Patent Publication Number: US-2023147782-A1

Title: Cleavable linker compositions and methods

Description:
CROSS-REFERENCE 
     This application is a continuation of U.S. patent application Ser. No. 17/398,500, filed on Aug. 10, 2021, which claims the benefit of U.S. Provisional Application No. 63/064,268, filed Aug. 11, 2020, which application is incorporated herein by reference. 
    
    
     SEQUENCE LISTING 
     The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on Oct. 18, 2022, is named 52426-720_301 SL.xml and is 42,044 bytes in size. 
     SUMMARY 
     Disclosed herein, in certain embodiments, are isolated polypeptides comprising a cleavable linker according to the amino acid sequence of SEQ ID NO: 1 (LSGRSDAG). In some embodiments, the cleavable linker comprises the amino acid sequence of SEQ ID NO: 3 (ISSGLLSGRSDAG). In some embodiments, the cleavable linker comprises the amino acid sequence of SEQ ID NO: 26 (AGLLAPPGGLSGRSDAG). In some embodiments, the cleavable linker comprises the amino acid sequence of SEQ ID NO: 4 (AAGLLAPPGGLSGRSDAG). In some embodiments, the cleavable linker comprises the amino acid sequence of SEQ ID NO: 5 (SPLGLSGRSDAG). In some embodiments, the cleavable linker comprises the amino acid sequence of SEQ ID NO: 6 (LSGRSDAGSPLGLAG). In some embodiments, the cleavable linker is cleavable by a protease. In some embodiments, the protease comprises a tumor specific protease. In some embodiments, the protease comprises a matrix metalloprotease (MMP) or a serine protease. In some embodiments, the matrix metalloprotease comprises MMP2, MMP7, MMP9, MMP13, or MMP14. In some embodiments, the serine protease comprises matriptase, urokinase, or hepsin. In some embodiments, the isolated polypeptide further comprises an antigen binding domain that binds to a target antigen. In some embodiments, the antigen binding domain is C-terminal to the cleavable linker. In some embodiments, the isolated polypeptide further comprises a cytokine or cytokine fragment that binds to a cytokine receptor. In some embodiments, the cytokine or cytokine fragment is C-terminal to the cleavable linker. In some embodiments, the cleavable linker connects a peptide to an antigen binding domain that binds to a target antigen or to a cytokine or cytokine fragment that binds to a cytokine receptor in a configuration according to Formula I: A 1 -L 1 -P 1  wherein A 1  comprises the antigen binding domain that binds to the target antigen or the cytokine or cytokine fragment that binds to the cytokine receptor; L 1  comprises the cleavable linker; P 1  comprises a peptide that impairs binding of the antigen binding domain to the target antigen or impairs binding of the cytokine to the cytokine receptor. In some embodiments, P 1  is connected N-terminal to the cleavable linker and A 1  is connected C-terminal to the cleavable linker. In some embodiments, P 1  is connected C-terminal to the cleavable linker and A 1  is connected N-terminal to the cleavable linker. In some embodiments, P 1  is bound to A 1  through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bonding interactions, or a combination thereof. In some embodiments, P 1  has less than 70% sequence homology to the target antigen or the cytokine receptor. In some embodiments, P 1  comprises a peptide sequence of at least 10 amino acids in length. In some embodiments, P 1  comprises a peptide sequence of at least 10 amino acids in length and no more than 20 amino acids in length. In some embodiments, P 1  comprises a peptide sequence of at least 16 amino acids in length. In some embodiments, P 1  comprises a peptide sequence of no more than 40 amino acids in length. In some embodiments, P 1  comprises a cyclic peptide or a linear peptide. In some embodiments, P 1  comprises a cyclic peptide. In some embodiments, P 1  is further linked to a half-life extending moiety. In some embodiments, the half-life extending moiety is a single-domain antibody. In some embodiments, the single domain antibody comprises 10G. In some embodiments, A 1  comprises an antibody, 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, a Fab, a Fab′, a Fab light chain polypeptide, or a Fab heavy chain polypeptide. In some embodiments, the target antigen comprises a tumor antigen. In some embodiments, A 1  comprises the Fab light chain polypeptide or the Fab heavy chain polypeptide. In some embodiments, A 1  comprises an epidermal growth factor receptor (EGFR) binding domain. In some embodiments, the target antigen comprises an effector cell antigen. In some embodiments, A 1  comprises the scFv. In some embodiments, the scFv