WO2022240637A2 - Compositions and methods related to tumor activated antibodies targeting egfr and effector cell antigens - Google Patents

Compositions and methods related to tumor activated antibodies targeting egfr and effector cell antigens Download PDF

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Publication number
WO2022240637A2
WO2022240637A2 PCT/US2022/027688 US2022027688W WO2022240637A2 WO 2022240637 A2 WO2022240637 A2 WO 2022240637A2 US 2022027688 W US2022027688 W US 2022027688W WO 2022240637 A2 WO2022240637 A2 WO 2022240637A2
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Prior art keywords
polypeptide
seq
isolated polypeptide
complex
amino acid
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PCT/US2022/027688
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English (en)
French (fr)
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WO2022240637A3 (en
Inventor
David Campbell
Thomas R. DIRAIMONDO
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Janux Therapeutics Inc
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Janux Therapeutics Inc
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Priority to EP22808066.9A priority Critical patent/EP4337697A4/en
Priority to US18/559,201 priority patent/US12161724B2/en
Priority to KR1020237042777A priority patent/KR20240035392A/ko
Priority to JP2023570121A priority patent/JP2024518539A/ja
Priority to MX2023013382A priority patent/MX2023013382A/es
Priority to AU2022272906A priority patent/AU2022272906A1/en
Priority to BR112023023650A priority patent/BR112023023650A2/pt
Priority to CN202280049696.XA priority patent/CN117642156A/zh
Application filed by Janux Therapeutics Inc filed Critical Janux Therapeutics Inc
Priority to CA3218661A priority patent/CA3218661A1/en
Publication of WO2022240637A2 publication Critical patent/WO2022240637A2/en
Publication of WO2022240637A3 publication Critical patent/WO2022240637A3/en
Anticipated expiration legal-status Critical
Priority to US18/919,094 priority patent/US12433953B2/en
Priority to US19/327,021 priority patent/US20260014267A1/en
Ceased legal-status Critical Current

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    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
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    • A61K47/6871—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting an enzyme
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
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    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
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    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
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    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
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    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
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Definitions

  • polypeptides or polypeptide complexes according to Formula I are polypeptides or polypeptide complexes according to Formula I:
  • Ai comprises a first antigen recognizing molecule that binds to an effector cell antigen
  • Pi comprises a peptide that binds to Ai
  • Li comprises a linking moiety that connects Ai to Pi and is a substrate for a tumor specific protease
  • Hi comprises a half-life extending molecule
  • A comprises a second antigen recognizing molecule that binds to epidermal growth factor receptor (EGFR).
  • the first antigen recognizing molecule comprises an antibody or antibody fragment.
  • first antigen recognizing molecule comprises an antibody or antibody fragment that is human or humanized.
  • Li is bound to N-terminus of the first antigen recognizing molecule. In some embodiments, A is bound to C-terminus of the first antigen recognizing molecule. In some embodiments, Li is bound to C-terminus of the first antigen recognizing molecule. In some embodiments, A is 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, Ai is the single chain variable fragment (scFv). In some embodiments, the scFv comprises a scFv heavy chain polypeptide and a scFv light chain polypeptide.
  • Ai is the single domain antibody.
  • 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.
  • Ai comprises an anti-CD3e single chain variable fragment.
  • Ai comprises an anti-CD3e single chain variable fragment that has a K D binding of 1 mM or less to CD3 on CD3 expressing cells.
  • the effector cell antigen comprises CD3.
  • Ai comprises a variable light chain and variable heavy chain each of which is capable of specifically binding to human CD3.
  • Ai comprises 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, FI 11-409, CLB-T3.4.2, TR-66, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII-87, 12F6, T3/RW2-8C8, T3/RW2-4B6, OKT3D, M-T301, SMC2, F101.01, UCHT-1, WT-31, 15865, 15865vl2, 15865vl6, and 15865vl9.
  • CDRs complementary determining regions
  • the isolated polypeptide or polypeptide complex of Formula I binds to an effector cell when Li is cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex of Formula I binds to an effector cell when Li is cleaved by the tumor specific protease and Aibinds to the effector cell. In some embodiments, the effector cell is a T cell. In some embodiments, Ai binds to a polypeptide that is part of a TCR-CD3 complex on the effector cell. In some embodiments, the polypeptide that is part of the TCR-CD3 complex is human CD3s.
  • the effector cell antigen comprises CD3, wherein the scFv comprises CDRs: LC-CDR1, LC- CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the scFv LC-CDR1: SEQ ID NO: 1, LC-CDR2: SEQ ID NO: 2, and LC-CDR3: SEQ ID NO: 3; and the scFv comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC- CDR1, the HC-CDR2, and the HC-CDR3 of the scFv: HC-CDR1: SEQ ID NO: 4, HC-CDR2: SEQ ID NO: 5, and HC-CDR3: SEQ ID NO: 6.
  • CDRs complementarity determining regions
  • the effector cell antigen comprises CD3, and the scFv comprises an amino acid sequence according to SEQ ID NO: 13.
  • the effector cell antigen comprises CD3, wherein the scFv comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the scFv LC-CDR1: SEQ ID NO: 7, LC-CDR2: SEQ ID NO: 8, and LC-CDR3: SEQ ID NO: 9; and the scFv comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the scFv: HC-CDR1: SEQ ID NO: 10, HC-CDR2: SEQ ID NO: 11, and HC-CDR
  • the effector cell antigen comprises CD3, and the scFv comprises an amino acid sequence according to SEQ ID NO: 14.
  • second antigen recognizing molecule comprises an antibody or antibody fragment.
  • the antibody or antibody fragment thereof comprises a single chain variable fragment, a single domain antibody, or a Fab.
  • 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.
  • the antibody or antibody fragment thereof is humanized or human.
  • A2 is the Fab.
  • the Fab comprises (a) a Fab light chain polypeptide and (b) a Fab heavy chain polypeptide.
  • the Fab comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the Fab comprise LC-CDR1: SEQ ID NO: 15, LC-CDR2: SEQ ID NO: 16, and LC-CDR3: SEQ ID NO: 17; and the Fab comprises complementarity determining regions (CDRs): HC- CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the Fab comprise: HC-CDR1: SEQ ID NO: 18, HC-CDR2: SEQ ID NO: 19, and HC-CDR3: SEQ ID NO: 20.
  • the Fab light chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 21. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 22. In some embodiments, Fab heavy chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 23. In some embodiments, Fab heavy chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 24. In some embodiments, the Fab light chain polypeptide of A 2 is bound to a C-terminus of the single chain variable fragment (scFv) of Ai. In some embodiments, the Fab heavy chain polypeptide of A 2 is bound to a C-terminus of the single chain variable fragment (scFv) Ai.
  • the Fab light chain polypeptide of A 2 is bound to a N-terminus of the single chain variable fragment (scFv) of Ai. In some embodiments, the Fab heavy chain polypeptide of A 2 is bound to a N-terminus of the single chain variable fragment (scFv) Ai. In some embodiments, the Fab heavy chain polypeptide of A 2 is bound to the scFv heavy chain polypeptide of Ai. In some embodiments, the Fab light chain polypeptide of A 2 is bound to the scFv heavy chain polypeptide of Ai. In some embodiments, the Fab heavy chain polypeptide of A 2 is bound to the scFv light chain polypeptide of Ai.
  • the Fab light chain polypeptide of A 2 is bound to the scFv light chain polypeptide of Ai.
  • a 2 further comprises P 2 and L 2 , wherein P 2 comprises a peptide that binds to A 2 ; and L 2 comprises a linking moiety that connects A 2 to P 2 and is a substrate for a tumor specific protease.
  • the isolated polypeptide or polypeptide complex is according to Formula la:
  • the Fab heavy chain polypeptide of A 2 is bound to the scFv heavy chain polypeptide of Ai and L 2 is bound to the Fab light chain polypeptide of A 2 .
  • the Fab light chain polypeptide of A 2 is bound to the scFv heavy chain polypeptide of Ai and L 2 is bound to the Fab heavy chain polypeptide of A 2 .
  • the Fab heavy chain polypeptide of A 2 is bound to the scFv light chain polypeptide of Ai and L 2 is bound to the Fab light chain polypeptide of A 2 .
  • the Fab light chain polypeptide of A 2 is bound to the scFv light chain polypeptide of Ai and L 2 is bound to the Fab heavy chain polypeptide of A 2 .
  • Pi impairs binding of Ai to the effector cell antigen.
  • Pi is bound to Ai through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bonding interactions, or a combination thereof.
  • Pi has less than 70% sequence homology to the effector cell antigen.
  • P 2 impairs binding of A 2 to EGFR.
  • P 2 is bound to A 2 through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bonding interactions, or a combination thereof. In some embodiments, P 2 is bound to A 2 at or near an antigen binding site. In some embodiments, P 2 has less than 70% sequence homology to EGFR. In some embodiments, Pi or P 2 comprises a peptide sequence of at least 10 amino acids in length. In some embodiments, Pi or P 2 comprises a peptide sequence of at least 10 amino acids in length and no more than 20 amino acids in length. In some embodiments, Pi or P2 comprises a peptide sequence of at least 16 amino acids in length.
  • Pi or P2 comprises a peptide sequence of no more than 40 amino acids in length. In some embodiments, Pi or P2 comprises at least two cysteine amino acid residues. In some embodiments, Pi or P2 comprises a cyclic peptide or a linear peptide. In some embodiments, Pi or P2 comprises a cyclic peptide. In some embodiments, Pi or P2 comprises a linear peptide. In some embodiments, Pi comprises at least two cysteine amino acid residues. In some embodiments, Pi comprises an amino acid sequence according to SEQ ID NO: 25. In some embodiments, P2 comprises an amino acid sequence according to SEQ ID NO: 26. In some embodiments, P2 comprises an amino acid sequence according to SEQ ID NO: 115.
  • P2 comprises an amino acid sequence according to SEQ ID NO: 116.
  • Pi comprises an amino acid sequence according to SEQ ID NO: 70.
  • Li is bound to N-terminus of Ai. In some embodiments, Li is bound to C- terminus of Ai. In some embodiments, L2 is bound to N-terminus of A2. In some embodiments, L2 is bound to C-terminus of A2. In some embodiments, Li or L2 is a peptide sequence having at least 5 to no more than 50 amino acids. In some embodiments, Li or L2IS a peptide sequence having at least 10 to no more than 30 amino acids. In some embodiments, Li or L2IS a peptide sequence having at least 10 amino acids.
  • Li or L2 is a peptide sequence having at least 18 amino acids. In some embodiments, Li or L2 is a peptide sequence having at least 26 amino acids. In some embodiments, Li or L2has a formula comprising (G2S) n , wherein n is an integer from 1 to 3 (SEQ ID NO: 840).
  • Li has a formula selected from the group consisting of (G2S) n , (GS) n , (GSGGS) n (SEQ ID NO: 836), (GGGS) n (SEQ ID NO: 837) , (GGGGS) context (SEQ ID NO: 838) , and (GSSGGS)force (SEQ ID NO: 839) , wherein n is an integer of at least 1.
  • Pi becomes unbound from Ai when Li is cleaved by the tumor specific protease thereby exposing Ai to the effector cell antigen.
  • P2 becomes unbound from A2 when L2 is cleaved by the tumor specific protease thereby exposing A2 to EGFR.
  • the tumor specific protease is selected from the group consisting of a matrix metalloprotease (MMP), serine protease, cysteine protease, threonine protease, and aspartic protease.
  • MMP matrix metalloprotease
  • the matrix metalloprotease comprises MMP2, MMP7, MMP9, MMP13, or MMP14.
  • the serine protease comprises matriptase (MTSP1), urokinase, or hepsin.
  • Li or L2 comprises 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.
  • Li or L2 comprises an amino acid sequence according to SEQ ID NO: 30 or 31.
  • Li or L2 comprises an amino acid sequence according to any one of SEQ ID NOs: 27-35.
  • Li or L2 comprises an amino acid sequence of Linker 4 (ISSGLLSGRSDAG) (SEQ ID NO: 66), Linker 5 (AAGLLAPPGGLSGRSDAG) (SEQ ID NO: 67), Linker 6 (SPLGLSGRSDAG) (SEQ ID NO: 68), or Linker 7 (LSGRSDAGSPLGLAG) (SEQ ID NO: 69), or an amino acid sequence that has 1, 2, or 3 amino acid substitutions, additions, or deletions relative to the amino acid sequence of Linker 4, Linker 5, Linker 6, or Linker 7.
  • Hi comprises a polymer.
  • the polymer is polyethylene glycol (PEG).
  • Hi comprises albumin.
  • Hi comprises an Fc domain.
  • the albumin is serum albumin.
  • the albumin is human serum albumin.
  • Hi comprises a polypeptide, a ligand, or a small molecule.
  • 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.
  • 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.
  • the circulating immunoglobulin molecule comprises IgGl, IgG2, IgG3, IgG4, slgA, IgM or IgD.
  • the serum protein is albumin.
  • the polypeptide is an antibody.
  • the antibody comprises a single domain antibody, a single chain variable fragment, or a Fab.
  • the single domain antibody comprises a single domain antibody that binds to albumin.
  • the single domain antibody is a human or humanized antibody.
  • the single domain antibody is 645gHlgLl. In some embodiments, the single domain antibody is 645dsgH5gL4.
  • 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-l 1-15. In some embodiments, the single domain antibody is Alb-1, Alb-8, or Alb-23. In some embodiments, the single domain antibody is 10E.
  • the single domain antibody comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC- CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the single domain antibody comprise: HC-CDR1 : SEQ ID NO: 36, HC-CDR2: SEQ ID NO: 37, and HC-CDR3: SEQ ID NO: 38.
  • CDRs complementarity determining regions
  • the single domain antibody comprises complementarity determining regions (CDRs): HC- CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the single domain antibody comprise: HC-CDR1: SEQ ID NO: 39, HC-CDR2: SEQ ID NO: 40, and HC-CDR3: SEQ ID NO: 41.
  • the single domain antibody is SA21.
  • 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.
  • the modified amino acid or modified non-natural amino acid comprises a post-translational modification.
  • Hi comprises a linking moiety (L3) that connects Hi to Pi .
  • L3 is a peptide sequence having at least 5 to no more than 50 amino acids.
  • L 3 is a peptide sequence having at least 10 to no more than 30 amino acids.
  • L 3 is a peptide sequence having at least 10 amino acids.
  • L 3 is a peptide sequence having at least 18 amino acids.
  • L 3 is a peptide sequence having at least 26 amino acids.
  • L 3 has a formula selected from the group consisting of (G 2 S) n , (GS) n , (GSGGS) n (SEQ ID NO: 836), (GGGS) n (SEQ ID NO: 837) , (GGGGS) context (SEQ ID NO: 838) , and (GSSGGS)trust (SEQ ID NO: 839) , wherein n is an integer of at least 1.
  • L 3 comprises an amino acid sequence according to SEQ ID NO: 29.
  • the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to any one of SEQ ID NOs: 44-61.
  • the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 50. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 51. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 44 and SEQ ID NO: 45. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 46 and SEQ ID NO: 47.
  • the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 48 and SEQ ID NO: 49. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 50 and SEQ ID NO: 51. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 52 and SEQ ID NO: 53. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 54 and SEQ ID NO: 55.
  • the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 56 and SEQ ID NO: 57. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 58 and SEQ ID NO: 59. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 60 and SEQ ID NO: 61. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 62 and SEQ ID NO: 63. In some embodiments, the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 64 and SEQ ID NO: 65.
  • compositions comprising: (a) the isolated polypeptide or polypeptide complex described herein; and (b) a pharmaceutically acceptable excipient.
  • polypeptide or polypeptide complexes are described herein, in certain embodiments, are polypeptide or polypeptide complexes according to
  • Li a comprises a tumor specific protease-cleaved linking moiety that when uncleaved connects Pi a to a first antigen recognizing molecule that binds to an effector cell antigen and the first antigen recognizing molecule is connected to a second antigen recognizing molecule that binds to EGFR; Pi a comprises a peptide that binds to the first antigen recognizing molecule when Li a is uncleaved; and Hi a comprises a half-life extending molecule.
  • Pi a when Li a is uncleaved impairs binding of the first antigen recognizing molecule to the effector cell antigen.
  • the first antigen recognizing molecule comprises an antibody or antibody fragment.
  • the effector cell antigen is an anti-CD3 effector cell antigen.
  • Pi a has less than 70% sequence homology to the effector cell antigen.
  • Pi a comprises a peptide sequence of at least 10 amino acids in length.
  • Pi a comprises a peptide sequence of at least 10 amino acids in length and no more than 20 amino acids in length.
  • Pi a comprises a peptide sequence of at least 16 amino acids in length.
  • Pi a comprises a peptide sequence of no more than 40 amino acids in length. In some embodiments, Pi a comprises at least two cysteine amino acid residues. In some embodiments, Pi a comprises a cyclic peptide or a linear peptide. In some embodiments, Pi a comprises a cyclic peptide. In some embodiments, Pi a comprises a linear peptide. In some embodiments, Pi a comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 25. In some embodiments, Pi comprises an amino acid sequence according to SEQ ID NO: 70. In some embodiments, Hi a comprises a polymer. In some embodiments, the polymer is polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • Hi a comprises albumin. In some embodiments, Hi a comprises an Fc domain. In some embodiments, the albumin is serum albumin. In some embodiments, the albumin is human serum albumin. In some embodiments, Hi a comprises 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.
  • 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.
  • the circulating immunoglobulin molecule comprises IgGl, IgG2, IgG3, IgG4, slgA, IgM or IgD.
  • the serum protein is albumin.
  • the polypeptide is an antibody.
  • the antibody comprises a single domain antibody, a single chain variable fragment or a Fab.
  • the antibody comprises a single domain antibody that binds to albumin.
  • the antibody is a human or humanized antibody.
  • the single domain antibody is 645gHlgLl. 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-l 1-15. In some embodiments, the single domain antibody is Alb-1, Alb-8, or Alb-23. In some embodiments, the single domain antibody is 10G.
  • the single domain antibody comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the single domain antibody comprise: HC-CDR1: SEQ ID NO: 36, HC-CDR2: SEQ ID NO: 37, and HC-CDR3: SEQ ID NO: 38.
  • CDRs complementarity determining regions
  • the single domain antibody comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC- CDR2, and the HC-CDR3 of the single domain antibody comprise: HC-CDR1: SEQ ID NO: 39, HC-CDR2: SEQ ID NO: 40, and HC-CDR3: SEQ ID NO: 41.
  • the single domain antibody is SA21.
  • Hi a comprises a linking moiety (Li a ) that connects Hi a to Pi a.
  • Li a is a peptide sequence having at least 5 to no more than 50 amino acids.
  • Li a is a peptide sequence having at least 10 to no more than 30 amino acids. In some embodiments, Li a is a peptide sequence having at least 10 amino acids. In some embodiments, Li a is a peptide sequence having at least 18 amino acids. In some embodiments, Li a is a peptide sequence having at least 26 amino acids.
  • Li a has a formula selected from the group consisting of (G2S) n , (GS) context, (GSGGS) context (SEQ ID NO: 836), (GGGS) friendship (SEQ ID NO: 837) , (GGGGS) connector (SEQ ID NO: 838) , and (GSSGGS) n (SEQ ID NO: 839) , wherein n is an integer of at least 1.
  • Li a comprises an amino acid sequence according to SEQ ID NO: 30 or 31.
  • Li or L2 comprises an amino acid sequence according to any one of SEQ ID NOs: 27-35.
  • Li or L2 comprises an amino acid sequence of Linker 4 (ISSGLLSGRSDAG) (SEQ ID NO: 66), Linker 5 (AAGLLAPPGGLSGRSDAG) (SEQ ID NO: 67), Linker 6 (SPLGLSGRSDAG) (SEQ ID NO: 68), or Linker 7 (LSGRSDAGSPLGLAG) (SEQ ID NO: 69), or an amino acid sequence that has 1, 2, or 3 amino acid substitutions, additions, or deletions relative to the amino acid sequence of Linker 4, Linker 5, Linker 6, or Linker 7.
  • polypeptide complexes comprising a structural arrangement according to Configuration 1 :
  • 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 (Pi) that impairs binding of the scFv to an effector cell antigen and Pi is linked to a N-terminus of the heavy chain variable domain of the scFv with a linking moiety (Li) that is a substrate for a tumor specific protease, and Pi is further linked to a half-life extending molecule; and a Fab that binds to epidermal growth factor receptor (EGFR), wherein the Fab comprises a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab light chain polypeptide is linked to a C terminus of the light chain variable domain of the scFv, and wherein the Fab is linked to P2 and L2, wherein P2 comprises a peptide that impairs binding
  • polypeptide complexes comprising a structural arrangement according to Configuration 2:
  • 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 (Pi) that impairs binding of the scFv to an effector cell antigen and Pi is linked to a N-terminus of the heavy chain variable domain of the scFv with a linking moiety (Li) that is a substrate for a tumor specific protease, and Pi is further linked to a half-life extending molecule; and a Fab that binds to epidermal growth factor receptor (EGFR), wherein the Fab comprises a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab heavy chain polypeptide is linked to a C terminus of the light chain variable domain of the scFv, and wherein the Fab is linked to P and L , wherein P comprises a peptide that impairs binding to
  • Figs. 1A-1B illustrate polypeptide complexes of this disclosure.
  • FIG. 2 illustrates binding to EGFR for several polypeptide complexes of this disclosure measured by ELISA.
  • Fig. 3 illustrates binding to CD3 for several polypeptide complexes of this disclosure measured by ELISA.
  • FIG. 4 illustrates polypeptide complexes of this disclosure mediated HCT116 tumor cell killing in the presence of CD8+ T cells.
  • Fig. 5 illustrates polypeptide complexes of this disclosure mediated A431 tumor cell killing in the presence of CD8+ T cells.
  • Fig. 6 illustrates kinetic binding and cross reactivity against human and cynomolgus monkey EGFR for polypeptide complexes of this disclosure.
  • Fig. 7 illustrates binding and cross reactivity against human and cynomolgus EGFR for polypeptide complexes of this disclosure equilibrium.
  • Fig. 8 illustrates kinetic binding and cross reactivity against human and cynomolgus CD3e for several polypeptide complexes.
  • Fig. 9 illustrates equilibrium binding and cross reactivity against human and cynomolgus CD3e for polypeptide complexes of this disclosure.
  • Figs. 10A-10B illustrate pharmacokinetics in cynomolgus monkeys after a single IV bolus injection for polypeptide complexes of this disclosure.
  • FIGs. 11A-11L illustrate cytokine release in cynomolgus monkeys after single IV bolus for polypeptide complexes of this disclosure.
  • Fig. 12 illustrates dosing of polypeptide complexes of the disclosure on inflammatory cytokine levels.
  • Figs. 13A-13D illustrate serum liver enzymes in cynomolgus monkeys after single IV bolus for polypeptide complexes of this disclosure.
  • Fig. 14 illustrates binding curves for binding of otEGFR IgG by peptides of the present disclosure as measured by ELISA.
  • Fig. 15 illustrates inhibition of otEGFR IgG binding to the EGFR antigen by peptides of the present disclosure as measured by ELISA.
  • Fig. 16 illustrates binding curves for binding of otEGFR Fab by peptides of the present disclosure as measured by ELISA.
  • Fig. 17 illustrates inhibition of otEGFR Fab binding to the EGFR antigen by peptides of the present disclosure as measured by ELISA.
  • Fig. 18 illustrates inhibition of otEGFR Fab binding to the EGFR antigen by alanine scanning peptides of peptide-8 as measured by ELISA.
  • FIG. 19 illustrates inhibition of otEGFR Fab binding to the EGFR antigen by alanine scanning peptides of peptide-8 as measured by ELISA.
  • Fig. 20 illustrates a consensus sequence of optimized otEGFR Fab peptide-8 generated using WebLogo 3.7.4.
  • Fig. 21 illustrates otEGFR Fab binding by peptides identified by phage display.
  • Fig. 22 illustrates otEGFR Fab binding by peptides identified by phage display.
  • Fig. 23 illustrates inhibition of otEGFR Fab binding to EGFR by peptides identified phage display.
  • Fig. 24 illustrates inhibition of otEGFR Fab binding to EGFR by peptides identified by phage display.
  • Fig. 25 shows in vivo tumor growth inhibition with different amounts of PC 1 in human PBMC engrafted NCG mice bearing HCT116 xenograft tumors.
  • Fig. 26 shows in vivo tumor growth inhibition with different amounts of PC5 in human PBMC engrafted NCG mice bearing HCT116 xenograft tumors.
  • Fig. 27 shows in vivo tumor growth inhibition with different amounts of PC7 in human PBMC engrafted NCG mice bearing HCT116 xenograft tumors.
  • Fig. 28 shows in vivo tumor growth inhibition with different amounts of PC6 in human PBMC engrafted NCG mice bearing HCT116 xenograft tumors.
  • Fig. 29 shows PCI and PC4 binding to human CD3s as measured by ELISA. PC4 was treated with protease where indicated.
  • Fig. 30 shows PCI and PC4 binding to cynomolgus monkey CD3s as measure by ELISA. PC4 was treated with protease where indicated.
  • Fig. 31 shows PCI and PC4 binding to human EGFR as measured by ELISA. PC4 was treated with protease where indicated.
  • Fig. 32 shows PCI and PC4 binding to cynomolgus EGFR as measured by ELISA. PC4 was treated with protease where indicated.
  • Fig. 33 shows PC4 binding to human albumin as measured by ELISA.
  • Fig. 34 shows PC4 binding to cynomolgus monkey albumin as measured by ELISA.
  • Fig. 35 shows PCI and PC4 mediated killing of A549 cells in the presence of peripheral blood mononuclear cells (PBMCs).
  • PBMCs peripheral blood mononuclear cells
  • Fig. 36 shows IFNy cytokine levels in EGFR positive HCT116 tumor cells with increasing concentrations of PC4 and PCI .
  • PC4 was protease treated where indicated.
  • Fig. 37 shows TNF cytokine levels in EGFR positive HCT116 tumor cells with increasing concentrations of PC4 and PCI .
  • PC4 was protease treated where indicated.
  • Fig. 38 shows IFNy cytokine levels in EGFR positive A549 tumor cells with increasing concentrations of PC4 and PCI .
  • PC4 was protease treated where indicated.
  • Fig. 39 shows TNF cytokine levels in EGFR positive A549 tumor cells with increasing concentrations of PC4 and PCI .
  • PC4 was protease treated where indicated.
  • Fig. 40 shows a schematic representation of a HCT116 mouse tumor model study of the present disclosure.
  • Fig. 41 shows mean tumor volume versus days of treatment with PCI, PC7, and PC6 in PBMC engrafted mice implanted with HCT116 tumor cells.
  • Fig. 42 shows tumor volume versus days of treatment with PC5 and vehicle in PBMC engrafted mice implanted with HCT116 tumor cells.
  • Fig. 43 shows percent survival in PBMC engrafted mice implanted with HCT116 tumor cells with treatment with PCI, PC5, and PC6.
  • Fig. 44 shows a schematic representation a human colorectal cancer (CRC) organoid study of the present disclosure.
  • Fig. 45 shows induction of activation markers CD25, CD69, 4-1BB, and Granzyme with PCI and PC5 in human colorectal cancer tumoroids.
  • Fig. 46 shows maximum tolerated dose (MTD) for PCI and no observed adverse effect level (NOAEL) for PC4 in cynomolgus monkeys.
  • Fig. 47 shows pharmacokinetics in cynomolgus monkeys with different dosing amounts of PC4 after IV bolus injections.
  • Fig. 48 shows PC 10 and PCI 1 binding to EGFR measured by ELISA.
  • Fig. 49 shows PC 10 and PCI 1 binding to CD3 measured by ELISA.
  • Fig. 50 shows a graph of PC10 and PCI 1 mediated HCT116 tumor cell killing in the presence of CD8+ T cells.
  • Figs. 51A-51F illustrate anti-CD3 scFv binding by alanine scanning peptides of anti-CD3 scFv Peptide-A and Peptide-B as measured by ELISA.
  • Figs. 52A-52F illustrate inhibition of anti-CD3 scFv binding to CD3 by alanine scanning peptides of anti-CD3 scFv Peptide-A and Peptide-B as measured by ELISA.
  • Figs. 53A-53B illustrate anti-CD3 scFv binding by optimized anti-CD3 scFv Peptide-B sequences as measured by ELISA.
