGSDIQLTQSPSSLSASVGDRVSITCKAS

(SEQ ID NO: 24) +

QDVSIA
VAWYQQKPGKAPKLLIYSASYRYTGVP

cleavable linker 2 +
DRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYI

TROP2 Fab LC

TPLT
FGAGTKVEIKRTVAAPSVFIFPPSDEQLKSG

TASVVCLLNNFYPREAKVQWKVDNALQSGNSQ

ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA

CEVTHQGLSSPVTKSFNRGEC

PC5: HC
EVQLVESGGGLVQPGGSLRLSCAASGSTFYTAV
83

Anti-albumin (SEQ ID
MGWVRQAPGKGLEWVAAIRWTALTTSYADSV

NO: 69) + Linker 3 +
KGRFTISRDGAKTTLYLQMNSLRPEDTAVYYCA

SP34.185 scFv

ARGTLGLFTTADSYDY
WGQGTLVTVSSGGGGS

mask (SEQ ID NO: 27) +
GGGSGGVYCGPEFDESVGCMGGGGSGGGLSGR

Cleavable Linker 1 +
SDAGSPLGLAGSGGGSEVQLVESGGGLVQPGGS

SP34.185 scFv (V-
LKLSCAASGFTFNKYAMNWVRQAPGKGLEWV

linker 1-VL) + Linker
ARIRSKYNNYATYYADSVKDRFTISRDDSKNTA

2 + TROP2 Fab HC
YLQMNNLKTEDTAVYYCVRHGNFGNSYISYW

AY
WGQGTLVTVSSGGGGSGGGGSGGGGSQTVV

TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNW

VQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLG

GKAALTLSGVQPEDEAEYYCVLWYSNRWVFG

GGTKLTVLGGGGSQVQLQQSGSELKKPGASVK

VSCKASGYTFTNYGMNWVKQAPGQGLKWMG

WINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQI

SSLKADDTAVYFCARGGFGSSYWYFDVWGQG

SLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL

VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG

LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD

KKVEPKSC

PC6: LC
GGSVLFCVKNLYCWVTGGGGSGGLSGRSDAGS
84

TROP2 Fab mask
PLGLAGSGGSDIQLTQSPSSLSASVGDRVSITCKA

(SEQ ID NO: 23) +
SQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGV

cleavable linker 2 +
PDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHY

TROP2 Fab LC

ITPLT
FGAGTKVEIKRTVAAPSVFIFPPSDEQLKS

GTASVVCLLNNFYPREAKVQWKVDNALQSGNS

QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY

ACEVTHQGLSSPVTKSFNRGEC

PC6: HC
EVQLVESGGGLVQPGGSLRLSCAASGSTFYTAV
85

Anti-albumin (SEQ ID
MGWVRQAPGKGLEWVAAIRWTALTTSYADSV

NO: 69) + Linker 3 +
KGRFTISRDGAKTTLYLQMNSLRPEDTAVYYCA

SP34.185 scFv

ARGTLGLFTTADSYDY
WGQGTLVTVSSGGGGS

mask (SEQ ID NO: 27) +
GGGSGGVYCGPEFDESVGCMGGGGSGGGLSGR

Cleavable Linker 1 +
SDAGSPLGLAGSGGGSEVQLVESGGGLVQPGGS

SP34.185 scFv (VH-
LKLSCAASGFTFNKYAMNWVRQAPGKGLEWV

linker 1-VL) + Linker
ARIRSKYNNYATYYADSVKDRFTISRDDSKNTA

2 + TROP2 Fab HC
YLQMNNLKTEDTAVYYCVRHGNFGNSYISYW

AY
WGQGTLVTVSSGGGGSGGGGSGGGGSQTVV

TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNW

VQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLG

GKAALTLSGVQPEDEAEYYCVLWYSNRWVFG

GGTKLTVLGGGGSQVQLQQSGSELKKPGASVK

VSCKASGYTFTNYGMNWVKQAPGQGLKWMG

WINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQI

SSLKADDTAVYFCARGGFGSSYWYFDVWGQG

SLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL

VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG

LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD

KKVEPKSC

PC7: LC
EVQLVESGGGLVQPGGSLRLSCAASGSTFYTAV
86

Anti-albumin (SEQ ID
MGWVRQAPGKGLEWVAAIRWTALTTSYADSV

NO: 69) + Linker 3 +
KGRFTISRDGAKTTLYLQMNSLRPEDTAVYYCA

SP34.185 scFv

ARGTLGLFTTADSYDY
WGQGTLVTVSSGGGGS

mask (SEQ ID NO: 27) +
GGGSGGVYCGPEFDESVGCMGGGGSGGGLSGR

Cleavable linker 1 +
SDAGSPLGLAGSGGGSEVQLVESGGGLVQPGGS

SP34.185 scFv (VH-
LKLSCAASGFTFNKYAMNWVRQAPGKGLEWV

linker 1-VL) + Linker
ARIRSKYNNYATYYADSVKDRFTISRDDSKNTA

2 + TROP2 Fab LC
YLQMNNLKTEDTAVYYCVRHGNFGNSYISYW

AY
WGQGTLVTVSSGGGGSGGGGSGGGGSQTVV

TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNW

VQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLG

GKAALTLSGVQPEDEAEYYCVLWYSNRWVFG

GGTKLTVLGGGGSDIQLTQSPSSLSASVGDRVSI

TCKASQDVSIAVAWYQQKPGKAPKLLIYSASYR

YTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYC

QQHYITPLT
FGAGTKVEIKRTVAAPSVFIFPPSD

EQLKSGTASVVCLLNNFYPREAKVQWKVDNAL

QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK

HKVYACEVTHQGLSSPVTKSFNRGEC

PC7: HC
GGVDFCKIYSWPVCHQGGGGSGGLSGRSDAGSP
87

TROP2 Fab mask
LGLAGSGGSQVQLQQSGSELKKPGASVKVSCKA

(SEQ ID NO: 24) +
SGYTFTNYGMNWVKQAPGQGLKWMGWINTY

cleavable linker 2 +

TGEP
TYTDDFKGRFAFSLDTSVSTAYLQISSLKA

TROP2 Fab HC
DDTAVYFCARGGFGSSYWYFDVWGQGSLVTV

SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY

FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS

SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP

KSC

PC8: LC
EVQLVESGGGLVQPGGSLRLSCAASGSTFYTAV
88

Anti-albumin (SEQ ID
MGWVRQAPGKGLEWVAAIRWTALTTSYADSV

NO: 69) + Linker 3 +
KGRFTISRDGAKTTLYLQMNSLRPEDTAVYYCA

SP34.185 scFv

ARGTLGLFTTADSYDY
WGQGTLVTVSSGGGGS

mask (SEQ ID NO: 27) +
GGGSGGVYCGPEFDESVGCMGGGGSGGGLSGR

Cleavable linker 1 +
SDAGSPLGLAGSGGGSEVQLVESGGGLVQPGGS

SP34.185 scFv (VH-
LKLSCAASGFTFNKYAMNWVRQAPGKGLEWV

linker 1-VL) + Linker
ARIRSKYNNYATYYADSVKDRFTISRDDSKNTA

2 + TROP2 Fab LC
YLQMNNLKTEDTAVYYCVRHGNFGNSYISYW

AY
WGQGTLVTVSSGGGGSGGGGSGGGGSQTVV

TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNW

VQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLG

GKAALTLSGVQPEDEAEYYCVLWYSNRWVFG

GGTKLTVLGGGGSDIQLTQSPSSLSASVGDRVSI

TCKASQDVSIAVAWYQQKPGKAPKLLIYSASYR

YTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYC

QQHYITPLT
FGAGTKVEIKRTVAAPSVFIFPPSD

EQLKSGTASVVCLLNNFYPREAKVQWKVDNAL

QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK

HKVYACEVTHQGLSSPVTKSFNRGEC

PC8: HC
GGSVLFCVKNLYCWVTGGGGSGGLSGRSDAGS
89

TROP2 Fab mask
PLGLAGSGGSQVQLQQSGSELKKPGASVKVSCK

(SEQ ID NO: 23) +
ASGYTFTNYGMNWVKQAPGQGLKWMGWINT

cleavable linker 2 +

YTGEP
TYTDDFKGRFAFSLDTSVSTAYLQISSLK

TROP2 Fab HC
ADDTAVYFCARGGFGSSYWYFDVWGQGSLVT

VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD

YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE

PKSC

PC9: LC
GGVDFCKIYSWPVCHQGGGGSGGLSGRSDAGSP
90

TROP2 Fab mask
LGLAGSGGSDIQLTQSPSSLSASVGDRVSITCKAS

(SEQ ID NO: 24) +

QDVSIA
VAWYQQKPGKAPKLLIYSASYRYTGVP

cleavable linker 2 +
DRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYI

TROP2 Fab LC

TPLT
FGAGTKVEIKRTVAAPSVFIFPPSDEQLKSG

TASVVCLLNNFYPREAKVQWKVDNALQSGNSQ

ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA

CEVTHQGLSSPVTKSFNRGEC

PC9: HC
EVQLVESGGGLVQPGGSLRLSCAASGSTFYTAV
91

Anti-albumin (SEQ ID
MGWVRQAPGKGLEWVAAIRWTALTTSYADSV

NO: 69) + Linker 3 +
KGRFTISRDGAKTTLYLQMNSLRPEDTAVYYCA

SP34.185 scFv

ARGTLGLFTTADSYDY
WGQGTLVTVSSGGGGS

mask (SEQ ID NO: 26) +
GGGSGGGSQCLGPEWEVCPYGGGGSGGGLSGR

Cleavable linker 1 +
SDAGSPLGLAGSGGGSEVQLVESGGGLVQPGGS

SP34.185 scFv (VH-
LKLSCAASGFTFNKYAMNWVRQAPGKGLEWV

linker 1-VL) + Linker
ARIRSKYNNYATYYADSVKDRFTISRDDSKNTA

2 + TROP2 Fab HC
YLQMNNLKTEDTAVYYCVRHGNFGNSYISYW

AY
WGQGTLVTVSSGGGGSGGGGSGGGGSQTVV

TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNW

VQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLG

GKAALTLSGVQPEDEAEYYCVLWYSNRWVFG

GGTKLTVLGGGGSQVQLQQSGSELKKPGASVK

VSCKASGYTFTNYGMNWVKQAPGQGLKWMG

WINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQI

SSLKADDTAVYFCARGGFGSSYWYFDVWGQG

SLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL

VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG

LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD

KKVEPKSC

PC10: LC
EVQLVESGGGLVQPGGSLRLSCAASGSTFYTAV
92

Anti-albumin (SEQ ID
MGWVRQAPGKGLEWVAAIRWTALTTSYADSV

NO: 69) + Linker 3 +
KGRFTISRDGAKTTLYLQMNSLRPEDTAVYYCA

SP34.185 scFv

ARGTLGLFTTADSYDY
WGQGTLVTVSSGGGGS

mask (SEQ ID NO: 26) +
GGGSGGGSQCLGPEWEVCPYGGGGSGGGLSGR

Cleavable linker 1 +
SDAGSPLGLAGSGGGSEVQLVESGGGLVQPGGS

SP34.185 scFv (VH-
LKLSCAASGFTFNKYAMNWVRQAPGKGLEWV

linker 1-VL) + Linker
ARIRSKYNNYATYYADSVKDRFTISRDDSKNTA

2 + TROP2 Fab LC
YLQMNNLKTEDTAVYYCVRHGNFGNSYISYW

AY
WGQGTLVTVSSGGGGSGGGGSGGGGSQTVV

TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNW

VQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLG

GKAALTLSGVQPEDEAEYYCVLWYSNRWVFG

GGTKLTVLGGGGSDIQLTQSPSSLSASVGDRVSI

TCKASQDVSIAVAWYQQKPGKAPKLLIYSASYR

YTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYC

QQHYITPLT
FGAGTKVEIKRTVAAPSVFIFPPSD

EQLKSGTASVVCLLNNFYPREAKVQWKVDNAL

QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK

HKVYACEVTHQGLSSPVTKSFNRGEC

PC10: HC
GGVDFCKIYSWPVCHQGGGGSGGLSGRSDAGSP
93

TROP2 Fab mask
LGLAGSGGSQVQLQQSGSELKKPGASVKVSCKA

(SEQ ID NO: 24) +
SGYTFTNYGMNWVKQAPGQGLKWMGWINTY

cleavable linker 2 +

TGEP
TYTDDFKGRFAFSLDTSVSTAYLQISSLKA

TROP2 Fab HC
DDTAVYFCARGGFGSSYWYFDVWGQGSLVTV

SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY

FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS

SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP

KSC

PC11: LC
GGSVLFCVKNLYCWVTGGGGSGGLSGRSDAGS
94

TROP2 Fab mask
PLGLAGSGGSDIQLTQSPSSLSASVGDRVSITCKA

(SEQ ID NO: 23) +
SQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGV

Cleavable linker 2 +
PDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHY

TROP2 Fab LC

ITPLT
FGAGTKVEIKRTVAAPSVFIFPPSDEQLKS

GTASVVCLLNNFYPREAKVQWKVDNALQSGNS

QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY

ACEVTHQGLSSPVTKSFNRGEC

PC11: HC
EVQLVESGGGLVQPGGSLRLSCAASGSTFYTAV
95

Anti-albumin (SEQ ID
MGWVRQAPGKGLEWVAAIRWTALTTSYADSV

NO: 69) + Linker 3 +
KGRFTISRDGAKTTLYLQMNSLRPEDTAVYYCA

SP34.185 scFv

ARGTLGLFTTADSYDY
WGQGTLVTVSSGGGGS

mask (SEQ ID NO: 26) +
GGGSGGGSQCLGPEWEVCPYGGGGSGGGLSGR

Cleavable linker 1 +
SDAGSPLGLAGSGGGSEVQLVESGGGLVQPGGS

SP34.185 scFv (VH-
LKLSCAASGFTFNKYAMNWVRQAPGKGLEWV

linker 1-VL) + Linker
ARIRSKYNNYATYYADSVKDRFTISRDDSKNTA

2 + TROP2 Fab HC
YLQMNNLKTEDTAVYYCVRHGNFGNSYISYW

AY
WGQGTLVTVSSGGGGSGGGGSGGGGSQTVV

TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNW

VQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLG

GKAALTLSGVQPEDEAEYYCVLWYSNRWVFG

GGTKLTVLGGGGSQVQLQQSGSELKKPGASVK

VSCKASGYTFTNYGMNWVKQAPGQGLKWMG

WINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQI

SSLKADDTAVYFCARGGFGSSYWYFDVWGQG

SLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL

VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG

LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD

KKVEPKSC

PC12: LC
EVQLVESGGGLVQPGGSLRLSCAASGSTFYTAV
96

Anti-albumin (SEQ ID
MGWVRQAPGKGLEWVAAIRWTALTTSYADSV

NO: 69) + Linker 3 +
KGRFTISRDGAKTTLYLQMNSLRPEDTAVYYCA

SP34.185 scFv

ARGTLGLFTTADSYDY
WGQGTLVTVSSGGGGS

mask (SEQ ID NO: 26) +
GGGSGGGSQCLGPEWEVCPYGGGGSGGGLSGR

Cleavable linker 1 +
SDAGSPLGLAGSGGGSEVQLVESGGGLVQPGGS

SP34.185 scFv (VH-
LKLSCAASGFTFNKYAMNWVRQAPGKGLEWV

linker 1-VL) + Linker
ARIRSKYNNYATYYADSVKDRFTISRDDSKNTA

2 + TROP2 Fab LC
YLQMNNLKTEDTAVYYCVRHGNFGNSYISYW

AY
WGQGTLVTVSSGGGGSGGGGSGGGGSQTVV

TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNW

VQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLG

GKAALTLSGVQPEDEAEYYCVLWYSNRWVFG

GGTKLTVLGGGGSDIQLTQSPSSLSASVGDRVSI

TCKASQDVSIAVAWYQQKPGKAPKLLIYSASYR

YTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYC

QQHYITPLT
FGAGTKVEIKRTVAAPSVFIFPPSD

EQLKSGTASVVCLLNNFYPREAKVQWKVDNAL

QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK

HKVYACEVTHQGLSSPVTKSFNRGEC

PC12: HC
GGSVLFCVKNLYCWVTGGGGSGGLSGRSDAGS
97

TROP2 Fab mask
PLGLAGSGGSQVQLQQSGSELKKPGASVKVSCK

(SEQ ID NO: 23) +
ASGYTFTNYGMNWVKQAPGQGLKWMGWINT

cleavable linker 2 +

YTGEP
TYTDDFKGRFAFSLDTSVSTAYLQISSLK

TROP2 Fab HC
ADDTAVYFCARGGFGSSYWYFDVWGQGSLVT

VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD

YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE

PKSC

PC13: LC
GGVDFCKIYSWPVCHQGGGGSGGLSGRSDAGSP
98

TROP2 Fab mask
LGLAGSGGSDIQLTQSPSSLSASVGDRVSITCKAS

(SEQ ID NO: 24) +

QDVSIA
VAWYQQKPGKAPKLLIYSASYRYTGVP

cleavable linker 2 +
DRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYI

TROP2 Fab LC

TPLT
FGAGTKVEIKRTVAAPSVFIFPPSDEQLKSG

TASVVCLLNNFYPREAKVQWKVDNALQSGNSQ

ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA

CEVTHQGLSSPVTKSFNRGEC

PC13: HC
EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFG
99

10G + Linker 3 +
MSWVRQAPGKGLEWVSSISGSGRDTLYADSVK

SP34.185 scFv
GRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTI

mask (SEQ ID NO: 27) +

GGSLSV
SSQGTLVTVSSGGGGSGGGSGGVYCGP

Cleavable linker 1 +
EFDESVGCMGGGGSGGGLSGRSDAGSPLGLAGS

SP34.185 scFv (VH-
GGGSEVQLVESGGGLVQPGGSLKLSCAASGFTF

linker 1-VL) + Linker

NKYA
MNWVRQAPGKGLEWVARIRSKYNNYAT

2 + TROP2 Fab HC
YYADSVKDRFTISRDDSKNTAYLQMNNLKTEDT

AVYYCVRHGNFGNSYISYWAYWGQGTLVTVS

SGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGT

VTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLI

GGTKFLAPGTPARFSGSLLGGKAALTLSGVQPE

DEAEYYCVLWYSNRWVFGGGTKLTVLGGGGS

QVQLQQSGSELKKPGASVKVSCKASGYTFTNY

G
MNWVKQAPGQGLKWMGWINTYTGEPTYTD

DFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFC

ARGGFGSSYWYFDV
WGQGSLVTVSSASTKGPS

VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW

NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS

LGTQTYICNVNHKPSNTKVDKKVEPKSC

PC14: LC
GGSVLFCVKNLYCWVTGGGGSGGLSGRSDAGS
100

TROP2 Fab mask
PLGLAGSGGSDIQLTQSPSSLSASVGDRVSITCKA

(SEQ ID NO: 23) +
SQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGV

cleavable linker 2 +
PDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHY

TROP2 Fab LC

ITPLT
FGAGTKVEIKRTVAAPSVFIFPPSDEQLKS

GTASVVCLLNNFYPREAKVQWKVDNALQSGNS

QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY

ACEVTHQGLSSPVTKSFNRGEC

PC14: HC
EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFG
101

10G + Linker 3 +
MSWVRQAPGKGLEWVSSISGSGRDTLYADSVK

SP34.185 scFv
GRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTI

mask (SEQ ID NO: 27) +

GGSLSV
SSQGTLVTVSSGGGGSGGGSGGVYCGP

Cleavable linker 1 +
EFDESVGCMGGGGSGGGLSGRSDAGSPLGLAGS

SP34.185 scFv (VH -
GGGSEVQLVESGGGLVQPGGSLKLSCAASGFTF

linker 1 - VL) + Linker

NKYA
MNWVRQAPGKGLEWVARIRSKYNNYAT

2 + TROP2 Fab HC
YYADSVKDRFTISRDDSKNTAYLQMNNLKTEDT

AVYYCVRHGNFGNSYISYWAYWGQGTLVTVS

SGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGT

VTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLI

GGTKFLAPGTPARFSGSLLGGKAALTLSGVQPE

DEAEYYCVLWYSNRWVFGGGTKLTVLGGGGS

QVQLQQSGSELKKPGASVKVSCKASGYTFTNY

G
MNWVKQAPGQGLKWMGWINTYTGEPTYTD

DFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFC

ARGGFGSSYWYFDV
WGQGSLVTVSSASTKGPS

VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW

NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS

LGTQTYICNVNHKPSNTKVDKKVEPKSC

PC15: LC
EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFG
102

10G + Linker 3 +
MSWVRQAPGKGLEWVSSISGSGRDTLYADSVK

SP34.185 scFv
GRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTI

mask (SEQ ID NO: 27) +

GGSLSV
SSQGTLVTVSSGGGGSGGGSGGVYCGP

Cleavable linker 1 +
EFDESVGCMGGGGSGGGLSGRSDAGSPLGLAGS

SP34.185 scFv (VH-
GGGSEVQLVESGGGLVQPGGSLKLSCAASGFTF

linker 1-VL) + Linker

NKYA
MNWVRQAPGKGLEWVARIRSKYNNYAT

2 + TROP2 Fab LC
YYADSVKDRFTISRDDSKNTAYLQMNNLKTEDT

AVYYCVRHGNFGNSYISYWAYWGQGTLVTVS

SGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGT

VTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLI

GGTKFLAPGTPARFSGSLLGGKAALTLSGVQPE

DEAEYYCVLWYSNRWVFGGGTKLTVLGGGGS

DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVA

WYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGS

GTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAG

TKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL

NNFYPREAKVQWKVDNALQSGNSQESVTEQDS

KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL

SSPVTKSFNRGEC

PC15: HC
GGVDFCKIYSWPVCHQGGGGSGGLSGRSDAGSP
103

TROP2 Fab mask
LGLAGSGGSQVQLQQSGSELKKPGASVKVSCKA

(SEQ ID NO: 24) +
SGYTFTNYGMNWVKQAPGQGLKWMGWINTY

cleavable linker 2 +

TGEP
TYTDDFKGRFAFSLDTSVSTAYLQISSLKA

TROP2 Fab HC
DDTAVYFCARGGFGSSYWYFDVWGQGSLVTV

SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY

FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS

SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP

KSC

PC16: LC
EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFG
104

10G + Linker 3 +
MSWVRQAPGKGLEWVSSISGSGRDTLYADSVK

SP34.185 scFv
GRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTI

mask (SEQ ID NO: 27) +

GGSLSV
SSQGTLVTVSSGGGGSGGGSGGVYCGP

Cleavable linker 1 +
EFDESVGCMGGGGSGGGLSGRSDAGSPLGLAGS

SP34.185 scFv (VH-
GGGSEVQLVESGGGLVQPGGSLKLSCAASGFTF

linker 1-VL) + Linker

NKYA
MNWVRQAPGKGLEWVARIRSKYNNYAT

2 + TROP2 Fab LC
YYADSVKDRFTISRDDSKNTAYLQMNNLKTEDT

AVYYCVRHGNFGNSYISYWAYWGQGTLVTVS

SGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGT

VTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLI

GGTKFLAPGTPARFSGSLLGGKAALTLSGVQPE

DEAEYYCVLWYSNRWVFGGGTKLTVLGGGGS

DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVA

WYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGS

GTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAG

TKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL

NNFYPREAKVQWKVDNALQSGNSQESVTEQDS

KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL

SSPVTKSFNRGEC

PC16: HC
GGSVLFCVKNLYCWVTGGGGSGGLSGRSDAGS
105

TROP2 Fab mask
PLGLAGSGGSQVQLQQSGSELKKPGASVKVSCK

(SEQ ID NO: 23) +
ASGYTFTNYGMNWVKQAPGQGLKWMGWINT

cleavable linker 2 +

YTGEP
TYTDDFKGRFAFSLDTSVSTAYLQISSLK

TROP2 Fab HC
ADDTAVYFCARGGFGSSYWYFDVWGQGSLVT

VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD

YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL

SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE

PKSC

PC17: LC
GGIDFCMLYNWPICAGGGGGSSGGSAAGLLAPP
106

GGLSGRSDAGGGGSDIQLTQSPSSLSASVGDRVS

ITCKASQDVSIAVAWYQQKPGKAPKLLIYSASY

RYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYY

CQQHYITPLTFGAGTKVEIKRTVAAPSVFIFPPS

DEQLKSGTASVVCLLNNFYPREAKVQWKVDNA

LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE

KHKVYACEVTHQGLSSPVTKSFNRGEC

PC17: HC
EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFG
107

MSWVRQAPGKGLEWVSSISGSGRDTLYADSVK

GRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTI

GGSLSV
SSQGTLVTVSSGGGGSGGGSGGVYCGP

EFDESVGCMGSSGGSAAGLLAPPGGLSGRSDAG

GGGSEVQLVESGGGLVQPGGSLKLSCAASGFTF

NKYA
MNWVRQAPGKGLEWVARIRSKYNNYAT

YYADSVKDRFTISRDDSKNTAYLQMNNLKTEDT

AVYYCVRHGNFGNSYISYWAYWGQGTLVTVS

SGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGT

VTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLI

GGTKFLAPGTPARFSGSLLGGKAALTLSGVQPE

DEAEYYCVLWYSNRWVFGGGTKLTVLGGGGS

QVQLQQSGSELKKPGASVKVSCKASGYTFTNY

G
MNWVKQAPGQGLKWMGWINTYTGEPTYTD

DFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFC

ARGGFGSSYWYFDV
WGQGSLVTVSSASTKGPS

VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW

NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS

LGTQTYICNVNHKPSNTKVDKKVEPKSC

PC18: LC
GGIDFCMLYNWPICAGGGGGSSGGSAAGLLAPP
108

GGLSGRSDAGGGGSDIQLTQSPSSLSASVGDRVS

ITCKASQDVSIAVAWYQQKPGKAPKLLIYSASY

RYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYY

CQQHYITPLTFGAGTKVEIKRTVAAPSVFIFPPS

DEQLKSGTASVVCLLNNFYPREAKVQWKVDNA

LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE

KHKVYACEVTHQGLSSPVTKSFNRGEC

PC18: HC
EVQLVESGGGLVQPGGSLRLSCAASGSTFYTAV
109

MGWVRQAPGKGLEWVAAIRWTALTTSYADSV

KGRFTISRDGAKTTLYLQMNSLRPEDTAVYYCA

ARGTLGLFTTADSYDY
WGQGTLVTVSSGGGGS

GGGSGGVYCGPEFDESVGCMGSSGGSAAGLLAP

PGGLSGRSDAGGGGSEVQLVESGGGLVQPGGSL

KLSCAASGFTFNKYAMNWVRQAPGKGLEWVA

RIRSKYNNYATYYADSVKDRFTISRDDSKNTAY

LQMNNLKTEDTAVYYCVRHGNFGNSYISYWA

Y
WGQGTLVTVSSGGGGSGGGGSGGGGSQTVVT

QEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWV

QQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGG

KAALTLSGVQPEDEAEYYCVLWYSNRWVFGG

GTKLTVLGGGGSQVQLQQSGSELKKPGASVKVS

CKASGYTFTNYGMNWVKQAPGQGLKWMGWI

NTYTGEP
TYTDDFKGRFAFSLDTSVSTAYLQISS

LKADDTAVYFCARGGFGSSYWYFDVWGQGSL

VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV

KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL

YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK

KVEPKSC

PC19: LC
EVQLVESGGGLVQPGGSLRLSCAASGSTFYTAV
110

MGWVRQAPGKGLEWVAAIRWTALTTSYADSV

KGRFTISRDGAKTTLYLQMNSLRPEDTAVYYCA

ARGTLGLFTTADSYDYWGQGTLVTVSSGGGGS

GGGSGGVYCGPEFDESVGCMGSSGGSAAGLLAP

PGGLSGRSDAGGGGSEVQLVESGGGLVQPGGSL

KLSCAASGFTFNKYAMNWVRQAPGKGLEWVA

RIRSKYNNYATYYADSVKDRFTISRDDSKNTAY

LQMNNLKTEDTAVYYCVRHGNFGNSYISYWA

Y
WGQGTLVTVSSGGGGSGGGGSGGGGSQTVVT

QEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWV

QQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGG

KAALTLSGVQPEDEAEYYCVLWYSNRWVFGG

GTKLTVLGGGGSDIQLTQSPSSLSASVGDRVSITC

KASQDVSIAVAWYQQKPGKAPKLLIYSASYRYT

GVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQ

HYITPLT
FGAGTKVEIKRTVAAPSVFIFPPSDEQL

KSGTASVVCLLNNFYPREAKVQWKVDNALQSG

NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV

YACEVTHQGLSSPVTKSFNRGEC

PC19: HC
GGIDFCMLYNWPICAGGGGGSSGGSAAGLLAPP
111

GGLSGRSDAGGGGSQVQLQQSGSELKKPGASVK

VSCKASGYTFTNYGMNWVKQAPGQGLKWMG

WINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQI

SSLKADDTAVYFCARGGFGSSYWYFDVWGQG

SLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL

VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG

LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD

KKVEPKSC

PC20: LC
EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFG
112

MSWVRQAPGKGLEWVSSISGSGRDTLYADSVK

GRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTI

GGSLSV
SSQGTLVTVSSGGGGSGGGSGGVYCGP

EFDESVGCMGSSGGSAAGLLAPPGGLSGRSDAG

GGGSEVQLVESGGGLVQPGGSLKLSCAASGFTF

NKYA
MNWVRQAPGKGLEWVARIRSKYNNYAT

YYADSVKDRFTISRDDSKNTAYLQMNNLKTEDT

AVYYCVRHGNFGNSYISYWAYWGQGTLVTVS

SGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGT

VTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLI

GGTKFLAPGTPARFSGSLLGGKAALTLSGVQPE

DEAEYYCVLWYSNRWVFGGGTKLTVLGGGGS

DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVA

WYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGS

GTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAG

TKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL

NNFYPREAKVQWKVDNALQSGNSQESVTEQDS

KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL

SSPVTKSFNRGEC

PC20: HC
GGIDFCMLYNWPICAGGGGGSSGGSAAGLLAPP
113

GGLSGRSDAGGGGSQVQLQQSGSELKKPGASVK

VSCKASGYTFTNYGMNWVKQAPGQGLKWMG

WINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQI

SSLKADDTAVYFCARGGFGSSYWYFDVWGQG

SLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL

VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG

LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD

KKVEPKSC

PC21: LC
GGVDFCKIYSWPVCHQGGGGSSGGSAAGLLAPP
114

GGLSGRSDAGGGGSDIQLTQSPSSLSASVGDRVS

ITCKASQDVSIAVAWYQQKPGKAPKLLIYSASY

RYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYY

CQQHYITPLTFGAGTKVEIKRTVAAPSVFIFPPS

DEQLKSGTASVVCLLNNFYPREAKVQWKVDNA

LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE

KHKVYACEVTHQGLSSPVTKSFNRGEC

PC21: HC
EVQLVESGGGLVQPGGSLRLSCAASGSTFYTAV
115

MGWVRQAPGKGLEWVAAIRWTALTTSYADSV

KGRFTISRDGAKTTLYLQMNSLRPEDTAVYYCA

ARGTLGLFTTADSYDY
WGQGTLVTVSSGGGGS

GGGSGGVYCGPEFDESVGCMGSSGGSAAGLLAP

PGGLSGRSDAGGGGSEVQLVESGGGLVQPGGSL

KLSCAASGFTFNKYAMNWVRQAPGKGLEWVA

RIRSKYNNYATYYADSVKDRFTISRDDSKNTAY

LQMNNLKTEDTAVYYCVRHGNFGNSYISYWA

Y
WGQGTLVTVSSGGGGSGGGGSGGGGSQTVVT

QEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWV

QQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGG

KAALTLSGVQPEDEAEYYCVLWYSNRWVFGG

GTKLTVLGGGGSQVQLQQSGSELKKPGASVKVS

CKASGYTFTNYGMNWVKQAPGQGLKWMGWI

NTYTGEP
TYTDDFKGRFAFSLDTSVSTAYLQISS

LKADDTAVYFCARGGFGSSYWYFDVWGQGSL

VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV

KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL

YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK

KVEPKSC

PC22: LC
GGIDFCMLYNWPICAGGGGGSGGLSGRSDAGSP
116

LGLAGSGGSDIQLTQSPSSLSASVGDRVSITCKAS

QDVSIA
VAWYQQKPGKAPKLLIYSASYRYTGVP

DRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYI

TPLT
FGAGTKVEIKRTVAAPSVFIFPPSDEQLKSG

TASVVCLLNNFYPREAKVQWKVDNALQSGNSQ

ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA

CEVTHQGLSSPVTKSFNRGEC

PC22: HC
EVQLVESGGGLVQPGGSLRLSCAASGSTFYTAV
117

MGWVRQAPGKGLEWVAAIRWTALTTSYADSV

KGRFTISRDGAKTTLYLQMNSLRPEDTAVYYCA

ARGTLGLFTTADSYDY
WGQGTLVTVSSGGGGS

GGGSGGVYCGPEFDESVGCMGGGGSGGGLSGR

SDAGSPLGLAGSGGGSEVQLVESGGGLVQPGGS

LKLSCAASGFTFNKYAMNWVRQAPGKGLEWV

ARIRSKYNNYATYYADSVKDRFTISRDDSKNTA

YLQMNNLKTEDTAVYYCVRHGNFGNSYISYW

AY
WGQGTLVTVSSGGGGSGGGGSGGGGSQTVV

TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNW

VQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLG

GKAALTLSGVQPEDEAEYYCVLWYSNRWVFG

GGTKLTVLGGGGSQVQLQQSGSELKKPGASVK

VSCKASGYTFTNYGMNWVKQAPGQGLKWMG

WINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQI

SSLKADDTAVYFCARGGFGSSYWYFDVWGQG

SLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL

VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG

LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD

KKVEPKSC

PC23: LC
GGIDFCMLYNWPICAGGGGGSGGLSGRSDAGSP
118

LGLAGSGGSDIQLTQSPSSLSASVGDRVSITCKAS

QDVSIA
VAWYQQKPGKAPKLLIYSASYRYTGVP

DRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYI

TPLT
FGAGTKVEIKRTVAAPSVFIFPPSDEQLKSG

TASVVCLLNNFYPREAKVQWKVDNALQSGNSQ

ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA

CEVTHQGLSSPVTKSFNRGEC

PC23: HC
EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFG
119

MSWVRQAPGKGLEWVSSISGSGRDTLYADSVK

GRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTI

GGSLSV
SSQGTLVTVSSGGGGSGGGSGGVYCGP

EFDESVGCMGGGGSGGGLSGRSDAGSPLGLAGS

GGGSEVQLVESGGGLVQPGGSLKLSCAASGFTF

NKYA
MNWVRQAPGKGLEWVARIRSKYNNYAT

YYADSVKDRFTISRDDSKNTAYLQMNNLKTEDT

AVYYCVRHGNFGNSYISYWAYWGQGTLVTVS

SGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGT

VTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLI

GGTKFLAPGTPARFSGSLLGGKAALTLSGVQPE

DEAEYYCVLWYSNRWVFGGGTKLTVLGGGGS

QVQLQQSGSELKKPGASVKVSCKASGYTFTNY

G
MNWVKQAPGQGLKWMGWINTYTGEPTYTD

DFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFC

ARGGFGSSYWYFDV
WGQGSLVTVSSASTKGPS

VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW

NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS

LGTQTYICNVNHKPSNTKVDKKVEPKSC

PC24: LC
GGIDFCMLYNWPICAGGGGGSGGGGGSGGGGS
120

GGASSGAGGSGGSDIQLTQSPSSLSASVGDRVSIT

CKASQDVSIAVAWYQQKPGKAPKLLIYSASYRY

TGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQ

QHYITPLT
FGAGTKVEIKRTVAAPSVFIFPPSDEQ

LKSGTASVVCLLNNFYPREAKVQWKVDNALQS

GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK

VYACEVTHQGLSSPVTKSFNRGEC

PC24: HC
EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFG
121

MSWVRQAPGKGLEWVSSISGSGRDTLYADSVK

GRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTI

GGSLSV
SSQGTLVTVSSGGGGSGGGSGGVYCGP

EFDESVGCMGGGGSGGGSGGGGSGGASSGAGG

SGGGSEVQLVESGGGLVQPGGSLKLSCAASGFT

FNKYA
MNWVRQAPGKGLEWVARIRSKYNNYA

T
YYADSVKDRFTISRDDSKNTAYLQMNNLKTED

TAVYYCVRHGNFGNSYISYWAYWGQGTLVTV

SSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGG

TVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRG

LIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQP

EDEAEYYCVLWYSNRWVFGGGTKLTVLGGGG

SQVQLQQSGSELKKPGASVKVSCKASGYTFTNY

G
MNWVKQAPGQGLKWMGWINTYTGEPTYTD

DFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFC

ARGGFGSSYWYFDV
WGQGSLVTVSSASTKGPS

VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW

NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS

LGTQTYICNVNHKPSNTKVDKKVEPKSC

PC25: LC
GGVDFCGLYHWPICYQGGGGSGGLSGRSDAGSP
123

LGLAGSGGSDIQLTQSPSSLSASVGDRVSITCKAS

QDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVP

DRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYIT

PLTFGAGTKVEIKRTVAAPSVFIFPPSDEQLKSGT

ASVVCLLNNFYPREAKVQWKVDNALQSGNSQE

SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC

EVTHQGLSSPVTKSFNRGEC

PC25: HC
EVQLVESGGGLVQPGGSLRLSCAASGSTFYTAV
124

MGWVRQAPGKGLEWVAAIRWTALTTSYADSV

KGRFTISRDGAKTTLYLQMNSLRPEDTAVYYCA

ARGTLGLFTTADSYDYWGQGTLVTVSSGGGGS

GGGSGGVYCGPEFDESVGCMGGGGSGGGLSGR

SDAGSPLGLAGSGGGSEVQLVESGGGLVQPGGS

LKLSCAASGFTFNKYAMNWVRQAPGKGLEWV

ARIRSKYNNYATYYADSVKDRFTISRDDSKNTA

YLQMNNLKTEDTAVYYCVRHGNFGNSYISYWA

YWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVT

QEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWV

QQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGK

AALTLSGVQPEDEAEYYCVLWYSNRWVFGGGT

KLTVLGGGGSQVQLQQSGSELKKPGASVKVSCK

ASGYTFTNYGMNWVKQAPGQGLKWMGWINTY

TGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKA

DDTAVYFCARGGFGSSYWYFDVWGQGSLVTVS

SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF

PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS

VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP

KSC

PC26: LC
EWVAAIRWTALTTSYADSVKGRFTISRDGAKTT
125

LYLQMNSLRPEDTAVYYCAARGTLGLFTTADSY

DYWGQGTLVTVSSGGGGSGGGSGGVYCGPEFD

ESVGCMGGGGSGGGLSGRSDAGSPLGLAGSGG

GSEVQLVESGGGLVQPGGSLKLSCAASGFTFNK

YAMNWVRQAPGKGLEWVARIRSKYNNYATYY

ADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAV

YYCVRHGNFGNSYISYWAYWGQGTLVTVSSGG

GGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTL

TCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGT

KFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAE

YYCVLWYSNRWVFGGGTKLTVLGGGGSDIQLT

QSPSSLSASVGDRVSITCKASQDVSIAVAWYQQK

PGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTL

TISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIKR

TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRE

AKVQWKVDNALQSGNSQESVTEQDSKDSTYSL

SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF

NRGEC

PC26: HC
GGVDFCGLYHWPICYQGGGGSGGLSGRSDAGSP
126

LGLAGSGGSQVQLQQSGSELKKPGASVKVSCKA

SGYTFTNYGMNWVKQAPGQGLKWMGWINTYT

GEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKAD

DTAVYFCARGGFGSSYWYFDVWGQGSLVTVSS

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP

EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV

VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS

C

PC27: LC
GGVDFCGLYHWPICYQGGGGSSGGSAAGLLAPP
127

GGLSGRSDAGGGGSDIQLTQSPSSLSASVGDRVS

ITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYR

YTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYC

QQHYITPLTFGAGTKVEIKRTVAAPSVFIFPPSDE

QLKSGTASVVCLLNNFYPREAKVQWKVDNALQ

SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH

KVYACEVTHQGLSSPVTKSFNRGEC

PC27: HC
EVQLVESGGGLVQPGGSLRLSCAASGSTFYTAV
128

MGWVRQAPGKGLEWVAAIRWTALTTSYADSV

KGRFTISRDGAKTTLYLQMNSLRPEDTAVYYCA

ARGTLGLFTTADSYDYWGQGTLVTVSSGGGGS

GGGSGGVYCGPEFDESVGCMGSSGGSAAGLLAP

PGGLSGRSDAGGGGSEVQLVESGGGLVQPGGSL

KLSCAASGFTFNKYAMNWVRQAPGKGLEWVA

RIRSKYNNYATYYADSVKDRFTISRDDSKNTAY

LQMNNLKTEDTAVYYCVRHGNFGNSYISYWAY

WGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQ

EPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQ

QKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKA

ALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTK

LTVLGGGGSQVQLQQSGSELKKPGASVKVSCKA

SGYTFTNYGMNWVKQAPGQGLKWMGWINTYT

GEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKAD

DTAVYFCARGGFGSSYWYFDVWGQGSLVTVSS

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP

EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV

VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS

C

PC28: LC
EVQLVESGGGLVQPGGSLRLSCAASGSTFYTAV
129

MGWVRQAPGKGLEWVAAIRWTALTTSYADSV

KGRFTISRDGAKTTLYLQMNSLRPEDTAVYYCA

ARGTLGLFTTADSYDYWGQGTLVTVSSGGGGS

GGGSGGVYCGPEFDESVGCMGSSGGSAAGLLAP

PGGLSGRSDAGGGGSEVQLVESGGGLVQPGGSL

KLSCAASGFTFNKYAMNWVRQAPGKGLEWVA

RIRSKYNNYATYYADSVKDRFTISRDDSKNTAY

LQMNNLKTEDTAVYYCVRHGNFGNSYISYWAY

WGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQ

EPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQ

QKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKA

ALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTK

LTVLGGGGSDIQLTQSPSSLSASVGDRVSITCKAS

QDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVP

DRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYIT

PLTFGAGTKVEIKRTVAAPSVFIFPPSDEQLKSGT

ASVVCLLNNFYPREAKVQWKVDNALQSGNSQE

SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC

EVTHQGLSSPVTKSFNRGEC

PC28: HC
GGVDFCGLYHWPICYQGGGGSSGGSAAGLLAPP
130

GGLSGRSDAGGGGSQVQLQQSGSELKKPGASVK

VSCKASGYTFTNYGMNWVKQAPGQGLKWMG

WINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQI

SSLKADDTAVYFCARGGFGSSYWYFDVWGQGS

LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL

VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG

LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD

KKVEPKSC

PC29: LC
GGVDFCGLYHWPICYQGGGGSGGGGGSGGGGS
241

GGASSGAGGSDIQLTQSPSSLSASVGDRVSITCK

ASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTG

VPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQH

YITPLTFGAGTKVEIKRTVAAPSVFIFPPSDEQLK

SGTASVVCLLNNFYPREAKVQWKVDNALQSGN

SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY

ACEVTHQGLSSPVTKSFNRGEC

PC29: HC
EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFG
131

MSWVRQAPGKGLEWVSSISGSGRDTLYADSVK

GRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIG

GSLSVSSQGTLVTVSSGGGGSGGGSGGVYCGPE

FDESVGCMGGGGSGGGSGGGGSGGASSGAGGS

GGGSEVQLVESGGGLVQPGGSLKLSCAASGFTF

NKYAMNWVRQAPGKGLEWVARIRSKYNNYAT

YYADSVKDRFTISRDDSKNTAYLQMNNLKTEDT

AVYYCVRHGNFGNSYISYWAYWGQGTLVTVSS

GGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTV

TLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIG

GTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDE

AEYYCVLWYSNRWVFGGGTKLTVLGGGGSQV

QLQQSGSELKKPGASVKVSCKASGYTFTNYGM

NWVKQAPGQGLKWMGWINTYTGEPTYTDDFK

GRFAFSLDTSVSTAYLQISSLKADDTAVYFCARG

GFGSSYWYFDVWGQGSLVTVSSASTKGPSVFPL

APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG

ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT

QTYICNVNHKPSNTKVDKKVEPKSC

PC30: LC
EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFG
132

MSWVRQAPGKGLEWVSSISGSGRDTLYADSVK

GRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIG

GSLSVSSQGTLVTVSSGGGGSGGGSGGVYCGPE

FDESVGCMGGGGSGGGSGGGGSGGASSGAGGS

GGGSEVQLVESGGGLVQPGGSLKLSCAASGFTF

NKYAMNWVRQAPGKGLEWVARIRSKYNNYAT

YYADSVKDRFTISRDDSKNTAYLQMNNLKTEDT

AVYYCVRHGNFGNSYISYWAYWGQGTLVTVSS

GGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTV

TLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIG

GTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDE

AEYYCVLWYSNRWVFGGGTKLTVLGGGGSDIQ

LTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQ

QKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDF

TLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEI

KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY

PREAKVQWKVDNALQSGNSQESVTEQDSKDST

YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV

TKSFNRGEC

PC30: HC
GGVDFCGLYHWPICYQGGGGSGGGGGSGGGGS
242

GGASSGAGGSQVQLQQSGSELKKPGASVKVSCK

ASGYTFTNYGMNWVKQAPGQGLKWMGWINTY

TGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKA

DDTAVYFCARGGFGSSYWYFDVWGQGSLVTVS

SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF

PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS

VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP

KSC

In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences according to SEQ ID NO: 76 and SEQ ID NO: 77. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 90% sequence identity to SEQ ID NO: 76 and SEQ ID NO: 77. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 95% sequence identity to SEQ ID NO: 76 and SEQ ID NO: 77. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 99% sequence identity to SEQ ID NO: 76 and SEQ ID NO: 77.

In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences according to SEQ ID NO: 78 and SEQ ID NO: 79. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 90% sequence identity to SEQ ID NO: 78 and SEQ ID NO: 79. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 95% sequence identity to SEQ ID NO: 78 and SEQ ID NO: 79. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 99% sequence identity to SEQ ID NO: 78 and SEQ ID NO: 79.

In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences according to SEQ ID NO: 80 and SEQ ID NO: 81. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 90% sequence identity to SEQ ID NO: 80 and SEQ ID NO: 81. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 95% sequence identity to SEQ ID NO: 80 and SEQ ID NO: 81. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 99% sequence identity to SEQ ID NO: 80 and SEQ ID NO: 81.

In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences according to SEQ ID NO: 82 and SEQ ID NO: 83. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 90% sequence identity to SEQ ID NO: 82 and SEQ ID NO: 83. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 95% sequence identity to SEQ ID NO: 82 and SEQ ID NO: 83. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 99% sequence identity to SEQ ID NO: 82 and SEQ ID NO: 83.

In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences according to SEQ ID NO: 84 and SEQ ID NO: 85. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 90% sequence identity to SEQ ID NO: 84 and SEQ ID NO: 85. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 95% sequence identity to SEQ ID NO: 84 and SEQ ID NO: 85. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 99% sequence identity to SEQ ID NO: 84 and SEQ ID NO: 85.

In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences according to SEQ ID NO: 86 and SEQ ID NO: 87. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 90% sequence identity to SEQ ID NO: 86 and SEQ ID NO: 87. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 95% sequence identity to SEQ ID NO: 86 and SEQ ID NO: 87. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 99% sequence identity to SEQ ID NO: 86 and SEQ ID NO: 87.

In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences according to SEQ ID NO: 88 and SEQ ID NO: 89. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 90% sequence identity to SEQ ID NO: 88 and SEQ ID NO: 89. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 95% sequence identity to SEQ ID NO: 88 and SEQ ID NO: 89. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 99% sequence identity to SEQ ID NO: 88 and SEQ ID NO: 89.

In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences according to SEQ ID NO: 90 and SEQ ID NO: 91. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 90% sequence identity to SEQ ID NO: 90 and SEQ ID NO: 91. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 95% sequence identity to SEQ ID NO: 90 and SEQ ID NO: 91. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 99% sequence identity to SEQ ID NO: 90 and SEQ ID NO: 91.

In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences according to SEQ ID NO: 92 and SEQ ID NO: 93. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 90% sequence identity to SEQ ID NO: 92 and SEQ ID NO: 93. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 95% sequence identity to SEQ ID NO: 92 and SEQ ID NO: 93. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 99% sequence identity to SEQ ID NO: 92 and SEQ ID NO: 93.

In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences according to SEQ ID NO: 94 and SEQ ID NO: 95. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 90% sequence identity to SEQ ID NO: 94 and SEQ ID NO: 95. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 95% sequence identity to SEQ ID NO: 94 and SEQ ID NO: 95. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 99% sequence identity to SEQ ID NO: 94 and SEQ ID NO: 95.

In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences according to SEQ ID NO: 96 and SEQ ID NO: 97. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 90% sequence identity to SEQ ID NO: 96 and SEQ ID NO: 97. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 95% sequence identity to SEQ ID NO: 96 and SEQ ID NO: 97. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 99% sequence identity to SEQ ID NO: 96 and SEQ ID NO: 97.

In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences according to SEQ ID NO: 98 and SEQ ID NO: 99. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 90% sequence identity to SEQ ID NO: 98 and SEQ ID NO: 99. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 95% sequence identity to SEQ ID NO: 98 and SEQ ID NO: 99. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 99% sequence identity to SEQ ID NO: 98 and SEQ ID NO: 99.

In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences according to SEQ ID NO: 100 and SEQ ID NO: 101. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 90% sequence identity to SEQ ID NO: 100 and SEQ ID NO: 101. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 95% sequence identity to SEQ ID NO: 100 and SEQ ID NO: 101. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 99% sequence identity to SEQ ID NO: 100 and SEQ ID NO: 101.

In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences according to SEQ ID NO: 102 and SEQ ID NO: 103. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 90% sequence identity to SEQ ID NO: 102 and SEQ ID NO: 103. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 95% sequence identity to SEQ ID NO: 102 and SEQ ID NO: 103. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 99% sequence identity to SEQ ID NO: 102 and SEQ ID NO: 103.

In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences according to SEQ ID NO: 104 and SEQ ID NO: 105. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 90% sequence identity to SEQ ID NO: 104 and SEQ ID NO: 105. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 95% sequence identity to SEQ ID NO: 104 and SEQ ID NO: 105. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 99% sequence identity to SEQ ID NO: 104 and SEQ ID NO: 105.

In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences according to SEQ ID NO: 106 and SEQ ID NO: 107. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 90% sequence identity to SEQ ID NO: 106 and SEQ ID NO: 107. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 95% sequence identity to SEQ ID NO: 106 and SEQ ID NO: 107. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 99% sequence identity to SEQ ID NO: 106 and SEQ ID NO: 107.

In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences according to SEQ ID NO: 108 and SEQ ID NO: 109. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 90% sequence identity to SEQ ID NO: 108 and SEQ ID NO: 109. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 95% sequence identity to SEQ ID NO: 108 and SEQ ID NO: 109. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 99% sequence identity to SEQ ID NO: 108 and SEQ ID NO: 109.

In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences according to SEQ ID NO: 110 and SEQ ID NO: 111. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 90% sequence identity to SEQ ID NO: 110 and SEQ ID NO: 111. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 95% sequence identity to SEQ ID NO: 110 and SEQ ID NO: 111. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 99% sequence identity to SEQ ID NO: 110 and SEQ ID NO: 111.

In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences according to SEQ ID NO: 112 and SEQ ID NO: 113. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 90% sequence identity to SEQ ID NO: 112 and SEQ ID NO: 113. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 95% sequence identity to SEQ ID NO: 112 and SEQ ID NO: 113. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 99% sequence identity to SEQ ID NO: 112 and SEQ ID NO: 113.

In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences according to SEQ ID NO: 114 and SEQ ID NO: 115. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 90% sequence identity to SEQ ID NO: 114 and SEQ ID NO: 115. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 95% sequence identity to SEQ ID NO: 114 and SEQ ID NO: 115. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 99% sequence identity to SEQ ID NO: 114 and SEQ ID NO: 115.

In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences according to SEQ ID NO: 116 and SEQ ID NO: 117. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 90% sequence identity to SEQ ID NO: 116 and SEQ ID NO: 117. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 95% sequence identity to SEQ ID NO: 116 and SEQ ID NO: 117. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 99% sequence identity to SEQ ID NO: 116 and SEQ ID NO: 117.

In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences according to SEQ ID NO: 118 and SEQ ID NO: 119. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 90% sequence identity to SEQ ID NO: 118 and SEQ ID NO: 119. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 95% sequence identity to SEQ ID NO: 118 and SEQ ID NO: 119. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 99% sequence identity to SEQ ID NO: 118 and SEQ ID NO: 119.

In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences according to SEQ ID NO: 120 and SEQ ID NO: 121. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 90% sequence identity to SEQ ID NO: 120 and SEQ ID NO: 121. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 95% sequence identity to SEQ ID NO: 120 and SEQ ID NO: 121. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 99% sequence identity to SEQ ID NO: 120 and SEQ ID NO: 121.

In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences according to SEQ ID NO: 123 and SEQ ID NO: 124. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 90% sequence identity to SEQ ID NO: 123 and SEQ ID NO: 124. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 95% sequence identity to SEQ ID NO: 123 and SEQ ID NO: 124. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 99% sequence identity to SEQ ID NO: 123 and SEQ ID NO: 124.

In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences according to SEQ ID NO: 125 and SEQ ID NO: 126. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 90% sequence identity to SEQ ID NO: 125 and SEQ ID NO: 126. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 95% sequence identity to SEQ ID NO: 125 and SEQ ID NO: 126. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 99% sequence identity to SEQ ID NO: 125 and SEQ ID NO: 126.

In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences according to SEQ ID NO: 127 and SEQ ID NO: 128. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 90% sequence identity to SEQ ID NO: 127 and SEQ ID NO: 128. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 95% sequence identity to SEQ ID NO: 127 and SEQ ID NO: 128. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 99% sequence identity to SEQ ID NO: 127 and SEQ ID NO: 128.

In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences according to SEQ ID NO: 129 and SEQ ID NO: 130. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 90% sequence identity to SEQ ID NO: 129 and SEQ ID NO: 130. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 95% sequence identity to SEQ ID NO: 129 and SEQ ID NO: 130. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 99% sequence identity to SEQ ID NO: 129 and SEQ ID NO: 130.

In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences according to SEQ ID NO: 241 and SEQ ID NO: 131. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 90% sequence identity to SEQ ID NO: 241 and SEQ ID NO: 131. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 95% sequence identity to SEQ ID NO: 241 and SEQ ID NO: 131. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 99% sequence identity to SEQ ID NO: 241 and SEQ ID NO: 131.

In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences according to SEQ ID NO: 132 and SEQ ID NO: 242. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 90% sequence identity to SEQ ID NO: 132 and SEQ ID NO: 242. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 95% sequence identity to SEQ ID NO: 132 and SEQ ID NO: 242. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 99% sequence identity to SEQ ID NO: 132 and SEQ ID NO: 242.

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: 74-132, 241-242. In some instances, the sequence comprises at least or about 95% homology to SEQ ID NOs: 74-132, 241-242. In some instances, the sequence comprises at least or about 97% homology to SEQ ID NOs: 74-132, 241-242. In some instances, the sequence comprises at least or about 99% homology to SEQ ID NOs: 74-132, 241-242. In some instances, the sequence comprises at least or about 100% homology to SEQ ID NOs: 74-132, 241-242. 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: 74, 77, 78, 81, 82, 84, 87, 89, 90, 93, 94, 97, 98, 100, 103, 105, 106, 108, 111, 113, 114, 116, 118, 120, 123, 126, 127, 130, 241, or 242. 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: 75, 76, 79, or 80. 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: 83, 85, 86, 88, 91, 92, 95, 96, 99, 101, 102, 104, 107, 109, 112, 115, 117, 119, 121, 124, 125, 128, 129, 131, or 132.