comprises an anti-CD3e single chain variable fragment. In some embodiments, A 1  comprises the cytokine. In some embodiments, the cytokine or cytokine fragment is a wild-type cytokine. In some embodiments, the cytokine or cytokine fragment is a mutein of the cytokine. In some embodiments, the cytokine receptor is an interferon receptor or an interleukin receptor. In some embodiments, the cytokine receptor comprises an interferon receptor, GM-CSF receptor, IL-2 receptor, IL-4 receptor, IL-6 receptor, IL-7 receptor, IL-10 receptor, IL-12 receptor, IL-15 receptor, IL-21 receptor, or TGF-β receptor. In some embodiments, the cytokine or cytokine fragment comprises an interferon, GM-CSF, IL-2, IL-7, IL-12, IL-15, or IL-21. In some embodiments, the cytokine or cytokine fragment comprises an IL-2, IL-12, IL-6, IL-4, IL-10, or TGFβ. In some embodiments, the isolated polypeptide is complexed with a second isolated polypeptide comprising a second antigen binding domain or a second cytokine or second cytokine fragment. In some embodiments, the second isolated polypeptide is in a configuration according to Formula II: A 2 -L 2 -P 2  wherein A 2  comprises the second antigen binding domain or the second cytokine; L 2  comprises a second cleavable linker; P 2  comprises a second peptide that impairs binding of the second antigen binding domain to a second target antigen or impairs binding of the second cytokine or second cytokine fragment to a second cytokine receptor. In some embodiments, the second cleavable linker comprises the amino acid sequence of SEQ ID NO: 1 (LSGRSDAG). In some embodiments, the second cleavable linker comprises the amino acid sequence of SEQ ID NO: 3 (ISSGLLSGRSDAG). In some embodiments, the second cleavable linker comprises the amino acid sequence of SEQ ID NO: 26 (AGLLAPPGGLSGRSDAG). In some embodiments, the second cleavable linker comprises the amino acid sequence of SEQ ID NO: 4 (AAGLLAPPGGLSGRSDAG). In some embodiments, the second cleavable linker comprises the amino acid sequence of SEQ ID NO: 5 (SPLGLSGRSDAG). In some embodiments, the second cleavable linker comprises the amino acid sequence of SEQ ID NO: 6 (LSGRSDAGSPLGLAG). In some embodiments, P 2  is connected N-terminal to the second cleavable linker and A 2  is connected C-terminal to the second cleavable linker. In some embodiments, P 2  is connected C-terminal to the second cleavable linker and A 2  is connected N-terminal to the second cleavable linker. In some embodiments, 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  has less than 70% sequence homology to the second target antigen or the second cytokine receptor. In some embodiments, P 2  comprises a peptide sequence of at least 10 amino acids in length. In some embodiments, 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, P 2  comprises a peptide sequence of at least 16 amino acids in length. In some embodiments, P 2  comprises a peptide sequence of no more than 40 amino acids in length. In some embodiments, P 2  comprises a cyclic peptide or a linear peptide. In some embodiments, P 2  comprises a cyclic peptide. In some embodiments, A 2  comprises an antibody, 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, a Fab, a Fab′, a Fab light chain polypeptide, or a Fab heavy chain polypeptide. In some embodiments, the second target antigen comprises a tumor antigen. In some embodiments, A 2  comprises the Fab light chain polypeptide or the Fab heavy chain polypeptide. In some embodiments, A 2  comprises an epidermal growth factor receptor (EGFR) binding domain. In some embodiments, the second target antigen comprises an effector cell antigen. In some embodiments, A 2  comprises the scFv. In some embodiments, the scFv comprises an anti-CD3e single chain variable fragment. In some embodiments, A 2  comprises the second cytokine. In some embodiments, the second cytokine or second cytokine fragment is a wild-type cytokine. In some embodiments, the second cytokine or second cytokine fragment is a mutein of the cytokine. In some embodiments, the second cytokine receptor is an interferon receptor or an interleukin receptor. In some embodiments, the second cytokine receptor comprises an interferon receptor, GM-CSF receptor, IL-2 receptor, IL-4 receptor, IL-6 receptor, IL-7 receptor, IL-10 receptor, IL-12 receptor, IL-15 receptor, IL-21 receptor, or TGF-β receptor. In some embodiments, the second cytokine or second cytokine fragment comprises an interferon, GM-CSF, IL-2, IL-7, IL-12, IL-15, or IL-21. In some embodiments, the second cytokine or second cytokine fragment comprises an IL-2, IL-12, IL-6, IL-4, IL-10, or TGFβ. 