  • Figs. 54A-54B illustrate inhibition of anti-CD3 scFv binding to CD3 by optimized anti-CD3 scFv Peptide-B sequences as measured by ELISA.
  • Fig. 55 illustrates the core sequence motif of optimized anti-CD3 scFv Peptide-B sequences generated using WebLogo 3.7.4.
  • 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.
  • EGFR epidermal growth factor receptor
  • EGFR epidermal growth factor receptor
  • EGFR is a transmembrane protein that is a receptor for members of the epidermal growth factor family of extracellular protein ligands. EGFR is the most commonly overexpressed membrane protein in cancer. However, EGFR expression is not limited to tumors and is widely expressed throughout the body, resulting in systemic toxicities with EGFR-directed therapies.
  • TCEs T cell engagers
  • CRS cytokine release syndrome
  • PK pharmacokinetics
  • 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.
  • IL-6 interleukin-6
  • 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.
  • polypeptide or polypeptide complexes that comprise binding domains that selectively bind to an effector cell antigen and EGFR, in which one or more of the binding domains is selectively activated in the tumor microenvironment and the isolated polypeptide or polypeptide complex comprises a half-life extending molecule.
  • binding domains that selectively bind to an effector cell antigen and EGFR
  • one or more of the binding domains is selectively activated in the tumor microenvironment and the isolated polypeptide or polypeptide complex comprises a half-life extending molecule.
  • Such modifications reduce CRS and on-target healthy tissue toxicity risk, improves stability in the bloodstream and serum half-life prior to activation.
  • the polypeptide or polypeptide complexes described herein have activity at low levels of target expression, and are easily manufactured.
  • the polypeptides or polypeptide complexes described herein are used in a method of treating cancer.
  • the cancer has cells that express EGFR.
  • the polypeptides or polypeptide complexes described herein are used in a method of treating colorectal cancer (CRC), squamous cell carcinoma of the head and Neck (SCCHN), non-small cell lung cancer (NSCLC), prostate cancer, breast cancer, colon/rectum cancer, head and neck cancer, esophagogastric cancer, liver cancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, kidney cancer, or pancreatic cancer.
  • CRC colorectal cancer
  • SCCHN squamous cell carcinoma of the head and Neck
  • NSCLC non-small cell lung cancer
  • prostate cancer breast cancer, colon/rectum cancer, head and neck cancer
  • esophagogastric cancer liver cancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, kidney cancer, or pancreatic cancer.
  • the polypeptides or polypeptide complexes described herein are used in a method of treating subjects who are resistant to EGFR inhibitor treatment. In some embodiments, the polypeptides or polypeptide complexes described herein are used in a method of treating subjects who harbor KRAS mutations. In some embodiments, the polypeptides or polypeptide complexes described herein are used in a method of treating subjects who are resistant to EGFR inhibitor treatment and harbor KRAS mutations. In some embodiments, are methods of treating cancer comprising administering to a subject in need thereof an isolated polypeptide or polypeptide complex according to Formula I:
  • Ai comprises a first antigen recognizing molecule that binds to an effector cell antigen
  • Pi comprises a peptide that binds to Ai
  • Li comprises a linking moiety that connects Ai to Pi and is a substrate for a tumor specific protease
  • Hi comprises a half-life extending molecule
  • A comprises a second antigen recognizing molecule that binds to epidermal growth factor receptor (EGFR).
  • EGFR epidermal growth factor receptor
  • polypeptides or polypeptide complexes according to Formula I are polypeptides or polypeptide complexes according to Formula I:
  • Ai comprises a first antigen recognizing molecule that binds to an effector cell antigen
  • Pi comprises a peptide that binds to Ai
  • Fi comprises a linking moiety that connects Ai to Pi and is a substrate for a tumor specific protease
  • Hi comprises a half-life extending molecule
  • A comprises a second antigen recognizing molecule that binds to epidermal growth factor receptor (EGFR).
  • EGFR epidermal growth factor receptor
  • polypeptides or polypeptide complexes according to Formula I are polypeptides or polypeptide complexes according to Formula I:
  • Ai is a first antigen recognizing molecule that binds to an effector cell antigen
  • Pi is a peptide that binds to Ai
  • Fi is a linking moiety that connects Ai to Pi and is a substrate for a tumor specific protease
  • Hi is a half-life extending molecule
  • A is a second antigen recognizing molecule that binds to epidermal growth factor receptor (EGFR).
  • polypeptides or polypeptide complexes comprising Formula I:
  • Ai comprises a first antigen recognizing molecule that binds to an effector cell antigen
  • Pi comprises a peptide that binds to Ai
  • Li comprises a linking moiety that connects Ai to Pi and is a substrate for a tumor specific protease
  • Hi comprises a half-life extending molecule
  • A comprises a second antigen recognizing molecule that binds to epidermal growth factor receptor (EGFR).
  • EGFR epidermal growth factor receptor
  • polypeptides or polypeptide complexes comprising Formula I:
  • Ai is a first antigen recognizing molecule that binds to an effector cell antigen
  • Pi is a peptide that binds to Ai
  • Li is a linking moiety that connects Ai to Pi and is a substrate for a tumor specific protease
  • Hi is a half-life extending molecule
  • A is a second antigen recognizing molecule that binds to epidermal growth factor receptor (EGFR).
  • polypeptides or polypeptide complexes according to Formula I are polypeptides or polypeptide complexes according to Formula I:
  • Ai comprises a first antigen recognizing molecule that binds to epidermal growth factor receptor (EGFR); Pi comprises a peptide that binds to Ai; Li comprises a linking moiety that connects Aito Pi and is a substrate for a tumor specific protease; Hi comprises a half-life extending molecule; and A2 comprises a second antigen recognizing molecule that binds to an effector cell antigen.
  • EGFR epidermal growth factor receptor
  • Pi comprises a peptide that binds to Ai
  • Li comprises a linking moiety that connects Aito Pi and is a substrate for a tumor specific protease
  • Hi comprises a half-life extending molecule
  • A2 comprises a second antigen recognizing molecule that binds to an effector cell antigen.
  • polypeptides or polypeptide complexes according to Formula I are polypeptides or polypeptide complexes according to Formula I:
  • Pi is a peptide that binds to Ai
  • Li is a linking moiety that connects Aito Pi and is a substrate for a tumor specific protease
  • Hi is a half-life extending molecule
  • A2 is a second antigen recognizing molecule that binds to effector cell antigen.
  • polypeptides or polypeptide complexes comprising Formula I:
  • Ai comprises a first antigen recognizing molecule that binds to epidermal growth factor receptor (EGFR); Pi comprises a peptide that binds to Ai; Li comprises a linking moiety that connects Aito Pi and is a substrate for a tumor specific protease; Hi comprises a half-life extending molecule; and A2 comprises a second antigen recognizing molecule that binds to an effector cell antigen.
  • EGFR epidermal growth factor receptor
  • Pi comprises a peptide that binds to Ai
  • Li comprises a linking moiety that connects Aito Pi and is a substrate for a tumor specific protease
  • Hi comprises a half-life extending molecule
  • A2 comprises a second antigen recognizing molecule that binds to an effector cell antigen.
  • polypeptides or polypeptide complexes comprising Formula I:
  • Ai is a first antigen recognizing molecule that binds epidermal growth factor receptor (EGFR); Pi is a peptide that binds to Ai; Li is a linking moiety that connects Aito Pi and is a substrate for a tumor specific protease; Hi is a half-life extending molecule; and A2 is a second antigen recognizing molecule that binds to an effector cell antigen.
  • EGFR epidermal growth factor receptor
  • Pi is a peptide that binds to Ai
  • Li is a linking moiety that connects Aito Pi and is a substrate for a tumor specific protease
  • Hi is a half-life extending molecule
  • A2 is a second antigen recognizing molecule that binds to an effector cell antigen.
  • A2 further comprises P2 and L2, wherein P2 comprises a peptide that binds to A2; and L2 comprises a linking moiety that connects A2to P2and is a substrate for a tumor specific protease.
  • polypeptides or polypeptide complexes according to Formula la: P 2 L 2 -A 2 -A I -L I -P 1 -H l
  • a 2 further comprises P 2 and L 2 , wherein P 2 is a peptide that binds to A 2 ; and L 2 is a linking moiety that connects A 2 to P 2 and is a substrate for a tumor specific protease.
  • polypeptides or polypeptide complexes comprising Formula la:
  • a 2 further comprises R 2 and L 2 , wherein R 2 comprises a peptide that binds to A 2 ; and L 2 comprises a linking moiety that connects A 2 to P 2 and is a substrate for a tumor specific protease.
  • polypeptides or polypeptide complexes comprising Formula la:
  • a 2 further comprises R 2 and L 2 , wherein R 2 is a peptide that binds to A 2 ; and L 2 is a linking moiety that connects A 2 to P 2 and is a substrate for a tumor specific protease.
  • polypeptides or polypeptide complexes according to Formula II are polypeptides or polypeptide complexes according to Formula II:
  • Li a comprises a tumor specific protease-cleaved linking moiety that when uncleaved connects Pi a to a first antigen recognizing molecule that binds to an effector cell antigen and the first antigen recognizing molecule is connected to a second antigen recognizing molecule that binds to epidermal growth factor receptor (EGFR);
  • Pi a comprises a peptide that binds to the first antigen recognizing molecule when Li a is uncleaved;
  • Hi a comprises a half-life extending molecule.
  • polypeptides or polypeptide complexes comprising Formula II:
  • Li a comprises a tumor specific protease-cleaved linking moiety that when uncleaved connects Pi a to a first antigen recognizing molecule that binds to an effector cell antigen and the first antigen recognizing molecule is connected to a second antigen recognizing molecule that binds to epidermal growth factor receptor (EGFR);
  • Pi a comprises a peptide that binds to the first antigen recognizing molecule when Li a is uncleaved;
  • Hi a comprises a half-life extending molecule.
  • polypeptides or polypeptide complexes according to Formula II are polypeptides or polypeptide complexes according to Formula II:
  • Li a is a tumor specific protease-cleaved linking moiety that when uncleaved connects Pi a to a first antigen recognizing molecule that binds to an effector cell antigen and the first antigen recognizing molecule is connected to a second antigen recognizing molecule that binds to epidermal growth factor receptor (EGFR); Pi a is a peptide that binds to the first antigen recognizing molecule when Li a is uncleaved; and Hi a is a half-life extending molecule.
  • EGFR epidermal growth factor receptor
  • polypeptides or polypeptide complexes comprising Formula II:
  • Li a is a tumor specific protease-cleaved linking moiety that when uncleaved connects Pi a to a first antigen recognizing molecule that binds to an effector cell antigen and the first antigen recognizing molecule is connected to a second antigen recognizing molecule that binds to epidermal growth factor receptor (EGFR); Pi a is a peptide that binds to the first antigen recognizing molecule when Li a is uncleaved; and Hi a is a half-life extending molecule.
  • EGFR epidermal growth factor receptor
  • polypeptides or polypeptide complexes wherein the first antigen recognizing molecule binds to an effector cell antigen and the second antigen recognizing molecule binds to EGFR.
  • the effector cell antigen comprises CD3.
  • Ai comprises a first antigen recognizing molecule that binds to an effector cell antigen.
  • Ai comprises an antibody or antibody fragment. In some embodiments, Ai comprises an antibody or antibody fragment that is human or humanized. In some embodiments, Li is bound to N-terminus of the antibody or antibody fragment. In some embodiments, Li is bound to N-terminus of the antibody or antibody fragment and A is bound to the other N-terminus of the antibody or antibody fragment. In some embodiments, A is bound to C-terminus of the antibody or antibody fragment. In some embodiments, Li is bound to C-terminus of the antibody or antibody fragment. In some embodiments, A is 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.
  • Ai is the single chain variable fragment (scFv).
  • the scFv comprises a scFv heavy chain polypeptide and a scFv light chain polypeptide.
  • Ai is the single domain antibody.
  • Ai comprises a variable light chain and variable heavy chain each of which is capable of specifically binding to human CD3.
  • the effector cell antigen comprises CD3.
  • Ai comprises an anti-CD3e single chain variable fragment.
  • Ai comprises an anti-CD3e single chain variable fragment that has a K D binding of 1 mM or less to CD3 on CD3 expressing cells.
  • Ai comprises complementary determining regions (CDRs) selected from the group consisting of muromonab-CD3 (OKT3), otelixizumab (TRX4), teplizumab (MGA031), visibzumab (Nuvion), SP34, X35, VIT3, BMA030 (BW264/56), CLB- T3/3, CRIS7, YTH12.5, FI 11-409, CLB-T3.4.2, TR-66, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII-87, 12F6, T3/RW2-8C8, T3/RW2-4B6, OKT3D, M-T301, SMC2, F101.01, UCHT-1, WT-31, 15865,
  • CDRs complementary determining regions
  • Ai comprises a first antigen recognizing molecule that binds EGFR.
  • Ai comprises a variable light chain and variable heavy chain each of which is capable of specifically binding to human EGFR.
  • the scFv that binds to CD3 comprises a scFv light chain variable domain and a scFv heavy chain variable domain.
  • 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).
  • 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).
  • CDR complementarity determining regions
  • 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).
  • CDR complementarity determining regions
  • the scFv light chain variable domain comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC- CDR3 of the scFv light chain variable domain comprise: LC-CDR1: SEQ ID NO: 1, LC-CDR2: SEQ ID NO: 2, and LC-CDR3: SEQ ID NO: 3, and wherein the CDRs comprise from 0-2 amino acid modifications in at least one of the LC-CDR1, LC-CDR2, or LC-CDR3.
  • CDRs complementarity determining regions
  • the scFv light chain variable domain comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC- CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the scFv light chain variable domain comprise: LC-CDR1: SEQ ID NO: 7, LC-CDR2: SEQ ID NO: 8, and LC-CDR3: SEQ ID NO: 9, and wherein the CDRs comprise from 0-2 amino acid modifications in at least one of the LC-CDR1, LC-CDR2, or LC-CDR3.
  • CDRs complementarity determining regions
  • the scFv heavy chain variable domain comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC- CDR2, and the HC-CDR3 of the scFv heavy chain variable domain comprise: HC-CDR1: SEQ ID NO: 4, HC-CDR2: SEQ ID NO: 5, and HC-CDR3: SEQ ID NO: 6, and wherein the CDRs comprise from 0-2 amino acid modifications in at least one of the HC-CDR1, HC-CDR2, or HC-CDR3.
  • CDRs complementarity determining regions
  • the scFv heavy chain variable domain comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the scFv heavy chain variable domain comprise: HC-CDR1: SEQ ID NO: 10, HC-CDR2: SEQ ID NO: 11, and HC-CDR3: SEQ ID NO: 12, and wherein the CDRs comprise from 0-2 amino acid modifications in at least one of the HC-CDR1, HC-CDR2, or HC-CDR3.
  • CDRs complementarity determining regions
  • the isolated polypeptide or polypeptide complex of Formula I binds to an effector cell when Li is cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex of Formula I binds to an effector cell when Li is cleaved by the tumor specific protease and Ai binds to the effector cell. In some embodiments, the effector cell is a T cell. In some embodiments, Ai binds to a polypeptide that is part of a TCR-CD3 complex on the effector cell. In some embodiments, the polypeptide that is part of the TCR-CD3 complex is human CD3s.
  • the effector cell antigen comprises CD3, wherein the effector cell antigen comprises CD3, wherein the scFv comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC- CDR2, and the LC-CDR3 of the scFv LC-CDR1: SEQ ID NO: 1, LC-CDR2: SEQ ID NO: 2, and LC- CDR3: SEQ ID NO: 3; and the scFv comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the scFv: HC- CDR1: SEQ ID NO: 4, HC-CDR2: SEQ ID NO: 5, and HC-CDR3: SEQ ID NO: 6.
  • CDRs complementarity determining regions
  • the effector cell antigen comprises CD3, and the scFv comprises an amino acid sequence according to SEQ ID NO: 13.
  • the effector cell antigen comprises CD3, wherein the effector cell antigen comprises CD3, wherein the scFv comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the scFv LC-CDR1: SEQ ID NO: 7, LC-CDR2: SEQ ID NO: 8, and LC-CDR3: SEQ ID NO: 9; and the scFv comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the scFv: HC-CDR1: SEQ ID NO: 10, HC-CDR2: S
  • the effector cell antigen comprises CD3, and Ai comprises CDRs: LC- CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of Ai comprise LC-CDR1: SEQ ID NO: 1, LC-CDR2: SEQ ID NO: 2, and LC-CDR3: SEQ ID NO: 3; and Ai comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC- CDR1, the HC-CDR2, and the HC-CDR3 of Ai comprise: HC-CDR1: SEQ ID NO: 4, HC-CDR2: SEQ ID NO: 5, and HC-CDR3: SEQ ID NO: 6.
  • the effector cell antigen comprises CD3, and Ai comprises CDRs: LC- CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of Ai comprise: LC-CDR1: SEQ ID NO: 7, LC-CDR2: SEQ ID NO: 8, and LC-CDR3: SEQ ID NO: 9; and Ai comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of Ai comprise: HC-CDR1: SEQ ID NO: 10, HC-CDR2: SEQ ID NO: 11, and HC-CDR3: SEQ ID NO: 12.
  • Ai comprises an amino acid sequence according to SEQ ID NO: 13. In some embodiments, Ai comprises an amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 13. In some embodiments, Ai comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 13. In some embodiments, Ai comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 13. In some embodiments, Ai comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 13. In some embodiments, Ai comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NO: 13.
  • Ai comprises an amino acid sequence according to SEQ ID NO: 14. In some embodiments, Ai comprises an amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 14. In some embodiments, Ai comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 14. In some embodiments, Ai comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 14. In some embodiments, Ai comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 14. In some embodiments, Ai comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NO: 14.
  • 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 Pi or Li. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 5X higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex that does not have Pi or Li.
  • the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 8X higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex that does not have Pi or Li. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 10X higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex that does not have Pi or Li.
  • the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 15X higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex that does not have Pi or Li. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 20X higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex that does not have Pi or Li.
  • the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 25X higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex that does not have Pi or Li. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 3 OX higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex that does not have Pi or Li.
  • the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 35X higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex that does not have Pi or Li. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 40X higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex that does not have Pi or Li.
  • the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 45X higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex that does not have Pi or Li. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 5 OX higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex that does not have Pi or Li.
  • the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 55X higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex that does not have Pi or Li. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 60X higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex that does not have Pi or Li.
  • the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 65X higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex that does not have Pi or Li. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 70X higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex that does not have Pi or Li.
  • the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 75X higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex that does not have Pi or Li. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 80X higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex that does not have Pi or Li.
  • the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 85X higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex that does not have Pi or Li. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 90X higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex that does not have Pi or Li.
  • the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 95X higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex that does not have Pi or Li. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 100X higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex that does not have Pi or Li.
  • the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 120X higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex that does not have Pi or Li. In some embodiments, the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 1000X higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex that does not have Pi or Li.
  • 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 Lihas 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 5X higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex in which Lihas been cleaved by the tumor specific protease.
  • the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 8X higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex in which Lihas 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 10X higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex in which Lihas been cleaved by the tumor specific protease.
  • the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 15X higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex in which Lihas 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 20X higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex in which Lihas been cleaved by the tumor specific protease.
  • the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 25X higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex in which Lihas 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 3 OX higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex in which Lihas been cleaved by the tumor specific protease.
  • the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 35X higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex in which Lihas 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 40X higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex in which Lihas been cleaved by the tumor specific protease.
  • the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 45X higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex in which Lihas 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 OX higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex in which Lihas been cleaved by the tumor specific protease.
  • the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 55X higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex in which Lihas 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 60X higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex in which Lihas been cleaved by the tumor specific protease.
  • the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 65X higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex in which Lihas 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 70X higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex in which Lihas been cleaved by the tumor specific protease.
  • the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 75X higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex in which Lihas 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 80X higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex in which Lihas been cleaved by the tumor specific protease.
  • the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 85X higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex in which Lihas 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 90X higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex in which Lihas been cleaved by the tumor specific protease.
  • the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 95X higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex in which Lihas 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 100X higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex in which Lihas been cleaved by the tumor specific protease.
  • the isolated polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 120X higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex in which Li has 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 1000X higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex in which Lihas been cleaved by the tumor specific protease.
  • the isolated polypeptide or polypeptide complex has an increased EC50 in an IFNy release T-cell activation assay as compared to the EC50 in an IENg release T-cell activation assay of an isolated polypeptide or polypeptide complex that does not have Pi or Li. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC50 in an IFNy release T-cell activation assay that is at least 10X higher than the EC50 in an IENg release T-cell activation assay of a form of the isolated polypeptide or polypeptide complex that does not have Pi or Li.
  • the isolated polypeptide or polypeptide complex has an increased EC50 in an IFNy release T-cell activation assay that is at least 20X higher than the EC50 in an IENg release T-cell activation assay of a form of the isolated polypeptide or polypeptide complex that does not have Pi or Li. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC50 in an IFNy release T-cell activation assay that is at least 30X higher than the EC50 in an IENg release T-cell activation assay of a form of the isolated polypeptide or polypeptide complex that does not have Pi or Li.
  • the isolated polypeptide or polypeptide complex has an increased EC50 in an IFNy release T-cell activation assay that is at least 40X higher than the EC50 in an IENg release T-cell activation assay of a form of the isolated polypeptide or polypeptide complex that does not have Pi or Li. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC50 in an IFNy release T-cell activation assay that is at least 50X higher than the EC50 in an IENg release T-cell activation assay of a form of the isolated polypeptide or polypeptide complex that does not have Pi or Li.
  • the isolated polypeptide or polypeptide complex has an increased EC50 in an IFNy release T-cell activation assay that is at least 60X higher than the EC50 in an IENg release T-cell activation assay of a form of the isolated polypeptide or polypeptide complex that does not have Pi or Li. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC50 in an IFNy release T-cell activation assay that is at least 70X higher than the EC50 in an IENg release T-cell activation assay of a form of the isolated polypeptide or polypeptide complex that does not have Pi or Li.
  • the isolated polypeptide or polypeptide complex has an increased EC50 in an IFNy release T-cell activation assay that is at least 80X higher than the EC50 in an IENg release T-cell activation assay of a form of the isolated polypeptide or polypeptide complex that does not have Pi or Li. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC50 in an IFNy release T-cell activation assay that is at least 90X higher than the EC50 in an IENg release T-cell activation assay of a form of the isolated polypeptide or polypeptide complex that does not have Pi or Li.
  • the isolated polypeptide or polypeptide complex has an increased EC50 in an IFNy release T-cell activation assay that is at least 100X higher than the EC50 in an IENg release T-cell activation assay of a form of the isolated polypeptide or polypeptide complex that does not have Pi or Li. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC50 in an IFNy release T-cell activation assay that is at least 1000X higher than the EC50 in an IFNy release T-cell activation assay of a form of the isolated polypeptide or polypeptide complex that does not have Pi or Li.
  • the isolated polypeptide or polypeptide complex has an increased EC 50 in an IFNy release T-cell activation assay as compared to the EC 50 in an IENg release T-cell activation assay of the isolated polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC 50 in an IFNy release T-cell activation assay that is at least 10X higher than the EC 50 in an IENg release T-cell activation assay of the isolated polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease.
  • the isolated polypeptide or polypeptide complex has an increased EC 50 in an IENg release T-cell activation assay that is at least 20X higher than the EC 50 in an IFNy release T-cell activation assay of the isolated polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC 50 in an IFNy release T-cell activation assay that is at least 30X higher than the EC 50 in an IENg release T-cell activation assay of the isolated polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease.
  • the isolated polypeptide or polypeptide complex has an increased EC 50 in an IENg release T-cell activation assay that is at least 40X higher than the EC 50 in an IFNy release T-cell activation assay of the isolated polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC 50 in an IFNy release T-cell activation assay that is at least 50X higher than the EC 50 in an IFNy release T-cell activation assay of the isolated polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease.
  • the isolated polypeptide or polypeptide complex has an increased EC 50 in an IFNy release T-cell activation assay that is at least 60X higher than the EC 50 in an IENg release T-cell activation assay of the isolated polypeptide or polypeptide complex in which Lihas been cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC 50 in an IENg release T-cell activation assay that is at least 70X higher than the EC 50 in an IENg release T-cell activation assay of the isolated polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease.
  • the isolated polypeptide or polypeptide complex has an increased EC 50 in an IENg release T-cell activation assay that is at least 80X higher than the EC 50 in an IENg release T-cell activation assay of the isolated polypeptide or polypeptide complex in which Lihas been cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC 50 in an IENg release T- cell activation assay that is at least 90X higher than the EC 50 in an IFNy release T-cell activation assay of the isolated polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease.
  • the isolated polypeptide or polypeptide complex has an increased EC 50 in an IFNy release T-cell activation assay that is at least 100X higher than the EC 50 in an IENg release T-cell activation assay of the isolated polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC 50 in an IENg release T-cell activation assay that is at least 1000X higher than the EC 50 in an IFNy release T-cell activation assay of the isolated polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease.
  • the isolated polypeptide or polypeptide complex has an increased EC 50 in a T-cell cytolysis assay as compared to the EC 50 in a T-cell cytolysis assay of an isolated polypeptide or polypeptide complex that does not have Pi or Li. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC 50 in a T-cell cytolysis assay that is at least 10X higher than the EC 50 in a T-cell cytolysis assay of a form of the isolated polypeptide or polypeptide complex that does not have Pi or Li.
  • the isolated polypeptide or polypeptide complex has an increased EC 50 in a T-cell cytolysis assay that is at least 20X higher than the EC 50 in a T-cell cytolysis assay of a form of the isolated polypeptide or polypeptide complex that does not have Pi or Li. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC 50 in a T-cell cytolysis assay that is at least 30X higher than the EC 50 in a T-cell cytolysis assay of a form of the isolated polypeptide or polypeptide complex that does not have Pi or Li.
  • the isolated polypeptide or polypeptide complex has an increased EC 50 in a T-cell cytolysis assay that is at least 40X higher than the EC 50 in a T-cell cytolysis assay of a form of the isolated polypeptide or polypeptide complex that does not have Pi or Li. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC 50 in a T-cell cytolysis assay that is at least 50X higher than the EC 50 in a T-cell cytolysis assay of a form of the isolated polypeptide or polypeptide complex that does not have Pi or Li.
  • the isolated polypeptide or polypeptide complex has an increased EC 50 in a T-cell cytolysis assay that is at least 60X higher than the EC 50 in a T-cell cytolysis assay of a form of the isolated polypeptide or polypeptide complex that does not have Pi or Li. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC 50 in a T-cell cytolysis assay that is at least 70X higher than the EC 50 in a T-cell cytolysis assay of a form of the isolated polypeptide or polypeptide complex that does not have Pi or Li.
  • the isolated polypeptide or polypeptide complex has an increased EC 50 in a T-cell cytolysis assay that is at least 80X higher than the EC 50 in a T-cell cytolysis assay of a form of the isolated polypeptide or polypeptide complex that does not have Pi or Li. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC 50 in a T-cell cytolysis assay that is at least 90X higher than the EC 50 in a T-cell cytolysis assay of a form of the isolated polypeptide or polypeptide complex that does not have Pi or Li.
  • the isolated polypeptide or polypeptide complex has an increased EC 50 in a T-cell cytolysis assay that is at least 100X higher than the EC 50 in a T-cell cytolysis assay of an isolated polypeptide or polypeptide complex that does not have Pi or Li. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC 50 in a T-cell cytolysis assay that is at least 1000X higher than the EC 50 in a T-cell cytolysis assay of an isolated polypeptide or polypeptide complex that does not have Pi or Li.
  • the isolated polypeptide or polypeptide complex has an increased EC 50 in a T-cell cytolysis assay as compared to the EC 50 in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex in which Lihas been cleaved by the tumor specific protease.
  • the isolated polypeptide or polypeptide complex has an increased EC 50 in a T-cell cytolysis assay that is at least 10X higher than the EC 50 in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease.
  • the isolated polypeptide or polypeptide complex has an increased EC 50 in a T-cell cytolysis assay that is at least 20X higher than the EC 50 in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC 50 in a T-cell cytolysis assay that is at least 30X higher than the EC 50 in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease.