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 isolated polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 1:

wherein the polypeptide complex comprises a single chain variable fragment (scFv) comprising a light chain variable domain and a heavy chain variable domain, wherein the scFv further comprises a peptide that impairs binding of the scFv to an effector cell antigen and the peptide is linked to a N-terminus of the heavy chain variable domain of the scFv with a linking moiety that is a substrate for a tumor specific protease, and the peptide further comprises a half-life extending molecule; and a Fab that binds to TROP2, 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 further comprises P₂and L₂, wherein P₂comprises a peptide that impairs binding to TROP2; and L₂comprises a linking moiety that connects the Fab light chain polypeptide to P₂and is a substrate for a tumor specific protease.

Disclosed herein, in some embodiments, are isolated polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 2:

wherein the polypeptide complex comprises a single chain variable fragment (scFv) comprising a light chain variable domain and a heavy chain variable domain, wherein the scFv further comprises a peptide that impairs binding of the scFv to an effector cell antigen and the peptide is linked to a N-terminus of the heavy chain variable domain of the scFv with a linking moiety that is a substrate for a tumor specific protease, and the peptide further comprises a half-life extending molecule; and a Fab that binds to TROP2, 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 further comprises P₂and L₂, wherein P₂comprises a peptide that impairs binding to TROP2; 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 polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 3:

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 that impairs binding of the scFv to an effector cell antigen and the peptide is linked to the light chain variable domain of the scFv with a linking moiety that is a substrate for a tumor specific protease, and the peptide is further linked to a half-life extending molecule; and a Fab that binds to TROP2, wherein the Fab comprises a Fab light chain polypeptide chain and a Fab heavy chain polypeptide chain, and wherein the Fab heavy chain polypeptide chain is linked to a C terminus of the heavy chain variable domain of the scFv.

Disclosed herein, in some embodiments, are isolated polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 4:

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 light chain variable domain of the scFv with a linking moiety 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 TROP2, 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 heavy chain variable domain of the scFv, and wherein the Fab is linked to P₂and L₂, wherein P₂comprises a peptide that impairs binding to TROP2; 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 polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 5:

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 further linked to a peptide that impairs binding of the scFv to an effector cell antigen and the peptide is linked to a N-terminus of the light chain variable domain of the scFv with a linking moiety that is a substrate for a tumor specific protease, and the peptide is further linked to a half-life extending molecule; and a Fab that binds to TROP2, 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 heavy chain variable domain of the scFv.

Disclosed herein, in some embodiments, are isolated polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 6:

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 TROP2, 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 TROP2; and L₂comprises a linking moiety that connects the Fab light chain polypeptide to P₂and is a substrate for a tumor specific protease.

Disclosed herein, in some embodiments, are isolated polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 7:

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 that impairs binding of the scFv to an effector cell antigen and the peptide is linked to the heavy chain variable domain of the scFv with a linking moiety that is a substrate for a tumor specific protease, and the peptide is further linked to a half-life extending molecule; and a Fab that binds to TROP2, wherein the Fab comprises a Fab light chain polypeptide chain and a Fab heavy chain polypeptide chain, and wherein the Fab heavy chain polypeptide chain is linked to a C terminus of the light chain variable domain of the scFv.

Disclosed herein, in some embodiments, are isolated polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 8:

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 that impairs binding of the scFv to an effector cell antigen and the peptide is linked to a N-terminus of the heavy chain variable domain of the scFv with a linking moiety that is a substrate for a tumor specific protease, and the peptide is further linked to a half-life extending molecule; and a Fab that binds to TROP2, 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.

Disclosed herein, in some embodiments, are isolated polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 9:

wherein the isolated polypeptide or polypeptide complex comprises a Fab that binds to TROP2, the Fab comprising a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab is linked to a peptide (P₁) that impairs binding of the Fab to TROP2 and P₁is linked to a N terminus of the Fab light chain polypeptide with a linking moiety (L₁) that is a substrate for a tumor specific protease, and the P₁is further linked to a half-life extending molecule; and a single chain variable fragment (scFv) that binds to an effector cell antigen, the scFv comprising a light chain variable domain and a heavy chain variable domain, wherein the heavy chain variable domain of the scFv is linked to an N terminus of the Fab heavy chain polypeptide, wherein the scFv is linked to P₂and L₂, wherein P₂comprises a peptide that impairs binding of the scFv to the effector cell antigen, and L₂comprises a linking moiety that connects the light chain variable domain of the scFv to P₂and is a substrate for a tumor specific protease.

Disclosed herein, in some embodiments, are isolated polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 10:

wherein the isolated polypeptide or polypeptide complex comprises a Fab that binds to TROP2, the Fab comprising a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab is linked to a peptide that impairs binding of the Fab to TROP2 and the peptide is linked to a N terminus of the Fab light chain polypeptide with a linking moiety that is a substrate for a tumor specific protease, and the peptide is further linked to half-life extending molecule; and a single chain variable fragment (scFv) that binds to an effector cell antigen, the scFv comprising a light chain variable domain and a heavy chain variable domain, wherein the heavy chain variable domain of the scFv is linked to an N terminus of the Fab heavy chain polypeptide.

Disclosed herein, in some embodiments, are isolated polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 11:

wherein the isolated polypeptide or polypeptide complex comprises a Fab that binds to TROP2, the Fab comprising a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab is linked to a peptide (P₁) that impairs binding of the Fab to TROP2 and P₁is linked to a N terminus of the Fab heavy chain polypeptide 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 single chain variable fragment (scFv) that binds to an effector cell antigen, the scFv comprising a light chain variable domain and a heavy chain variable domain, wherein the heavy chain variable domain of the scFv is linked to an N terminus of the Fab light chain polypeptide, wherein the scFv further is linked to P₂and L₂, wherein P₂comprises a peptide that impairs binding of the scFv to the effector cell antigen, and L₂comprises a linking moiety that connects the light chain variable domain of the scFv to P₂and is a substrate for a tumor specific protease.

Disclosed herein, in some embodiments, are isolated polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 12:

wherein the isolated polypeptide or polypeptide complex comprises a Fab that binds to TROP2, the Fab comprising a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab is linked to a peptide that impairs binding of the Fab to TROP2 and the peptide is linked to a N terminus of the Fab heavy chain polypeptide with a linking moiety that is a substrate for a tumor specific protease, and the peptide is further linked to a half-life extending molecule; and a single chain variable fragment (scFv) that binds to an effector cell antigen, the scFv comprising a light chain variable domain and a heavy chain variable domain, wherein the heavy chain variable domain of the scFv is linked to an N terminus of the Fab light chain polypeptide.

Disclosed herein, in some embodiments, are isolated polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 13:

wherein the isolated polypeptide or polypeptide complex comprises a Fab that binds to TROP2, the Fab comprising a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab is linked to a peptide (P₁) that impairs binding of the Fab to TROP2 and P₁is linked to a N terminus of the Fab light chain polypeptide 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 single chain variable fragment (scFv) that binds to an effector cell antigen, the scFv comprising a light chain variable domain and a heavy chain variable domain, wherein the light chain variable domain of the scFv is linked to an N terminus of the Fab heavy chain polypeptide, wherein the scFv is linked to P₂and L₂, wherein P₂comprises a peptide that impairs binding of the scFv to the effector cell antigen, and L₂comprises a linking moiety that connects the heavy chain variable domain of the scFv to P₂and is a substrate for a tumor specific protease.

Disclosed herein, in some embodiments, are isolated polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 14:

wherein the isolated polypeptide or polypeptide complex comprises a Fab that binds to TROP2, the Fab comprising a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab is linked to a peptide that impairs binding of the Fab to TROP2 and the peptide is linked to a N terminus of the Fab light chain polypeptide with a linking moiety that is a substrate for a tumor specific protease, and the peptide is further linked to a half-life extending molecule; and a single chain variable fragment (scFv) that binds to an effector cell antigen, the scFv comprising a light chain variable domain and a heavy chain variable domain, wherein the light chain variable domain of the scFv is linked to an N terminus of the Fab heavy chain polypeptide.

Disclosed herein, in some embodiments, are isolated polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 15:

wherein the isolated polypeptide or polypeptide complex comprises a Fab that binds to TROP2, the Fab comprising a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab is linked to a (P₁) that impairs binding of the Fab to TROP2 and P₁is linked to a N terminus of the Fab heavy chain polypeptide 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 single chain variable fragment (scFv) that binds to an effector cell antigen, the scFv comprising a light chain variable domain and a heavy chain variable domain, wherein the light chain variable domain of the scFv is linked to an N terminus of the Fab light chain polypeptide, wherein the scFv is linked to P₂and L₂, wherein P₂comprises a peptide that impairs binding of the scFv to the effector cell antigen, and L₂comprises a linking moiety that connects the heavy chain variable domain of the scFv to P₂and is a substrate for a tumor specific protease.

Disclosed herein, in some embodiments, are isolated polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 16:

wherein the isolated polypeptide or polypeptide complex comprises a Fab that binds to TROP2, the Fab comprising a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab is linked to a peptide that impairs binding of the Fab to TROP2 and the peptide is linked to a N terminus of the Fab heavy chain polypeptide with a linking moiety that is a substrate for a tumor specific protease, and the peptide is further linked to a half-life extending molecule; and a single chain variable fragment (scFv) that binds to an effector cell antigen, the scFv comprising a light chain variable domain and a heavy chain variable domain, wherein the light chain variable domain of the scFv is linked to an N terminus of the Fab light chain polypeptide.

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:

A₂-A₁-L₁-P₁-H₁ (Formula I)

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

A₂-A₁-L₁-P₁-H₁ (Formula I)

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

A₂-A₁-L₁-P₁-H₁ (Formula I)

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

A₂-A₁-L₁-P₁-H₁ (Formula I)

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

P₂-L₂-A₂-A₁-L₁-P₁-H₁ (Formula Ia).

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

L_1a-P_1a-H_1a (Formula II)

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

L_1a-P_1a-H_1a (Formula II)

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

L_1a-P_1a-H_1a (Formula II)

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

L_1a-P_1a-H_1a (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:

Disclosed herein, in some embodiments, are isolated nucleic acid molecules encoding polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 2:

wherein the polypeptide complex comprises a single chain variable fragment (scFv) comprising a light chain variable domain and a heavy chain variable domain, wherein the scFv further comprises a peptide that impairs binding of the scFv to an effector cell antigen and the peptide is linked to a N-terminus of the heavy chain variable domain of the scFv with a linking moiety that is a substrate for a tumor specific protease, and the peptide further comprises a half-life extending molecule; and a Fab that binds to TROP2, 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 further comprises P₂and L₂, wherein P₂comprises a peptide that impairs binding to TROP2; and L₂comprises a linking moiety that connects the Fab heavy 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 isolated polypeptides or polypeptide complexes as disclosed herein; and (b) a pharmaceutically acceptable excipient.

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

A₂-A₁-L₁-P₁-H₁ (Formula I)

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

A₂-A₁-L₁-P₁-H₁ (Formula I)

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

A₂-A₁-L₁-P₁-H₁ (Formula I)

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

A₂-A₁-L₁-P₁-H₁ (Formula I)

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

P₂-L₂-A₂-A₁-L₁-P₁-H₁ (Formula Ia);

and (b) a pharmaceutically acceptable excipient.

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

L_1a-P_1a-H_1a (Formula II)

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

L_1a-P_1a-H_1a (Formula II)

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

L_1a-P_1a-H_1a (Formula II)

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

L_1a-P_1a-H_1a (Formula II)

Disclosed herein, in some embodiments, the pharmaceutical composition comprises (a) isolated polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 1:

Disclosed herein, in some embodiments, the pharmaceutical composition comprises (a) isolated polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 2:

wherein the polypeptide complex comprises a single chain variable fragment (scFv) comprising a light chain variable domain and a heavy chain variable domain, wherein the scFv further comprises a peptide that impairs binding of the scFv to an effector cell antigen and the peptide is linked to a N-terminus of the heavy chain variable domain of the scFv with a linking moiety that is a substrate for a tumor specific protease, and the peptide further comprises a half-life extending molecule; and a Fab that binds to TROP2, 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 further comprises P₂and L₂, wherein P₂comprises a peptide that impairs binding to TROP2; 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.

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-TROP2 Binding Domains to TROP2

Disclosed herein are isolated polypeptide or polypeptide complexes comprising an anti-TROP2 binding domain that is linked to a peptide that impairs binding of the anti-TROP2 binding to TROP2 wherein the peptide comprises an amino acid sequence according to X₁-X₂-X₃-X₄-C-X₆-X₇-X₈-X₉-X₁₀-C-X₁₂-X₁₃-X₁₄and X₁is selected from N, D, S, Y, A, F, H, T, L, and V; X₂is selected from S, T, D, A, H, V, Y, N, F, I, and L; X₃is selected from L, I, and V; X₄is selected from F, L, V, M, W, I, Y, and H; X₆is selected from V, F, L, I, and W; X₇is selected from K, R, Q, N, H, and M; X₈is selected from N and K; X₉is selected from L, V, and I; X₁₀is selected from Y, W, F, Q, and L; X₁₂is selected from W and V; X₁₃is selected from I, N, H, T, V, Y, and D; X₁₄is selected from D, V, A, S, I, T, N, Y, H, and P; or the peptide comprises an amino acid according to J₁-J₂-J₃-C-J₈-J₆-J₇-J₈-W-J₁₀-J₁₁-C-J₁₃-J₁₄and J₁is selected from V, I, L, P, E, F, and M; J₂is selected from D and N; J₃is selected from F and W; J₈is selected from A, E, S, R, K, Y, L, Q, G, M, F, T, W, and D; J₆is selected from L, M, I, V, F, T, R, and S; J₇is selected from Y, F, and N; J₈is selected from N, R, D, H, K, Q, S, G, A, E, and M; J₁₀is selected from P and R; J₁₁is selected from V and I; J₁₃is selected from D, G, N, R, S, Q, Y, T, A, E, L, V, K, M, I, H, F, and W; J₁₄is selected from T, S, Q, L, D, N, A, E, K, M, V, R, I, H, P, V, and W; or the peptide comprises an amino acid sequence according to B₁-B₂-B₃-C-B₅-B₆-B₇-B₈-W-B₁₀-B₁₁-C-B₁₃-B₁₄and B₁is selected from V, I, L, P, E, F, and M; B₂is selected from D and N; B₃is selected from F and W; B₈is selected from A, E, S, R, K, Y, L, Q, G, M, F, T, W, and D; B₆is selected from L, M, I, V, F, T, R, and S; B₇is selected from Y, F, and N; B₈is selected from N, R, D, H, K, Q, S, G, A, E, and M; B₁₀is selected from P and R; B₁₁is selected from V and I; B₁₃is selected from D, G, N, R, S, Q, Y, T, A, E, L, V, K, M, I, H, F, and W; and B₁₄is selected from T, S, Q, L, D, N, A, E, K, M, V, R, I, H, P, V, G, and W. In some embodiments, X₁is selected from N, D, S, Y, A, F, and T; X₂is selected from S, T, D, A, H, V, Y, and N; X₃is L; X₄is selected from F, L, V, M, and W; X₆is selected from V, F, and L; X₇is selected from K, R, Q, and N; X₈is selected from N and K; X₉is selected from L, V, and I; X₁₀is selected from Y, W, and F; X₁₂is W; X₁₃is selected from I, N, H, and T; and X₁₄is selected from D, V, A, S, I, T, and N. In some embodiments, X₁is selected from N, D, S, and Y; X₂is selected from S, T, and D; X₃is L; X₄is selected from F, L, and V; X₆is selected from V and F; X₇is selected from K, R, and Q; X₈is N; X₉is selected from L and V; X₁₀is selected from Y and W; X₁₂is W; X₁₃is selected from I, N, and H; and X₁₄is selected from D, V, A, and S. In some embodiments, J₁is selected from V, I, and L; J₂is D; J₃is F; J₈is selected from A, E, S, R, K, Y, and L; J₆is selected from L, M, and I; J₇is Y; J₈is selected from N, R, D, H, K, Q, S, and G; J₁₀is P; J₁₁is selected from V and I; J₁₃is selected from D, G, N, R, S, Q, Y, T, and A; and J₁₄is selected from T, S, Q, L, D, N, A, and E. In some embodiments, J₁is selected from V, I, and L; J₂is D; J₃is F; J₈is selected from A, E, S, and R; J₆is selected from L, M, and I; J₇is Y; J₈is selected from N, R, and D; J₁₀is P; J₁₁is selected from V and I; J₁₃is selected from D, G, N, R, S, and Q; J₁₄is selected from T, S, Q, and L. In some embodiments, B₁is selected from V, I, and L; B₂is D; B₃is F; B₈is selected from A, E, S, R, K, Y, M, G, and L; B₆is selected from L, M, and I; B₇is Y; B₈is selected from N, R, D, H, K, Q, S, and G; B₁₀is P; B₁₁is selected from V and I; B₁₃is selected from D, G, N, R, S, Q, Y, T, H, and A; B₁₄is selected from T, S, Q, L, D, N, A, G, and E. In some embodiments, B₁is selected from V, I, and L; B₂is D; B₃is F; B₈is selected from A, E, S, K, M, G, and R; B₆is selected from L, M, and I; B₇is Y; B₈is selected from N, R, S, H, and D; B₁₀is P; B₁₁is selected from V and I; B₁₃is selected from D, G, N, R, S, H, A, Y, and Q; and B₁₄is selected from T, S, Q, G, and L.

In some embodiments, the peptide comprises the amino acid sequences according to SEQ ID NOs: 133-145.

In some embodiments, the peptide comprises the amino acid sequences according to SEQ ID NOs: 146-158.

In some embodiments, the peptide comprises an amino acid sequence according to any of the sequences of Table 27.

In some embodiments, the peptide comprises the amino acid sequences according to SEQ ID NOs: 159-178.

In some embodiments, the peptide comprises an amino acid sequences according to any of the sequences of Table 29.

In some embodiments, the peptide comprises the amino acid sequences according to SEQ ID NOs: 179-201.

In some embodiments, the peptide comprises the amino acid sequence according to SEQ ID NO: 24 or a peptide sequence that has 1, 2, or 3, amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 24.

In some embodiments, the peptide comprises the amino acid sequence according to SEQ ID NO: 181 or a peptide sequence that has 1, 2, or 3, amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 181.

In some embodiments, the peptide comprises the amino acid sequence according to SEQ ID NO: 186 or a peptide sequence that has 1, 2, or 3, amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 186.

In some embodiments, the peptide comprises the amino acid sequence according to SEQ ID NO: 24.

In some embodiments, the peptide comprises the amino acid sequence according to SEQ ID NO: 181.

In some embodiments, the peptide comprises the amino acid sequence according to SEQ ID NO: 186.

In some embodiments, the anti-TROP2 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-TROP2 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-TROP2 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-TROP2 binding domain comprises amino acid sequences according to SEQ ID NOs: 21-22. 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 A10m3 or a fragment thereof. In some embodiments, the single domain antibody is DOM7r-31. In some embodiments, the single domain antibody is DOM7h-11-15. In some embodiments, the single domain antibody is Alb-1, Alb-8, or Alb-23. In some embodiments, the single domain antibody is 10E. In some embodiments, the single domain antibody comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the single domain antibody comprise: HC-CDR1: SEQ ID NO: 66, HC-CDR2: SEQ ID NO: 67, and HC-CDR3: SEQ ID NO: 68. 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: 70, HC-CDR2: SEQ ID NO: 71, and HC-CDR3: SEQ ID NO: 72. In some embodiments, the single domain antibody comprises an amino acid sequence according to SEQ ID NO: 73. 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: 62), (GGGS)_n(SEQ ID NO: 63), (GGGGS)_n(SEQ ID NO: 64), and (GSSGGS)_n(SEQ ID NO: 65), wherein n is an integer of at least 1. In some embodiments, L₃comprises an amino acid sequence according to SEQ ID NO: 30.

Methods of Treatment

In some embodiments, are methods of treating cancer in a subject need in need thereof comprising administering to the subject an isolated polypeptide or polypeptide complex as described herein. In some embodiments, the cancer has cells that express TROP2. In some instances, the cancer is a solid tumor cancer. In some embodiments, the cancer is lung, breast (e.g. HER2+; ER/PR+; TNBC), cervical, ovarian, colorectal, pancreatic or gastric.

In some embodiments, are methods of treating triple-negative breast cancer (TNBC), urothelial cancer (UC), non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), gastric cancer, esophageal cancer, head and neck cancer, prostate cancer, or endometrial cancer in a subject need in need thereof comprising administering to the subject an isolated polypeptide or polypeptide complex as described herein. In some embodiments, are methods of treating breast cancer, lung cancer, urothelial cancer, endometrial cancer, ovarian cancer, prostate cancer, pancreatic cancer, gastric cancer, colon cancer, head and neck cancer, and glioma in a subject need in need thereof comprising administering to the subject an isolated polypeptide or polypeptide complex as described herein.

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, 119 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 isolated polypeptide or polypeptide complexes administered as once weekly. In some embodiments, the isolated polypeptide or polypeptide complexes are administered once weekly by intravenous, intramuscular, intralesional, topical, subcutaneous, infusion, or oral. In some embodiments, the isolated polypeptide or polypeptide complexes are administered once weekly by bolus injection. In some embodiments, the isolated 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 TROP2 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 fragment thereof 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 TECH 11(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 Pichi pastoris vector, pGAPZA, B, & C Pichia pastoris vector, pPIC3.5K Pichia vector, pPIC6 A, B, & C Pichia vector, pPIC9K Pichia vector, pTEF1/Zeo, pYES2 yeast vector, pYES2/CT yeast vector, pYES2/NT A, B, & C yeast vector, or pYES3/CT yeast vector.

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, p3×FLAG-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, p3×FLAG-CMV 7.1, pFLAG-CMV 20, p3×FLAG-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, p3×FLAG-CMV 9, p3×FLAG-CMV 13, pFLAG-Myc-CMV 21, p3×FLAG-Myc-CMV 25, pFLAG-CMV 4, p3×FLAG-CMV 10, p3×FLAG-CMV 14, pFLAG-Myc-CMV 22, p3×FLAG-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, Stbl2™, Stbl3™, or Stbl4™.

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 baili, Cryptococcus neoformans, Cryptococcus gattii, or Saccharomyces boulardii.

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

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 PPC7942.

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 TROP2 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 TROP2.

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

Embodiment 3 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 1-2, wherein the first antigen recognizing molecule comprises an antibody or antibody fragment that is human or humanized.

Embodiment 4 comprises an 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 an isolated polypeptide or polypeptide complex of any one of embodiments 1-4, wherein A₂is bound to C-terminus of the first antigen recognizing molecule.

Embodiment 6 comprises an 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 an isolated polypeptide or polypeptide complex of any one of embodiments 1-3 and 6, wherein A₂is bound to N-terminus of the first antigen recognizing molecule.

Embodiment 8 comprises an isolated polypeptide or polypeptide complex of embodiment 2, wherein the antibody or antibody fragment comprises a single chain variable fragment, a single domain antibody, or a Fab fragment.

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

Embodiment 10 comprises an 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 an isolated polypeptide or polypeptide complex of embodiment 8, wherein A₁is the single domain antibody.

Embodiment 12 comprises an isolated polypeptide or polypeptide complex of embodiment 2, 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 an isolated polypeptide or polypeptide complex of any one of embodiments 1-10 and 12, wherein A₁comprises an anti-CD3e single chain variable fragment.

Embodiment 14 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 1-10 and 12-13, wherein A₁comprises an anti-CD3e single chain variable fragment that has a K_Dbinding of 1 μM or less to CD3 on CD3 expressing cells.

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

Embodiment 16 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 1-15, wherein A₁comprises a variable light chain and variable heavy chain each of which is capable of specifically binding to human CD3.

Embodiment 17 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 1-16, 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 an isolated polypeptide or polypeptide complex of any one of embodiments 1-17, 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 an isolated polypeptide or polypeptide complex of any one of embodiments 1-17, 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 an isolated polypeptide or polypeptide complex of any one of embodiments 18-19, wherein the effector cell is a T cell.

Embodiment 21 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 18-20, wherein A₁binds to a polypeptide that is part of a TCR-CD3 complex on the effector cell.

Embodiment 22 comprises an isolated polypeptide or polypeptide complex of embodiment 21, wherein the polypeptide that is part of the TCR-CD3 complex is human CD3ε.

Embodiment 23 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 9-10 and 12-22, wherein the effector cell antigen comprises CD3, wherein 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 comprise: HC-CDR1: SEQ ID NO: 1, HC-CDR2: SEQ ID NO: 2, and HC-CDR3: SEQ ID NO: 3; and 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 comprise LC-CDR1: SEQ ID NO: 4, LC-CDR2: SEQ ID NO:5, and LC-CDR3: SEQ ID NO: 6.

Embodiment 24 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 1-23, wherein the effector cell antigen comprises CD3, wherein 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: 1, HC-CDR2: SEQ ID NO: 2, and HC-CDR3: SEQ ID NO: 3; 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: 4, LC-CDR2: SEQ ID NO:5, and LC-CDR3: SEQ ID NO: 6.

Embodiment 25 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 9-10 and 12-22, wherein the effector cell antigen comprises CD3, and the scFv comprises an amino acid sequence according to SEQ ID NO: 13.

Embodiment 26 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 9-10 and 12-22, wherein the effector cell antigen comprises CD3, wherein 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 comprise: HC-CDR1: SEQ ID NO: 7, HC-CDR2: SEQ ID NO: 8, and HC-CDR3: SEQ ID NO: 9; and 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 comprise LC-CDR1: SEQ ID NO: 10, LC-CDR2: SEQ ID NO: 11, and LC-CDR3: SEQ ID NO: 12.

Embodiment 27 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 1-22, wherein the effector cell antigen comprises CD3, wherein 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: 7, HC-CDR2: SEQ ID NO: 8, and HC-CDR3: SEQ ID NO: 9; 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: 10, LC-CDR2: SEQ ID NO: 11, and LC-CDR3: SEQ ID NO: 12.

Embodiment 28 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 9-10 and 12-22, wherein the effector cell antigen comprises CD3, and the scFv comprises an amino acid sequence according to SEQ ID NO: 14.

Embodiment 29 comprises an 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 an 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 an isolated polypeptide or polypeptide complex of embodiment 30, 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 an isolated polypeptide or polypeptide complex of any one of embodiments 29-31, wherein the antibody or antibody fragment thereof is humanized or human.

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

Embodiment 34 comprises an 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 an isolated polypeptide or polypeptide complex of any one of embodiments 33-34, wherein 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: 15, HC-CDR2: SEQ ID NO: 16, and HC-CDR3: SEQ ID NO: 17; and 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: 18, LC-CDR2: SEQ ID NO: 19, and LC-CDR3: SEQ ID NO: 20.

Embodiment 36 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 1-35, wherein 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: 15, HC-CDR2: SEQ ID NO: 16, and HC-CDR3: SEQ ID NO: 17; and 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: 18, LC-CDR2: SEQ ID NO: 19, and LC-CDR3: SEQ ID NO: 20.

Embodiment 37 comprises an 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 an isolated polypeptide or polypeptide complex of embodiment 34 and 37, wherein Fab heavy chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 22.

Embodiment 39 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 34 and 37-38, wherein the Fab light chain polypeptide of A₂is bound to a C-terminus of the single chain variable fragment (scFv) of A₁.

Embodiment 40 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 34 and 37-38, wherein the Fab heavy chain polypeptide of A₂is bound to a C-terminus of the single chain variable fragment (scFv) A₁.

Embodiment 41 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 34, 37-38 and 40, wherein the Fab light chain polypeptide of A₂is bound to a N-terminus of the single chain variable fragment (scFv) of A₁.

Embodiment 42 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 34 and 37-39, wherein the Fab heavy chain polypeptide of A₂is bound to a N-terminus of the single chain variable fragment (scFv) A₁.

Embodiment 43 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 34-38, wherein the Fab heavy chain polypeptide of A₂is bound to the scFv heavy chain polypeptide of A₁.

Embodiment 44 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 34-38, wherein the Fab light chain polypeptide of A₂is bound to the scFv heavy chain polypeptide of A₁.

Embodiment 45 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 34-48, wherein the Fab heavy chain polypeptide of A₂is bound to the scFv light chain polypeptide of A₁.

Embodiment 46 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 34-38, wherein the Fab light chain polypeptide of A₂is bound to the scFv light chain polypeptide of A₁.

Embodiment 47 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 1-46, 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 48 comprises an isolated polypeptide or polypeptide complex of embodiment 47, wherein the isolated polypeptide or polypeptide complex is according to Formula Ia: P₂-L₂-A₂-A₁-L₁-P₁-H₁.

Embodiment 49 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 47-48, 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 50 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 47-48, 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 51 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 47-48, 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 52 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 47-48, 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 53 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 1-52, wherein P₁impairs binding of A₁to the effector cell antigen.

Embodiment 54 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 1-53, wherein P₁is bound to A₁through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bonding interactions, or a combination thereof.

Embodiment 55 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 1-54, wherein P₁has less than 70% sequence homology to the effector cell antigen.

Embodiment 56 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 47-55, wherein P₂impairs binding of A₂to TROP2.

Embodiment 57 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 47-56, wherein P₂is bound to A₂through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bonding interactions, or a combination thereof.

Embodiment 58 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 47-57, wherein P₂is bound to A₂at or near an antigen binding site.

Embodiment 59 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 47-58, wherein P₂has less than 70% sequence homology to TROP2.

Embodiment 60 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 47-59, wherein P₁or P₂comprises a peptide sequence of at least 10 amino acids in length.

Embodiment 61 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 47-60, wherein P₁or P₂comprises a peptide sequence of at least 10 amino acids in length and no more than 20 amino acids in length.

Embodiment 62 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 47-59, wherein P₁or P₂comprises a peptide sequence of at least 16 amino acids in length.

Embodiment 63 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 47-60 and 62, wherein P₁or P₂comprises a peptide sequence of no more than 40 amino acids in length.

Embodiment 64 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 47-63, wherein P₁or P₂comprises at least two cysteine amino acid residues.

Embodiment 65 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 47-64, wherein P₁or P₂comprises a cyclic peptide or a linear peptide.

Embodiment 66 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 47-65, wherein P₁or P₂comprises a cyclic peptide.

Embodiment 67 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 47-65, wherein P₁or P₂comprises a linear peptide.

Embodiment 68 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 1-67, wherein P₁comprises at least two cysteine amino acid residues.

Embodiment 69 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 1-68, wherein P₁comprises an amino acid sequence according to SEQ ID NO: 26, 27, or 122.

Embodiment 70 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 47-68, wherein P₂comprises an amino acid sequence according to any one of SEQ ID NOs: 23-25.

Embodiment 71 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 47-68, wherein P₂comprises an amino acid sequence according to X1-X₂-X₃-X₄-C-X₆-X₇-X₈-X₉-X₁₀-C-X₁₂-X₁₃-X₁₄and X₁is selected from N, D, S, Y, A, F, H, T, L, and V; X₂is selected from S, T, D, A, H, V, Y, N, F, I, and L; X₃is selected from L, I, and V; X₄is selected from F, L, V, M, W, I, Y, and H; X₆is selected from V, F, L, I, and W; X₇is selected from K, R, Q, N, H, and M; X₈is selected from N and K; X₉is selected from L, V, and I; X₁₀is selected from Y, W, F, Q, and L; X₁₂is selected from W and V; X₁₃is selected from I, N, H, T, V, Y, and D; X₁₄is selected from D, V, A, S, I, T, N, Y, H, and P.

Embodiment 72 comprises an isolated polypeptide or polypeptide complex of embodiment 71, wherein X₁is selected from N, D, S, Y, A, F, and T; X₂is selected from S, T, D, A, H, V, Y, and N; X₃is L; X₄is selected from F, L, V, M, and W; X₆is selected from V, F, and L; X₇is selected from K, R, Q, and N; X₈is selected from N and K; X₉is selected from L, V, and I; X₁₀is selected from Y, W, and F; X₁₂is W; X₁₃is selected from I, N, H, and T; X₁₄is selected from D, V, A, S, I, T, and N.

Embodiment 73 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 71-72, wherein X₁is selected from N, D, S, and Y; X₂is selected from S, T, and D; X₃is L; X₄is selected from F, L, and V; X₆is selected from V and F; X₇is selected from K, R, and Q; X₈is N; X₉is selected from L and V; X₁₀is selected from Y and W; X₁₂is W; X₁₃is selected from I, N, and H; X₁₄is selected from D, V, A, and S.

Embodiment 74 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 47-68, wherein P₂comprises an amino acid sequence according to an amino acid sequence according to J₁-J₂-J₃-C-J₅-J₆-J₇-J₈-W-J₁₀-J₁₁-C-J₁₃-J₁₄and J₁is selected from V, I, L, P, E, F, and M; J₂is selected from D and N; J₃is selected from F and W; J₈is selected from A, E, S, R, K, Y, L, Q, G, M, F, T, W, and D; J₆is selected from L, M, I, V, F, T, R, and S; J₇is selected from Y, F, and N; J₈is selected from N, R, D, H, K, Q, S, G, A, E, and M; J₁₀is selected from P and R; J₁₁is selected from V and I; J₁₃is selected from D, G, N, R, S, Q, Y, T, A, E, L, V, K, M, I, H, F, and W; J₁₄is selected from T, S, Q, L, D, N, A, E, K, M, V, R, I, H, P, V, and W; or P₂comprises an amino acid sequence according to B₁-B₂-B₃-C-B₈-B₆-B₇-B₈-W-B₁₀-B₁₁-C-B₁₃-B₁₄and B₁is selected from V, I, L, P, E, F, and M; B₂is selected from D and N; B₃is selected from F and W; B₈is selected from A, E, S, R, K, Y, L, Q, G, M, F, T, W, and D; B₆is selected from L, M, I, V, F, T, R, and S; B₇is selected from Y, F, and N; B₈is selected from N, R, D, H, K, Q, S, G, A, E, and M; B₁₀is selected from P and R; B₁₁is selected from V and I; B₁₃is selected from D, G, N, R, S, Q, Y, T, A, E, L, V, K, M, I, H, F, and W; B₁₄is selected from T, S, Q, L, D, N, A, E, K, M, V, R, I, H, P, V, G, and W.

Embodiment 75 comprises an isolated polypeptide or polypeptide complex of embodiment 74, wherein J₁is selected from V, I, and L; J₂is D; J₃is F; J₈is selected from A, E, S, R, K, Y, and L; J₆is selected from L, M, and I; J₇is Y; J₈is selected from N, R, D, H, K, Q, S, and G; J₁₀is P; J₁, is selected from V and I; J₁₃is selected from D, G, N, R, S, Q, Y, T, and A; J₁₄is selected from T, S, Q, L, D, N, A, and E; and B₁is selected from V, I, and L; B₂is D; B₃is F; B₈is selected from A, E, S, R, K, Y, M, G, and L; B₆is selected from L, M, and I; B₇is Y; B₈is selected from N, R, D, H, K, Q, S, and G; B₁₀is P; B₁₁is selected from V and I; B₁₃is selected from D, G, N, R, S, Q, Y, T, H, and A; B₁₄is selected from T, S, Q, L, D, N, A, G, and E.

Embodiment 76 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 74-75, wherein J₁is selected from V, I, and L; J₂is D; J₃is F; J₈is selected from A, E, S, and R; J₆is selected from L, M, and I; J₇is Y; J₈is selected from N, R, and D; J₁₀is P; J₁₁is selected from V and I; J₁₃is selected from D, G, N, R, S, and Q; J₁₄is selected from T, S, Q, and L; and B₁is selected from V, I, and L; B₂is D; B₃is F; B₈is selected from A, E, S, K, M, G, and R; B₆is selected from L, M, and I; B₇is Y; B₈is selected from N, R, S, H, and D; B₁₀is P; B₁₁is selected from V and I; B₁₃is selected from D, G, N, R, S, H, A, Y, and Q; B₁₄is selected from T, S, Q, G, and L.

Embodiment 77 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 47-68, wherein P₂comprises the amino acid sequences according to SEQ ID NOs: 133-145.

Embodiment 78 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 47-68, wherein P₂comprises the amino acid sequences according to SEQ ID NOs: 146-158.

Embodiment 79 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 47-68, wherein P₂comprises an amino acid sequence according to any of the sequences of Table 27.

Embodiment 80 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 71-73, or 79, wherein P₂comprises the amino acid sequences according to SEQ ID NOs: 159-178.

Embodiment 81 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 47-68, wherein P₂comprises an amino acid sequence according to any of the sequences of Table 29.

Embodiment 82 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 74-76, or 81, wherein P₂comprises the amino acid sequences according to SEQ ID NOs: 179-201.

Embodiment 83 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 74-76, or 81, wherein P₂comprises the amino acid sequence according to SEQ ID NO: 24 or a peptide sequence that has 1, 2, or 3, amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 24.

Embodiment 84 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 74-76, or 81, wherein P₂comprises the amino acid sequence according to SEQ ID NO: 181 or a peptide sequence that has 1, 2, or 3, amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 181.

Embodiment 85 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 74-76, or 81, wherein P₂comprises the amino acid sequence according to SEQ ID NO: 186 or a peptide sequence that has 1, 2, or 3, amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 186.

Embodiment 86 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 74-76, or 81, wherein P₂comprises the amino acid sequence according to SEQ ID NO: 24.

Embodiment 87 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 74-76, or 81, wherein P₂comprises the amino acid sequence according to SEQ ID NO: 181.

Embodiment 88 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 74-76, or 81, wherein P₂comprises the amino acid sequence according to SEQ ID NO: 186.

Embodiment 89 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 1-68 and 70-88, 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; Z₁₄is selected from D, Y, N, F, I, P, V, A, T, H, L and S; or 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; U₁₄is selected from D, Y, N, F, I, P, V, A, T, H, L, M, and S.

Embodiment 90 comprises an isolated polypeptide or polypeptide complex of embodiment 89, 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; Z₁is selected from I, Y, F, V, L, and T; Z₁₂is selected from F, D, Y, L, I, V, A, and N; Z₁₄is selected from D, Y, N, F, I, and P; and 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; U₁₄is selected from D, Y, N, F, I, M, and P.

Embodiment 91 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 89-90, wherein Z₁is selected 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; Z₁is selected from I, Y, and F; Z₁₂is selected from F, D, Y, and L; Z₁₄is selected from D, Y, and N; and 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; U₁₄is selected from D, Y, M, and N.

Embodiment 92 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 1-68 and 70-88, wherein P₁comprises the amino acid sequences according to SEQ ID NOs: 202-228.

Embodiment 93 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 1-68 and 70-88, wherein P₁comprises an amino acid sequences according to any of the sequences of Table 35.

Embodiment 94 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 89-91 or 93, wherein P₁comprises the amino acid sequences according to SEQ ID NOs: 229-240.

Embodiment 95 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 89-91 or 93, wherein P₁comprises the amino acid sequence according to SEQ ID NO: 239 or a peptide sequence that has 1, 2, or 3, amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 239.

Embodiment 96 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 89-91 or 93, wherein P₁comprises the amino acid sequence according to SEQ ID NO: 27 or a peptide sequence that has 1, 2, or 3, amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 27.

Embodiment 97 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 1-68 and 70-88, wherein P₁comprises the amino acid sequence according to SEQ ID NO: 26 or a peptide sequence that has 1, 2, or 3, amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 26.

Embodiment 98 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 89-91 or 93, wherein P₁comprises the amino acid sequence according to SEQ ID NO: 239.

Embodiment 99 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 89-91 or 93, wherein P₁comprises the amino acid sequence according to SEQ ID NO: 27.

Embodiment 100 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 1-68 and 70-88, wherein P₁comprises the amino acid sequence according to SEQ ID NO: 26.

Embodiment 101 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 1-5, 8-40, and 43-100, wherein L₁is bound to N-terminus of A₁.

Embodiment 102 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 1-3, 6-38, and 41-100, wherein L₁is bound to C-terminus of A₁.

Embodiment 103 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 1-38, and 41-100, wherein L₂is bound to N-terminus of A₂.

Embodiment 104 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 1-40, and 43-100, wherein L₂is bound to C-terminus of A₂.

Embodiment 105 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 1-104, wherein L₁or L₂is a peptide sequence having at least 5 to no more than 50 amino acids.

Embodiment 106 comprises an 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 107 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 1-106, wherein L₁or L₂is a peptide sequence having at least 10 amino acids.

Embodiment 108 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 1-107, wherein L₁or L₂is a peptide sequence having at least 18 amino acids.

Embodiment 109 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 1-108, wherein L₁or L₂is a peptide sequence having at least 26 amino acids.

Embodiment 110 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 1-109, wherein L₁or L₂has a formula comprising (G₂S)_n(SEQ ID NO: 243), wherein n is an integer from 1 to 3.

Embodiment 111 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 1-109, wherein L₁has a formula selected from the group consisting of (G₂S)_n, (GS)_n, (GSGGS)_n(SEQ ID NO: 62), (GGGS)_n(SEQ ID NO: 63), (GGGGS)_n(SEQ ID NO: 64), and (GSSGGS)_n(SEQ ID NO: 65), wherein n is an integer of at least 1.

Embodiment 112 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 1-111, wherein P₁becomes unbound from A₁when L₁is cleaved by the tumor specific protease thereby exposing A₁to the effector cell antigen.

Embodiment 113 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 1-112, wherein P₂becomes unbound from A₂when L₂is cleaved by the tumor specific protease thereby exposing A₂to TROP2.

Embodiment 114 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 1-113, 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 115 comprises an isolated polypeptide or polypeptide complex of embodiment 114, wherein the matrix metalloprotease comprises MMP2, MMP7, MMP9, MMP13, or MMP14.

Embodiment 116 comprises an isolated polypeptide or polypeptide complex of embodiment 114, wherein the serine protease comprises matriptase (MTSP1), urokinase, or hepsin.

Embodiment 117 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 1-116, 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 118 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 1-117, wherein L₁or L₂comprises an amino acid sequence according to SEQ ID NO: 31 or 32.

Embodiment 119 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 1-117, wherein L₁or L₂comprises an amino acid sequence according to SEQ ID NO: 58 or 59.

Embodiment 120 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 1-117, wherein L₁or L₂comprises an amino acid sequence according to any one of SEQ ID NOs: 28-61.

Embodiment 121 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 1-117, wherein L₁or L₂comprises an amino acid sequence of Linker 25 (ISSGLLSGRSDAG) (SEQ ID NO: 54), Linker 26 (AAGLLAPPGGLSGRSDAG) (SEQ ID NO: 55), Linker 27 (SPLGLSGRSDAG) (SEQ ID NO: 56), or Linker 28 (LSGRSDAGSPLGLAG) (SEQ ID NO: 57), 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 25, Linker 26, Linker 27, or Linker 28.

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

Embodiment 123 comprises an isolated polypeptide or polypeptide complex of embodiment 122, wherein the polymer is polyethylene glycol (PEG).

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

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

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

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

Embodiment 128 comprises an 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 an 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 an 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 an isolated polypeptide or polypeptide complex of any one of embodiments 129, wherein the circulating immunoglobulin molecule comprises IgG1, IgG2, IgG3, IgG4, slgA, IgM or IgD.

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

Embodiment 133 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 128-132, wherein the polypeptide is an antibody.

Embodiment 134 comprises an 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 an 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 an isolated polypeptide or polypeptide complex of any one of embodiments 134-135, wherein the single domain antibody is a human or humanized antibody.

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

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

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

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

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

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

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

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

Embodiment 145 comprises an 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: 66, HC-CDR2: SEQ ID NO: 67, and HC-CDR3: SEQ ID NO: 68.

Embodiment 146 comprises an isolated polypeptide or polypeptide complex of embodiment 134, wherein the single domain antibody comprises an amino acid sequence according to SEQ ID NO: 69.

Embodiment 147 comprises an 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: 70, HC-CDR2: SEQ ID NO: 71, and HC-CDR3: SEQ ID NO: 72.

Embodiment 148 comprises an isolated polypeptide or polypeptide complex of embodiment 134, wherein the single domain antibody comprises an amino acid sequence according to SEQ ID NO: 73.

Embodiment 149 comprises an isolated polypeptide or polypeptide complex of embodiment 134, wherein the single domain antibody is SA21.

Embodiment 150 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 1-149, 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 151 comprises an isolated polypeptide or polypeptide complex of embodiment 150, wherein the modified amino acid or modified non-natural amino acid comprises a post-translational modification.

Embodiment 152 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 1-151, wherein H₁comprises a linking moiety (L₃) that connects H₁to P₁.

Embodiment 153 comprises an isolated polypeptide or polypeptide complex of embodiment 152, wherein L₃is a peptide sequence having at least 5 to no more than 50 amino acids.

Embodiment 154 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 152-153, wherein L₃is a peptide sequence having at least 10 to no more than 30 amino acids.

Embodiment 155 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 152-154, wherein L₃is a peptide sequence having at least 10 amino acids.

Embodiment 156 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 152-155, wherein L₃is a peptide sequence having at least 18 amino acids.

Embodiment 157 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 152-156, wherein L₃is a peptide sequence having at least 26 amino acids.

Embodiment 158 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 152-157, wherein L₃has a formula selected from the group consisting of (G₂S)_n, (GS)_n, (GSGGS)_n(SEQ ID NO: 62), (GGGS)_n(SEQ ID NO: 63), (GGGGS)_n(SEQ ID NO: 64), and (GSSGGS)_n(SEQ ID NO: 65), wherein n is an integer of at least 1.

Embodiment 159 comprises an isolated polypeptide or polypeptide complex of embodiment 152, wherein L₃comprises an amino acid sequence according to SEQ ID NO: 30.

Embodiment 160 comprises an 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 NOs: 74-132, 241-242.

Embodiment 161 comprises an 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: 82.

Embodiment 162 comprises an 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: 83.

Embodiment 163 comprises an 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: 74 and SEQ ID NO: 75.

Embodiment 164 comprises an 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: 76 and SEQ ID NO: 77.

Embodiment 165 comprises an 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: 78 and SEQ ID NO: 79.

Embodiment 166 comprises an 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: 80 and SEQ ID NO: 81.

Embodiment 167 comprises an 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: 82 and SEQ ID NO: 83.

Embodiment 168 comprises an 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: 84 and SEQ ID NO: 85.

Embodiment 169 comprises an 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: 86 and SEQ ID NO: 87.

Embodiment 170 comprises an 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: 88 and SEQ ID NO: 89.

Embodiment 171 comprises an 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: 90 and SEQ ID NO: 91.

Embodiment 172 comprises an 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: 92 and SEQ ID NO: 93.

Embodiment 173 comprises an 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: 94 and SEQ ID NO: 95.

Embodiment 174 comprises an 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: 96 and SEQ ID NO: 97.

Embodiment 175 comprises an 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: 98 and SEQ ID NO: 99.

Embodiment 176 comprises an 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: 100 and SEQ ID NO: 101.

Embodiment 177 comprises an 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: 102 and SEQ ID NO: 103.

Embodiment 178 comprises an 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: 104 and SEQ ID NO: 105.

Embodiment 179 comprises an 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: 106 and SEQ ID NO: 107.

Embodiment 180 comprises an 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: 108 and SEQ ID NO: 109.

Embodiment 181 comprises an 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: 110 and SEQ ID NO: 111.

Embodiment 182 comprises an 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: 112 and SEQ ID NO: 113.

Embodiment 183 comprises an 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: 114 and SEQ ID NO: 115.

Embodiment 184 comprises an 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: 116 and SEQ ID NO: 117.

Embodiment 185 comprises an 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: 118 and SEQ ID NO: 119.

Embodiment 186 comprises an 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: 120 and SEQ ID NO: 121.

Embodiment 187 comprises an 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: 123 and SEQ ID NO: 124.

Embodiment 188 comprises an 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: 125 and SEQ ID NO: 126.

Embodiment 189 comprises an 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: 127 and SEQ ID NO: 128.

Embodiment 190 comprises an 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: 129 and SEQ ID NO: 130.

Embodiment 191 comprises an 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: 241 and SEQ ID NO: 131.

Embodiment 192 comprises an 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: 132 and SEQ ID NO: 242.

Embodiment 193 comprises a pharmaceutical composition comprising: (a) the isolated polypeptide or polypeptide complex of any one of embodiments 1-193; and (b) a pharmaceutically acceptable excipient.

Embodiment 194 comprises an isolated recombinant nucleic acid molecule encoding the isolated polypeptide or polypeptide complex of any one of embodiments 1-Error! Reference source not found.

Embodiment 195 comprises an isolated polypeptide or polypeptide complex according to Formula II: L_1a-P_1a-H₁a 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 TROP2; 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 196 comprises an isolated polypeptide or polypeptide complex of embodiment 195, wherein P_1awhen L_1ais uncleaved impairs binding of the first antigen recognizing molecule to the effector cell antigen.

Embodiment 197 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 195-196, wherein the first antigen recognizing molecule comprises an antibody or antibody fragment.

Embodiment 198 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 195-197, wherein the effector cell antigen is an anti-CD3 effector cell antigen.

Embodiment 199 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 195-198, wherein P_1ahas less than 70% sequence homology to the effector cell antigen.

Embodiment 200 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 195-199, wherein P_1acomprises a peptide sequence of at least 10 amino acids in length.

Embodiment 201 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 195-200, wherein P_1acomprises a peptide sequence of at least 10 amino acids in length and no more than 20 amino acids in length.

Embodiment 202 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 195-201, wherein P_1acomprises a peptide sequence of at least 16 amino acids in length.

Embodiment 203 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 195-200 and 202, wherein P_1acomprises a peptide sequence of no more than 40 amino acids in length.

Embodiment 204 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 195-203, wherein P_1acomprises at least two cysteine amino acid residues.

Embodiment 205 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 195-204, wherein P_1acomprises a cyclic peptide or a linear peptide.

Embodiment 206 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 195-205, wherein P_1acomprises a cyclic peptide.

Embodiment 207 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 195-205, wherein P_1acomprises a linear peptide.

Embodiment 208 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 195-201 and 203-207, wherein P_1acomprises an amino acid sequence selected from the group consisting of SEQ ID NO: 26, 27, or 122.

Embodiment 209 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 195-201 and 203-207, 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; Z₁₄is selected from D, Y, N, F, I, P, V, A, T, H, L and S; or 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; U₁₄is selected from D, Y, N, F, I, P, V, A, T, H, L, M, and S.

Embodiment 210 comprises an isolated polypeptide or polypeptide complex of embodiment 209, 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; Z₁₄is selected from D, Y, N, F, I, and P; and 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; U₁₄is selected from D, Y, N, F, I, M, and P.

Embodiment 211 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 209-210, 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; Z₁is selected from I, Y, and F; Z₁₂is selected from F, D, Y, and L; Z₁₄is selected from D, Y, and N; and U₁is selected 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; U₁₄is selected from D, Y, M, and N.

Embodiment 212 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 195-201 and 203-207, wherein P_1acomprises the amino acid sequences according to SEQ ID NOs: 202-228.

Embodiment 213 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 195-201 and 203-207, wherein P_1acomprises an amino acid sequence according to any of the sequences of Table 35.

Embodiment 214 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 209-211 or 213, wherein P_1acomprises the amino acid sequences according to SEQ ID NOs: 229-240.

Embodiment 215 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 209-211 or 213, wherein P_1acomprises the amino acid sequence according to SEQ ID NO: 239 or a peptide sequence that has 1, 2, or 3, amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 239.

Embodiment 216 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 209-211 or 213, wherein P_1acomprises the amino acid sequence according to SEQ ID NO: 27 or a peptide sequence that has 1, 2, or 3, amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 27.

Embodiment 216 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 195-201 and 203-207 wherein P_1acomprises the amino acid sequence according to SEQ ID NO: 26 or a peptide sequence that has 1, 2, or 3, amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 26.

Embodiment 218 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 209-211 or 213, wherein P_1acomprises the amino acid sequence according to SEQ ID NO: 239.

Embodiment 219 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 209-211 or 213, wherein P_1acomprises the amino acid sequence according to SEQ ID NO: 27.

Embodiment 220 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 195-201 and 203-207, wherein P_1acomprises the amino acid sequence according to SEQ ID NO: 26.

Embodiment 221 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 195-220, wherein H_1acomprises a polymer.

Embodiment 222 comprises an isolated polypeptide or polypeptide complex of embodiment 221, wherein the polymer is polyethylene glycol (PEG).

Embodiment 223 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 195-221, wherein H_1acomprises albumin.

Embodiment 224 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 195-221, wherein H_1acomprises an Fc domain.

Embodiment 225 comprises an isolated polypeptide or polypeptide complex of embodiment 223, wherein the albumin is serum albumin.

Embodiment 226 comprises an isolated polypeptide or polypeptide complex of embodiment 223, wherein the albumin is human serum albumin.

Embodiment 227 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 195-220, wherein H_1acomprises a polypeptide, a ligand, or a small molecule.

Embodiment 228 comprises an isolated polypeptide or polypeptide complex of embodiment 227, 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 229 comprises an isolated polypeptide or polypeptide complex of embodiment 228, 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 230 comprises an isolated polypeptide or polypeptide complex of embodiment 228, wherein the circulating immunoglobulin molecule comprises IgG1, IgG2, IgG3, IgG4, slgA, IgM or IgD.

Embodiment 231 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 228-229, wherein the serum protein is albumin.

Embodiment 232 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 227-231, wherein the polypeptide is an antibody.

Embodiment 233 comprises an isolated polypeptide or polypeptide complex of embodiment 232, wherein the antibody comprises a single domain antibody, a single chain variable fragment or a Fab.

Embodiment 234 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 232-233, wherein the antibody comprises a single domain antibody that binds to albumin.

Embodiment 235 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 232-234, wherein the antibody is a human or humanized antibody.

Embodiment 236 comprises an isolated polypeptide or polypeptide complex of embodiment 233, wherein the single domain antibody is 645gH1gL1.

Embodiment 237 comprises an isolated polypeptide or polypeptide complex of embodiment 233, wherein the single domain antibody is 645dsgH5gL4.

Embodiment 238 comprises an isolated polypeptide or polypeptide complex of embodiment 233, wherein the single domain antibody is 23-13-A01-sc02.

Embodiment 239 comprises an isolated polypeptide or polypeptide complex of embodiment 233, wherein the single domain antibody is A10m3 or a fragment thereof.

Embodiment 240 comprises an isolated polypeptide or polypeptide complex of embodiment 233, wherein the single domain antibody is DOM7r-31.

Embodiment 241 comprises an isolated polypeptide or polypeptide complex of embodiment 233, wherein the single domain antibody is DOM7h-11-15.

Embodiment 242 comprises an isolated polypeptide or polypeptide complex of embodiment 233, wherein the single domain antibody is Alb-1, Alb-8, or Alb-23.

Embodiment 243 comprises an isolated polypeptide or polypeptide complex of embodiment 233, wherein the single domain antibody is 10E.

Embodiment 244 comprises an isolated polypeptide or polypeptide complex of embodiment 233, 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: 66, HC-CDR2: SEQ ID NO: 67, and HC-CDR3: SEQ ID NO: 68.

Embodiment 245 comprises an isolated polypeptide or polypeptide complex of embodiment 233, wherein the single domain antibody comprises an amino acid sequence according to SEQ ID NO: 69.

Embodiment 246 comprises an isolated polypeptide or polypeptide complex of embodiment 233, 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: 70, HC-CDR2: SEQ ID NO: 71, and HC-CDR3: SEQ ID NO: 72.

Embodiment 247 comprises an isolated polypeptide or polypeptide complex of embodiment 233, wherein the single domain antibody comprises an amino acid sequence according to SEQ ID NO: 73.