     Disclosed herein are pharmaceutical compositions comprising: the isolated polypeptide comprising a cleavable linker according to any of the above embodiments; and a pharmaceutically acceptable excipient. 
     Disclosed herein are isolated recombinant nucleic acid molecule encoding the isolated polypeptide comprising a cleavable linker according to any of the above embodiments. 
     Disclosed herein are vectors comprising the recombinant nucleic acid molecule according to the above embodiment. 
     Disclosed herein are methods of producing an isolated polypeptide comprising a cleavable linker comprising culturing a cell under conditions that lead to expression of the polypeptide, wherein the cell comprises the vector of the above embodiment. 
     Disclosed herein are methods of manufacturing an isolated polypeptide comprising a cleavable linker, the method comprising: (a) culturing a cell comprising the recombinant nucleic acid molecule of the above embodiments under conditions that lead to expression of the polypeptide, and (b) isolating the polypeptide. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which: 
         FIGS.  1 A- 1 B  illustrate binding of polypeptide complexes PC-1, PC-2, PC-3, PC-4, and PC-5 comprising EGFR masking ( FIG.  1 A ) and followed by cleavage by the tumor protease MTSP1 ( FIG.  1 B ). 
         FIGS.  2 A- 2 B  illustrate of polypeptide complexes PC-1, PC-2, PC-3, PC-4, and PC-5 comprising CD3ε masking ( FIG.  2 A ) and followed by cleavage by the tumor protease MTSP1 ( FIG.  2 B ). 
         FIGS.  3 A- 3 B  illustrate binding of polypeptide complexes PC-2, PC-3, PC-4, PC-5, PC-1, and PC-6 to EGFR-biotin ( FIG.  3 A ) and CD3ε-biotin ( FIG.  3 B ) measured by ELISA. 
         FIGS.  4 A- 4 E  illustrate cytotoxicity against tumor target cells HCT116 for polypeptide complexes PC-2, PC-3, PC-4, PC-5, PC-1, and PC-6. 
         FIGS.  5 A- 5 D  illustrate pharmacokinetics of polypeptide PC-1, PC-2, PC-3, PC-7, PC-4, and PC-5 in cynomolgus monkey. 
         FIGS.  6 A- 6 D  illustrate cytokine release of polypeptide complexes PC-1, PC-2, PC-3, PC-7, PC-4, and PC-5 molecules in cynomolgus monkey. 
         FIG.  7 A- 7 B  illustrates graphs of AST and ALT levels of polypeptide complexes PC-1, PC-2, PC-3, PC-4, and PC-5 in cynomolgus monkey. 
     
    
    
     DETAILED DESCRIPTION 
     Protein-based therapies such as antibodies, T cell receptors (TCR), and cytokine therapies have proven effective for a variety diseases and disorders. As with any therapy, there is a need to minimize off-target effects of the protein-based therapy in healthy tissue while maintaining activity of the protein-based therapy in disease tissue. One such strategy is to create an inactive form of the protein-based therapy in which a necessary binding site on the protein-based therapy is blocked with a peptide linked to the protein-based therapy, thereby preventing the protein-based therapy from binding or interacting with its cognate receptor or target antigen when in healthy tissue. For activating the protein-based therapy in the desired disease-state microenvironment, the peptide is linked to the protein-based therapy with a linker that is cleavable by a protease that is specific to the disease-state microenvironment. The peptide is then released from the protein-based therapy when in the disease-state microenvironment. 
     Accordingly, disclosed herein, are cleavable linkers which can be applied to a variety of protein-based therapy formats, for use in reducing off-target effects of the protein-based therapy in healthy tissue, while maintaining activity of the protein-based therapy in disease tissue. The cleavable linkers, as disclosed herein, have desirable properties, which include, for example but are not limited to, increased rates of proteolysis by tumor proteases or cleavable by an expanded panel of tumor proteases while also having comparable safety profiles relative to control linkers. 