  • the isolated polypeptide or polypeptide complex has an increased EC 50 in a T-cell cytolysis assay that is at least 40X higher than the EC 50 in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC 50 in a T-cell cytolysis assay that is at least 50X higher than the EC 50 in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex in which Lihas been cleaved by the tumor specific protease.
  • the isolated polypeptide or polypeptide complex has an increased EC 50 in a T-cell cytolysis assay that is at least 60X higher than the EC 50 in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex in which Lihas been cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC 50 in a T-cell cytolysis assay that is at least 70X higher than the EC 50 in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease.
  • the isolated polypeptide or polypeptide complex has an increased EC 50 in a T-cell cytolysis assay that is at least 80X higher than the EC 50 in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC 50 in a T-cell cytolysis assay that is at least 90X higher than the EC 50 in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease.
  • the isolated polypeptide or polypeptide complex has an increased EC 50 in a T-cell cytolysis assay that is at least 100X higher than the EC 50 in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex in which Li has been cleaved by the tumor specific protease. In some embodiments, the isolated polypeptide or polypeptide complex has an increased EC 50 in a T-cell cytolysis assay that is at least 1,000X higher than the EC 50 in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex in which Lihas been cleaved by the tumor specific protease.
  • the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1-H1 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 la that does not have Pi, Li, P2, or L2.
  • the isolated polypeptide or polypeptide complex P2-L2-A2-A1-L1-P1- Hi has weaker binding affinity for the tumor cell antigen that is at least 10X higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex of Formula la that does not have Pi, Li, P 2 , or L 2 .
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least 5 OX higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex of Formula la that does not have Pi, Li, P 2 , or L 2 .
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least 75X higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex of Formula la that does not have Pi, Li, P 2 , or L 2 .
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 - L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least 100X higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex of Formula la that does not have Pi, Li, P 2 , or L 2 .
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least 120X higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex of Formula la that does not have Pi, Li, P 2 , or L 2 .
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least 200X higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex of Formula la that does not have Pi, Li, P 2 , or L 2 .
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 - L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least 300X higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex of Formula la that does not have Pi, Li, P 2 , or L 2 .
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least 400X higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex of Formula la that does not have Pi, Li, P 2 , or L 2 .
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least 500X higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex of Formula la that does not have Pi, Li, P 2 , or L 2 .
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 - L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least 600X higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex of Formula la that does not have Pi, Li, P 2 , or L 2 .
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least 700X higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex of Formula la that does not have Pi, Li, P 2 , or L 2 .
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least 800X higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex of Formula la that does not have Pi, Li, P 2 , or L 2 .
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 - L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least 900X higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex of Formula la that does not have Pi, Li, P 2 , or L 2 .
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least 1000X higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex of Formula la that does not have Pi, Li, P 2 , or L 2 .
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least IO,OOOC higher than the binding affinity for the tumor cell antigen of a form of the isolated polypeptide or polypeptide complex of Formula la that does not have Pi, Li, P 2 , or L 2 .
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 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 la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least 10X higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex of Formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 - P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least 50X higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex of Formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least 75X higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex of Formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 - A 1 -L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least 100X higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex of Formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least 120X higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex of Formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 - A 1 -L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least 200X higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex of Formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least 300X higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex of Formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 - A 1 -L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least 400X higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex of Formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least 500X higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex of Formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 - A 1 -L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least 600X higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex of Formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least 700X higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex of Formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 - A 1 -L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least 800X higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex of Formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least 900X higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex of Formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 - A 1 -L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least 1000X higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex of Formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has weaker binding affinity for the tumor cell antigen that is at least IO,OOOC higher than the binding affinity for the tumor cell antigen of the isolated polypeptide or polypeptide complex of Formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in an IFNy release T-cell activation assay as compared to the EC 50 in an IFNy release T-cell activation assay of an isolated polypeptide or polypeptide complex of Formula la that does not have Pi, Li, P 2 , or L 2 .
  • the isolated polypeptide or polypeptide complex P 2 - L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in an IFNy release T-cell activation assay that is at least 10X higher than the EC 50 in an IFNy release T-cell activation assay of a form of the isolated polypeptide or polypeptide complex of Formula la that does not have Pi, Li, P 2 , or L 2 .
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in an IFNy release T-cell activation assay that is at least 50X higher than the EC 50 in an IFNy release T-cell activation assay of a form of the isolated polypeptide or polypeptide complex of Formula la that does not have Pi, Li, P 2 , or L 2 .
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in an IFNy release T-cell activation assay that is at least 75X higher than the EC 50 in an IFNy release T-cell activation assay of a form of the isolated polypeptide or polypeptide complex of Formula la that does not have Pi, Li, P 2 , or L 2 .
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in an IFNy release T-cell activation assay that is at least 100X higher than the EC 50 in an IFNy release T-cell activation assay of a form of the isolated polypeptide or polypeptide complex of Formula la that does not have Pi, Li, P 2 , or L 2 .
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in an IFNy release T-cell activation assay that is at least 200X higher than the EC 50 in an IFNy release T-cell activation assay of a form of the isolated polypeptide or polypeptide complex of Formula la that does not have Pi, Li, P 2 , or L 2 .
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in an IFNy release T-cell activation assay that is at least 300X higher than the EC 50 in an IFNy release T-cell activation assay of a form of the isolated polypeptide or polypeptide complex of Formula la that does not have Pi, Li, P 2 , or L 2 .
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in an IFNy release T-cell activation assay that is at least 400X higher than the EC 50 in an IFNy release T-cell activation assay of a form of the isolated polypeptide or polypeptide complex of Formula la that does not have Pi, Li, P 2 , or L 2 .
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in an IFNy release T-cell activation assay that is at least 500X higher than the EC 50 in an IFNy release T-cell activation assay of a form of the isolated polypeptide or polypeptide complex of Formula la that does not have Pi, Li, P 2 , or L 2 .
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in an IFNy release T-cell activation assay that is at least 600X higher than the EC 50 in an IFNy release T-cell activation assay of a form of the isolated polypeptide or polypeptide complex of Formula la that does not have Pi, Li, P 2 , or L 2 .
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in an IFNy release T-cell activation assay that is at least 700X higher than the EC 50 in an IFNy release T-cell activation assay of a form of the isolated polypeptide or polypeptide complex of Formula la that does not Pi, Li, P 2 , or L 2 .
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in an IFNy release T-cell activation assay that is at least 800X higher than the EC 50 in an IFNy release T-cell activation assay of a form of the isolated polypeptide or polypeptide complex of Formula la that does not have Pi, Li, P 2 , or L 2 .
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in an IFNy release T-cell activation assay that is at least 900X higher than the EC 50 in an IFNy release T-cell activation assay of a form of the isolated polypeptide or polypeptide complex of Formula la that does not have Pi, Fi, P 2 , or F 2 .
  • the isolated polypeptide or polypeptide complex P 2 -F 2 -A 2 -A 1 -F 1 -P 1 -H 1 has an increased EC 50 in an IFNy release T-cell activation assay that is at least 1000X higher than the EC 50 in an IFNy release T-cell activation assay of a form of the isolated polypeptide or polypeptide complex of Formula la that does not have Pi, Fi, P 2 , or F 2 .
  • the isolated polypeptide or polypeptide complex P 2 -F 2 -A 2 -A 1 -F 1 -P 1 -H 1 has an increased EC 50 in an IFNy release T-cell activation assay that is at least IO,OOOC higher than the EC 50 in an IENg release T-cell activation assay of a form of the isolated polypeptide or polypeptide complex of Formula la that does not have Pi, Fi, P 2 , or F 2 .
  • the isolated polypeptide or polypeptide complex P 2 -F 2 -A 2 -A 1 -F 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay as compared to the EC 50 in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula la in which Fi and F 2 have been cleaved by the tumor specific proteases.
  • the isolated polypeptide or polypeptide complex P 2 -F 2 -A 2 -A 1 -F 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 10X higher than the EC 50 in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula la in which Fi and F 2 have been cleaved by the tumor specific proteases.
  • the isolated polypeptide or polypeptide complex P 2 -F 2 -A 2 -A 1 -F 1 -P 1 -H 1 (Formula la) has an increased EC 50 in a T-cell cytolysis assay that is at least 50X higher than the EC 50 in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula la in which Fi and F 2 have been cleaved by the tumor specific proteases.
  • the isolated polypeptide or polypeptide complex P 2 -F 2 -A 2 -A 1 -L 1 - P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 75X higher than the EC 50 in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula la in which Fi and F 2 have been cleaved by the tumor specific proteases.
  • the isolated polypeptide or polypeptide complex P 2 -F 2 -A 2 -A 1 -F 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 100X higher than the EC 50 in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula la in which Fi and F 2 have been cleaved by the tumor specific proteases.
  • the isolated polypeptide or polypeptide complex P 2 -F 2 -A 2 -A 1 -F 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 200X higher than the EC 50 in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula la in which Fi and F 2 have been cleaved by the tumor specific proteases.
  • the isolated polypeptide or polypeptide complex P 2 -F 2 -A 2 -A 1 -F 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 300X higher than the EC 50 in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula la in which Fi and F 2 have been cleaved by the tumor specific proteases.
  • the isolated polypeptide or polypeptide complex P 2 -F 2 -A 2 -A 1 -F 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 400X higher than the EC 50 in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula la in which Fi and F 2 have been cleaved by the tumor specific proteases.
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 - P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 500X higher than the EC 50 in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 600X higher than the EC 50 in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 700X higher than the EC 50 in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 800X higher than the EC 50 in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 900X higher than the EC 50 in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 - P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 1000X higher than the EC 50 in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least IO,OOOC higher than the EC 50 in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay as compared to the EC 50 in a T-cell cytolysis assay of an isolated polypeptide or polypeptide complex that does not have Pi, Li, P 2 , or L 2 .
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 10X higher than the EC 50 in a T-cell cytolysis assay of a form of the isolated polypeptide or polypeptide complex of Formula la that does not have Pi, Li, P 2 , or L 2 .
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 50X higher than the EC 50 in a T-cell cytolysis assay of a form of the isolated polypeptide or polypeptide complex of Formula la that does not have Pi, Li, P 2 , or L 2 .
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 - L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 75X higher than the EC 50 in a T-cell cytolysis assay of a form of the isolated polypeptide or polypeptide complex of Formula la that does not have Pi, Li, P 2 , or L 2 .
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 100X higher than the EC 50 in a T-cell cytolysis assay of an isolated polypeptide or polypeptide complex that does not have Pi, Li, P 2 , or L 2 .
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 - A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 200X higher than the EC 50 in a T-cell cytolysis assay of an isolated polypeptide or polypeptide complex that does not have Pi, Li, P 2 , or L 2 .
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 300X higher than the EC 50 in a T-cell cytolysis assay of an isolated polypeptide or polypeptide complex that does not have Pi, Li, P 2 , or L 2 .
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 400X higher than the EC 50 in a T-cell cytolysis assay of an isolated polypeptide or polypeptide complex that does not have Pi, Li, P 2 , or L 2 .
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 500X higher than the EC 50 in a T-cell cytolysis assay of an isolated polypeptide or polypeptide complex that does not have Pi, Li, P 2 , or L 2 .
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 600X higher than the EC 50 in a T-cell cytolysis assay of an isolated polypeptide or polypeptide complex that does not have Pi, Li, P 2 , or L 2 .
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 700X higher than the EC 50 in a T-cell cytolysis assay of an isolated polypeptide or polypeptide complex that does not have Pi, Li, P 2 , or L 2 .
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 800X higher than the EC 50 in a T-cell cytolysis assay of an isolated polypeptide or polypeptide complex that does not have Pi, Li, P 2 , or L 2 .
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 900X higher than the EC 50 in a T-cell cytolysis assay of an isolated polypeptide or polypeptide complex that does not have Pi, Li, P 2 , or L 2 .
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 1000X higher than the EC 50 in a T-cell cytolysis assay of an isolated polypeptide or polypeptide complex that does not have Pi, Li, P 2 , or L 2 .
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least IO,OOOC higher than the EC 50 in a T-cell cytolysis assay of an isolated polypeptide or polypeptide complex that does not have Pi, Li, P 2 , or L 2 .
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay as compared to the EC 50 in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 10X higher than the EC 50 in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula la in which Li and L 2 have been cleaved by the tumor specific proteases.
  • the isolated polypeptide or polypeptide complex P 2 -L 2 -A 2 -A 1 -L 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 50X higher than the EC 50 in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula la in which Fi and F 2 have been cleaved by the tumor specific proteases.
  • the isolated polypeptide or polypeptide complex P 2 -F 2 -A 2 -A 1 -L 1 - P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 75X higher than the EC 50 in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula la in which Fi and F 2 have been cleaved by the tumor specific proteases.
  • the isolated polypeptide or polypeptide complex P 2 -F 2 -A 2 -A 1 -F 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 100X higher than the EC 50 in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula la in which Fi and F 2 have been cleaved by the tumor specific proteases.
  • the isolated polypeptide or polypeptide complex P 2 -F 2 -A 2 -A 1 -F 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 200X higher than the EC 50 in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula la in which Fi and F 2 have been cleaved by the tumor specific proteases.
  • the isolated polypeptide or polypeptide complex P 2 -F 2 -A 2 -A 1 -F 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 300X higher than the EC 50 in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula la in which Fi and F 2 have been cleaved by the tumor specific proteases.
  • the isolated polypeptide or polypeptide complex P 2 -F 2 -A 2 -A 1 -F 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 400X higher than the EC 50 in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula la in which Fi and F 2 have been cleaved by the tumor specific proteases.
  • the isolated polypeptide or polypeptide complex P 2 -F 2 -A 2 -A 1 -L 1 - P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 500X higher than the EC 50 in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula la in which Fi and F 2 have been cleaved by the tumor specific proteases.
  • the isolated polypeptide or polypeptide complex P 2 -F 2 -A 2 -A 1 -F 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 600X higher than the EC 50 in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula la in which Fi and F 2 have been cleaved by the tumor specific proteases.
  • the isolated polypeptide or polypeptide complex P 2 -F 2 -A 2 -A 1 -F 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 700X higher than the EC 50 in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula la in which Fi and F 2 have been cleaved by the tumor specific proteases.
  • the isolated polypeptide or polypeptide complex P 2 -F 2 -A 2 -A 1 -F 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 800X higher than the EC 50 in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula la in which Fi and F 2 have been cleaved by the tumor specific proteases.
  • the isolated polypeptide or polypeptide complex P 2 -F 2 -A 2 -A 1 -F 1 -P 1 -H 1 has an increased EC 50 in a T-cell cytolysis assay that is at least 900X higher than the EC50 in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula la in which Li and L2 have been cleaved by the tumor specific proteases.
  • the isolated polypeptide or polypeptide complex P2-F2-A2-A1-L1- P1-H1 has an increased EC50 in a T-cell cytolysis assay that is at least 1000X higher than the EC50 in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula la in which Fi and F2 have been cleaved by the tumor specific proteases.
  • the isolated polypeptide or polypeptide complex P2-F2-A2-A1-F1-P1-H1 has an increased EC50 in a T-cell cytolysis assay that is at least IO,OOOC higher than the EC50 in a T-cell cytolysis assay of the isolated polypeptide or polypeptide complex of Formula la in which Fi and F2 have been cleaved by the tumor specific proteases.
  • A2 comprises an antibody or antibody fragment.
  • the antibody or antibody fragment thereof comprises a single chain variable fragment, a single domain antibody, a Fab, or a Fab’.
  • 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 (VF domain), a variable domain (VHH) of a camelid derived single domain antibody.
  • the antibody or antibody fragment thereof is humanized or human.
  • A2 is the Fab or Fab’.
  • the Fab or Fab’ comprises (a) a Fab light chain polypeptide and (b) a Fab heavy chain polypeptide.
  • the antibody or antibody fragment thereof comprises a EGFR binding domain.
  • the antigen binding fragment (Fab) or Fab' that binds to EGFR comprises a Fab light chain polypeptide chain and a Fab heavy chain polypeptide.
  • the Fab light chain polypeptide comprises a Fab light chain variable domain.
  • the Fab heavy chain polypeptide comprises a Fab heavy chain variable domain.
  • 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%,
  • 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).
  • CDR complementarity determining regions
  • 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).
  • CDR complementarity determining regions
  • the Fab comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the Fab comprise LC-CDR1: SEQ ID NO: 15, LC-CDR2: SEQ ID NO: 16, and LC-CDR3: SEQ ID NO: 17; and the Fab comprises CDRs: HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the Fab comprise: HC-CDR1: SEQ ID NO: 18, HC-CDR2: SEQ ID NO: 19, and HC-CDR3: SEQ ID NO: 20.
  • CDRs complementarity determining regions
  • the Fab comprises complementarity determining regions (CDRs): LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the Fab comprise LC-CDR1: SEQ ID NO: 15, LC-CDR2: SEQ ID NO: 16, and LC-CDR3: SEQ ID NO: 17 and wherein the CDRs comprise from 0-2 amino acid modifications in at least one of the HC-CDR1, HC-CDR2, or HC-CDR3; and the Fab comprises CDRs: HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the Fab comprise: HC-CDR1: SEQ ID NO: 18, HC-CDR2: SEQ ID NO: 19, and HC-CDR3: SEQ ID NO: 20 and wherein the CDRs
  • A2 comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC- CDR1, the LC-CDR2, and the LC-CDR3 of A 2 comprise LC-CDR1: SEQ ID NO: 15, LC-CDR2: SEQ ID NO: 16, and LC-CDR3: SEQ ID NO: 17; and A 2 comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of A 2 comprise: HC-CDR1: SEQ ID NO: 18, HC-CDR2: SEQ ID NO: 19, and HC-CDR3: SEQ ID NO: 20.
  • CDRs complementarity determining regions
  • the Fab light chain polypeptide comprises the amino acid sequence according to SEQ ID NO: 21. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 80% sequence identity according to SEQ ID NO: 21. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 85% sequence identity according to SEQ ID NO: 21. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 90% sequence identity according to SEQ ID NO: 21. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 95% sequence identity according to SEQ ID NO: 21. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 99% sequence identity according to SEQ ID NO: 21.
  • the Fab light chain polypeptide comprises the amino acid sequence according to SEQ ID NO: 22. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 80% sequence identity according to SEQ ID NO: 22. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 85% sequence identity according to SEQ ID NO: 22. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 90% sequence identity according to SEQ ID NO: 22. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 95% sequence identity according to SEQ ID NO: 22. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 99% sequence identity according to SEQ ID NO: 22.
  • the Fab heavy chain polypeptide comprises the amino acid sequence according to SEQ ID NO: 23. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 80% sequence identity according to SEQ ID NO: 23. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 85% sequence identity according to SEQ ID NO: 23. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 90% sequence identity according to SEQ ID NO: 23. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 95% sequence identity according to SEQ ID NO: 23. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 99% sequence identity according to SEQ ID NO: 23.
  • the Fab heavy chain polypeptide comprises the amino acid sequence according to SEQ ID NO: 24. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 80% sequence identity according to SEQ ID NO: 24. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 85% sequence identity according to SEQ ID NO: 24. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 90% sequence identity according to SEQ ID NO: 24. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 95% sequence identity according to SEQ ID NO: 24. In some embodiments, the Fab light chain polypeptide comprises an amino acid sequence that has at least 99% sequence identity according to SEQ ID NO: 24.
  • the Fab light chain polypeptide of A2 is bound to a C-terminus of the single chain variable fragment (scFv) of Ai. In some embodiments, the Fab heavy chain polypeptide of A2 is bound to a C-terminus of the single chain variable fragment (scFv) Ai. In some embodiments, the Fab light chain polypeptide of A2 is bound to a N-terminus of the single chain variable fragment (scFv) of Ai. In some embodiments, the Fab heavy chain polypeptide of A2 is bound to a N-terminus of the single chain variable fragment (scFv) Ai.
  • the Fab heavy chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of Ai. In some embodiments, Fab light chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of Ai. In some embodiments, the Fab heavy chain polypeptide of A2 is bound to the scFv light chain polypeptide of AI. In some embodiments, the Fab light chain polypeptide of A2 is bound to the scFv light chain polypeptide of Ai.
  • A2 further comprises P2 and F2, wherein P2 comprises a peptide that binds to A2; and F2 comprises a linking moiety that connects A2to P2 and is a substrate for a tumor specific protease.
  • the Fab heavy chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of Ai and F2 is bound to the Fab light chain polypeptide of A2.
  • the Fab light chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of Ai and F2 is bound to the Fab heavy chain polypeptide of A2.
  • the Fab heavy chain polypeptide of A2 is bound to the scFv light chain polypeptide of Ai and F2 is bound to the Fab light chain polypeptide of A2. In some embodiments, the Fab light chain polypeptide of A2 is bound to the scFv light chain polypeptide of Ai and F2 is bound to the Fab heavy chain polypeptide of A2.
  • the Fab heavy chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of Ai and F2 is bound to the Fab light chain polypeptide of A2. In some embodiments, the Fab light chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of Ai and F2 is bound to the Fab heavy chain polypeptide of A2. In some embodiments, the Fab heavy chain polypeptide of A2 is bound to the scFv light chain polypeptide of Ai and F2 is bound to the Fab light chain polypeptide of A2. In some embodiments, the Fab light chain polypeptide of A is bound to the scFv light chain polypeptide of Ai and L is bound to the Fab heavy chain polypeptide of A .
  • Pi, P2, or Pi a comprises 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).
  • Pi impairs binding of Ai to a first target antigen. In some embodiments, Pi impairs binding of Ai to the effector cell antigen. In some embodiments, Pi is bound to Ai through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bonding interactions, or a combination thereof. In some embodiments, Pi is bound to Ai at or near an antigen binding site. In some embodiments, Pi becomes unbound from Ai when Li is cleaved by the tumor specific protease thereby exposing Ai to the effector cell antigen. In some embodiments, the protease comprises a tumor specific protease.
  • the protease comprises a matrix metalloprotease (MMP) or a serine protease.
  • MMP matrix metalloprotease
  • the matrix metalloprotease comprises MMP2, MMP7, MMP9, MMP13, or MMP14.
  • the serine protease comprises matriptase (MTSP1), urokinase, or hepsin.
  • MTSP1 matriptase
  • Pi has less than 70% sequence identity to the effector cell antigen.
  • Pi has less than 75% sequence identity to the effector cell antigen.
  • Pi has less than 80% sequence identity to the effector cell antigen.
  • Pi has less than 85% sequence identity to the effector cell antigen.
  • Pi has less than 90% sequence identity to the effector cell antigen. In some embodiments, Pi has less than 95% sequence identity to the effector cell antigen. In some embodiments, Pi has less than 98% sequence identity to the effector cell antigen. In some embodiments, Pi has less than 99% sequence identity to the effector cell antigen. In some embodiments, Pi comprises a de novo amino acid sequence that shares less than 10% sequence identity to the effector cell antigen. In some embodiments, Pi comprises an amino acid sequence according to SEQ ID NO: 25.
  • Ai comprises a first antigen recognizing molecule that comprises an antibody or antibody binding fragment that binds to CD3.
  • Pi comprises an amino acid sequence according to any one of SEQ ID NOs: 25, 797-835, or 843-1690 or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 25, 797- 835, or 843-1690.
  • Pi comprises an amino acid sequence according to Z1-Z2-C-Z4-P-Z6-Z7-Z8- Z9-Z10-Z11- Z12-C-Z14 and Zi is selected from D, Y, F, I, N, V, H, L, A, T, S, and P; Z is selected from D, Y, L, F, I, N, A, V, H, T, and S; Z 4 is selected from G and W; Zb is selected from E, D, V, and P; Z 7 is selected from W, L, F, V, G, M, I, and Y; Zg is selected from E, D, P, and Q; Z 9 is selected from E, D, Y, V, F, W, P, L, and Q; Z 10 is selected from S, D, Y, T, I, F, V, N, A, P, L, and H; Zn is selected from I, Y, F, V, L, T, N, S, D, A,
  • Zi is selected from D, Y, F, I, and N;
  • Z 2 is selected from D, Y, L, F, I, and N;
  • Z 4 is selected from G and W;
  • Zb is selected from E and D;
  • Z 7 is selected from W, L, F, and V;
  • Zs is selected from E and D;
  • Z 9 is selected from E, D, Y, and V;
  • Z 10 is selected from S, D, Y, T, and I;
  • Zn is selected from I, Y, F, V, L, and T;
  • Z 12 is selected from F, D, Y, L, I, V, A, and N;
  • Zi4 is selected from D, Y, N, F, I, and P.
  • Zi is selected from D, Y, and F;
  • Z 2 is selected from D, Y, L, and F;
  • Z 4 is selected from G and W;
  • Zb is selected from E and D;
  • Z 7 is selected from W, L, and F;
  • Zs is selected from E and D;
  • Z 9 is selected from E and D;
  • Z 10 is selected from S, D, and Y;
  • Zn is selected from I, Y, and F;
  • Z 12 is selected from F, D, Y, and L; and Zu is selected from D, Y, and N.
  • Pi comprises an amino acid sequence according to U 1 -U 2 -C-U 4 -P-U6-U 7 -U8- U9-U 10 - U 11 - U 12 -C-U 14 and Ui is selected from D, Y, F, I, N, V, H, L, A, T, S, and P;
  • U 2 is selected from D, Y, L, F, I, N, A, V, H, T, and S;
  • U 4 is selected from G and W;
  • Ur is selected from E, D, V, and P;
  • U 7 is selected from W, L, F, V, G, M, I, and Y;
  • Us is selected from E, D, P, and Q;
  • U 9 is selected from E, D, Y, V, F, W, P, L, and Q;
  • U 10 is selected from S, D, Y, T, I, F, V, N, A, P, L, and H;
  • Un is selected from I, Y, F
  • Ui is selected from D, Y, F, I, V, and N;
  • U 2 is selected from D, Y, L, F, I, and N;
  • U 4 is selected from G and W;
  • U 3 ⁇ 4 is selected from E and D;
  • U 7 is selected from W, L, F, G, and V;
  • Us is selected from E and D;
  • U 9 is selected from E, D, Y, and V;
  • U 10 is selected from S, D, Y, T, and I;
  • Un is selected from I, Y, F, V, L, and T;
  • U 12 is selected from F, D, Y, L, I, V, A, G, and N;
  • U 14 is selected from D, Y, N, F, I, M, and P.
  • Ui is selected from D, Y, V, and F;
  • U 2 is selected from D, Y, L, and F;
  • U 4 is selected from G and W;
  • U 3 ⁇ 4 is selected from E and D;
  • U 7 is selected from W, L, G, and F;
  • Us is selected from E and D;
  • U 9 is selected from E and D;
  • U 10 is selected from S, D, T, and Y ;
  • Un is selected from I, Y, V, L, and F;
  • U 12 is selected from F, D, Y, G, A, and L;
  • Un is selected from D, Y, M, and N.
  • Pi comprises the amino acid sequences according to any one of SEQ ID NOs: 797-823.
  • Pi comprises an amino acid sequences according to any one of SEQ ID Nos: 25, 824-835 and 843-1690.
  • Pi comprises the amino acid sequences according to any of SEQ ID NOs: 824-835.
  • Pi comprises the amino acid sequence according to SEQ ID NO: 810 or an amino acid sequence that has 1, 2, or 3, amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 810.
  • Pi comprises the amino acid sequence according to SEQ ID NO: 811 or an amino acid sequence that has 1, 2, or 3, amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 811.
  • Pi comprises the amino acid sequence according to SEQ ID NO: 834 or an amino acid sequence that has 1, 2, or 3, amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 834.
  • Pi comprises the amino acid sequence according to SEQ ID NO: 810.
  • Pi comprises the amino acid sequence according to SEQ ID NO: 811.
  • Pi comprises the amino acid sequence according to SEQ ID NO: 834.
  • P2 impairs binding of A2to the second target antigen. In some embodiments, wherein P2 impairs binding of A2 to EGFR. In some embodiments, P2 is bound to A2 through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bonding interactions, or a combination thereof. In some embodiments, P2 is bound to A2 at or near an antigen binding site. In some embodiments, P2 becomes unbound from A2 when L2 is cleaved by the tumor specific protease thereby exposing A2 to the EGFR. In some embodiments, the protease comprises a tumor specific protease.
  • the protease comprises a matrix metalloprotease (MMP) or a serine protease.
  • MMP matrix metalloprotease
  • the matrix metalloprotease comprises MMP2, MMP7, MMP9, MMP13, or MMP14.