Embodiment 248 comprises an isolated polypeptide or polypeptide complex of embodiment 233, wherein the single domain antibody is SA21.

Embodiment 249 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 195-248, wherein H_1acomprises a linking moiety (L_1a) that connects H_1ato Pia.

Embodiment 250 comprises an isolated polypeptide or polypeptide complex of embodiment 249, wherein L_1ais a peptide sequence having at least 5 to no more than 50 amino acids.

Embodiment 251 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 249-250, wherein L_1ais a peptide sequence having at least 10 to no more than 30 amino acids.

Embodiment 252 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 249-251, wherein L_1ais a peptide sequence having at least 10 amino acids.

Embodiment 253 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 249-252, wherein L_1ais a peptide sequence having at least 18 amino acids.

Embodiment 254 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 249-253, wherein L_1ais a peptide sequence having at least 26 amino acids.

Embodiment 255 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 249-254, wherein L_1ahas a formula selected from the group consisting of (G₂S)_n, (GS)_n, (GSGGS)_n(SEQ ID NO: 62), (GGGS)_n(SEQ ID NO: 63), (GGGGS)_n(SEQ ID NO: 64), and (GSSGGS)_n(SEQ ID NO: 65), wherein n is an integer of at least 1.

Embodiment 256 comprises an isolated polypeptide or polypeptide complex of embodiment 249, wherein L_1acomprises an amino acid sequence according to any one of SEQ ID NOs: 28-61.

Embodiment 257 comprises a polypeptide complex comprising a structural arrangement according to Configuration 1:

Embodiment 258 comprises a polypeptide complex comprising a structural arrangement according to Configuration 2:

wherein the polypeptide complex comprises a single chain variable fragment (scFv) comprising a light chain variable domain and a heavy chain variable domain, wherein the scFv further comprises a peptide that impairs binding of the scFv to an effector cell antigen and the peptide is linked to a N-terminus of the heavy chain variable domain of the scFv with a linking moiety that is a substrate for a tumor specific protease, and the peptide further comprises a half-life extending molecule; and a Fab that binds to TROP2, 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 further comprises P₂and L₂, wherein P₂comprises a peptide that impairs binding to TROP2; and L₂comprises a linking moiety that connects the Fab heavy chain polypeptide to P₂and is a substrate for a tumor specific protease.

Embodiment 259 comprises an isolated polypeptide or polypeptide complex comprising an anti-TROP2 binding domain that is linked to a peptide that impairs binding of the anti-TROP2 binding to TROP2 wherein the peptide comprises an amino acid sequence according to X₁-X₂-X₃-X₄-C-X₆-X₇-X₈-X₉-X₁₀-C-X₁₂-X₁₃-X₁₄and X₁is selected from N, D, S, Y, A, F, H, T, L, and V; X₂is selected from S, T, D, A, H, V, Y, N, F, I, and L; X₃is selected from L, I, and V; X₄is selected from F, L, V, M, W, I, Y, and H; X₆is selected from V, F, L, I, and W; X₇is selected from K, R, Q, N, H, and M; X₈is selected from N and K; X₉is selected from L, V, and I; X₁₀is selected from Y, W, F, Q, and L; X₁₂is selected from W and V; X₁₃is selected from I, N, H, T, V, Y, and D; X₁₄is selected from D, V, A, S, I, T, N, Y, H, and P; or the peptide comprises an amino acid according to J₁-J₂-J₃-C-J₅-J₆-J₇-J₈-W-J₁₀-J₁₁-C-J₁₃-J₁₄and J₁is selected from V, I, L, P, E, F, and M; J₂is selected from D and N; J₃is selected from F and W; J₅is selected from A, E, S, R, K, Y, L, Q, G, M, F, T, W, and D; J₆is selected from L, M, I, V, F, T, R, and S; J₇is selected from Y, F, and N; J₈is selected from N, R, D, H, K, Q, S, G, A, E, and M; J₁₀is selected from P and R; J₁₁is selected from V and I; J₁₃is selected from D, G, N, R, S, Q, Y, T, A, E, L, V, K, M, I, H, F, and W; J₁₄is selected from T, S, Q, L, D, N, A, E, K, M, V, R, I, H, P, V, and W; or the peptide comprises an amino acid sequence according to B₁-B₂-B₃-C-B₅-B₆-B₇-Bg-W-B₁₀-B₁₁-C-B₁₃-B₁₄and B₁is selected from V, I, L, P, E, F, and M; B₂is selected from D and N; B₃is selected from F and W; B₅is selected from A, E, S, R, K, Y, L, Q, G, M, F, T, W, and D; B₆is selected from L, M, I, V, F, T, R, and S; B₇is selected from Y, F, and N; B₈is selected from N, R, D, H, K, Q, S, G, A, E, and M; B₁₀is selected from P and R; B₁₁is selected from V and I; B₁₃is selected from D, G, N, R, S, Q, Y, T, A, E, L, V, K, M, I, H, F, and W; B₁₄is selected from T, S, Q, L, D, N, A, E, K, M, V, R, I, H, P, V, G, and W.

Embodiment 260 comprises an isolated polypeptide or polypeptide complex of embodiment 259, wherein X₁is selected from N, D, S, Y, A, F, and T; X₂is selected from S, T, D, A, H, V, Y, and N; X₃is L; X₄is selected from F, L, V, M, and W; X₆is selected from V, F, and L; X₇is selected from K, R, Q, and N; X₈is selected from N and K; X₉is selected from L, V, and I; X₁₀is selected from Y, W, and F; X₁₂is W; X₁₃is selected from I, N, H, and T; X₁₄is selected from D, V, A, S, I, T, and N.

Embodiment 261 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 259-260, wherein X₁is selected from N, D, S, and Y; X₂is selected from S, T, and D; X₃is L; X₄is selected from F, L, and V; X₆is selected from V and F; X₇is selected from K, R, and Q; X₈is N; X₉is selected from L and V; X₁₀is selected from Y and W; X₁₂is W; X₁₃is selected from I, N, and H; X₁₄is selected from D, V, A, and S.

Embodiment 262 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 259-261, wherein J₁is selected V, I, and L; J₂is D; J₃is F; J₈is selected from A, E, S, R, K, Y, and L; J₆is selected from L, M, and I; J₇is Y; J₈is selected from N, R, D, H, K, Q, S, and G; J₁₀is P; J₁₁is selected from V and I; J₁₃is selected from D, G, N, R, S, Q, Y, T, and A; J₁₄is selected from T, S, Q, L, D, N, A, and E.

Embodiment 263 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 259-262, wherein J₁is selected from V, I, and L; J₂is D; J₃is F; J₈is selected from A, E, S, and R; J₆is selected from L, M, and I; J₇is Y; J₈is selected from N, R, and D; J₁₀is P; J₁₁is selected from V and I; J₁₃is selected from D, G, N, R, S, and Q; J₁₄is selected from T, S, Q, and L.

Embodiment 264 comprises an isolated polypeptide or polypeptide complex of embodiment 259, wherein B₁is selected from V, I, and L; B₂is D; B₃is F; B₅is selected from A, E, S, R, K, Y, M, G, and L; B₆is selected from L, M, and I; B₇is Y; B₈is selected from N, R, D, H, K, Q, S, and G; B₁₀is P; B₁₁is selected from V and I; B₁₃is selected from D, G, N, R, S, Q, Y, T, H, and A; B₁₄is selected from T, S, Q, L, D, N, A, G, and E.

Embodiment 265 comprises an isolated polypeptide or polypeptide complex of embodiment 264, wherein B₁is selected from V, I, and L; B₂is D; B₃is F; B₅is selected from A, E, S, K, M, G, and R; B₆is selected from L, M, and I; B₇is Y; B₈is selected from N, R, S, H, and D; B₁₀is P; B₁₁is selected from V and I; B₁₃is selected from D, G, N, R, S, H, A, Y, and Q; B₁₄is selected from T, S, Q, G, and L.

Embodiment 266 comprises an isolated polypeptide or polypeptide complex of embodiment 259, wherein the peptide comprises an amino acid sequence according to any of the sequences of Table 27.

Embodiment 267 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 259-261, or 266, wherein the peptide comprises the amino acid sequences according to SEQ ID NOs: 159-178.

Embodiment 268 comprises an isolated polypeptide or polypeptide complex of embodiment 259, wherein the peptide comprises an amino acid sequences according to any of the sequences of Table 29.

Embodiment 269 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 259, 262-265, wherein the peptide comprises the amino acid sequences according to SEQ ID NOs: 179-201.

Embodiment 270 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 259, 264-265, wherein the peptide comprises the amino acid sequence according to SEQ ID NO: 24 or a peptide sequence that has 1, 2, or 3, amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 24.

Embodiment 271 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 259, 264-265, wherein the peptide comprises the amino acid sequence according to SEQ ID NO: 181 or a peptide sequence that has 1, 2, or 3, amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 181.

Embodiment 272 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 259, 264-265, wherein the peptide comprises the amino acid sequence according to SEQ ID NO: 186 or a peptide sequence that has 1, 2, or 3, amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 186.

Embodiment 273 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 259, 264-265, wherein the peptide comprises the amino acid sequence according to SEQ ID NO: 24.

Embodiment 274 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 259, 264-265, wherein the peptide comprises the amino acid sequence according to SEQ ID NO: 181.

Embodiment 275 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 259, 264-265, wherein the peptide comprises the amino acid sequence according to SEQ ID NO: 186.

Embodiment 276 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 259-275, wherein the anti-TROP2 binding domain comprises an antibody or an antibody fragment.

Embodiment 277 comprises an isolated polypeptide or polypeptide complex of embodiment 276, wherein the antibody or antibody fragment comprises a single chain variable fragment, a single domain antibody, Fab, or Fab′.

Embodiment 278 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 276-277, wherein the anti-TROP2 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-TROP2 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 279 comprises an isolated polypeptide or polypeptide complex of embodiment 277, wherein the antibody or antibody fragment comprises the Fab.

Embodiment 280 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 259-279, wherein the anti-TROP2 binding domain comprises amino acid sequences according to SEQ ID NOs: 21-22.

Embodiment 281 comprises an isolated polypeptide or polypeptide complex of embodiment 280, wherein the antibody or antibody fragment comprises the Fab or Fab′.

Embodiment 282 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 259-280, wherein the isolated polypeptide or polypeptide complex further comprises a half-life extending molecule (H₁).

Embodiment 283 comprises an isolated polypeptide or polypeptide complex of embodiment 282, wherein the half-life extending molecule is linked to the peptide.

Embodiment 284 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 282-283, wherein H₁comprises a polymer.

Embodiment 285 comprises an isolated polypeptide or polypeptide complex of embodiment 284, wherein the polymer is polyethylene glycol (PEG).

Embodiment 286 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 282-284, wherein H₁comprises albumin.

Embodiment 287 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 282-284, wherein H₁comprises an Fc domain.

Embodiment 288 comprises an isolated polypeptide or polypeptide complex of embodiment 286, wherein the albumin is serum albumin.

Embodiment 289 comprises an isolated polypeptide or polypeptide complex of embodiment 286, wherein the albumin is human serum albumin.

Embodiment 290 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 282-283, wherein H₁comprises a polypeptide, a ligand, or a small molecule.

Embodiment 291 comprises an isolated polypeptide or polypeptide complex of embodiment 290, 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 292 comprises an isolated polypeptide or polypeptide complex of embodiment 291, 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 293 comprises an isolated polypeptide or polypeptide complex of embodiment 291, wherein the circulating immunoglobulin molecule comprises IgG1, IgG2, IgG3, IgG4, slgA, IgM or IgD.

Embodiment 294 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 291-292, wherein the serum protein is albumin.

Embodiment 295 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 290-294, wherein the polypeptide is an antibody.

Embodiment 296 comprises an isolated polypeptide or polypeptide complex of embodiment 295, wherein the antibody comprises a single domain antibody, a single chain variable fragment, or a Fab.

Embodiment 297 comprises an isolated polypeptide or polypeptide complex of embodiment 296, wherein the single domain antibody comprises a single domain antibody that binds to albumin.

Embodiment 298 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 296-297, wherein the single domain antibody is a human or humanized antibody.

Embodiment 299 comprises an isolated polypeptide or polypeptide complex of embodiment 296, wherein the single domain antibody is 645gH1gL1.

Embodiment 300 comprises an isolated polypeptide or polypeptide complex of embodiment 296, wherein the single domain antibody is 645dsgH5gL4.

Embodiment 301 comprises an isolated polypeptide or polypeptide complex of embodiment 296, wherein the single domain antibody is 23-13-A01-sc02.

Embodiment 302 comprises an isolated polypeptide or polypeptide complex of embodiment 296, wherein the single domain antibody is A10m3 or a fragment thereof.

Embodiment 303 comprises an isolated polypeptide or polypeptide complex of embodiment 296, wherein the single domain antibody is DOM7r-31.

Embodiment 304 comprises an isolated polypeptide or polypeptide complex of embodiment 296, wherein the single domain antibody is DOM7h-11-15.

Embodiment 305 comprises an isolated polypeptide or polypeptide complex of embodiment 296, wherein the single domain antibody is Alb-1, Alb-8, or Alb-23.

Embodiment 306 comprises an isolated polypeptide or polypeptide complex of embodiment 296, wherein the single domain antibody is 10E.

Embodiment 307 comprises an isolated polypeptide or polypeptide complex of embodiment 296, 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: 66, HC-CDR2: SEQ ID NO: 67, and HC-CDR3: SEQ ID NO: 68.

Embodiment 308 comprises an isolated polypeptide or polypeptide complex of embodiment 296, wherein the single domain antibody comprises an amino acid sequence according to SEQ ID NO: 69.

Embodiment 309 comprises an isolated polypeptide or polypeptide complex of embodiment 296, 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: 70, HC-CDR2: SEQ ID NO: 71, and HC-CDR3: SEQ ID NO: 72.

Embodiment 310 comprises an isolated polypeptide or polypeptide complex of embodiment 296, wherein the single domain antibody comprises an amino acid sequence according to SEQ ID NO: 73.

Embodiment 311 comprises an isolated polypeptide or polypeptide complex of embodiment 296, wherein the single domain antibody is SA21.

Embodiment 312 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 259-311, 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 313 comprises an isolated polypeptide or polypeptide complex of embodiment 312, wherein the modified amino acid or modified non-natural amino acid comprises a post-translational modification.

Embodiment 314 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 282-313, wherein H₁comprises a linking moiety (L₃) that connects H₁to the peptide.

Embodiment 315 comprises an isolated polypeptide or polypeptide complex of embodiment 314, wherein L₃is a peptide sequence having at least 5 to no more than 50 amino acids.

Embodiment 316 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 314-315, wherein L₃is a peptide sequence having at least 10 to no more than 30 amino acids.

Embodiment 317 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 314-315, wherein L₃is a peptide sequence having at least 10 amino acids.

Embodiment 318 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 314-317, wherein L₃is a peptide sequence having at least 18 amino acids.

Embodiment 319 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 314-318, wherein L₃is a peptide sequence having at least 26 amino acids.

Embodiment 320 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 314-319, wherein L₃has a formula selected from the group consisting of (G₂S)_n, (GS)_n, (GSGGS)_n(SEQ ID NO: 62), (GGGS)_n(SEQ ID NO: 63), (GGGGS)_n(SEQ ID NO: 64), and (GSSGGS) (SEQ ID NO: 65), wherein n is an integer of at least 1.

Embodiment 321 comprises an isolated polypeptide or polypeptide complex of any one of embodiments 314-315, wherein L₃comprises an amino acid sequence according to SEQ ID NO: 30.

Embodiment 322 comprises a method of treating triple-negative breast cancer comprising administering to a subject in need thereof an isolated polypeptide or polypeptide complex according to Embodiments 1-192, or 257-321.

Embodiment 323 comprises a method of treating urothelial cancer comprising administering to a subject in need thereof an isolated polypeptide or polypeptide complex according to Embodiments 1-192, or 257-321.

Embodiment 324 comprises a method of treating non-small cell lung cancer (NSCLC) comprising administering to a subject in need thereof an isolated polypeptide or polypeptide complex according to Embodiments 1-192, or 257-321.

Embodiment 325 comprises a method of treating small cell lung cancer (SCLC) comprising administering to a subject in need thereof an isolated polypeptide or polypeptide complex according to Embodiments 1-192, or 257-321.

Embodiment 326 comprises a method of treating gastric cancer comprising administering to a subject in need thereof an isolated polypeptide or polypeptide complex according to Embodiments 1-192, or 257-321.

Embodiment 327 comprises a method of treating esophageal cancer comprising administering to a subject in need thereof an isolated polypeptide or polypeptide complex according to Embodiments 1-192, or 257-321.

Embodiment 328 comprises a method of treating head and neck cancer comprising administering to a subject in need thereof an isolated polypeptide or polypeptide complex according to Embodiments 1-192, or 257-321.

Embodiment 329 comprises a method of treating prostate cancer comprising administering to a subject in need thereof an isolated polypeptide or polypeptide complex according to Embodiments 1-192, or 257-321.

Embodiment 330 comprises a method of treating endometrial cancer comprising administering to a subject in need thereof an isolated polypeptide or polypeptide complex according to Embodiments 1-192, or 257-321.

Embodiment 331 comprises a method of treating breast cancer comprising administering to a subject in need thereof an isolated polypeptide or polypeptide complex according to Embodiments 1-192, or 257-321.

Embodiment 332 comprises a method of treating colon cancer comprising administering to a subject in need thereof an isolated polypeptide or polypeptide complex according to Embodiments 1-192, or 257-321.

Embodiment 333 comprises a method of treating glioma comprising administering to a subject in need thereof an isolated polypeptide or polypeptide complex according to Embodiments 1-192, or 257-321.

EXAMPLES
Example 1: TROP2 Polypeptide Complex Binding

The TROP2-CD3 polypeptide complexes of Table 7 were evaluated for TROP2 and CD3ε binding.

TABLE 7

Polypeptide complexes

Polypeptide

CD3

complex
Form
Fab Mask
CD3
Mask
Cleavable linker
sdA

PC1
Vh
Non masked, SEQ ID NO. 13 (normal orientation)

PC5
Vh
SEQ ID
SEQ ID NO.
SEQ ID
LSGRSDAGSPLGLAG
SEQ ID

NO. 24
13
NO. 27
(SEQ ID NO: 57)
NO: 69

PC6
Vh
SEQ ID
SEQ ID NO.
SEQ ID
LSGRSDAGSPLGLAG
SEQ ID

NO. 23
13
NO. 27
(SEQ ID NO: 57)
NO: 69

PC2
Vl
Non masked, SEQ ID NO. 13 (flipped orientation)

PC7
Vl
SEQ ID
SEQ ID NO.
SEQ ID
LSGRSDAGSPLGLAG
SEQ ID

NO. 24
13
NO. 27
(SEQ ID NO: 57)
NO: 69

PC8
Vl
SEQ ID
SEQ ID NO.
SEQ ID
LSGRSDAGSPLGLAG
SEQ ID

NO. 23
13
NO. 27
(SEQ ID NO: 57)
NO: 69

PC13
Vh
SEQ ID
SEQ ID NO.
SEQ ID
LSGRSDAGSPLGLAG
SEQ ID

NO. 24
13
NO. 27
(SEQ ID NO: 57)
NO: 73

PC14
Vh
SEQ ID
SEQ ID NO.
SEQ ID
LSGRSDAGSPLGLAG
SEQ ID

NO. 23
13
NO. 27
(SEQ ID NO: 57)
NO: 73

PC15
Vl
SEQ ID
SEQ ID NO.
SEQ ID
LSGRSDAGSPLGLAG
SEQ ID

NO. 24
13
NO. 27
(SEQ ID NO: 57)
NO: 73

PC16
Vl
SEQ ID
SEQ ID NO.
SEQ ID
LSGRSDAGSPLGLAG
SEQ ID

NO. 23
13
NO. 27
(SEQ ID NO: 57)
NO: 73

The polypeptide complex molecules of Table 7 were evaluated for their ability to bind TROP2 as well as CD3 in a standard enzyme linked immunosorbent assay (ELISA) format. Polypeptide complex binding kinetics of TROP2 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.

FIGS. 2A and 2B show representative ELISAs of TROP2 binding. This data is summarized in Tables 8 and 9. The masked polypeptide complexes of PC13 and PC5 have EC50s about 400 fold higher than the unmasked polypeptide complex of PC1, protease treatment rescues the EC50s of PC13 and PC5 to only about 4 fold higher than the unmasked polypeptide complex. Similarly, the masked polypeptide complex of PC6 has an EC50 300 fold higher than PC1, and protease treatment rescues this to about 4.5 fold higher than PC1. The masked polypeptide complex of PC14 has an EC50 about 200 fold higher than PC1, and protease treatment rescues this to about 5 fold higher than PC1.

The polypeptide complexes with the flipped orientation show a similar pattern. PC7 and PC8 have EC50s greater than 100 fold and 60 fold higher than PC2 respectively. PC15 and PC16 have EC50s greater than 50 fold and 30 fold higher than PC2 respectively. Protease treatment rescued binding of all four complexes such that the EC50s were close to that of PC2.

TABLE 8

TROP2 binding

PC13 +

PC5 +

PC14 +

PC6 +

TROP2
PC1
PC13
MTSP1
PC5
MTSP1
PC14
MTSP1
PC6
MTSP1

EC50 nM
0.1477
58.42
0.5907
60.25
0.6337
30.88
0.7302
44.37
0.6686

Fold
1×
395.5×
4×
407.9×
4.3×
209.1×
4.9×
300.4×
4.5×

shift

TABLE 9

TROP2 binding

PC15 +

PC7 +

PC16 +

PC 8 +

TROP2
PC2
PC15
MTSP1
PC7
MTSP1
PC16
MTSP1
PC8
MTSP1

EC50 nM
0.3697
19.39
0.5306
39.38
0.4087
11.98
0.4572
23.25
0.3289

Fold
1×
52.4×
1.4×
106.5×
1.1×
32.4×
1.2×
62.9×
0.9×

shift

FIGS. 3A and 3B show representative ELISAs of CD3 binding. This data is summarized in Tables 10 and 11. The masked polypeptide complexes of PC13 and PC14 respectively had an EC50s about 1313 and 1723 fold higher than the unmasked polypeptide complex of PC1, protease treatment rescued the EC50s to under 2 fold higher than the unmasked polypeptide complex. Similarly, the masked polypeptide complexes of PC5 and PC6 respectively had EC50s about 321 and 267 fold higher than PC1, and protease treatment rescued the EC50s of both to about 1.5 fold higher than PC1. The polypeptide complexes with the flipped orientation show a similar pattern. PC15 and PC16 had EC50s 390 fold and 442 fold higher than PC2 respectively. PC7 and PC8 have EC50s 138 fold and 165 fold higher than PC2 respectively. Protease treatment rescued the EC50s of all four complexes such that the EC50s are close to that of PC2.

TABLE 10

CD3 binding

PC13 +

PC5 +

PC14 +

PC6 +

CD3e
PC1
PC13
MTSP1
PC5
MTSP1
PC14
MTSP1
PC6
MTSP1

EC50 nM
0.05402
70.96
0.07024
17.34
0.07634
93.10
0.09582
14.46
0.07916

Fold
1×
1313.6×
1.3×
321×
1.4×
1723.4×
1.8×
267.7×
1.5×

shift

TABLE 11

CD3 binding

PC15 +

PC7 +

PC16 +

PC8 +

CD3e
PC2
PC15
MTSP1
PC7
MTSP1
PC16
MTSP1
PC8
MTSP1

EC50 nM
0.08100
31.62
0.09884
11.21
0.07748
35.79
0.07767
15.02
0.06267

Fold
1×
390.4×
1.2×
138.4×
1×
441.9×
1×
185.4×
0.8×

shift

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 TROP2 positive tumor cell lines HCT116 and MDAMB231. Tumor cell killing was measured using a real time cell analyzer from Acea Biosciences 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. 25,000 tumor cells were added per well and allowed to adhere overnight. The following day polypeptide complexes titrated in human serum supplemented medium along with 75,000 CD8+ T cells were added to the wells. Cell index measurements were taken every 10 minutes for an additional 96 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.

The HCT116 tumor cell line has a TROP2 density of <10,000 copies per cell. FIGS. 4A and 4B show representative viability data for HCT116. This data is summarized in Tables 12 and 13. The masked polypeptide complex of PC5 has an IC50 greater than 6000 fold higher than the unmasked polypeptide complex of PC1, protease treatment rescues the IC50 to only about 3 fold higher than the unmasked polypeptide complex. Similarly, the masked polypeptide complex of PC6 has an IC50 about 5750 fold higher than PC1, and protease treatment rescues this to about 8.6 fold higher than PC1. The polypeptide complexes with the flipped orientation show a similar pattern. PC7 and PC8 have IC50s 4567 fold and 1707 fold higher than PC2 respectively. Protease treatment rescues both of these complexes such that the IC50s are less than 3 fold that of PC2.

TABLE 12

HCT116 cell assay

HCT116

PC13 +

PC5 +

PC14 +

PC6 +

72 hr
PC1
PC13
MTSP1
PC5
MTSP1
PC14
MTSP1
PC6
MTSP1

IC50 pM
8.604
98.305
45.06
56.038
25.69
58.112
34.62
49.533
74.25

Fold
1×
11,425.5×
5.2×
6.513×
3×
6.754.1×
4×
5.757×
8.6×

shift

TABLE 13

HCT116 cell assay

HCT116

PC15 +

PC7 +

PC16 +

PC8 +

72 hr
PC2
PC15
MTSP1
PC7
MTSP1
PC16
MTSP1
PC8
MTSP1

IC50 pM
1.523
3.253
2.46
6.955
4.488
1.715
2.34
2.601
3.676

Fold
1×
2,135.9×
1.6×
4,566.6×
2.9×
1,126.1×
1.5×
1,707.8×
2.4×

shift

The MDAMB231 tumor cell line has a TROP2 density of about 32,000 copies per cell. FIGS. 4C and 4D show representative viability data for MDAMB231. This data is summarized in Table 14. The masked polypeptide complex of PC5 has an IC50 greater than 12000 fold higher than the unmasked polypeptide complex of PC1, protease treatment rescues the IC50 to less than 2 fold higher than the unmasked polypeptide complex. The masked polypeptide complex of PC6 has an IC50 about 24,000 fold higher than PC1. The polypeptide complexes with the flipped orientation show a similar pattern. PC7 has an IC50 6864 fold higher than PC2.

TABLE 14

MDAMB231 cell assay

MDAMB231

PC5 +

72 hr
PC1
PC5
MTSP1
PC6
PC7
PC2

IC50 pM
1.514
18.951
2.868
36.549
1.817
0.2647

Fold shift
1×
12.517×
1.9×
24.141×
6.864×
1×

Some further polypeptide complexes with different Cd3 masking sequences are shown in Table 14. The TROP2-CD3 polypeptide complexes of Table 15 were evaluated for TROP2 and CD3ε binding as described above.

TABLE 15

Polypeptide complexes

Polypeptide

complex
Fab Mask
CD3
CD3 Mask
Cleavable linker
sdA

PC1
Non masked TCE, SEQ ID NO. 13 (normal orientation)

PC9
SEQ ID NO.
SEQ ID NO.
SEQ ID NO.
LSGRSDAGSPLGLAG
SEQ ID

24
13
26
(SEQ ID NO: 57)
NO: 69

PC11
SEQ ID NO.
SEQ ID NO.
SEQ ID NO.
LSGRSDAGSPLGLAG
SEQ ID

23
13
26
(SEQ ID NO: 57)
NO: 69

PC2
Non masked TCE, SEQ ID NO. 13 (flipped orientation)

PC10
SEQ ID NO.
SEQ ID NO.
SEQ ID NO.
LSGRSDAGSPLGLAG
SEQ ID

24
13
26
(SEQ ID NO: 57)
NO: 69

PC12
SEQ ID
SEQ ID
SEQ ID
LSGRSDAGSPLGLAG
SEQ ID

NO. 23
NO. 13
NO. 26
(SEQ ID NO: 57)
NO: 69

FIGS. 5A and 5B show representative ELISAs of TROP2 binding. This data is summarized in Tables 16 and 17. The masked polypeptide complex of PC9 has an EC50 greater than 450 fold higher than the unmasked polypeptide complex of PC1, protease treatment rescues the EC50 to only about 3.3 fold higher than the unmasked polypeptide complex. Similarly, the masked polypeptide complex of PC11 has an EC50 greater than 200 fold higher than PC1, and protease treatment rescues this to about 8 fold higher than PC1. The polypeptide complexes with the flipped orientation show a similar pattern. PC10 and PC12 have EC50s about 128 fold and 37 fold higher than PC2 respectively. Protease treatment rescues both of these complexes such that the EC50s are about twice that of PC2.

TABLE 16

TROP2 binding

PC9 +

PC11 +

TROP2
PC1
PC9
MTSP1
PC11
MTSP1

EC50
0.1344
62.57
0.4432
28.90
1.023

nM

Fold
1x
465.6x
3.3x
215x
7.6x

shift

TABLE 17

TROP2 binding

PC10 +

PC12 +

TROP2
PC2
PC10
MTSP1
PC12
MTSP1

EC50
0.2833
36.46
0.5404
10.37
0.6020

nM

Fold
1x
128.7x
1.9x
36.6x
2.1x

shift

FIGS. 6A and 6B show representative ELISAs of CD3e binding. This data is summarized in Tables 18 and 19. The masked polypeptide complex of PC9 has an EC50 greater than 78 fold higher than the unmasked polypeptide complex of PC1, protease treatment rescues the EC50 to only about the same as the unmasked polypeptide complex. Similarly, the masked polypeptide complex of PC11 has an EC50 about 70 fold higher than PC1, and protease treatment rescues this to about 2.5 fold higher than PC1. The polypeptide complexes with the flipped orientation show a similar pattern. PC10 and PC12 have EC50s about 85 fold and 50 fold higher than PC2 respectively. Protease treatment rescues both of these complexes such that the EC50s are about twice that of PC2.

TABLE 18

CD3 binding

PC9 +

PC11 +

CD3e
PC1
PC9
MTSP1
PC11
MTSP1

EC50 nM
0.05085
3.976
0.05538
3.526
0.1286

Fold shift
1x
78.2x
1.1x
69.3x
2.5x

TABLE 19

CD3 binding

PC10 +

PC12 +

CD3e
PC2
PC10
MTSP1
PC12
MTSP1

EC50 nM
0.05690
4.829
0.1019
2.819
0.09992

Fold shift
1x
84.9x
1.8x
49.5x
1.8x

The polypeptide complexes of Table 14 were also evaluated in a functional in vitro tumor cell killing assay using HCT116 as described above. The HCT116 tumor cell line has a TROP2 density of <10,000 copies per cell. FIGS. 7A and 7B show representative viability data for HCT116. This data is summarized in Table 20. The masked polypeptide complexes of PC9 and PC11 had IC50s respectively about 1259 and 488 fold higher than the unmasked polypeptide complex of PC1, protease treatment rescues the IC50s to about 2.5 and 5 fold higher than the unmasked polypeptide complex. The masked polypeptide complexes of PC10 and PC12 had IC50s respectively about 2337 and 935 fold higher than the unmasked polypeptide complex of PC2, protease treatment rescues the IC50s to about 2 and 3 fold higher than the unmasked polypeptide complex.

TABLE 20

HCT116 cell assay

HCT116

PC9 +

PC11 +

PC10 +

PC12 +

72 hr
PC1
PC9
MTSP1
PC11
MTSP1
PC2
PC10
MTSP1
PC12
MTSP1

IC50 pM
12.34
15.533
30.21
6.022
60.18
1.119
2.615
2.101
935.2
3.36

Fold shift
1×
1.259×
2.45×
488.0×
4.88×
1
2.337×
1.88×
835.7×
3.0×

Example 3: Polypeptide Complex Mediated Tumor Cell Killing

Polypeptide complexes were evaluated in a functional in vitro tumor cell killing assay using the TROP2 positive tumor cell lines HCT116, NCI-H292, and MDAMB231. 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. Data for NCI-H292 (H292) is seen in FIGS. 8A-8C. Data for HCT116 is seen in FIGS. 9A-9D. Data for MDMAB231 is seen in FIGS. 10A-10C.

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 pharmacokinetics enabled the calculation of Cmax, half-life, volume of distribution, clearance, and 7 day area under the curve (AUC) shown in Table 21 for TROP2 TCE polypeptide complexes and Tables 22-24 for TROP2 TRACTr polypeptide complexes and FIGS. 11A-11D.

TABLE 21

PC1 3 ug/kg
Units

C_MAX
0.40
nM

t_1/2
1.02
hr

Vd
0.30
L

VSS
0.30
L

CL
67.31
mL/hr/kg

BW
3.00
kg

7 day
49
nM · min

AUC

TABLE 22

PC5 100 ug/kg
Units

C_MAX
34.32
nM

t_1/2
90.16
hr

Vd
0.09
L

VSS
0.19
L

CL
0.23
mL/hr/kg

BW
3.00
kg

7 day
142,182
nM · min

AUC

TABLE 23

PC18 100 ug/kg
Units

C_MAX
37.94
nM

t_1/2
97.31
hr

Vd
0.08
L

VSS
0.33
L

CL
0.19
mL/hr/kg

BW
3.00
kg

7 day
127,094
nM · min

AUC

TABLE 24

PC21 100 ug/kg
Units

C_MAX
42.12
nM

t_1/2
100.73
hr

Vd
0.07
L

VSS
0.39
L

CL
0.17
mL/hr/kg

BW
3.00
kg

7 day
137,581
nM · min

AUC

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. 12A-12D.

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: Optimized Phage Library Construction—TROP2 Fab Peptides

Sequence activity relationships were established for TROP2 Fab Peptide-1 and TROP2 Fab Peptide-2 by mutating each individual residue within the peptide to alanine and measuring binding and inhibition against TROP2 Fab. Peptide residues whose alanine mutations significantly weakened binding and inhibition were considered key residues where mutations were not tolerated. Peptide residues whose alanine mutations performed similarly to the non-mutated sequence were considered non-critical sites where mutations were indeed tolerated. Using the peptide sequence activity relationships (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. FIG. 14A and FIG. 14B demonstrate TROP2 Fab inhibition of alanine scanning peptides of TROP2 Fab Peptide-1, the sequences of which are shown in Table 25. FIG. 15A and FIG. 15B demonstrate TROP2 Fab binding of alanine scanning peptides of TROP2 Fab Peptide-2. FIG. 16A and FIG. 16B demonstrate TROP2 Fab inhibition of alanine scanning peptides of TROP2 Fab Peptide-2, the sequences of which are shown in Table 26.

TABLE 25

TROP2 Fab Peptide-1 Ala Scan Peptide Sequences

TROP2 Fab

Peptide-1 Ala

Scan Peptide
Amino acid sequence
SEQ ID NO:

Peptide-4
AVLFCVKNLYCWVT
133

Peptide-5
SALFCVKNLYCWVT
134

Peptide-6
SVAFCVKNLYCWVT
135

Peptide-7
SVLACVKNLYCWVT
136

Peptide-8
SVLFCAKNLYCWVT
137

Peptide-9
SVLFCVANLYCWVT
138

Peptide-10
SVLFCVKALYCWVT
139

Peptide-11
SVLFCVKNAYCWVT
140

Peptide-12
SVLFCVKNLACWVT
141

Peptide-13
SVLFCVKNLYCAVT
142

Peptide-14
SVLFCVKNLYCWAT
143

Peptide-15
SVLFCVKNLYCWVA
144

Peptide-16
SVLFCVKNLYCWVT
145

TABLE 26

TROP2 Fab Peptide-2 Ala Scan Peptide Sequences

TROP2 Fab

Peptide-2 Ala

Scan Peptide
Amino Acid Sequence
SEQ ID NO:

Peptide-17
ADFCKIYSWPVCHQ
146

Peptide-18
VAFCKIYSWPVCHQ
147

Peptide-19
VDACKIYSWPVCHQ
148

Peptide-20
VDFCAIYSWPVCHQ
149

Peptide-21
VDFCKAYSWPVCHQ
150

Peptide-22
VDFCKIASWPVCHQ
151

Peptide-23
VDFCKIYAWPVCHQ
152

Peptide-24
VDFCKIYSAPVCHQ
153

Peptide-25
VDFCKIYSWAVCHQ
154

Peptide-26
VDFCKIYSWPACHQ
155

Peptide-27
VDFCKIYSWPVCAQ
156

Peptide-28
VDFCKIYSWPVCHA
157

Peptide-29
VDFCKIYSWPVCHQ
158

Example 8: Panning of the Optimized Phage Library—TROP2 Fab Peptides

Once the phage optimization libraries were completed, phage libraries were bio-panned using TROP2 Fab loaded beads. Multiple rounds of panning were performed where bacteriophage was allowed to bind to TROP2 Fab loaded beads, washed, eluted, and amplified. Additional selective pressure was included during each round of panning using a fixed concentration of TROP2 soluble protein, TROP2 Fab Peptide-1, or TROP2 Fab Peptide-2. 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 TROP2 Fab coated plates in the presence or absence of saturating concentration of TROP2 soluble protein. Phage able to bind TROP2 Fab were selected for sequence analysis if the binding signal was reduced in the presence of TROP2 soluble protein.

Example 9: Panning ELISAs—TROP2 Fab Peptides

Clonal phage were harvested as crude supernatants and screened via standard enzyme linked immunosorbent assays (ELISAs). Briefly, biotinylated TROP2 Fab was captured on neutravidin coated plates. Prior to the addition of clonal phage, wells were incubated with blocking buffer and TROP2 soluble protein 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 were then sent for sequence analysis.

Phage panning results of TROP2 Fab Peptide-1 library sequences are shown in Table 27. 979 clonal phage sequences were identified. 20 of 979 clonal phage were selected for peptide synthesis and evaluated for peptide inhibition of TROP2 Fab. The sequences of those peptides selected for synthesis are shown in Table 28, and further evaluated for inhibition of TROP2 Fab as shown in FIGS. 17A through 17C. The consensus sequence calculated from all the sequences of Table 27 is shown in FIG. 18 and was generated using WebLogo 3.7.4.

Phage panning results of TROP2 Fab Peptide-2 library sequences are shown in Table 29. 596 clonal phage sequences were identified. 23 of 596 were selected for peptide synthesis and evaluated for peptide binding to TROP2 Fab (FIGS. 19A-19C), and peptide inhibition of TROP2 Fab (FIGS. 20A-20C). The sequences of those peptides selected for synthesis are shown in Table 30. The consensus sequence calculated from all the sequences of Table 29 is shown in FIG. 21 and was generated using WebLogo 3.7.4.

TABLE 27

Phage panning results of TROP2 Fab Peptide-1 library sequences. (—) indicates same amino

acid as in TROP2 Fab Peptide-1 corresponding position (e.g. Phage-1 position).

Amino acid position sequence

2
1
2
3
4
5
6
7
8
9
10
11
12
13
14

Phage-1
S
V
L
F
C
V
K
N
L
Y
C
W
V
T

Phage-2
D
D
—
I
—
—
—
—
—
F
—
—
T
S

Phage-3
D
N
—
I
—
—
—
—
—
W
—
—
I
A

Phage-4
D
T
—
—
—
—
—
—
—
—
—
—
I
V

Phage-5
D
T
—
S
—
F
R
—
—
—
—
—
I
—

Phage-6
D
T
—
W
—
—
—
—
—
—
—
—
—
A

Phage-7
D
—
—
—
—
—
R
—
—
—
—
—
T
S

Phage-8
F
D
—
L
—
—
R
—
—
—
—
—
N
V

Phage-9
F
S
—
V
—
—
R
—
—
—
—
—
N
V

Phage-
F
T
—
—
—
—
Q
—
—
—
—
—
N
V

10

Phage-
H
D
—
Q
—
F
Q
—
—
F
—
—
I
V

11

Phage-
L
S
—
—
—
—
—
—
—
—
—
—
N
V

12

Phage-
N
Y
—
L
—
—
—
—
—
—
—
—
I
V

13

Phage-
—
T
—
E
—
—
R
—
—
—
—
—
I
S

14

Phage-
—
T
—
—
—
—
—
—
—
—
—
—
—
A

15

Phage-
—
T
—
—
—
—
R
—
—
—
—
—
T
V

16

Phage-
—
Y
—
V
—
—
—
—
V
—
—
—
T
A

17

Phage-
T
A
—
—
—
F
—
—
V
—
—
—
N
V

18

Phage-
T
S
—
I
—
F
R
—
V
—
—
—
N
V

19

Phage-
Y
D
—
V
—
L
R
—
—
F
—
—
T
A

20

Phage-
Y
S
—
V
—
—
—
—
—
—
—
—
N
L

21

Phage-
A
T
—
—
—
—
R
—
—
—
—
—
T
A

22

Phage-
F
S
—
L
—
—
R
—
—
—
—
—
N
V

23

Phage-
N
A
—
V
—
—
R
—
—
—
—
—
I
Y

24

Phage-
T
S
—
W
—
—
R
—
—
—
—
Y
N
V

25

Phage-
Y
T
—
Y
—
—
Q
—
V
—
—
—
T
L

26

Phage-
D
T
—
—
—
—
—
—
—
—
—
—
I
N

27

Phage-
Y
S
—
V
—
—
N
—
—
—
—
—
N
—

28

Phage-
T
D
—
V
—
—
M
—
R
W
—
—
H
Y

29

Phage-
N
T
—
—
—
—
Q
—
—
—
—
—
I
H

30

Phage-
N
T
—
—
—
—
R
—
—
—
—
—
I
N

31

Phage-
T
D
—
L
—
F
Q
—
I
—
—
—
H
—

32

Phage-
N
S
—
—
—
L
H
—
—
V
—
—
I
D

33

Phage-
N
T
—
V
—
—
Q
—
V
—
—
—
I
N

34

Phage-
N
S
—
—
—
F
Q
—
—
L
—
—
T
D

35

Phage-
N
H
—
I
—
—
—
—
—
W
—
—
I
N

36

Phage-
—
D
—
—
—
L
—
—
I
—
—
—
H
V

37

Phage-
N
I
—
V
—
—
N
—
I
—
—
—
I
D

38

Phage-
N
T
—
W
—
—
—
—
—
—
—
—
I
D

39

Phage-
D
T
—
—
—
—
—
—
—
F
—
—
T
H

40

Phage-
A
T
—
—
—
F
Q
—
—
—
—
—
N
I

41

Phage-
Y
A
—
—
—
—
N
—
I
—
—
—
I
S

42

Phage-
—
D
—
—
—
—
R
—
—
—
—
—
N
S

43

Phage-
D
—
—
I
—
—
—
—
—
—
—
—
I
H

44

Phage-
N
T
—
L
—
F
R
—
—
—
—
—
—
D

45

Phage-
—
H
—
M
—
—
R
—
—
—
—
—
I
S

46

Phage-
D
H
—
V
—
—
—
—
—
—
—
—
I
D

47

Phage-
N
S
—
—
—
—
—
—
—
—
—
—
—
D

48

Phage-
A
S
—
—
—
—
Q
—
—
W
—
—
T
S

49

Phage-
A
S
—
—
—
F
Q
—
—
—
—
—
N
Y

50

Phage-
L
S
—
—
—
—
H
—
I
—
—
—
T
D

51

Phage-
T
T
—
Y
—
F
Q
—
—
—
—
—
S
V

52

Phage-
P
A
—
V
—
—
R
—
—
—
—
—
I
V

53

Phage-
N
S
—
—
—
—
Q
—
—
—
—
—
I
I

54

Phage-
A
S
—
L
—
—
—
—
—
—
—
—
I
D

55

Phage-
N
S
—
—
—
L
—
—
—
—
—
—
I
D

56

Phage-
N
S
—
L
—
—
—
—
—
W
—
—
I
D

57

Phage-
—
D
—
—
—
W
R
—
—
F
—
—
H
I

58

Phage-
N
N
—
V
—

—
—
—
W
—
—
—
D

59

Phage-
Y
H
—
M
—
—
R
—
—
—
—
—
—
V

60

Phage-
—
S
—
V
—
—
R
—
—
—
—
—
I
D

61

Phage-
T
T
—
L
—
—
—
—
—
—
—
—
H
S

62

Phage-
T
S
—
—
—
F
Q
—
V
—
—
—
I
D

63

Phage-
T
S
—
I
—
—
R
—
I
L
—
—
H
S

64

Phage-
D
—
—
L
—
F
R
—
—
W
—
—
H
—

65

Phage-
T
D
—
I
—
—
M
—
—
—
—
—
H
—

66

Phage-
L
D
—
I
—
—
R
—
—
—
—
—
I
N

67

Phage-
N
Y
—
V
—
E
—
—
—
—
—
R
I
D

68

Phage-
N
T
—
I
—
—
R
—
—
—
—
—
—
I

69

Phage-
N
A
—
L
—
W
Q
—
I
—
—
—
I
D

70

Phage-
N
T
—
L
—
—
N
—
—
W
—
—
I
D

71

Phage-
L
S
—
—
—
—
—
—
—
—
—
—
N
A

72

Phage-
N
N
—
Y
—
F
Q
—
—
—
—
—
I
D

73

Phage-
L
A
—
—
—
—
—
—
I
—
—
—
T
S

74

Phage-
T
H
—
V
—
—
Y
—
V
—
—
—
H
A

75

Phage-
N
T
—
I
—
L
N
—
—
W
—
—
I
D

76

Phage-
N
N
—
L
—
—
R
—
—
—
—
—
I
D

77

Phage-
N
T
—
V
—
—
N
—
—
F
—
—
I
N

78

Phage-
T
S
—
M
—
—
—
—
—
F
—
—
H
—

79

Phage-
—
S
F
S
—
W
H
—
—
—
—
—
D
V

80

Phage-
D
A
—
—
—
—
R
—
—
—
—
—
I
D

81

Phage-
V
D
—
V
—
I
R
—
—
—
—
—
N
D

82

Phage-
—
T
—
—
—
—
—
—
—
—
—
—
T
N

83

Phage-
A
N
—
V
—
—
Q
—
—
—
—
—
I
—

84

Phage-
A
A
—
—
—
—
—
—
—
—
—
—
I
D

85

Phage-
—
S
—
—
—
—
—
—
—
—
—
—
—
D

86

Phage-
H
D
—
V
—
F
—
—
—
W
—
—
N
D

87

Phage-
N
H
—
L
—
L
N
—
—
—
—
—
I
D

88

Phage-
Y
D
—
V
—
—
R
—
—
—
—
—
H
D

89

Phage-
—
S
—
V
—
—
N
—
—
—
—
—
N
—

90

Phage-
—
D
—
—
—
—
M
—
—
—
—
—
H
Y

91

Phage-
D
S
—
L
—
F
Q
—
—
W
—
—
I
V

92

Phage-
N
H
—
L
—
—
R
—
—
—
—
—
—
—

93

Phage-
A
D
—
—
—
—
—
—
—
W
—
—
H
S

94

Phage-
N
A
—
—
—
F
N
—
—
—
—
—
I
D

95

Phage-
N
S
—
—
—
—
Q
—
V
—
—
—
N
—

96

Phage-
T
N
—
M
—
—
R
—
—
—
—
—
N
D

97

Phage-
I
H
—
I
—
—
E
—
I
—
—
—
H
V

98

Phage-
A
D
—
L
—
—
R
—
—
—
—
—
H
D

99

Phage-
H
S
—
—
—
—
—
—
—
—
—
—
H
—

100

Phage-
—
—
—
—
—
—
R
—
—
—
—
—
T
D

101

Phage-
T
N
—
—
—
—
R
—
—
—
—
—
T
D

102

Phage-
—
T
—
Y
—
—
—
—
—
—
—
—
T
D

103

Phage-
N
H
—
L
—
—
D
—
—
W
—
—
I
D

104

Phage-
D
A
—
L
—
W
R
—
—
—
—
—
T
N

105

Phage-
D
H
—
V
—
—
Q
—
—
F
—
—
I
D

106

Phage-
A
T
—
S
—
F
Q
—
—
—
—
—
H
I

107

Phage-
—
N
—
—
—
—
—
—
—
F
—
—
T
D

108

Phage-
D
T
—
—
—
—
—
—
—
F
—
—
I
—

109

Phage-
—
—
—
W
—
—
R
—
—
—
—
—
N
D

110

Phage-
D
H
—
M
—
—
—
—
—
W
—
—
—
D

111

Phage-
N
T
—
—
—
M
D
—
I
W
—
—
H
D

112

Phage-
V
S
—
L
—
—
—
—
—
W
—
—
H
I

113

Phage-
H
S
—
—
—
F
—
—
—
—
—
—
N
V

114

Phage-
D
H
—
—
—
—
Q
—
—
W
—
—
I
S

115

Phage-
—
N
—
V
—
—
N
—
—
W
—
—
I
D

116

Phage-
N
H
—
V
—
—
N
—
—
W
—
—
H
D

117

Phage-
T
N
—
V
—
—
N
—
—
W
—
—
L
D

118

Phage-
D
T
—
—
—
—
R
—
—
—
—
—
I
D

119

Phage-
N
A
—
L
—
L
—
—
—
—
—
—
I
D

120

Phage-
T
S
—
E
—
W
—
—
—
W
—
—
H
S

121

Phage-
—
H
—
—
—
—
—
—
V
F
—
—
D
D

122

Phage-
N
A
—
W
—
I
Q
—
—
—
—
Y
T
D

123

Phage-
N
S
—
L
—
F
Q
—
—
F
—
—
H
S

124

Phage-
—
D
—
V
—
—
—
—
—
—
—
—
N
I

125

Phage-
H
S
—
W
—
I
N
—
—
—
—
—
H
D

126

Phage-
Y
A
—
—
—
—
—
—
—
W
—
—
N
A

127

Phage-
A
H
—
W
—
W
—
—
—
W
—
—
—
D

128

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130

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131

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132

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137

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138

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139

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140

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148

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149

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150

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151

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152

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153

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154

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155

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156

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157

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158

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159

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160

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161

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162

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163

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164

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165

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166

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167

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168

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170

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171

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172

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173

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174

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175

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176

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177

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178

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179

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180

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181

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182

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183

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184

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185

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186

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187

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188

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189

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190

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191

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192

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193

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194

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195

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196

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197

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198

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199

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200

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201

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202

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203

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204

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205

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206

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207

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208

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209

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210

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211

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212

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213

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214

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215

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216

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217

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218

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219

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220

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221

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222

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223

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224

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225

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226

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227

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228

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229

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230

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231

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232

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233

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234

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235

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236

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237

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238

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239

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240

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241

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242

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243

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244

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245

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246

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247

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248

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249

Phage-
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250

Phage-
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251

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252

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253

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254

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255

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256

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257

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258

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259

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260

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261

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262

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263

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264

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265

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266

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267

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268

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269

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270

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271

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272

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273

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274

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275

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276

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277

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278

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279

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280

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281

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282

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283

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284

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285

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286

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287

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288

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289

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290

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291

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292

Phage-
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293

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294

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295

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296

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297

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298

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299

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300

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301

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302

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303

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304

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305

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306

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307

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308

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309

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310

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311

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312

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313

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314

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315

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316

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317

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318

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319

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320

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321

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322

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323

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324

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325

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326

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327

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328

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329

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330

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331

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332

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333

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334

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335

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336

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337

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338

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339

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340

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341

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342

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343

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344

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345

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346

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347

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348

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349

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350

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351

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352

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353

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354

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355

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356

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357

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358

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359

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360

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361

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362

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363

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364

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365

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366

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367

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368

Phage-
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369

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370

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371

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372

Phage-
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373

Phage-
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374

Phage-
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375

Phage-
N
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376

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—
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377

Phage-
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378

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379

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N
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380

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381

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—
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382

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383

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384

Phage-
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385

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N
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386

Phage-
P
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—
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387

Phage-
N
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—
—
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388

Phage-
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—
—
—
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—
—
—
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389

Phage-
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—
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—
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—
—
—
—
—
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390

Phage-
N
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—
—
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—
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391

Phage-
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S
—
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—
—
—
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392

Phage-
N
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—
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—
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—
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393

Phage-
Y
T
—
L
—
—
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—
—
—
—
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394

Phage-
T
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—
L
—
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Q
—
—
—
—
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395

Phage-
—
T
—
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—
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—
—
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—
—
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396

Phage-
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—
—
—
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—
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—
—
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397

Phage-
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—
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—
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—
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—

398

Phage-
D
T
—
—
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—
—
—
—
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399

Phage-
—
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—
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—
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—
—
—
—
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400

Phage-
N
S
—
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—
—
—
—
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401

Phage-
N
S
—
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—
—
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—
—
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402

Phage-
N
S
—
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—
—
—
—
—
—
—
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403

Phage-
N
A
—
—
—
—
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—
—
—
—
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404

Phage-
H
T
—
—
—
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—
—
W
—
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405

Phage-
N
Y
—
—
—
—
—
—
—
—
—
—
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—

406

Phage-
N
S
—
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—
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—
—
—
—
—
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407

Phage-
N
—
—
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—
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—
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—
—
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408

Phage-
N
D
—
—
—
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—
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—
—
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D

409

Phage-
Y
S
—
M
—
—
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—
—
—
—
—
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L

410

Phage-
N
T
—
—
—
—
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—
—
—
—
—
—
N

411

Phage-
D
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—
L
—
—
—
—
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—
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412

Phage-
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D
—
L
—
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—
—
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413

Phage-
D
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—
—
—
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Q
—
—
W
—
—
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D

414

Phage-
F
T
—
—
—
—
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—
—
—
—
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415

Phage-
A
A
—
—
—
—
—
—
—
—
—
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416

Phage-
Y
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—
—
—
—
—
—
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—
—
—
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417

Phage-
N
H
—
L
—
—
—
—
—
—
—
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N

418

Phage-
F
S
—
M
—
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—
—
—
—
—
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D

419

Phage-
A
S
—
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—
—
M
—
I
—
—
—
T
I

420

Phage-
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D
—
W
—
—
D
—
—
W
—
—
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D

421

Phage-
A
D
—
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—
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—
—
—
—
—
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422

Phage-
—
D
—
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—
—
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—
—
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—
—
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—

423

Phage-
N
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—
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—
—
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—

424

Phage-
—
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—
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—
—
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—
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425

Phage-
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T
—
—
—
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—
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—
—
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426

Phage-
F
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—
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—
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—
—
—
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427

Phage-
H
D
—
—
—
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—
—
—
—
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D

428

Phage-
N
Y
—
V
—
L
N
—
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—
—
—
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A

429

Phage-
D
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—
—
—
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—
—
—
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—
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D

430

Phage-
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—
—
—
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—
I
—
—
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S

431

Phage-
Y
S
—
—
—
—
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—
—
—
—
—
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V

432

Phage-
T
D
—
L
—
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—
—
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—
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I

433

Phage-
L
A
—
—
—
—
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—
—
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434

Phage-
N
A
—
—
—
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—
—
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—
—
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435

Phage-
L
S
—
M
—
—
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—
—
—
—
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I

436

Phage-
F
T
M
—
—
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—
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—
—
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A

437

Phage-
N
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—
—
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—
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438

Phage-
H
A
—
I
—
—
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—
—
W
—
—
N
—

439

Phage-
N
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—
M
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—
—
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440

Phage-
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—
—
—
—
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441

Phage-
N
A
—
M
—
—
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—
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W
—
—
I
D

442

Phage-
N
T
—
—
—
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—
—
—
—
—
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V

443

Phage-
N
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—
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—
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—
—
—
—
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444

Phage-
N
L
—
—
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—
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—
—
—
—
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D

445

Phage-
Y
S
—
M
—
—
N
—
—
W
—
—
N
—

446

Phage-
G
S
—
L
—
—
—
—
—
—
—
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D

447

Phage-
L
D
—
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—
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—
—
—
—
—
I
N

448

Phage-
N
A
—
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—
—
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—
—
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D

449

Phage-
Y
T
—
—
—
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Q
—
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—
—
Y
H
I

450

Phage-
N
Y
—
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—
—
—
—
—
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—
—
I
V