     Certain Definitions 
     The terminology used herein is for the purpose of describing particular cases only and is not intended to be limiting. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.” 
     The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the given value. Where particular values are described in the application and claims, unless otherwise stated the term “about” should be assumed to mean an acceptable error range for the particular value. 
     “Fragment” as used herein refers to a peptide or a polypeptide that comprises less than the full length amino acid sequence. 
     “Peptide”, “P 1 ”, or “P 2 ” as used herein refers to an amino acid sequence of less than 50 amino acids and specifically excludes a cytokine ligand binding domain, fragments, or muteins thereof, a cytokine receptor, fragments, or muteins thereof, and any antibody or antibody binding fragments (for example, a single domain antibody, Fab, or scFv) that binds to a cytokine, or binds to a cognate cytokine receptor. 
     As disclosed herein, in some embodiments, are isolated polypeptides comprising a cleavable linker according to the amino acid sequence of SEQ ID NO: 1 (LSGRSDAG) 
     In some embodiments, the cleavable linker comprises the amino acid sequence of SEQ ID NO: 3 (ISSGLLSGRSDAG). In some embodiments, the cleavable linker comprises the amino acid sequence of SEQ ID NO: 26 (AGLLAPPGGLSGRSDAG). In some embodiments, the cleavable linker comprises the amino acid sequence of SEQ ID NO: 4 (AAGLLAPPGGLSGRSDAG). In some embodiments, the cleavable linker comprises the amino acid sequence of SEQ ID NO: 5 (SPLGLSGRSDAG). In some embodiments, the cleavable linker comprises the amino acid sequence of SEQ ID NO: 6 (LSGRSDAGSPLGLAG). 
     In some embodiments, the cleavable linker consists of the amino acid sequence of SEQ ID NO: 1 (LSGRSDAG). In some embodiments, the cleavable linker consists of the amino acid sequence of SEQ ID NO: 3 (ISSGLLSGRSDAG). In some embodiments, the cleavable linker consists of the amino acid sequence of SEQ ID NO: 26 (AGLLAPPGGLSGRSDAG). In some embodiments, the cleavable linker consists of the amino acid sequence of SEQ ID NO: 4 (AAGLLAPPGGLSGRSDAG). In some embodiments, the cleavable linker consists of the amino acid sequence of SEQ ID NO: 5 (SPLGLSGRSDAG). In some embodiments, the cleavable linker consists of the amino acid sequence of SEQ ID NO: 6 (LSGRSDAGSPLGLAG). 
     In some embodiments, the cleavable linker comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 3, 4, 5, and 6. 
     In some embodiments, are isolated polypeptides comprising a cleavable linker according to the amino acid sequence of Linker 1 (ISSGLLSGRSDAG) SEQ ID NO: 3, Linker 2 (AAGLLAPPGGLSGRSDAG) SEQ ID NO:4, Linker 3 (SPLGLSGRSDAG) SEQ ID NO: 5, or Linker 4 (LSGRSDAGSPLGLAG) SEQ ID NO: 6 or an isolated polypeptide comprising a cleavable linker that has 1, 2, or 3 amino acid substitutions, additions, or deletions relative to the amino acid sequence of Linker 1, Linker 2, Linker 3, or Linker 4. 
     In some embodiments, the cleavable linker comprises the amino acid sequence of Linker 1. In some embodiments, the cleavable linker consists of the amino acid sequence of Linker 1. In some embodiments, the cleavable linker has 1 amino acid substitution, addition, or deletion relative to the amino acid sequence of Linker 1. In some embodiments, the cleavable linker has 2 amino acid substitutions, additions, or deletions relative to the amino acid sequence of Linker 1. In some embodiments, the cleavable linker has 3 amino acid substitutions, additions, or deletions relative to the amino acid sequence of Linker 1. 
     In some embodiments, the cleavable linker comprises the amino acid sequence of Linker 2. In some embodiments, the cleavable linker consists of the amino acid sequence of Linker 2. In some embodiments, the cleavable linker has 1 amino acid substitution, addition, or deletion relative to the amino acid sequence of Linker 2. In some embodiments, the cleavable linker has 2 amino acid substitutions, additions, or deletions relative to the amino acid sequence of Linker 2. In some embodiments, the cleavable linker has 3 amino acid substitutions, additions, or deletions relative to the amino acid sequence of Linker 2. 