  • the serine protease comprises matriptase (MTSP1), urokinase, or hepsin.
  • MTSP1 matriptase
  • P2 has less than 70% sequence identity to the EGFR.
  • P2 has less than 75% sequence identity to the EGFR.
  • P2 has less than 80% sequence identity to the EGFR.
  • P2 has less than 85% sequence identity to the EGFR.
  • P2 has less than 90% sequence identity to the EGFR. In some embodiments, P2 has less than 95% sequence identity to the EGFR. In some embodiments, P2 has less than 98% sequence identity to the EGFR. In some embodiments, P2 has less than 99% sequence identity to the EGFR. In some embodiments, P2 comprises a de novo amino acid sequence that shares less than 10% sequence identity to the EGFR. In some embodiments, P2 comprises an amino acid sequence according to SEQ ID NO: 26. In some embodiments, P2 comprises an amino acid sequence according to SEQ ID NO: 115. In some embodiments, P2 comprises an amino acid sequence according to SEQ ID NO: 116.
  • P2 comprises an amino acid sequence according to any one of SEQ ID NOs: 26, 71-96, 98-776, or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 26, 71-96, 98-776.
  • P2 comprises an an amino acid sequence according to X1-C-X2-X3-X4-X5-D-X6-A-X7-P-X8-C-X9 wherein Xi is selected from P and L; X2 is selected from R, L, T, A, N, I, V, S, H, and P; X3 is selected from S, P, F, and Y; X4 is selected from H, L, Q, P, R, F, N; X5 is selected from I, F, Y, H, N, T, S, D, A, L, and V; Xe is selected from T, P, N, L, I, V, S, D, H, A, and Y; X7 is selected from K and Y; Xs is selected from I, P, L, and M; and X9 is selected from A, V, I, T, L, S, D, F, V, and H (SEQ ID NO: 841).
  • P2 comprises an amino acid sequence according to any one of SEQ ID NOs: 26, 86-96, or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of 26, 86-96. In some embodiments, P2 comprises an amino acid sequence according to any one of SEQ ID NOs: 26, 86-96.
  • P2 comprises an amino acid sequence according to any one of SEQ ID NOs:
  • P2 comprises an amino acid sequence according to any one of SEQ ID NOs: SEQ ID NOs: 98-776.
  • P2 comprises an amino acid sequence according to any one of SEQ ID NOs:
  • P2 comprises an amino acid sequence according to any one of SEQ ID NOs: 99-118.
  • P2 comprises an amino acid sequence according to SEQ ID NO: 26 or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to SEQ ID NO: 26. In some embodiments, P2 comprises the amino acid sequence according to SEQ ID NO: 26.
  • P2 comprises an amino acid sequence according to SEQ ID NO: 115 or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to SEQ ID NO: 115. In some embodiments, P2 comprises the amino acid sequence according to SEQ ID NO: 115. [0141] In some embodiments, P2 comprises an amino acid sequence according to SEQ ID NO: 115 or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to SEQ ID NO: 116. In some embodiments, P2 comprises the amino acid sequence according to SEQ ID NO: 116.
  • the antigen recognizing molecule comprises an antibody or antibody fragment.
  • the target antigen is an anti-CD3 effector cell antigen.
  • the target antigen is epidermal growth factor receptor (EGFR).
  • EGFR epidermal growth factor receptor
  • Pi a has less than 70% sequence identity to the target antigen. In some embodiments, Pi a has less than 75% sequence identity to the target antigen. In some embodiments, Pi a has less than 80% sequence identity to the target antigen. In some embodiments, Pi a has less than 85% sequence identity to the target antigen.
  • Pi a has less than 90% sequence identity to the target antigen. In some embodiments, Pi a has less than 95% sequence identity to the target antigen. In some embodiments, Pi a has less than 98% sequence identity to the target antigen. In some embodiments, Pi a has less than 99% sequence identity to the target antigen. In some embodiments, Pi a comprises a de novo amino acid sequence that shares less than 10% sequence identity to the second target antigen.
  • Pi a comprises an amino acid sequence according to any one of SEQ ID NOs: 25, 797-835, or 843-1690 or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 25, 797-835, or 843-1690.
  • Pi a comprises an amino acid sequence according to Z 1 -Z 2 -C-Z 4 -P-Z 6 -Z 7 -Z 8 - Z 9 -Z 10 -Z 11 -Z 12 -C-Z 14 and Zi is selected from D, Y, F, I, N, V, H, L, A, T, S, and P;
  • Z 2 is selected from D, Y, L, F, I, N, A, V, H, T, and S;
  • Z 4 is selected from G and W;
  • Zb is selected from E, D, V, and P;
  • Z 7 is selected from W, L, F, V, G, M, I, and Y;
  • Zs is selected from E, D, P, and Q;
  • Z 9 is selected from E, D, Y, V, F, W, P, L, and Q;
  • Z 10 is selected from S, D, Y, T, I, F, V, N, A, P, L, and H;
  • Zn is selected
  • Zi is selected from D, Y, F, I, and N; Z 2 is selected from D, Y, F, F, I, and N; Z 4 is selected from G and W; Zb is selected from E and D; Z 7 is selected from W, F, F, and V; Zs is selected from E and D; Z 9 is selected from E, D, Y, and V; Z 10 is selected from S, D, Y, T, and I; Zn is selected from I, Y, F, V, F, and T; Z 12 is selected from F, D, Y, F, I, V, A, and N; and Z 14 is selected from D, Y, N, F, I, and P. In some embodiments, Zi is selected from D, Y, and F; Z 2 is selected from D, Y, and F; Z 2 is selected from D, Y,
  • Z 4 is selected from G and W;
  • Zb is selected from E and D;
  • Z 7 is selected from W, F, and F;
  • Zs is selected from E and D;
  • Z 9 is selected from E and D;
  • Z 10 is selected from S, D, and Y;
  • Zn is selected from I, Y, and F; Zuis selected from F, D, Y, and F;
  • Z 14 is selected from D, Y, and N.
  • Pi comprises an amino acid sequence according to U 1 -U 2 -C-U 4 -P-U6-U7-U8- U 9 -U 10 -U 11 - U 12 -C-U 14 and Ui is selected from D, Y, F, I, N, V, H, F, A, T, S, and P;
  • U 2 is selected from D, Y, F, F, I, N, A, V, H, T, and S;
  • U 4 is selected from G and W;
  • Ue is selected from E, D, V, and P;
  • U 7 is selected from W, F, F, V, G, M, I, and Y;
  • Us is selected from E, D, P, and Q;
  • U 9 is selected from E, D, Y, V, F, W, P, F, and Q;
  • U 10 is selected from S, D, Y, T, I, F, V, N, A, P, F, and H;
  • Un is selected from I, Y,
  • Ui is selected from D, Y, F, I, V, and N;
  • U 2 is selected from D, Y, F, F, I, and N;
  • U 4 is selected from G and W;
  • U 3 ⁇ 4 is selected from E and D;
  • U 7 is selected from W, F, F, G, and V;
  • Us is selected from E and D;
  • U 9 is selected from E, D, Y, and V;
  • U 10 is selected from S, D, Y, T, and I;
  • Un is selected from I, Y, F, V, F, and T;
  • U 12 is selected from F, D, Y, F, I, V, A, G, and N;
  • U 14 is selected from D, Y, N, F, I, M, and P.
  • Ui is selected from D, Y, V, and F;
  • U 2 is selected from D, Y, F, and F;
  • U 4 is selected from G and W;
  • Ue is selected from E and D;
  • U 7 is selected from W, F, G, and F;
  • Us is selected from E and D;
  • U 9 is selected from E and D;
  • U 10 is selected from S, D, T, and Y;
  • Un is selected from I, Y, V, F, and F;
  • U 12 is selected from F, D, Y, G, A, and F; and Un is selected from D, Y, M, and N.
  • Pi a comprises the amino acid sequences according to any one of SEQ ID NOs: 797-823.
  • Pi a comprises an amino acid sequences according to any one of SEQ ID NOs: 25, 824-835 or 843-1690. [0147] In some embodiments, Pi a comprises the amino acid sequences according to any of SEQ ID NOs: 824-835.
  • Pi a comprises the amino acid sequence according to SEQ ID NO: 810 or an amino acid sequence that has 1, 2, or 3, amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 810.
  • Pi a comprises the amino acid sequence according to SEQ ID NO: 811 or an amino acid sequence that has 1, 2, or 3, amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 811.
  • Pi a comprises the amino acid sequence according to SEQ ID NO: 834 or an amino acid sequence that has 1, 2, or 3, amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 834.
  • Pi a comprises the amino acid sequence according to SEQ ID NO: 810.
  • Pi a comprises the amino acid sequence according to SEQ ID NO: 811.
  • Pi a comprises the amino acid sequence according to SEQ ID NO: 834.
  • Pi, P2, or Pi a comprises a peptide sequence of at least 5 amino acids in length.
  • Pi, P2, or Pi a comprises a peptide sequence of at least 6 amino acids in length. In some embodiments, Pi, P2, or Pi a comprises a peptide sequence of at least 10 amino acids in length. In some embodiments, Pi, P2, or Pi a comprises a peptide sequence of at least 10 amino acids in length and no more than 20 amino acids in length. In some embodiments, Pi, P2, or Pi a comprises a peptide sequence of at least 16 amino acids in length. In some embodiments, Pi, P2, or Pi a comprises a peptide sequence of no more than 40 amino acids in length. In some embodiments, Pi, P2, or Pi a comprises at least two cysteine amino acid residues.
  • Pi, P2, or Pi a comprises a cyclic peptide or a linear peptide. In some embodiments, Pi, P2, or Pi a comprises a cyclic peptide. In some embodiments, Pi, P2, or Pi a comprises a linear peptide.
  • Pi, P2, or Pi a or Pi, P2, and Pi a comprise a modified amino acid or non-natural amino acid, or a modified non-natural amino acid, or a combination thereof.
  • the modified amino acid or a modified non-natural amino acid comprises a post-translational modification.
  • Pi, P2, or Pi a or Pi, P2, and Pi a comprise 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 argin
  • Pi, P2, or Pi a or Pi, P2, and Pi a including the peptide backbone, the amino acid side chains, and the terminus.
  • Pi, P2, or Pi a does not comprise albumin or an albumin fragment. In some embodiments, Pi, P2, or Pi a does not comprise an albumin binding domain.
  • Linking Moiety Li, L 2 , L 3 , and Li a
  • Li, L2, L3, or Li a is a peptide sequence having at least 5 to no more than 50 amino acids. In some embodiments Li, L2, L3, or Li a is a peptide sequence having at least 10 to no more than 30 amino acids. In some embodiments, Li, L2, L3, or Li a is a peptide sequence having at least 10 amino acids. In some embodiments, Li, L2, L3, or Li a is a peptide sequence having at least 18 amino acids. In some embodiments, Li, L2, L3, or Li a is a peptide sequence having at least 26 amino acids. In some embodiments, Li, L2, L3, or Lia has a formula comprising (G2S) n , wherein n is an integer from 1 to 3 (SEQ ID NO: 840).
  • Li, L2, L3, or Li a has a formula comprising (G2S) n , wherein n is an integer of at least 1.
  • Li, L2, L3, or Li a has a formula selected from the group consisting of (G2S) n , (GS) n , (GSGGS) context (SEQ ID NO: 836), (GGGS) dislike (SEQ ID NO: 837) , (GGGGS) connector (SEQ ID NO: 838) , and(GSSGGS)trust (SEQ ID NO: 839) , wherein n is an integer of at least 1.
  • the tumor specific protease is selected from the group consisting of metalloprotease, serine protease, cysteine protease, threonine protease, and aspartic protease.
  • Li, L2, L3, or Li a comprises a urokinase cleavable amino acid sequence, amatriptase (MTSP1) cleavable amino acid sequence, a legumain cleavable amino acid sequence, or a matrix metalloprotease cleavable amino acid sequence.
  • the matrix metalloprotease comprises MMP2, MMP7, MMP9, MMP13, or MMP14.
  • the serine protease comprises matriptase (MTSP1), urokinase, or hepsin.
  • Li, L2, L3, or Li a comprises 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).
  • Li comprises an amino acid sequence of Linker 4 (ISSGLLSGRSDAG)
  • Li comprises an amino acid sequence of Linker 4 (ISSGLLSGRSDAG) (SEQ ID NO: 66). In some embodiments, Li comprises an amino acid sequence of Linker 5 (AAGLLAPPGGLSGRSDAG) (SEQ ID NO: 67). In some embodiments, Li comprises an amino acid sequence of Linker 6 (SPLGLSGRSDAG) (SEQ ID NO: 68). In some embodiments, Li comprises an amino acid sequence of Linker 7 (LSGRSDAGSPLGLAG) (SEQ ID NO: 66), Linker 5 (AAGLLAPPGGLSGRSDAG) (SEQ ID NO: 67), Linker 6 (SPLGLSGRSDAG) (SEQ ID NO: 68). In some embodiments, Li comprises an amino acid sequence of Linker 7 (LSGRSDAGSPLGLAG) (SEQ ID NO:
  • L2 comprises an amino acid sequence of Linker 4 (ISSGLLSGRSDAG)
  • L2 comprises an amino acid sequence of Linker 4 (ISSGLLSGRSDAG) (SEQ ID NO: 66).
  • Li comprises an amino acid sequence of Linker 5 (AAGLLAPPGGLSGRSDAG) (SEQ ID NO: 67).
  • L2 comprises an amino acid sequence of Linker 6 (SPLGLSGRSDAG) (SEQ ID NO: 68). In some embodiments, L2 comprises an amino acid sequence of Linker 7 (LSGRSDAGSPLGLAG) (SEQ ID NO: 69).
  • Li is bound to N-terminus of Ai. In some embodiments, Li is bound to C- tenninus of Ai. In some embodiments, L2IS bound to N-terminus of A2. In some embodiments, L2IS bound to C-terminus of A2. In some embodiments, Pi becomes unbound from Ai when Li is cleaved by the tumor specific protease thereby exposing Ai to the effector cell antigen. In some embodiments, P2 becomes unbound from A2 when L2 is cleaved by the tumor specific protease thereby exposing A2 to EGFR.
  • Li, L 2 , L 3 , or Li a comprise a modified amino acid or non-natural amino acid, or a modified non-natural amino acid, or a combination thereof.
  • the modified amino acid or a modified non-natural amino acid comprises a post-translational modification.
  • Li, L 2 , L 3 , or Li a comprise 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 arginy
  • the cleavable linker is cleavable by a protease.
  • 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.
  • the protease comprises a tumor specific protease.
  • the protease comprises a matrix metalloprotease (MMP) or a serine protease.
  • MMP matrix metalloprotease
  • the matrix metalloprotease comprises MMP2, MMP7, MMP9, MMP 13, or MMP 14.
  • 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 MMP 13. In some embodiments, the matrix metalloprotease comprises MMP14. In some embodiments, the serine protease comprises matriptase (MTSP1), urokinase, or hepsin.
  • MTSP1 matriptase
  • urokinase urokinase
  • hepsin hepsin.
  • 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.
  • Hi does not block Ai binding to the effector cell antigen.
  • Hi comprises a linking moiety (L3) that connects Hi to Pi.
  • Hi a does not block the first antigen recognizing molecule binding to the effector cell antigen.
  • Hi a comprises a linking moiety (L3) that connects Hi a to Pi a .
  • half-life extending molecule does not have binding affinity to antigen recognizing molecule.
  • half-life extending molecule does not have binding affinity to the effector cell antigen.
  • half-life extending molecule does not shield antigen recognizing molecule from the effector cell antigen. In some embodiments, half-life extending molecule (Hi or Hi a ) is not directly linked to antigen recognizing molecule.
  • Hi or Hi a comprises 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).
  • Hi or Hi a comprise an amino acid sequence that has repetitive sequence motifs. In some embodiments, Hi or Hi a comprises 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 Hi or Hi a contribute to secondary structure, as measured or determined by means, including, but not limited to, spectrophotometry (e.g. by circular dichroism spectroscopy in the “far-UV” spectral region (190-250 nm), and computer programs or algorithms, such as the Chou-Fasman algorithm and the Gamier-Osguthorpe-Robson (“GOR”) algorithm.
  • spectrophotometry e.g. by circular dichroism spectroscopy in the “far-UV” spectral region (190-250 nm
  • computer programs or algorithms such as the Chou-Fasman algorithm and the Gamier-Osguthorpe-Robson (“GOR”) algorithm.
  • Hi or Hi a comprises a polymer.
  • the polymer is polyethylene glycol (PEG).
  • Hi or Hi a comprises albumin.
  • Hi or Hi a comprises an Fc domain.
  • the albumin is serum albumin.
  • the albumin is human serum albumin.
  • Hi or Hi a comprises a polypeptide, a ligand, or a small molecule.
  • 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.
  • 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.
  • the circulating immunoglobulin molecule comprises IgGl, IgG2, IgG3, IgG4, slgA, IgM or IgD.
  • the serum protein is albumin.
  • the polypeptide is an antibody.
  • the antibody comprises a single domain antibody, a single chain variable fragment or a Fab.
  • the single domain antibody comprises a single domain antibody that binds to albumin.
  • the single domain antibody is a human or humanized antibody. In some embodiments, the single domain antibody is selected from the group consisting of 645gHlgLl, 645dsgH5gL4, 23-13-A01 -sc02, A10m3 or a fragment thereof, DOM7r-31, DOM7h-ll-15, Alb-1, Alb-8, Alb-23, 10G, 10E and SA21.
  • the single domain antibody comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the single domain antibody comprise: HC- CDR1: SEQ ID NO: 36, HC-CDR2: SEQ ID NO: 37, and HC-CDR3: SEQ ID NO: 38.
  • CDRs complementarity determining regions
  • the single domain antibody comprises complementarity determining regions (CDRs): HC- CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the single domain antibody comprise: HC-CDR1: SEQ ID NO: 36, HC-CDR2: SEQ ID NO: 37, and HC-CDR3: SEQ ID NO: 38; and wherein the CDRs comprise from 0-2 amino acid modifications in at least one of the HC- CDR1, HC-CDR2, or HC-CDR3.
  • CDRs complementarity determining regions
  • the single domain antibody comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC- CDR1, the HC-CDR2, and the HC-CDR3 of the single domain antibody comprise: HC-CDR1: SEQ ID NO: 39, HC-CDR2: SEQ ID NO: 40, and HC-CDR3: SEQ ID NO: 41.
  • CDRs complementarity determining regions
  • the single domain antibody comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the single domain antibody comprise: HC- CDR1: SEQ ID NO: 39, HC-CDR2: SEQ ID NO: 40, and HC-CDR3: SEQ ID NO: 41; and wherein the CDRs comprise from 0-2 amino acid modifications in at least one of the HC-CDR1, HC-CDR2, or HC- CDR3.
  • CDRs complementarity determining regions
  • Hi comprises an amino acid sequence according to SEQ ID NO: 42. In some embodiments, Hi comprises an amino acid sequence that has at least 80% sequence identity to SEQ ID NO:
  • Hi comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 42. In some embodiments, Hi comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 42. In some embodiments, Hi comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 42. In some embodiments, Hi comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NO: 42.
  • Hi a comprises an amino acid sequence according to SEQ ID NO: 42. In some embodiments, Hi a comprises an amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 42. In some embodiments, Hi a comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 42. In some embodiments, Hi a comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 42. In some embodiments, Hi a comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 42. In some embodiments, Hi a comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NO: 42.
  • Hi comprises an amino acid sequence according to SEQ ID NO: 43. In some embodiments, Hi comprises an amino acid sequence that has at least 80% sequence identity to SEQ ID NO:
  • Hi comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 43. In some embodiments, Hi comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 43. In some embodiments, Hi comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 43. In some embodiments, Hi comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NO: 43. [0171] In some embodiments, Hi a comprises an amino acid sequence according to SEQ ID NO: 43. In some embodiments, Hi a comprises an amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 43.
  • Hi a comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 43. In some embodiments, Hi a comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 43. In some embodiments, Hi a comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 43. In some embodiments, Hi a comprises an amino acid sequence that has at least 99% sequence identity to SEQ ID NO: 43.
  • Hi or Hi a or Hi and Hi a comprise a modified amino acid or non-natural amino acid, or a modified non-natural amino acid, or a combination thereof.
  • the modified amino acid or a modified non-natural amino acid comprises a post-translational modification.
  • Hi or Hi a or Hi and Hi a comprise 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 ubi
  • Hi comprises a linking moiety (L3) that connects Hi to Pi .
  • L is a peptide sequence having at least 5 to no more than 50 amino acids. In some embodiments, L is a peptide sequence having at least 10 to no more than 30 amino acids. In some embodiments, L is a peptide sequence having at least 10 amino acids. In some embodiments, L is a peptide sequence having at least 18 amino acids. In some embodiments, L is a peptide sequence having at least 26 amino acids.
  • L has a formula selected from the group consisting of (G S) n , (GS) n , (GSGGS) context (SEQ ID NO: 836), (GGGS) protest , (SEQ ID NO: 837) (GGGGS) format (SEQ ID NO: 838) , and (GSSGGS)fact (SEQ ID NO: 839) , wherein n is an integer of at least 1.
  • L comprises an amino acid sequence according to SEQ ID NO: 29.
  • Hi a comprises a linking moiety (Li a ) that connects Hi a to Pi a.
  • Li a is a peptide sequence having at least 5 to no more than 50 amino acids.
  • Li a is a peptide sequence having at least 10 to no more than 30 amino acids.
  • Li a is a peptide sequence having at least 10 amino acids.
  • Li a is a peptide sequence having at least 18 amino acids.
  • Li a is a peptide sequence having at least 26 amino acids.
  • Li a has a formula selected from the group consisting of (G S) n , (GS) context, (GSGGS) context (SEQ ID NO: 836), (GGGS) format (SEQ ID NO: 837) , (GGGGS) connector (SEQ ID NO: 838) , and (GSSGGS) n (SEQ ID NO: 839) , wherein n is an integer of at least 1.
  • Li a comprises an amino acid sequence according to SEQ ID NO: 30 or 31.
  • 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).
  • the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NOs: 44-61.
  • the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 50.
  • the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 51.
  • the isolated polypeptide or polypeptide complex comprises amino acid sequences according to SEQ ID NO: 44 and SEQ ID NO: 45. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 90% sequence identity to SEQ ID NO: 44 and SEQ ID NO: 45. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 95% sequence identity to SEQ ID NO: 44 and SEQ ID NO: 45. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 99% sequence identity to SEQ ID NO: 44 and SEQ ID NO: 45.
  • the isolated polypeptide or polypeptide complex comprises amino acid sequences according to SEQ ID NO: 46 and SEQ ID NO: 47. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 90% sequence identity to SEQ ID NO: 46 and SEQ ID NO: 47. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 95% sequence identity to SEQ ID NO: 46 and SEQ ID NO: 47. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 99% sequence identity to SEQ ID NO: 46 and SEQ ID NO: 47.
  • the isolated polypeptide or polypeptide complex comprises amino acid sequences according to SEQ ID NO: 48 and SEQ ID NO: 49. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 90% sequence identity to SEQ ID NO: 48 and SEQ ID NO: 49. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 95% sequence identity to SEQ ID NO: 48 and SEQ ID NO: 49. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 99% sequence identity to SEQ ID NO: 48 and SEQ ID NO: 49.
  • the isolated polypeptide or polypeptide complex comprises amino acid sequences according to SEQ ID NO: 50 and SEQ ID NO: 51. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 90% sequence identity to SEQ ID NO: 50 and SEQ ID NO: 51. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 95% sequence identity to SEQ ID NO: 50 and SEQ ID NO: 51. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 99% sequence identity to SEQ ID NO: 50 and SEQ ID NO: 51.
  • the isolated polypeptide or polypeptide complex comprises amino acid sequences according to SEQ ID NO: 52 and SEQ ID NO: 53. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 90% sequence identity to SEQ ID NO: 52 and SEQ ID NO: 53. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 95% sequence identity to SEQ ID NO: 52 and SEQ ID NO: 53. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 99% sequence identity to SEQ ID NO: 52 and SEQ ID NO: 53.
  • the isolated polypeptide or polypeptide complex comprises amino acid sequences according to SEQ ID NO: 54 and SEQ ID NO: 55. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 90% sequence identity to SEQ ID NO: 54 and SEQ ID NO: 55. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 95% sequence identity SEQ ID NO: 54 and SEQ ID NO: 55. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 99% sequence identity to SEQ ID NO: 54 and SEQ ID NO: 55.
  • the isolated polypeptide or polypeptide complex comprises amino acid sequences according to SEQ ID NO: 56 and SEQ ID NO: 57. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 90% sequence identity to SEQ ID NO: 56 and SEQ ID NO: 57. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 95% sequence identity SEQ ID NO: 56 and SEQ ID NO: 57. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 99% sequence identity to SEQ ID NO: 56 and SEQ ID NO: 57.
  • the isolated polypeptide or polypeptide complex comprises amino acid sequences according to SEQ ID NO: 58 and SEQ ID NO: 59. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 90% sequence identity to SEQ ID NO: 58 and SEQ ID NO: 59. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 95% sequence identity SEQ ID NO: 58 and SEQ ID NO: 59. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 99% sequence identity to SEQ ID NO: 58 and SEQ ID NO: 59.
  • the isolated polypeptide or polypeptide complex comprises amino acid sequences according to SEQ ID NO: 60 and SEQ ID NO: 61. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 90% sequence identity to SEQ ID NO: 60 and SEQ ID NO: 61. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 95% sequence identity SEQ ID NO: 60 and SEQ ID NO: 61. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 99% sequence identity to SEQ ID NO: 60 and SEQ ID NO: 61.
  • the isolated polypeptide or polypeptide complex comprises amino acid sequences according to SEQ ID NO: 62 and SEQ ID NO: 63. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 90% sequence identity to SEQ ID NO: 62 and SEQ ID NO: 63. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 95% sequence identity SEQ ID NO: 62 and SEQ ID NO: 63. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 99% sequence identity to SEQ ID NO: 62 and SEQ ID NO: 63.
  • the isolated polypeptide or polypeptide complex comprises amino acid sequences according to SEQ ID NO: 64 and SEQ ID NO: 65. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 90% sequence identity to SEQ ID NO: 64 and SEQ ID NO: 65. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 95% sequence identity SEQ ID NO: 64 and SEQ ID NO: 65. In some embodiments, the isolated polypeptide or polypeptide complex comprises amino acid sequences with at least 99% sequence identity to SEQ ID NO: 64 and SEQ ID NO: 65.
  • Polypeptides or polypeptide complexes comprise a sequence set forth in Table 6.
  • the sequence comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 44-65.
  • the sequence comprises at least or about 95% homology to SEQ ID NOs: 44-65.
  • the sequence comprises at least or about 97% homology to SEQ ID NOs: 44-65.
  • the sequence comprises at least or about 99% homology to SEQ ID NOs: 44-65.
  • the sequence comprises at least or about 100% homology to SEQ ID NOs: 44-65. In some instances, the sequence comprises at least a portion having at least or about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, or more than 210 amino acids of any one of SEQ ID NOs: 44, 46, 48, 50, 52, 54, 56, 58, 60, 63, or 65. In some instances, the sequence comprises at least a portion having at least or about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170,
  • 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,
  • 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.
  • the % amino acid sequence identity of a given amino acid sequence A 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.
  • polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 1 :
  • 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 (Pi) that impairs binding of the scFv to an effector cell antigen and Pi is linked to a N-terminus of the heavy chain variable domain of the scFv with a linking moiety (Li) that is a substrate for a tumor specific protease, and Pi is further linked to a half-life extending molecule; and a Fab that binds to epidermal growth factor receptor (EGFR), wherein the Fab comprises a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab light chain polypeptide is linked to a C terminus of the light chain variable domain of the scFv, and wherein the Fab is linked to P and L , wherein P comprises a peptide that impairs binding to
  • polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 2:
  • 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 (Pi) that impairs binding of the scFv to an effector cell antigen and Pi is linked to a N-terminus of the heavy chain variable domain of the scFv with a linking moiety (Li) that is a substrate for a tumor specific protease, and Pi is further linked to a half-life extending molecule; and a Fab that binds to epidermal growth factor receptor (EGFR), wherein the Fab comprises a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab heavy chain polypeptide is linked to a C terminus of the light chain variable domain of the scFv, and wherein the Fab is linked to P and L , wherein P comprises a peptide that impairs binding to
  • polypeptides or polypeptide complexes as disclosed herein.