451

Phage-
—
F
—
T
—
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—
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—
—
—
—
S

452

Phage-
L
A
—
—
—
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—
—
F
—
—
I
N

453

Phage-
A
H
—
M
—
—
—
—
—
—
—
—
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V

454

Phage-
—
D
—
I
—
—
—
—
—
—
—
—
Y
D

455

Phage-
H
A
—
—
—
—
N
—
—
W
—
—
N
—

456

Phage-
Y
T
—
—
—
I
Q
—
V
H
—
—
T
V

457

Phage-
D
N
—
R
—
—
R
—
—
—
—
—
I
D

458

Phage-
D
F
—
R
—
T
—
—
—
F
—
—
I
D

459

Phage-
D
H
—
L
—
F
R
—
V
—
—
—
H
I

460

Phage-
Y
—
—
L
—
—
—
—
V
—
—
—
N
E

461

Phage-
F
S
—
M
—
I
—
—
—
—
—
—
T
—

462

Phage-
T
H
—
L
—
—
—
—
I
—
—
—
H
V

463

Phage-
A
S
—
—
—
L
Q
—
—
—
—
—
N
Y

464

Phage-
H
D
—
V
—
—
N
—
—
W
—
—
N
D

465

Phage-
A
A
—
E
—
F
Q
—
—
W
—
—
—
I

466

Phage-
L
D
—
I
—
F
Q
—
V
—
—
—
Y
A

467

Phage-
N
D
—
H
—
—
M
—
—
—
—
—
—
Y

468

Phage-
N
T
—
L
—
I
E
—
—
—
—
—
I
D

469

Phage-
A
S
—
—
—
I
Q
—
—
—
—
—
T
D

470

Phage-
N
Y
—
V
—
—
N
—
—
W
—
—
I
S

471

Phage-
N
—
—
—
—
—
—
—
Y
—
—
—
I
I

472

Phage-
N
Y
—
—
L
Q
—
V
—
—
—
—
—
D

473

Phage-
F
S
—
H
—
F
—
—
V
—
—
—
—
V

474

Phage-
A
S
—
—
—
I
—
—
—
W
—
—
N
N

475

Phage-
Y
S
—
—
—
—
—
—
—
—
—
—
N
A

476

Phage-
T
Y
—
Y
—
F
Q
—
—
—
—
—
H
V

477

Phage-
N
—
—
—
—
I
N
—
—
W
—
—
T
—

478

Phage-
N
D
—
—
—
I
M
—
V
—
—
—
N
—

479

Phage-
N
T
—
—
—
—
R
—
—
—
—
—
N
S

480

Phage-
F
D
—
—
—
—
R
—
—
—
—
—
T
N

481

Phage-
N
F
—
W
—
—
N
—
V
—
—
—
I
D

482

Phage-
T
Y
—
—
—
F
Q
—
—
—
—
—
N
L

483

Phage-
N
S
—
M
—
—
M
—
—
—
—
—
I
—

484

Phage-
A
S
—
—
—
—
M
—
—
—
—
—
N
V

485

Phage-
N
T
—
—
—
—
—
—
—
—
—
—
I
A

486

Phage-
H
S
—
—
—
I
E
—
—
—
—
—
N
D

487

Phage-
A
D
—
I
—
—
M
—
V
—
—
—
—
S

488

Phage-
H
A
—
M
—
—
R
—
—
—
—
—
N
A

489

Phage-
Y
P
—
V
—
F
—
—
—
—
—
—
N
H

490

Phage-
—
H
—
—
—
Y
—
—
—
—
—
—
N
Y

491

Phage-
—
D
—
W
—
—
—
—
—
—
—
—
H
V

492

Phage-
F
S
—
—
—
—
—
—
—
—
—
—
H
A

493

Phage-
H
T
—
M
—
F
Q
—
—
—
—
—
H
N

494

Phage-
Y
S
—
—
—
—
N
—
—
W
—
—
—
D

495

Phage-
Y
P
—
—
—
—
R
—
—
—
—
—
N
D

496

Phage-
N
T
—
L
—
F
Q
—
—
—
—
—
I
D

497

Phage-
N
S
—
L
—
I
H
—
—
W
—
—
I
D

498

Phage-
T
N
—
W
—
—
R
—
V
L
—
—
H
S

499

Phage-
H
A
—
—
—
—
—
—
—
—
—
—
N
S

500

Phage-
H
T
—
V
—
—
R
—
—
—
—
—
H
N

501

Phage-
N
H
—
V
—
F
—
—
—
—
—
—
—
S

502

Phage-
A
T
—
L
—
—
—
—
—
W
—
—
H
—

503

Phage-
D
H
—
L
—
—
—
—
—
F
—
—
I
A

504

Phage-
L
D
—
—
—
W
R
—
—
—
—
—
N
A

505

Phage-
F
D
—
—
—
—
R
—
—
—
—
R
N
D

506

Phage-
Y
S
—
—
—
F
H
—
—
—
—
—
I
D

507

Phage-
N
T
—
M
—
—
Q
—
V
—
—
—
I
I

508

Phage-
N
A
—
—
—
—
R
—
—
—
—
—
N
F

509

Phage-
—
S
—
M
—
—
R
—
—
—
—
—
I
I

510

Phage-
H
—
—
—
—
—
Q
—
I
—
—
—
T
V

511

Phage-
—
S
—
M
—
—
N
—
—
—
—
—
T
V

512

Phage-
H
S
—
W
—
F
Q
—
—
—
—
—
N
A

513

Phage-
N
A
—
Y
—
—
—
—
—
W
—
—
I
D

514

Phage-
T
S
M
L
—
F
Q
—
—
—
—
—
H
V

515

Phage-
Y
D
—
I
—
—
—
—
—
W
—
—
T
—

516

Phage-
A
D
—
—
—
—
—
—
—
—
—
—
H
V

517

Phage-
A
S
—
—
—
—
—
—
—
—
—
—
N
A

518

Phage-
H
S
—
—
—
—
R
—
—
—
—
—
N
D

519

Phage-
N
Y
—
V
—
—
—
—
—
W
—
Y
I
N

520

Phage-
N
D
—
L
—
F
Q
—
I
—
—
—
N
V

521

Phage-
T
D
—
—
—
Y
R
—
—
W
—
—
H
D

522

Phage-
I
A
—
L
—
F
Q
—
V
—
—
—
H
I

523

Phage-
I
D
—
V
—
F
R
—
I
W
—
—
N
—

524

Phage-
N
A
—
—
—
F
H
—
—
—
—
—
I
D

525

Phage-
N
N
—
—
—
—
R
—
—
—
—
—
N
I

526

Phage-
D
T
—
—
—
—
Q
—
I
W
—
—
T
A

527

Phage-
Y
H
—
—
—
—
R
—
—
—
—
—
—
S

528

Phage-
F
S
—
L
—
—
R
—
—
—
—
—
T
V

529

Phage-
D
T
—
V
—
—
Q
—
—
—
—
—
I
S

530

Phage-
D
H
—
V
—
—
—
—
—
L
—
—
I
N

531

Phage-
N
Y
—
—
—
—
—
—
—
—
—
—
I
D

532

Phage-
H
S
—
—
—
—
N
—
—
—
—
—
T
S

533

Phage-
N
F
—
M
—
—
—
—
—
S
—
—
I
D

534

Phage-
F
A
—
—
—
—
N
—
—
—
—
—
N
D

535

Phage-
—
Y
—
Y
—
L
R
—
I
—
—
—
N
—

536

Phage-
T
S
—
L
—
—
—
—
—
W
—
—
N
V

537

Phage-
F
S
—
M
—
—
R
—
—
—
—
—
H
—

538

Phage-
N
Y
—
Y
—
L
—
—
—
—
—
—
N
I

539

Phage-
N
H
—
L
—
—
R
—
—
—
—
—
I
—

540

Phage-
N
Y
—
—
—
—
Q
—
V
—
—
—
I
A

541

Phage-
N
T
—
M
—
—
—
—
—
F
—
—
I
D

542

Phage-
D
Y
—
L
—
F
Q
—
V
—
—
—
H
I

543

Phage-
H
A
—
—
—
—
R
—
—
—
—
—
—
D

544

Phage-
—
D
—
—
—
—
M
—
V
—
—
—
H
I

545

Phage-
N
—
—
—
—
—
—
—
—
—
—
—
I
D

546

Phage-
—
S
—
—
—
F
Q
—
—
—
—
—
—
D

547

Phage-
N
T
—
—
—
—
—
—
—
—
—
—
T
S

548

Phage-
P
H
—
M
—
—
Q
—
I
—
—
—
I
I

549

Phage-
D
L
—
V
—
L
R
—
W
F
—
—
N
D

550

Phage-
H
D
—
—
—
—
R
—
—
W
—
—
N
A

551

Phage-
N
L
—
M
—
—
Q
—
—
—
—
—
I
A

552

Phage-
D
T
—
W
—
—
R
—
—
—
—
—
I
N

553

Phage-
N
—
—
V
—
—
N
—
—
W
—
—
I
D

554

Phage-
—
Y
—
—
—
F
—
—
—
—
—
—
N
D

555

Phage-
N
N
—
W
—
F
Q
—
—
—
—
—
H
V

556

Phage-
L
Y
—
M
—
—
—
—
—
—
—
—
I
N

557

Phage-
N
D
—
I
—
—
R
—
—
—
—
—
H
Y

558

Phage-
N
H
—
—
—
—
R
—
—
—
—
—
T
—

559

Phage-
N
A
—
L
—
—
—
—
—
—
—
—
I
D

560

Phage-
L
A
—
—
—
—
—
—
—
—
—
—
H
D

561

Phage-
N
A
—
V
—
—
Y
—
I
—
—
—
H
V

562

Phage-
N
N
—
Y
—
—
—
—
—
W
—
—
—
A

563

Phage-
Y
S
—
M
—
—
—
—
—
—
—
—
H
A

564

Phage-
N
A
—
L
—
—
N
—
—
W
—
—
I
D

565

Phage-
D
T
—
L
—
—
H
—
—
F
—
—
I
D

566

Phage-
N
N
—
Y
—
F
Q
—
V
F
—
—
I
A

567

Phage-
A
S
—
M
—
—
—
—
—
—
—
—
H
A

568

Phage-
T
A
—
—
—
F
Q
—
V
—
—
—
H
D

569

Phage-
N
—
—
V
—
—
Q
—
I
—
—
—
—
S

570

Phage-
N
N
—
M
—
F
—
—
—
—
—
—
H
I

571

Phage-
—
S
—
L
—
—
N
—
—
W
—
—
T
—

572

Phage-
N
S
—
—
—
—
M
—
I
—
—
—
H
D

573

Phage-
N
H
—
—
—
I
—
—
—
—
—
—
H
A

574

Phage-
F
S
—
—
—
—
—
—
—
—
—
—
T
N

575

Phage-
N
T
—
—
—
—
Q
—
V
F
—
—
T
Y

576

Phage-
N
A
—
—
—
F
—
—
—
—
—
—
T
Y

577

Phage-
N
T
—
L
—
—
—
—
—
—
—
—
I
D

578

Phage-
—
H
—
—
—
F
Q
—
—
—
—
—
N
A

579

Phage-
Y
T
—
—
—
F
Q
—
—
—
—
—
N
V

580

Phage-
H
S
—
—
—
—
—
—
—
—
—
—
T
I

581

Phage-
D
Y
—
L
—
F
R
—
—
—
—
—
Y
D

582

Phage-
Y
N
—
—
—
—
R
—
—
—
—
—
—
A

583

Phage-
Y
S
—
V
—
—
—
—
—
W
—
—
N
I

584

Phage-
F
N
—
—
—
—
—
—
—
—
—
—
N
D

585

Phage-
V
A
—
—
—
—
—
—
I
—
—
—
N
F

586

Phage-
D
Y
—
L
—
—
H
—
—
F
—
—
I
V

587

Phage-
D
T
—
L
—
—
—
—
—
F
—
—
I
D

588

Phage-
D
—
—
V
—
—
N
—
—
F
—
—
I
D

589

Phage-
N
Y
—
Y
—
—
Q
—
V
—
—
—
I
I

590

Phage-
N
S
—
Q
—
—
R
—
I
—
—
—
I
N

591

Phage-
Y
S
—
—
—
—
Q
—
—
—
—
—
N
A

592

Phage-
F
S
—
—
—
—
—
—
—
—
—
—
N
—

593

Phage-
N
N
—
V
—
—
R
—
—
—
—
—
I
A

594

Phage-
N
S
—
L
—
—
—
—
I
—
—
—
I
D

595

Phage-
N
F
—
V
—
—
R
—
—
—
—
—
I
D

596

Phage-
H
—
—
—
—
—
R
—
—
—
—
—
T
N

597

Phage-
—
I
—
L
—
Y
Q
—
—
L
—
—
I
D

598

Phage-
N
F
—
—
—
—
R
—
—
—
—
—
I
D

599

Phage-
D
T
—
V
—
—
—
—
—
L
—
—
T
F

600

Phage-
Y
S
—
L
—
—
—
—
V
—
—
—
H
I

601

Phage-
D
T
—
I
—
—
R
—
—
—
—
—
I
N

602

Phage-
A
H
—
L
—
—
—
—
—
W
—
—
S
V

603

Phage-
F
S
—
—
—
—
—
—
V
—
—
—
—
S

604

Phage-
D
T
—
—
—
—
R
—
—
W
—
—
N
A

605

Phage-
D
T
—
V
—
—
R
—
—
F
—
—
P
I

606

Phage-
—
D
—
—
—
—
—
—
—
W
—
—
H
D

607

Phage-
F
H
—
L
—
—
Q
—
—
—
—
—
H
D

608

Phage-
N
S
—
—
—
—
—
—
V
F
—
—
I
V

609

Phage-
D
S
—
V
—
—
—
—
—
F
—
—
—
V

610

Phage-
Y
—
—
—
—
—
—
—
I
—
—
—
H
V

611

Phage-
—
S
—
W
—
—
—
—
—
W
—
Y
H
V

612

Phage-
N
—
—
—
—
—
Q
—
I
—
—
—
N
D

613

Phage-
Y
S
—
—
—
—
Q
—
—
W
—
—
H
D

614

Phage-
F
D
—
—
—
—
R
—
—
—
—
—
N
Y

615

Phage-
P
A
—
K
—
—
—
—
—
F
—
—
I
D

616

Phage-
F
S
—
—
—
—
—
—
—
—
—
—
N
D

617

Phage-
A
S
—
L
—
—
—
—
—
W
—
—
H
V

618

Phage-
N
S
—
W
—
—
N
—
—
F
—
—
T
D

619

Phage-
T
Y
—
L
—
F
Q
—
—
—
—
—
N
V

620

Phage-
F
S
—
—
—
—
Q
—
—
—
—
—
T
—

621

Phage-
Y
A
—
—
—
—
Q
—
—
—
—
—
N
—

622

Phage-
N
Y
—
V
—
—
N
—
I
H
—
—
I
A

623

Phage-
L
S
—
—
—
—
N
—
—
—
—
—
T
A

624

Phage-
N
Y
—
I
—
—
—
—
—
—
—
—
I
D

625

Phage-
T
T
—
—
—
—
—
—
V
F
—
—
H
V

626

Phage-
F
D
—
—
—
—
R
—
—
—
—
—
N
A

627

Phage-
D
—
—
—
—
W
R
—
—
W
—
—
N
N

628

Phage-
—
T
—
V
—
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R
—
—
—
—
—
N
D

629

Phage-
—
D
—
L
—
Y
—
—
—
—
—
—
N
Y

630

Phage-
L
S
—
—
—
—
Q
—
—
—
—
—
H
A

631

Phage-
H
Y
—
M
—
W
Q
—
—
W
—
—
T
V

632

Phage-
F
H
—
V
—
W
R
—
—
H
—
—
I
D

633

Phage-
V
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—
—
—
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—
—
—
—
—
N
D

634

Phage-
T
A
—
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—
—
R
—
—
—
—
—
H
V

635

Phage-
L
S
—
—
—
—
—
—
—
—
—
—
N
D

636

Phage-
—
S
—
—
—
—
—
—
—
—
—
—
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A

637

Phage-
D
—
—
I
—
—
H
—
—
F
—
—
T
V

638

Phage-
Y
A
—
—
—
—
R
—
—
—
—
—
N
—

639

Phage-
A
S
—
—
—
—
—
—
I
—
—
—
T
V

640

Phage-
A
D
—
V
—
—
—
—
—
W
—
—
Y
D

641

Phage-
N
T
—
—
—
F
—
—
V
F
—
—
N
V

642

Phage-
Y
N
—
—
—
L
Q
—
V
—
—
—
T
I

643

Phage-
A
T
—
M
—
—
—
—
—
—
—
—
I
N

644

Phage-
H
A
—
L
—
—
E
—
I
—
—
—
Y
A

645

Phage-
Y
A
—
—
—
—
N
—
I
—
—
—
T
V

646

Phage-
N
T
—
—
—
—
—
—
—
—
—
—
I
V

647

Phage-
D
T
—
—
—
—
—
—
—
F
—
—
—
V

648

Phage-
N
H
—
V
—
—
R
—
—
W
—
—
N
V

649

Phage-
A
S
—
—
—
L
Q
—
—
—
—
—
T
A

650

Phage-
F
S
—
—
—
F
—
—
—
—
—
—
N
D

651

Phage-
A
S
—
M
—
—
R
—
—
—
—
—
N
A

652

Phage-
—
H
—
L
—
F
Q
—
—
—
—
—
T
I

653

Phage-
F
S
—
—
—
—
Q
—
—
W
—
—
N
D

654

Phage-
—
Y
—
—
—
—
R
—
—
—
—
—
N
S

655

Phage-
F
S
—
—
—
—
Q
—
V
F
—
—
T
D

656

Phage-
N
H
—
L
—
—
—
—
—
W
—
—
—
V

657

Phage-
Y
S
—
—
—
—
E
—
—
—
—
—
N
A

658

Phage-
—
H
—
V
—
W
H
—
—
—
—
—
N
D

659

Phage-
H
S
—
—
—
—
—
—
—
—
—
—
T
S

660

Phage-
D
L
—
W
—
F
Q
—
—
—
—
—
H
V

661

Phage-
H
D
—
K
—
F
Q
—
—
—
—
—
Y
D

662

Phage-
N
T
—
L
—
W
H
—
—
—
—
—
—
D

663

Phage-
F
D
—
V
—
—
R
—
—
—
—
—
T
V

664

Phage-
A
T
—
—
—
—
—
—
—
—
—
—
I
D

665

Phage-
F
A
—
L
—
F
Q
—
V
—
—
—
H
I

666

Phage-
—
S
—
—
—
L
Q
—
—
—
—
—
T
D

667

Phage-
N
A
—
W
—
—
—
—
—
—
—
—
I
D

668

Phage-
F
A
—
—
—
—
—
—
V
—
—
—
T
N

669

Phage-
—
D
—
—
—
—
—
—
I
—
—
—
H
F

670

Phage-
N
H
—
L
—
F
Q
—
—
W
—
—
T
V

671

Phage-
D
S
—
M
—
W
—
—
—
W
—
—
I
D

672

Phage-
V
—
—
—
—
W
Q
—
V
—
—
—
H
S

673

Phage-
Y
T
—
Q
—
F
H
—
—
—
—
—
H
I

674

Phage-
Y
H
—
L
—
—
—
—
—
—
—
—
I
N

675

Phage-
Y
F
—
H
—
F
R
—
—
—
—
—
D
—

676

Phage-
D
T
—
L
—
—
—
—
—
F
—
—
I
V

677

Phage-
T
L
—
H
—
Y
—
—
—
—
—
—
H
D

678

Phage-
Y
L
F
C
I
K
N
L
Y
C
W
N
D
G

679

Phage-
L
S
—
—
—
F
—
—
—
—
—
—
N
S

680

Phage-
Y
S
—
—
—
—
R
—
—
—
—
—
N
D

681

Phage-
N
T
—
—
—
—
—
—
I
—
—
—
—
D

682

Phage-
D
S
—
—
—
—
M
—
—
—
—
—
T
D

683

Phage-
—
S
—
—
—
I
H
—
—
—
—
—
N
D

684

Phage-
—
N
—
V
—
—
R
—
—
—
—
—
I
N

685

Phage-
N
S
—
—
—
—
Q
—
—
—
—
—
—
D

686

Phage-
A
S
—
—
—
—
H
—
—
—
—
—
T
A

687

Phage-
F
S
—
V
—
—
—
—
—
W
—
—
T
V

688

Phage-
T
D
—
M
—
F
R
—
—
W
—
—
N
—

689

Phage-
—
I
—
L
—
F
Q
—
—
—
—
—
T
—

690

Phage-
Y
D
—
—
—
—
R
—
—
W
—
—
N
D

691

Phage-
—
N
—
M
—
F
R
—
—
—
—
—
L
D

692

Phage-
Y
S
—
H
—
—
R
—
—
—
—
—
H
D

693

Phage-
H
T
—
—
—
—
R
—
—
—
—
—
H
D

694

Phage-
N
I
—
—
—
—
D
—
—
—
—
—
I
D

695

Phage-
A
T
—
M
—
—
M
—
V
—
—
—
Y
A

696

Phage-
N
A
—
M
—
—
—
—
V
—
—
—
I
N

697

Phage-
D
T
—
V
—
L
R
—
—
F
—
—
I
N

698

Phage-
Y
A
—
L
—
L
—
—
I
—
—
—
N
I

699

Phage-
N
S
—
V
—
—
M
—
I
—
—
—
H
—

700

Phage-
N
—
—
—
—
M
D
—
—
W
—
—
I
D

701

Phage-
H
N
—
V
—
L
Q
—
—
—
—
—
N
D

702

Phage-
N
A
—
M
—
F
—
—
V
—
—
—
I
D

703

Phage-
V
F
—
M
—
—
R
—
—
—
—
—
H
D

704

Phage-
D
Y
—
V
—
—
R
—
—
—
—
—
I
N

705

Phage-
F
S
—
M
—
E
—
—
—
W
—
—
H
V

706

Phage-
Y
T
—
—
—
—
N
—
I
—
—
—
T
D

707

Phage-
N
F
—
V
—
—
—
—
—
—
—
—
I
D

708

Phage-
F
D
—
—
—
—
R
—
—
—
—
—
N
D

709

Phage-
H
T
—
—
—
—
R
—
—
—
—
—
T
I

710

Phage-
D
—
—
L
—
—
—
—
—
F
—
—
T
V

711

Phage-
N
T
—
V
—
F
Q
—
F
F
—
—
I
D

712

Phage-
N
S
—
—
—
—
—
—
—
W
—
—
I
A

713

Phage-
F
A
—
—
—
—
—
—
—
—
—
—
H
I

714

Phage-
D
N
—
—
—
I
R
—
—
—
—
—
I
D

715

Phage-
H
S
—
—
—
F
Q
—
—
W
—
—
H
D

716

Phage-
—
I
—
W
—
F
Q
—
—
—
—
—
H
V

717

Phage-
—
D
—
V
—
—
M
—
I
—
—
F
H
—

718

Phage-
D
H
—
V
—
W
R
—
V
—
—
—
H
—

719

Phage-
F
S
—
—
—
—
—
—
—
—
—
—
T
D

720

Phage-
D
H
—
V
—
—
—
—
—
L
—
—
I
V

721

Phage-
N
I
—
L
—
—
—
—
—
—
—
—
N
D

722

Phage-
D
Y
—
M
—
—
—
—
—
F
—
—
I
I

723

Phage-
N
F
—
V
—
—
—
—
—
—
—
—
I
S

724

Phage-
T
H
—
L
—
—
M
—
I
—
—
Y
H
I

725

Phage-
Y
T
F
—
—
—
—
—
—
—
—
—
N
D

726

Phage-
D
—
—
—
—
—
Q
—
V
—
—
—
H
I

727

Phage-
D
L
—
V
—
—
—
—
—
F
—
—
T
V

728

Phage-
D
I
—
—
—
F
Q
—
I
—
—
—
H
—

729

Phage-
Y
N
—
L
—
—
—
—
I
—
—
—
H
D

730

Phage-
N
F
—
V
—
W
—
—
T
L
—
—
I
A

731

Phage-
D
S
—
—
—
L
R
—
—
—
—
—
T
A

732

Phage-
Y
S
—
M
—
—
—
—
—
—
—
—
N
D

733

Phage-
D
S
—
—
—
—
R
—
—
F
—
—
T
—

734

Phage-
D
A
—
—
—
I
—
—
—
W
—
—
N
S

735

Phage-
N
Y
—
I
—
—
—
—
—
—
—
—
I
N

736

Phage-
F
D
—
V
—
—
R
—
—
W
—
—
H
—

737

Phage-
Y
D
—
L
—
F
—
—
—
W
—
—
N
—

738

Phage-
D
F
—
R
—
—
—
—
—
—
—
—
I
D

739

Phage-
N
A
—
V
—
I
—
—
—
W
—
—
H
V

740

Phage-
A
A
—
V
—
—
—
—
I
—
—
—
I
H

741

Phage-
N
S
—
L
—
—
R
—
—
—
—
—
I
A

742

Phage-
N
L
—
—
—
F
Q
—
—
—
—
—
I
D

743

Phage-
T
T
—
—
—
—
—
—
—
—
—
—
H
A

744

Phage-
D
F
M
V
—
—
—
—
—
F
—
—
I
D

745

Phage-
T
S
—
V
—
W
R
—
V
—
—
—
H
D

746

Phage-
Y
S
—
—
—
—
—
—
—
—
—
—
N
D

747

Phage-
—
Y
—
—
—
F
Q
—
—
—
—
—
N
I

748

Phage-
D
T
—
V
—
I
R
—
—
—
—
—
I
D

749

Phage-
N
T
—
—
—
—
—
—
—
—
—
—
T
A

750

Phage-
N
L
—
—
—
I
N
—
—
F
—
—
N
D

751

Phage-
F
S
—
N
—
—
—
—
I
—
—
—
H
—

752

Phage-
H
A
—
—
—
—
—
—
—
W
—
—
H
D

753

Phage-
N
H
—
V
—
F
Q
—
V
K
—
—
I
A

754

Phage-
N
D
—
—
—
—
Q
—
I
W
—
—
H
D

755

Phage-
N
D
—
I
—
—
—
—
—
W
—
—
H
—

756

Phage-
D
T
—
V
—
—
R
—
—
F
—
—
I
Y

757

Phage-
Y
I
—
H
—
F
R
—
—
—
—
—
N
—

758

Phage-
H
—
—
L
—
W
R
—
V
—
—
—
H
S

759

Phage-
F
A
—
—
—
—
R
—
I
—
—
—
H
A

760

Phage-
N
T
—
M
—
—
—
—
I
—
—
—
I
I

761

Phage-
Y
S
—
—
—
—
H
—
—
—
—
—
H
V

762

Phage-
N
T
—
V
—
—
R
—
—
—
—
—
I
H

763

Phage-
N
N
—
—
—
—
—
—
—
—
—
—
T
A

764

Phage-
—
D
—
—
—
—
R
—
—
—
—
—
H
Y

765

Phage-
H
A
—
—
—
—
—
—
—
—
—
—
T
A

766

Phage-
D
T
—
L
—
—
R
—
—
W
—
—
I
D

767

Phage-
D
S
—
L
—
—
—
—
—
F
—
—
I
V

768

Phage-
D
H
—
V
—
—
R
—
—
F
—
—
I
—

769

Phage-
D
T
—
L
—
—
—
—
—
F
—
—
T
Y

770

Phage-
D
S
—
—
—
—
—
—
—
F
—
—
P
V

771

Phage-
Y
D
—
—
—
W
—
—
—
—
—
—
N
—

772

Phage-
N
D
—
—
—
—
R
—
—
—
—
—
N
A

773

Phage-
N
I
—
—
—
F
Q
—
—
—
—
—
H
A

774

Phage-
—
H
—
L
—
—
—
—
I
F
—
—
T
A

775

Phage-
Y
A
—
—
—
L
—
—
—
—
—
—
N
V

776

Phage-
N
A
—
M
—
—
—
—
—
—
—
—
I
H

777

Phage-
A
N
—
L
—
F
H
—
—
W
—
—
H
D

778

Phage-
F
S
—
—
—
—
Q
—
V
—
—
—
T
S

779

Phage-
L
H
—
—
—
—
—
—
—
—
—
—
H
A

780

Phage-
H
N
—
I
—
—
—
—
—
W
—
—
H
N

781

Phage-
N
T
—
V
—
F
N
—
—
—
—
Y
I
D

782

Phage-
F
S
—
—
—
—
N
—
—
W
—
—
N
D

783

Phage-
—
Y
—
—
—
—
—
—
I
—
—
—
N
F

784

Phage-
H
S
—
—
—
—
—
—
—
—
—
—
T
A

785

Phage-
L
S
—
—
—
—
R
—
—
—
—
—
T
A

786

Phage-
A
A
—
I
—
—
—
—
—
—
—
—
N
—

787

Phage-
—
S
—
W
—
F
Q
—
V
—
—
—
—
V

788

Phage-
A
—
—
W
—
F
Q
—
V
—
—
—
N
I

789

Phage-
N
S
—
—
—
—
R
—
—
—
—
—
I
A

790

Phage-
Y
Y
—
N
—
I
—
—
—
—
—
—
T
D

791

Phage-
Y
S
—
—
—
L
R
—
—
—
—
—
T
D

792

Phage-
F
A
—
—
—
—
N
—
—
—
—
—
T
S

793

Phage-
N
F
—
K
—
F
Q
—
V
—
—
—
H
V

794

Phage-
D
S
—
M
—
L
R
—
—
F
—
—
T
Y

795

Phage-
N
Y
—
V
—
—
—
—
I
—
—
—
I
H

796

Phage-
Y
S
—
V
—
—
—
—
—
—
—
—
N
V

797

Phage-
—
F
—
W
—
F
Q
—
—
—
—
—
N
D

798

Phage-
N
T
—
—
—
—
R
—
—
—
—
—
I
D

799

Phage-
D
S
—
R
—
—
N
—
—
—
—
—
I
D

800

Phage-
A
D
—
V
—
I
M
—
I
—
—
Y
N
—

801

Phage-
N
N
—
V
—
—
—
—
I
—
—
—
I
F

802

Phage-
—
D
—
M
—
L
N
—
—
W
—
—
H
—

803

Phage-
N
F
—
W
—
—
—
—
—
W
—
—
T
S

804

Phage-
Y
L
V
—
—
—
D
K
R
K
—
M
—
L

805

Phage-
A
A
—
—
—
W
—
—
—
—
—
—
H
I

806

Phage-
D
L
—
L
—
F
R
—
—
W
—
—
H
V

807

Phage-
D
—
—
V
—
L
R
—
—
F
—
—
I
D

808

Phage-
F
S
—
H
—
—
M
—
—
—
—
—
H
V

809

Phage-
N
A
—
I
—
—
—
—
—
W
—
—
N
—

810

Phage-
—
D
—
—
—
F
R
—
—
—
—
—
H
D

811

Phage-
D
A
—
V
—
—
—
—
—
F
—
—
I
D

812

Phage-
N
F
—
M
—
F
H
—
—
—
—
—
I
N

813

Phage-
A
S
—
Y
—
—
Q
—
—
F
—
—
H
S

814

Phage-
N
S
—
—
—
—
R
—
—
—
—
—
T
D

815

Phage-
N
H
—
M
—
I
—
—
—
—
—
—
I
D

816

Phage-
N
H
—
—
—
I
N
—
—
—
—
—
H
S

817

Phage-
F
S
—
—
—
I
—
—
—
—
—
—
H
—

818

Phage-
N
A
—
—
—
M
E
—
—
—
—
—
I
D

819

Phage-
—
A
—
—
—
F
Q
—
—
—
—
—
H
F

820

Phage-
D
S
—
V
—
—
R
—
—
L
—
—
I
V

821

Phage-
L
A
—
—
—
L
Q
—
I
—
—
—
H
S

822

Phage-
Y
T
—
M
—
—
—
—
—
—
—
—
H
V

823

Phage-
D
T
—
W
—
—
H
—
V
—
—
—
I
I

824

Phage-
N
S
—
—
—
—
—
—
I
—
—
—
N
I

825

Phage-
N
D
—
L
—
F
R
—
—
W
—
—
Y
D

826

Phage-
T
D
—
L
—
F
R
—
—
W
—
—
H
S

827

Phage-
N
T
—
L
—
L
N
—
—
F
—
—
I
D

828

Phage-
—
H
—
—
—
L
R
—
—
—
—
—
H
V

829

Phage-
N
—
—
—
—
F
Q
—
I
—
—
—
N
F

830

Phage-
V
H
—
M
—
—
—
—
—
W
—
—
H
A

831

Phage-
D
L
—
V
—
—
R
—
—
F
—
—
I
D

832

Phage-
N
T
—
L
—
—
—
—
—
—
—
—
I
V

833

Phage-
D
T
—
I
—
—
R
—
—
—
—
—
I
D

834

Phage-
Y
A
—
—
—
—
R
—
—
—
—
Y
N
A

835

Phage-
N
A
—
V
—
F
Q
—
I
—
—
—
I
D

836

Phage-
N
A
—
—
—
—
—
—
I
F
—
—
N
S

837

Phage-
—
T
—
—
—
W
R
—
—
V
—
—
T
A

838

Phage-
N
D
—
L
—
F
Q
—
—
W
—
—
H
A

839

Phage-
N
F
—
V
—
—
—
—
F
—
—
—
I
D

840

Phage-
—
I
—
M
—
—
—
—
—
W
—
—
I
D

841

Phage-
D
H
—
—
—
F
R
—
—
W
—
—
N
I

842

Phage-
Y
D
—
I
—
F
R
—
I
W
—
—
N
D

843

Phage-
D
A
—
—
—
—
—
—
—
F
—
—
T
Y

844

Phage-
N
D
—
I
—
—
—
—
I
—
—
—
H
F

845

Phage-
F
S
—
—
—
—
—
—
—
—
—
—
N
A

846

Phage-
I
F
A
L
—
W
L
K
—
W
—
L
I
N

847

Phage-
D
T
—
—
—
L
R
—
V
L
—
—
T
I

848

Phage-
A
T
—
Q
—
F
—
—
I
—
—
—
I
I

849

Phage-
H
S
—
—
—
I
—
—
—
W
—
—
N
H

850

Phage-
—
A
—
Y
—
F
R
—
V
F
—
—
H
V

851

Phage-
D
S
—
V
—
—
—
—
—
L
—
—
I
Y

852

Phage-
A
—
—
Y
—
F
Q
—
I
—
—
—
H
I

853

Phage-
—
D
—
—
—
W
R
—
—
W
—
—
N
D

854

Phage-
A
S
—
—
—
—
—
—
—
—
—
—
T
S

855

Phage-
D
A
—
M
—
—
—
—
—
L
—
—
I
V

856

Phage-
D
T
—
R
—
—
M
—
I
—
—
—
I
D

857

Phage-
D
T
—
—
—
—
—
—
—
F
—
—
T
Y

858

Phage-
F
S
—
—
—
—
N
—
V
—
—
—
T
D

859

Phage-
N
S
—
M
—
—
M
—
—
—
—
—
T
I

860

Phage-
H
I
—
—
—
Q
H
K
R
A
—
Q
I
S

861

Phage-
D
H
—
M
—
—
N
—
W
F
—
—
I
N

862

Phage-
D
S
—
—
—
F
R
—
—
W
—
—
—
I

863

Phage-
—
S
—
M
—
—
R
—
V
—
—
—
I
N

864

Phage-
Y
H
—
—
—
—
—
—
I
—
—
—
H
V

865

Phage-
D
S
—
—
—
I
R
—
—
F
—
—
T
A

866

Phage-
N
T
—
L
—
W
H
—
—
—
—
—
I
N

867

Phage-
T
A
—
I
—
—
—
—
—
—
—
—
I
D

868

Phage-
F
S
—
L
—
F
Q
—
I
—
—
—
T
V

869

Phage-
D
T
—
H
—
—
—
—
I
F
—
—
T
D

870

Phage-
N
T
—
V
—
F
—
—
V
H
—
—
I
D

871

Phage-
—
N
—
L
—
—
M
—
I
—
—
—
H
V

872

Phage-
A
S
—
I
—
—
N
—
—
—
—
—
I
D

873

Phage-
N
D
—
V
—
—
—
—
—
W
—
—
N
S

874

Phage-
H
F
—
M
—
—
H
—
V
F
—
—
I
S

875

Phage-
A
T
P
S
—
—
Q
—
V
Q
—
Y
I
V

876

Phage-
T
H
—
—
—
—
R
—
—
—
—
—
H
A

877

Phage-
Y
D
—
—
—
I
R
—
I
—
—
—
T
Y

878

Phage-
F
D
—
L
—
—
—
—
I
—
—
—
H
D

879

Phage-
D
S
—
Y
—
—
—
—
—
F
—
—
T
F

880

Phage-
N
H
—
V
—
—
—
—
—
W
—
—
I
Y

881

Phage-
N
H
—
V
—
—
R
—
—
—
—
—
I
—

882

Phage-
—
F
—
V
—
—
—
—
—
—
—
—
T
D

883

Phage-
H
S
—
—
—
—
R
—
—
—
—
—
N
A

884

Phage-
N
—
—
Y
—
—
—
—
I
—
—
—
N
S

885

Phage-
D
Y
—
—
—
F
Q
—
V
—
—
—
I
A

886

Phage-
N
Y
—
—
—
L
R
—
—
—
—
—
T
A

887

Phage-
L
S
—
—
—
W
H
—
—
—
—
—
N
I

888

Phage-
N
T
—
—
—
F
Q
—
—
—
—
—
I
N

889

Phage-
F
S
—
—
—
—
N
—
—
W
—
—
T
D

890

Phage-
F
S
—
—
—
L
N
—
—
—
—
—
N
A

891

Phage-
Y
S
—
M
—
—
—
—
—
W
—
—
N
D

892

Phage-
N
A
—
W
—
—
—
—
V
—
—
—
T
S

893

Phage-
N
P
—
Q
—
S
W
K
Y
Q
—
Q
—
F

894

Phage-
N
H
—
—
—
—
M
—
I
—
—
—
N
—

895

Phage-
N
H
—
H
—
—
M
—
V
—
—
—
I
V

896

Phage-
F
D
—
I
—
W
—
—
—
W
—
—
N
D

897

Phage-
—
F
—
Q
—
L
M
—
I
—
—
—
H
D

898

Phage-
Y
S
—
—
—
L
—
—
—
—
—
—
N
V

899

Phage-
N
A
—
—
—
—
R
—
—
—
—
—
T
—

900

Phage-
—
D
—
—
—
F
R
—
—
W
—
—
N
S

901

Phage-
V
—
—
V
—
L
—
—
—
Q
—
F
D
D

902

Phage-
A
D
—
L
—
W
R
—
—
W
—
—
S
A

903

Phage-
F
S
—
—
—
—
—
—
I
—
—
—
—
A

904

Phage-
D
S
—
—
—
I
—
—
V
F
—
—
T
Y

905

Phage-
—
T
—
Y
—
I
—
—
I
W
—
—
N
V

906

Phage-
Y
S
—
—
—
—
—
—
V
—
—
—
T
S

907

Phage-
N
T
—
I
—
—
R
—
—
—
—
—
—
Y

908

Phage-
D
N
—
V
—
—
—
—
—
F
—
—
I
V

909

Phage-
D
S
—
W
—
—
R
—
—
—
—
—
I
V

910

Phage-
D
Y
—
H
—
—
R
—
—
F
—
—
I
—

911

Phage-
H
I
—
S
—
W
H
—
I
—
—
—
Y
D

912

Phage-
Y
S
—
—
—
L
R
—
—
—
—
—
N
Y

913

Phage-
—
P
Y
L
—
K
Q
K
Q
L
—
G
S
I

914

Phage-
F
S
—
—
—
—
—
—
V
—
—
—
T
A

915

Phage-
N
A
—
—
—
—
R
—
—
—
—
—
I
S

916

Phage-
Y
S
—
—
—
—
—
—
—
W
—
—
N
A

917

Phage-
Y
S
—
—
—
—
—
—
—
W
—
—
T
D

918

Phage-
N
H
P
Q
—
—
N
K
—
Q
—
—
D
H

919

Phage-
H
D
—
—
—
F
R
—
—
W
—
—
T
V

920

Phage-
A
Y
—
L
—
—
M
—
—
W
—
T
N
V

921

Phage-
Y
A
—
Y
—
F
Q
—
—
W
—
—
H
N

922

Phage-
D
H
—
W
—
F
R
—
—
W
—
—
T
I

923

Phage-
D
T
—
W
—
—
R
—
—
F
—
—
T
—

924

Phage-
H
T
—
W
—
F
R
—
—
—
—
—
N
I

925

Phage-
H
S
—
—
—
—
H
—
—
W
—
—
T
I

926

Phage-
N
Y
—
L
—
F
H
—
—
—
—
—
I
S

927

Phage-
V
—
—
V
—
—
D
H
R
Q
—
—
H
D

928

Phage-
N
S
—
—
—
—
N
—
I
—
—
—
N
A

929

Phage-
—
H
—
W
—
F
R
—
I
W
—
—
—
—

930

Phage-
F
S
—
—
—
—
—
—
—
—
—
—
N
S

931

Phage-
L
L
P
V
L
C
Q
E
Y
V
V
C
G
R

932

Phage-
—
Y
—
—
—
F
Q
—
V
—
—
—
N
V

933

Phage-
H
N
—
L
—
G
Q
K
—
—
—
Y
D
L

934

Phage-
D
H
R
Y
—
—
N
D
Q
Q
—
—
—
P

935

Phage-
H
A
—
—
—
L
—
—
—
W
—
—
N
I

936

Phage-
Y
H
—
—
—
F
R
—
V
—
—
—
H
V

937

Phage-
T
F
—
W
—
F
Q
—
V
—
—
—
N
A

938

Phage-
F
S
—
—
—
F
Q
—
—
W
—
—
N
S

939

Phage-
L
I
—
—
—
—
T
—
—
Q
—
S
I
H

940

Phage-
D
Y
—
M
—
L
R
—
V
F
—
—
T
V

941

Phage-
D
A
F
Q
—
G
E
R
—
Q
—
—
N
D

942

Phage-
H
S
—
—
—
—
R
—
—
W
—
—
T
V

943

Phage-
L
D
—
I
—
—
—
—
—
—
—
—
I
N

944

Phage-
F
D
—
—
—
R
R
K
—
W
—
P
—
N

945

Phage-
Y
T
—
—
—
W
R
—
V
H
—
—
H
I

946

Phage-
—
H
—
W
—
F
R
—
—
—
—
—
D
V

947

Phage-
D
T
—
—
—
—
R
—
I
—
—
—
H
I

948

Phage-
H
N
—
L
—
W
R
—
I
—
—
—
I
D

949

Phage-
D
I
—
—
—
L
R
—
V
F
—
—
T
A

950

Phage-
P
—
V
L
—
L
H
K
H
Q
—
L
S
Y

951

Phage-
N
I
—
L
—
—
N
K
M
Q
—
F
Y
H

952

Phage-
A
D
—
W
—
—
R
—
—
—
—
—
I
I

953

Phage-
H
—
—
—
—
W
H
—
V
—
—
—
H
—

954

Phage-
A
F
—
M
—
W
H
—
—
—
—
—
H
—

955

Phage-
V
I
—
L
—
S
D
Q
—
—
—
K
D
—

956

Phage-
V
Y
—
—
—
—
Q
E
—
K
—
—
F
S

957

Phage-
I
H
—
W
—
F
R
—
V
—
—
—
H
V

958

Phage-
D
I
—
—
—
W
R
—
—
—
—
—
T
S

959

Phage-
F
D
—
—
—
—
R
—
—
W
—
—
N
A

960

Phage-
Y
S
—
—
—
—
—
—
—
W
—
—
N
V

961

Phage-
N
Y
—
W
—
W
—
—
—
—
—
—
T
D

962

Phage-
Y
—
—
H
—
W
H
—
—
—
—
—
N
—

963

Phage-
D
—
S
—
—
Q
Q
K
—
Q
—
S
Y
D

964

Phage-
N
D
—
V
—
W
R
—
I
—
—
—
I
Y

965

Phage-
N
T
V
P
—
A
M
—
—
W
—
R
D
F

966

Phage-
—
H
F
—
—
W
R
—
—
—
—
—
N
D

967

Phage-
D
F
—
—
—
L
R
—
V
F
—
—
T
A

968

Phage-
N
D
—
—
—
—
Q
—
—
—
—
—
H
—

969

Phage-
D
A
—
—
—
F
R
—
—
W
—
—
N
L

970

Phage-
Y
N
—
V
—
F
Q
—
I
F
—
—
H
D

971

Phage-
V
P
—
—
—
—
L
K
I
F
—
G
H
N

972

Phage-
Y
S
—
—
—
—
H
—
—
—
—
—
N
A

973

Phage-
T
N
—
—
—
G
—
S
—
Q
—
R
N
I

974

Phage-
H
I
V
—
—
—
N
Y
—
Q
—
G
H
F

975

Phage-
N
H
—
L
—
A
S
Q
F
Q
—
—
N
A

976

Phage-
F
I
—
—
—
G
Q
H
—
Q
—
L
D
A

977

Phage-
A
D
—
L
—
F
R
—
—
W
—
—
H
N

978

Phage-
T
T
—
W
—
Y
R
—
V
—
—
—
N
I

979

Phage-
D
S
—
W
—
L
—
—
—
V
—
Q
S
N

980

Phage binding ELISA

TROP2 Fab

TROP2
signal in

Backgroud
Fab
presence of
SEQ

signal
signal
TROP2
ID NO.