     In some embodiments, the cleavable linker comprises the amino acid sequence of Linker 3. In some embodiments, the cleavable linker consists of the amino acid sequence of Linker 3. In some embodiments, the cleavable linker has 1 amino acid substitution, addition, or deletion relative to the amino acid sequence of Linker 3. In some embodiments, the cleavable linker has 2 amino acid substitutions, additions, or deletions relative to the amino acid sequence of Linker 3. In some embodiments, the cleavable linker has 3 amino acid substitutions, additions, or deletions relative to the amino acid sequence of Linker 3. 
     In some embodiments, the cleavable linker comprises the amino acid sequence of Linker 4. In some embodiments, the cleavable linker consists of the amino acid sequence of Linker 4. In some embodiments, the cleavable linker has 1 amino acid substitution, addition, or deletion relative to the amino acid sequence of Linker 4. In some embodiments, the cleavable linker has 2 amino acid substitutions, additions, or deletions relative to the amino acid sequence of Linker 4. In some embodiments, the cleavable linker has 3 amino acid substitutions, additions, or deletions relative to the amino acid sequence of Linker 4. 
     In some embodiments, the amino acid substitution, addition, or deletion results in an amino acid sequence that is at least 75% identical, e.g., 77%, 80%, 82%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any protein described herein. In some embodiments, the amino acid substitution is a conservative amino acid substitution. Among the common amino acids, for example, a “conservative amino acid substitution” is illustrated by a substitution among amino acids within each of the following groups: (1) glycine, alanine, valine, leucine, and isoleucine, (2) phenylalanine, tyrosine, and tryptophan, (3) serine and threonine, (4) aspartate and glutamate, (5) glutamine and asparagine, and (6) lysine, arginine and histidine. 
     In some embodiments, the cleavable linker comprises a modified amino acid or non-natural amino acid, or a modified non-natural amino acid, or a combination thereof. In some embodiments, the modified amino acid or a modified non-natural amino acid comprises a post-translational modification. In some embodiments, the cleavable linker comprises a modification including, but not limited, to acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. Modifications are made anywhere to the cleavable linker including the peptide backbone, or the amino acid side chains. 
     In some embodiments, the cleavable linker is cleavable by a protease. In some embodiments, the protease is present in higher levels in a disease-state microenvironment relative to levels in healthy tissue or a microenvironment that is not the disease-state microenvironment. In some embodiments, the protease comprises a tumor specific protease. In some embodiments, the protease comprises a matrix metalloprotease (MMP) or a serine protease. In some embodiments, the matrix metalloprotease comprises MMP2, MMP7, MMP9, MMP13, or MMP14. In some embodiments, the matrix metalloprotease is selected from the group consisting of MMP2, MMP7, MMP9, MMP13, and MMP14. In some embodiments, the matrix metalloprotease comprises MMP2. In some embodiments, the matrix metalloprotease comprises MMP7. In some embodiments, the matrix metalloprotease comprises MMP9. In some embodiments, the matrix metalloprotease comprises MMP13. In some embodiments, the matrix metalloprotease comprises MMP14. In some embodiments, the serine protease comprises matriptase, urokinase, or hepsin. In some embodiments, the serine protease is selected from the group consisting of matriptase, urokinase, and hepsin. In some embodiments, the serine protease comprises matriptase. 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. 