  • the polypeptides or polypeptide complexes comprise an antibody or an antibody fragment.
  • the polypeptides or polypeptide complexes comprise a Fab and a single chain variable fragment (scFv).
  • nucleic acid molecules encoding polypeptides or polypeptide complexes according to Formula I:
  • A2-AI-LI-P l-Hi (Formula I) wherein: Ai comprises a first antigen recognizing molecule that binds to an effector cell antigen; Pi comprises a peptide that binds to Ai; Li comprises a linking moiety that connects Ai to Pi and is a substrate for a tumor specific protease; Hi comprises a half-life extending molecule; and A comprises a second antigen recognizing molecule that binds to epidermal growth factor receptor (EGFR).
  • EGFR epidermal growth factor receptor
  • nucleic acid molecules encoding polypeptides or polypeptide complexes according to Formula I:
  • Ai is a first antigen recognizing molecule that binds to an effector cell antigen
  • Pi is a peptide that binds to Ai
  • Li is a linking moiety that connects Ai to Pi and is a substrate for a tumor specific protease
  • Hi is a half-life extending molecule
  • A is a second antigen recognizing molecule that binds to epidermal growth factor receptor (EGFR).
  • nucleic acid molecules encoding polypeptides or polypeptide complexes comprising Formula I:
  • Ai comprises a first antigen recognizing molecule that binds to an effector cell antigen
  • Pi comprises a peptide that binds to Ai
  • Li comprises a linking moiety that connects Ai to Pi and is a substrate for a tumor specific protease
  • Hi comprises a half-life extending molecule
  • A comprises a second antigen recognizing molecule that binds to epidermal growth factor receptor (EGFR).
  • EGFR epidermal growth factor receptor
  • nucleic acid molecules encoding polypeptides or polypeptide complexes comprising Formula I:
  • Ai is a first antigen recognizing molecule that binds to an effector cell antigen
  • Pi is a peptide that binds to Ai
  • Li is a linking moiety that connects Ai to Pi and is a substrate for a tumor specific protease
  • Hi is a half-life extending molecule
  • A2 is a second antigen recognizing molecule that binds to epidermal growth factor receptor (EGFR).
  • nucleic acid molecules encoding polypeptides or polypeptide complexes according to Formula la:
  • nucleic acid molecules encoding polypeptides or polypeptide complexes according to Formula II:
  • Fi a comprises a tumor specific protease-cleaved linking moiety that when uncleaved connects Pi a to 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 epidermal growth factor receptor (EGFR);
  • Pi a comprises a peptide that binds to the first antigen recognizing molecule when Fi a is uncleaved;
  • Hi a comprises a half-life extending molecule.
  • nucleic acid molecules encoding polypeptides or polypeptide complexes comprising Formula II:
  • Fi a comprises a tumor specific protease-cleaved linking moiety that when uncleaved connects Pi a to a first antigen recognizing molecule that binds to an effector cell antigen and the first antigen recognizing molecule is connected to a second antigen recognizing molecule that binds to epidermal growth factor receptor (EGFR);
  • Pi a comprises a peptide that binds to the first antigen recognizing molecule when Li a is uncleaved;
  • Hi a comprises a half-life extending molecule.
  • nucleic acid molecules encoding polypeptides or polypeptide complexes according to Formula II:
  • Li a is a tumor specific protease-cleaved linking moiety that when uncleaved connects Pi a to a first antigen recognizing molecule that binds to an effector cell antigen and the first antigen recognizing molecule is connected to a second antigen recognizing molecule that binds to epidermal growth factor receptor (EGFR); Pi a is a peptide that binds to the first antigen recognizing molecule when Li a is uncleaved; and Hi a is a half-life extending molecule.
  • EGFR epidermal growth factor receptor
  • nucleic acid molecules encoding polypeptides or polypeptide complexes comprising Formula II: L a -P la -H a
  • Li a is a tumor specific protease-cleaved linking moiety that when uncleaved connects Pi a to a first antigen recognizing molecule that binds to an effector cell antigen and the first antigen recognizing molecule is connected to a second antigen recognizing molecule that binds to epidermal growth factor receptor (EGFR); Pi a is a peptide that binds to the first antigen recognizing molecule when Li a is uncleaved; and Hi a is a half-life extending molecule.
  • EGFR epidermal growth factor receptor
  • nucleic acid molecules encoding polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 1:
  • 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 (Pi) that impairs binding of the scFv to an effector cell antigen and Pi is linked to a N-terminus of the heavy chain variable domain of the scFv with a linking moiety (Fi) that is a substrate for a tumor specific protease, and Pi is further linked to a half-life extending molecule; and a Fab that binds to epidermal growth factor receptor (EGFR), wherein the Fab comprises a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab light chain polypeptide is linked to a C terminus of the light chain variable domain of the scFv, and wherein the Fab is linked to P 2 and F 2 , wherein P 2 comprises a peptide that impairs
  • nucleic acid molecules encoding polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 2:
  • 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 (Pi) that impairs binding of the scFv to an effector cell antigen and Pi is linked to a N-terminus of the heavy chain variable domain of the scFv with a linking moiety (Li) that is a substrate for a tumor specific protease, and Pi is further linked to a half-life extending molecule; and a Fab that binds to epidermal growth factor receptor (EGFR), wherein the Fab comprises a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab heavy chain polypeptide is linked to a C terminus of the light chain variable domain of the scFv, and wherein the Fab is linked to P2 and L2, wherein P2 comprises a peptide that impairs binding
  • compositions comprising: (a) the polypeptides or polypeptide complexes as disclosed herein; and (b) a pharmaceutically acceptable excipient.
  • the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes according to Formula I:
  • A2-AI-LI-P l-Hi (Formula I) wherein: Ai comprises a first antigen recognizing molecule that binds to an effector cell antigen; Pi comprises a peptide that binds to Ai; Li comprises a linking moiety that connects Ai to Pi and is a substrate for a tumor specific protease; Hi comprises a half-life extending molecule; and A2 comprises a second antigen recognizing molecule that binds to epidermal growth factor receptor (EGFR); and (b) a pharmaceutically acceptable excipient.
  • EGFR epidermal growth factor receptor
  • the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes according to Formula I:
  • Ai is a first antigen recognizing molecule that binds to an effector cell antigen
  • Pi is a peptide that binds to Ai
  • Li is a linking moiety that connects Ai to Pi and is a substrate for a tumor specific protease
  • Hi is a half-life extending molecule
  • A2 is a second antigen recognizing molecule that binds to epidermal growth factor receptor (EGFR); and (b) a pharmaceutically acceptable excipient.
  • the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes comprising Formula I:
  • Ai comprises a first antigen recognizing molecule that binds to an effector cell antigen
  • Pi comprises a peptide that binds to Ai
  • Li comprises a linking moiety that connects Ai to Pi and is a substrate for a tumor specific protease
  • Hi comprises a half-life extending molecule
  • A2 comprises a second antigen recognizing molecule that binds to epidermal growth factor receptor (EGFR); and (b) a pharmaceutically acceptable excipient.
  • the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes comprising Formula I:
  • Ai is a first antigen recognizing molecule that binds to an effector cell antigen
  • Pi is a peptide that binds to Ai
  • Fi is a linking moiety that connects Ai to Pi and is a substrate for a tumor specific protease
  • Hi is a half-life extending molecule
  • A2 is a second antigen recognizing molecule that binds to epidermal growth factor receptor (EGFR); and (b) a pharmaceutically acceptable excipient.
  • the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes according to Formula la:
  • the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes according to Formula II:
  • Fi a comprises a tumor specific protease-cleaved linking moiety that when uncleaved connects Pi a to a first antigen recognizing molecule that binds to an effector cell antigen and the first antigen recognizing molecule is connected to a second antigen recognizing molecule that binds to epidermal growth factor receptor (EGFR);
  • Pi a comprises a peptide that binds to the first antigen recognizing molecule when Li a is uncleaved; and
  • Hi a comprises a half-life extending molecule; and (b) a pharmaceutically acceptable excipient.
  • the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes comprising Formula II:
  • Li a comprises a tumor specific protease-cleaved linking moiety that when uncleaved connects Pi a to a first antigen recognizing molecule that binds to an effector cell antigen and the first antigen recognizing molecule is connected to a second antigen recognizing molecule that binds to epidermal growth factor receptor (EGFR); Pi a comprises a peptide that binds to the first antigen recognizing molecule when Li a is uncleaved; and Hi a comprises a half-life extending molecule; and (b) a pharmaceutically acceptable excipient.
  • EGFR epidermal growth factor receptor
  • the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes according to Formula II: Lla Pla Hia
  • Li a is a tumor specific protease-cleaved linking moiety that when uncleaved connects Pi a to a first antigen recognizing molecule that binds to an effector cell antigen and the first antigen recognizing molecule is connected to a second antigen recognizing molecule that binds to epidermal growth factor receptor (EGFR); Pi a is a peptide that binds to the first antigen recognizing molecule when Li a is uncleaved; and Hi a is a half-life extending molecule; and (b) a pharmaceutically acceptable excipient.
  • EGFR epidermal growth factor receptor
  • the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes comprising Formula II:
  • Li a is a tumor specific protease-cleaved linking moiety that when uncleaved connects Pi a to a first antigen recognizing molecule that binds to an effector cell antigen and the first antigen recognizing molecule is connected to a second antigen recognizing molecule that binds to epidermal growth factor receptor (EGFR); Pi a is a peptide that binds to the first antigen recognizing molecule when Li a is uncleaved; and Hi a is a half-life extending molecule; and (b) a pharmaceutically acceptable excipient.
  • EGFR epidermal growth factor receptor
  • the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 1:
  • 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 (Pi) that impairs binding of the scFv to an effector cell antigen and Pi is linked to a N-terminus of the heavy chain variable domain of the scFv with a linking moiety (Li) that is a substrate for a tumor specific protease, and Pi is further linked to a half-life extending molecule; and a Fab that binds to epidermal growth factor receptor (EGFR), wherein the Fab comprises a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab light chain polypeptide is linked to a C terminus of the light chain variable domain of the scFv, and wherein the Fab is linked to P and L , wherein P comprises a peptide that impairs binding to
  • the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes comprising a structural arrangement according to Configuration 2:
  • 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 (Pi) that impairs binding of the scFv to an effector cell antigen and Pi is linked to a N-terminus of the heavy chain variable domain of the scFv with a linking moiety (Li) that is a substrate for a tumor specific protease, and Pi is further linked to a half-life extending molecule; and a Fab that binds to epidermal growth factor receptor (EGFR), wherein the Fab comprises a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab heavy chain polypeptide is linked to a C terminus of the light chain variable domain of the scFv, and wherein the Fab is linked to P and L , wherein P comprises a peptide that impairs binding to
  • the isolated polypeptide or polypeptide complex further comprises a detectable label, a therapeutic agent, or a pharmacokinetic modifying moiety.
  • the detectable label comprises a fluorescent label, a radiolabel, an enzyme, a nucleic acid probe, or a contrast agent.
  • 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.
  • 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.
  • suitable pharmaceutical carriers 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.
  • 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. [0218]
  • 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.
  • polypeptide or polypeptide complex comprising an anti-EGFR binding domain that are linked to a peptide that impairs binding of the anti-EGFR binding domain to EGFR wherein the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 26, 71-96, 98-776 or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 26, 71-96, 98- 776.
  • the peptide comprises an amino acid sequence according to any one of 26, 71-96, 98-776.
  • the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 26, 86-96 or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 26, 86-96.
  • the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 26, 86-96.
  • the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 98-776 or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any of SEQ ID NOs: 98-776.
  • the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 98-776.
  • the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 99-118 or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 99-118.
  • the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 99-118.
  • the peptide comprises the amino acid sequence according to SEQ ID NO: 71 or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to SEQ ID NO: 71. [0229] In some embodiments, the peptide comprises the amino acid sequence according to SEQ ID NO: 71.
  • the peptide comprises the amino acid sequence according to SEQ ID NO: 26 or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to SEQ ID NO: 26. [0231] In some embodiments, the peptide comprises the amino acid sequence according to SEQ ID NO: 26. [0232] In some embodiments, the peptide comprises the amino acid sequence according to SEQ ID NO: 115 or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to SEQ ID NO:
  • the peptide comprises the amino acid sequence according to SEQ ID NO: 115.
  • the peptide comprises the amino acid sequence according to SEQ ID NO: 116 or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to SEQ ID NO:
  • the peptide comprises the amino acid sequence according to SEQ ID NO: 116.
  • polypeptide or polypeptide complex comprising an anti-EGFR binding domain that are linked to a peptide that impairs binding of the anti-EGFR binding to EGFR
  • the peptide comprises an amino acid sequence according to X1-C-X2-X3-X4-X5-D-X6-A-X7-P-X8-C-X9 wherein Xi is selected from P and L; X2 is selected from R, L, T, A, N, I, V, S, H, and P; X3 is selected from S, P, F, and Y; X4 is selected from H, L, Q, P, R, F, N; X5 is selected from I, F, Y, H, N, T, S, D, A, L, and V; Xe is selected from T, P, N, L, I, V, S, D, H, A, and Y; X7 is selected from K and Y; Xs is selected from I,
  • the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 98-776. In some embodiments, the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 99-118. In some embodiments, the peptide comprises the amino acid sequence according to SEQ ID NO: 26. In some embodiments, the peptide comprises the amino acid sequence according to SEQ ID NO: 115. In some embodiments, the peptide comprises the amino acid sequence according to SEQ ID NO: 116.
  • the anti-EGFR binding domain comprises an antibody or an antibody fragment.
  • the antibody or antibody fragment comprises a single chain variable fragment, a single domain antibody, Fab, or Fab’.
  • the anti-EGFR binding domain comprises heavy chain complementarity determining regions HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC- CDR2, and the HC-CDR3 comprise: HC-CDR1: SEQ ID NO: 15, HC-CDR2: SEQ ID NO: 16, and HC-CDR3:
  • the anti-EGFR binding domain comprises light chain complementarity determining regions CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the Fab comprise FC-CDR1: SEQ ID NO: 18, FC-CDR2: SEQ ID NO: 19, and LC-CDR3: SEQ ID NO: 20.
  • the antibody or antibody fragment comprises the Fab.
  • the anti-EGFR binding domain comprises an amino acid sequences according to any of SEQ ID NOs: 21-24.
  • the anti-EGFR binding domain is linked to the peptide through a linking moiety (Li).
  • Li is a substrate for a tumor specific protease.
  • Li is bound to N- terminus of the anti-EGFR binding domain.
  • Li is bound to C-terminus of the anti-EGFR binding domain.
  • Li is a peptide sequence having at least 5 to no more than 50 amino acids.
  • Li is a peptide sequence having at least 10 to no more than 30 amino acids. In some embodiments, Li is a peptide sequence having at least 10 amino acids. In some embodiments, Li is a peptide sequence having at least 18 amino acids. In some embodiments, Li is a peptide sequence having at least 26 amino acids. In some embodiments, Li has a formula comprising (G2S) n , wherein n is an integer from 1 to 3 (SEQ ID NO: 840).
  • Li has a formula selected from the group consisting of (G2S) n , (GS) n , (GSGGS) n (SEQ ID NO: 836), (GGGS)format (SEQ ID NO: 837) , (GGGGS) thread (SEQ ID NO: 838) , and (GSSGGS)trust (SEQ ID NO: 839) , wherein n is an integer of at least 1.
  • the peptide becomes unbound from the anti-EGFR binding domain when Li is cleaved by the tumor specific protease thereby exposing the anti-EGFR binding domain to EGFR.
  • the tumor specific protease is selected from the group consisting of a matrix metalloprotease (MMP), serine protease, cysteine protease, threonine protease, and aspartic protease.
  • MMP2 matrix metalloprotease
  • the matrix metalloprotease comprises MMP2, MMP7, MMP9, MMP13, or MMP14.
  • the serine protease comprises matriptase (MTSP1), urokinase, or hepsin.
  • Li comprises 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.
  • Li comprises an amino acid sequence according to any of SEQ ID NO: 30 or 31.
  • Li comprises an amino acid sequence according to SEQ ID NO: 27-35.
  • Li comprises an amino acid sequence of of Linker 4 (ISSGLLSGRSDAG) (SEQ ID NO: 66), Linker 5 (AAGLLAPPGGLSGRSDAG) (SEQ ID NO: 67), Linker 6 (SPLGLSGRSDAG) (SEQ ID NO: 68), or Linker 7 (LSGRSDAGSPLGLAG) (SEQ ID NO: 69), or an amino acid sequence that has 1, 2, or 3 amino acid substitutions, additions, or deletions relative to the amino acid sequence of Linker 4, Linker 5, Linker 6, or Linker 7.
  • the isolated polypeptide or polypeptide complex further comprises a half-life extending molecule (Hi).
  • Hi comprises a polymer.
  • the polymer is polyethylene glycol (PEG).
  • Hi comprises albumin.
  • Hi comprises an Fc domain.
  • the albumin is serum albumin.
  • the albumin is human serum albumin.
  • Hi comprises a polypeptide, a ligand, or a small molecule.
  • 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.
  • 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.
  • the circulating immunoglobulin molecule comprises IgGl, IgG2, IgG3, IgG4, slgA, IgM or IgD.
  • the serum protein is albumin.
  • the polypeptide is an antibody.
  • the antibody comprises a single domain antibody, a single chain variable fragment, or a Fab.
  • the single domain antibody comprises a single domain antibody that binds to albumin.
  • the single domain antibody is a human or humanized antibody.
  • the single domain antibody is 645gHlgLl.
  • the single domain antibody is 645dsgH5gL4.
  • the single domain antibody is 23-13-A01 -sc02.
  • the single domain antibody is A10m3 or a fragment thereof.
  • the single domain antibody is DOM7r-31.
  • the single domain antibody is DOM7h-ll-15.
  • the single domain antibody is Alb-1, Alb-8, or Alb-23. In some embodiments, the single domain antibody is 10E. In some embodiments, the single domain antibody comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the single domain antibody comprise: HC-CDR1: SEQ ID NO: 36, HC-CDR2: SEQ ID NO: 37, and HC-CDR3: SEQ ID NO: 38. In some embodiments, the single domain antibody comprises an amino acid sequence according to SEQ ID NO: 69.
  • the single domain antibody comprises complementarity determining regions (CDRs): HC- CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the single domain antibody comprise: HC-CDR1: SEQ ID NO: 36, HC-CDR2: SEQ ID NO: 37, and HC-CDR3: SEQ ID NO: 38.
  • the single domain antibody comprises an amino acid sequence according to SEQ ID NO: 42.
  • the single domain antibody is SA21.
  • 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.
  • the modified amino acid or modified non-natural amino acid comprises a post-translational modification.
  • Hi comprises a linking moiety (L3) that connects Hi to the peptide. .
  • L 3 is a peptide sequence having at least 5 to no more than 50 amino acids.
  • L 3 is a peptide sequence having at least 10 to no more than 30 amino acids.
  • L 3 is a peptide sequence having at least 10 amino acids.
  • L 3 is a peptide sequence having at least 18 amino acids.
  • L 3 is a peptide sequence having at least 26 amino acids.
  • L3 has a formula selected from the group consisting of (G 2 S) n , (GS) n , (GSGGS) n (SEQ ID NO: 836), (GGGS)format (SEQ ID NO: 837) , (GGGGS) connector (SEQ ID NO: 838) , and(GSSGGS)trust (SEQ ID NO: 839) , wherein n is an integer of at least 1.
  • L 3 comprises an amino acid sequence according to SEQ ID NO: 29.
  • the polypeptides or polypeptide complexes described herein are used in a method of treating cancer.
  • the cancer has cells that express EGFR.
  • the polypeptides or polypeptide complexes described herein are used in a method of treating colorectal cancer (CRC), squamous cell carcinoma of the head and Neck (SCCHN), non-small cell lung cancer (NSCLC), prostate cancer, breast cancer, colon/rectum cancer, head and neck cancer, esophagogastric cancer, liver cancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, kidney cancer, or pancreatic cancer.
  • CRC colorectal cancer
  • SCCHN squamous cell carcinoma of the head and Neck
  • NSCLC non-small cell lung cancer
  • prostate cancer breast cancer, colon/rectum cancer, head and neck cancer
  • esophagogastric cancer liver cancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, kidney cancer, or pancreatic cancer.
  • the polypeptides or polypeptide complexes described herein are used in a method of treating subjects who are resistant to EGFR inhibitor treatment. In some embodiments, the polypeptides or polypeptide complexes described herein are used in a method of treating subjects who harbor KRAS mutations. In some embodiments, the polypeptides or polypeptide complexes described herein are used in a method of treating subjects who are resistant to EGFR inhibitor treatment and harbor KRAS mutations.
  • polypeptides or polypeptide complexes wherein the polypeptides or polypeptide complexes comprise a long half-life.
  • the half-life of the polypeptides or polypeptide complexes is at least or about 12 hours, 24 hours 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, 100 hours, 108 hours, 120 hours, 140 hours, 160 hours, 180 hours, 200 hours, or more than 200 hours.
  • 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.
  • polypeptide or polypeptide complexes administered as once weekly.
  • the polypeptide or polypeptide complexes are administered once weekly by intravenous, intramuscular, intralesional, topical, subcutaneous, infusion, or oral.
  • the polypeptide or polypeptide complexes are administered once weekly by bolus injection.
  • the polypeptide or polypeptide complexes are administered once weekly by continuous infusion.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • polypeptides described herein 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.
  • 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.
  • chemically synthesized oligonucleotides e.g., as described in Kutmeier et al., 1994, BioTechniques 17:242
  • 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.
  • a suitable source e.g., an antibody cDNA library, or cDNA library generated from any tissue or cells expressing the immunoglobulin
  • an antibody or its binding is optionally generated by immunizing an animal, such as a mouse, to generate polyclonal antibodies or, more preferably, by generating monoclonal antibodies, e.g., as described by Kohler and Milstein (1975, Nature 256:495-497) or, as described by Kozbor et al. (1983, Immunology Today 4:72) or Cole et al. (1985 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
  • 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).
  • chimeric antibodies 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.
  • single chain antibodies 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).
  • 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.
  • the expression of the antibody is regulated by a constitutive, an inducible or a tissue, specific promoter.
  • host-expression vector systems is utilized to express an antibody, or its binding fragment described herein.
  • 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.
  • 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.
  • 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
  • cell lines that stably express an antibody are optionally engineered.
  • 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.
  • appropriate expression control elements e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
  • 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.
  • 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 (Fowy et al., 1980, Cell 22:817) genes are employed in tk-, hgprt- or aprt- cells, respectively.
  • 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.
  • 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)).
  • 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)
  • 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 ak, 1983, Mol. Cell Biol. 3:257).
  • 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.
  • chromatography e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography
  • centrifugation e.g., centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
  • vectors include any suitable vectors derived from either a eukaryotic or prokaryotic sources.
  • 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-l, 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 Mil, pVL1393 Ml 2, FLAG vectors such as pPolh-FLAGl or pPolh-MAT 2, or MAT vectors such as pPolh-MAT 1, or pPolh-MAT2.
  • yeast vectors include Gateway® pDESTTM 14 vector, Gateway® pDESTTM 15 vector, Gateway® pDESTTM 17 vector, Gateway® pDESTTM 24 vector, Gateway® pYES-DEST52 vector, pBAD-DEST49 Gateway® destination vector, pA0815 Pichia vector, pFLDl Pichi pastoris vector, pGAPZA,B, & C Pichia pastoris vector, pPIC3.5K Pichia vector, pPIC6 A, B, & C Pichia vector, pPIC9K Pichia vector, pTEFl/Zeo, pYES2 yeast vector, pYES2/CT yeast vector, pYES2/NT A, B, & C yeast vector, or pYES3/CT yeast vector.
  • Exemplary algae vectors include pChlamy-4 vector or MCS vector.
  • Examples of mammalian vectors include transient expression vectors or stable expression vectors. Mammalian transient expression vectors may include pRK5, p3xFLAG-CMV 8, pFLAG-Myc-CMV 19, pFFAG-Myc-CMV 23, pFFAG-CMV 2, pFFAG-CMV 6a,b,c, pFFAG-CMV 5.1, pFFAG-CMV 5a,b,c, p3xFFAG-CMV 7.1, pFFAG-CMV 20, p3xFFAG-Myc-CMV 24, pCMV -FFAG-MAT 1 , pCMV-FFAG- MAT2, pBICEP-CMV 3, or pBICEP-CMV 4.
  • Mammalian stable expression vector may include pFFAG- CMV 3, p3xFFAG-CMV 9, p3xFFAG-CMV 13, pFFAG-Myc-CMV 21, p3xFFAG-Myc-CMV 25, pFFAG-CMV 4, p3xFFAG-CMV 10, p3xFFAG-CMV 14, pFFAG-Myc-CMV 22, p3xFFAG-Myc-CMV 26, pBICEP-CMV 1, or pBICEP-CMV 2.
  • 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.
  • a cell-free system utilizes either prokaryotic cell components or eukaryotic cell components.
  • a nucleic acid synthesis is obtained in a cell-free system based on for example Drosophila cell, Xenopus egg, or HeFa 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
  • a host cell includes any suitable cell such as a naturally derived cell or a genetically modified cell.
  • a host cell is a production host cell.
  • a host cell is a eukaryotic cell.
  • a host cell is a prokaryotic cell.
  • a eukaryotic cell includes fungi (e.g., yeast cells), animal cell or plant cell.
  • 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.
  • gram-positive bacteria include Actinobacteria, Firmicutes or Tenericutes.
  • gram-negative bacteria include Aquificae, Deinococcus-Thermus, Fibrobacteres- Chlorobi/Bacteroidetes (FCB group), Fusobacteria, Gemmatimonadetes, Nitrospirae, Planctomycetes- Verrucomicrobia/ Chlamydiae (PVC group), Proteobacteria, Spirochaetes or Synergistetes.
  • 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, MachlTM, DH10BTM, TOP10, DH5a, DHlOBacTM, OmniMaxTM, MegaXTM, DH12STM, INV110, TOP10F’, INVaF, TOP10/P3, ccdB Survival, PIR1, PIR2, Stbl2TM, Stbl3TM, or Stbl4TM.
  • animal cells include a cell from a vertebrate or from an invertebrate.
  • an animal cell includes a cell from a marine invertebrate, fish, insects, amphibian, reptile, or mammal.
  • 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.
  • yeast includes Ascomycota or Basidiomycota.
  • Ascomycota includes Saccharomycotina (true yeasts, e.g. Saccharomyces cerevisiae (baker’s yeast)) or Taphrinomycotina (e.g. Schizosaccharomycetes (fission yeasts)).
  • 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 ory
  • Schizosaccharomyces pombe Torulaspora delbrueckii, Zygosaccharomyces bailii, Cryptococcus neoformans, Cryptococcus gattii, or Saccharomyces boulardii.
  • Exemplary yeast host cells include, but are not limited to, Pichia pastoris yeast strains such as GS115, KM71H, SMD1168, SMD1168H, and X-33; and Saccharomyces cerevisiae yeast strain such as INVScl.
  • additional animal cells include cells obtained from a mollusk, arthropod, annelid or sponge.
  • an additional animal cell is a mammalian cell, e.g., from a primate, ape, equine, bovine, porcine, canine, feline or rodent.
  • 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, Expi293FTM cells, Flp-InTM T-RExTM 293 cell line, Flp-InTM-293 cell line, Flp-InTM-3T3 cell line, Flp-InTM-BHK cell line, Flp-InTM-CHO cell line, Flp-InTM-CV-l cell line, Flp-InTM-Jurkat cell line, FreeStyleTM 293-F cells, FreeStyleTM CHO-S cells, GripTiteTM 293 MSR cell line, GS-CHO cell line, HepaRGTM cells, T-RExTM Jurkat cell line, Per.C6 cells, T-RExTM-293 cell line, T-RExTM-CHO cell line, and T-RExTM-HeFa cell line.
  • 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.
  • 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 FiveTM cells, and expresSF+® cells.
  • plant cells include a cell from algae.
  • Exemplary insect cell lines include, but are not limited to, strains from Chlamydomonas reinhardtii 137c, or Synechococcus elongatus PPC 7942.
  • an article of manufacture containing materials useful for the treatment, prevention and/or diagnosis of the disorders described above comprises a container and a label or package insert on or associated with the container.
  • Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper that is pierceable by a hypodermic injection needle).