Phage-1
0.091
1.227
0.085
23

Phage-2
0.072
2.622
0.125
159

Phage-3
0.110
2.763
0.142
160

Phage-4
0.073
2.739
0.215
161

Phage-5
0.083
2.746
0.343
162

Phage-6
0.067
2.512
0.167
163

Phage-7
0.077
2.742
0.451
164

Phage-8
0.078
2.729
0.339
165

Phage-9
0.072
2.761
0.289
166

Phage-
0.114
2.798
0.086
167

10

Phage-
0.098
2.717
0.173
168

11

Phage-
0.097
2.737
0.221
169

12

Phage-
0.135
2.713
0.168
170

13

Phage-
0.102
2.737
0.327
171

14

Phage-
0.122
2.694
0.161
172

15

Phage-
0.089
2.674
0.186
173

16

Phage-
0.072
2.704
0.283
174

17

Phage-
0.184
2.820
0.379
175

18

Phage-
0.071
2.710
0.237
176

19

Phage-
0.082
2.784
0.344
177

20

Phage-
0.122
2.711
0.134
178

21

Phage-
0.221
2.717
0.247
244

22

Phage-
0.112
2.714
0.296
245

23

Phage-
0.081
2.721
0.258
246

24

Phage-
0.178
2.728
0.211
247

25

Phage-
0.067
2.814
0.156
248

26

Phage-
0.089
2.824
0.083
249

27

Phage-
0.142
2.777
0.134
250

28

Phage-
0.541
2.760
0.419
251

29

Phage-
0.293
2.748
0.225
252

30

Phage-
0.269
2.747
0.265
253

31

Phage-
0.219
2.744
0.143
254

32

Phage-
0.104
2.742
0.087
255

33

Phage-
0.191
2.740
0.138
256

34

Phage-
0.078
2.739
0.078
257

35

Phage-
0.239
2.739
0.194
258

36

Phage-
0.106
2.738
0.104
259

37

Phage-
0.257
2.738
0.228
260

38

Phage-
0.204
2.736
0.137
261

39

Phage-
0.317
2.736
0.166
262

40

Phage-
0.171
2.735
0.148
263

41

Phage-
0.383
2.735
0.324
264

42

Phage-
0.207
2.731
0.200
265

43

Phage-
0.183
2.729
0.144
266

44

Phage-
0.236
2.728
0.216
267

45

Phage-
0.349
2.728
0.306
268

46

Phage-
0.151
2.726
0.137
269

47

Phage-
0.269
2.724
0.111
270

48

Phage-
0.375
2.724
0.357
271

49

Phage-
0.231
2.723
0.218
272

50

Phage-
0.611
2.722
0.297
273

51

Phage-
0.159
2.722
0.136
274

52

Phage-
0.169
2.722
0.147
275

53

Phage-
0.329
2.721
0.128
276

54

Phage-
0.181
2.721
0.108
277

55

Phage-
0.297
2.719
0.169
278

56

Phage-
0.379
2.719
0.167
279

57

Phage-
2.696
2.719
2.689
280

58

Phage-
0.225
2.718
0.216
281

59

Phage-
0.321
2.716
0.319
282

60

Phage-
0.215
2.714
0.195
283

61

Phage-
0.338
2.713
0.208
284

62

Phage-
0.392
2.712
0.270
285

63

Phage-
0.223
2.711
0.168
286

64

Phage-
0.566
2.711
0.556
287

65

Phage-
0.470
2.711
0.208
288

66

Phage-
0.342
2.710
0.310
289

67

Phage-
0.267
2.710
0.133
290

68

Phage-
0.313
2.709
0.226
291

69

Phage-
0.301
2.706
0.159
292

70

Phage-
0.264
2.705
0.093
293

71

Phage-
0.444
2.704
0.418
294

72

Phage-
0.245
2.702
0.122
295

73

Phage-
0.456
2.702
0.313
296

74

Phage-
0.329
2.701
0.287
297

75

Phage-
0.337
2.701
0.214
298

76

Phage-
0.358
2.701
0.222
299

77

Phage-
0.133
2.699
0.123
300

78

Phage-
0.246
2.697
0.138
301

79

Phage-
0.220
2.694
0.196
302

80

Phage-
0.349
2.693
0.130
303

81

Phage-
0.234
2.691
0.135
304

82

Phage-
0.316
2.691
0.167
305

83

Phage-
0.324
2.687
0.202
306

84

Phage-
0.145
2.681
0.136
307

85

Phage-
0.138
2.674
0.138
308

86

Phage-
0.343
2.672
0.204
309

87

Phage-
0.185
2.672
0.082
310

88

Phage-
0.180
2.666
0.155
311

89

Phage-
0.132
2.664
0.119
312

90

Phage-
0.200
2.657
0.195
313

91

Phage-
0.247
2.655
0.149
314

92

Phage-
0.204
2.652
0.136
315

93

Phage-
0.235
2.631
0.195
316

94

Phage-
0.160
2.626
0.153
317

95

Phage-
0.178
2.623
0.160
318

96

Phage-
0.177
2.621
0.111
319

97

Phage-
0.145
2.594
0.131
320

98

Phage-
0.341
2.593
0.148
321

99

Phage-
0.347
2.577
0.171
322

100

Phage-
0.259
2.540
0.226
323

101

Phage-
0.162
2.538
0.124
324

102

Phage-
0.338
2.487
0.173
325

103

Phage-
0.087
2.479
0.074
326

104

Phage-
0.296
2.437
0.263
327

105

Phage-
0.266
2.413
0.163
328

106

Phage-
0.193
2.313
0.139
329

107

Phage-
0.140
2.290
0.128
330

108

Phage-
0.091
2.217
0.078
331

109

Phage-
0.167
2.036
0.162
332

110

Phage-
0.089
2.029
0.083
333

111

Phage-
0.372
2.011
0.154
334

112

Phage-
0.290
2.001
0.266
335

113

Phage-
0.191
1.935
0.143
336

114

Phage-
0.215
1.805
0.172
337

115

Phage-
0.234
1.730
0.120
338

116

Phage-
0.174
1.648
0.110
339

117

Phage-
0.124
1.475
0.118
340

118

Phage-
0.203
1.442
0.070
341

119

Phage-
0.183
1.441
0.134
342

120

Phage-
0.150
1.332
0.132
343

121

Phage-
0.150
0.814
0.148
344

122

Phage-
0.107
0.698
0.099
345

123

Phage-
0.084
0.636
0.078
346

124

Phage-
0.182
0.508
0.064
347

125

Phage-
0.185
0.488
0.131
348

126

Phage-
0.110
0.480
0.108
349

127

Phage-
0.196
0.302
0.153
350

128

Phage-
0.126
0.194
0.095
351

129

Phage-
0.146
0.143
0.166
352

130

Phage-
0.166
0.105
2.655
353

131

Phage-
0.268
0.099
2.674
354

132

Phage-
0.117
0.098
0.125
355

133

Phage-
0.113
0.095
0.134
356

134

Phage-
0.234
0.090
2.682
357

135

Phage-
0.113
0.089
2.695
358

136

Phage-
0.097
0.088
0.099
359

137

Phage-
0.082
0.088
0.073
360

138

Phage-
0.087
0.085
0.095
361

139

Phage-
0.087
0.084
2.843
362

140

Phage-
0.094
0.083
0.100
363

141

Phage-
0.121
0.082
0.133
364

142

Phage-
0.087
0.078
0.138
365

143

Phage-
0.092
0.077
0.098
366

144

Phage-
0.083
0.077
0.092
367

145

Phage-
0.090
0.076
0.111
368

146

Phage-
0.098
0.076
0.101
369

147

Phage-
0.090
0.076
0.109
370

148

Phage-
0.070
0.075
0.069
371

149

Phage-
0.079
0.075
0.088
372

150

Phage-
0.081
0.075
0.112
373

151

Phage-
0.083
0.075
0.092
374

152

Phage-
0.138
0.073
0.252
375

153

Phage-
0.096
0.073
0.098
376

154

Phage-
0.119
0.072
0.280
377

155

Phage-
0.074
0.072
0.149
378

156

Phage-
0.078
0.072
0.113
379

157

Phage-
0.081
0.071
0.136
380

158

Phage-
0.077
0.070
0.087
381

159

Phage-
0.089
0.070
0.110
382

160

Phage-
0.085
0.070
0.107
383

161

Phage-
0.083
0.069
0.191
384

162

Phage-
0.085
0.069
0.089
385

163

Phage-
0.094
0.068
0.124
386

164

Phage-
0.084
0.068
2.688
387

165

Phage-
0.229
0.067
2.668
388

166

Phage-
0.070
0.067
0.389
389

167

Phage-
0.073
0.066
0.089
390

168

Phage-
0.084
0.065
0.130
391

169

Phage-
0.082
0.064
0.150
392

170

Phage-
0.065
0.064
0.212
393

171

Phage-
0.141
0.063
0.371
394

172

Phage-
0.096
0.062
0.110
395

173

Phage-
0.072
0.062
0.086
396

174

Phage-
0.185
0.062
0.251
397

175

Phage-
0.167
0.061
0.308
398

176

Phage-
0.066
0.061
0.181
399

177

Phage-
0.186
0.061
0.403
400

178

Phage-
0.067
0.061
0.149
401

179

Phage-
0.074
0.057
0.082
402

180

Phage-
0.169
2.685
0.169
403

181

Phage-
0.096
2.715
0.097
404

182

Phage-
0.110
2.671
0.111
405

183

Phage-
0.150
2.408
0.151
406

184

Phage-
0.150
2.515
0.151
407

185

Phage-
0.234
2.711
0.236
408

186

Phage-
0.379
2.712
0.381
409

187

Phage-
0.131
2.710
0.134
410

188

Phage-
0.093
2.697
0.097
411

189

Phage-
0.146
2.601
0.150
412

190

Phage-
0.144
2.565
0.149
413

191

Phage-
0.170
2.513
0.175
414

192

Phage-
0.212
2.706
0.218
415

193

Phage-
0.118
2.054
0.124
416

194

Phage-
0.202
2.626
0.209
417

195

Phage-
0.104
2.786
0.113
418

196

Phage-
0.148
2.745
0.157
419

197

Phage-
0.182
2.701
0.191
420

198

Phage-
0.076
1.157
0.079
421

199

Phage-
0.080
2.690
0.089
422

200

Phage-
0.142
1.423
0.147
423

201

Phage-
0.086
2.755
0.098
424

202

Phage-
0.062
2.685
0.075
425

203

Phage-
0.070
2.639
0.083
426

204

Phage-
0.163
2.636
0.175
427

205

Phage-
0.088
2.686
0.102
428

206

Phage-
0.076
2.729
0.090
429

207

Phage-
0.093
2.754
0.109
430

208

Phage-
0.099
2.013
0.110
431

209

Phage-
0.144
2.664
0.159
432

210

Phage-
0.167
2.511
0.182
433

211

Phage-
0.104
2.774
0.121
434

212

Phage-
0.132
2.746
0.148
435

213

Phage-
0.095
2.597
0.112
436

214

Phage-
0.252
2.703
0.269
437

215

Phage-
0.091
2.822
0.110
438

216

Phage-
0.204
2.710
0.222
439

217

Phage-
0.210
2.711
0.228
440

218

Phage-
0.108
2.666
0.128
441

219

Phage-
0.150
2.461
0.168
442

220

Phage-
0.130
2.710
0.152
443

221

Phage-
0.077
1.498
0.089
444

222

Phage-
0.104
2.740
0.128
445

223

Phage-
0.087
2.805
0.112
446

224

Phage-
0.073
0.666
0.078
447

225

Phage-
0.096
2.436
0.118
448

226

Phage-
0.117
2.752
0.141
449

227

Phage-
0.067
2.324
0.088
450

228

Phage-
0.104
2.050
0.123
451

229

Phage-
0.154
2.810
0.180
452

230

Phage-
0.078
2.368
0.101
453

231

Phage-
0.075
1.658
0.091
454

232

Phage-
0.176
2.738
0.203
455

233

Phage-
0.072
2.693
0.099
456

234

Phage-
0.125
2.662
0.151
457

235

Phage-
0.073
2.358
0.097
458

236

Phage-
0.082
2.397
0.107
459

237

Phage-
0.070
2.653
0.099
460

238

Phage-
0.308
2.740
0.335
461

239

Phage-
0.138
1.860
0.157
462

240

Phage-
0.123
2.555
0.150
463

241

Phage-
0.076
2.684
0.106
464

242

Phage-
0.108
2.754
0.138
465

243

Phage-
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466

244

Phage-
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467

245

Phage-
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468

246

Phage-
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469

247

Phage-
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470

248

Phage-
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471

249

Phage-
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472

250

Phage-
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473

251

Phage-
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474

252

Phage-
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475

253

Phage-
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476

254

Phage-
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477

255

Phage-
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478

256

Phage-
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479

257

Phage-
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480

258

Phage-
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481

259

Phage-
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482

260

Phage-
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483

261

Phage-
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484

262

Phage-
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485

263

Phage-
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486

264

Phage-
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487

265

Phage-
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488

266

Phage-
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489

267

Phage-
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0.127
490

268

Phage-
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491

269

Phage-
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492

270

Phage-
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493

271

Phage-
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494

272

Phage-
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495

273

Phage-
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496

274

Phage-
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0.274
497

275

Phage-
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498

276

Phage-
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0.197
499

277

Phage-
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2.656
0.208
500

278

Phage-
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501

279

Phage-
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502

280

Phage-
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0.165
503

281

Phage-
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0.209
504

282

Phage-
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0.371
505

283

Phage-
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506

284

Phage-
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0.434
507

285

Phage-
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0.148
508

286

Phage-
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0.131
509

287

Phage-
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510

288

Phage-
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511

289

Phage-
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0.294
512

290

Phage-
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0.186
513

291

Phage-
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0.135
514

292

Phage-
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2.698
0.184
515

293

Phage-
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2.748
0.202
516

294

Phage-
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0.129
517

295

Phage-
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0.097
518

296

Phage-
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2.740
0.180
519

297

Phage-
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0.742
0.162
520

298

Phage-
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0.142
521

299

Phage-
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0.171
522

300

Phage-
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0.136
523

301

Phage-
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0.128
524

302

Phage-
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2.754
0.270
525

303

Phage-
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526

304

Phage-
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2.341
0.163
527

305

Phage-
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2.759
0.129
528

306

Phage-
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0.105
529

307

Phage-
0.123
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0.176
530

308

Phage-
0.138
2.750
0.192
531

309

Phage-
0.096
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0.141
532

310

Phage-
0.273
2.656
0.323
533

311

Phage-
0.077
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534

312

Phage-
0.064
0.214
0.068
535

313

Phage-
0.095
2.798
0.154
536

314

Phage-
0.068
0.455
0.077
537

315

Phage-
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2.722
0.246
538

316

Phage-
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2.722
0.229
539

317

Phage-
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0.141
540

318

Phage-
0.066
2.001
0.110
541

319

Phage-
0.094
2.815
0.156
542

320

Phage-
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0.159
543

321

Phage-
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0.159
544

322

Phage-
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0.153
545

323

Phage-
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2.579
0.300
546

324

Phage-
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2.701
0.134
547

325

Phage-
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2.817
0.161
548

326

Phage-
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0.136
549

327

Phage-
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550

328

Phage-
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0.254
551

329

Phage-
0.094
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552

330

Phage-
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0.134
553

331

Phage-
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0.168
554

332

Phage-
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0.160
555

333

Phage-
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0.131
556

334

Phage-
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2.725
0.175
557

335

Phage-
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0.115
558

336

Phage-
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0.384
559

337

Phage-
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0.189
560

338

Phage-
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561

339

Phage-
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0.152
562

340

Phage-
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0.133
563

341

Phage-
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0.141
564

342

Phage-
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565

343

Phage-
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566

344

Phage-
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0.186
567

345

Phage-
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0.115
568

346

Phage-
0.104
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569

347

Phage-
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0.196
570

348

Phage-
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0.254
571

349

Phage-
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0.127
572

350

Phage-
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0.211
573

351

Phage-
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0.160
574

352

Phage-
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575

353

Phage-
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576

354

Phage-
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0.113
577

355

Phage-
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578

356

Phage-
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0.148
579

357

Phage-
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0.163
580

358

Phage-
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0.247
581

359

Phage-
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0.133
582

360

Phage-
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583

361

Phage-
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0.238
584

362

Phage-
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2.362
0.268
585

363

Phage-
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586

364

Phage-
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587

365

Phage-
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588

366

Phage-
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589

367

Phage-
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590

368

Phage-
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591

369

Phage-
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592

370

Phage-
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593

371

Phage-
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594

372

Phage-
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595

373

Phage-
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596

374

Phage-
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0.364
597

375

Phage-
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598

376

Phage-
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599

377

Phage-
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600

378

Phage-
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601

379

Phage-
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602

380

Phage-
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0.257
603

381

Phage-
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604

382

Phage-
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605

383

Phage-
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606

384

Phage-
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0.273
607

385

Phage-
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608

386

Phage-
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0.158
609

387

Phage-
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0.196
610

388

Phage-
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611

389

Phage-
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0.202
612

390

Phage-
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0.229
613

391

Phage-
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614

392

Phage-
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0.562
615

393

Phage-
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616

394

Phage-
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0.212
617

395

Phage-
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618

396

Phage-
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0.244
619

397

Phage-
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0.185
620

398

Phage-
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0.381
621

399

Phage-
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0.286
622

400

Phage-
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0.169
623

401

Phage-
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2.669
0.278
624

402

Phage-
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0.170
625

403

Phage-
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0.188
626

404

Phage-
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627

405

Phage-
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628

406

Phage-
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0.223
629

407

Phage-
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0.269
630

408

Phage-
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0.255
631

409

Phage-
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0.216
632

410

Phage-
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0.227
633

411

Phage-
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0.181
634

412

Phage-
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0.150
635

413

Phage-
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636

414

Phage-
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0.246
637

415

Phage-
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638

416

Phage-
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0.393
639

417

Phage-
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0.179
640

418

Phage-
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641

419

Phage-
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0.251
642

420

Phage-
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643

421

Phage-
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0.311
644

422

Phage-
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645

423

Phage-
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646

424

Phage-
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647

425

Phage-
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648

426

Phage-
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649

427

Phage-
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650

428

Phage-
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651

429

Phage-
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652

430

Phage-
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653

431

Phage-
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654

432

Phage-
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655

433

Phage-
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656

434

Phage-
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657

435

Phage-
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658

436

Phage-
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659

437

Phage-
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660

438

Phage-
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661

439

Phage-
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662

440

Phage-
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663

441

Phage-
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664

442

Phage-
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665

443

Phage-
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666

444

Phage-
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667

445

Phage-
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668

446

Phage-
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669

447

Phage-
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670

448

Phage-
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671

449

Phage-
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672

450

Phage-
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673

451

Phage-
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674

452

Phage-
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675

453

Phage-
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676

454

Phage-
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677

455

Phage-
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678

456

Phage-
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679

457

Phage-
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680

458

Phage-
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681

459

Phage-
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682

460

Phage-
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683

461

Phage-
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684

462

Phage-
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685

463

Phage-
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686

464

Phage-
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687

465

Phage-
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688

466

Phage-
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689

467

Phage-
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690

468

Phage-
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691

469

Phage-
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692

470

Phage-
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693

471

Phage-
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694

472

Phage-
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695

473

Phage-
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696

474

Phage-
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697

475

Phage-
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698

476

Phage-
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699

477

Phage-
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700

478

Phage-
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701

479

Phage-
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702

480

Phage-
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703

481

Phage-
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704

482

Phage-
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705

483

Phage-
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706

484

Phage-
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707

485

Phage-
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708

486

Phage-
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709

487

Phage-
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710

488

Phage-
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711

489

Phage-
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712

490

Phage-
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713

491

Phage-
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714

492

Phage-
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715

493

Phage-
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716

494

Phage-
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717

495

Phage-
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718

496

Phage-
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719

497

Phage-
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720

498

Phage-
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721

499

Phage-
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722

500

Phage-
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723

501

Phage-
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724

502

Phage-
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725

503

Phage-
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726

504

Phage-
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727

505

Phage-
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728

506

Phage-
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729

507

Phage-
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730

508

Phage-
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731

509

Phage-
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732

510

Phage-
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733

511

Phage-
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734

512

Phage-
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735

513

Phage-
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736

514

Phage-
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737

515

Phage-
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738

516

Phage-
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739

517

Phage-
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740

518

Phage-
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741

519

Phage-
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742

520

Phage-
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743

521

Phage-
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744

522

Phage-
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745

523

Phage-
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746

524

Phage-
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747

525

Phage-
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748

526

Phage-
0.091
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749

527

Phage-
0.172
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750

528

Phage-
0.111
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751

529

Phage-
0.069
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752

530

Phage-
0.084
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753

531

Phage-
0.087
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754

532

Phage-
0.141
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755

533

Phage-
0.077
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756

534

Phage-
0.153
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757

535

Phage-
0.210
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758

536

Phage-
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759

537

Phage-
0.150
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760

538

Phage-
0.384
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761

539

Phage-
0.092
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762

540

Phage-
0.186
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0.362
763

541

Phage-
0.072
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0.256
764

542

Phage-
0.154
2.718
0.330
765

543

Phage-
0.094
2.736
0.277
766

544

Phage-
0.102
2.755
0.285
767

545

Phage-
0.074
2.836
0.265
768

546

Phage-
0.096
2.750
0.282
769

547

Phage-
0.090
2.725
0.274
770

548

Phage-
0.103
2.838
0.296
771

549

Phage-
0.110
2.362
0.269
772

550

Phage-
0.124
2.723
0.308
773

551

Phage-
0.065
1.332
0.155
774

552

Phage-
0.073
2.702
0.259
775

553

Phage-
0.260
2.703
0.434
776

554

Phage-
0.151
2.837
0.344
777

555

Phage-
0.134
2.802
0.326
778

556

Phage-
0.198
2.676
0.377
779

557

Phage-
0.072
2.820
0.272
780

558

Phage-
0.142
2.696
0.329
781

559

Phage-
0.088
2.654
0.277
782

560

Phage-
0.092
2.746
0.287
783

561

Phage-
0.187
2.740
0.375
784

562

Phage-
0.069
2.321
0.236
785

563

Phage-
0.142
2.737
0.336
786

564

Phage-
0.079
2.679
0.273
787

565

Phage-
0.074
2.733
0.273
788

566

Phage-
0.163
2.695
0.353
789

567

Phage-
0.079
2.697
0.276
790

568

Phage-
0.090
2.738
0.288
791

569

Phage-
0.070
2.004
0.216
792

570

Phage-
0.431
2.750
0.606
793

571

Phage-
0.084
1.931
0.223
794

572

Phage-
0.176
2.753
0.370
795

573

Phage-
0.230
2.742
0.421
796

574

Phage-
0.139
2.755
0.340
797

575

Phage-
0.082
2.676
0.282
798

576

Phage-
0.105
2.697
0.305
799

577

Phage-
0.082
2.748
0.288
800

578

Phage-
0.124
2.833
0.335
801

579

Phage-
0.101
2.731
0.305
802

580

Phage-
0.082
2.732
0.288
803

581

Phage-
0.237
2.715
0.430
804

582

Phage-
0.327
2.697
0.512
805

583

Phage-
0.411
2.750
0.594
806

584

Phage-
0.132
2.588
0.326
807

585

Phage-
0.188
2.806
0.395
808

586

Phage-
0.102
2.192
0.272
809

587

Phage-
0.084
2.822
0.307
810

588

Phage-
0.072
2.743
0.289
811

589

Phage-
0.077
2.724
0.293
812

590

Phage-
0.076
2.132
0.244
813

591

Phage-
0.116
2.755
0.334
814

592

Phage-
0.182
2.829
0.401
815

593

Phage-
0.193
2.716
0.402
816

594

Phage-
0.064
1.493
0.182
817

595

Phage-
0.595
2.722
0.771
818

596

Phage-
0.151
2.827
0.375
819

597

Phage-
0.074
2.775
0.300
820

598

Phage-
0.108
2.656
0.323
821

599

Phage-
0.089
2.735
0.313
822

600

Phage-
0.173
2.760
0.392
823

601

Phage-
0.282
2.739
0.490
824

602

Phage-
0.250
2.708
0.459
825

603

Phage-
0.220
2.835
0.443
826

604

Phage-
0.117
2.698
0.337
827

605

Phage-
0.076
2.713
0.302
828

606

Phage-
0.070
2.740
0.299
829

607

Phage-
0.239
2.161
0.405
830

608

Phage-
0.220
2.763
0.444
831

609

Phage-
0.148
2.693
0.373
832

610

Phage-
0.156
2.742
0.386
833

611

Phage-
0.173
2.730
0.401
834

612

Phage-
0.074
2.752
0.313
835

613

Phage-
0.115
2.673
0.347
836

614

Phage-
0.135
2.832
0.381
837

615

Phage-
0.086
2.745
0.328
838

616

Phage-
0.121
2.747
0.361
839

617

Phage-
0.169
2.743
0.405
840

618

Phage-
0.103
2.042
0.281
841

619

Phage-
0.079
2.723
0.322
842

620

Phage-
0.094
2.843
0.346
843

621

Phage-
0.173
2.737
0.411
844

622

Phage-
0.130
2.734
0.372
845

623

Phage-
0.103
2.735
0.348
846

624

Phage-
0.113
2.740
0.360
847

625

Phage-
0.098
2.784
0.351
848

626

Phage-
0.076
2.844
0.337
849

627

Phage-
0.405
2.705
0.622
850

628

Phage-
0.204
2.764
0.445
851

629

Phage-
0.092
2.027
0.275
852

630

Phage-
0.132
2.714
0.376
853

631

Phage-
0.760
2.768
0.955
854

632

Phage-
0.258
2.738
0.499
855

633

Phage-
0.224
2.708
0.469
856

634

Phage-
0.213
2.697
0.461
857

635

Phage-
0.142
2.700
0.401
858

636

Phage-
0.146
2.718
0.409
859

637

Phage-
0.133
2.700
0.395
860

638

Phage-
0.225
2.846
0.494
861

639

Phage-
0.082
2.751
0.356
862

640

Phage-
0.068
2.180
0.287
863

641

Phage-
0.145
2.775
0.419
864

642

Phage-
0.134
2.536
0.386
865

643

Phage-
0.107
2.721
0.382
866

644

Phage-
0.069
1.074
0.176
867

645

Phage-
0.176
2.696
0.445
868

646

Phage-
0.074
2.821
0.368
869

647

Phage-
0.110
2.735
0.392
870

648

Phage-
0.203
2.737
0.475
871

649

Phage-
0.096
2.853
0.394
872

650

Phage-
0.193
2.683
0.463
873

651

Phage-
0.078
2.849
0.379
874

652

Phage-
0.103
2.743
0.392
875

653

Phage-
0.147
2.741
0.433
876

654

Phage-
0.121
2.750
0.411
877

655

Phage-
0.188
2.725
0.469
878

656

Phage-
0.120
2.647
0.400
879

657

Phage-
0.073
2.735
0.370
880

658

Phage-
0.170
2.712
0.455
881

659

Phage-
0.106
2.705
0.398
882

660

Phage-
0.166
2.713
0.453
883

661

Phage-
0.096
2.672
0.386
884

662

Phage-
0.182
2.674
0.463
885

663

Phage-
0.087
2.813
0.396
886

664

Phage-
0.092
2.730
0.391
887

665

Phage-
0.325
2.741
0.599
888

666

Phage-
0.110
2.721
0.407
889

667

Phage-
0.163
2.680
0.451
890

668

Phage-
0.282
2.609
0.550
891

669

Phage-
0.088
2.739
0.394
892

670

Phage-
0.147
2.853
0.461
893

671

Phage-
0.124
2.724
0.426
894

672

Phage-
0.198
2.824
0.503
895

673

Phage-
0.182
2.799
0.486
896

674

Phage-
0.097
0.831
0.183
897

675

Phage-
0.419
2.697
0.689
898

676

Phage-
0.130
2.764
0.443
899

677

Phage-
0.150
2.816
0.471
900

678

Phage-
0.115
2.720
0.430
901

679

Phage-
0.218
2.740
0.522
902

680

Phage-
0.145
2.714
0.456
903

681

Phage-
0.153
2.712
0.464
904

682

Phage-
0.083
2.696
0.403
905

683

Phage-
0.068
2.706
0.391
906

684

Phage-
0.087
2.709
0.409
907

685

Phage-
0.073
1.763
0.280
908

686

Phage-
0.113
2.824
0.446
909

687

Phage-
0.109
2.741
0.435
910

688

Phage-
0.088
2.754
0.419
911

689

Phage-
0.310
2.739
0.621
912

690

Phage-
0.150
2.735
0.482
913

691

Phage-
0.127
2.713
0.460
914

692

Phage-
0.141
2.732
0.475
915

693

Phage-
0.080
2.700
0.421
916

694

Phage-
0.316
2.691
0.625
917

695

Phage-
0.151
2.758
0.493
918

696

Phage-
0.082
2.625
0.419
919

697

Phage-
0.089
2.739
0.441
920

698

Phage-
0.342
2.683
0.654
921

699

Phage-
0.157
2.697
0.497
922

700

Phage-
0.136
2.723
0.487
923

701

Phage-
0.108
0.383
0.146
924

702

Phage-
0.105
2.755
0.470
925

703

Phage-
0.109
1.973
0.366
926

704

Phage-
0.147
2.683
0.497
927

705

Phage-
0.094
2.729
0.458
928

706

Phage-
0.156
2.704
0.508
929

707

Phage-
0.098
2.847
0.478
930

708

Phage-
0.094
2.711
0.456
931

709

Phage-
0.154
2.753
0.514
932

710

Phage-
0.075
2.731
0.444
933

711

Phage-
0.109
2.710
0.471
934

712

Phage-
0.153
2.845
0.529
935

713

Phage-
0.313
2.662
0.641
936

714

Phage-
0.352
2.710
0.684
937

715

Phage-
0.195
2.807
0.563
938

716

Phage-
0.180
2.727
0.541
939

717

Phage-
0.083
2.713
0.457
940

718

Phage-
0.125
2.746
0.498
941

719

Phage-
0.166
2.719
0.530
942

720

Phage-
0.274
2.707
0.626
943

721

Phage-
0.070
0.666
0.156
944

722

Phage-
0.131
2.696
0.505
945

723

Phage-
0.077
2.710
0.461
946

724

Phage-
0.300
2.749
0.658
947

725

Phage-
0.123
2.719
0.507
948

726

Phage-
0.073
2.656
0.455
949

727

Phage-
0.167
2.739
0.552
950

728

Phage-
0.126
2.709
0.512
951

729

Phage-
0.083
2.594
0.459
952

730

Phage-
0.104
2.728
0.498
953

731

Phage-
0.139
2.717
0.526
954

732

Phage-
0.086
2.745
0.486
955

733

Phage-
0.226
2.714
0.602
956

734

Phage-
0.131
2.723
0.524
957

735

Phage-
0.070
2.723
0.474
958

736

Phage-
0.159
2.753
0.555
959

737

Phage-
0.218
2.696
0.598
960

738

Phage-
0.078
2.741
0.488
961

739

Phage-
0.167
2.753
0.567
962

740

Phage-
0.349
2.716
0.717
963

741

Phage-
0.099
2.849
0.528
964

742

Phage-
0.202
2.721
0.598
965

743

Phage-
0.089
2.746
0.508
966

744

Phage-
0.086
0.545
0.159
967

745

Phage-
0.105
2.839
0.539
968

746

Phage-
0.106
2.851
0.543
969

747

Phage-
0.097
2.832
0.541
970

748

Phage-
0.194
2.721
0.605
971

749

Phage-
0.098
2.739
0.530
972

750

Phage-
0.077
2.547
0.483
973

751

Phage-
0.183
2.681
0.595
974

752

Phage-
0.310
2.838
0.734
975

753

Phage-
0.071
2.736
0.526
976

754

Phage-
0.063
0.767
0.183
977

755

Phage-
0.075
2.853
0.550
978

756

Phage-
0.150
2.711
0.589
979

757

Phage-
0.291
2.755
0.715
980

758

Phage-
0.441
2.714
0.839
981

759

Phage-
0.195
2.673
0.629
982

760

Phage-
0.123
2.737
0.583
983

761

Phage-
0.311
2.725
0.738
984

762

Phage-
0.095
2.737
0.563
985

763

Phage-
0.090
2.706
0.554
986

764

Phage-
0.102
2.753
0.573
987

765

Phage-
0.228
2.753
0.678
988

766

Phage-
0.171
2.719
0.627
989

767

Phage-
0.182
2.700
0.637
990

768

Phage-
0.112
2.753
0.590
991

769

Phage-
0.085
2.737
0.566
992

770

Phage-
0.085
2.744
0.568
993

771

Phage-
0.239
2.826
0.710
994

772

Phage-
0.067
2.724
0.557
995

773

Phage-
0.142
2.833
0.640
996

774

Phage-
0.192
2.780
0.671
997

775

Phage-
0.462
2.755
0.887
998

776

Phage-
0.095
2.724
0.583
999

777

Phage-
0.187
2.858
0.687
1000

778

Phage-
0.176
2.759
0.659
1001

779

Phage-
0.250
2.831
0.735
1002

780

Phage-
0.109
0.631
0.207
1003

781

Phage-
0.093
2.780
0.599
1004

782

Phage-
0.433
2.737
0.869
1005

783

Phage-
0.406
2.818
0.862
1006

784

Phage-
0.121
2.837
0.639
1007

785

Phage-
0.111
2.754
0.619
1008

786

Phage-
0.092
2.743
0.603
1009

787

Phage-
0.097
2.849
0.630
1010

788

Phage-
0.139
2.839
0.663
1011

789

Phage-
0.080
2.788
0.612
1012

790

Phage-
0.171
2.724
0.672
1013

791

Phage-
0.094
2.708
0.612
1014

792

Phage-
0.386
2.858
0.877
1015

793

Phage-
0.343
2.832
0.846
1016

794

Phage-
0.103
2.869
0.663
1017

795

Phage-
0.105
2.832
0.660
1018

796

Phage-
0.099
2.729
0.635
1019

797

Phage-
0.291
2.732
0.791
1020

798

Phage-
0.098
2.823
0.658
1021

799

Phage-
0.145
2.691
0.682
1022

800

Phage-
0.073
2.735
0.639
1023

801

Phage-
0.136
2.735
0.697
1024

802

Phage-
0.074
2.721
0.646
1025

803

Phage-
0.709
2.687
1.137
1026

804

Phage-
0.115
0.066
0.105
1027

805

Phage-
0.824
2.717
1.236
1028

806

Phage-
0.330
2.727
0.851
1029

807

Phage-
0.093
2.824
0.689
1030

808

Phage-
0.506
2.727
0.995
1031

809

Phage-
0.142
2.721
0.719
1032

810

Phage-
0.084
2.740
0.678
1033

811

Phage-
0.079
2.760
0.681
1034

812

Phage-
1.432
2.725
1.725
1035

813

Phage-
0.090
2.413
0.617
1036

814

Phage-
0.075
2.843
0.711
1037

815

Phage-
0.070
2.719
0.680
1038

816

Phage-
0.090
2.718
0.698
1039

817

Phage-
0.761
2.730
1.218
1040

818

Phage-
0.079
0.186
0.103
1041

819

Phage-
0.621
2.743
1.115
1042

820

Phage-
0.131
2.708
0.732
1043

821

Phage-
0.121
2.687
0.729
1044

822

Phage-
0.099
0.088
0.096
1045

823

Phage-
0.181
2.751
0.799
1046

824

Phage-
0.288
2.742
0.881
1047

825

Phage-
0.106
2.737
0.742
1048

826

Phage-
0.154
2.760
0.790
1049

827

Phage-
0.078
2.641
0.704
1050

828

Phage-
0.870
2.714
1.321
1051

829

Phage-
0.578
2.745
1.115
1052

830

Phage-
0.369
2.819
0.978
1053

831

Phage-
0.116
2.845
0.796
1054

832

Phage-
0.108
2.748
0.766
1055

833

Phage-
0.104
2.741
0.765
1056

834

Phage-
0.083
0.755
0.255
1057

835

Phage-
0.077
2.849
0.795
1058

836

Phage-
0.084
2.733
0.772
1059

837

Phage-
0.259
2.752
0.908
1060

838

Phage-
0.154
2.746
0.830
1061

839

Phage-
0.085
2.745
0.781
1062

840

Phage-
0.294
2.098
0.768
1063

841

Phage-
2.648
2.751
2.675
1064

842

Phage-
0.183
2.746
0.860
1065

843

Phage-
0.281
2.762
0.947
1066

844

Phage-
0.207
2.717
0.881
1067

845

Phage-
0.625
2.711
1.186
1068

846

Phage-
0.727
0.580
0.686
1069

847

Phage-
0.141
2.764
0.863
1070

848

Phage-
0.193
2.736
0.901
1071

849

Phage-
0.409
2.621
1.042
1072

850

Phage-
0.234
2.827
0.979
1073

851

Phage-
0.086
0.066
0.080
1074

852

Phage-
0.116
2.838
0.927
1075

853

Phage-
0.147
2.815
0.951
1076

854

Phage-
0.078
2.754
0.888
1077

855

Phage-
0.086
2.717
0.887
1078

856

Phage-
0.099
2.729
0.902
1079

857

Phage-
0.103
2.870
0.948
1080

858

Phage-
0.082
2.774
0.923
1081

859

Phage-
0.117
2.736
0.940
1082

860

Phage-
0.184
0.066
0.147
1083

861

Phage-
0.116
2.729
0.938
1084

862

Phage-
0.506
2.748
1.220
1085

863

Phage-
0.066
2.329
0.787
1086

864

Phage-
0.162
2.753
0.999
1087

865

Phage-
0.098
2.750
0.957
1088

866

Phage-
0.488
2.730
1.221
1089

867

Phage-
0.089
0.076
0.084
1090

868

Phage-
0.187
2.811
1.059
1091

869

Phage-
0.089
2.731
0.974
1092

870

Phage-
0.065
2.730
0.960
1093

871

Phage-
0.713
2.748
1.397
1094

872

Phage-
0.068
2.722
0.962
1095

873

Phage-
0.103
2.723
0.992
1096

874

Phage-
0.365
2.707
1.161
1097

875

Phage-
0.186
0.093
0.154
1098

876

Phage-
0.116
2.725
1.020
1099

877

Phage-
0.320
2.752
1.168
1100

878

Phage-
0.100
2.727
1.017
1101

879

Phage-
0.092
0.078
0.087
1102

880

Phage-
0.288
2.839
1.183
1103

881

Phage-
0.081
2.837
1.056
1104

882

Phage-
0.076
2.709
1.015
1105

883

Phage-
0.145
2.727
1.069
1106

884

Phage-
0.133
2.721
1.063
1107

885

Phage-
0.093
2.754
1.055
1108

886

Phage-
0.272
2.681
1.151
1109

887

Phage-
2.625
2.705
2.654
1110

888

Phage-
0.160
2.709
1.097
1111

889

Phage-
0.362
2.716
1.249
1112

890

Phage-
0.107
0.079
0.096
1113

891

Phage-
0.098
2.719
1.136
1114

892

Phage-
0.103
2.706
1.139
1115

893

Phage-
0.133
0.088
0.114
1116

894

Phage-
0.414
2.732
1.375
1117

895

Phage-
0.138
2.753
1.227
1118

896

Phage-
0.444
2.746
1.404
1119

897

Phage-
2.674
2.737
2.701
1120

898

Phage-
0.233
2.708
1.286
1121

899

Phage-
0.098
2.807
1.254
1122

900

Phage-
0.141
2.834
1.298
1123

901

Phage-
0.291
0.077
0.198
1124

902

Phage-
0.567
2.835
1.582
1125

903

Phage-
0.448
2.791
1.507
1126

904

Phage-
0.202
2.747
1.388
1127

905

Phage-
0.593
2.743
1.595
1128

906

Phage-
0.184
2.716
1.369
1129

907

Phage-
0.091
0.078
0.085
1130

908

Phage-
0.079
2.712
1.373
1131

909

Phage-
0.107
0.078
0.092
1132

910

Phage-
0.205
2.852
1.509
1133

911

Phage-
2.238
2.758
2.500
1134

912

Phage-
0.064
0.106
0.085
1135

913

Phage-
0.363
0.074
0.214
1136

914

Phage-
0.131
2.847
1.543
1137

915

Phage-
0.096
0.079
0.087
1138

916

Phage-
0.792
2.762
1.822
1139

917

Phage-
0.356
2.688
1.580
1140

918

Phage-
0.193
0.076
0.131
1141

919

Phage-
2.542
2.739
2.649
1142

920

Phage-
2.282
2.794
2.572
1143

921

Phage-
0.636
2.717
1.844
1144

922

Phage-
2.598
2.730
2.679
1145

923

Phage-
0.184
2.742
1.803
1146

924

Phage-
0.816
2.745
2.039
1147

925

Phage-
0.735
2.756
2.019
1148

926

Phage-
0.479
2.699
1.911
1149

927

Phage-
0.375
0.074
0.172
1150

928

Phage-
0.169
0.067
0.100
1151

929

Phage-
2.316
2.823
2.660
1152

930

Phage-
0.765
2.845
2.181
1153

931

Phage-
0.163
0.080
0.106
1154

932

Phage-
0.295
2.745
1.989
1155

933

Phage-
0.192
0.082
0.114
1156

934

Phage-
0.075
0.066
0.069
1157

935

Phage-
1.474
2.816
2.447
1158

936

Phage-
0.769
2.717
2.218
1159

937

Phage-
0.609
2.745
2.200
1160

938

Phage-
1.479
2.733
2.413
1161

939

Phage-
0.220
0.079
0.113
1162

940

Phage-
0.106
2.863
2.262
1163

941

Phage-
0.161
0.064
0.085
1164

942

Phage-
1.234
2.743
2.430
1165

943

Phage-
0.073
0.115
0.106
1166

944

Phage-
0.117
0.064
0.075
1167

945

Phage-
0.967
2.861
2.503
1168

946

Phage-
0.731
2.735
2.357
1169

947

Phage-
0.246
0.082
0.113
1170

948

Phage-
0.479
2.690
2.290
1171

949

Phage-
0.274
2.747
2.304
1172

950

Phage-
0.117
0.065
0.074
1173

951

Phage-
0.200
0.066
0.085
1174

952

Phage-
0.064
0.378
0.335
1175

953

Phage-
2.463
2.777
2.734
1176

954

Phage-
1.900
2.747
2.634
1177

955

Phage-
0.070
0.065
0.065
1178

956

Phage-
0.449
0.072
0.120
1179

957

Phage-
1.318
2.708
2.533
1180

958

Phage-
2.109
2.706
2.632
1181

959

Phage-
1.469
2.850
2.688
1182

960

Phage-
2.104
2.733
2.665
1183

961

Phage-
0.823
2.672
2.471
1184

962

Phage-
0.765
2.831
2.645
1185

963

Phage-
0.202
0.067
0.079
1186

964

Phage-
1.392
2.711
2.605
1187

965

Phage-
0.467
0.081
0.098
1188

966

Phage-
0.215
2.726
2.636
1189

967

Phage-
0.748
2.719
2.701
1190

968

Phage-
0.092
0.692
0.781
1191

969

Phage-
0.074
0.381
1.203
1192

970

Phage-
0.317
0.294
0.100
1193

971

Phage-
0.105
0.112
0.133
1194

972

Phage-
0.138
0.107
0.099
1195

973

Phage-
0.063
0.086
0.096
1196

974

Phage-
0.079
0.081
0.113
1197

975

Phage-
0.071
0.074
0.081
1198

976

Phage-
0.068
0.072
0.109
1199

977

Phage-
0.234
0.068
0.066
1200

978

Phage-
0.064
0.065
0.076
1201

979

Phage-
0.104
0.065
0.065
1202

980

TABLE 28

Sequences of those peptides selected for

synthesis (TROP2 Fab Peptide-1 Optimization)

Construct
Amino

Description
Acid Sequence
SEQ ID NO:

Peptide-30
DALICVKNLFCWTS
159

Peptide-31
DNLICVKNLWCWIA
160

Peptide-32
DTLFCVKNLYCWIV
161

Peptide-33
DTLSCFRNLYCWIT
162

Peptide-34
DTLWCVKNLYCWVA
163

Peptide-35
DVLFCVRNLYCWTS
164

Peptide-36
FDLLCVRNLYCWNV
165

Peptide-37
FSLVCVRNLYCWNV
166

Peptide-38
FTLFCVQNLYCWNV
167

Peptide-39
HDLQCFQNLFCWIV
168

Peptide-40
LSLFCVKNLYCWNV
169

Peptide-41
NYLLCVKNLYCWIV
170

Peptide-42
STLECVRNLYCWIS
171

Peptide-43
STLFCVKNLYCWVA
172

Peptide-44
STLFCVRNLYCWTV
173

Peptide-45
SYLVCVKNVYCWTA
174

Peptide-46
TALFCFKNVYCWNV
175

Peptide-47
TSLICFRNVYCWNV
176

Peptide-48
YDLVCLRNLFCWTA
177

Peptide-49
YSLVCVKNLYCWNL
178

TABLE 29

Phage panning results of TROP2 Fab Peptide-2 library sequences.

Phage binding ELISA

TROP2

Fab

signal in

TROP2
presence
SEQ

Amino acid position sequence
Backgroud
Fab
of
ID

Phage ID
1
2
3
4
5
6
7
8
9
10
11
12
13
14
signal
signal
TROP2
NO:

Phage-981
V
D
F
C
K
I
Y
S
W
P
V
C
H
Q
0.055
2.832
0.067
24

Phage-982
I
—
—
—
A
M
—
Q
—
—
I
—
D
T
0.058
2.945
0.064
179

Phage-983
I
—
—
—
A
V
—
K
—
—
—
—
Q
V
0.059
2.885
0.064
180

Phage-984
I
—
—
—
M
L
—
N
—
—
I
—
A
G
0.061
2.933
0.064
25

Phage-985
—
—
—
—
—
—
—
A
—
—
I
—
G
S
0.066
2.868
0.068
182

Phage-986
—
—
—
—
—
L
—
N
—
—
—
—
Q
T
0.061
2.874
0.067
183

Phage-987
I
—
—
—
L
—
—
N
—
—
—
—
D
T
0.060
2.849
0.068
184

Phage-988
E
—
—
—
—
L
—
N
—
—
I
—
Y
—
0.054
2.864
0.099
185

Phage-989
—
—
—
—
G
L
—
H
—
—
I
—
Y
—
0.079
2.878
0.097
186

Phage-990
—
—
—
—
Y
L
—
N
—
—
—
—
S
K
0.057
2.892
0.063
187

Phage-991
I
—
—
—
A
—
—
Q
—
—
—
—
R
S
0.066
2.799
0.067
188

Phage-992
—
—
—
—
A
L
—
N
—
—
—
—
E
T
0.056
2.752
0.058
189

Phage-993
P
—
—
—
A
V
—
R
—
—
I
—
Y
—
0.062
2.746
0.073
190

Phage-994
—
—
—
—
E
L
—
R
—
—
I
—
N
S
0.066
2.756
0.057
191

Phage-995
L
—
—
—
—
—
—
D
—
—
I
—
—
L
0.082
2.760
0.083
192

Phage-996
L
—
—
—
—
L
—
Q
—
—
—
—
F
T
0.080
2.763
0.079
193

Phage-997
I
—
—
—
L
L
—
D
—
—
—
—
A
S
0.058
2.873
0.065
194

Phage-998
I
—
—
—
L
L
—
D
—
—
I
—
G
R
0.170
2.774
0.205
195

Phage-999
M
—
—
—
Q
—
—
D
—
—
I
—
R
L
0.057
2.589
0.062
196

Phage-1000
P
—
—
—
Q
L
—
N
—
—
—
—
A
G
0.067
2.523
0.062
197

Phage-1001
—
—
—
—
S
F
—
R
—
—
I
—
E
T
0.063
2.697
0.070
198

Phage-1002
I
—
—
—
S
L
—
Q
—
—
—
—
G
T
0.063
2.828
0.091
199

Phage-1003
—
—
—
—
T
—
—
K
—
—
—
—
E
G
0.055
2.760
0.059
200

Phage-1004
—
—
—
—
Y
Q
—
G
—
—
I
—
S
R
0.060
2.696
0.060
201

Phage-1005
—
—
—
—
A
—
F
N
—
—
—
—
S
S
0.063
2.742
0.068
1203

Phage-1006
L
—
—
—
A
—
—
K
—
—
—
—
F
E
0.057
2.901
0.063
1204

Phage-1007
—
—
—
—
A
—
—
—
—
—
I
—
K
I
0.066
2.834
0.064
1205

Phage-1008
I
—
—
—
A
—
—
P
—
—
I
—
D
T
0.059
2.852
0.058
1206

Phage-1009
I
—
—
—
A
—
—
H
—
—
—
—
T
S
0.057
2.707
0.063
1207

Phage-1010
I
—
—
—
A
—
—
Q
—
—
—
—
K
S
0.108
2.810
0.096
1208

Phage-1011
M
—
—
—
A
—
—
H
—
—
—
—
N
Y
0.061
2.760
0.062
1209

Phage-1012
E
—
—
—
A
—
—
R
—
—
—
—
L
T
0.055
2.742
0.056
1210

Phage-1013
—
—
—
—
A
—
—
G
—
—
—
—
D
I
0.068
1.940
0.067
1211

Phage-1014
F
—
—
—
A
—
—
R
—
—
—
—
Y
D
0.072
1.407
0.080
1212

Phage-1015
—
—
—
—
A
K
—
E
—
—
I
—
S
T
0.054
2.391
0.056
1213

Phage-1016
—
—
—
—
A
L
—
A
—
—
—
—
N
V
0.060
2.810
0.057
1214

Phage-1017
I
—
—
—
A
L
—
—
—
—
—
—
N
S
0.057
2.855
0.071
1215

Phage-1018
L
—
—
—
A
L
—
D
—
—
—
—
R
N
0.062
2.717
0.069
1216

Phage-1019
—
—
—
—
A
L
—
H
—
—
—
—
G
L
0.065
2.770
0.065
1217

Phage-1020
L
—
—
—
A
L
—
A
—
—
—
—
N
M
0.059
2.649
0.071
1218

Phage-1021
L
—
—
—
A
L
N
A
—
—
—
—
N
M
0.062
2.241
0.064
1219

Phage-1022
—
—
—
—
A
L
—
G
—
—
—
—
Y
L
0.065
2.710
0.062
1220

Phage-1023
L
—
—
—
A
L
—
G
—
—
I
—
S
A
0.055
2.208
0.059
1221

Phage-1024
—
—
—
—
A
L
—
N
—
—
—
—
Q
T
0.061
2.848
0.059
1222

Phage-1025
I
—
—
—
A
L
—
R
—
—
—
—
Y
M
0.136
2.760
0.148
1223

Phage-1026
—
—
—
—
A
L
—
E
—
—
I
—
M
M
0.068
2.709
0.078
1224

Phage-1027
L
—
—
—
A
L
—
N
—
—
—
—
N
I
0.063
2.625
0.073
1225

Phage-1028
—
—
—
—
A
L
—
N
—
—
—
—
D
T
0.056
2.625
0.058
1226

Phage-1029
—
—
—
—
A
L
—
A
—
—
—
—
M
Y
0.066
2.553
0.066
1227

Phage-1030
L
—
—
—
A
L
—
A
—
—
—
—
L
G
0.059
2.401
0.064
1228

Phage-1031
—
—
—
—
A
L
F
E
—
—
I
—
G
A
0.057
2.354
0.065
1229

Phage-1032
—
—
—
—
A
L
—
M
—
—
—
—
R
D
0.066
2.270
0.073
1230

Phage-1033
—
—
W
—
A
L
—
K
—
—
—
—
S
T
0.079
2.239
0.097
1231

Phage-1034
—
—
W
—
A
L
—
—
—
—
—
—
S
H
0.069
2.091
0.070
1232

Phage-1035
L
G
—
—
A
L
N
G
—
—
I
—
S
A
0.082
1.816
0.077
1233

Phage-1036
L
—
—
—
A
L
N
D
—
—
—
—
K
E
0.055
1.662
0.062
1234

Phage-1037
I
—
—
—
A
L
—
G
—
—
—
—
D
T
0.073
1.576
0.068
1235

Phage-1038
—
—
—
—
A
L
—
L
—
—
I
—
N
—
0.054
1.567
0.062
1236

Phage-1039
—
—
—
—
A
L
—
M
—
—
—
—
Q
R
0.055
1.464
0.059
1237

Phage-1040
L
—
—
—
A
L
N
G
—
—
I
—
S
A
0.058
1.072
0.056
1238

Phage-1041
E
—
—
—
A
L
—
K
—
—
—
—
R
T
0.059
0.658
0.059
1239

Phage-1042
I
—
—
—
A
M
—
H
—
—
—
—
D
T
0.067
2.733
0.062
1240

Phage-1043
I
—
—
—
A
M
F
—
—
—
—
—
F
T
0.085
2.836
0.082
1241

Phage-1044
—
—
—
—
A
M
—
H
—
—
I
—
Y
—
0.062
2.771
0.067
1242

Phage-1045
1
—
—
—
A
M
—
Q
—
—
I
—
Y
T
0.056
2.674
0.062
1243

Phage-1046
I
—
—
—
A
M
—
Q
—
—
I
—
V
A
0.072
2.525
0.070
1244

Phage-1047
E
—
—
—
A
M
—
Y
—
—
I
—
G
L
0.054
2.112
0.061
1245

Phage-1048
—
—
—
—
A
M
—
K
—
—
—
—
I
G
0.081
2.053
0.066
1246

Phage-1049
I
—
—
—
A
M
—
Q
—
—
I
—
Q
T
0.054
1.944
0.056
1247

Phage-1050
E
—
—
—
A
M
—
Q
—
—
—
—
I
T
0.054
1.549
0.055
1248

Phage-1051
—
—
—
—
A
M
—
—
—
—
—
—
A
E
0.056
1.123
0.058
1249

Phage-1052
L
—
—
—
A
M
—
D
—
—
—
—
G
P
0.067
0.939
0.059
1250

Phage-1053
—
—
—
—
A
Q
—
G
—
—
I
—
V
T
0.058
1.548
0.059
1251

Phage-1054
I
—
—
—
A
T
—
N
—
—
—
—
G
N
0.055
2.674
0.056
1252

Phage-1055
—
—
—
—
A
T
—
G
—
—
—
—
F
T
0.068
2.290
0.061
1253

Phage-1056
I
—
—
—
A
T
—
—
—
—
—
—
A
S
0.057
2.083
0.056
1254

Phage-1057
W
T
—
—
A
T
S
M
—
R
Y
—
V
D
0.150
1.312
0.226
1255

Phage-1058
W
K
—
—
A
T
S
M
—
R
Y
—
V
D
0.130
2.690
0.194
1256

Phage-1059
—
—
—
—
A
V
—
E
—
—
I
—
R
H
0.054
2.645
0.068
1257

Phage-1060
P
—
—
—
A
V
—
N
—
—
—
—
T
N
0.055
2.628
0.058
1258

Phage-1061
—
—
—
—
A
V
—
K
—
—
—
—
L
E
0.060
2.520
0.056
1259

Phage-1062
L
—
—
—
A
V
—
R
—
—
—
—
N
M
0.059
2.380
0.062
1260

Phage-1063
—
—
—
—
A
V
—
K
—
—
—
—
S
I
0.056
2.338
0.056
1261

Phage-1064
I
—
W
—
A
V
—
K
—
—
I
—
L
D
0.067
2.277
0.075
1262

Phage-1065
—
—
—
—
A
V
—
G
—
—
—
—
Q
T
0.065
2.139
0.075
1263

Phage-1066
I
—
—
—
A
V
—
K
—
—
—
—
A
R
0.091
1.866
0.084
1264

Phage-1067
—
—
—
—
A
V
—
G
—
—
I
—
D
A
0.056
1.853
0.056
1265

Phage-1068
L
V
—
—
A
V
—
D
—
—
—
—
S
N
0.061
0.975
0.061
1266

Phage-1069
—
—
—
—
A
V
W
R
—
—
—
—
D
S
0.058
0.154
0.060
1267

Phage-1070
I
—
—
—
A
V
—
N
—
—
—
—
G
S
0.059
2.735
0.082
1268

Phage-1071
E
—
—
—
D
—
—
H
—
—
—
—
R
M
0.099
1.583
0.064
1269

Phage-1072
—
—
—
—
D
L
—
R
—
—
I
—
L
L
0.056
2.822
0.061
1270

Phage-1073
—
—
—
—
D
L
F
—
—
—
I
—
N
R
0.067
1.993
0.077
1271

Phage-1074
—
—
—
—
D
L
—
D
—
—
—
—
S
T
0.056
1.157
0.058
1272

Phage-1075
P
Y
—
—
D
M
—
N
—
—
—
—
V
T
0.090
2.564
0.073
1273

Phage-1076
—
—
—
—
E
E
—
H
—
—
—
—
D
S
0.056
1.112
0.057
1274

Phage-1077
I
—
—
—
E
—
—
R
—
—
—
—
S
R
0.070
2.689
0.074
1275

Phage-1078
—
—
—
—
E
—
—
G
—
—
I
—
F
D
0.055
2.696
0.055
1276

Phage-1079
I
—
—
—
E
—
—
—
—
—
—
—
I
—
0.053
2.687
0.055
1277

Phage-1080
—
—
—
—
E
—
—
E
—
—
I
—
N
D
0.054
2.634
0.060
1278

Phage-1081
P
N
—
—
E
—
—
R
—
—
—
—
N
V
0.055
1.858
0.058
1279

Phage-1082
L
N
—
—
E
—
N
R
—
—
—
—
L
G
0.060
0.701
0.064
1280

Phage-1083
I
—
—
—
E
K
—
Q
—
—
—
—
L
T
0.056
1.625
0.056
1281

Phage-1084
F
—
—
—
E
K
—
K
—
—
I
—
I
P
0.060
2.651
0.062
1282

Phage-1085
F
—
—
—
E
K
—
K
—
—
I
—
N
T
0.057
2.236
0.057
1283

Phage-1086
I
—
—
—
E
L
—
Q
—
—
—
—
G
H
0.055
2.700
0.057
1284

Phage-1087
—
—
—
—
E
L
—
K
—
—
—
—
F
K
0.059
2.460
0.060
1285

Phage-1088
I
—
—
—
E
L
—
H
—
—
—
—
S
S
0.057
2.767
0.062
1286

Phage-1089
—
—
—
—
E
L
—
K
—
—
—
—
M
G
0.060
2.766
0.063
1287

Phage-1090
I
—
—
—
E
L
—
—
—
—
—
—
G
D
0.068
2.530
0.092
1288

Phage-1091
—
—
—
—
E
L
—
H
—
—
—
—
D
—
0.056
2.745
0.057
1289

Phage-1092
L
—
—
—
E
L
—
H
—
—
—
—
T
L
0.076
2.684
0.065
1290

Phage-1093
—
—
—
—
E
L
—
—
—
—
—
—
Y
L
0.060
2.753
0.058
1291

Phage-1094
I
—
—
—
E
L
—
H
—
—
—
—
K
W
0.079
2.725
0.104
1292

Phage-1095
P
—
—
—
E
L
—
R
—
—
I
—
R
L
0.071
2.364
0.073
1293

Phage-1096
—
—
—
—
E
L
—
R
—
—
—
—
M
I
0.075
2.156
0.073
1294

Phage-1097
F
—
—
—
E
L
—
N
—
—
—
—
Q
S
0.080
2.130
0.071
1295

Phage-1098
L
—
—
—
E
L
—
—
—
—
I
—
D
I
0.056
2.093
0.064
1296

Phage-1099
—
—
—
—
E
L
—
E
—
—
I
—
F
K
0.078
2.052
0.075
1297

Phage-1100
—
—
—
—
E
L
—
R
—
—
—
—
Y
K
0.054
1.841
0.058
1298

Phage-1101
L
—
—
—
E
L
—
R
—
—
—
—
G
I
0.062
1.329
0.056
1299

Phage-1102
—
—
—
—
E
L
—
R
—
—
—
—
N
G
0.067
1.215
0.056
1300

Phage-1103
P
—
—
—
E
L
—
Q
—
—
—
—
D
R
0.054
1.120
0.065
1301

Phage-1104
—
—
—
—
E
M
—
N
—
—
—
—
R
M
0.057
2.726
0.055
1302

Phage-1105
L
—
—
—
E
M
—
G
—
—
—
—
K
V
0.062
2.712
0.068
1303

Phage-1106
L
—
—
—
E
M
—
D
—
—
—
—
R
L
0.056
1.989
0.059
1304

Phage-1107
L
—
—
—
E
M
—
N
—
—
—
—
K
D
0.059
1.416
0.063
1305

Phage-1108
E
—
—
—
E
M
—
N
—
—
—
—
W
K
0.055
1.334
0.057
1306

Phage-1109
—
—
—
—
E
M
—
R
—
—
—
—
A
S
0.053
1.289
0.055
1307

Phage-1110
P
—
—
—
E
M
—
K
—
—
—
—
D
G
0.054
1.244
0.055
1308

Phage-1111
—
—
—
—
E
V
—
H
—
—
—
—
Q
R
0.058
2.621
0.057
1309

Phage-1112
—
—
—
—
E
V
—
H
—
—
—
—
D
R
0.054
2.016
0.056
1310

Phage-1113
L
—
—
—
E
V
—
R
—
—
I
—
G
D
0.062
0.064
0.052
1311

Phage-1114
—
—
—
—
F
—
—
—
—
—
—
—
T
E
0.055
2.306
0.058
1312

Phage-1115
I
—
—
—
F
—
—
N
—
—
—
—
N
T
0.259
2.747
0.192
1313

Phage-1116
L
—
W
—
F
—
—
N
—
—
—
—
Q
T
0.158
2.720
0.155
1314

Phage-1117
—
—
—
—
F
L
—
—
—
—
—
—
S
T
0.058
2.364
0.062
1315

Phage-1118
I
—
—
—
F
L
—
—
—
—
—
—
R
E
0.061
2.181
0.059
1316

Phage-1119
I
—
—
—
F
L
—
R
—
—
—
—
S
S
0.084
2.678
0.106
1317

Phage-1120
P
—
—
—
F
L
—
D
—
—
—
—
Y
E
0.054
1.113
0.056
1318

Phage-1121
L
—
—
—
F
M
—
D
—
—
—
—
R
G
0.056
2.678
0.060
1319

Phage-1122
—
—
—
—
F
M
F
H
—
—
—
—
A
E
0.071
2.481
0.072
1320

Phage-1123
—
—
—
—
F
M
—
R
—
—
—
—
E
T
0.065
1.765
0.058
1321

Phage-1124
E
—
—
—
F
M
—
T
—
—
—
—
—
N
0.057
0.227
0.057
1322

Phage-1125
—
—
—
—
F
R
—
N
—
—
I
—
M
R
0.065
2.265
0.070
1323

Phage-1126
—
—
—
—
F
R
—
G
—
—
—
—
G
T
0.072
0.827
0.081
1324

Phage-1127
—
—
—
—
G
—
—
R
—
—
—
—
N
M
0.059
2.526
0.062
1325

Phage-1128
—
—
—
—
G
—
—
R
—
—
—
—
M
T
0.055
2.087
0.061
1326

Phage-1129
I
—
—
—
G
—
—
Q
—
—
I
—
—
N
0.153
2.773
0.295
1327

Phage-1130
L
—
—
—
G
—
—
R
—
—
—
—
E
T
0.060
2.310
0.059
1328

Phage-1131
—
—
—
—
G
L
—
N
—
—
—
—
A
S
0.059
2.788
0.056
1329

Phage-1132
—
—
—
—
G
L
—
N
—
—
—
—
Q
E
0.066
2.373
0.074
1330

Phage-1133
—
—
—
—
G
L
—
H
—
—
I
—
D
K
0.083
2.723
0.081
1331

Phage-1134
—
—
—
—
G
L
—
N
—
—
I
—
A
T
0.056
1.438
0.057
1332

Phage-1135
—
—
—
—
G
L
—
R
—
—
—
—
N
—
0.055
0.872
0.056
1333

Phage-1136
E
—
—
—
G
M
—
H
—
—
—
—
D
I
0.075
1.887
0.058
1334

Phage-1137
I
—
—
—
G
M
—
E
—
—
I
—
E
M
0.054
1.827
0.062
1335

Phage-1138
E
—
—
—
G
M
—
H
—
—
I
—
S
G
0.057
1.626
0.060
1336

Phage-1139
—
—
—
—
G
M
—
H
—
—
I
—
Y
K
0.083
2.731
0.097
1337

Phage-1140
F
—
—
—
G
T
—
H
—
—
—
—
E
S
0.067
2.212
0.061
1338

Phage-1141
I
—
—
—
G
V
—
K
—
—
—
—
T
L
0.066
1.604
0.061
1339

Phage-1142
—
E
—
—
H
L
—
N
—
—
I
—
S
K
0.059
2.849
0.061
1340

Phage-1143
—
—
—
—
H
M
—
R
—
—
—
—
D
M
0.062
2.419
0.060
1341

Phage-1144
—
—
—
—
I
—
—
N
—
—
—
—
R
D
0.075
2.649
0.074
1342

Phage-1145
I
—
—
—
I
L
—
D
—
—
—
—
E
M
0.057
1.498
0.056
1343

Phage-1146
—
—
—
—
—
E
—
N
—
—
I
—
S
D
0.071
2.476
0.070
1344

Phage-1147
I
—
—
—
—
E
—
—
—
—
I
—
L
N
0.078
1.470
0.079
1345

Phage-1148
L
—
—
—
—
—
—
D
—
—
I
—
G
H
0.076
2.670
0.084
1346

Phage-1149
—
—
—
—
—
—
—
—
—
—
—
—
S
N
0.064
2.623
0.063
1347

Phage-1150
L
—
—
—
—
—
N
D
—
—
I
—
—
L
0.063
2.436
0.078
1348

Phage-1151
—
—
—
—
—
—
—
E
—
—
I
—
R
M
0.055
2.418
0.066
1349

Phage-1152
L
—
—
—
—
—
—
G
—
—
—
—
Y
T
0.064
1.291
0.060
1350

Phage-1153
I
—
—
—
—
L
—
E
—
—
—
—
F
I
0.079
2.696
0.090
1351

Phage-1154
P
—
—
—
—
L
N
K
—
—
—
—
I
—
0.064
2.291
0.066
1352

Phage-1155
—
—
—
—
—
L
—
H
—
—
—
—
T
G
0.062
2.216
0.074
1353

Phage-1156
—
—
—
—
—
L
—
K
—
—
—
—
E
G
0.060
2.201
0.062
1354

Phage-1157
P
—
—
—
—
L
—
Q
—
—
I
—
V
R
0.058
2.201
0.059
1355

Phage-1158
L
—
—
—
—
L
—
H
—
—
—
—
R
D
0.065
2.118
0.068
1356

Phage-1159
—
E
—
—
—
L
—
Q
—
—
I
—
Q
S
0.056
1.273
0.064
1357

Phage-1160
L
—
—
—
—
L
—
H
—
—
—
—
S
D
0.071
1.062
0.075
1358

Phage-1161
—
—
—
—
—
M
—
A
—
—
—
—
K
L
0.056
2.162
0.063
1359

Phage-1162
—
—
—
—
—
M
—
A
—
—
—
—
E
M
0.081
2.518
0.071
1360

Phage-1163
—
—
—
—
—
M
F
A
—
—
—
—
N
T
0.084
2.476
0.093
1361

Phage-1164
M
—
—
—
—
M
—
R
—
—
I
—
G
M
0.067
2.340
0.072
1362

Phage-1165
—
—
—
—
—
M
—
D
—
—
—
—
T
N
0.067
1.732
0.074
1363

Phage-1166
F
—
—
—
—
M
—
D
—
—
—
—
R
L
0.089
1.728
0.092
1364

Phage-1167
I
N
—
—
—
M
—
E
—
—
I
—
S
T
0.067
1.694
0.064
1365

Phage-1168
—
—
—
—
—
V
—
N
—
—
—
—
W
L
0.119
2.605
0.143
1366

Phage-1169
I
—
—
—
—
V
F
A
—
—
—
—
T
A
0.096
2.212
0.097
1367

Phage-1170
I
—
—
—
L
—
—
Q
—
—
I
—
V
N
0.060
2.883
0.062
1368

Phage-1171
—
—
—
—
L
—
—
Q
—
—
—
—
N
S
0.054
2.722
0.057
1369

Phage-1172
I
—
—
—
L
—
—
N
—
—
—
—
Y
T
0.060
2.581
0.065
1370

Phage-1173
—
—
—
—
L
—
—
K
—
—
—
—
G
L
0.055
2.117
0.062
1371

Phage-1174
I
—
—
—
L
—
—
E
—
—
—
—
T
A
0.066
2.039
0.076
1372

Phage-1175
—
—
—
—
L
K
—
N
—
—
—
—
D
G
0.078
0.436
0.073
1373

Phage-1176
L
—
—
—
L
L
—
D
—
—
—
—
Q
T
0.055
2.588
0.057
1374

Phage-1177
I
—
—
—
L
L
—
K
—
—
—
—
E
L
0.056
2.770
0.060
1375

Phage-1178
L
N
—
—
L
L
—
D
—
—
—
—
A
S
0.072
1.096
0.079
1376

Phage-1179
—
—
—
—
L
L
—
E
—
—
—
—
N
L
0.054
0.629
0.066
1377

Phage-1180
I
—
—
—
L
M
—
N
—
—
—
—
T
T
0.061
2.759
0.061
1378

Phage-1181
—
—
—
—
L
M
—
Q
—
—
I
—
—
M
0.057
2.774
0.062
1379

Phage-1182
I
—
—
—
L
M
—
H
—
—
—
—
Y
E
0.058
2.766
0.068
1380

Phage-1183
I
—
—
—
L
M
—
N
—
—
—
—
M
S
0.055
2.384
0.058
1381

Phage-1184
L
—
—
—
L
M
—
K
—
—
—
—
D
F
0.082
2.134
0.073
1382

Phage-1185
I
—
—
—
L
M
—
—
—
—
—
—
T
A
0.082
1.634
0.086
1383

Phage-1186
I
N
—
—
L
V
—
D
—
—
—
—
K
—
0.061
2.403
0.067
1384

Phage-1187
F
—
—
—
L
V
—
—
—
—
—
—
N
S
0.059
1.424
0.061
1385

Phage-1188
I
—
—
—
L
V
—
N
—
—
—
—
R
S
0.053
0.659
0.063
1386

Phage-1189
L
—
—
—
L
W
—
K
—
—
—
—
E
—
0.168
2.176
0.226
1387

Phage-1190
I
—
—
—
M
F
—
E
—
—
I
—
G
T
0.062
2.678
0.058
1388

Phage-1191
L
V
—
—
M
F
N
N
—
—
—
—
Q
H
0.054
0.062
0.054
1389

Phage-1192
M
—
—
—
M
—
—
N
—
—
—
—
A
—
0.057
2.791
0.056
1390

Phage-1193
L
—
—
—
M
—
—
A
—
—
I
—
A
—
0.057
2.522
0.070
1391

Phage-1194
I
—
—
—
M
—
—
R
—
—
—
—
Q
V
0.091
2.729
0.089
1392

Phage-1195
L
—
—
—
M
—
—
K
—
—
I
—
K
G
0.072
2.621
0.086
1393

Phage-1196
L
—
—
—
M
—
N
D
—
—
—
—
S
K
0.055
1.578
0.057
1394

Phage-1197
—
—
—
—
M
L
—
K
—
—
I
—
K
T
0.068
2.714
0.078
1395

Phage-1198
—
—
—
—
M
L
—
N
—
—
—
—
M
E
0.105
2.751
0.056
1396

Phage-1199
I
—
—
—
M
L
—
Q
—
—
—
—
—
K
0.081
2.496
0.090
1397

Phage-1200
I
—
—
—
M
L
—
A
—
—
—
—
N
S
0.058
1.542
0.065
1398

Phage-1201
—
—
—
—
M
L
—
—
—
—
—
—
L
T
0.059
0.073
0.061
1399

Phage-1202
I
—
—
—
M
L
—
N
—
—
I
—
A
E
0.094
2.803
0.145
1400

Phage-1203
P
—
—
—
M
M
—
D
—
—
—
—
R
I
0.117
2.260
0.059
1401

Phage-1204
F
V
—
—
N
—
L
A
M
R
N
—
V
S
0.055
2.712
1.924
1402

Phage-1205
—
—
—
—
N
—
—
N
—
—
—
—
L
D
0.054
2.166
0.060
1403

Phage-1206
—
—
—
—
N
L
—
N
—
—
—
—
Q
N
0.083
2.488
0.063
1404

Phage-1207
Y
W
L
—
N
Q
G
K
—
L
A
—
E
—
0.067
0.068
0.086
1405

Phage-1208
F
V
L
—
N
T
L
T
M
R
E
—
V
S
0.065
1.426
2.552
1406

Phage-1209
I
—
—
—
N
V
—
D
—
—
—
—
R
T
0.055
2.663
0.061
1407

Phage-1210
—
T
—
—
P
T
S
Q
—
R
Y
—
I
I
0.063
0.102
0.090
1408

Phage-1211
I
—
—
—
Q
E
—
N
—
—
—
—
V
N
0.056
2.141
0.058
1409

Phage-1212
—
—
—
—
Q
—
—
—
—
—
—
—
N
W
0.060
2.336
0.062
1410

Phage-1213
—
—
—
—
Q
—
—
K
—
—
—
—
E
—
0.060
2.583
0.060
1411

Phage-1214
—
—
—
—
Q
—
—
N
—
—
—
—
T
D
0.059
2.573
0.057
1412

Phage-1215
F
—
—
—
Q
—
—
N
—
—
I
—
G
R
0.081
1.795
0.093
1413

Phage-1216
—
—
—
—
Q
—
—
A
—
—
—
—
G
N
0.058
1.796
0.065
1414

Phage-1217
N
E
Q
—
Q
—
—
I
M
A
—
—
R
A
0.074
0.065
0.074
1415

Phage-1218
—
—
—
—
Q
L
—
H
—
—
—
—
N
A
0.056
2.830
0.062
1416

Phage-1219
P
—
—
—
Q
L
N
N
—
—
—
—
A
G
0.062
2.428
0.057
1417

Phage-1220
—
—
—
—
Q
L
—
A
—
—
—
—
G
T
0.056
2.428
0.057
1418

Phage-1221
I
—
—
—
Q
L
—
—
—
—
—
—
V
N
0.062
2.046
0.058
1419

Phage-1222
L
—
—
—
Q
L
—
R
—
—
—
—
E
V
0.060
1.887
0.059
1420

Phage-1223
—
—
—
—
Q
L
—
R
—
—
—
—
G
N
0.067
1.583
0.062
1421

Phage-1224
—
—
W
—
Q
L
—
N
—
—
—
—
R
E
0.060
0.211
0.074
1422

Phage-1225
—
—
—
—
Q
M
—
D
—
—
I
—
M
A
0.063
2.464
0.063
1423

Phage-1226
I
—
—
—
Q
M
—
—
—
—
—
—
G
—
0.069
2.089
0.069
1424

Phage-1227
—
—
—
—
Q
M
—
M
—
—
—
—
L
S
0.243
2.619
0.257
1425

Phage-1228
I
—
—
—
Q
V
—
—
—
—
—
—
E
—
0.054
2.449
0.055
1426

Phage-1229
M
—
—
—
Q
V
—
A
—
—
—
—
N
M
0.054
2.130
0.057
1427

Phage-1230
—
—
—
—
Q
V
—
N
—
—
—
—
L
S
0.057
1.133
0.057
1428

Phage-1231
L
—
—
—
Q
V
—
N
—
—
I
—
A
W
0.065
0.720
0.074
1429

Phage-1232
L
E
S
—
Q
V
—
E
—
H
G
—
G
A
0.066
0.059
0.059
1430

Phage-1233
I
—
—
—
R
E
—
N
—
—
—
—
S
W
0.072
1.660
0.076
1431

Phage-1234
—
—
—
—
R
F
—
D
—
—
—
—
D
T
0.055
1.646
0.063
1432

Phage-1235
L
—
—
—
R
—
—
R
—
—
—
—
E
T
0.070
1.819
0.063
1433

Phage-1236
—
—
—
—
R
—
—
H
—
—
—
—
E
D
0.065
2.723
0.062
1434

Phage-1237
—
—
—
—
R
—
—
Q
—
—
—
—
G
S
0.053
0.345
0.054
1435

Phage-1238
—
—
—
—
R
—
—
N
—
—
—
—
I
N
0.078
2.287
0.079
1436

Phage-1239
—
—
—
—
R
—
—
Q
—
—
—
—
T
Y
0.059
1.675
0.061
1437

Phage-1240
—
—
—
—
R
—
—
—
—
—
—
—
T
L
0.059
1.442
0.061
1438

Phage-1241
L
—
—
—
R
—
—
N
—
—
—
—
A
R
0.090
1.189
0.092
1439

Phage-1242
L
—
—
—
R
—
N
D
—
—
—
—
Q
S
0.061
0.899
0.056
1440

Phage-1243
—
—
—
—
R
L
—
H
—
—
—
—
G
D
0.076
2.276
0.076
1441

Phage-1244
I
—
—
—
R
L
—
D
—
—
—
—
G
L
0.067
2.435
0.062
1442

Phage-1245
—
—
—
—
R
L
—
D
—
—
—
—
Q
D
0.054
1.729
0.056
1443

Phage-1246
—
—
W
—
R
L
—
Q
—
—
—
—
Y
—
0.255
2.541
0.334
1444

Phage-1247
I
—
—
—
R
L
—
N
—
—
I
—
P
G
0.072
2.698
0.089
1445

Phage-1248
L
—
—
—
R
L
—
K
—
—
I
—
S
V
0.226
2.666
0.267
1446

Phage-1249
L
—
—
—
R
L
—
N
—
—
—
—
S
M
0.107
2.576
0.124
1447

Phage-1250
L
—
—
—
R
L
—
N
—
—
—
—
G
V
0.081
2.527
0.073
1448

Phage-1251
—
—
—
—
R
L
—
N
—
—
—
—
S
V
0.056
2.276
0.059
1449

Phage-1252
—
—
—
—
R
L
—
—
—
—
—
—
Q
E
0.059
2.032
0.059
1450

Phage-1253
—
—
—
—
R
L
—
T
—
—
I
—
Q
S
0.074
1.979
0.102
1451

Phage-1254
P
—
—
—
R
L
—
D
—
—
—
—
A
K
0.065
1.823
0.057
1452

Phage-1255
L
—
—
—
R
L
N
D
—
—
—
—
Q
T
0.056
1.551
0.070
1453

Phage-1256
—
—
—
—
R
L
—
D
—
—
—
—
I
E
0.078
0.978
0.074
1454

Phage-1257
I
—
W
—
R
M
—
E
—
—
—
—
K
H
0.080
1.130
0.098
1455

Phage-1258
—
—
—
—
R
V
—
N
—
—
—
—
Q
V
0.067
2.726
0.069
1456

Phage-1259
I
—
—
—
R
V
—
E
—
—
I
—
Y
G
0.056
2.433
0.058
1457

Phage-1260
I
—
—
—
R
V
—
D
—
—
—
—
N
R
0.063
2.155
0.056
1458

Phage-1261
I
—
—
—
R
V
—
D
—
—
—
—
N
W
0.084
2.489
0.079
1459

Phage-1262
D
W
—
—
R
V
—
—
—
—
I
—
Q
D
0.316
2.348
0.404
1460

Phage-1263
I
—
—
—
R
V
—
—
—
—
—
—
G
T
0.059
1.879
0.055
1461

Phage-1264
—
—
W
—
R
Y
—
D
—
—
—
—
S
T
0.075
0.652
0.095
1462

Phage-1265
—
—
—
—
S
—
—
H
—
—
—
—
Y
V
0.067
2.862
0.078
1463

Phage-1266
—
—
—
—
S
—
—
R
—
—
—
—
T
G
0.065
1.384
0.068
1464

Phage-1267
I
—
—
—
S
—
—
—
—
—
—
—
R
H
0.072
2.321
0.105
1465

Phage-1268
L
—
—
—
S
—
N
D
—
—
—
—
N
V
0.061
1.436
0.059
1466

Phage-1269
I
—
W
—
S
L
—
A
—
—
—
—
E
L
0.066
2.508
0.069
1467

Phage-1270
E
—
—
—
S
L
—
K
—
—
I
—
N
S
0.073
2.525
0.089
1468

Phage-1271
—
—
—
—
S
L
—
D
—
—
—
—
N
—
0.055
2.115
0.053
1469

Phage-1272
F
—
—
—
S
L
—
K
—
—
—
—
E
T
0.059
1.805
0.068
1470

Phage-1273
—
—
—
—
S
L
—
D
—
—
—
—
S
G
0.053
2.153
0.057
1471

Phage-1274
—
—
—
—
S
L
—
N
—
—
—
—
Q
N
0.059
2.725
0.063
1472

Phage-1275
—
—
—
—
S
L
—
N
—
—
—
—
K
M
0.059
2.714
0.064
1473

Phage-1276
I
—
—
—
S
L
—
—
—
—
—
—
M
S
0.056
2.694
0.056
1474

Phage-1277
—
—
—
—
S
L
—
N
—
—
—
—
M
R
0.082
2.694
0.090
1475

Phage-1278
—
—
—
—
S
L
—
H
—
—
—
—
M
—
0.058
2.653
0.062
1476

Phage-1279
—
—
—
—
S
L
—
Q
—
—
—
—
D
V
0.080
2.335
0.077
1477

Phage-1280
I
—
—
—
S
L
—
K
—
—
I
—
G
A
0.056
2.197
0.060
1478

Phage-1281
M
—
—
—
S
L
—
N
—
—
—
—
R
L
0.071
1.469
0.067
1479

Phage-1282
L
—
—
—
S
L
—
A
—
—
I
—
A
K
0.063
1.323
0.067
1480

Phage-1283
—
—
—
—
S
L
—
D
—
—
—
—
L
S
0.062
1.281
0.060
1481

Phage-1284
I
—
—
—
S
L
—
K
—
—
I
—
G
G
0.065
2.760
0.086
1482

Phage-1285
—
—
—
—
S
M
—
N
—
—
—
—
A
R
0.054
2.428
0.067
1483

Phage-1286
—
—
—
—
S
M
—
N
—
—
—
—
V
—
0.059
2.611
0.061
1484

Phage-1287
L
—
—
—
S
M
—
H
—
—
—
—
G
D
0.057
2.570
0.056
1485

Phage-1288
L
—
—
—
S
M
—
N
—
—
—
—
R
E
0.057
2.200
0.062
1486

Phage-1289
—
—
—
—
S
M
—
Q
—
—
—
—
D
G
0.074
1.991
0.073
1487

Phage-1290
—
—
—
—
S
M
—
M
—
—
—
—
Y
—
0.057
1.808
0.072
1488

Phage-1291
L
—
—
—
S
M
—
D
—
—
—
—
A
R
0.058
0.939
0.059
1489

Phage-1292
P
—
—
—
S
M
—
Q
—
—
—
—
Q
E
0.081
0.409
0.064
1490

Phage-1293
—
—
—
—
S
M
—
A
—
—
—
—
K
T
0.053
0.327
0.065
1491

Phage-1294
I
—
—
—
S
V
—
N
—
—
—
—
D
P
0.081
2.329
0.069
1492

Phage-1295
M
—
—
—
S
V
—
Q
—
—
I
—
G
S
0.070
2.495
0.089
1493

Phage-1296
—
—
—
—
S
V
—
Q
—
—
I
—
Y
K
0.060
1.925
0.065
1494

Phage-1297
—
—
W
—
S
V
—
D
—
—
—
—
R
—
0.057
0.453
0.059
1495

Phage-1298
L
—
—
—
S
Y
—
D
—
—
—
—
R
T
0.058
2.921
0.065
1496

Phage-1299
L
—
—
—
T
F
—
D
—
—
—
—
I
G
0.057
0.658
0.058
1497

Phage-1300
—
—
—
—
T
—
—
H
—
—
—
—
F
T
0.062
2.800
0.064
1498

Phage-1301
L
—
—
—
T
—
—
N
—
—
I
—
N
R
0.060
0.955
0.058
1499

Phage-1302
L
—
—
—
T
L
—
K
—
—
—
—
K
I
0.064
1.606
0.066
1500

Phage-1303
—
—
—
—
T
L
—
H
—
—
—
—
T
V
0.069
1.880
0.088
1501

Phage-1304
I
—
—
—
T
M
—
H
—
—
I
—
D
T
0.057
2.853
0.066
1502

Phage-1305
L
—
—
—
T
M
—
D
—
—
—
—
G
H
0.055
2.583
0.065
1503

Phage-1306
I
—
—
—
T
M
—
Q
—
—
I
—
D
T
0.076
2.761
0.071
1504

Phage-1307
—
—
—
—
T
V
—
N
—
—
—
—
Q
T
0.061
2.843
0.059
1505

Phage-1308
I
—
—
—
T
V
—
K
—
—
—
—
Q
I
0.059
2.833
0.074
1506

Phage-1309
—
—
—
—
T
V
—
R
—
—
I
—
S
N
0.059
2.799
0.081
1507

Phage-1310
F
—
—
—
T
V
—
—
—
—
—
—
R
—
0.058
0.385
0.062
1508

Phage-1311
L
—
L
—
T
V
Q
—
G
—
—
—
R
S
0.055
0.056
0.056
1509

Phage-1312
I
—
—
—
V
—
—
H
—
—
—
—
R
R
0.058
2.750
0.064
1510

Phage-1313
I
—
—
—
V
L
—
D
—
—
—
—
Q
G
0.054
2.606
0.060
1511

Phage-1314
L
—
—
—
V
L
—
R
—
—
I
—
S
T
0.072
2.459
0.072
1512

Phage-1315
—
—
—
—
V
V
—
Q
—
—
I
—
G
—
0.056
2.367
0.074
1513

Phage-1316
—
—
—
—
W
—
—
R
—
—
—
—
L
A
0.467
2.853
0.514
1514

Phage-1317
—
—
—
—
W
—
—
E
—
—
I
—
L
N
0.059
2.146
0.070
1515

Phage-1318
T
—
—
—
W
—
—
D
—
—
—
—
—
—
0.055
0.474
0.067
1516

Phage-1319
—
—
—
—
W
L
—
K
—
—
—
—
F
—
0.288
2.659
0.327
1517

Phage-1320
—
—
—
—
W
L
—
K
—
—
—
—
E
N
0.065
1.993
0.062
1518

Phage-1321
E
—
—
—
W
L
—
K
—
—
—
—
T
L
0.060
1.750
0.057
1519

Phage-1322
—
—
—
—
W
L
—
E
—
—
—
—
Q
N
0.055
1.633
0.060
1520

Phage-1323
—
—
—
—
W
M
—
D
—
—
—
—
R
E
0.055
2.656
0.064
1521

Phage-1324
E
—
—
—
W
M
—
A
—
—
—
—
G
I
0.059
1.958
0.062
1522

Phage-1325
—
—
—
—
Y
E
—
D
—
—
—
—
—
T
0.057
0.797
0.059
1523

Phage-1326
P
—
—
—
Y
—
N
N
—
—
—
—
V
T
0.057
2.321
0.057
1524

Phage-1327
P
—
—
—
Y
—
—
K
—
—
I
—
T
E
0.065
2.157
0.064
1525

Phage-1328
I
—
—
—
Y
—
—
D
—
—
—
—
Y
T
0.056
2.157
0.068
1526

Phage-1329
L
—
—
—
Y
—
N
K
—
—
—
—
W
E
0.057
1.901
0.076
1527

Phage-1330
—
—
—
—
Y
L
—
—
—
—
I
—
T
K
0.063
2.858
0.062
1528

Phage-1331
—
—
—
—
Y
L
F
N
—
—
—
—
W
E
0.150
2.833
0.134
1529

Phage-1332
—
—
—
—
Y
L
—
D
—
—
—
—
N
S
0.053
2.601
0.064
1530

Phage-1333
—
—
—
—
Y
L
—
—
—
—
—
—
V
—
0.088
2.495
0.073
1531

Phage-1334
—
—
—
—
Y
L
F
D
—
—
—
—
P
N
0.073
2.620
0.078
1532

Phage-1335
F
—
—
—
Y
L
—
D
—
—
—
—
W
G
0.066
2.600
0.075
1533

Phage-1336
M
—
—
—
Y
L
—
R
—
—
I
—
G
M
0.071
1.341
0.075
1534

Phage-1337
P
—
—
—
Y
M
—
N
—
—
—
—
V
T
0.070
2.778
0.072
1535

Phage-1338
P
—
—
—
Y
M
—
G
—
—
I
—
D
S
0.055
2.835
0.056
1536

Phage-1339
—
—
—
—
Y
M
—
R
—
—
—
—
L
N
0.063
2.740
0.076
1537

Phage-1340
P
—
—
—
Y
M
—
R
—
—
—
—
M
S
0.059
2.646
0.063
1538

Phage-1341
P
—
—
—
Y
M
—
G
—
—
I
—
D
T
0.053
2.742
0.075
1539

Phage-1342
—
—
—
—
Y
M
—
G
—
—
—
—
R
M
0.055
2.664
0.057
1540

Phage-1343
—
—
—
—
Y
M
—
N
—
—
—
—
V
P
0.055
2.663
0.067
1541

Phage-1344
—
—
—
—
Y
M
—
G
—
—
—
—
A
A
0.066
2.624
0.057
1542

Phage-1345
L
—
—
—
Y
M
—
G
—
—
I
—
A
S
0.056
2.598
0.060
1543

Phage-1346
—
—
—
—
Y
M
—
D
—
—
—
—
V
R
0.080
2.573
0.061
1544

Phage-1347
—
—
—
—
Y
M
—
A
—
—
—
—
M
A
0.056
2.563
0.058
1545

Phage-1348
E
—
—
—
Y
M
—
R
—
—
—
—
Y
R
0.058
2.441
0.058
1546

Phage-1349
—
—
—
—
Y
M
—
G
—
—
—
—
V
—
0.071
2.041
0.058
1547

Phage-1350
A
—
—
—
Y
M
—
D
—
—
—
—
S
K
0.054
1.289
0.068
1548

Phage-1351
E
—
—
—
Y
R
—
N
—
—
—
—
K
L
0.054
2.581
0.056
1549

Phage-1352
F
—
—
—
Y
R
—
N
—
—
I
—
T
K
0.274
2.176
0.336
1550

Phage-1353
—
—
—
—
Y
R
—
N
—
—
L
—
R
V
0.095
1.623
0.127
1551

Phage-1354
I
—
—
—
Y
R
—
G
—
—
—
—
G
T
0.060
1.592
0.066
1552

Phage-1355
I
—
—
—
Y
R
—
G
—
—
—
—
Q
T
0.058
1.535
0.059
1553

Phage-1356
I
—
—
—
Y
R
—
G
—
—
—
—
Y
T
0.082
1.336
0.074
1554

Phage-1357
—
—
—
—
Y
V
—
D
—
—
—
—
S
S
0.055
2.162
0.058
1555

Phage-1358
I
—
—
—
Q
L
—
H
—
—
—
—
R
A
0.110
2.717
0.699
1556

Phage-1359
—
—
—
—
A
L
—
N
—
—
—
—
A
I
0.070
2.762
0.426
1557

Phage-1360
F
—
—
—
F
M
—
H
—
—
—
—
K
D
0.225
2.870
0.398
1558

Phage-1361
I
—
—
—
E
—
—
R
—
—
—
—
Y
D
0.068
2.837
0.134
1559

Phage-1362
—
—
—
—
T
M
—
E
—
—
I
—
E
K
0.116
2.415
0.144
1560

Phage-1363
—
—
W
—
Y
M
—
A
—
—
—
—
S
—
0.079
2.820
0.161
1561

Phage-1364
I
—
—
—
F
—
—
R
—
—
—
—
S
Y
0.432
2.722
0.527
1562

Phage-1365
I
—
—
—
T
L
—
N
—
—
I
—
D
G
0.093
2.747
0.121
1563

Phage-1366
L
—
—
—
A
—
—
D
—
—
—
—
E
—
0.095
2.706
0.088
1564

Phage-1367
L
—
—
—
A
L
—
D
—
—
—
—
K
E
0.178
1.820
0.118
1565

Phage-1368
L
—
—
—
E
V
—
R
—
—
I
—
G
V
0.064
2.833
0.357
1566

Phage-1369
L
—
—
—
—
—
—
R
—
—
—
—
T
P
0.146
2.734
0.184
1567

Phage-1370
L
—
—
—
—
L
—
K
—
—
I
—
T
T
0.121
2.772
0.193
1568

Phage-1371
L
—
—
—
R
V
—
K
—
—
—
—
D
M
0.071
2.638
0.088
1569

Phage-1372
L
—
—
—
S
L
—
D
—
—
I
—
R
S
0.069
2.725
0.070
1570

Phage-1373
L
—
—
—
S
M
—
N
—
—
—
—
S
V
0.098
2.683
0.083
1571

Phage-1374
P
—
—
—
A
M
—
—
—
—
—
—
V
K
0.071
2.718
0.079
1572

Phage-1375
P
—
—
—
—
L
—
K
—
—
—
—
I
—
0.072
2.825
0.199
1573

Phage-1376
T
—
—
—
L
L
—
N
—
—
—
—
Q
H
0.098
2.706
0.142
1574

Phage-1377
—
—
—
—
A
F
—
D
—
—
—
—
D
M
0.080
2.740
0.087
1575

Phage-1378
—
—
—
—
Y
H
—
N
—
—
—
—
N
F
0.077
2.710
0.099
1576

Phage-1379
—
—
—
—
Y
L
F
N
—
—
—
—
G
K
0.130
2.757
0.191
1577

Phage-1380
A
—
—
—
A
M
—
H
—
—
M
—
D
—
0.085
2.757
0.097
1578

Phage-1381
A
—
—
—
Y
L
—
H
—
—
—
—
Y
V
0.084
2.322
0.154
1579

Phage-1382
D
E
—
—
I
Q
—
R
—
—
I
—
E
S
0.060
2.135
0.094
1580

Phage-1383
E
—
—
—
A
L
—
R
—
—
—
—
L
S
0.074
1.190
0.096
1581

Phage-1384
E
—
—
—
E
W
—
H
—
—
—
—
A
N
0.061
2.728
0.991
1582

Phage-1385
E
—
—
—
F
F
—
N
—
—
—
—
A
A
0.064
2.737
0.241
1583

Phage-1386
E
—
—
—
L
—
—
D
—
—
—
—
V
—
0.088
2.104
0.078
1584

Phage-1387
E
—
—
—
M
L
—
R
—
—
—
—
V
S
0.073
2.730
0.816
1585

Phage-1388
E
N
—
—
A
M
—
N
—
—
—
—
N
L
0.077
2.714
0.253
1586

Phage-1389
F
—
—
—
A
M
—
M
—
—
—
—
L
M
0.344
2.703
0.548
1587

Phage-1390
F
—
—
—
D
V
—
Y
—
—
—
—
—
A
0.089
2.741
0.089
1588

Phage-1391
F
—
—
—
E
V
—
K
—
—
—
—
Y
L
0.113
0.642
0.126
1589

Phage-1392
F
—
—
—
S
K
—
K
—
—
I
—
D
G
0.079
1.162
0.208
1590

Phage-1393
G
N
—
—
A
T
—
A
—
—
I
—
V
N
0.121
2.710
0.594
1591

Phage-1394
I
—
—
—
A
—
—
Q
—
—
—
—
W
N
0.080
2.691
0.080
1592

Phage-1395
I
—
—
—
A
M
—
H
—
—
I
—
D
K
0.075
2.806
0.156
1593

Phage-1396
I
—
—
—
A
M
—
Q
—
—
—
—
K
S
0.103
2.733
0.086
1594

Phage-1397
I
—
—
—
A
M
—
R
—
—
I
—
N
I
0.076
2.735
0.919
1595

Phage-1398
I
—
—
—
A
V
—
K
—
—
M
—
Q
I
0.065
1.530
0.105
1596

Phage-1399
I
—
—
—
A
V
—
—
—
—
—
—
Y
L
0.075
2.704
0.120
1597

Phage-1400
I
—
—
—
D
—
—
R
—
—
—
—
S
—
0.066
2.751
0.068
1598

Phage-1401
I
—
—
—
D
M
—
Q
—
—
I
—
D
T
0.153
2.728
0.292
1599

Phage-1402
I
—
—
—
E
—
F
—
—
—
—
—
R
P
0.084
2.823
0.267
1600

Phage-1403
I
—
—
—
F
L
—
Q
—
—
I
—
D
T
0.073
2.718
0.073
1601

Phage-1404
I
—
—
—
I
—
—
R
—
—
—
—
L
V
0.118
0.627
0.127
1602

Phage-1405
I
—
—
—
—
—
—
V
—
—
I
—
N
S
0.093
2.837
0.317
1603

Phage-1406
I
—
—
—
—
V
—
K
—
—
—
—
M
N
0.086
2.478
0.126
1604

Phage-1407
I
—
—
—
M
L
—
H
—
—
I
—
D
G
0.121
2.738
0.144
1605

Phage-1408
I
—
—
—
M
M
F
D
—
—
I
—
T
K
0.116
2.695
0.117
1606

Phage-1409
I
—
—
—
P
M
—
Q
—
T
I
—
I
T
0.080
2.705
0.081
1607

Phage-1410
I
—
—
—
Q
M
—
R
—
—
—
—
Y
G
0.132
2.671
0.167
1608

Phage-1411
I
—
—
—
R
M
—
K
—
—
—
—
Y
G
0.284
2.830
0.431
1609

Phage-1412
I
—
—
—
R
V
—
D
—
—
—
—
S
L
0.072
2.704
0.128
1610

Phage-1413
I
—
—
—
S
—
—
W
—
—
—
—
Q
E
0.160
2.731
0.177
1611

Phage-1414
I
—
—
—
S
L
—
E
—
—
—
—
W
S
1.574
2.721
1.471
1612

Phage-1415
I
—
—
—
S
L
—
T
—
—
—
—
Q
T
0.094
2.739
0.074
1613

Phage-1416
I
—
—
—
S
V
—
Y
—
—
—
—
N
S
0.087
0.808
0.141
1614

Phage-1417
I
—
—
—
V
L
—
E
—
—
—
—
G
T
0.061
2.687
0.122
1615

Phage-1418
I
—
—
—
W
M
—
Q
—
—
—
—
D
Y
0.106
2.690
0.212
1616

Phage-1419
I
—
—
—
W
Q
—
G
—
—
—
—
Y
G
0.126
2.666
0.118
1617

Phage-1420
I
—
—
—
Y
L
—
Q
—
—
I
—
T
K
0.064
2.748
0.067
1618

Phage-1421
I
N
—
—
R
—
—
D
—
—
—
—
—
D
0.098
0.552
0.122
1619

Phage-1422
K
—
—
—
Y
W
—
Q
—
—
I
—
G
D
0.071
2.855
0.219
1620

Phage-1423
L
—
—
—
A
L
N
R
—
—
—
—
Q
A
0.127
2.426
0.169
1621

Phage-1424
L
—
—
—
A
L
—
R
—
—
—
—
Q
A
0.082
2.484
0.175
1622

Phage-1425
L
—
—
—
A
L
—
R
—
—
—
—
S
V
0.109
1.974
0.135
1623

Phage-1426
L
—
—
—
A
M
—
K
—
—
—
—
R
S
0.113
2.763
0.159
1624

Phage-1427
L
—
—
—
E
L
—
R
—
—
—
—
W
M
0.250
1.784
0.314
1625

Phage-1428
L
—
—
—
E
M
—
Q
—
—
—
—
R
H
0.069
2.685
0.089
1626

Phage-1429
L
—
—
—
E
R
—
A
—
—
I
—
Y
G
0.080
0.553
0.091
1627

Phage-1430
L
—
—
—
—
—
N
D
—
—
I
—
G
H
0.068
2.712
0.068
1628

Phage-1431
L
—
—
—
—
—
N
G
—
—
—
—
Y
T
0.087
2.817
0.134
1629

Phage-1432
L
—
—
—
—
—
—
D
—
—
I
—
D
H
0.088
2.692
0.090
1630

Phage-1433
L
—
—
—
—
L
—
R
—
—
—
—
I
—
0.205
2.503
0.287
1631

Phage-1434
L
—
—
—
—
V
—
—
—
—
I
—
R
V
0.218
1.602
0.240
1632

Phage-1435
L
—
—
—
L
M
—
N
—
—
—
—
S
H
0.143
2.484
0.151
1633

Phage-1436
L
—
—
—
M
F
—
N
—
—
—
—
Q
H
0.255
2.711
0.275
1634

Phage-1437
L
—
—
—
Q
—
—
K
—
—
—
—
T
—
0.060
0.874
0.081
1635

Phage-1438
L
—
—
—
R
F
—
D
—
—
—
—
Q
I
0.075
2.682
0.130
1636

Phage-1439
L
—
—
—
R
—
—
R
—
—
—
—
V
S
0.263
2.708
0.329
1637

Phage-1440
L
—
—
—
S
—
—
H
—
—
—
—
G
D
0.078
1.102
0.090
1638

Phage-1441
L
—
—
—
S
M
—
D
—
—
—
—
N
V
0.109
1.130
0.082
1639

Phage-1442
L
—
—
—
S
M
—
R
—
—
—
—
V
G
0.108
0.851
0.114
1640

Phage-1443
L
—
—
—
Y
L
—
G
—
—
—
—
Y
R
0.240
2.705
0.508
1641

Phage-1444
L
N
—
—
D
T
—
H
—
—
I
—
G
G
0.072
0.620
0.076
1642

Phage-1445
L
N
—
—
E
R
—
H
—
—
I
—
G
D
0.106
2.806
0.138
1643

Phage-1446
M
—
—
—
F
—
—
G
—
—
I
—
R
Y
0.205
2.723
0.359
1644

Phage-1447
M
—
—
—
F
L
—
N
—
—
—
—
D
A
0.066
1.048
0.114
1645

Phage-1448
M
—
—
—
G
Y
—
H
—
—
—
—
D
E
0.081
2.709
0.265
1646

Phage-1449
M
—
—
—
—
M
—
M
—
—
I
—
A
T
0.123
2.753
0.515
1647

Phage-1450
M
—
—
—
L
L
—
D
—
—
—
—
R
M
0.104
2.680
0.206
1648

Phage-1451
M
—
—
—
Q
V
—
A
—
—
I
—
N
M
0.083
2.748
0.121
1649

Phage-1452
M
—
—
—
R
—
—
N
—
—
—
—
N
E
0.070
2.704
0.084
1650

Phage-1453
M
—
—
—
S
F
—
H
—
—
I
—
K
S
0.172
2.728
0.283
1651

Phage-1454
P
—
—
—
A
M
—
R
—
—
—
—
V
G
0.086
2.000
0.074
1652

Phage-1455
P
—
—
—
H
L
—
R
—
—
—
—
E
W
0.087
2.597
0.145
1653

Phage-1456
P
—
—
—
L
L
—
G
—
—
—
—
M
T
0.073
0.621
0.080
1654

Phage-1457
P
—
—
—
R
L
—
H
—
—
—
—
L
A
0.094
2.748
0.099
1655

Phage-1458
P
—
—
—
Y
—
—
A
—
—
I
—
D
S
0.081
2.741
0.099
1656

Phage-1459
P
—
—
—
Y
M
—
M
—
—
I
—
A
—
0.065
2.850
0.091
1657

Phage-1460
T
—
—
—
G
—
—
R
—
—
—
—
F
H
0.096
2.628
0.086
1658

Phage-1461
—
—
—
—
A
—
F
A
—
—
—
—
M
M
0.186
2.446
0.321
1659

Phage-1462
—
—
—
—
A
—
—
H
—
—
—
—
M
G
0.112
2.841
0.363
1660

Phage-1463
—
—
—
—
A
—
—
—
—
—
—
—
R
T
0.075
2.721
0.078
1661

Phage-1464
—
—
—
—
A
L
—
Q
—
—
—
—
W
—
0.077
2.725
0.115
1662

Phage-1465
—
—
—
—
A
M
—
R
—
—
—
—
T
S
0.144
2.702
0.232
1663

Phage-1466
—
—
—
—
A
V
—
A
—
—
—
—
A
T
0.063
2.772
0.148
1664

Phage-1467
—
—
—
—
A
V
—
E
—
—
—
—
D
T
0.084
2.707
0.108
1665

Phage-1468
—
—
—
—
A
V
—
N
—
—
—
—
G
L
0.098
2.675
0.108
1666

Phage-1469
—
—
—
—
A
V
—
R
—
—
—
—
—
A
0.070
2.766
0.096
1667

Phage-1470
—
—
—
—
E
F
—
H
—
—
I
—
Y
—
0.153
2.663
0.160
1668

Phage-1471
—
—
—
—
E
L
—
R
—
—
—
—
Q
V
0.129
2.679
0.121
1669

Phage-1472
—
—
—
—
E
L
—
—
—
—
—
—
Y
—
0.060
2.728
0.093
1670

Phage-1473
—
—
—
—
E
L
—
W
—
—
—
—
D
R
0.105
1.800
0.191
1671

Phage-1474
—
—
—
—
E
M
F
H
—
—
—
—
L
R
0.067
0.996
0.157
1672

Phage-1475
—
—
—
—
E
M
—
H
—
—
I
—
Y
—
0.096
2.833
0.114
1673

Phage-1476
—
—
—
—
E
M
—
R
—
—
—
—
T
G
0.086
2.624
0.081
1674

Phage-1477
—
—
—
—
E
V
F
H
—
—
—
—
D
—
0.122
2.782
0.095
1675

Phage-1478
—
—
—
—
E
V
—
H
—
—
—
—
L
G
0.086
2.724
0.098
1676

Phage-1479
—
—
—
—
E
V
—
R
—
—
—
—
L
G
0.074
2.705
0.080
1677

Phage-1480
—
—
—
—
F
L
—
P
—
—
I
—
Y
—
0.084
2.754
0.116
1678

Phage-1481
—
—
—
—
G
F
—
H
—
—
I
—
Y
S
0.097
2.706
0.377
1679

Phage-1482
—
—
—
—
G
—
—
Q
—
—
—
—
I
—
0.071
2.689
0.124
1680

Phage-1483
—
—
—
—
G
—
—
R
—
—
I
—
E
N
0.071
2.749
0.156
1681

Phage-1484
—
—
—
—
G
L
F
R
—
—
I
—
T
D
0.104
2.722
0.180
1682

Phage-1485
—
—
—
—
G
L
—
H
—
—
—
—
W
T
0.102
2.703
0.179
1683

Phage-1486
—
—
—
—
—
—
F
Q
—
—
—
—
V
D
0.083
0.508
0.105
1684

Phage-1487
—
—
—
—
—
—
—
A
—
—
I
—
E
S
0.076
2.824
0.123
1685

Phage-1488
—
—
—
—
—
—
—
—
—
—
—
—
S
Y
0.072
2.684
0.102
1686

Phage-1489
—
—
—
—
—
L
F
Q
—
—
—
—
A
G
0.131
2.703
0.192
1687

Phage-1490
—
—
—
—
—
L
—
K
—
—
—
—
A
V
0.074
2.707
0.156
1688

Phage-1491
—
—
—
—
—
L
—
K
—
—
—
—
T
A
0.067
2.685
0.154
1689

Phage-1492
—
—
—
—
—
L
—
N
—
—
—
—
P
G
0.082
2.828
0.218
1690

Phage-1493
—
—
—
—
—
L
—
R
—
—
—
—
G
L
0.086
2.728
0.093
1691

Phage-1494
—
—
—
—
—
V
—
R
—
—
—
—
L
A
0.114
0.865
0.096
1692

Phage-1495
—
—
—
—
L
—
—
H
—
—
—
—
Y
V
0.087
2.707
0.180
1693

Phage-1496
—
—
—
—
L
—
—
N
—
—
—
—
L
—
0.094
1.112
0.115
1694

Phage-1497
—
—
—
—
L
L
—
D
—
—
—
—
N
G
0.065
2.819
0.208
1695

Phage-1498
—
—
—
—
L
L
—
D
—
—
—
—
S
K
0.076
2.844
0.194
1696

Phage-1499
—
—
—
—
L
L
—
R
—
—
—
—
V
—
0.071
1.580
0.102
1697

Phage-1500
—
—
—
—
L
M
—
H
—
—
—
—
S
—
0.075
2.740
0.131
1698

Phage-1501
—
—
—
—
L
V
—
R
—
—
I
—
T
—
0.093
2.840
0.148
1699

Phage-1502
—
—
—
—
M
L
—
A
—
—
—
—
—
—
0.062
2.682
0.114
1700

Phage-1503
—
—
—
—
M
L
—
H
—
—
I
—
Y
—
0.120
2.745
0.104
1701

Phage-1504
—
—
—
—
M
V
—
R
—
—
—
—
D
N
0.075
2.700
0.074
1702

Phage-1505
—
—
—
—
Q
L
—
D
—
—
—
—
N
S
0.070
2.672
0.075
1703

Phage-1506
—
—
—
—
Q
L
—
N
—
—
—
—
L
K
0.079
0.938
0.090
1704

Phage-1507
—
—
—
—
Q
M
—
R
—
—
—
—
Y
G
0.129
2.725
0.117
1705

Phage-1508
—
—
—
—
Q
M
—
—
—
—
—
—
A
T
0.061
2.711
0.148
1706

Phage-1509
—
—
—
—
R
—
—
H
—
—
—
—
K
H
0.188
2.298
0.291
1707

Phage-1510
—
—
—
—
R
L
—
D
—
—
—
—
M
T
0.080
2.827
0.147
1708

Phage-1511
—
—
—
—
R
L
—
D
—
—
—
—
S
G
0.078
2.730
0.103
1709

Phage-1512
—
—
—
—
R
L
—
M
—
—
I
—
L
E
0.083
2.734
0.123
1710

Phage-1513
—
—
—
—
R
L
—
N
—
—
—
—
I
T
0.094
2.703
0.128
1711

Phage-1514
—
—
—
—
R
L
—
R
—
—
I
—
Q
H
0.165
2.714
0.284
1712

Phage-1515
—
—
—
—
R
L
—
—
—
—
—
—
D
P
0.064
1.575
0.084
1713

Phage-1516
—
—
—
—
R
M
—
A
—
—
I
—
D
P
0.105
2.738
0.161
1714

Phage-1517
—
—
—
—
R
M
—
A
—
—
—
—
S
H
0.084
2.713
0.119
1715

Phage-1518
—
—
—
—
R
M
—
D
—
—
—
—
T
S
0.073
1.039
0.080
1716

Phage-1519
—
—
—
—
R
M
—
—
—
—
—
—
T
T
0.111
1.917
0.133
1717

Phage-1520
—
—
—
—
S
—
—
R
—
—
—
—
N
G
0.076
2.686
0.069
1718

Phage-1521
—
—
—
—
S
L
—
H
—
—
—
—
M
R
0.090
2.592
0.146
1719

Phage-1522
—
—
—
—
S
L
—
—
—
—
—
—
E
A
0.066
2.711
0.210
1720

Phage-1523
—
—
—
—
S
M
—
A
—
—
—
—
N
Y
0.080
2.708
0.163
1721

Phage-1524
—
—
—
—
S
M
—
D
—
—
—
—
T
A
0.067
2.649
0.079
1722

Phage-1525
—
—
—
—
S
M
—
R
—
—
—
—
Y
E
0.080
2.733
0.079
1723

Phage-1526
—
—
—
—
S
V
—
G
—
—
—
—
N
L
0.080
0.754
0.094
1724

Phage-1527
—
—
—
—
S
V
—
Y
—
—
I
—
Q
N
0.068
2.730
0.116
1725

Phage-1528
—
—
—
—
T
—
F
H
—
—
—
—
Q
S
0.150
2.730
0.195
1726

Phage-1529
—
—
—
—
T
—
—
Q
—
—
I
—
R
S
0.098
2.830
0.175
1727

Phage-1530
—
—
—
—
T
L
—
Q
—
—
—
—
L
K
0.083
2.619
0.093
1728

Phage-1531
—
—
—
—
W
L
—
Q
—
—
I
—
N
G
0.154
2.823
0.196
1729

Phage-1532
—
—
—
—
W
L
—
Q
—
—
—
—
N
S
0.151
2.725
0.139
1730

Phage-1533
—
—
—
—
W
L
—
R
—
—
—
—
T
—
0.624
2.695
0.795
1731

Phage-1534
—
—
—
—
W
M
—
D
—
—
—
—
R
V
0.166
2.067
0.210
1732

Phage-1535
—
—
—
—
Y
L
—
G
—
—
—
—
M
L
0.139
2.476
0.131
1733

Phage-1536
—
—
—
—
Y
L
—
Q
—
—
—
—
G
K
0.061
2.559
0.243
1734

Phage-1537
—
—
—
—
Y
L
—
V
—
—
—
—
L
R
0.107
2.667
0.157
1735

Phage-1538
—
—
—
—
Y
M
—
G
—
—
—
—
N
P
0.078
2.780
0.162
1736

Phage-1539
—
—
—
—
Y
M
—
G
—
—
—
—
R
L
0.075
2.735
0.070
1737

Phage-1540
—
—
W
—
A
L
—
D
—
—
—
—
E
—
0.094
2.714
0.116
1738

Phage-1541
—
—
W
—
H
—
—
R
—
—
—
—
D
M
0.131
2.001
0.222
1739

Phage-1542
—
—
W
—
T
L
—
A
—
—
—
—
Q
—
0.146
2.466
0.195
1740

Phage-1543
—
N
—
—
Q
—
—
E
—
—
I
—
T
S
0.187
1.474
0.138
1741

Phage-1544
W
K
—
—
A
T
S
M
—
—
N
—
V
D
0.133
2.852
0.223
1742

Phage-1545
I
—
—
—
I
L
—
D
—
—
—
—
E
G
0.146
2.661
0.121
1743

Phage-1546
A
—
L
—
Y
L
Q
P
Q
—
—
—
E
T
0.065
0.058
0.084
1744

Phage-1547
E
—
—
—
M
R
—
N
—
—
—
—
R
E
0.074
0.098
0.088
1745

Phage-1548
E
—
—
—
Q
T
—
R
—
—
I
—
I
Y
0.072
0.094
0.100
1746

Phage-1549
G
T
—
—
P
T
S
Q
—
R
Y
—
I
I
0.108
0.106
0.421
1747

Phage-1550
I
—
—
—
E
R
—
—
—
—
—
—
V
T
0.077
0.184
0.093
1748

Phage-1551
L
—
—
—
W
L
—
R
—
—
—
—
E
N
0.464
1.967
0.712
1749

Phage-1552
L
E
—
—
A
W
Q
L
—
S
—
—
D
D
0.081
0.075
0.136
1750

Phage-1553
L
N
—
—
A
F
—
D
—
—
I
—
—
T
0.872
2.693
1.387
1751

Phage-1554
P
—
—
—
E
M
—
N
—
—
—
—
D
R
0.071
0.493
0.072
1752

Phage-1555
P
—
—
—
L
L
—
K
—
—
I
—
P
S
0.074
0.381
0.082
1753

Phage-1556
—
—
—
—
A
L
—
H
—
—
I
—
D
T
1.367
2.725
1.630
1754

Phage-1557
—
—
—
—
G
L
—
K
—
—
—
—
S
M
0.102
0.480
0.108
1755

Phage-1558
—
—
—
—
G
M
—
K
—
—
I
—
N
K
0.075
0.249
0.082
1756

Phage-1559
—
—
—
—
S
V
—
D
—
—
—
—
W
G
0.074
0.332
0.077
1757

Phage-1560
—
—
—
—
W
—
—
G
—
—
—
—
Q
S
0.124
0.298
0.191
1758

Phage-1561
—
—
—
—
W
L
—
R
—
—
—
—
F
D
1.299
2.660
1.660
1759

Phage-1562
—
—
W
—
G
L
—
N
—
—
—
—
T
W
0.468
2.082
0.674
1760

Phage-1563
—
E
Q
—
G
L
Q
—
L
R
G
—
K
V
0.070
0.071
0.097
1761

Phage-1564
W
—
—
—
F
L
—
—
—
—
—
—
D
S
0.329
0.858
0.465
1762

Phage-1565
I
—
—
—
Q
—
—
G
—
—
—
—
L
G
0.080
2.478
0.078
1763

Phage-1566
I
—
—
—
L
L
—
K
—
—
—
—
D
L
0.070
2.731
0.332
1764

Phage-1567
—
—
—
—
E
L
—
H
—
—
I
—
Y
—
0.114
2.730
0.332
1765

Phage-1568
—
—
—
—
T
L
—
D
—
—
—
—
R
K
0.080
2.642
0.088
1766

Phage-1569
L
—
—
—
E
V
N
R
—
—
I
—
G
V
0.080
2.698
0.075
1767

Phage-1570
I
—
—
—
M
M
F
D
—
—
—
—
N
K
0.122
2.062
0.152
1768

Phage-1571
L
—
—
—
F
F
—
D
—
—
—
—
R
N
0.072
2.841
0.221
1769

Phage-1572
P
—
—
—
V
L
—
H
—
—
I
—
A
A
0.075
2.715
0.070
1770

Phage-1573
—
—
—
—
A
—
—
—
—
—
I
—
K
T
0.134
2.694
0.169
1771

Phage-1574
—
—
—
—
L
F
—
R
—
—
I
—
T
E
0.125
2.717
0.154
1772

Phage-1575
—
—
—
—
L
—
—
G
—
—
I
—
S
S
0.075
2.718
0.124
1773

Phage-1576
W
I
—
V
G
L
M
L
A
L
—
G
T
I
0.067
2.717
0.092
1774

(—) indicates same amino acid as in TROP2 Fab Peptide-2 corresponding position (e.g. Phage-981 position).

TABLE 30

Sequences of those peptides selected for synthesis

(TROP2 Fab Peptide-2 Optimization)

Construct

Description
Amino Acid Sequence
SEQ ID NO:

Peptide-50
IDFCAMYQWPICDT
179

Peptide-51
IDFCAVYKWPVCQV
180

Peptide-52
IDFCMLYNWPICAG
181

Peptide-53
VDFCKIYAWPICGS
182

Peptide-54
VDFCKLYNWPVCQT
183

Peptide-55
IDFCLIYNWPVCDT
184

Peptide-56
EDFCKLYNWPICYQ
185

Peptide-57
VDFCGLYHWPICYQ
186

Peptide-58
VDFCYLYNWPVCSK
187

Peptide-59
IDFCAIYQWPVCRS
188

Peptide-60
VDFCALYNWPVCET
189

Peptide-61
PDFCAVYRWPICYQ
190

Peptide-62
VDFCELYRWPICNS
191

Peptide-63
LDFCKIYDWPICHL
192

Peptide-64
LDFCKLYQWPVCFT
193

Peptide-65
IDFCLLYDWPVCAS
194

Peptide-66
IDFCLLYDWPICGR
195

Peptide-67
MDFCQIYDWPICRL
196

Peptide-68
PDFCQLYNWPVCAG
197

Peptide-69
VDFCSFYRWPICET
198

Peptide-70
IDFCSLYQWPVCGT
199

Peptide-71
VDFCTIYKWPVCEG
200

Peptide-72
VDFCYQYGWPICSR
201

Example 10: TROP2 Polypeptide Complex Binding (PC25. PC26, PC27, PC28, PC29, and PC30)

PC25, PC26, PC27, PC28, PC29, and PC30, the sequences of which are provided in Table 6 were evaluated for TROP2 and CD3ε binding according to the methods of Example 1. FIGS. 22 and 23 show representative ELISAs of TROP2 and CD3ε binding, respectively.

Example 11: Polypeptide Complex Mediated Tumor Cytotoxicity

Polypeptide complexes were evaluated in a functional in vitro tumor cell killing assay using the TROP2 positive tumor cell lines HCT116, NCI-1H292, and MDAMB231. 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. 24A illustrates PC25 mediated HCT116 tumor cell killing in the presence of CD8+ T cells. FIG. 24B illustrates PC26 mediated HCT116 tumor cell killing in the presence of CD8+ T cells. FIG. 24C illustrates PC25 mediated MDAMB231 tumor cell killing in the presence of CD8+ T cells.

Example 12: Polypeptide Complex-22 (PC22) 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 pharmacokinetics enabled the calculation of Cmax, half-life, volume of distribution, clearance, and 7 day area under the curve (AUC) shown in Table 31 and in FIG. 25. Measured pharmacokinetics in cyno support once weekly dosing in humans.

TABLE 31

PC22 PK calculations

PC22 100 ug/kg
Units

C_MAX
17.38
nM

t_1/2
68.97
hr

Vd
0.18
L

VSS
0.44
L

CL
0.61
mL/hr/kg

BW
3.00
kg

7 day
45640
nM · min

AUC

Example 13: PC22 in Cynomolgus Cytokine Release

Example 14: PC22 in Cynomolgus Toxicity

Systemic liver enzymes after PC22 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. FIGS. 27A-27B illustrate serum liver enzyme levels in cynomolgus monkeys after single IV bolus of PC22.

Example 15: Anti-Tumor Efficacy in a Mouse Model of Human Triple Negative Breast Cancer

Female NCG mice were subcutaneously implanted with 5 million MDA-MB-231 triple negative tumor cells. When tumors reached 50-80 mm³, 20 million human PBMCs were engrafted via the tail vein. When tumors reached 275 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 TROP2 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. Shown are PC3 (FIG. 28A), PC17 (FIG. 28B), PC23 (FIG. 28C), PC24 (FIG. 28D).