  • At least one active agent in the composition is a bispecific antibody comprising a first antigen-binding site that specifically binds to CD3 and a second antigen-binding site that specifically binds to EGFR as defined herein before.
  • the label or package insert indicates that the composition is used for treating the condition of choice.
  • 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.
  • 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.
  • BWFI bacteriostatic water for injection
  • phosphate-buffered saline such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution.
  • BWFI bacteriostatic water for injection
  • phosphate-buffered saline such as phosphate-buffered saline, Ringer's solution and dextrose solution.
  • dextrose solution such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer'
  • 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.
  • CDR complementarity determining 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.
  • Fab refers to a protein that contains the constant domain of the light chain and the first constant domain (CHI) 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 CHI 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 (VF) 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 VF, 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).
  • antibody fragments comprise single chain polypeptides having the characteristics of a VH domain, namely being able to assemble together with a VF domain, or of a VF 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.
  • Embodiment 1 comprises an isolated polypeptide or polypeptide complex according to Formula I: A2-A1-L1-P1-H1 wherein Ai comprises a first antigen recognizing molecule that binds to an effector cell antigen; Pi comprises a peptide that binds to Ai; Li comprises a linking moiety that connects Ai to Pi and is a substrate for a tumor specific protease; Hi comprises a half-life extending molecule; and A2 comprises a second antigen recognizing molecule that binds to epidermal growth factor receptor (EGFR).
  • Embodiment 2 comprises the isolated polypeptide or polypeptide complex of embodiment 1, wherein the first antigen recognizing molecule comprises an antibody or antibody fragment.
  • Embodiment 3 comprises the isolated polypeptide or polypeptide complex of embodiment 1, wherein first antigen recognizing molecule comprises an antibody or antibody fragment that is human or humanized.
  • Embodiment 4 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-3, wherein Li is bound to N-terminus of the first antigen recognizing molecule.
  • Embodiment 5 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-3, wherein A is bound to C-terminus of the first antigen recognizing molecule.
  • Embodiment 6 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-3, wherein Li is bound to C-terminus of the first antigen recognizing molecule.
  • Embodiment 7 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-3, wherein A is bound to N-terminus of the first antigen recognizing molecule.
  • Embodiment 8 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 2-7, wherein the antibody or antibody fragment comprises a single chain variable fragment, a single domain antibody, or a Fab fragment.
  • Embodiment 9 comprises the isolated polypeptide or polypeptide complex of embodiment 8, wherein Ai is the single chain variable fragment (scFv).
  • Embodiment 10 comprises the isolated polypeptide or polypeptide complex of embodiment 9, wherein the scFv comprises a scFv heavy chain polypeptide and a scFv light chain polypeptide.
  • Embodiment 11 comprises the isolated polypeptide or polypeptide complex of embodiment 8, wherein Ai is the single domain antibody,
  • Embodiment 12 comprises the isolated polypeptide or polypeptide complex of embodiment 8, wherein the antibody or antibody fragment comprises a single chain variable fragment (scFv), a heavy chain variable domain (VH domain), a light chain variable domain (VL domain), or a variable domain (VHH) of a came lid derived single domain antibody.
  • scFv single chain variable fragment
  • VH domain heavy chain variable domain
  • VL domain light chain variable domain
  • VHH variable domain
  • Embodiment 13 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-12, wherein Ai comprises an anti-CD3e single chain variable fragment.
  • Embodiment 14 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-12, wherein Ai comprises an anti-CD3e single chain variable fragment that has a KD binding of 1 mM or less to CD3 on CD3 expressing cells.
  • Embodiment 15 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-14, wherein the effector cell antigen comprises CD3.
  • Embodiment 16 comprises the isolated polypeptide or polypeptide complex of embodiment 1, wherein Ai comprises a variable light chain and variable heavy chain each of which is capable of specifically binding to human CD3.
  • Embodiment 17 comprises the isolated polypeptide or polypeptide complex of embodiment 1, wherein Ai comprises 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, FI 11-409, CLB-T3.4.2, TR-66, WT32, SPv- T3b, 11D8, XIII- 141, XIII-46, XIII-87, 12F6, T3/RW2-8C8, T3/RW2-4B6, OKT3D, M-T301
  • CDRs
  • Embodiment 18 comprises the isolated polypeptide or polypeptide complex of embodiment 1, wherein the isolated polypeptide or polypeptide complex of Formula I binds to an effector cell when Fi is cleaved by the tumor specific protease.
  • Embodiment 19 comprises the isolated polypeptide or polypeptide complex of embodiment 1, wherein the isolated polypeptide or polypeptide complex of Formula I binds to an effector cell when Fi is cleaved by the tumor specific protease and Albinds to the effector cell.
  • Embodiment 20 comprises the isolated polypeptide or polypeptide complex of embodiment 19, wherein the effector cell is a T cell.
  • Embodiment 21 comprises the isolated polypeptide or polypeptide complex of embodiment 1, wherein Ai binds to a polypeptide that is part of a TCR-CD3 complex on the effector cell.
  • Embodiment 22 comprises the isolated polypeptide or polypeptide complex of embodiment 21, wherein the polypeptide that is part of the TCR-CD3 complex is human CD3s.
  • Embodiment 23 comprises the isolated polypeptide or polypeptide complex of embodiment 1, wherein the effector cell antigen comprises CD3, wherein the scFv comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the scFv LC-CDR1: SEQ ID NO: 1, LC-CDR2: SEQ ID NO: 2, and LC-CDR3: SEQ ID NO: 3; and the scFv comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC- CDR2, and the HC-CDR3 of the scFv: HC-CDR1: SEQ ID NO: 4, HC-CDR2: SEQ ID NO: 5, and HC- CDR3: SEQ ID NO: 6.
  • the effector cell antigen comprises
  • Embodiment 24 comprises the isolated polypeptide or polypeptide complex of embodiment 1, wherein the effector cell antigen comprises CD3, and Ai comprises CDRs: LC-CDR1, LC-CDR2, and LC- CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of Ai comprise LC-CDR1: SEQ ID NO: 1, LC-CDR2: SEQ ID NO: 2, and LC-CDR3: SEQ ID NO: 3; and Ai comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of Ai comprise: HC-CDR1: SEQ ID NO: 4, HC-CDR2: SEQ ID NO: 5, and HC-CDR3: SEQ ID NO: 6.
  • CDRs complementarity determining regions
  • Embodiment 25 comprises the isolated polypeptide or polypeptide complex of embodiment 1, wherein the effector cell antigen comprises CD3, and the scFv comprises an amino acid sequence according to SEQ ID NO: 13.
  • Embodiment 26 comprises the isolated polypeptide or polypeptide complex of embodiment 1, wherein the effector cell antigen comprises CD3, wherein the scFv comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the scFv LC-CDR1: SEQ ID NO: 7, LC-CDR2: SEQ ID NO: 8, and LC-CDR3: SEQ ID NO: 9; and the scFv comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC- CDR2, and the HC-CDR3 of the scFv: HC-CDR1: SEQ ID NO: 10, HC-CDR2: SEQ ID NO: 11, and HC- CDR3: SEQ ID NO: 12.
  • the effector cell antigen comprises
  • Embodiment 27 comprises the isolated polypeptide or polypeptide complex of embodiment 1, wherein the effector cell antigen comprises CD3, and Ai comprises CDRs: LC-CDR1, LC-CDR2, and LC- CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of Ai comprise: LC-CDR1: SEQ ID NO: 7, LC-CDR2: SEQ ID NO: 8, and LC-CDR3: SEQ ID NO: 9; and Ai comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC- CDR2, and the HC-CDR3 of Ai comprise: HC-CDR1: SEQ ID NO: 10, HC-CDR2: SEQ ID NO: 11, and HC-CDR3: SEQ ID NO: 12.
  • Embodiment 28 comprises the isolated polypeptide or polypeptide complex of embodiment 1, wherein the effector cell antigen comprises CD3, and the scFv comprises an amino acid sequence according to SEQ ID NO: 14.
  • Embodiment 29 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-28, wherein second antigen recognizing molecule comprises an antibody or antibody fragment.
  • Embodiment 30 comprises the isolated polypeptide or polypeptide complex of embodiment 29, wherein the antibody or antibody fragment thereof comprises a single chain variable fragment, a single domain antibody, or a Fab.
  • Embodiment 31 comprises the isolated polypeptide or polypeptide complex of embodiment 29, wherein the antibody or antibody fragment thereof comprises a single chain variable fragment (scFv), a heavy chain variable domain (VH domain), a light chain variable domain (VL domain), a variable domain (VHH) of a camelid derived single domain antibody.
  • scFv single chain variable fragment
  • VH domain heavy chain variable domain
  • VL domain light chain variable domain
  • VHH variable domain
  • Embodiment 32 comprises the isolated polypeptide or polypeptide complex of embodiment 29, wherein the antibody or antibody fragment thereof is humanized or human.
  • Embodiment 33 comprises the isolated polypeptide or polypeptide complex of embodiment 30, wherein A2 is the Fab.
  • Embodiment 34 comprises the isolated polypeptide or polypeptide complex of embodiment 33, wherein the Fab comprises (a) a Fab light chain polypeptide and (b) a Fab heavy chain polypeptide.
  • Embodiment 35 comprises the isolated polypeptide or polypeptide complex of embodiment 33, wherein the Fab comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC- CDR2, and the LC-CDR3 of the Fab comprise LC-CDR1: SEQ ID NO: 15, LC-CDR2: SEQ ID NO: 16, and LC-CDR3: SEQ ID NO: 17; and the Fab comprises complementarity determining regions (CDRs): HC- CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the Fab comprise: HC-CDR1: SEQ ID NO: 18, HC-CDR2: SEQ ID NO: 19, and HC-CDR3: SEQ ID NO: 20.
  • the Fab comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR
  • Embodiment 36 comprises the isolated polypeptide or polypeptide complex of embodiment 29, wherein A2 comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of A 2 comprise LC-CDR1: SEQ ID NO: 15, LC-CDR2: SEQ ID NO: 16, and LC-CDR3: SEQ ID NO: 17; and A 2 comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of A2 comprise: HC-CDR1: SEQ ID NO: 18, HC-CDR2: SEQ ID NO: 19, and HC-CDR3: SEQ ID NO: 20.
  • A2 comprises CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC
  • Embodiment 37 comprises the isolated polypeptide or polypeptide complex of embodiment 34, wherein the Fab light chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 21.
  • Embodiment 38 comprises the isolated polypeptide or polypeptide complex of embodiment 34, wherein the Fab light chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 22.
  • Embodiment 39 comprises the isolated polypeptide or polypeptide complex of embodiment 34, wherein Fab heavy chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 23.
  • Embodiment 40 comprises the isolated polypeptide or polypeptide complex of embodiment 34, wherein Fab heavy chain polypeptide comprises an amino acid sequence according to SEQ ID NO: 24.
  • Embodiment 41 comprises the isolated polypeptide or polypeptide complex of embodiment 34, wherein the Fab light chain polypeptide of A 2 is bound to a C-terminus of the single chain variable fragment (scFv) of Ai.
  • Embodiment 42 comprises the isolated polypeptide or polypeptide complex of embodiment 34, wherein the Fab heavy chain polypeptide of A 2 is bound to a C-terminus of the single chain variable fragment (scFv) Ai.
  • Embodiment 43 comprises the isolated polypeptide or polypeptide complex of embodiment 34, wherein the Fab light chain polypeptide of A 2 is bound to a N-terminus of the single chain variable fragment (scFv) of Ai.
  • Embodiment 44 comprises the isolated polypeptide or polypeptide complex of embodiment 34, wherein the Fab heavy chain polypeptide of A 2 is bound to a N-terminus of the single chain variable fragment (scFv) Ai.
  • Embodiment 45 comprises the isolated polypeptide or polypeptide complex of embodiment 34, wherein the Fab heavy chain polypeptide of A 2 is bound to the scFv heavy chain polypeptide of Ai .
  • Embodiment 46 comprises the isolated polypeptide or polypeptide complex of embodiment 34, wherein the Fab light chain polypeptide of A 2 is bound to the scFv heavy chain polypeptide of Ai.
  • Embodiment 47 comprises the isolated polypeptide or polypeptide complex of embodiment 34, wherein the Fab heavy chain polypeptide of A 2 is bound to the scFv light chain polypeptide of Ai.
  • Embodiment 48 comprises the isolated polypeptide or polypeptide complex of embodiment 34, wherein the Fab light chain polypeptide of A 2 is bound to the scFv light chain polypeptide of Ai.
  • Embodiment 49 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-48, wherein A 2 further comprises P 2 and L 2 , wherein P 2 comprises a peptide that binds to A 2 ; and L 2 comprises a linking moiety that connects A 2 to P 2 and is a substrate for a tumor specific protease.
  • Embodiment 50 comprises the isolated polypeptide or polypeptide complex of embodiment 49, wherein the isolated polypeptide or polypeptide complex is according to Formula la: P2-L2-A2-A1-L1-P1-H1.
  • Embodiment 51 comprises the isolated polypeptide or polypeptide complex of embodiment 50, wherein the Fab heavy chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of Ai and L2 is bound to the Fab light chain polypeptide of A2.
  • Embodiment 52 comprises the isolated polypeptide or polypeptide complex of embodiment 50, wherein the Fab light chain polypeptide of A2 is bound to the scFv heavy chain polypeptide of Ai and F2 is bound to the Fab heavy chain polypeptide of A2.
  • Embodiment 53 comprises the isolated polypeptide or polypeptide complex of embodiment 50, wherein the Fab heavy chain polypeptide of A2 is bound to the scFv light chain polypeptide of Ai and F2 is bound to the Fab light chain polypeptide of A2.
  • Embodiment 54 comprises the isolated polypeptide or polypeptide complex of embodiment 50, wherein the Fab light chain polypeptide of A2 is bound to the scFv light chain polypeptide of Ai and F2 is bound to the Fab heavy chain polypeptide of A2.
  • Embodiment 55 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-54, wherein Pi impairs binding of Ai to the effector cell antigen.
  • Embodiment 56 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-55, wherein Pi is bound to Ai through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bonding interactions, or a combination thereof.
  • Embodiment 57 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-56, wherein Pi has less than 70% sequence homology to the effector cell antigen.
  • Embodiment 58 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 49-57, wherein P2 impairs binding of A2 to EGFR.
  • Embodiment 59 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 49-58, wherein P2 is bound to A2 through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bonding interactions, or a combination thereof.
  • Embodiment 60 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 49-59, wherein P2 is bound to A2 at or near an antigen binding site.
  • Embodiment 61 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 49-60, wherein P2 has less than 70% sequence homology to EGFR.
  • Embodiment 62 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-61, wherein Pi or P2 comprises a peptide sequence of at least 10 amino acids in length.
  • Embodiment 63 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-62, wherein Pi or P2 comprises a peptide sequence of at least 10 amino acids in length and no more than 20 amino acids in length.
  • Embodiment 64 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-63, wherein Pi or P2 comprises a peptide sequence of at least 16 amino acids in length.
  • Embodiment 65 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-61, wherein Pi or P comprises a peptide sequence of no more than 40 amino acids in length.
  • Embodiment 66 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-61, wherein Pi or P comprises at least two cysteine amino acid residues.
  • Embodiment 67 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-61, wherein Pi or P comprises a cyclic peptide or a linear peptide.
  • Embodiment 68 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-61, wherein Pi or P comprises a cyclic peptide.
  • Embodiment 69 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-61, wherein Pi or P comprises a linear peptide.
  • Embodiment 70 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-61, wherein Pi comprises at least two cysteine amino acid residues.
  • Embodiment 71 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-61, wherein Pi comprises an amino acid sequence according to SEQ ID NO: 25.
  • Embodiment 72 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-61, wherein P comprises an amino acid sequence according to SEQ ID NO: 26.
  • Embodiment 73 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-61, wherein Pi comprises an amino acid sequence according to SEQ ID NO: 70.
  • Embodiment 74 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-61, wherein P comprises an amino acid sequence according to any one of SEQ ID NOs: 26, 71-96, 98-776, or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 26, 71-96, 98-776.
  • Embodiment 75 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-61, wherein P 2 comprises an an amino acid sequence according to X 1 -C-X 2 -X 3 -X 4 -X 5 -D-X 6 - A-X 7 -P-X 8 -C-X 9 wherein Xi is selected from P and L; X 2 is selected from R, L, T, A, N, I, V, S, H, and P;
  • X 3 is selected from S, P, F, and Y;
  • X 4 is selected from H, L, Q, P, R, F, N;
  • X 5 is selected from I, F, Y, H, N, T, S, D, A, L, and V;
  • Xe is selected from T, P, N, L, I, V, S, D, H, A, and Y;
  • X 7 is selected from K and Y ;
  • Xs is selected from I, P, L, and M;
  • X 9 is selected from A, V, I, T, L, S, D, F, V, and H (SEQ ID NO: 841).
  • Embodiment 76 comprises the isolated polypeptide or polypeptide complex of embodiment 75, wherein Xi is selected from P and L; X 2 is selected from R, L, T, A, and N; X 3 is selected from S, P, and F; X 4 is selected from H, L, Q, and P; X 5 is selected from I, F, Y, H, N, and T; Xe is selected from T, P, N, L, I, and V; X 7 is K; Xs is I; and X 9 is selected from A, V, I, T, L, and S.
  • Embodiment 77 comprises the isolated polypeptide or polypeptide complex of embodiment 76, wherein Xi is P; X 2 is selected from R, L, and T; X 3 is S; X 4 is selected from H, L, Q, and P; X 5 is selected from I, F, Y, and T; X ( , is selected from T, P, N, and V; X 7 is K; Xs is I; and X 9 is selected from A, V, and I.
  • Embodiment 78 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-77, wherein P comprises an amino acid sequence according to any one of SEQ ID NOs: 26, 86-96, or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 26, 86-96.
  • Embodiment 79 comprises the isolated polypeptide or polypeptide complex of embodiment 78, wherein P 2 comprises an amino acid sequence according to any one of SEQ ID NOs: 26, 86-96.
  • Embodiment 80 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-77, wherein P 2 comprises an amino acid sequence according to any one of SEQ ID NOs: 98- 776 or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 98-776.
  • Embodiment 81 comprises the isolated polypeptide or polypeptide complex of embodiment 80, wherein P 2 comprises an amino acid sequence according to any one of SEQ ID NOs: 98-776.
  • Embodiment 82 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-77, wherein P 2 comprises an amino acid sequence according to any one of SEQ ID NOs: 99- 118 or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 99-118.
  • Embodiment 83 comprises the isolated polypeptide or polypeptide complex of embodiment 82, wherein P 2 comprises an amino acid sequence according to any one of SEQ ID NOs: 99-118.
  • Embodiment 84 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-77, wherein P 2 comprises an amino acid sequence according to SEQ ID NO: 26 or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to SEQ ID NO: 26.
  • Embodiment 85 comprises the isolated polypeptide or polypeptide complex of embodiment 84, wherein P 2 comprises the amino acid sequence according to SEQ ID NO: 26.
  • Embodiment 86 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-85, wherein Pi comprises an amino acid sequence according to any one of SEQ ID NOs: 25, 797-835, or 843-1690 or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 25, 797-835, or 843-1690.
  • Embodiment 87 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-85, wherein Pi comprises an amino acid sequence according to Z 1 -Z 2 -C-Z 4 -P-Z 6 -Z 7 -Z 8 - Z 9 - Z 10 -Z 11 -Z 12 -C-Z 14 and Zi is selected from D, Y, F, I, N, V, H, L, A, T, S, and P; Z 2 is selected from D, Y, L, F, I, N, A, V, H, T, and S; Z 4 is selected from G and W; Zb is selected from E, D, V, and P; Z 7 is selected from W, L, F, V, G, M, I, and Y; Zs is selected from E, D, P, and Q; Z 9 is selected from E, D, Y, V, F, W, P, L, and Q; Z 10 is selected from S, D, Y, T, I, F, V, N,
  • Embodiment 88 comprises the isolated polypeptide or polypeptide complex of embodiment 87, wherein Z ⁇ is selected from D, Y, F, I, and N; Z 2 is selected from D, Y, L, F, I, and N; Z 4 is selected from G and W; Zb is selected from E and D; Z 7 is selected from W, L, F, and V; Zs is selected from E and D; Z 9 is selected from E, D, Y, and V; Zio is selected from S, D, Y, T, and I; Zn is selected from I, Y, F, V, L, and T; Z 12 IS selected from F, D, Y, F, I, V, A, and N; and Z 14 is selected from D, Y, N, F, I, and P.
  • Embodiment 89 comprises the isolated polypeptide or polypeptide complex of embodiment 88, wherein Z ⁇ is selected from D, Y, and F; Z 2 is selected from D, Y, F, and F; Z 4 is selected from G and W; Zb is selected from E and D; Z 7 is selected from W, F, and F; Zg is selected from E and D; Z 9 is selected from E and D; Z 10 is selected from S, D, and Y; Zn is selected from I, Y, and F; Z 12 is selected from F, D, Y, and F; and Zn is selected from D, Y, and N.
  • Embodiment 90 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-85, wherein Pi comprises an amino acid sequence according to U 1 -U 2 -C-U 4 -P-U 6 -U 7 -U 8 - U 9 - U 10 -U 11 -U 12 -C-U 14 and Ui is selected from D, Y, F, I, N, V, H, F, A, T, S, and P; U 2 is selected from D, Y, F, F, I, N, A, V, H, T, and S; U 4 is selected from G and W; U 3 ⁇ 4 is selected from E, D, V, and P; U 7 is selected from W, F, F, V, G, M, I, and Y; Us is selected from E, D, P, and Q; U 9 is selected from E, D, Y, V, F, W, P, F, and Q; U 10 is selected from S, D, Y, T, I, F, V, N
  • Embodiment 91 comprises the isolated polypeptide or polypeptide complex of embodiment 90, wherein Ui is selected from D, Y, F, I, V, and N; U 2 is selected from D, Y, F, F, I, and N; U 4 is selected from G and W: Ur, is selected from E and D; U 7 is selected from W, F, F, G, and V; Us is selected from E and D; U 9 is selected from E, D, Y, and V; U 10 is selected from S, D, Y, T, and I; Un is selected from I, Y, F, V, F, and T; U 12 is selected from F, D, Y, F, I, V, A, G, and N; and Un is selected from D, Y, N, F, I, M, and P.
  • Embodiment 92 comprises the isolated polypeptide or polypeptide complex of embodiment 91, wherein Ui is selected from D, Y, V, and F; U 2 is selected from D, Y, F, and F; U 4 is selected from G and W; Ue is selected from E and D; U 7 is selected from W, F, G, and F; Us is selected from E and D; U 9 is selected from E and D; U 10 is selected from S, D, T, and Y; Un is selected from I, Y, V, F, and F; Unis selected from F, D, Y, G, A, and F; and Un is selected from D, Y, M, and N.
  • Embodiment 93 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-92, wherein Pi comprises the amino acid sequences according to any one of SEQ ID NOs: 797-823.
  • Embodiment 94 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-92, wherein Pi comprises an amino acid sequences according to any one of SEQ ID NOs:
  • Embodiment 95 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-92, wherein Pi comprises the amino acid sequences according to any of SEQ ID NOs: 824- 835.
  • Embodiment 96 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-92, wherein Pi comprises the amino acid sequence according to SEQ ID NO: 810 or an amino acid sequence that has 1, 2, or 3, amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 810.
  • Embodiment 97 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-92, wherein Pi comprises the amino acid sequence according to SEQ ID NO: 811 or an amino acid sequence that has 1, 2, or 3, amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 811.
  • Embodiment 98 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-92, wherein Pi comprises the amino acid sequence according to SEQ ID NO: 834 or an amino acid sequence that has 1, 2, or 3, amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 834.
  • Embodiment 99 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-92, wherein Pi comprises the amino acid sequence according to SEQ ID NO: 810.
  • Embodiment 100 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-92, wherein Pi comprises the amino acid sequence according to SEQ ID NO: 811.
  • Embodiment 101 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-92, wherein Pi comprises the amino acid sequence according to SEQ ID NO: 834.
  • Embodiment 102 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-101, wherein Li is bound to N-terminus of Ai.
  • Embodiment 103 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-101, wherein Li is bound to C-terminus of Ai.
  • Embodiment 104 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 49-101, wherein L IS bound to N-terminus of A .
  • Embodiment 105 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 49-101, wherein L IS bound to C-terminus of A .
  • Embodiment 106 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-105, wherein Li or L is a peptide sequence having at least 5 to no more than 50 amino acids.
  • Embodiment 107 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-105, wherein Li or L is a peptide sequence having at least 10 to no more than 30 amino acids.
  • Embodiment 108 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-105, wherein Li or L is a peptide sequence having at least 10 amino acids.
  • Embodiment 109 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-105, wherein Li or L is a peptide sequence having at least 18 amino acids.
  • Embodiment 110 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-105, wherein Li or L is a peptide sequence having at least 26 amino acids.
  • Embodiment 111 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-105, wherein Li or L2 has a formula comprising (G2S) n , wherein n is an integer from 1 to 3 (SEQ ID NO: 840).
  • Embodiment 112 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-105, wherein Li has a formula selected from the group consisting of (G 2 S) n , (GS) n , (GSGGS) friendship (SEQ ID NO: 836), (GGGS) dislike(SEQ ID NO: 837), (GGGGS) format (SEQ ID NO: 838), and (GSSGGS) n (SEQ ID NO: 839), wherein n is an integer of at least 1.
  • Embodiment 113 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-105, wherein Pi becomes unbound from Ai when Li is cleaved by the tumor specific protease thereby exposing Ai to the effector cell antigen.
  • Embodiment 114 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-105, wherein P2 becomes unbound from A2 when L2IS cleaved by the tumor specific protease thereby exposing A2 to EGFR.
  • Embodiment 115 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-105, wherein the tumor specific protease is selected from the group consisting of a matrix metalloprotease (MMP), serine protease, cysteine protease, threonine protease, and aspartic protease.
  • MMP matrix metalloprotease
  • Embodiment 116 comprises the isolated polypeptide or polypeptide complex of embodiment 115, wherein the matrix metalloprotease comprises MMP2, MMP7, MMP9, MMP 13, or MMP 14.
  • Embodiment 117 comprises the isolated polypeptide or polypeptide complex of embodiment 115, wherein the serine protease comprises matriptase (MTSP1), urokinase, or hepsin.
  • MTSP1 matriptase
  • urokinase urokinase
  • hepsin hepsin
  • Embodiment 118 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-105, wherein Li or L2 comprises a urokinase cleavable amino acid sequence, a matriptase cleavable amino acid sequence, matrix metalloprotease cleavable amino acid sequence, or a legumain cleavable amino acid sequence.
  • Embodiment 119 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-105, wherein Li or L2 comprises an amino acid sequence according to SEQ ID NO: 30 or 31.
  • Embodiment 120 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-105, wherein Li or L2 comprises an amino acid sequence according to any one of SEQ ID NOs: 27-35.
  • Embodiment 121 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-105, wherein Li or L2 comprises an amino acid sequence of Linker 4 (ISSGLLSGRSDAG) (SEQ ID NO: 66), Linker 5 (AAGLLAPPGGLSGRSDAG) (SEQ ID NO: 67), Linker 6 (SPLGLSGRSDAG) (SEQ ID NO: 68), or Linker 7 (LSGRSDAGSPLGLAG) (SEQ ID NO: 69), or an amino acid sequence that has 1, 2, or 3 amino acid substitutions, additions, or deletions relative to the amino acid sequence of Linker 4, Linker 5, Linker 6, or Linker 7.
  • Embodiment 122 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-121, wherein Hi comprises a polymer.
  • Embodiment 123 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-121, wherein the polymer is polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • Embodiment 124 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-121, wherein Hi comprises albumin.
  • Embodiment 125 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-121, wherein Hi comprises an Fc domain.
  • Embodiment 126 comprises the isolated polypeptide or polypeptide complex of embodiment 124, wherein the albumin is serum albumin.
  • Embodiment 127 comprises the isolated polypeptide or polypeptide complex of embodiment 124, wherein the albumin is human serum albumin.
  • Embodiment 128 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-121, wherein Hi comprises a polypeptide, a ligand, or a small molecule.
  • Embodiment 129 comprises the isolated polypeptide or polypeptide complex of embodiment 128, wherein the polypeptide, the ligand or the small molecule binds serum protein or a fragment thereof, a circulating immunoglobulin or a fragment thereof, or CD35/CR1.
  • Embodiment 130 comprises the isolated polypeptide or polypeptide complex of embodiment 129, wherein the serum protein comprises a thyroxine-binding protein, a transthyretin, a 1-acid glycoprotein, a transferrin, transferrin receptor or a transferrin-binding portion thereof, a fibrinogen, or an albumin.
  • the serum protein comprises a thyroxine-binding protein, a transthyretin, a 1-acid glycoprotein, a transferrin, transferrin receptor or a transferrin-binding portion thereof, a fibrinogen, or an albumin.
  • Embodiment 131 comprises the isolated polypeptide or polypeptide complex of embodiment 129, wherein the circulating immunoglobulin molecule comprises IgGl, IgG2, IgG3, IgG4, slgA, IgM or IgD.
  • Embodiment 132 comprises the isolated polypeptide or polypeptide complex of embodiment 129, wherein the serum protein is albumin.
  • Embodiment 133 comprises the isolated polypeptide or polypeptide complex of embodiment 128, wherein the polypeptide is an antibody.
  • Embodiment 134 comprises the isolated polypeptide or polypeptide complex of embodiment 133, wherein the antibody comprises a single domain antibody, a single chain variable fragment, or a Fab.
  • Embodiment 135 comprises the isolated polypeptide or polypeptide complex of embodiment 134, wherein the single domain antibody comprises a single domain antibody that binds to albumin.
  • Embodiment 136 comprises the isolated polypeptide or polypeptide complex of embodiment 134, wherein the single domain antibody is a human or humanized antibody.
  • Embodiment 137 comprises the isolated polypeptide or polypeptide complex of embodiment 134, wherein the single domain antibody is 645gHlgLl.
  • Embodiment 138 comprises the isolated polypeptide or polypeptide complex of embodiment 134, wherein the single domain antibody is 645dsgH5gL4.
  • Embodiment 139 comprises the isolated polypeptide or polypeptide complex of embodiment 134, wherein the single domain antibody is 23-13-A01 -sc02.
  • Embodiment 140 comprises the isolated polypeptide or polypeptide complex of embodiment 134, wherein the single domain antibody is A10m3 or a fragment thereof.
  • Embodiment 141 comprises the isolated polypeptide or polypeptide complex of embodiment 134, wherein the single domain antibody is DOM7r-31.
  • Embodiment 142 comprises the isolated polypeptide or polypeptide complex of embodiment 134, wherein the single domain antibody is DOM7h-l 1-15.
  • Embodiment 143 comprises the isolated polypeptide or polypeptide complex of embodiment 134, wherein the single domain antibody is Alb-1, Alb-8, or Alb-23.
  • Embodiment 144 comprises the isolated polypeptide or polypeptide complex of embodiment 134, wherein the single domain antibody is 10E.
  • Embodiment 145 comprises the isolated polypeptide or polypeptide complex of embodiment 134, wherein the single domain antibody comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the single domain antibody comprise: HC-CDR1: SEQ ID NO: 36, HC-CDR2: SEQ ID NO: 37, and HC-CDR3: SEQ ID NO: 38.
  • CDRs complementarity determining regions
  • Embodiment 146 comprises the isolated polypeptide or polypeptide complex of embodiment 134, wherein the single domain antibody comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the single domain antibody comprise: HC-CDR1: SEQ ID NO: 39, HC-CDR2: SEQ ID NO: 40, and HC-CDR3: SEQ ID NO: 41.
  • CDRs complementarity determining regions
  • Embodiment 147 comprises the isolated polypeptide or polypeptide complex of embodiment 134, wherein the single domain antibody is SA21.
  • Embodiment 148 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-147, wherein the isolated polypeptide or polypeptide complex comprises a modified amino acid, a non-natural amino acid, a modified non-natural amino acid, or a combination thereof.
  • Embodiment 149 comprises the isolated polypeptide or polypeptide complex of embodiment 148, wherein the modified amino acid or modified non-natural amino acid comprises a post-translational modification.
  • Embodiment 150 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-149, wherein Hi comprises a linking moiety (L3) that connects Hi to Pi .
  • Hi comprises a linking moiety (L3) that connects Hi to Pi .
  • Embodiment 151 comprises the isolated polypeptide or polypeptide complex of embodiment 150, wherein L3 is a peptide sequence having at least 5 to no more than 50 amino acids.
  • Embodiment 152 comprises the isolated polypeptide or polypeptide complex of embodiment 150, wherein L3 is a peptide sequence having at least 10 to no more than 30 amino acids.
  • Embodiment 153 comprises the isolated polypeptide or polypeptide complex of embodiment 150, wherein L is a peptide sequence having at least amino acids.
  • Embodiment 154 comprises the isolated polypeptide or polypeptide complex of embodiment 150, wherein L is a peptide sequence having at least 18 amino acids.
  • Embodiment 155 comprises the isolated polypeptide or polypeptide complex of embodiment 150, wherein L is a peptide sequence having at least 26 amino acids.
  • Embodiment 156 comprises the isolated polypeptide or polypeptide complex of embodiment 150, wherein L has a formula selected from the group consisting of (G S) n , (GS) n , (GSGGS) n (SEQ ID NO: 836), (GGGS) friendship (SEQ ID NO: 837), (GGGGS) connector (SEQ ID NO: 838), and (GSSGGS) worship(SEQ ID NO: 839), wherein n is an integer of at least .
  • Embodiment 157 comprises the isolated polypeptide or polypeptide complex of embodiment 150, wherein L comprises an amino acid sequence according to SEQ ID NO: 29.
  • Embodiment 158 comprises the isolated polypeptide or polypeptide complex of embodiment 1, wherein the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to any one of SEQ ID NOs: 44-61.
  • Embodiment 159 comprises the isolated polypeptide or polypeptide complex of embodiment 1, wherein the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 50.
  • Embodiment 160 comprises the isolated polypeptide or polypeptide complex of embodiment 1, wherein the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 51.
  • Embodiment 161 comprises the isolated polypeptide or polypeptide complex of embodiment 1, wherein the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 44 and SEQ ID NO: 45.
  • Embodiment 162 comprises the isolated polypeptide or polypeptide complex of embodiment 1, wherein the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 46 and SEQ ID NO: 47.
  • Embodiment 163 comprises the isolated polypeptide or polypeptide complex of embodiment 1, wherein the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 48 and SEQ ID NO: 49.
  • Embodiment 164 comprises the isolated polypeptide or polypeptide complex of embodiment 1, wherein the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 50 and SEQ ID NO: 51.
  • Embodiment 165 comprises the isolated polypeptide or polypeptide complex of embodiment 1, wherein the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 52 and SEQ ID NO: 53.
  • Embodiment 166 comprises the isolated polypeptide or polypeptide complex of embodiment 1, wherein the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 54 and SEQ ID NO: 55.
  • Embodiment 167 comprises the isolated polypeptide or polypeptide complex of embodiment 1, wherein the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 56 and SEQ ID NO: 57.
  • Embodiment 168 comprises the isolated polypeptide or polypeptide complex of embodiment 1, wherein the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 58 and SEQ ID NO: 59.
  • Embodiment 169 comprises the isolated polypeptide or polypeptide complex of embodiment 1, wherein the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 60 and SEQ ID NO: 61.
  • Embodiment 170 comprises the isolated polypeptide or polypeptide complex of embodiment 1, wherein the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 62 and SEQ ID NO: 63.
  • Embodiment 171 comprises the isolated polypeptide or polypeptide complex of embodiment 1, wherein the isolated polypeptide or polypeptide complex comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 64 and SEQ ID NO: 65.
  • Embodiment 172 comprises a pharmaceutical composition comprising: (a) the isolated polypeptide or polypeptide complex of any one of embodiments 1-171; and (b) a pharmaceutically acceptable excipient.
  • Embodiment 173 comprises an isolated recombinant nucleic acid molecule encoding the isolated polypeptide or polypeptide complex of any one of embodiments 1-171.
  • Embodiment 174 an isolated polypeptide or polypeptide complex according to Formula II: Li a -Pi - Hi a
  • Li a comprises a tumor specific protease -cleaved linking moiety that when uncleaved connects Pi a to a first antigen recognizing molecule that binds to an effector cell antigen and the first antigen recognizing molecule is connected to a second antigen recognizing molecule that binds to EGFR
  • Pi a comprises a peptide that binds to the first antigen recognizing molecule when Li a is uncleaved
  • Hi a comprises a half-life extending molecule.
  • Embodiment 175 comprises the isolated polypeptide or polypeptide complex of embodiment 174, wherein Pi a when Li a is uncleaved impairs binding of the first antigen recognizing molecule to the effector cell antigen.
  • Embodiment 176 comprises the isolated polypeptide or polypeptide complex of embodiment 174, wherein the first antigen recognizing molecule comprises an antibody or antibody fragment.
  • Embodiment 177 comprises the isolated polypeptide or polypeptide complex of embodiment 174, wherein the effector cell antigen is an anti-CD3 effector cell antigen.
  • Embodiment 178 comprises the isolated polypeptide or polypeptide complex of embodiment 174, wherein Pi a has less than 70% sequence homology to the effector cell antigen.
  • Embodiment 179 comprises the isolated polypeptide or polypeptide complex of embodiment 174, wherein Pi a comprises a peptide sequence of at least 10 amino acids in length.
  • Embodiment 180 comprises the isolated polypeptide or polypeptide complex of embodiment 174, wherein Pi a comprises a peptide sequence of at least 10 amino acids in length and no more than 20 amino acids in length.
  • Embodiment 181 comprises the isolated polypeptide or polypeptide complex of embodiment 174, wherein Pi a comprises a peptide sequence of at least 16 amino acids in length.
  • Embodiment 182 comprises the isolated polypeptide or polypeptide complex of embodiment 174, wherein Pi a comprises a peptide sequence of no more than 40 amino acids in length.
  • Embodiment 183 comprises the isolated polypeptide or polypeptide complex of embodiment 174, wherein Pi a comprises at least two cysteine amino acid residues.
  • Embodiment 184 comprises the isolated polypeptide or polypeptide complex of embodiment 174, wherein Pi a comprises a cyclic peptide or a linear peptide.
  • Embodiment 185 comprises the isolated polypeptide or polypeptide complex of embodiment 174, wherein Pi a comprises a cyclic peptide.
  • Embodiment 186 comprises the isolated polypeptide or polypeptide complex of embodiment 174, wherein Pi a comprises a linear peptide.
  • Embodiment 187 comprises the isolated polypeptide or polypeptide complex of embodiment 174, wherein Pi a comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 25.
  • Embodiment 188 comprises the isolated polypeptide or polypeptide complex of embodiment 174, wherein Pi a comprises an amino acid sequence according to SEQ ID NO: 70.
  • Embodiment 189 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 174-187, wherein Hi a comprises a polymer.
  • Embodiment 190 comprises the isolated polypeptide or polypeptide complex of embodiment 189, wherein the polymer is polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • Embodiment 191 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 174-190, wherein Pi a comprises an amino acid sequence according to any one of SEQ ID NOs: 25, 797-835, or 843-1690, or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOS: 25, 797-835, or 843-1690.
  • Embodiment 192 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 174-190, wherein Pi a comprises an amino acid sequence according to Z 1 -Z 2 -C-Z 4 -P-Z 6 -Z 7 -Z 8 - Z 9 -Z 10 -Z 11 - Z 12 -C-Z 14 and Zi is selected from D, Y, F, I, N, V, H, L, A, T, S, and P; Z 2 is selected from D, Y, L, F, I, N, A, V, H, T, and S; Z 4 is selected from G and W; Z is selected from E, D, V, and P; Z 7 is selected from W, L, F, V, G, M, I, and Y; Zs is selected from E, D, P, and Q; Z 9 is selected from E, D, Y, V, F, W, P, L, and Q; Z 10 is selected from S, D, Y, T, I, F, V, V,
  • Embodiment 193 comprises the isolated polypeptide or polypeptide complex of embodiment 192, wherein Z ⁇ is selected from D, Y, F, I, and N; Z2 is selected from D, Y, L, F, I, and N; Z4 is selected from G and W; Zb is selected from E and D; Z7 is selected from W, F, F, and V; Zg is selected from E and D; Z9 is selected from E, D, Y, and V; Z10 is selected from S, D, Y, T, and I; Zn is selected from I, Y, F, V, F, and T; Z12IS selected from F, D, Y, F, I, V, A, and N; and Z14 is selected from D, Y, N, F, I, and P.
  • Embodiment 194 comprises the isolated polypeptide or polypeptide complex of embodiment 193, wherein Zi is selected from D, Y, and F; Z2 is selected from D, Y, F, and F; Z4 is selected from G and W; Zb is selected from E and D; Z7 is selected from W, F, and F; Zs is selected from E and D; Z9 is selected from E and D; Z10 is selected from S, D, and Y; Zn is selected from I, Y, and F; Z12 is selected from F, D, Y, and F; and Zn is selected from D, Y, and N.
  • Embodiment 195 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 174-190, wherein Pi a comprises an amino acid sequence according to U1-U2-C-U4-P-U6-U7- U 8 - U9-U10-U11-U12-C-U14 and Ui is selected from D, Y, F, I, N, V, H, F, A, T, S, and P; U2 is selected from D, Y, F, F, I, N, A, V, H, T, and S; U4 is selected from G and W; Ue is selected from E, D, V, and P; U7 is selected from W, F, F, V, G, M, I, and Y; Us is selected from E, D, P, and Q; U9 is selected from E, D, Y, V, F, W, P, F, and Q; U10 is selected from S, D, Y, T, I, F, V, N, A, P, F,
  • Embodiment 196 comprises the isolated polypeptide or polypeptide complex of embodiment 195, wherein Ui is selected from D, Y, F, I, V, and N; U2 is selected from D, Y, F, F, I, and N; U4IS selected from G and W; Ur, is selected from E and D; U7 is selected from W, F, F, G, and V; Us is selected from E and D; U9 is selected from E, D, Y, and V; U10 is selected from S, D, Y, T, and I; Un is selected from I, Y, F, V, F, and T; U12 is selected from F, D, Y, F, I, V, A, G, and N; and Un is selected from D, Y, N, F, I, M, and P.
  • Embodiment 197 comprises the isolated polypeptide or polypeptide complex of embodiment 196, wherein Ui is selected from D, Y, V, and F; U2 is selected from D, Y, F, and F; U4 is selected from G and W; Ue is selected from E and D; U7 is selected from W, F, G, and F; Us is selected from E and D; U9 is selected from E and D; U10 is selected from S, D, T, and Y; Un is selected from I, Y, V, F, and F; Unis selected from F, D, Y, G, A, and F; and Unis selected from D, Y, M, and N.
  • Embodiment 198 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 174-197, wherein Pi a comprises an amino acid sequence according to any one of SEQ ID NOs: 797-823.
  • Embodiment 199 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 174-197, wherein Pi a comprises an amino acid sequence according to any one of SEQ ID NOs: 25, 824-835 or 843-1690.
  • Embodiment 200 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 174-197, wherein Pi a comprises an amino acid sequence according to any one of SEQ ID NOs: 824-835.
  • Embodiment 201 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 174-197, wherein Pi a comprises the amino acid sequence according to SEQ ID NO: 810 or an amino acid sequence that has 1, 2, or 3, amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 810.
  • Embodiment 202 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 174-197, wherein Pi a comprises the amino acid sequence according to SEQ ID NO: 811 or an amino acid sequence that has 1, 2, or 3, amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 811.
  • Embodiment 203 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 174-197, wherein Pi a comprises the amino acid sequence according to SEQ ID NO: 834 or an amino acid sequence that has 1, 2, or 3, amino acid substitutions, additions, or deletions relative to the amino acid sequence of SEQ ID NO: 834.
  • Embodiment 204 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 174-197, wherein Pi a comprises the amino acid sequence according to SEQ ID NO: 810.
  • Embodiment 205 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 174-197, wherein Pi a comprises the amino acid sequence according to SEQ ID NO: 811.
  • Embodiment 206 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 174-197, wherein Pi a comprises the amino acid sequence according to SEQ ID NO: 834.
  • Embodiment 207 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 174-206, wherein Hi a comprises albumin.
  • Embodiment 208 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 174-206, wherein Hi a comprises an Fc domain.
  • Embodiment 209 comprises the isolated polypeptide or polypeptide complex of embodiment 207, wherein the albumin is serum albumin.
  • Embodiment 210 comprises the isolated polypeptide or polypeptide complex of embodiment 209, wherein the albumin is human serum albumin.
  • Embodiment 211 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 174-206, wherein Hi a comprises a polypeptide, a ligand, or a small molecule.
  • Embodiment 212 comprises the isolated polypeptide or polypeptide complex of embodiment 211, wherein the polypeptide, the ligand or the small molecule binds a serum protein or a fragment thereof, a circulating immunoglobulin or a fragment thereof, or CD35/CR1.
  • Embodiment 213 comprises the isolated polypeptide or polypeptide complex of embodiment 212, wherein the serum protein comprises a thyroxine-binding protein, a transthyretin, a 1-acid glycoprotein, a transferrin, transferrin receptor or a transferrin-binding portion thereof, a fibrinogen, or an albumin.
  • the serum protein comprises a thyroxine-binding protein, a transthyretin, a 1-acid glycoprotein, a transferrin, transferrin receptor or a transferrin-binding portion thereof, a fibrinogen, or an albumin.
  • Embodiment 214 comprises the isolated polypeptide or polypeptide complex of embodiment 212, wherein the circulating immunoglobulin molecule comprises IgGl, IgG2, IgG3, IgG4, slgA, IgM or IgD.
  • Embodiment 215 comprises the isolated polypeptide or polypeptide complex of embodiment 212, wherein the serum protein is albumin.
  • Embodiment 216 comprises the isolated polypeptide or polypeptide complex of embodiment 211, wherein the polypeptide is an antibody.
  • Embodiment 217 comprises the isolated polypeptide or polypeptide complex of embodiment 216, wherein the antibody comprises a single domain antibody, a single chain variable fragment or a Fab.
  • Embodiment 218 comprises the isolated polypeptide or polypeptide complex of embodiment 216, wherein the antibody comprises a single domain antibody that binds to albumin.
  • Embodiment 219 comprises the isolated polypeptide or polypeptide complex of embodiment 216, wherein the antibody is a human or humanized antibody.
  • Embodiment 220 comprises the isolated polypeptide or polypeptide complex of embodiment 217, wherein the single domain antibody is 645gHlgLl.
  • Embodiment 221 comprises the isolated polypeptide or polypeptide complex of embodiment 217, wherein the single domain antibody is 645dsgH5gL4.
  • Embodiment 222 comprises the isolated polypeptide or polypeptide complex of embodiment 217, wherein the single domain antibody is 23-13-A01 -sc02.
  • Embodiment 223 comprises the isolated polypeptide or polypeptide complex of embodiment 217, wherein the single domain antibody is A10m3 or a fragment thereof.
  • Embodiment 224 comprises the isolated polypeptide or polypeptide complex of embodiment 217, wherein the single domain antibody is DOM7r-31.
  • Embodiment 225 comprises the isolated polypeptide or polypeptide complex of embodiment 217, wherein the single domain antibody is DOM7h-l 1-15.
  • Embodiment 226 comprises the isolated polypeptide or polypeptide complex of embodiment 217, wherein the single domain antibody is Alb-1, Alb-8, or Alb-23.
  • Embodiment 227 comprises the isolated polypeptide or polypeptide complex of embodiment 217, wherein the single domain antibody is 10G.
  • Embodiment 228 comprises the isolated polypeptide or polypeptide complex of embodiment 217, wherein the single domain antibody comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the single domain antibody comprise: HC-CDR1: SEQ ID NO: 36, HC-CDR2: SEQ ID NO: 37, and HC-CDR3: SEQ ID NO: 38.
  • CDRs complementarity determining regions
  • Embodiment 229 comprises the isolated polypeptide or polypeptide complex of embodiment 217, wherein the single domain antibody comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the single domain antibody comprise: HC-CDR1: SEQ ID NO: 39, HC-CDR2: SEQ ID NO: 40, and HC-CDR3: SEQ ID NO: 41.
  • Embodiment 230 comprises the isolated polypeptide or polypeptide complex of embodiment 217, wherein the single domain antibody is SA21.
  • Embodiment 231 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 174-230, wherein Hi a comprises a linking moiety (Li a ) that connects Hi a to Pi a .
  • Embodiment 232 comprises the isolated polypeptide or polypeptide complex of embodiment 231, wherein Li a is a peptide sequence having at least 5 to no more than 50 amino acids.
  • Embodiment 233 comprises the isolated polypeptide or polypeptide complex of embodiment 231, wherein Li a is a peptide sequence having at least 10 to no more than 30 amino acids.
  • Embodiment 234 comprises the isolated polypeptide or polypeptide complex of embodiment 231, wherein Li a is a peptide sequence having at least 10 amino acids.
  • Embodiment 235 comprises the isolated polypeptide or polypeptide complex of embodiment 231, wherein Li a is a peptide sequence having at least 18 amino acids.
  • Embodiment 236 comprises the isolated polypeptide or polypeptide complex of embodiment 231, wherein Li a is a peptide sequence having at least 26 amino acids.
  • Embodiment 237 comprises the isolated polypeptide or polypeptide complex of embodiment 231, wherein Li a has a formula selected from the group consisting of (G2S) n , (GS) n , (GSGGS )n (SEQ ID NO: 836), (GGGS) friendship (SEQ ID NO: 837), (GGGGS) connector (SEQ ID NO: 838), and (GSSGGS)trust (SEQ ID NO: 839), wherein n is an integer of at least 1.
  • Embodiment 238 comprises the isolated polypeptide or polypeptide complex of embodiment 231, wherein Li a comprises an amino acid sequence according to SEQ ID NO: 30 or 31.
  • Embodiment 239 comprises the isolated polypeptide or polypeptide complex of embodiment 231, wherein Li or L2 comprises an amino acid sequence according to any one of SEQ ID NOs: 27-35.
  • Embodiment 240 comprises the isolated polypeptide or polypeptide complex of embodiment 231, wherein Li or L2 comprises an amino acid sequence of Linker 4 (ISSGLLSGRSDAG) (SEQ ID NO: 66), Linker 5 (AAGLLAPPGGLSGRSDAG) (SEQ ID NO: 67), Linker 6 (SPLGLSGRSDAG) (SEQ ID NO: 68), or Linker 7 (LSGRSDAGSPLGLAG) (SEQ ID NO: 69), or an amino acid sequence that has 1, 2, or 3 amino acid substitutions, additions, or deletions relative to the amino acid sequence of Linker 4, Linker 5, Linker 6, or Linker 7.
  • Embodiment 241 comprises an isolated polypeptide complex comprising a structural arrangement according to Configuration 1 :
  • 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 (Pi) that impairs binding of the scFv to an effector cell antigen and Pi is linked to a N-terminus of the heavy chain variable domain of the scFv with a linking moiety (Li) that is a substrate for a tumor specific protease, and Pi is further linked to a half-life extending molecule; and a Fab that binds to epidermal growth factor receptor (EGFR), wherein the Fab comprises a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab light chain polypeptide is linked to a C terminus of the light chain variable domain of the scFv, and wherein the Fab is linked to P2 and L2, wherein P2 comprises a peptide that impairs binding
  • Embodiment 242 comprises an isolated polypeptide complex comprising a structural arrangement according to Configuration 2:
  • 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 (Pi) that impairs binding of the scFv to an effector cell antigen and Pi is linked to a N-terminus of the heavy chain variable domain of the scFv with a linking moiety (Li) that is a substrate for a tumor specific protease, and Pi is further linked to a half-life extending molecule; and a Fab that binds to epidermal growth factor receptor (EGFR), wherein the Fab comprises a Fab light chain polypeptide and a Fab heavy chain polypeptide, wherein the Fab heavy chain polypeptide is linked to a C terminus of the light chain variable domain of the scFv, and wherein the Fab is linked to P2 and L2, wherein P2 comprises a peptide that impairs binding
  • Embodiment 243 comprises an isolated polypeptide or polypeptide complex comprising an anti- EGFR binding domain that is linked to a peptide that impairs binding of the anti-EGFR binding domain to EGFR wherein the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 26, 71- 96, 98-776 or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 26, 71-96, 98-776.
  • Embodiment 244 comprises the isolated polypeptide or polypeptide complex of embodiment 243, wherein the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 26, 71-96, 98- 776.
  • Embodiment 245 comprises the isolated polypeptide or polypeptide complex of embodiment 243, wherein the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 26, 86-96 or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 26, 86-96.
  • Embodiment 246 comprises the isolated polypeptide or polypeptide complex of embodiment 245, wherein the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 26, 86-96.
  • Embodiment 247 comprises the isolated polypeptide or polypeptide complex of embodiment 243, wherein the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 98-776 or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any of SEQ ID NOs: 98-776.
  • Embodiment 248 comprises the isolated polypeptide or polypeptide complex of embodiment 247, wherein the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 98-776.
  • Embodiment 249 comprises the isolated polypeptide or polypeptide complex of embodiment 243, wherein the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 99-118 or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to any one of SEQ ID NOs: 99-118.
  • Embodiment 250 comprises the isolated polypeptide or polypeptide complex of embodiment 249, wherein the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 99-118.
  • Embodiment 251 comprises the isolated polypeptide or polypeptide complex of embodiment 243, wherein the peptide comprises the amino acid sequence according to SEQ ID NO: 71 or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to SEQ ID NO: 71.
  • Embodiment 252 comprises the isolated polypeptide or polypeptide complex of embodiment 251, wherein the peptide comprises the amino acid sequence according to SEQ ID NO: 71.
  • Embodiment 253 comprises the isolated polypeptide or polypeptide complex of embodiment 243, wherein the peptide comprises the amino acid sequence according to SEQ ID NO: 26 or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to SEQ ID NO: 26.
  • Embodiment 254 comprises the isolated polypeptide or polypeptide complex of embodiment 253, wherein the peptide comprises the amino acid sequence according to SEQ ID NO: 26.
  • Embodiment 255 comprises an isolated polypeptide or polypeptide complex comprising an anti- EGFR binding domain that is linked to a peptide that impairs binding of the anti-EGFR binding to EGFR wherein the peptide comprises an amino acid sequence according to X 1 -C-X 2 -X 3 -X 4 -X 5 -D-X 6 -A-X 7 -P-X 8 -C- X 9 wherein Xi is selected from P and L; X 2 is selected from R, L, T, A, N, I, V, S, H, and P; X 3 is selected from S, P, F, and Y; X is selected from H, L, Q, P, R, F, N; X is selected from I, F, Y, H, N, T, S, D, A, L, and V; Xe is selected from T, P, N, L, I, V, S, D, H, A, and Y; X IS selected from K and
  • Embodiment 256 comprises the isolated polypeptide or polypeptide complex of embodiment 255, wherein Xi is selected from P and L; X 2 is selected from R, L, T, A, and N; X 3 is selected from S, P, and F; X4 is selected from H, L, Q, and P; X5 is selected from I, F, Y, H, N, and T; Xe is selected from T, P, N, L, I, and V; X7 is K; Xs is I; and X9 is selected from A, V, I, T, L, and S.
  • Embodiment 257 comprises the isolated polypeptide or polypeptide complex of embodiment 256, wherein Xi is P; X2 is selected from R, L, and T; X3 is S; X4 is selected from H, L, Q, and P; X5 is selected from I, F, Y, and T; Xe is selected from T, P, N, and V; X7 is K; Xs is I; and X9 is selected from A, V, and F [0539] Embodiment 258 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 255-257, wherein the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 98-776.
  • Embodiment 259 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 255-258, wherein the peptide comprises an amino acid sequence according to any one of SEQ ID NOs: 99-118.
  • Embodiment 260 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 255-257, wherein the peptide comprises the amino acid sequence according to SEQ ID NO:
  • Embodiment 261 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 243-260, wherein the anti-EGFR binding domain comprises an antibody or an antibody fragment.
  • Embodiment 262 comprises the isolated polypeptide or polypeptide complex of embodiment 261, wherein the antibody or antibody fragment comprises a single chain variable fragment, a single domain antibody, Fab, or Fab’.
  • Embodiment 263 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 243-262, wherein the anti-EGFR binding domain comprises heavy chain complementarity determining regions HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 comprise: HC-CDR1: SEQ ID NO: 15, HC-CDR2: SEQ ID NO: 16, and HC-CDR3: SEQ ID NO: 17; and the anti-EGFR binding domain comprises light chain complementarity determining regions CDRs: LC-CDR1, LC-CDR2, and LC-CDR3, wherein the LC-CDR1, the LC-CDR2, and the LC-CDR3 of the Fab comprise LC-CDR1: SEQ ID NO: 18, LC-CDR2: SEQ ID NO: 19, and LC-CDR3: SEQ ID NO: 20.