Example 16: Optimized Phage 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, quench 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 EC50 was calculated using Graphpad prism. Data is shown in FIGS. 29A-29F and summarized in Tables 32A-32D. Peptide Sequences of CD3 Ala Scan Peptides for Peptide A and Peptide-B are shown in Table 34.

TABLE 32A

Summary of FIG. 29B

ELISA
Peptide-A
Peptide-C
Peptide-D
Peptide-E
Peptide-F
Peptide-G
Peptide-H

EC50 nM
1.013
0.9429
1.018
0.9738
1.27
47.5
346.2

TABLE 32B

Summary of FIG. 29C

ELISA
Peptide-A
Peptide-I
Peptide-J
Peptide-K
Peptide-L
Peptide-M
Peptide-N

EC50 nM
0.986
310.8
3.134
1.960
4.363
2.76
1.546

TABLE 32C

Summary of FIG. 29E

ELISA
Peptide-O
Peptide-P
Peptide-Q
Peptide-R
Peptide-S
Peptide-T

EC50 nM
1.356
2.359
30.04
47.50
457.1
4.762

TABLE 32D

Summary of FIG. 29F

ELISA
Peptide-U
Peptide-V
Peptide-W
Peptide-X
Peptide-Y
Peptide-Z

EC50 nM
39.90
2168
1.916
1.948
2.012
1.833

Peptides were evaluated for their ability to inhibit SP34.185 scFv from binding CD3e 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. 30A-30F and summarized in Tables 33A-33D.

TABLE 33A

Summary of FIG. 30B

ELISA
Peptide-A
Peptide-C
Peptide-D
Peptide-E
Peptide-F
Peptide-G
Peptide-H

IC50 uM
0.1926
0.1025
0.2318
0.1905
5.484
>100
>100

TABLE 33B

Summary of FIG. 30C

ELISA
Peptide-A
Peptide-I
Peptide-10
Peptide-K
Peptide-L
Peptide-M
Peptide-N

IC50 uM
0.1138
>100
63.18
>100
86.78
36.66
3.009

TABLE 33C

Summary of FIG. 30E

ELISA
Peptide-O
Peptide-P
Peptide-Q
Peptide-R
Peptide-S
Peptide-T

IC50 uM
0.1473
3.333
>100
>100
>100
41.46

TABLE 33D

Summary of FIG. 30F

ELISA
Peptide-U
Peptide-V
Peptide-W
Peptide-X
Peptide-Y
Peptide-Z

IC50 uM
>100
>100
1.912
0.6992
1.456
0.1180

TABLE 34

CD3 Ala Scan Sequences-Peptide A and Peptide-B

SEQ

anti-CD3

ID

Peptide-ID
Panned target
Sequence
NO:

Peptide-A
SP34.185
GSQCLGPEWEVCPY
202

Peptide-C
SP34.185
ASQCLGPEWEVCPY
203

Peptide-D
SP34.185
GAQCLGPEWEVCPY
204

Peptide-E
SP34.185
GSACLGPEWEVCPY
205

Peptide-F
SP34.185
GSQCAGPEWEVCPY
206

Peptide-G
SP34.185
GSQCLAPEWEVCPY
207

Peptide-H
SP34.185
GSQCLGAEWEVCPY
208

Peptide-I
SP34.185
GSQCLGPAWEVCPY
209

Peptide-J
SP34.185
GSQCLGPEAEVCPY
210

Peptide-K
SP34.185
GSQCLGPEWAVCPY
211

Peptide-L
SP34.185
GSQCLGPEWEACPY
212

Peptide-M
SP34.185
GSQCLGPEWEVCAY
213

Peptide-N
SP34.185
GSQCLGPEWEVCPA
214

Peptide-A
SP34.185
GSQCLGPEWEVCPY
215

Peptide-B
SP34.185
VYCGPEFDESVGCM
216

Peptide-O
SP34.185
AYCGPEFDESVGCM
217

Peptide-P
SP34.185
VACGPEFDESVGCM
218

Peptide-Q
SP34.185
VYCAPEFDESVGCM
219

Peptide-R
SP34.185
VYCGAEFDESVGCM
220

Peptide-S
SP34.185
VYCGPAFDESVGCM
221

Peptide-T
SP34.185
VYCGPEADESVGCM
222

Peptide-U
SP34.185
VYCGPEFAESVGCM
223

Peptide-V
SP34.185
VYCGPEFDASVGCM
224

Peptide-W
SP34.185
VYCGPEFDEAVGCM
225

Peptide-X
SP34.185
VYCGPEFDESAGCM
226

Peptide-Y
SP34.185
VYCGPEFDESVACM
227

Peptide-Z
SP34.185
VYCGPEFDESVGCA
228

Example 17: 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 18: 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-A library sequences are shown in Table 35. The sequences of those peptides selected for synthesis are shown in Table 36, and further evaluated for binding to anti-CD3 scFv (FIGS. 31A-31B) and inhibition of anti-CD3 scFv binding to CD3 (FIGS. 32A-32B). The consensus sequence shown in FIG. 33 was calculated from all the sequences shown in Table 35 and was generated using WebLogo 3.7.4.

TABLE 35

Clonal Phage Peptide Sequences from the Peptide-B Optimization Library Panning

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
1Q
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
27

Peptide B

Phage-2
D
D
—
W
—
D
W
E
F
D
F
A
—
A
0.08
2.75
0.09
229

Phage-3
Y
I
—
—
L
D
—
P
D
F
L
Y
—
D
0.08
2.88
0.10
230

Phage-4
F
D
—
W
—
D
W
E
—
Y
F
V
—
D
0.08
2.79
0.09
231

Phage-5
Y
I
—
W
—
D
W
E
—
Y
F
D
—
D
0.08
2.74
0.09
232

Phage-6
N
I
—
W
—
D
W
E
D
D
Y
F
—
F
0.09
2.54
0.09
233

Phage-7
N
F
—
W
—
D
W
E
Y
I
Y
P
—
I
0.07
2.77
0.09
234

Phage-8
—
D
—
W
—
D
W
E
—
D
F
L
—
I
0.08
2.54
0.08
235

Phage-9
H
A
—
W
—
D
W
E
—
Y
F
P
—
N
0.08
2.85
0.09
236

Phage-10
Y
D
—
—
—
D
V
—
—
—
Y
V
—
V
0.09
2.63
0.10
237

Phage-11
I
D
—
W
—
D
W
E
D
D
T
F
—
Y
0.09
2.73
0.08
238

Phage-12
Y
L
—
—
—
D
G
—
—
T
L
A
—
Y
0.08
2.66
0.15
239

Phage-13
—
D
—
—
—
D
G
—
—
—
I
L
—
Y
0.11
2.13
0.08
240

Phage-14
F
I
—
W
—
D
W
E
—
D
Y
F
—
A
0.07
2.44
0.09
1775

Phage-15
G
D
—
W
—
D
W
E
W
D
F
Y
—
D
0.07
2.71
0.07
1776

Phage-16
Y
L
—
W
—
D
W
E
Y
I
D
L
—
D
0.12
2.67
0.08
1777

Phage-17
S
F
—
W
—
D
W
E
—
Y
F
D
—
D
0.10
2.60
0.07
1778

Phage-18
D
D
—
W
—
D
W
E
—
Y
A
S
—
D
0.09
2.57
0.07
1779

Phage-19
N
L
—
W
—
D
W
E
Y
P
F
F
—
D
0.09
2.52
0.09
1780

Phage-20
F
D
—
W
—
D
W
E
—
—
F
V
—
D
0.08
2.34
0.09
1781

Phage-21
D
I
—
—
—
D
G
—
—
T
I
I
—
D
0.13
2.30
0.10
1782

Phage-22
D
D
—
W
—
D
W
E
Y
Y
A
V
—
D
0.09
2.28
0.09
1783

Phage-23
Y
D
—
W
—
D
W
E
—
Y
S
N
—
D
0.10
2.17
0.08
1784

Phage-24
I
N
—
W
—
D
W
E
D
Y
F
F
—
D
0.07
2.16
0.07
1785

Phage-25
N
I
—
W
—
D
W
E
D
D
T
F
—
F
0.06
2.87
0.07
1786

Phage-26
N
I
—
W
—
D
W
E
P
N
S
F
—
F
0.09
2.87
0.08
1787

Phage-27
Y
D
—
—
—
—
M
—
—
—
I
D
—
F
0.09
2.39
0.08
1788

Phage-28
D
F
—
W
—
D
W
E
F
P
F
I
—
H
0.11
2.73
0.12
1789

Phage-29
D
F
—
—
—
—
M
—
—
—
I
T
—
I
0.07
2.36
0.08
1790

Phage-30
Y
D
—
—
—
—
—
—
—
—
T
V
—
I
0.10
2.32
0.08
1791

Phage-31
H
D
—
W
—
D
W
E
W
D
I
F
—
I
0.07
2.26
0.08
1792

Phage-32
H
A
—
W
—
D
W
E
—
Y
N
P
—
N
0.11
2.71
0.11
1793

Phage-33
D
V
—
W
—
D
W
E
W
D
F
F
—
N
0.08
2.65
0.08
1794

Phage-34
N
—
—
W
—
D
W
E
Y
Y
I
P
—
N
0.10
2.57
0.08
1795

Phage-35
I
I
—
W
—
D
W
E
F
I
D
Y
—
N
0.08
2.10
0.07
1796

Phage-36
S
L
—
W
—
D
W
E
Y
D
I
A
—
P
0.07
2.53
0.08
1797

Phage-37
D
L
—
—
—
—
L
—
—
—
I
F
—
P
0.08
2.49
0.09
1798

Phage-38
T
N
—
W
—
D
W
E
W
V
L
P
—
P
0.14
2.47
0.10
1799

Phage-39
I
E
—
W
—
D
W
E
P
N
Y
F
—
P
0.13
2.29
0.09
1800

Phage-40
I
F
—
W
—
D
W
E
D
Y
—
D
—
P
0.07
2.28
0.07
1801

Phage-41
I
D
—
W
—
D
W
E
Y
D
F
F
—
P
0.07
2.26
0.08
1802

Phage-42
L
F
—
W
—
D
W
E
D
—
F
F
—
P
0.18
2.11
0.13
1803

Phage-43
—
D
—
W
—
D
W
E
D
Y
A
D
—
T
0.11
2.20
0.10
1804

Phage-44
—
I
—
W
—
D
W
E
Q
Y
F
P
—
V
0.11
2.34
0.09
1805

Phage-45
I
E
—
W
—
D
W
E
P
I
Y
P
—
Y
0.09
2.85
0.09
1806

Phage-46
I
T
—
W
—
D
W
E
V
Y
F
P
—
Y
0.07
2.55
0.08
1807

Phage-47
I
D
—
W
—
D
W
E
Y
I
H
P
—
Y
0.06
2.51
0.09
1808

Phage-48
I
D
—
W
—
D
W
E
Y
I
N
P
—
—
0.12
2.50
0.12
1809

Phage-49
A
D
—
W
—
D
W
E
—
A
F
P
—
Y
0.09
2.44
0.09
1810

Phage-50
I
D
—
W
—
D
W
E
Y
I
Y
P
—
Y
0.09
2.31
0.07
1811

Phage-51
N
I
—
W
—
D
W
E
D
D
N
F
—
F
0.09
2.08
0.09
1812

Phage-52
Y
D
—
W
—
D
W
E
Y
V
D
A
—
Y
0.09
2.06
0.09
1813

Phage-53
F
—
—
—
—
D
G
—
—
—
Y
V
—
D
0.09
2.03
0.11
1814

Phage-54
D
I
—
W
—
D
W
E
Y
I
N
I
—
S
0.11
2.02
0.11
1815

Phage-55
F
V
—
W
—
D
W
E
D
F
N
F
—
D
0.07
2.01
0.08
1816

Phage-56
F
A
—
W
—
D
W
E
D
Y
—
A
—
D
0.07
2.01
0.09
1817

Phage-57
D
N
—
W
—
D
W
E
Y
D
F
F
—
V
0.08
1.99
0.09
1818

Phage-58
Y
D
—
W
—
D
W
E
—
Y
N
D
—
A
0.09
1.96
0.11
1819

Phage-59
D
D
—
—
—
D
G
—
—
T
I
I
—
V
0.07
1.91
0.09
1820

Phage-60
F
P
—
W
—
D
W
E
—
Y
A
I
—
D
0.10
1.89
0.10
1821

Phage-61
P
D
—
—
—
D
G
—
—
—
L
F
—
T
0.12
1.86
0.07
1822

Phage-62
D
N
—
W
—
D
W
E
Y
D
Y
F
—
V
0.07
1.83
0.07
1823

Phage-63
I
F
—
W
—
D
W
E
—
F
Y
D
—
Y
0.12
1.82
0.08
1824

Phage-64
A
D
—
W
—
D
W
E
—
Y
F
P
—
N
0.08
1.82
0.08
1825

Phage-65
H
T
—
W
—
D
W
E
D
D
I
F
—
N
0.12
1.81
0.10
1826

Phage-66
F
A
—
W
—
D
W
E
—
A
F
L
—
L
0.09
1.80
0.09
1827

Phage-67
Y
D
—
—
—
—
—
—
—
—
I
A
—
D
0.08
1.77
0.08
1828

Phage-68
N
S
—
W
—
D
W
E
Y
D
I
I
—
D
0.08
1.77
0.10
1829

Phage-69
F
A
—
W
—
D
W
E
—
V
A
P
—
Y
0.07
1.75
0.07
1830

Phage-70
L
D
—
—
—
D
G
—
—
T
L
T
—
Y
0.10
1.75
0.12
1831

Phage-71
—
L
—
W
—
D
W
E
—
F
Y
D
—
P
0.07
1.74
0.09
1832

Phage-72
H
A
—
W
—
V
W
E
—
Y
F
P
—
N
0.07
1.72
0.08
1833

Phage-73
N
E
—
W
—
N
G
E
P
T
F
P
—
T
0.08
1.71
0.07
1834

Phage-74
L
T
—
—
—
D
G
—
—
T
L
Y
—
D
0.08
1.70
0.07
1835

Phage-75
Y
D
—
—
—
—
Y
—
—
—
—
P
—
I
0.13
1.67
0.09
1836

Phage-76
I
E
—
W
—
D
W
E
P
N
S
F
—
D
0.09
1.66
0.08
1837

Phage-77
Y
D
—
—
—
—
L
—
—
—
I
H
—
Y
0.12
1.66
0.09
1838

Phage-78
I
—
—
—
—
—
—
—
—
—
T
I
—
N
0.08
1.63
0.08
1839

Phage-79
I
—
—
—
—
—
V
E
—
A
Y
L
—
Y
0.09
1.62
0.10
1840

Phage-80
F
D
—
—
—
D
G
—
—
T
—
Y
—
D
0.09
1.61
0.08
1841

Phage-81
I
D
—
—
—
D
G
—
—
T
I
S
—
Y
0.08
1.57
0.11
1842

Phage-82
N
—
—
—
—
—
—
—
—
—
S
T
—
L
0.10
1.55
0.11
1843

Phage-83
Y
D
—
—
—
D
G
—
—
—
Y
F
—
D
0.08
1.53
0.08
1844

Phage-84
N
F
—
W
—
D
W
E
Y
F
N
D
—
N
0.09
1.53
0.09
1845

Phage-85
—
L
—
W
—
D
W
E
A
F
F
D
—
D
0.07
1.47
0.07
1846

Phage-86
I
—
—
—
—
—
W
E
W
P
—
A
—
N
0.16
1.47
0.10
1847

Phage-87
—
F
—
W
—
D
W
E
D
N
F
F
—
N
0.08
1.46
0.10
1848

Phage-88
—
V
—
W
—
D
W
E
T
F
F
P
—
D
0.08
1.46
0.08
1849

Phage-89
D
N
—
—
—
D
G
—
—
T
Y
I
—
N
0.10
1.45
0.09
1850

Phage-90
D
N
—
W
—
D
W
E
Y
N
F
F
—
V
0.07
1.45
0.08
1851

Phage-91
F
—
—
—
—
—
V
E
—
D
Y
L
—
I
0.10
1.43
0.10
1852

Phage-92
D
N
—
W
—
D
W
E
Y
D
I
F
—
V
0.07
1.43
0.07
1853

Phage-93
I
D
—
—
—
—
—
—
—
—
I
A
—
P
0.08
1.42
0.08
1854

Phage-94
Y
F
—
—
—
—
V
E
—
Y
T
L
—
F
0.10
1.42
0.10
1855

Phage-95
F
—
—
—
—
—
—
—
—
—
A
P
—
N
0.06
1.37
0.08
1856

Phage-96
F
D
—
—
—
—
V
E
—
Y
F
Y
—
A
0.11
1.36
0.08
1857

Phage-97
D
F
—
W
—
D
W
E
D
F
F
F
—
A
0.18
1.35
0.12
1858

Phage-98
F
F
—
—
—
D
G
—
—
T
L
S
—
N
0.08
1.35
0.09
1859

Phage-99
F
I
—
—
—
—
—
—
—
—
—
A
—
L
0.14
1.35
0.09
1860

Phage-
Y
D
—
—
—
—
—
—
—
A
I
—
—
Y
0.09
1.32
0.10
1861

100

Phage-
Y
I
—
W
—
D
W
E
—
Y
L
Y
—
P
0.10
1.32
0.15
1862

101

Phage-
F
D
—
W
—
D
W
E
—
P
T
T
—
H
0.08
1.31
0.08
1863

102

Phage-
Y
D
—
W
—
D
W
E
D
F
P
I
—
D
0.14
1.31
0.10
1864

103

Phage-
—
V
—
W
—
D
W
E
Y
I
D
D
—
S
0.08
1.30
0.07
1865

104

Phage-
I
N
—
W
—
D
W
E
V
I
S
F
—
D
0.12
1.30
0.08
1866

105

Phage-
L
S
—
W
—
D
W
E
—
V
T
P
—
L
0.10
1.29
0.10
1867

106

Phage-
F
A
—
W
—
D
W
E
—
V
D
I
—
Y
0.09
1,28
0.08
1868

107

Phage-
Y
D
—
—
—
—
M
—
—
—
I
V
—
D
0.10
1.25
0.08
1869

108

Phage-
Y
D
—
W
—
D
W
E
V
F
I
V
—
D
0.06
1.25
0.07
1870

109

Phage-
D
N
—
W
—
D
W
E
H
N
F
F
—
V
0.10
1.25
0.08
1871

110

Phage-
Y
D
—
—
—
D
G
—
—
—
I
Y
—
P
0.07
1.23
0.08
1872

111

Phage-
Y
D
—
—
—
—
—
E
F
P
Y
Y
—
F
0.12
1.23
0.12
1873

112

Phage-
A
D
—
—
—
—
Y
—
—
—
—
P
—
V
0.11
1.22
0.09
1874

113

Phage-
F
L
—
—
—
—
V
E
—
V
H
Y
—
S
0.08
1.22
0.10
1875

114

Phage-
T
D
—
W
—
D
W
E
Y
I
T
S
—
S
0.08
1.22
0.08
1876

115

Phage-
A
F
—
—
—
—
L
—
—
—
I
T
—
D
0.09
1.21
0.09
1877

116

Phage-
N
D
—
W
—
D
W
E
—
Y
F
S
—
Y
0.09
1.19
0.09
1878

117

Phage-
F
D
—
—
—
—
W
E
T
V
T
D
—
Y
0.08
1.19
0.09
1879

118

Phage-
N
L
—
—
—
—
M
—
—
—
I
I
—
P
0.13
1.19
0.11
1880

119

Phage-
D
L
—
—
—
—
M
—
—
—
I
Y
—
D
0.10
1.19
0.14
1881

120

Phage-
F
D
—
—
—
D
G
V
—
D
Y
I
—
D
0.09
1.18
0.09
1882

121

Phage-
Y
A
—
W
—
D
W
E
—
D
F
A
—
Y
0.11
1.18
0.08
1883

122

Phage-
H
D
—
—
—
—
M
—
—
—
I
V
—
V
0.10
1.17
0.10
1884

123

Phage-
—
F
—
—
—
—
—
E
F
I
F
L
—
A
0.07
1.17
0.08
1885

124

Phage-
Y
D
—
—
—
—
L
—
—
—
I
L
—
D
0.08
1.16
0.09
1886

125

Phage-
S
V
—
W
—
D
W
E
—
F
Y
S
—
D
0.11
1.16
0.10
1887

126

Phage-
P
—
—
—
—
D
G
—
—
T
A
I
—
T
0.13
1.16
0.10
1888

127

Phage-
D
D
—
—
—
—
L
E
W
Y
Y
P
—
Y
0.09
1.16
0.08
1889

128

Phage-
F
I
—
—
—
—
—
—
—
—
L
P
—
N
0.08
1.14
0.09
1890

129

Phage-
T
D
—
—
—
—
—
—
—
—
L
P
—
D
0.11
1.14
0.33
1891

130

Phage-
F
L
—
—
—
—
—
E
—
D
A
P
—
Y
0.08
1.13
0.08
1892

131

Phage-
I
F
—
—
—
D
G
—
—
T
H
I
—
H
0.10
1.13
0.07
1893

132

Phage-
—
F
—
W
—
D
W
E
Y
I
D
F
—
N
0.10
1.11
0.21
1894

133

Phage-
I
F
—
—
—
—
Y
—
—
—
L
H
—
I
0.12
1.11
0.11
1895

134

Phage-
H
L
—
W
—
D
W
E
W
Y
—
D
—
P
0.08
1.11
0.10
1896

135

Phage-
F
I
—
—
—
—
M
—
—
—
I
A
—
N
0.08
1.11
0.09
1897

136

Phage-
I
F
—
—
—
—
V
E
M
I
F
L
—
N
0.09
1.10
0.08
1898

137

Phage-
Y
D
—
—
—
—
W
E
F
P
—
D
—
I
0.11
1.09
0.11
1899

138

Phage-
N
L
—
—
—
—
L
—
—
—
I
T
—
F
0.10
1.09
0.08
1900

139

Phage-
F
—
—
—
—
—
V
E
D
F
Y
F
—
Y
0.08
1.09
0.08
1901

140

Phage-
D
—
—
—
—
—
—
—
—
—
L
—
—
N
0.11
1.07
0.11
1902

141

Phage-
D
—
—
—
—
—
—
—
—
—
L
P
—
D
0.08
1.07
0.08
1903

142

Phage-
A
I
—
—
—
—
L
—
—
—
I
A
—
P
0.09
1.07
0.09
1904

143

Phage-
—
I
—
—
—
—
V
E
D
Y
N
L
—
Y
0.08
1.07
0.09
1905

144

Phage-
H
T
—
W
—
D
W
E
D
Y
T
V
—
P
0.10
1.06
0.09
1906

145

Phage-
S
D
—
W
—
D
W
E
Y
F
Y
D
—
N
0.10
1.06
0.08
1907

146

Phage-
—
F
—
—
—
D
G
—
—
T
—
H
—
D
0.09
1.05
0.08
1908

147

Phage-
D
—
—
—
—
—
Y
—
—
—
—
H
—
I
0.09
1.05
0.08
1909

148

Phage-
A
D
—
—
—
D
G
—
—
—
I
I
—
H
0.07
1.05
0.08
1910

149

Phage-
F
—
—
—
—
—
L
—
—
—
L
T
—
V
0.10
1.05
0.08
1911

150

Phage-
I
L
—
—
—
—
V
E
—
D
Y
Y
—
Y
0.11
1.04
0.09
1912

151

Phage-
H
L
—
W
—
D
W
E
—
Y
H
S
—
D
0.09
1.04
0.09
1913

152

Phage-
I
F
—
W
—
D
W
E
D
Y
N
F
—
T
0.08
1.04
0.11
1914

153

Phage-
I
V
—
—
—
D
G
—
—
T
L
T
—
H
0.12
1.04
0.11
1915

154

Phage-
A
D
—
W
—
D
W
E
W
D
Y
T
—
D
0.12
1.03
0.11
1916

155

Phage-
I
T
—
—
—
—
—
—
—
—
T
T
—
N
0.20
1.02
0.21
1917

156

Phage-
Y
H
—
W
—
D
W
E
—
Y
T
S
—
D
0.20
1.02
0.09
1918

157

Phage-
N
—
—
—
—
—
V
E
—
Y
A
L
—
T
0.11
1.01
0.10
1919

158

Phage-
F
I
—
—
—
—
M
—
—
—
I
H
—
D
0.15
1.00
0.19
1920

159

Phage-
D
N
—
W
—
D
W
E
—
F
A
V
—
P
0.14
1.00
0.10
1921

160

Phage-
Y
D
—
—
—
—
L
—
—
T
—
V
—
D
0.10
1.00
0.09
1922

161

Phage-
Y
D
—
—
—
—
—
—
—
—
I
A
—
Y
0.08
0.99
0.08
1923

162

Phage-
I
D
—
W
—
D
W
E
Y
T
—
H
—
D
0.07
0.97
0.09
1924

163

Phage-
D
D
—
—
—
—
L
—
—
—
I
I
—
I
0.09
0.96
0.09
1925

164

Phage-
—
—
—
—
—
—
Y
—
—
—
S
F
—
F
0.09
0.91
0.08
1926

165

Phage-
F
N
—
W
—
D
W
E
D
P
Y
F
—
V
0.09
0.86
0.07
1927

166

Phage-
Y
D
—
—
—
—
Y
—
—
—
S
Y
—
S
0.08
0.82
0.07
1928

167

Phage-
—
A
—
W
—
D
W
E
Y
T
D
S
—
F
0.13
0.79
0.09
1929

168

Phage-
T
D
—
—
—
—
—
—
—
—
—
A
—
Y
0.10
0.77
0.09
1930

169

Phage-
T
D
—
W
—
D
W
E
F
Y
A
D
—
D
0.07
0.75
0.08
1931

170

Phage-
Y
D
—
—
—
—
L
—
—
—
—
I
—
H
0.09
0.69
0.09
1932

171

Phage-
S
D
—
—
—
D
G
—
—
—
I
I
—
T
0.07
0.69
0.07
1933

172

Phage-
Y
—
—
—
—
—
—
—
—
—
I
D
—
D
0.08
0.67
0.09
1934

173

Phage-
F
F
—
—
—
—
I
—
—
—
I
A
—
V
0.08
0.62
0.09
1935

174

Phage-
D
—
—
—
—
—
—
—
—
—
T
F
—
D
0.16
0.60
0.10
1936

175

Phage-
Y
D
—
—
—
—
W
E
W
P
I
D
—
V
0.10
0.59
0.10
1937

176

Phage-
F
—
—
—
—
—
T
E
L
F
S
F
—
Y
0.13
0.59
0.11
1938

177

Phage-
Y
—
—
—
—
—
V
—
—
—
I
T
—
P
0.15
0.42
0.11
1939

178

Phage-
I
L
—
—
—
—
—
—
—
—
I
N
—
N
0.09
0.37
0.25
1940

179

Phage-
—
V
—
—
—
A
M
G
Q
H
Y
L
—
D
0.08
0.09
0.08
1941

180

Phage-
—
V
—
—
T
K
M
G
—
H
Y
L
—
S
0.08
0.08
0.08
1942

181

Phage-
Y
D
—
W
—
D
W
E
Y
V
Y
A
—
Y
0.08
0.98
0.08
1943

182

Phage-
D
L
—
—
—
—
L
—
—
—
—
N
—
D
0.09
0.98
0.08
1944

183

Phage-
Y
—
—
—
—
—
—
—
—
—
T
V
—
Y
0.14
0.97
0.16
1945

184

Phage-
L
D
—
W
—
D
W
E
W
P
Y
S
—
N
0.08
0.96
0.09
1946

185

Phage-
F
I
—
W
—
D
W
E
D
D
F
F
—
Y
0.08
0.96
0.09
1947

186

Phage-
D
L
—
—
—
—
V
E
W
Y
F
F
—
N
0.11
0.95
0.10
1948

187

Phage-
Y
D
—
—
—
—
L
—
—
—
I
V
—
F
0.07
0.94
0.08
1949

188

Phage-
L
N
—
W
—
V
W
E
D
D
—
F
—
Y
0.09
0.92
0.09
1950

189

Phage-
F
N
—
W
—
D
W
E
D
P
N
F
—
V
0.09
0.91
0.09
1951

190

Phage-
—
I
—
W
—
D
W
E
D
D
Y
F
—
P
0.10
0.91
0.13
1952

191

Phage-
F
L
—
—
—
—
—
—
—
—
S
V
—
Y
0.10
0.91
0.08
1953

192

Phage-
Y
D
I
—
—
—
L
—
—
—
I
F
—
Y
0.10
0.91
0.09
1954

193

Phage-
H
L
—
—
—
D
G
—
—
—
F
T
—
F
0.11
0.90
0.10
1955

194

Phage-
Y
F
—
—
—
—
M
—
—
—
L
Y
—
I
0.08
0.90
0.08
1956

195

Phage-
Y
—
—
—
—
—
V
E
—
Y
A
N
—
Y
0.07
0.90
0.07
1957

196

Phage-
N
T
—
—
—
—
—
—
—
—
T
A
—
Y
0.16
0.90
0.58
1958

197

Phage-
I
D
—
W
—
D
W
E
—
A
F
N
—
Y
0.09
0.90
0.08
1959

198

Phage-
A
—
—
—
—
—
L
E
—
F
F
L
—
T
0.09
0.89
0.08
1960

199

Phage-
I
—
—
—
—
—
V
E
—
V
H
H
—
Y
0.08
0.89
0.08
1961

200

Phage-
F
F
—
—
—
—
—
—
—
—
—
A
—
D
0.10
0.89
0.11
1962

201

Phage-
Y
D
—
—
—
—
L
—
—
T
I
I
—
Amino acid position sequence
0.08
0.89
0.08
1963

202

Phage-
I
L
—
—
—
—
W
E
Y
P
L
D
—
S
0.09
0.89
0.10
1964

203

Phage-
F
I
—
—
—
—
—
—
—
—
T
—
—
N
0.09
0.88
0.10
1965

204

Phage-
F
—
—
—
—
—
L
—
—
—
—
S
—
D
0.17
0.88
0.15
1966

205

Phage-
H
L
—
—
—
—
L
—
—
—
—
T
—
F
0.10
0.87
0.10
1967

206

Phage-
L
I
—
—
—
—
V
E
D
Y
S
L
—
H
0.09
0.87
0.09
1968

207

Phage-
Y
F
—
—
—
—
M
—
—
—
—
Y
—
D
0.08
0.87
0.08
1969

208

Phage-
H
—
—
—
—
—
M
—
—
—
I
Y
—
I
0.13
0.87
0.09
1970

209

Phage-
F
D
—
—
—
—
L
—
—
—
I
N
—
D
0.08
0.87
0.09
1971

210

Phage-
Y
—
—
—
—
—
V
E
—
Y
I
Y
—
T
0.07
0.87
0.08
1972

211

Phage-
L
A
—
W
—
V
R
E
—
I
N
A
—
I
0.08
0.85
0.07
1973

212

Phage-
I
D
—
W
—
D
W
E
D
I
T
F
—
D
0.08
0.85
0.08
1974

213

Phage-
I
V
—
—
—
—
L
I
—
—
I
T
—
P
0.11
0.85
0.15
1975

214

Phage-
F
—
—
—
—
—
—
E
L
P
A
D
—
D
0.08
0.85
0.09
1976

215

Phage-
F
D
—
—
—
—
—
—
—
—
N
P
—
F
0.10
0.85
0.09
1977

216

Phage-
D
A
—
W
—
D
W
E
—
Y
S
S
—
D
0.10
0.83
0.10
1978

217

Phage-
D
H
—
W
—
D
W
E
P
N
Y
F
—
V
0.08
0.83
0.09
1979

218

Phage-
D
—
—
W
—
D
W
E
I
N
Y
I
—
F
0.09
0.83
0.10
1980

219

Phage-
I
—
—
W
—
D
W
E
Y
V
Y
A
—
N
0.10
0.82
0.09
1981

220

Phage-
D
F
—
—
—
—
V
E
—
D
Y
L
—
D
0.07
0.82
0.08
1982

221

Phage-
H
D
—
—
—
D
G
R
—
D
Y
D
—
A
0.11
0.82
0.09
1983

222

Phage-
L
A
—
W
—
D
W
E
D
D
Y
F
—
V
0.08
0.82
0.09
1984

223

Phage-
D
I
—
W
—
D
W
E
D
Y
L
P
—
V
0.10
0.82
0.10
1985

224

Phage-
I
L
—
—
—
—
I
E
V
Y
A
L
—
P
0.08
0.81
0.10
1986

225

Phage-
I
F
—
—
—
—
W
E
F
—
—
L
—
N
0.10
0.81
0.11
1987

226

Phage-
T
—
—
—
—
—
V
E
D
F
S
L
—
V
0.07
0.80
0.08
1988

227

Phage-
F
I
—
—
—
—
W
E
F
V
D
A
—
F
0.11
0.80
0.09
1989

228

Phage-
F
A
—
W
—
D
W
E
—
D
S
P
—
D
0.06
0.80
0.07
1990

229

Phage-
I
L
—
—
—
—
V
E
—
L
I
F
—
P
0.12
0.80
0.08
1991

230

Phage-
F
—
—
—
—
—
V
E
—
Y
I
Y
—
Y
0.08
0.80
0.08
1992

231

Phage-
D
S
—
—
—
—
L
—
—
—
I
I
—
D
0.10
0.79
0.09
1993

232

Phage-
F
L
—
—
—
D
G
—
—
T
S
V
—
D
0.11
0.79
0.08
1994

233

Phage-
F
N
—
W
—
N
G
E
P
T
Y
F
—
V
0.11
0.79
0.08
1995

234

Phage-
L
A
—
W
—
V
W
E
Y
P
—
T
—
I
0.09
0.78
0.09
1996

235

Phage-
D
—
—
—
—
—
V
E
—
D
—
Y
—
Y
0.09
0.78
0.09
1997

236

Phage-
I
T
—
W
—
D
W
E
—
Y
A
N
—
T
0.08
0.77
0.07
1998

237

Phage-
F
F
—
—
—
D
G
—
—
T
Y
S
—
I
0.15
0.77
0.13
1999

238

Phage-
T
D
—
W
—
D
W
E
Y
A
T
S
—
D
0.09
0.76
0.09
2000

239

Phage-
F
N
—
—
—
D
G
Y
—
D
Y
L
—
D
0.10
0.76
0.11
2001

240

Phage-
Y
D
—
W
—
D
W
E
V
D
F
H
—
P
0.11
0.76
0.08
2002

241

Phage-
N
I
—
W
—
D
W
E
D
D
S
F
—
F
0.08
0.76
0.08
2003

242

Phage-
A
T
—
—
—
—
—
—
—
—
I
—
—
S
0.13
0.75
0.09
2004

243

Phage-
S
—
—
—
—
—
—
—
—
—
T
F
—
D
0.10
0.74
0.09
2005

244

Phage-
P
I
—
—
—
—
Y
—
—
—
D
V
—
A
0.08
0.74
0.08
2006

245

Phage-
Y
—
—
—
—
D
G
—
—
Y
N
S
—
I
0.11
0.74
0.11
2007

246

Phage-
—
D
—
W
—
D
W
E
V
F
I
A
—
D
0.11
0.74
0.10
2008

247

Phage-
D
L
—
—
—
—
V
E
—
V
N
L
—
L
0.12
0.74
0.10
2009

248

Phage-
F
D
—
—
—
—
M
—
—
—
T
T
—
F
0.09
0.74
0.09
2010

249

Phage-
N
F
—
W
—
D
W
E
P
I
Y
F
—
T
0.13
0.74
0.14
2011

250

Phage-
—
D
—
—
—
D
G
—
—
—
F
F
—
L
0.08
0.73
0.08
2012

251

Phage-
—
D
—
—
—
D
G
—
—
T
A
F
—
I
0.10
0.73
0.09
2013

252

Phage-
N
I
—
—
—
—
M
—
—
—
L
V
—
I
0.10
0.73
0.09
2014

253

Phage-
I
I
—
—
—
—
—
—
—
—
F
F
—
F
0.08
0.73
0.09
2015

254

Phage-
N
F
—
—
—
—
Y
—
—
—
I
S
—
I
0.10
0.73
0.38
2016

255

Phage-
H
L
—
—
—
—
I
E
—
A
D
—
—
N
0.11
0.73
0.51
2017

256

Phage-
D
—
—
—
—
—
V
E
—
D
Y
L
—
D
0.08
0.72
0.09
2018

257

Phage-
D
—
—
—
—
—
L
—
—
—
I
N
—
D
0.11
0.72
0.10
2019

258

Phage-
F
—
—
—
—
—
L
—
—
—
L
F
—
V
0.09
0.72
0.08
2020

259

Phage-
P
D
—
—
—
—
W
E
F
Y
—
T
—
N
0.12
0.72
0.08
2021

260

Phage-
F
D
—
—
—
—
—
E
Y
I
Y
A
—
T
0.09
0.72
0.08
2022

261

Phage-
D
—
—
—
—
—
—
—
—
—
S
I
—
N
0.12
0.72
0.11
2023

262

Phage-
D
F
—
—
—
—
V
E
—
Y
I
F
—
F
0.08
0.72
0.07
2024

263

Phage-
P
V
—
W
—
D
W
E
Y
V
S
S
—
D
0.08
0.71
0.08
2025

264

Phage-
Y
I
—
—
—
—
R
—
—
—
N
L
—
L
0.09
0.71
0.09
2026

265

Phage-
Y
D
—
—
—
—
L
—
—
—
I
V
—
D
0.11
0.71
0.11
2027

266

Phage-
H
D
—
W
—
D
W
E
D
F
Y
F
—
V
0.09
0.71
0.08
2028

267

Phage-
H
—
—
—
—
—
Y
—
—
—
I
D
—
Y
0.12
0.71
0.10
2029

268

Phage-
L
F
—
—
—
—
M
P
—
D
I
F
—
N
0.08
0.71
0.08
2030

269

Phage-
H
D
—
—
—
—
L
E
F
H
Y
A
—
Y
0.10
0.71
0.12
2031

270

Phage-
D
F
—
—
—
—
L
—
—
—
—
N
—
F
0.08
0.70
0.08
2032

271

Phage-
Y
F
—
—
—
—
L
—
—
—
I
A
—
N
0.10
0.70
0.10
2033

272

Phage-
T
D
—
W
—
D
W
E
D
D
T
I
—
D
0.10
0.70
0.08
2034

273

Phage-
Y
D
—
—
—
—
L
—
—
—
I
Y
—
F
0.09
0.70
0.08
2035

274

Phage-
Y
—
—
W
—
D
W
W
—
Y
—
T
—
D
0.10
0.70
0.11
2036

275

Phage-
P
I
—
—
—
—
L
E
—
—
Y
L
—
N
0.13
0.69
0.52
2037

276

Phage-
F
D
—
—
—
—
—
—
—
—
I
V
—
Y
0.12
0.69
0.11
2038

277

Phage-
—
L
—
—
—
D
G
I
—
F
F
D
—
P
0.09
0.68
0.07
2039

278

Phage-
A
—
—
—
—
—
Y
—
—
—
L
T
—
V
0.07
0.68
0.07
2040

279

Phage-
D
F
—
—
—
—
L
—
—
—
I
I
—
A
0.14
0.68
0.14
2041

280

Phage-
Y
D
—
—
—
—
—
—
—
—
L
D
—
N
0.08
0.68
0.08
2042

281

Phage-
A
I
—
—
—
—
—
—
—
—
—
A
—
D
0.14
0.67
0.08
2043

282

Phage-
L
L
—
—
—
D
G
V
—
D
F
F
—
D
0.10
0.67
0.10
2044

283

Phage-
N
F
—
—
—
—
L
P
—
D
I
F
—
F
0.12
0.66
0.13
2045

284

Phage-
F
—
—
—
—
—
V
E
—
V
S
L
—
N
0.08
0.66
0.08
2046

285

Phage-
Y
D
—
—
—
D
G
Y
—
A
F
Y
—
H
0.12
0.66
0.10
2047

286

Phage-
N
F
—
—
—
—
T
E
F
D
Y
L
—
D
0.08
0.65
0.08
2048

287

Phage-
D
—
—
—
—
D
G
V
—
D
F
I
—
N
0.06
0.65
0.08
2049

288

Phage-
T
D
—
W
—
D
W
E
Y
I
Y
S
—
S
0.08
0.65
0.07
2050

289

Phage-
F
—
—
—
—
—
—
E
—
I
T
N
—
I
0.14
0.65
0.11
2051

290

Phage-
F
D
—
W
—
D
W
E
—
—
F
F
—
H
0.07
0.64
0.08
2052

291

Phage-
D
F
—
—
—
D
G
—
—
—
—
F
—
P
0.08
0.63
0.08
2053

292

Phage-
H
N
—
—
—
—
L
—
—
—
L
V
—
D
0.13
0.63
0.09
2054

293

Phage-
T
—
—
—
—
D
G
A
—
D
Y
T
—
D
0.07
0.63
0.07
2055

294

Phage-
F
D
—
—
—
—
—
E
F
P
—
I
—
F
0.08
0.62
0.08
2056

295

Phage-
Y
N
—
—
—
—
L
—
—
—
—
T
—
D
0.09
0.62
0.08
2057

296

Phage-
F
D
—
—
—
—
L
—
—
—
I
H
—
A
0.07
0.62
0.08
2058

297

Phage-
D
I
—
—
—
—
V
E
—
Y
F
L
—
F
0.15
0.61
0.10
2059

298

Phage-
F
D
—
—
—
—
V
—
—
—
L
T
—
F
0.10
0.61
0.09
2060

299

Phage-
F
D
—
—
—
—
I
E
—
F
H
L
—
F
0.08
0.61
0.08
2061

300

Phage-
A
—
—
—
—
—
L
—
—
—
I
I
—
D
0.12
0.61
0.10
2062

301

Phage-
Y
N
—
—
—
—
L
—
—
—
I
T
—
N
0.09
0.61
0.10
2063

302

Phage-
F
D
—
W
—
D
W
E
—
P
—
D
—
L
0.08
0.61
0.07
2064

303

Phage-
D
V
—
—
—
—
L
—
—
—
I
L
—
P
0.08
0.60
0.08
2065

304

Phage-
N
—
—
—
—
—
L
—
—
—
L
P
—
P
0.09
0.60
0.08
2066

305

Phage-
Y
—
—
W
—
D
W
E
Y
D
I
F
—
S
0.08
0.60
0.10
2067

306

Phage-
D
D
—
—
—
—
—
—
—
—
T
Y
—
N
0.08
0.60
0.09
2068

307

Phage-
F
—
—
—
—
—
—
E
—
V
F
H
—
Y
0.09
0.60
0.09
2069

308

Phage-
T
D
—
W
—
D
W
E
—
Y
F
L
—
D
0.07
0.60
0.09
2070

309

Phage-
—
—
—
—
—
—
W
E
—
—
Y
L
—
P
0.09
0.60
0.09
2071

310

Phage-
D
D
—
I
—
N
G
Y
A
T
F
I
—
Y
0.06
0.59
0.08
2072

311

Phage-
F
L
—
—
—
—
I
E
D
D
T
H
—
Y
0.16
0.59
0.38
2073

312

Phage-
F
A
—
W
—
D
W
E
—
T
I
P
—
H
0.08
0.59
0.08
2074

313

Phage-
—
L
—
—
—
—
—
—
—
—
Y
N
—
Y
0.10
0.58
0.08
2075

314

Phage-
Y
D
—
—
—
—
—
—
—
—
I
S
—
I
0.09
0.58
0.09
2076

315

Phage-
Y
D
—
—
—
—
—
—
—
—
—
N
—
Y
0.12
0.57
0.10
2077

316

Phage-
A
I
—
W
—
D
W
E
—
F
—
D
—
Y
0.10
0.57
0.09
2078

317

Phage-
L
T
—
W
—
V
R
E
—
I
F
A
—
D
0.07
0.57
0.08
2079

318

Phage-
—
L
—
—
—
—
—
—
—
—
Y
Y
—
N
0.09
0.57
0.09
2080

319

Phage-
N
V
—
—
—
—
Y
—
—
—
A
P
—
N
0.07
0.56
0.08
2081

320

Phage-
H
D
—
—
—
—
—
—
—
—
I
S
—
V
0.11
0.55
0.09
2082

321

Phage-
F
D
—
—
—
—
L
—
—
T
—
D
—
N
0.12
0.55
0.41
2083

322

Phage-
Y
F
—
—
—
—
V
E
—
H
F
Y
—
Y
0.09
0.55
0.08
2084

323

Phage-
D
—
—
—
—
—
L
—
—
—
I
I
—
H
0.09
0.54
0.09
2085

324

Phage-
D
D
—
—
—
—
V
P
—
D
I
T
—
Y
0.11
0.54
0.08
2086

325

Phage-
D
N
—
—
—
—
L
—
—
—
—
V
—
D
0.10
0.54
0.08
2087

326

Phage-
—
H
—
W
—
D
W
E
P
N
Y
V
—
D
0.10
0.54
0.08
2088

327

Phage-
D
—
—
—
—
—
L
—
—
—
L
F
—
L
0.09
0.53
0.09
2089

328

Phage-
D
D
—
—
—
—
L
—
—
—
—
V
—
A
0.08
0.53
0.11
2090

329

Phage-
A
A
—
—
—
—
L
—
—
—
I
V
—
D
0.13
0.53
0.09
2091

330

Phage-
D
F
—
—
—
—
—
E
—
I
N
N
—
F
0.14
0.52
0.48
2092

331

Phage-
Y
—
—
—
—
—
—
—
—
—
N
A
—
Y
0.08
0.52
0.07
2093

332

Phage-
—
L
—
—
—
—
—
—
—
—
N
S
—
Y
0.10
0.52
0.11
2094

333

Phage-
Y
D
—
—
—
—
—
—
—
—
I
D
—
D
0.09
0.52
0.09
2095

334

Phage-
D
S
—
—
—
—
—
E
F
Y
Y
V
—
F
0.11
0.52
0.14
2096

335

Phage-
Y
I
—
—
—
—
L
—
—
—
L
I
—
H
0.10
0.52
0.08
2097

336

Phage-
F
D
—
—
—
—
V
E
—
D
Y
F
—
Y
0.11
0.52
0.10
2098

337

Phage-
F
D
—
—
—
—
Y
—
—
—
L
Y
—
F
0.08
0.52
0.07
2099

338

Phage-
—
L
—
—
—
D
G
—
—
Y
S
F
—
H
0.10
0.51
0.10
2100

339

Phage-
I
P
—
—
—
—
M
—
—
—
—
V
—
N
0.12
0.51
0.09
2101

340

Phage-
I
I
—
—
—
D
G
Y
—
D
F
T
—
D
0.12
0.51
0.09
2102

341

Phage-
I
F
—
—
—
—
L
—
—
—
I
I
—
Y
0.11
0.51
0.08
2103

342

Phage-
—
D
—
—
—
—
—
—
—
—
Y
D
—
Y
0.18
0.51
0.18
2104

343

Phage-
L
S
—
—
—
—
M
—
—
—
L
Y
—
D
0.12
0.51
0.08
2105

344

Phage-
Y
D
—
W
—
D
W
E
Y
N
I
D
—
T
0.08
0.51
0.09
2106

345

Phage-
N
H
—
—
—
D
G
—
—
T
I
V
—
F
0.09
0.51
0.08
2107

346

Phage-
N
F
—
—
—
—
L
—
—
—
I
P
—
H
0.11
0.50
0.12
2108

347

Phage-
F
H
—
—
—
—
I
E
—
Y
A
L
—
D
0.08
0.50
0.09
2109

348

Phage-
—
—
—
—
—
—
V
E
D
Y
N
L
—
Y
0.07
0.50
0.08
2110

349

Phage-
—
—
—
—
—
D
G
—
—
L
A
N
—
Y
0.09
0.50
0.08
2111

350

Phage-
L
I
—
—
—
V
I
A
—
D
L
P
—
N
0.17
0.50
0.26
2112

351

Phage-
D
I
—
—
—
—
I
P
—
D
—
S
—
D
0.10
0.50
0.08
2113

352

Phage-
I
—
—
—
—
—
W
E
—
A
D
Y
—
D
0.11
0.50
0.45
2114

353

Phage-
I
D
—
W
—
D
W
E
D
D
S
I
—
Y
0.10
0.50
0.10
2115

354

Phage-
—
L
—
—
—
—
V
E
D
F
T
L
—
D
0.09
0.50
0.11
2116

355

Phage-
L
—
—
—
—
V
I
E
—
I
Y
Y
—
Y
0.09
0.49
0.09
2117

356

Phage-
F
F
—
—
—
—
—
E
V
H
S
D
—
N
0.14
0.49
0.38
2118

357

Phage-
N
D
—
—
—
—
V
E
L
V
S
D
—
N
0.10
0.49
0.08
2119

358

Phage-
D
L
—
—
—
—
L
—
—
—
T
V
—
D
0.09
0.49
0.08
2120

359

Phage-
I
P
—
—
—
—
V
E
D
Y
N
L
—
N
0.08
0.49
0.08
2121

360

Phage-
Y
—
—
—
—
—
L
E
W
P
—
V
—
N
0.10
0.49
0.10
2122

361

Phage-
Y
D
—
—
—
—
L
—
—
—
—
I
—
N
0.08
0.49
0.10
2123

362

Phage-
—
—
—
—
—
D
G
—
—
—
F
D
—
A
0.08
0.49
0.08
2124

363

Phage-
N
D
—
—
—
—
W
E
D
T
Y
F
—
L
0.08
0.49
0.10
2125

364

Phage-
P
—
—
—
—
—
M
E
—
L
S
N
—
S
0.13
0.48
0.16
2126

365

Phage-
D
D
—
—
—
—
—
E
V
I
S
D
—
Y
0.15
0.48
0.10
2127

366

Phage-
D
L
—
—
—
—
—
P
—
D
—
P
—
D
0.08
0.48
0.08
2128

367

Phage-
I
—
—
—
—
—
—
—
—
—
F
V
—
Y
0.11
0.48
0.10
2129

368

Phage-
A
—
—
—
—
—
Y
E
V
F
A
D
—
N
0.10
0.48
0.11
2130

369

Phage-
I
D
—
—
—
—
Y
—
—
—
—
D
—
L
0.09
0.48
0.09
2131

370

Phage-
H
I
—
W
—
D
W
E
—
F
H
D
—
N
0.07
0.48
0.08
2132

371

Phage-
Y
D
—
—
—
—
L
—
—
T
I
T
—
L
0.08
0.48
0.08
2133

372

Phage-
Y
L
—
—
—
—
L
—
—
T
I
L
—
N
0.09
0.48
0.08
2134

373

Phage-
F
F
—
—
—
—
—
E
—
A
F
L
—
F
0.10
0.48
0.14
2135

374

Phage-
I
L
—
—
—
—
L
—
—
—
F
T
—
A
0.07
0.47
0.08
2136

375

Phage-
F
H
—
—
—
—
V
E
L
Y
T
D
—
N
0.09
0.47
0.08
2137

376

Phage-
N
L
—
—
—
—
V
E
—
Y
N
F
—
Y
0.08
0.47
0.08
2138

377

Phage-
F
D
—
—
—
—
V
E
—
T
Y
Y
—
F
0.21
0.47
0.09
2139

378

Phage-
—
F
—
—
—
—
—
E
—
D
H
Y
—
Y
0.12
0.47
0.37
2140

379

Phage-
A
I
—
—
—
—
W
E
V
V
A
D
—
N
0.11
0.47
0.11
2141

380

Phage-
F
I
—
W
—
D
W
E
—
D
N
Y
—
N
0.12
0.47
0.25
2142

381

Phage-
I
—
—
—
—
—
—
—
—
—
F
I
—
D
0.08
0.47
0.10
2143

382

Phage-
N
L
—
—
—
—
V
E
D
V
Y
D
—
H
0.12
0.47
0.43
2144

383

Phage-
H
—
—
—
—
—
V
E
—
Y
H
N
—
N
0.09
0.47
0.09
2145

384

Phage-
D
I
—
—
—
—
Y
—
—
—
Y
S
—
T
0.09
0.47
0.09
2146

385

Phage-
D
—
—
—
—
—
L
—
—
T
L
I
—
A
0.13
0.46
0.10
2147

386

Phage-
I
A
—
—
—
—
M
P
—
D
I
D
—
Y
0.12
0.46
0.09
2148

387

Phage-
I
D
—
—
—
—
L
—
—
—
I
F
—
D
0.10
0.46
0.10
2149

388

Phage-
Y
F
—
—
—
D
V
E
—
D
F
A
—
D
0.11
0.46
0.11
2150

389

Phage-
Y
N
—
—
—
—
W
E
Y
A
I
L
—
D
0.12
0.46
0.34
2151

390

Phage-
I
—
—
—
—
—
V
E
D
Y
I
V
—
N
0.14
0.46
0.22
2152

391

Phage-
Y
D
—
—
—
—
I
—
—
—
T
P
—
A
0.07
0.46
0.07
2153

392

Phage-
D
T
—
W
—
D
W
E
H
I
Y
A
—
D
0.09
0.46
0.09
2154

393

Phage-
D
I
—
—
—
—
M
—
—
—
—
T
—
N
0.13
0.45
0.12
2155

394

Phage-
Y
D
—
W
—
D
W
E
R
Y
F
P
—
I
0.10
0.45
0.09
2156

395

Phage-
H
L
—
—
—
—
L
—
—
—
—
A
—
S
0.13
0.45
0.11
2157

396

Phage-
Y
D
—
—
—
D
G
—
—
T
T
I
—
A
0.09
0.45
0.09
2158

397

Phage-
Y
—
—
—
—
—
Y
E
D
V
L
D
—
F
0.07
0.45
0.08
2159

398

Phage-
D
F
—
—
—
—
M
—
—
T
I
S
—
D
0.14
0.45
0.10
2160

399

Phage-
I
L
—
—
—
—
L
—
I
—
L
V
—
D
0.08
0.44
0.07
2161

400

Phage-
L
I
—
—
—
—
W
E
V
I
T
N
—
D
0.12
0.44
0.40
2162

401

Phage-
Y
D
—
—
—
—
—
—
—
Y
F
—
—
P
0.09
0.44
0.09
2163

402

Phage-
A
L
—
—
—
—
V
E
V
Y
D
V
—
V
0.08
0.44
0.08
2164

403

Phage-
Y
H
—
W
—
D
W
E
D
V
N
F
—
Y
0.10
0.44
0.09
2165

404

Phage-
F
L
I
—
—
M
G
G
L
T
F
Y
—
Y
0.09
0.44
0.08
2166

405

Phage-
I
I
—
—
—
—
—
—
—
Y
—
—
—
F
0.09
0.43
0.19
2167

406

Phage-
F
F
—
—
—
—
M
—
—
—
—
H
—
F
0.11
0.43
0.09
2168

407

Phage-
A
F
—
—
—
—
—
—
—
—
L
F
—
A
0.09
0.43
0.09
2169

408

Phage-
N
—
—
—
—
D
G
—
—
T
N
I
—
D
0.08
0.43
0.08
2170

409

Phage-
Y
L
—
—
—
—
W
E
W
V
H
N
—
L
0.13
0.43
0.09
2171

410

Phage-
A
T
—
—
—
D
G
—
—
—
H
I
—
A
0.08
0.43
0.09
2172

411

Phage-
—
—
—
—
—
—
V
E
V
L
D
Y
—
D
0.07
0.42
0.08
2173

412

Phage-
I
H
—
—
—
—
W
E
F
Y
T
D
—
D
0.08
0.42
0.08
2174

413

Phage-
D
D
—
—
—
—
L
—
—
T
—
A
—
D
0.13
0.42
0.32
2175

414

Phage-
Y
L
—
—
—
—
—
—
—
—
I
D
—
N
0.11
0.42
0.17
2176

415

Phage-
L
L
—
—
—
—
V
E
D
V
F
A
—
Y
0.09
0.42
0.09
2177

416

Phage-
Y
D
—
—
—
—
L
—
—
—
L
H
—
D
0.08
0.42
0.08
2178

417

Phage-
F
D
—
—
—
—
L
—
—
T
—
N
—
Y
0.09
0.41
0.09
2179

418

Phage-
F
A
—
W
—
D
W
E
—
I
N
D
—
H
0.08
0.41
0.09
2180

419

Phage-
Y
—
—
—
—
—
Y
E
—
D
I
Y
—
N
0.09
0.41
0.09
2181

420

Phage-
N
V
—
—
—
—
V
E
D
Y
T
F
—
Y
0.09
0.40
0.08
2182

421

Phage-
A
—
—
—
—
—
L
E
—
Y
D
F
—
T
0.12
0.40
0.08
2183

422

Phage-
F
D
—
—
—
—
I
—
—
—
T
I
—
T
0.08
0.40
0.09
2184

423

Phage-
N
L
—
—
—
—
L
—
—
T
L
V
—
A
0.10
0.40
0.09
2185

424

Phage-
Y
S
—
W
—
D
W
E
—
Y
L
A
—
N
0.08
0.40
0.08
2186

425

Phage-
G
I
—
—
—
—
V
E
D
Y
N
Y
—
D
0.09
0.40
0.10
2187

426

Phage-
F
F
—
—
—
—
L
—
—
—
—
N
—
H
0.07
0.40
0.07
2188

427

Phage-
Y
—
—
—
—
—
Y
E
—
D
F
Y
—
F
0.11
0.40
0.09
2189

428

Phage-
L
—
—
—
—
—
Y
—
—
—
T
D
—
Y
0.11
0.40
0.10
2190

429

Phage-
D
—
—
—
—
—
V
E
—
D
F
L
—
Y
0.10
0.40
0.08
2191

430

Phage-
T
L
—
—
—
—
V
E
L
Y
I
F
—
D
0.08
0.40
0.09
2192

431

Phage-
F
—
—
—
—
—
—
E
Q
I
A
D
—
Y
0.11
0.40
0.09
2193

432

Phage-
D
D
—
—
—
—
V
E
—
Y
H
L
—
D
0.11
0.40
0.18
2194

433

Phage-
D
—
—
—
—
—
L
E
D
V
T
L
—
H
0.13
0.39
0.09
2195

434

Phage-
—
L
—
—
—
—
V
E
D
V
N
L
—
Y
0.09
0.39
0.08
2196

435

Phage-
D
I
—
—
—
—
L
—
—
T
I
D
—
Y
0.09
0.39
0.09
2197

436

Phage-
T
—
—
—
—
—
V
E
—
D
I
N
—
Y
0.08
0.39
0.08
2198

437

Phage-
I
V
—
W
—
D
W
E
—
Y
P
N
—
D
0.08
0.39
0.08
2199

438

Phage-
S
D
—
—
—
—
L
—
—
—
I
I
—
T
0.11
0.39
0.10
2200

439

Phage-
Y
D
—
—
—
—
L
P
—
D
Y
D
—
N
0.15
0.39
0.10
2201

440

Phage-
N
L
—
W
—
D
W
E
—
Y
Y
A
—
D
0.12
0.39
0.17
2202

441

Phage-
D
D
—
—
—
—
L
—
—
I
L
P
—
H
0.10
0.39
0.08
2203

442

Phage-
S
L
—
—
—
D
G
Q
—
D
Y
T
—
F
0.08
0.39
0.08
2204

443

Phage-
L
I
—
W
—
D
W
E
—
Y
N
F
—
T
0.13
0.39
0.11
2205

444

Phage-
F
H
—
—
—
D
G
—
—
T
—
P
—
I
0.08
0.39
0.08
2206

445

Phage-
F
D
—
—
—
—
W
E
W
I
Y
D
—
F
0.08
0.38
0.08
2207

446

Phage-
I
—
—
—
—
—
W
—
—
—
L
D
—
D
0.08
0.38
0.09
2208

447

Phage-
L
I
—
—
—
—
I
—
—
—
A
S
—
N
0.10
0.38
0.11
2209

448

Phage-
T
S
—
W
V
D
W
E
—
F
S
D
—
I
0.11
0.38
0.34
2210

449

Phage-
Y
—
—
—
—
—
V
E
—
D
Y
V
—
D
0.10
0.38
0.08
2211

450

Phage-
D
D
—
—
—
—
Q
E
F
I
Y
A
—
I
0.09
0.38
0.08
2212

451

Phage-
A
D
—
W
—
D
W
E
—
Y
A
D
—
Y
0.11
0.38
0.10
2213

452

Phage-
Y
—
—
—
—
—
—
—
—
—
I
H
—
I
0.08
0.38
0.07
2214

453

Phage-
S
E
—
W
—
D
W
E
P
F
F
D
—
N
0.08
0.37
0.09
2215

454

Phage-
Y
H
—
—
—
—
M
—
—
—
L
I
—
T
0.07
0.37
0.07
2216

455

Phage-
S
D
—
W
—
D
W
E
D
A
Y
F
—
I
0.09
0.37
0.07
2217

456

Phage-
—
D
—
—
—
—
V
E
—
Y
Y
H
—
D
0.08
0.37
0.08
2218

457

Phage-
D
N
—
—
—
—
Y
—
—
—
I
A
—
N
0.10
0.37
0.10
2219

458

Phage-
L
—
—
—
—
V
—
E
F
Y
D
Y
—
Y
0.08
0.37
0.10
2220

459

Phage-
Y
T
—
—
—
—
M
—
—
—
—
T
—
I
0.09
0.37
0.08
2221

460

Phage-
N
F
—
W
—
D
W
E
V
N
S
F
—
D
0.09
0.37
0.08
2222

461

Phage-
N
A
—
W
—
D
W
E
Y
I
D
F
—
N
0.12
0.36
0.09
2223

462

Phage-
Y
N
—
—
—
—
M
—
—
—
I
F
—
S
0.09
0.36
0.07
2224

463

Phage-
L
D
—
—
—
—
L
—
—
—
I
T
—
Y
0.08
0.36
0.09
2225

464

Phage-
H
—
—
—
—
—
—
—
—
—
I
N
—
D
0.11
0.36
0.08
2226

465

Phage-
I
I
—
—
—
—
L
P
—
D
Y
V
—
T
0.08
0.36
0.08
2227

466

Phage-
D
I
—
—
—
—
—
—
—
—
I
D
—
S
0.08
0.36
0.08
2228

467

Phage-
P
—
—
—
—
—
—
—
—
—
—
L
—
F
0.10
0.36
0.09
2229

468

Phage-
—
F
—
—
—
—
—
—
—
—
—
D
—
Y
0.07
0.36
0.08
2230

469

Phage-
Y
D
—
W
—
D
W
E
—
A
L
P
—
A
0.08
0.36
0.08
2231

470

Phage-
D
D
—
W
—
D
W
E
D
Y
—
F
—
F
0.10
0.36
0.10
2232

471

Phage-
H
F
—
—
—
—
W
E
L
F
S
D
—
Y
0.11
0.36
0.11
2233

472

Phage-
I
T
—
W
—
D
W
E
V
N
F
P
—
Y
0.07
0.35
0.07
2234

473

Phage-
P
D
—
—
—
—
L
—
—
—
I
T
—
N
0.20
0.35
0.16
2235

474

Phage-
N
L
—
W
—
D
W
E
A
F
F
P
—
Y
0.08
0.35
0.07
2236

475

Phage-
F
—
—
—
—
—
—
E
Y
I
R
D
—
Y
0.08
0.35
0.07
2237

476

Phage-
F
F
—
—
—
—
—
—
—
—
I
I
—
D
0.09
0.35
0.10
2238

477

Phage-
—
L
—
—
—
K
G
G
P
T
Y
N
—
S
0.08
0.35
0.10
2239

478

Phage-
L
A
—
W
—
V
W
E
—
P
G
H
—
D
0.11
0.35
0.10
2240

479

Phage-
D
—
—
—
—
—
V
E
D
V
N
D
—
Y
0.07
0.35
0.08
2241

480

Phage-
D
—
—
—
—
—
—
E
—
A
H
Y
—
N
0.08
0,35
0.07
2242

481

Phage-
—
L
—
—
—
—
L
—
—
T
L
T
—
T
0.08
0.35
0.07
2243

482

Phage-
Y
—
—
—
—
—
I
E
D
Y
N
L
—
N
0.10
0.34
0.09
2244

483

Phage-
Y
I
—
—
—
—
V
E
—
Y
Y
N
—
F
0.13
0.34
0.14
2245

484

Phage-
D
I
—
—
—
—
L
—
—
—
I
F
—
F
0.08
0.34
0.09
2246

485

Phage-
D
I
—
—
—
—
V
E
—
D
Y
L
—
Y
0.07
0.34
0.08
2247

486

Phage-
T
L
—
—
—
—
—
E
—
D
A
P
—
I
0.10
0.34
0.08
2248

487

Phage-
N
—
—
W
—
D
W
E
Y
I
N
S
—
V
0.14
0.34
0.09
2249

488

Phage-
N
D
—
—
—
—
V
E
—
Y
Y
Y
—
T
0.07
0.34
0.09
2250

489

Phage-
I
T
—
—
—
—
M
—
—
—
I
D
—
N
0.08
0.34
0.08
2251

490

Phage-
Y
—
—
—
—
—
M
A
—
D
L
I
—
D
0.10
0.34
0.29
2252

491

Phage-
I
D
—
—
—
—
L
—
—
—
I
V
—
T
0.11
0,33
0.09
2253

492

Phage-
H
T
—
W
—
D
W
E
W
D
—
Y
—
D
0.08
0.33
0.07
2254

493

Phage-
I
H
—
—
—
—
W
E
L
I
D
D
—
L
0.08
0.33
0.10
2255

494

Phage-
Y
T
—
—
—
—
L
—
—
—
I
T
—
T
0.08
0.33
0.08
2256

495

Phage-
F
H
—
—
—
—
V
E
—
T
—
Y
—
F
0.11
0.33
0.09
2257

496

Phage-
D
—
—
—
—
—
L
—
—
—
L
I
—
N
0.07
0.33
0.08
2258

497

Phage-
I
—
—
—
—
—
—
—
—
—
D
Y
—
I
0.08
0.33
0.10
2259

498

Phage-
D
L
—
—
—
—
I
E
—
D
L
V
—
T
0.09
0.33
0.09
2260

499

Phage-
N
I
—
—
—
—
L
Q
—
D
I
V
—
P
0.09
0.33
0.09
2261

500

Phage-
N
N
—
—
—
—
M
—
—
—
I
T
—
Y
0.08
0.33
0.08
2262

501

Phage-
H
T
—
—
—
—
L
—
—
—
I
V
—
V
0.08
0.33
0.08
2263

502

Phage-
Y
—
—
—
—
—
I
E
D
I
L
V
—
T
0.12
0.33
0.23
2264

503

Phage-
N
T
—
—
—
—
—
E
F
V
H
L
—
P
0.07
0.33
0.11
2265

504

Phage-
D
I
—
—
—
—
M
—
—
—
T
V
—
D
0.10
0.33
0.09
2266

505

Phage-
A
I
—
—
—
—
V
E
I
V
N
Y
—
Y
0.09
0.32
0.07
2267

506

Phage-
H
L
—
—
—
—
V
E
D
P
T
A
—
V
0.10
0.32
0.28
2268

507

Phage-
A
D
—
—
—
—
L
—
—
—
I
S
—
T
0.10
0.32
0.09
2269

508

Phage-
F
D
—
—
—
—
L
—
—
—
—
I
—
D
0.07
0.32
0.09
2270

509

Phage-
D
V
—
—
—
—
—
—
—
—
I
D
—
N
0.10
0.32
0.08
2271

510

Phage-
Y
L
—
—
—
—
V
E
—
I
S
I
—
F
0.08
0.32
0.07
2272

511

Phage-
S
A
—
—
—
—
—
—
—
—
L
H
—
V
0.10
0.31
0.30
2273

512

Phage-
H
L
—
W
—
D
W
E
—
D
S
A
—
N
0.08
0.31
0,08
2274

513

Phage-
H
T
—
W
—
D
W
E
Y
D
Y
D
—
F
0.10
0.31
0.08
2275

514

Phage-
Y
D
—
—
—
—
W
E
—
V
A
L
—
N
0.10
0.31
0.10
2276

515

Phage-
T
L
I
—
—
—
I
E
—
Y
I
V
—
Y
0.09
0.31
0.23
2277

516

Phage-
S
—
—
—
—
—
—
—
—
—
I
F
—
T
0.09
0.31
0.09
2278

517

Phage-
D
I
—
—
—
—
—
—
—
—
L
H
—
Y
0.08
0.31
0.08
2279

518

Phage-
L
F
—
—
—
—
—
E
—
A
Y
L
—
I
0.08
0.31
0.08
2280

519

Phage-
I
F
—
—
—
—
I
E
—
D
F
V
—
T
0.10
0.31
0.10
2281

520

Phage-
D
D
—
—
—
—
W
E
Y
Y
—
A
—
V
0.08
0.31
0.08
2282

521

Phage-
D
D
—
—
—
—
L
—
—
T
T
I
—
Y
0.10
0.31
0.09
2283

522

Phage-
A
S
—
—
—
—
L
—
—
—
I
A
—
D
0.10
0.31
0.09
2284

523

Phage-
Y
L
—
—
—
—
V
E
D
Y
D
Y
—
Y
0.08
0.31
0.09
2285

524

Phage-
D
L
—
—
—
—
W
E
—
T
I
F
—
A
0.12
0.30
0.09
2286

525

Phage-
D
F
—
—
—
D
G
E
—
F
Y
I
—
P
0.12
0.30
0.11
2287

526

Phage-
—
—
—
—
—
—
V
E
—
N
I
L
—
H
0.15
0.30
0.17
2288

527

Phage-
D
—
—
—
—
—
V
E
—
N
Y
F
—
F
0.12
0.30
0.21
2289

528

Phage-
N
D
—
W
—
D
W
Y
—
F
L
S
—
D
0.10
0.30
0.10
2290

529

Phage-
R
D
—
W
—
D
W
E
V
P
Y
F
—
D
0.08
0.30
0.09
2291

530

Phage-
N
L
—
—
—
—
V
E
—
A
—
Y
—
Y
0.10
0.30
0.13
2292

531

Phage-
F
D
—
W
—
D
G
E
L
N
Y
L
—
T
0.23
0.30
0.08
2293

532

Phage-
Y
—
—
—
—
—
V
E
D
V
N
L
—
I
0.18
0.30
0.10
2294

533

Phage-
D
N
—
—
—
—
Y
—
—
—
I
T
—
L
0.11
0.29
0.09
2295

534

Phage-
L
N
—
W
—
D
W
E
—
D
Y
S
—
N
0.09
0.29
0.09
2296

535

Phage-
P
T
—
—
—
—
V
E
—
L
L
S
—
N
0.12
0.29
0.26
2297

536

Phage-
D
H
—
—
—
—
V
E
L
I
F
Y
—
H
0.11
0.29
0.08
2298

537

Phage-
F
H
—
—
—
—
L
—
—
—
I
F
—
Y
0.08
0.29
0.08
2299

538

Phage-
F
D
—
—
—
—
L
E
—
T
—
V
—
P
0.16
0.29
0.16
2300

539

Phage-
D
F
—
—
—
—
L
—
—
—
L
P
—
A
0.08
0.29
0.08
2301

540

Phage-
D
S
—
—
—
—
L
—
—
—
I
Y
—
D
0.09
0.29
0.09
2302

541

Phage-
A
D
—
W
—
D
W
E
—
F
L
L
—
F
0.12
0.29
0.10
2303

542

Phage-
I
L
—
—
—
—
V
E
—
L
D
F
—
N
0.10
0.28
0.08
2304

543

Phage-
F
F
—
—
—
—
—
E
—
I
F
L
—
Y
0.09
0.28
0.07
2305

544

Phage-
Y
N
—
—
—
D
G
—
—
—
Y
D
—
H
0.07
0.28
0.07
2306

545

Phage-
D
N
—
—
—
—
L
—
—
T
I
T
—
F
0.11
0.28
0.11
2307

546

Phage-
H
N
—
—
—
D
G
—
—
A
F
I
—
N
0.09
0.28
0.23
2308

547

Phage-
D
—
—
—
—
—
V
E
—
D
L
V
—
P
0.10
0.28
0.23
2309

548

Phage-
D
—
—
—
—
—
L
—
—
—
L
N
—
F
0.08
0.28
0.07
2310

549

Phage-
D
—
—
—
—
—
—
Q
—
D
F
H
—
H
0.11
0.28
0.27
2311

550

Phage-
Y
D
—
—
—
—
W
E
F
T
D
D
—
I
0.10
0.28
0.18
2312

551

Phage-
D
I
—
—
—
—
Y
E
—
D
I
I
—
Y
0.14
0.28
0.19
2313

552

Phage-
F
D
—
—
—
—
L
—
—
T
—
P
—
P
0.11
0.28
0.08
2314

553

Phage-
N
—
—
—
—
—
L
—
—
T
S
V
—
D
0.09
0.28
0.26
2315

554

Phage-
Y
—
—
—
—
—
W
E
F
—
F
D
—
D
0.17
0.28
0.11
2316

555

Phage-
Y
A
—
—
—
—
L
—
—
—
—
T
—
D
0.09
0.28
0.08
2317

556

Phage-
F
L
—
—
—
—
V
E
Q
D
Y
F
—
V
0.08
0.28
0.10
2318

557

Phage-
D
N
—
—
—
—
—
—
—
—
—
R
—
D
0.09
0.27
0.26
2319

558

Phage-
N
D
—
—
—
D
G
I
—
T
—
D
—
Y
0.10
0.27
0.24
2320

559

Phage-
Y
F
—
—
—
—
V
E
D
Y
N
D
—
F
0.08
0.27
0.09
2321

560

Phage-
N
L
—
—
—
—
—
—
—
—
I
F
—
Y
0.10
0.27
0.07
2322

561

Phage-
I
D
—
W
—
D
W
E
—
Y
I
P
—
T
0.10
0.27
0.08
2323

562

Phage-
I
—
—
—
—
D
G
—
—
—
F
I
—
A
0.07
0.27
0.08
2324

563

Phage-
D
—
—
—
—
—
—
—
—
—
—
V
—
Y
0.08
0.27
0.07
2325

564

Phage-
—
F
—
—
—
—
W
E
D
I
T
D
—
D
0.13
0.27
0.09
2326

565

Phage-
L
D
—
—
—
—
V
—
—
T
F
T
—
H
0.08
0.26
0.08
2327

566

Phage-
D
D
—
—
—
—
Y
—
—
—
F
A
—
H
0.13
0.26
0.10
2328

567

Phage-
I
—
—
—
—
—
Y
Q
—
D
L
P
—
N
0.12
0.26
0.11
2329

568

Phage-
L
D
—
—
—
—
V
E
—
Y
N
Y
—
V
0.09
0.26
0.08
2330

569

Phage-
Y
V
—
—
—
—
—
—
—
—
S
A
—
N
0.14
0.26
0.08
2331

570

Phage-
T
P
—
—
—
—
L
E
—
A
I
—
—
Y
0.10
0.26
0.10
2332

571

Phage-
Y
F
—
—
—
—
—
—
—
—
A
D
—
N
0.08
0.26
0.08
2333

572

Phage-
D
D
—
—
—
—
—
E
—
D
I
I
—
D
0.12
0.26
0.25
2334

573

Phage-
L
—
—
—
—
V
V
E
—
L
N
H
—
N
0.08
0.26
0.09
2335

574

Phage-
—
I
—
—
—
D
G
E
—
L
I
A
—
A
0.09
0.26
0.27
2336

575

Phage-
F
A
—
W
—
D
W
Q
—
T
Y
V
—
N
0.09
0.25
0.08
2337

576

Phage-
Y
I
—
—
—
—
V
E
F
L
F
F
—
N
0.08
0.25
0.08
2338

577

Phage-
T
Q
—
—
—
K
G
E
P
T
Y
H
—
Y
0.12
0.25
0.12
2339

578

Phage-
N
—
—
—
—
—
V
E
—
Y
H
N
—
D
0.10
0.25
0.17
2340

579

Phage-
—
—
—
—
—
—
W
E
F
F
S
D
—
A
0.08
0.25
0.07
2341

580

Phage-
F
—
—
—
—
—
L
E
—
—
F
F
—
Y
0.10
0.25
0.22
2342

581

Phage-
D
—
—
—
=
—
I
E
—
N
F
Y
—
Y
0.13
0.25
0.09
2343

582

Phage-
Y
A
—
—
—
—
V
E
—
Y
—
Y
—
A
0.08
0.25
0.09
2344

583

Phage-
—
D
—
—
—
—
L
—
—
—
I
I
—
D
0.08
0.25
0.07
2345

584

Phage-
N
D
—
—
—
—
M
—
—
—
I
A
—
Y
0.07
0.25
0.07
2346

585

Phage-
—
—
—
—
—
—
—
—
—
Y
L
A
—
A
0.09
0.25
0.21
2347

586

Phage-
—
I
—
—
—

—
—
—
—
A
N
—
D
0.08
0.25
0.09
2348

587

Phage-
L
T
—
W
—
D
W
E
—
D
F
F
—
N
0.07
0.24
0.07
2349

588

Phage-
F
—
—
—
—
—
Q
E
—
I
N
Y
—
Y
0.10
0.24
0.23
2350

589

Phage-
T
—
—
—
—
—
L
E
—
F
F
L
—
Y
0.13
0.24
0.08
2351

590

Phage-
P
D
—
—
—
—
L
—
—
—
—
A
—
H
0.12
0.24
0.09
2352

591

Phage-
N
D
—
—
—
—
—
E
—
I
I
F
—
V
0.09
0.24
0.24
2353

592

Phage-
Y
I
—
—
—
—
—
—
—
—
F
Y
—
N
0.25
0.24
0.08
2354

593

Phage-
H
A
—
—
—
—
L
—
—
—
L
L
—
N
0.20
0.24
0.07
2355

594

Phage-
Y
—
—
—
—
—
W
E
—
A
—
L
—
A
0.09
0.24
0.21
2356

595

Phage-
A
F
—
—
—
—
V
E
—
Y
D
L
—
N
0.10
0.24
0.16
2357

596

Phage-
Y
N
—
—
—
—
—
—
—
—
—
A
—
S
0.15
0.23
0.09
2358

597

Phage-
Y
L
—
—
—
—
V
E
D
D
T
L
—
A
0.08
0.23
0.09
2359

598

Phage-
A
V
—
—
—
—
—
—
—
—
—
N
—
D
0.08
0.23
0.08
2360

599

Phage-
N
—
—
—
—
—
W
E
V
Y
S
L
—
P
0.13
0.23
0.08
2361

600

Phage-
D
F
—
—
—
—
V
E
—
D
T
Y
—
H
0.07
0.23
0.07
2362

601

Phage-
I
S
—
—
—
—
Y
E
W
D
Y
A
—
N
0.08
0.23
0.10
2363

602

Phage-
—
N
—
—
—
—
L
—
—
—
I
I
—
Y
0.08
0.23
0.08
2364

603

Phage-
Y
D
—
—
—
—
—
—
—
T
A
P
—
Y
0.07
0.23
0.08
2365

604

Phage-
Y
L
—
—
—
—
—
E
—
N
F
L
—
T
0.09
0.23
0.22
2366

605

Phage-
F
D
—
—
—
—
V
—
—
—
—
D
—
A
0.08
0.22
0.09
2367

606

Phage-
Y
D
—
—
—
—
Q
E
—
I
S
F
—
N
0.09
0.22
0.09
2368

607

Phage-
I
D
—
—
—
—
I
E
L
Y
D
D
—
F
0.09
0.22
0.09
2369

608

Phage-
T
F
—
—
—
—
L
—
—
—
—
Y
—
Y
0.08
0.22
0.07
2370

609

Phage-
F
F
—
—
—
—
I
—
—
—
N
A
—
V
0.09
0.22
0.07
2371

610

Phage-
Y
H
—
W
—
D
W
E
P
I
Y
I
—
I
0.12
0.22
0.10
2372

611

Phage-
A
I
—
—
—
—
Y
E
—
D
H
Y
—
Y
0.08
0.22
0.08
2373

612

Phage-
P
L
—
—
—
D
G
F
—
N
Y
N
—
F
0.12
0.22
0.08
2374

613

Phage-
F
P
—
W
—
D
W
E
W
D
N
N
—
H
0.09
0.22
0.09
2375

614

Phage-
—
D
—
—
—
D
G
—
—
L
A
A
—
H
0.10
0.22
0.11
2376

615

Phage-
—
D
—
W
—
D
W
E
—
Y
Y
S
—
D
0.08
0.22
0.07
2377

616

Phage-
—
—
—
—
—
—
Y
—
—
—
Y
D
—
T
0.07
0.21
0.10
2378

617

Phage-
N
L
—
—
—
—
W
E
N
F
A
D
—
F
0.08
0.21
0.08
2379

618

Phage-
Y
L
—
—
—
—
L
E
V
F
F
V
—
D
0.12
0.21
0.10
2380

619

Phage-
I
F
—
—
—
—
L
E
D
Y
S
I
—
F
0.09
0.21
0.08
2381

620

Phage-
D
—
—
—
—
—
L
E
Q
Y
D
L
—
F
0.09
0.21
0.08
2382

621

Phage-
L
L
—
—
—
V
N
E
D
P
L
D
—
Y
0.11
0.21
0.13
2383

622

Phage-
I
D
—
—
—
—
—
—
—
—
—
F
—
Y
0.08
0.21
0.08
2384

623

Phage-
I
I
—
—
—
—
V
E
—
I
D
I
—
S
0.08
0.21
0.08
2385

624

Phage-
I
A
—
W
—
D
W
E
D
Y
S
S
—
P
0.08
0.21
0.11
2386

625

Phage-
Y
—
—
—
—
—
V
E
D
I
N
D
—
I
0.09
0.21
0.07
2387

626

Phage-
N
I
—
—
—
—
M
—
—
—
I
D
—
I
0.08
0.21
0.07
2388

627

Phage-
F
D
—
W
—
D
W
E
—
L
—
S
—
Y
0.07
0.21
0.08
2389

628

Phage-
Y
F
I
—
—
—
W
E
D
H
F
F
—
D
0.09
0.21
0.19
2390

629

Phage-
T
—
—
—
—
—
—
E
—
D
S
Y
—
D
0.12
0.20
0.09
2391

630

Phage-
N
L
—
—
—
—
V
E
L
I
D
I
—
S
0.11
0.20
0.09
2392

631

Phage-
D
N
—
—
—
—
W
E
—
V
Y
L
—
N
0.08
0.20
0.08
2393

632

Phage-
F
L
—
—
—
—
—
—
—
—
D
L
—
F
0.08
0.20
0.09
2394

633

Phage-
H
I
—
—
—
—
Q
—
—
—
I
—
—
T
0.09
0.20
0.19
2395

634

Phage-
F
D
—
W
—
D
W
E
D
N
S
Y
—
D
0.10
0.20
0.09
2396

635

Phage-
T
A
—
—
—
—
W
E
F
D
F
N
—
D
0.08
0.20
0.07
2397

636

Phage-
H
H
—
W
—
D
W
E
D
Y
S
T
—
P
0.10
0.20
0.11
2398

637

Phage-
Y
—
—
—
—
—
—
—
—
—
—
N
—
F
0.07
0.20
0.08
2399

638

Phage-
L
H
—
W
—
D
W
E
—
I
D
I
—
D
0.08
0.20
0.09
2400

639

Phage-
D
I
—
—
—
D
G
Q
—
D
F
V
—
S
0.08
0.20
0.09
2401

640

Phage-
D
V
—
W
—
D
W
E
V
N
Y
F
—
D
0.09
0.20
0.07
2402

641

Phage-
—
N
—
—
—
—
M
—
—
—
I
D
—
A
0.12
0.20
0.10
2403

642

Phage-
D
N
—
—
—
—
—
—
—
A
T
V
—
N
0.11
0.19
0.19
2404

643

Phage-
D
L
—
—
—
—
—
E
—
V
H
N
—
N
0.08
0.19
0.08
2405

644

Phage-
—
N
—
—
—
—
—
—
—
—
S
Y
—
F
0.13
0.19
0.09
2406

645

Phage-
N
I
—
W
—
D
W
E
—
D
N
F
—
S
0.08
0.19
0.08
2407

646

Phage-
F
V
—
—
—
—
W
E
V
Y
D
D
—
D
0.08
0.19
0.08
2408

647

Phage-
A
—
—
—
—
—
L
E
V
V
H
L
—
V
0.10
0.19
0.17
2409

648

Phage-
P
F
—
—
—
—
M
—
—
T
I
D
—
Y
0.07
0.19
0.09
2410

649

Phage-
L
L
—
—
—
V
M
E
D
V
F
A
—
Y
0.08
0.19
0.08
2411

650

Phage-
D
L
—
—
—
—
—
—
—
—
T
N
—
Y
0.07
0.19
0.08
2412

651

Phage-
H
D
—
—
—
—
M
E
—
Y
Y
L
—
P
0.10
0.18
0.10
2413

652

Phage-
T
D
—
—
—
—
Y
—
—
—
I
I
—
P
0.08
0.18
0.09
2414

653

Phage-
—
L
—
W
—
D
W
E
D
Y
A
D
—
N
0.09
0.18
0.08
2415

654

Phage-
N
D
—
—
—
—
L
—
—
—
L
T
—
D
0.07
0.18
0.09
2416

655

Phage-
I
—
—
—
—
—
L
—
—
—
I
A
—
Y
0.11
0.18
0.08
2417

656

Phage-
N
—
—
—
—
—
V
E
—
F
N
F
—
H
0.11
0.18
0.14
2418

657

Phage-
D
V
—
—
—
—
I
E
—
Y
S
F
—
I
0.08
0.18
0.09
2419

658

Phage-
D
L
—
—
—
—
V
E
—
I
T
D
—
A
0.10
0.18
0.12
2420

659

Phage-
H
D
—
—
—
—
—
—
—
—
—
F
—
I
0.12
0.18
0.12
2421

660

Phage-
P
L
—
—
—
V
L
E
—
D
I
Y
—
Y
0.10
0.18
0.13
2422

661

Phage-
D
L
—
—
—
—
—
E
D
I
I
D
—
N
0.10
0.18
0.11
2423

662

Phage-
D
—
—
—
—
—
V
E
V
P
S
N
—
N
0.10
0.18
0.18
2424

663

Phage-
I
I
—
—
—
—
L
—
—
—
T
A
—
D
0.10
0.18
0.09
2425

664

Phage-
D
H
—
—
—
—
—
—
—
—
—
N
—
D
0.10
0.18
0.14
2426

665

Phage-
F
D
—
—
—
—
—
—
—
—
L
Y
—
S
0.07
0.18
0.07
2427

666

Phage-
F
A
—
W
—
D
W
E
—
V
Y
I
—
Y
0.08
0.18
0.08
2428

667

Phage-
L
—
—
—
—
—
—
—
—
—
L
D
—
S
0.08
0.18
0.09
2429

668

Phage-
D
L
—
—
—
—
L
E
—
A
F
L
—
A
0.09
0.18
0.08
2430

669

Phage-
F
A
—
—
—
—
L
—
—
T
L
T
—
L
0.10
0.18
0.08
2431

670

Phage-
F
D
—
—
—
—
V
E
—
I
S
N
—
D
0.17
0.18
0.10
2432

671

Phage-
—
H
—
—
—
—
L
E
Y
P
F
D
—
N
0.09
0.17
0.16
2433

672

Phage-
A
—
—
—
—
—
—
E
—
H
T
T
—
N
0.10
0.17
0.15
2434

673

Phage-
L
—
—
—
—
V
S
E
Q
F
T
F
—
I
0.08
0.17
0.08
2435

674

Phage-
D
—
—
—
—
—
L
—
—
—
Y
D
—
N
0.10
0.17
0.09
2436

675

Phage-
F
—
—
—
—
—
W
E
—
F
D
V
—
I
0.13
0.17
0.15
2437

676

Phage-
F
T
—
—
—
—
V
E
—
Y
D
H
—
I
0.08
0.17
0.09
2438

677

Phage-
Y
N
—
—
—
—
—
—
—
—
—
T
—
F
0.12
0.17
0.11
2439

678

Phage-
A
V
—
—
—
—
N
—
—
—
N
S
—
A
0.08
0.17
0.08
2440

679

Phage-
I
—
—
W
—
D
W
E
V
P
N
D
—
A
0.10
0.17
0.09
2441

680

Phage-
Y
F
—
—
—
—
—
E
—
F
F
H
—
Y
0.12
0.17
0.12
2442

681

Phage-
Y
V
—
—
—
D
G
—
—
—
S
F
—
D
0.12
0.17
0.12
2443

682

Phage-
I
S
—
—
—
—
V
E
—
F
F
Y
—
Y
0.10
0.17
0.08
2444

683

Phage-
L
I
—
—
—
V
—
E
—
D
—
Y
—
D
0.17
0.17
0.15
2445

684

Phage-
—
—
—
—
—
—
V
E
D
H
N
Y
—
A
0.14
0.17
0.16
2446

685

Phage-
L
D
—
—
—
—
—
E
F
V
Y
I
—
A
0.08
0.17
0.10
2447

686

Phage-
Y
D
—
—
—
—
—
E
—
D
L
P
—
I
0.17
0.17
0.11
2448

687

Phage-
D
V
—
—
—
—
V
E
—
D
Y
Y
—
D
0.10
0.17
0.14
2449

688

Phage-
N
D
—
W
—
D
W
E
Y
D
N
V
—
V
0.08
0.17
0.10
2450

689

Phage-
D
L
—
—
—
—
—
E
V
A
N
D
—
N
0.10
0.16
0.16
2451

690

Phage-
H
D
—
—
—
—
L
—
—
—
I
S
—
N
0.09
0.16
0.07
2452

691

Phage-
L
D
—
W
—
D
W
E
—
T
T
H
—
D
0.08
0.16
0.08
2453

692

Phage-
I
I
—
—
—
—
V
E
—
D
D
Y
—
L
0.09
0.16
0.09
2454

693

Phage-
Y
—
—
W
—
D
W
E
—
V
I
I
—
D
0.09
0.16
0.09
2455

694

Phage-
F
D
—
—
—
—
I
—
—
Y
T
N
—
N
0.12
0.16
0.09
2456

695

Phage-
I
T
—
—
—
—
L
—
—
T
I
N
—
D
0.08
0.16
0.11
2457

696

Phage-
D
S
—
—
—
—
V
E
—
D
I
Y
—
I
0.07
0.16
0.08
2458

697

Phage-
Y
L
—
—
—
—
—
—
—
—
G
N
—
H
0.07
0.16
0.08
2459

698

Phage-
D
N
—
—
—
—
L
P
—
D
Y
F
—
D
0.08
0.16
0.10
2460

699

Phage-
—
L
—
—
—
—
—
E
—
V
S
N
—
N
0.11
0.16
0.08
2461

700

Phage-
—
D
—
W
—
D
W
E
—
D
I
V
—
D
0.10
0.16
0.09
2462

701

Phage-
—
—
—
W
—
D
W
E
D
N
F
P
—
Y
0.07
0.16
0.07
2463

702

Phage-
D
—
—
—
—
—
V
E
—
H
F
N
—
H
0.08
0.16
0.08
2464

703

Phage-
A
D
—
—
—
—
I
E
—
D
A
Y
—
Y
0.12
0.16
0.09
2465

704

Phage-
I
L
—
W
—
D
W
E
D
A
T
F
—
Y
0.09
0.16
0.07
2466

705

Phage-
I
H
—
W
—
D
W
E
D
F
N
I
—
P
0.09
0.16
0.08
2467

706

Phage-
T
I
—
—
—
—
V
E
D
Y
N
D
—
I
0.07
0.16
0.07
2468

707

Phage-
D
D
—
—
—
—
L
—
—
—
—
A
—
I
0.08
0.16
0.08
2469

708

Phage-
D
D
—
W
—
D
W
E
D
H
I
F
—
F
0.13
0.16
0.08
2470

709

Phage-
—
N
—
—
—
—
V
E
—
I
I
F
—
D
0.12
0.15
0.12
2471

710

Phage-
I
F
—
W
—
D
W
E
D
D
T
V
—
I
0.08
0.15
0.09
2472

711

Phage-
I
I
—
—
—
—
—
E
—
I
S
D
—
L
0.12
0.15
0.15
2473

712

Phage-
F
D
—
—
—
—
V
E
—
Y
N
D
—
D
0.11
0.15
0.09
2474

713

Phage-
N
D
—
—
—
—
L
—
—
T
L
Y
—
I
0.08
0.15
0.10
2475

714

Phage-
A
I
—
—
—
—
L
E
—
D
I
S
—
N
0.12
0.15
0.17
2476

715

Phage-
H
L
—
—
—
—
—
—
—
—
T
N
—
Y
0.07
0.15
0.07
2477

716

Phage-
S
—
—
—
—
—
L
—
—
—
—
A
—
I
0.10
0.15
0.11
2478

717

Phage-
L
—
—
—
—
—
—
E
—
L
A
D
—
T
0.08
0.15
0.08
2479

718

Phage-
I
D
—
—
—
—
L
—
—
—
I
A
—
N
0.07
0.15
0.08
2480

719

Phage-
F
D
—
—
—
D
G
Q
—
D
L
V
—
N
0.10
0.15
0.08
2481

720

Phage-
N
L
—
—
—
—
—
E
—
F
F
D
—
Y
0.09
0.15
0.15
2482

721

Phage-
S
I
—
—
—
—
L
Q
—
D
I
V
—
P
0.09
0.14
0.14
2483

722

Phage-
N
P
—
—
—
—
Y
—
—
—
A
H
—
D
0.08
0.14
0.08
2484

723

Phage-
Y
D
—
—
—
—
L
—
—
Y
Y
N
—
N
0.12
0.14
0.11
2485

724

Phage-
—
D
—
—
—
—
L
—
—
T
I
F
—
D
0.07
0.14
0.08
2486

725

Phage-
D
N
—
—
—
—
L
—
—
—
—
T
—
T
0.09
0.14
0.10
2487

726

Phage-
—
D
—
—
—
—
L
—
—
—
S
Y
—
D
0.10
0.14
0.13
2488

727

Phage-
Y
—
—
—
—
—
—
E
F
I
D
F
—
F
0.07
0.14
0.07
2489

728

Phage-
Y
D
—
W
—
D
W
E
V
I
T
Y
—
N
0.08
0.14
0.09
2490

729

Phage-
F
D
—
—
—
—
I
E
—
D
F
F
—
V
0.06
0.14
0.07
2491

730

Phage-
I
F
—
W
—
D
W
—
D
I
N
F
—
D
0.10
0.14
0.14
2492

731

Phage-
N
F
—
—
—
—
L
P
—
D
I
T
—
Y
0.37
0.14
0.09
2493

732

Phage-
S
L
—
—
—
—
—
E
—
Y
Y
H
—
L
0.09
0.14
0.07
2494

733

Phage-
A
S
—
—
—
—
L
—
—
—
L
D
—
L
0.12
0.14
0.13
2495

734

Phage-
S
F
—
—
—
—
R
E
W
D
L
A
—
Y
0.09
0.14
0.08
2496

735

Phage-
H
L
—
—
—
—
—
E
D
V
L
D
—
I
0.08
0.14
0.12
2497

736

Phage-
L
D
—
—
—
D
G
—
—
F
Y
Y
—
L
0.20
0.14
0.09
2498

737

Phage-
D
N
—
W
—
D
W
E
—
D
I
A
—
T
0.16
0.14
0.11
2499

738

Phage-
S
D
—
—
—
—
L
—
—
T
I
H
—
I
0.09
0.14
0.08
2500

739

Phage-
N
S
—
—
—
D
G
—
—
—
—
D
—
L
0.08
0.14
0.08
2501

740

Phage-
D
L
—
—
—
—
L
—
—
—
T
L
—
I
0.07
0.14
0.08
2502

741

Phage-
F
D
—
—
—
—
S
—
—
—
F
N
—
Y
0.09
0.14
0.11
2503

742

Phage-
D
L
—
—
—
—
—
E
—
D
D
I
—
Y
0.12
0.14
0.13
2504

743

Phage-
H
A
—
—
—
—
—
E
—
D
T
Y
—
F
0.10
0.14
0.14
2505

744

Phage-
S
D
—
—
—
—
L
—
—
—
—
A
—
T
0.11
0.14
0.08
2506

745

Phage-
I
V
—
—
—
—
L
P
—
D
Y
N
—
Y
0.09
0.13
0.11
2507

746

Phage-
D
L
—
—
—
—
—
—
—
I
F
I
—
F
0.09
0.13
0.08
2508

747

Phage-
Y
D
—
—
—
—
—
—
—
T
L
T
—
N
0.13
0.13
0.08
2509

748

Phage-
Y
D
—
—
—
—
V
E
—
T
—
N
—
D
0.11
0.13
0.12
2510

749

Phage-
—
F
—
—
—
—
I
E
D
D
H
V
—
I
0.07
0.13
0.07
2511

750

Phage-
F
I
—
—
—
—
W
E
D
D
Y
A
—
S
0.12
0.13
0.12
2512

751

Phage-
N
F
—
—
—
—
—
—
—
—
—
—
—
N
0.10
0.13
0.09
2513

752

Phage-
N
L
—
—
—
—
V
E
—
I
L
I
—
D
0.07
0.13
0.07
2514

753

Phage-
D
S
—
—
—
—
V
E
—
Y
D
L
—
N
0.11
0.13
0.08
2515

754

Phage-
T
L
—
—
—
—
—
E
—
T
T
D
—
N
0.09
0.13
0.08
2516

755

Phage-
T
V
—
—
—
K
M
E
M
N
S
T
—
D
0.09
0.13
0.09
2517

756

Phage-
—
H
—
W
—
D
W
E
D
A
—
S
—
N
0.10
0.12
0.11
2518

757

Phage-
D
L
—
—
—
D
G
N
—
L
D
F
—
F
0.08
0.12
0.08
2519

758

Phage-
D
L
—
—
—
D
G
E
—
H
Y
Y
—
D
0.09
0.12
0.07
2520

759

Phage-
F
N
—
—
—
—
V
E
—
I
L
L
—
T
0.11
0.12
0.12
2521

760

Phage-
H
—
—
—
—
—
V
E
N
I
N
D
—
I
0.09
0.12
0.07
2522

761

Phage-
I
D
—
—
—
—
L
—
—
—
—
I
—
D
0.09
0.12
0.08
2523

762

Phage-
I
F
—
—
—
—
I
E
Q
P
A
L
—
Y
0.08
0.12
0.09
2524

763

Phage-
Y
D
—
—
—
—
—
Q
—
D
L
V
—
P
0.10
0.12
0.08
2525

764

Phage-
H
A
—
W
—
D
W
E
—
P
N
Y
—
D
0.13
0.12
0.09
2526

765

Phage-
N
V
—
W
—
D
W
E
—
D
Y
N
—
Y
0.11
0.12
0.10
2527

766

Phage-
S
F
—
—
Q
—
L
G
D
N
Y
D
—
I
0.09
0.12
0.12
2528

767

Phage-
D
D
—
—
—
—
L
—
—
T
T
V
—
Y
0.08
0.12
0.09
2529

768

Phage-
N
F
—
—
—
—
W
E
V
A
T
L
—
L
0.09
0.12
0.14
2530

769

Phage-
D
L
—
—
—
—
V
E
—
D
T
Y
—
N
0.09
0.11
0.07
2531

770

Phage-
A
L
—
—
—
—
V
E
Q
V
D
L
—
T
0.08
0.11
0.08
2532

771

Phage-
D
D
—
—
—
—
L
—
—
—
—
N
—
N
0.08
0.11
0.08
2533

772

Phage-
I
F
—
—
—
—
—
E
Q
I
I
Y
—
D
0.09
0.11
0.11
2534

773

Phage-
S
D
—
W
—
D
W
E
—
V
Y
Y
—
S
0.09
0.11
0.10
2535

774

Phage-
F
F
—
—
—
D
G
—
—
V
A
I
—
D
0.09
0.11
0.07
2536

775

Phage-
D
D
—
W
—
D
W
E
D
D
—
Y
—
Y
0.11
0.11
0.07
2537

776

Phage-
Y
D
—
—
—
—
—
—
—
T
—
V
—
P
0.08
0.11
0.08
2538

777

Phage-
S
—
—
—
—
—
L
—
—
—
—
N
—
V
0.10
0.11
0.08
2539

778

Phage-
A
F
—
V
S
F
Q
Q
S
L
P
H
—
D
0.09
0.11
0.10
2540

779

Phage-
—
N
—
—
—
—
V
E
—
Y
F
V
—
F
0.09
0.11
0.09
2541

780

Phage-
T
N
—
W
—
D
W
E
—
D
F
A
—
V
0.07
0.11
0.08
2542

781

Phage-
D
D
—
—
—
—
—
E
—
I
I
L
—
F
0.08
0.10
0.08
2543

782

Phage-
N
S
—
—
—
—
L
E
D
Y
H
L
—
P
0.08
0.10
0.10
2544

783

Phage-
I
H
—
—
—
D
S
—
G
F
D
F
—
D
0.09
0.10
0.08
2545

784

Phage-
F
D
—
—
—
—
L
E
—
D
H
L
—
F
0.08
0.10
0.08
2546

785

Phage-
T
V
—
W
—
D
—
E
—
Y
A
D
—
D
0.10
0.10
0.08
2547

786

Phage-
Y
F
—
W
—
D
W
E
—
A
A
D
—
L
0.09
0.10
0.09
2548

787

Phage-
—
S
—
—
—
—
—
—
—
—
—
D
—
I
0.08
0.10
0.07
2549

788

Phage-
A
V
—
—
—
—
L
P
—
D
I
V
—
Y
0.08
0.10
0.08
2550

789

Phage-
—
L
—
—
—
—
V
E
—
Y
H
L
—
A
0.08
0.10
0.30
2551

790

Phage-
S
L
—
W
—
D
W
E
—
V
D
N
—
F
0.12
0.10
0.11
2552

791

Phage-
T
I
—
—
—
D
G
Q
—
D
Y
N
—
H
0.07
0.10
0.07
2553

792

Phage-
F
L
—
—
—
—
G
E
P
T
Y
L
—
T
0.12
0.10
0.09
2554

793

Phage-
D
D
—
W
L
—
Q
H
D
I
Y
V
—
A
0.10
0.10
0.08
2555

794

Phage-
I
F
—
—
—
—
V
E
—
V
A
F
—
F
0.07
0.10
0.08
2556

795

Phage-
A
L
—
—
—
—
V
E
D
D
Y
D
—
L
0.09
0.10
0.09
2557

796

Phage-
D
D
—
—
—
—
I
E
L
Y
L
T
—
A
0.08
0.10
0.08
2558

797

Phage-
T
D
—
W
—
D
W
E
D
D
S
I
—
D
0.07
0.10
0.07
2559

798

Phage-
S
I
—
—
—
—
L
E
—
I
F
L
—
N
0.08
0.10
0.08
2560

799

Phage-
N
D
—
—
—
—
L
E
—
D
I
L
—
F
0.07
0.10
0.09
2561

800

Phage-
D
D
—
—
—
—
L
—
—
T
Y
S
—
Y
0.09
0.09
0.08
2562

801

Phage-
F
V
—
—
—
—
W
E
—
I
D
L
—
I
0.10
0.09
0.09
2563

802

Phage-
Y
D
—
—
—
—
L
—
—
—
L
S
—
P
0.07
0.09
0.07
2564

803

Phage-
N
L
—
—
—
—
L
—
—
—
I
I
—
P
0.08
0.09
0.08
2565

804

Phage-
I
D
—
W
—
D
W
E
—
F
N
N
—
F
0.08
0.09
0.09
2566

805

Phage-
A
—
—
—
—
—
L
E
—
H
D
Y
—
Y
0.08
0.09
0.09
2567

806

Phage-
D
D
—
S
—
Q
—
—
Q
I
D
L
—
D
0.14
0.09
0.09
2568

807

Phage-
S
—
—
—
—
—
S
—
—
—
I
Y
—
Y
0.08
0.09
0.07
2569

808

Phage-
S
L
—
—
—
—
—
Q
—
D
A
P
—
N
0.07
0.09
0.07
2570

809

Phage-
D
A
—
—
—
—
L
—
—
T
T
H
—
D
0.09
0.09
0.08
2571

810

Phage-
N
D
—
—
—

V
E
—
V
A
D
—
F
0.07
0.09
0.08
2572

811

Phage-
Y
I
—
—
—
—
—
E
Q
D
Y
F
—
F
0.08
0.09
0.08
2573

812

Phage-
T
D
—
—
—
D
G
—
—
T
N
Y
—
F
0.11
0.09
0.08
2574

813

Phage-
Y
D
—
—
—
—
V
—
—
—
—
L
—
P
0.08
0.09
0.08
2575

814

Phage-
I
D
—
—
—
—
—
—
—
—
A
Y
—
T
0.08
0.09
0.08
2576

815

Phage-
F
D
—
—
—
—
—
E
—
F
F
H
—
Y
0.09
0.09
0.09
2577

816

Phage-
F
P
—
—
—
—
I
E
—
Y
D
Y
—
V
0.09
0.09
0.08
2578

817

Phage-
A
D
—
—
—
I
—
E
S
I
D
I
—
V
0.09
0.09
0.07
2579

818

Phage-
I
S
—
—
—
D
L
W
P
T
D
I
—
T
0.10
0.09
0.10
2580

819

Phage-
D
D
—
—
—
D
G
—
—
V
H
T
—
N
0.09
0.09
0.07
2581

820

Phage-
I
D
—
W
—
D
W
E
G
—
F
A
—
N
0.09
0.09
0.08
2582

821

Phage-
L
N
—
—
—
D
G
—
—
T
F
Y
—
D
0.08
0.08
0.07
2583

822

Phage-
I
H
—
—
—
—
L
G
A
Y
I
S
—
S
0.08
0.08
0.09
2584

823

Phage-
T
I
—
W
—
D
W
E
—
D
Y
F
—
Y
0.08
0.08
0.08
2585

824

Phage-
H
S
—
L
A
Q
—
—
Q
D
L
V
—
I
0.07
0.08
0.07
2586

825

Phage-
N
N
—
A
S
D
L
S
—
D
N
S
—
I
0.07
0.08
0.08
2587

826

Phage-
—
N
—
W
—
D
W
E
—
D
—
A
—
N
0.09
0.08
0.07
2588

827

Phage-
—
—
—
—
—
—
L
—
—
—
F
Y
—
F
0.08
0.08
0.07
2589

828

Phage-
L
F
—
—
T
V
L
—
—
F
F
D
—
D
0.07
0.08
0.07
2590

829

Phage-
F
D
—
—
—
—
L
—
—
—
—
S
—
A
0.08
0.08
0.07
2591

830

Phage-
Y
—
—
—
—
—
V
E
—
N
—
Y
—
T
0.07
0.08
0.08
2592

831

Phage-
D
—
—
—
—
—
L
—
D
—
T
I
—
H
0.12
0.08
0.10
2593

832

Phage-
—
F
—
—
—
Q
W
A
—
A
N
A
—
F
0.09
0.08
0.07
2594

833

Phage-
F
T
—
—
—
—
Y
—
—
—
I
T
—
P
0.09
0.08
0.09
2595

834

Phage-
L
N
—
—
—
V
N
—
—
—
S
V
—
T
0.07
0.08
0.08
2596

835

Phage-
A
I
—
W
—
D
W
E
—
F
S
D
—
H
0.07
0.08
0.07
2597

836

Phage-
Y
—
—
—
V
D
L
G
A
N
—
Y
—
Y
0.10
0.08
0.09
2598

837

Phage-
H
—
—
—
—
—
V
E
—
D
Y
H
—
D
0.07
0.08
0.07
2599

838

Phage-
N
D
—
—
S
L
Q
Y
D
I
P
T
—
V
0.08
0.08
0.07
2600

839

Phage-
Y
V
—
R
—
Q
L
—
V
Y
H
Y
—
N
0.15
0.08
0.07
2601

840

Phage-
H
D
—
—
—
D
G
—
—
—
I
I
—
S
0.08
0.08
0.07
2602

841

Phage-
F
D
—
—
—
—
L
—
—
T
I
I
—
P
0.08
0.08
0.08
2603

842

Phage-
—
D
—
—
S
D
R
G
—
N
A
A
—
H
0.07
0.08
0.07
2604

843

Phage-
N
I
—
L
A
Q
—
N
—
D
P
T
—
N
0.07
0.08
0.08
2605

844

Phage-
T
N
—
—
S
K
S
Q
V
—
D
H
—
I
0.10
0.08
0.09
2606

845

Phage-
N
H
—
H
—
Q
—
W
—
L
T
N
—
N
0.09
0.07
0.07
2607

846

Phage-
L
L
—
H
—
Q
G
—
L
Y
H
L
—
H
0.09
0.07
0.08
2608

847

Phage-
T
N
—
D
S
K
L
E
G
D
D
N
—
F
0.09
0.07
0.07
2609

848

Phage-
N
D
—
—
—
—
M
—
—
—
L
L
—
D
0.08
0.07
0.07
2610

849

Phage-
F
H
—
—
—
—
V
—
—
—
I
N
—
N
0.07
0.07
0.07
2611

850

Phage-
D
F
—
—
—
D
G
—
—
T
Y
V
—
S
0.07
0.07
0.08
2612

851

Phage-
—
S
—
—
—
Q
—
—
—
N
N
T
—
N
0.07
0.07
0.08
2613

852

Phage-
—
—
—
H
L
—
S
E
Q
F
D
I
—
I
0.08
0.07
0.08
2614

853

Phage-
D
D
—
—
—
—
W
E
F
V
F
F
—
D
0.08
0.07
0.08
2615

854

Phage-
Y
N
—
E
Q
Q
Q
—
—
D
P
S
—
I
0.07
0.07
0.07
2616

855

Phage-
N
T
—
—
T
—
Q
H
—
F
N
—
—
L
0.08
0.07
0.08
2617

856

Phage-
H
P
—
Q
—
G
—
E
—
V
D
Y
—
V
0.08
0.07
0.08
2618

857

Phage-
—
A
—
S
R
Q
L
G
—
D
A
Y
—
N
0.07
0.07
0.07
2619

858

Phage-
D
I
—
—
A
Q
E
V
H
V
Y
T
—
P
0.07
0.07
0.07
2620

859

Phage-
F
F
—
E
G
N
L
—
A
Y
L
L
—
L
0.08
0.07
0.08
2621

860

Phage-
Y
—
—
—
—
D
G
E
—
N
I
V
—
D
0.07
0.07
0.07
2622

861

(—) indicates same amino acid as in CD3 scFv Peptide-B corresponding position (e.g. Phage-1 position).

TABLE 36

Sequences of those peptides selected for synthesis

(CD3 scFv Peptide-B Optimization)

SEQ ID

Peptide-ID
Sequence
NO:

Peptide-AA
DDCWPDWEFDFACA
229

Peptide-AB
YICGLDFPDFLYCD
230

Peptide-AC
FDCWPDWEEYFVCD
231

Peptide-AD
YICWPDWEEYFDCD
232

Peptide-AE
NICWPDWEDDYFCF
233

Peptide-AF
NFCWPDWEYIYPCI
234

Peptide-AG
VDCWPDWEEDFLCI
235

Peptide-AH
HACWPDWEEYFPCN
236

Peptide-AI
YDCGPDVDESYVCV
237

Peptide-AJ
IDCWPDWEDDTFCY
238

Peptide-AK
YLCGPDGDETLACY
239

Peptide-AL
VDCGPDGDESILCY
240

	Number	Date	Country
	63187719	May 2021	US
	63123327	Dec 2020	US

COMPOSITIONS AND METHODS RELATED TO TUMOR ACTIVATED ANTIBODIES TARGETING TROP2 AND EFFECTOR CELL ANTIGENS

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