  • Embodiment 264 Embodiment
  • Embodiment 265 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 243-264, wherein the anti-EGFR binding domain comprises an amino acid sequence according to any of SEQ ID NOs: 21-24.
  • Embodiment 266 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 243-265, wherein the anti-EGFR binding domain is linked to the peptide through a linking moiety (Li).
  • Embodiment 267 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 243-266, wherein Li is a substrate for a tumor specific protease.
  • Embodiment 268 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 243-267, wherein Li is bound to N-terminus of the anti-EGFR binding domain.
  • Embodiment 269 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 243-267, wherein Li is bound to C-terminus of the anti-EGFR binding domain.
  • Embodiment 270 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 243-267, wherein Li is a peptide sequence having at least 5 to no more than 50 amino acids.
  • Embodiment 271 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 243-267, wherein Li is a peptide sequence having at least 10 to no more than 30 amino acids.
  • Embodiment 272 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 243-267, wherein Li is a peptide sequence having at least 10 amino acids.
  • Embodiment 273 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 243-267, wherein Li is a peptide sequence having at least 18 amino acids.
  • Embodiment 274 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 243-267, wherein Li is a peptide sequence having at least 26 amino acids.
  • Embodiment 275 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 243-267, wherein Li has a formula comprising (G2S) n, wherein n is an integer from 1 to 3 (SEQ ID NO: 840).
  • Embodiment 276 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 243-267, wherein Li has a formula selected from the group consisting of (G2S) n, (GS) n, (GSGGS) friendship (SEQ ID NO: 836), (GGGS) dislike (SEQ ID NO: 837), (GGGGS) format (SEQ ID NO: 838), and (GSSGGS) n (SEQ ID NO: 839), wherein n is an integer of at least 1.
  • Embodiment 277 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 243-276, wherein the peptide becomes unbound from the anti-EGFR binding domain when Li is cleaved by the tumor specific protease thereby exposing the anti-EGFR binding domain to EGFR.
  • Embodiment 278 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 267-277, wherein the tumor specific protease is selected from the group consisting of a matrix metalloprotease (MMP), serine protease, cysteine protease, threonine protease, and aspartic protease.
  • MMP matrix metalloprotease
  • Embodiment 279 comprises the isolated polypeptide or polypeptide complex of embodiment 278, wherein the matrix metalloprotease comprises MMP2, MMP7, MMP9, MMP 13, or MMP 14.
  • Embodiment 280 comprises the isolated polypeptide or polypeptide complex of embodiment 278, wherein the serine protease comprises matriptase (MTSP1), urokinase, or hepsin.
  • MTSP1 matriptase
  • urokinase urokinase
  • hepsin hepsin
  • Embodiment 281 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 267-277, wherein Li comprises a urokinase cleavable amino acid sequence, a matriptase cleavable amino acid sequence, matrix metalloprotease cleavable amino acid sequence, or a legumain cleavable amino acid sequence.
  • Embodiment 282 comprises the isolated polypeptide or polypeptide complex of embodiment 266, wherein Li comprises an amino acid sequence according to any of SEQ ID NO: 30 or 31.
  • Embodiment 283 comprises the isolated polypeptide or polypeptide complex of embodiment 266, wherein Li comprises an amino acid sequence according to SEQ ID NO: 27-35.
  • Embodiment 284 comprises the isolated polypeptide or polypeptide complex of embodiment 266, wherein Li comprises an amino acid sequence of Linker 4 (ISSGLLSGRSDAG) (SEQ ID NO: 66), Linker 5 (AAGLLAPPGGLSGRSDAG) (SEQ ID NO: 67), Linker 6 (SPLGLSGRSDAG) (SEQ ID NO: 68), or Linker 7 (LSGRSDAGSPLGLAG) (SEQ ID NO: 69), or an amino acid sequence that has 1, 2, or 3 amino acid substitutions, additions, or deletions relative to the amino acid sequence of Linker 4, Linker 5, Linker 6, or Linker 7.
  • Embodiment 285 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 243-284, wherein the isolated polypeptide or polypeptide complex further comprises a half- life extending molecule (Hi)
  • Embodiment 286 comprises the isolated polypeptide or polypeptide complex of embodiment 285, wherein the half-life extending molecule is linked to the peptide.
  • Embodiment 287 comprises the isolated polypeptide or polypeptide complex of embodiment 285, wherein Hi comprises a polymer.
  • Embodiment 288 comprises the isolated polypeptide or polypeptide complex of embodiment 287, wherein the polymer is polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • Embodiment 289 comprises the isolated polypeptide or polypeptide complex of embodiment 285, wherein Hi comprises albumin.
  • Embodiment 290 comprises the isolated polypeptide or polypeptide complex of embodiment 285, wherein Hi comprises an Fc domain.
  • Embodiment 291 comprises the isolated polypeptide or polypeptide complex of embodiment 289, wherein the albumin is serum albumin.
  • Embodiment 292 comprises the isolated polypeptide or polypeptide complex of embodiment 289, wherein the albumin is human serum albumin.
  • Embodiment 293 comprises the isolated polypeptide or polypeptide complex of embodiment 285, wherein Hi comprises a polypeptide, a ligand, or a small molecule.
  • Embodiment 294 comprises the isolated polypeptide or polypeptide complex of embodiment 293, wherein the polypeptide, the ligand or the small molecule binds serum protein or a fragment thereof, a circulating immunoglobulin or a fragment thereof, or CD35/CR1.
  • Embodiment 295 comprises the isolated polypeptide or polypeptide complex of embodiment 294, wherein the serum protein comprises a thyroxine-binding protein, a transthyretin, a 1-acid glycoprotein, a transferrin, transferrin receptor or a transferrin-binding portion thereof, a fibrinogen, or an albumin.
  • the serum protein comprises a thyroxine-binding protein, a transthyretin, a 1-acid glycoprotein, a transferrin, transferrin receptor or a transferrin-binding portion thereof, a fibrinogen, or an albumin.
  • Embodiment 296 comprises the isolated polypeptide or polypeptide complex of embodiment 294, wherein the circulating immunoglobulin molecule comprises IgGl, IgG2, IgG3, IgG4, slgA, IgM or IgD.
  • Embodiment 297 comprises the isolated polypeptide or polypeptide complex of embodiment 294, wherein the serum protein is albumin.
  • Embodiment 298 comprises the isolated polypeptide or polypeptide complex of embodiment 294, wherein the polypeptide is an antibody.
  • Embodiment 299 comprises the isolated polypeptide or polypeptide complex of embodiment 298, wherein the antibody comprises a single domain antibody, a single chain variable fragment, or a Fab.
  • Embodiment 300 comprises the isolated polypeptide or polypeptide complex of embodiment 299, wherein the single domain antibody comprises a single domain antibody that binds to albumin.
  • Embodiment 301 comprises the isolated polypeptide or polypeptide complex of embodiment 300, wherein the single domain antibody is a human or humanized antibody.
  • Embodiment 302 comprises the isolated polypeptide or polypeptide complex of embodiment 299, wherein the single domain antibody is 645gHlgLl.
  • Embodiment 303 comprises the isolated polypeptide or polypeptide complex of embodiment 299, wherein the single domain antibody is 645dsgH5gL4.
  • Embodiment 304 comprises the isolated polypeptide or polypeptide complex of embodiment 299, wherein the single domain antibody is 23-13-A01 -sc02.
  • Embodiment 305 comprises the isolated polypeptide or polypeptide complex of embodiment 299, wherein the single domain antibody is A10m3 or a fragment thereof.
  • Embodiment 306 comprises the isolated polypeptide or polypeptide complex of embodiment 299, wherein the single domain antibody is DOM7r-31.
  • Embodiment 307 comprises the isolated polypeptide or polypeptide complex of embodiment 299, wherein the single domain antibody is DOM7h-l 1-15.
  • Embodiment 308 comprises the isolated polypeptide or polypeptide complex of embodiment 299, wherein the single domain antibody is Alb-1, Alb-8, or Alb-23.
  • Embodiment 309 comprises the isolated polypeptide or polypeptide complex of embodiment 299, wherein the single domain antibody is 10E.
  • Embodiment 310 comprises the isolated polypeptide or polypeptide complex of embodiment 299, wherein the single domain antibody comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the single domain antibody comprise: HC-CDR1: SEQ ID NO: 36, HC-CDR2: SEQ ID NO: 37, and HC-CDR3: SEQ ID NO: 38.
  • CDRs complementarity determining regions
  • Embodiment 311 comprises the isolated polypeptide or polypeptide complex of embodiment 299, wherein the single domain antibody comprises an amino acid sequence according to SEQ ID NO: 69.
  • Embodiment 312 comprises the isolated polypeptide or polypeptide complex of embodiment 299, wherein the single domain antibody comprises complementarity determining regions (CDRs): HC-CDR1, HC-CDR2, and HC-CDR3, wherein the HC-CDR1, the HC-CDR2, and the HC-CDR3 of the single domain antibody comprise: HC-CDR1: SEQ ID NO: 36, HC-CDR2: SEQ ID NO: 37, and HC-CDR3: SEQ ID NO: 38.
  • Embodiment 313 comprises the isolated polypeptide or polypeptide complex of embodiment 299, wherein the single domain antibody comprises an amino acid sequence according to SEQ ID NO: 42.
  • Embodiment 314 comprises the isolated polypeptide or polypeptide complex of embodiment 299, wherein the single domain antibody is SA21.
  • Embodiment 315 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-314, wherein the isolated polypeptide or polypeptide complex comprises a modified amino acid, a non-natural amino acid, a modified non-natural amino acid, or a combination thereof.
  • Embodiment 316 comprises the isolated polypeptide or polypeptide complex of embodiment 315, wherein the modified amino acid or modified non-natural amino acid comprises a post-translational modification.
  • Embodiment 317 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 285-316, wherein Hi comprises a linking moiety (L3) that connects Hi to Pi.
  • Hi comprises a linking moiety (L3) that connects Hi to Pi.
  • Embodiment 318 comprises the isolated polypeptide or polypeptide complex of embodiment 317, wherein L3 is a peptide sequence having at least 5 to no more than 50 amino acids.
  • Embodiment 319 comprises the isolated polypeptide or polypeptide complex of embodiment 317, wherein L3 is a peptide sequence having at least 10 to no more than 30 amino acids.
  • Embodiment 320 comprises the isolated polypeptide or polypeptide complex of embodiment 317, wherein L3 is a peptide sequence having at least 10 amino acids.
  • Embodiment 321 comprises the isolated polypeptide or polypeptide complex of embodiment 317, wherein L3 is a peptide sequence having at least 18 amino acids.
  • Embodiment 322 comprises the isolated polypeptide or polypeptide complex of embodiment 317, wherein L3 is a peptide sequence having at least 26 amino acids.
  • Embodiment 323 comprises the isolated polypeptide or polypeptide complex of embodiment 317, wherein L3 has a formula selected from the group consisting of (G2S) n , (GS) n , (GSGGS) n (SEQ ID NO: 836), (GGGS) friendship (SEQ ID NO: 837), (GGGGS) pertain (SEQ ID NO: 838), and (GSSGGS)trust (SEQ ID NO: 839), wherein n is an integer of at least 1.
  • Embodiment 324 comprises the isolated polypeptide or polypeptide complex of embodiment 317, wherein L3 comprises an amino acid sequence according to SEQ ID NO: 29.
  • Embodiment 325 comprises a method of treating cancer comprising administering the polypeptides or polypeptide complexes of any of the above embodiments to a subject in need thereof.
  • Embodiment 326 comprises the method of embodiment 325, wherein the cancer has cells that express EGFR.
  • Embodiment 327 comprises a method of treating colorectal cancer (CRC), squamous cell carcinoma of the head and Neck (SCCHN), non-small cell lung cancer (NSCLC), prostate cancer, breast cancer, colon/rectum cancer, head and neck cancer, esophagogastric cancer, liver cancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, kidney cancer, or pancreatic cancer comprising administering the polypeptides or polypeptide complexes of any of the above embodiments to a subject in need thereof.
  • Embodiment 328 comprises the method of embodiment 327, wherein the subject in need thereof is resistant to EGFR inhibitor treatment.
  • Embodiment 329 comprises the method of embodiment 327, wherein the subject in need thereof harbors KRAS mutations.
  • Embodiment 330 comprises the method of embodiment 327, wherein the subject in need thereof is resistant to EGFR inhibitor treatment and harbors KRAS mutations.
  • Embodiment 331 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-77, wherein P comprises the amino acid sequence according to SEQ ID NO: 115 or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to SEQ ID NO: 115.
  • Embodiment 332 comprises the isolated polypeptide or polypeptide complex of embodiment 331, wherein P comprises the amino acid sequence according to SEQ ID NOs: 115.
  • Embodiment 333 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 1-77, wherein P comprises the amino acid sequence according to SEQ ID NO: 116 or an amino acid sequence that has 1, 2, or 3 amino acid mutations, substitutions, or deletions relative to SEQ ID NO: 116.
  • Embodiment 334 comprises the isolated polypeptide or polypeptide complex of embodiment 333, wherein P comprises the amino acid sequence according to SEQ ID NOs: 116.
  • Embodiment 335 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 255-257, wherein the peptide comprises the amino acid sequence according to SEQ ID NO:
  • Embodiment 336 comprises the isolated polypeptide or polypeptide complex of any one of embodiments 255-257, wherein the peptide comprises the amino acid sequence according to SEQ ID NO:
  • polypeptide complex molecules were evaluated for their ability to bind EGFR as well as CD3 in a standard enzyme linked immunosorbent assay (ELISA) format.
  • ELISA enzyme linked immunosorbent assay
  • Polypeptide complex binding of EGFR or CD3 were measured before and after protease treatment.
  • biotinylated antigen was captured on neutravidin coated plates.
  • Polypeptide complex molecules were treated with active matriptase (MTSP1) where indicated.
  • MTSP1 active matriptase
  • Polypeptide complex molecules diluted in buffer were then added to the antigen coated plates.
  • Bound polypeptide complex was detected using a standard horse radish peroxidase conjugate secondary antibody.
  • the concentration of polypeptide complex required to achieve 50% maximal signal (EC50) was calculated using Graphpad Prism software.
  • Fig. 2 shows representative polypeptide molecule binding to EGFR measured by ELISA.
  • Fig. 3 shows representative polypeptide binding to CD3 measured by ELISA.
  • Example 2 Polypeptide complex mediated tumor cytotoxicity and T cell activation
  • Polypeptide complexes were evaluated in a functional in vitro tumor cell killing assay using the EGFR positive tumor cell lines HCT116 and A431. Tumor cell killing was measured using an xCelligence real time cell analyzer from Agilent that relies on sensor impedance measurements (cell index) that increased as tumor cells adhere, spread, and expand on the surface of the sensor. Likewise, as the tumor cells were killed the impedance decreased. 10,000 tumor cells were added per well and allowed to adhere overnight on a 96 well E-Plate. The following day polypeptide complexes titrated in human serum supplemented medium along with 30,000 CD8+ T cells were added to the wells.
  • Fig. 4 shows a graph of polypeptide complex mediated HCT116 tumor cell killing in the presence of CD8+ T cells.
  • Fig. 5 shows a graph of polypeptide complex mediated A431 tumor cell killing in the presence of CD8+ T cells.
  • polypeptide complex molecules were also evaluated for their ability to bind human and cynomolgus monkey antigen EGFR in a standard enzyme linked immunosorbent assay (ELISA) format. Briefly, antigens fused to a human Fc domain were directly coated on high binding ELISA plates. Polypeptide complex molecules diluted in buffer were then added to the antigen coated plates. Bound polypeptide complex was detected using a standard horse radish peroxidase conjugate secondary antibody. The concentration of polypeptide complex required to achieve 50% maximal signal (EC50) was calculated using Graphpad Prism software. Data is seen in Fig. 7.
  • ELISA enzyme linked immunosorbent assay
  • polypeptide complex molecules were evaluated for their ability to bind human and cynomolgus monkey antigen CD3 in a standard enzyme linked immunosorbent assay (ELISA) format. Briefly, antigens fused to a human Fc domain were directly coated on high binding ELISA plates. Polypeptide complex molecules diluted in buffer were then added to the antigen coated plates. Bound polypeptide complex was detected using a standard horse radish peroxidase conjugate secondary antibody. The concentration of polypeptide complex required to achieve 50% maximal signal (EC50) was calculated using Graphpad Prism software. Data is seen in Fig. 9.
  • ELISA enzyme linked immunosorbent assay
  • FIG. 10A demonstrates that the polypeptide complexes described herein comprising a mask was over 8,500 less potent at T cell-mediated killing of EGFR-expressing tumor cells than an equivalent unmasked TCE in an in vitro assay.
  • PC4 masked
  • Fig. 10B and Table 9 below PC4 (masked) exhibited a half-life of approximately 94 hours, which supports once-weekly dosing in humans, as compared to an unmasked polypeptide complex that exhibited a half-life of about an hour.
  • Example 5 Polypeptide complexes in cvnomolgus cytokine release
  • Cytokine release after polypeptide molecule administration by IV bolus was evaluated in cynomolgus monkeys. Briefly, cynomolgus monkeys of approximately 3kg 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 Thl/Th2 bead array kit from BD biosciences following the manufacturer’s instructions.
  • Fig. 12 shows dosing of polypeptide complexes (EGFR TRACTr) described herein at 10()pg/kg resulted in minimal levels of inflammatory cytokine release, relative to an unmasked EGFR-TCE at 10pg/kg, which led to a greater than 20-fold expression of IL-6.
  • EGFR TRACTr polypeptide complexes
  • Example 6 Polypeptide complexes in cvnomolgus toxicity
  • cynomolgus monkeys Systemic liver enzymes after polypeptide molecule administration by IV bolus was evaluated in cynomolgus monkeys. Briefly, cynomolgus monkeys of approximately 3kg 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 aspartate transaminase
  • ALT alanine aminotransferase
  • AST and ALT levels were remained within the normal ranges for all timepoints tested after dosing suggesting a lack of liver toxicity.
  • AST and ALT were quantified following the instructions provided in a commercially available kit from Millipore. AST and ALT levels were calculated according to manufacturer’s instructions relative to a positive control reference standard. Data is seen in Figs. 13A-13D.
  • Example 7 Peptides bind EGFR IgG and inhibit EGFR IgG from binding to the EGFR antigen [0631]
  • the peptide sequences (peptide- 1 to peptide-6) of Table 11 were evaluated for binding to EGFR IgG (see Table 11) in an ELISA format. Briefly, biotinylated peptides were captured on neutravidin coated plates. EGFR IgG diluted in buffer was then added to the peptide coated plates. Bound EGFR IgG was detected using a standard horse radish peroxidase conjugate secondary antibody. The ELISA signal was plotted versus the log-scale antibody concentration.
  • EC50S concentrations of antibody required to observe half maximal binding signal
  • Fig. 14 shows binding curves for peptide binding to EGFR IgG
  • Table 12 provides the calculated EC50S for peptide binding to EGFR IgG.
  • Peptide-6 of SEQ ID NO: 76 showed the strongest binding to EGFR IgG (lowest EC50) among the peptides tested in this series.
  • a peptide For a peptide to function as a mask, it must also inhibit the antibody of interest from binding to its cognate antigen. As such, ELISA-based competitive inhibition studies were also used to test the ability of each peptide to inhibit EGFR IgG from binding to the EGFR antigen. Biotinylated antigen was captured on neutravidin coated plates, quenched using biocytin, and washed. Inhibitory peptides were titrated in a dilution series and pre-incubated with a constant concentration of antibody. Inhibitory peptide and antibody mixtures were then incubated on the antigen captured plates. A horseradish peroxidase conjugate secondary antibody was then used to detect the antibody binding to the plate-bound antigen.
  • the ELISA signal was plotted versus log-scale peptide concentration (see Fig. 15). A dose dependent decrease of signal was indicative of peptides that compete for antibody binding to the cognate antigen. Graphpad Prism software was used to calculate the inhibitory concentrations of peptide required to achieve 50% maximal signal (IC50S). The IC50 data is provided in Table 13. Of the peptides tested in this series, peptide-6 was the most effective at inhibiting EGFR IgG binding to the EGFR antigen.
  • Example 8 Peptides bind EGFR Fab and inhibit EGFR Fab from binding to the EGFR antigen [0633]
  • the peptide sequences of Table 14 were evaluated for binding to EGFR Fab in an ELISA format. Binding studies were carried out as described above in Example 7. Binding curves are shown in Fig. 16 and EC50S for binding are provided in Tables 15-16. Peptide-8 and peptide-15 showed the strongest binding affinities (lowest EC50S) for EGFR Fab among the peptides tested in this series.
  • Sequence activity relationships were established for peptide-8 which was found to exhibit strong binding to EGFR Fab and inhibition of EGFR Fab binding to the EFGR antigen.
  • the sequence activity relationships were established by mutating individual residues within the peptide to alanine and measuring the influence of each mutation on the ability of the peptide to inhibit EGFR Fab binding to the EGFR antigen.
  • the alanine scanning sequences are provided in Table 19.
  • DNA oligo libraries were constructed where codons encoding critical residues within peptide-8 were minimally mutated, and codons encoding non-critical residues that were tolerated in the alanine scanning mutation studies (e.g., Arg-3, Ile-6, Val-8, and Val-14) were heavily mutated.
  • the resulting oligos were cloned into bacteriophage vectors used to display the SAR guided peptides via fusion to the pill filament of the bacteriophage.
  • the relevant vectors were then used to produce the phage optimization libraries via amplification in bacteria using standard techniques in the field.
  • Example 11 Panning of the optimized phage library
  • phage libraries were bio-panned using EGFR Fab loaded beads. Multiple rounds of panning were performed where bacteriophage was allowed to bind to EGFR Fab loaded beads, washed, eluted, and amplified. Additional selective pressure was included during each round of panning using a fixed concentration of EGFR antigen or peptide-8. After panning, phage infected bacteria were plated out and colonies picked into 96 well blocks. Clonal phage was then amplified and separated from bacterial cells via centrifugation. Phage containing supernatants were tested in binding ELISAs against EGFR Fab coated plates in the presence or absence of saturating concentrations of EGFR antigen. Phage able to bind EGFR Fab were selected for sequence analysis if the binding signal was reduced in the presence of EGFR.
  • Clonal phage were harvested as crude supernatants and screened via standard EFISAs. Briefly, biotinylated EGFR Fab was captured on neutravidin coated plates. Prior to the addition of clonal phage, wells were incubated with blocking buffer and EGFR antigen or blocking buffer alone. Without washing or aspirating, clonal phage supernatants were then added to the wells and incubated for a short time. Wells were then washed followed by detection of bound phage using a horse radish conjugated anti-M13 antibody. Clonal phage of interest were then sent for sequence analysis.
  • Phage panning results of peptide-8 library sequences are shown in Table 22. 679 clonal phage sequences were identified. A consensus sequence was calculated from all the sequences of Table 22 using WebFogo 3.7.4. The consensus sequence is shown in Fig. 20.
  • Example 13 Anti-tumor efficacy in a mouse model of colorectal cancer
  • mice Female mice were subcutaneously implanted with 5 million HCT116 colorectal tumor cells in 50% matrigel. 20 million human PBMCs were engrafted via the tail vein the same day as HCT116 inoculation. When tumors reached 250 mm 3 , mice were randomized into groups and compounds were dosed intravenously every day for 10 days. Tumor volume was measured every two to three days and plotted overtime. The tumor volume growth kinetics indicate anti -tumor activity of masked EGFR targeted bispecific compounds. The anti-tumor activity observed was protease dependent in that the compound lacking the protease substrate within the cleavable linker was equivalent to vehicle controls.
  • Figs-25-28 show the in vivo tumor growth inhibition data with different dosing amounts of PCI (Fig. 25), PC5 (Fig. 26), PC7 (Fig. 27), and PC6 (Fig. 28).
  • Example 14 Binding of PCI and PC4 to human and cvnomolgus CD3e, EGFR. and albumin
  • Polypeptide complexes PCI and PC4 were evaluated for binding to human and cynomolgus monkey CD3s, EGFR, and albumin by ELISA. Briefly, biotinylated antigen was captured on neutravidin coated plates. Polypeptide complex molecules were treated with active protease where indicated. Polypeptide complex molecules diluted in buffer were then added to the antigen coated plates. Bound polypeptide complex was detected using a standard horse radish peroxidase conjugate secondary antibody. Binding curves were fit using Graphpad Prism software. Binding curves for PCI and PC4 binding to human and cynomolgus monkey CD3s are shown in Figs. 29 and 30, respectively.
  • Binding curves for PCI and PC4 binding to human and cynomolgus monkey EGFR are shown in Figs. 31 and 32, respectively. As can be seen from the curves, target engagement is cleavage dependent as masking reduces CD3s and EGFR binding by more than 1000-fold and more than 300-fold, respectively. Treatment of PC4 with protease enzyme enables potent antigen binding comparable to non-masked PCI. Binding curves for PC4 binding to human and cynomolgus monkey albumin are shown in Figs. 33 and 34, respectively. As can be seen, PC4 exhibits potent binding to both human and monkey albumin.
  • Example 15 Polypeptide complex mediated tumor cytotoxicity and T cell activation (A549 cells)
  • Polypeptide complexes were evaluated in a functional in vitro tumor cell killing assay using the EGFR positive tumor cell line A549. Tumor cell killing was measured using an xCelligence real time cell analyzer from Agilent that relies on sensor impedance measurements (cell index) that increased as tumor cells adhere, spread, and expand on the surface of the sensor. Likewise, as the tumor cells were killed the impedance decreased. Tumor cells were added per well and allowed to adhere overnight on a 96 well E- Plate. The following day polypeptide complexes titrated in human serum supplemented medium along with human PBMCs were added to the wells. Cell index measurements were taken every 10 minutes for an additional 72hours.
  • Fig. 35 shows a graph of PCI and PC4 mediated A549 tumor cell killing in the presence of PBMCs.
  • PC4 was treated with protease where indicated.
  • Protease treatment increased the activity of PC4 by about 43000-fold relative to untreated PC4.
  • the activity level of protease treated PC4 was comparable to that of non-masked PCI (see Fig. 35).

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WO2022094299A2 (en) 2020-10-30 2022-05-05 Janux Therapeutics, Inc. Multispecific antibodies for targeting cd28 and pd-l1 and methods of use thereof
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WO2024102723A2 (en) 2022-11-10 2024-05-16 Janux Therapeutics, Inc. Antibodies targeting egfr and cd3 and uses thereof
EP4615879A2 (en) 2022-11-11 2025-09-17 Janux Therapeutics, Inc. Compositions and uses of tumor activated antibodies targeting egfr and effector cell antigens

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US12161724B2 (en) 2020-04-04 2024-12-10 Janux Therapeutics, Inc. Compositions and methods related to tumor activated antibodies targeting EGFR and effector cell antigens
US12433953B2 (en) 2020-04-04 2025-10-07 Janux Therapeutics, Inc. Compositions and methods related to tumor activated antibodies targeting EGFR and effector cell antigens
EP4259177A4 (en) * 2020-12-08 2024-11-06 Janux Therapeutics, Inc. Peptide compositions and methods for anti-cd3 binding domains
WO2025195306A1 (en) * 2024-03-16 2025-09-25 Vibrant Pharma Limited Multispecific antigen-binding proteins and uses thereof
WO2025195305A1 (en) * 2024-03-16 2025-09-25 Vibrant Pharma Limited Multispecific antigen binding proteins and uses thereof
WO2025201240A1 (en) * 2024-03-25 2025-10-02 Tavotek Biotherapeutics (Hong Kong) Limited Antibodies targeting cd3, cd28, and pd-l1 and uses thereof
WO2025201560A1 (en) * 2024-03-29 2025-10-02 Vibrant Pharma Limited Multispecific antigen-binding proteins and uses thereof

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MX2023013382A (es) 2024-01-30
JP2024518539A (ja) 2024-05-01
AU2022272906A9 (en) 2023-12-07
BR112023023650A2 (pt) 2024-01-30
EP4337697A4 (en) 2025-05-14
TW202311289A (zh) 2023-03-16
US20260014267A1 (en) 2026-01-15
AR125758A1 (es) 2023-08-09
US12433953B2 (en) 2025-10-07
WO2022240637A3 (en) 2022-12-15
US12161724B2 (en) 2024-12-10
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