     In some embodiments, the cleavable linker has increased rates of proteolysis as compared to the rates of proteolysis for linkers without the cleavable linker sequences. In some embodiments, the cleavable linker has increased rates of proteolysis that is at least 5× higher than the rates of proteolysis for linkers without the cleavable linker sequences. In some embodiments, the cleavable linker has increased rates of proteolysis that is at least 8× higher than the rates of proteolysis for linkers without the cleavable linker sequences. In some embodiments, the cleavable linker has increased rates of proteolysis that is at least 10× higher than the rates of proteolysis for linkers without the cleavable linker sequences. In some embodiments, the cleavable linker has increased rates of proteolysis that is at least 15× higher than the rates of proteolysis for linkers without the cleavable linker sequences. In some embodiments, the cleavable linker has increased rates of proteolysis that is at least 20× higher than the rates of proteolysis for linkers without the cleavable linker sequences. In some embodiments, the cleavable linker has increased rates of proteolysis that is at least 25× higher than the rates of proteolysis for linkers without the cleavable linker sequences. In some embodiments, the cleavable linker has increased rates of proteolysis that is at least 30× higher than the rates of proteolysis for linkers without the cleavable linker sequences. In some embodiments, the cleavable linker has increased rates of proteolysis that is at least 40× higher than the rates of proteolysis for linkers without the cleavable linker sequences. In some embodiments, the cleavable linker has increased rates of proteolysis that is at least 50× higher than the rates of proteolysis for linkers without the cleavable linker sequences. In some embodiments, the cleavable linker has increased rates of proteolysis that is at least 60× higher than the rates of proteolysis for linkers without the cleavable linker sequences. In some embodiments, the cleavable linker has increased rates of proteolysis that is at least 70× higher than the rates of proteolysis for linkers without the cleavable linker sequences. In some embodiments, the cleavable linker has increased rates of proteolysis that is at least 75× higher than the rates of proteolysis for linkers without the cleavable linker sequences. In some embodiments, the cleavable linker has increased rates of proteolysis that is at least 80× higher than the rates of proteolysis for linkers without the cleavable linker sequences. In some embodiments, the cleavable linker has increased rates of proteolysis that is at least 90× higher than the rates of proteolysis for linkers without the cleavable linker sequences. In some embodiments, the cleavable linker has increased rates of proteolysis that is at least 100× higher than the rates of proteolysis for linkers without the cleavable linker sequences. In some embodiments, the cleavable linker has increased rates of proteolysis that is at least 120× higher than the rates of proteolysis for linkers without the cleavable linker sequences. In some embodiments, the cleavable linker is cleaved by a protease. In some embodiments, the protease comprises a tumor specific protease. In some embodiments, the protease comprises a matrix metalloprotease (MMP) or a serine protease. In some embodiments, the matrix metalloprotease comprises MMP2, MMP7, MMP9, MMP13, or MMP14. In some embodiments, the serine protease comprises matriptase, urokinase, or hepsin. 
     In some embodiments, the cleavable linker has improved stability in human serum as compared to the stability in human serum without the cleavable linker sequences. In some embodiments, the cleavable linker has improved stability in human serum that is at least 5× higher than the stability in human serum for linkers without the cleavable linker sequences. In some embodiments, the cleavable linker has improved stability in human serum that is at least 8× higher than the stability in human serum for linkers without the cleavable linker sequences. In some embodiments, the cleavable linker has improved stability in human serum that is at least 10× higher than the stability in human serum for linkers without the cleavable linker sequences. In some embodiments, the cleavable linker has improved stability in human serum that is at least 15× higher than the stability in human serum for linkers without the cleavable linker sequences. In some embodiments, the cleavable linker has improved stability in human serum that is at least 20× higher than the stability in human serum for linkers without the cleavable linker sequences. In some embodiments, the cleavable linker has improved stability in human serum that is at least 25× higher than the stability in human serum for linkers without the cleavable linker sequences. In some embodiments, the cleavable linker has improved stability in human serum that is at least 30× higher than the stability in human serum for linkers without the cleavable linker sequences. In some embodiments, the cleavable linker has improved stability in human serum that is at least 40× higher than the stability in human serum for linkers without the cleavable linker sequences. In some embodiments, the cleavable linker has improved stability in human serum that is at least 50× higher than the stability in human serum for linkers without the cleavable linker sequences. In some embodiments, the cleavable linker has improved stability in human serum that is at least 60× higher than the stability in human serum for linkers without the cleavable linker sequences. In some embodiments, the cleavable linker has improved stability in human serum that is at least 70× higher than the stability in human serum for linkers without the cleavable linker sequences. In some embodiments, the cleavable linker has improved stability in human serum that is at least 75× higher than the stability in human serum for linkers without the cleavable linker sequences. In some embodiments, the cleavable linker has improved stability in human serum that is at least 80× higher than the stability in human serum for linkers without the cleavable linker sequences. In some embodiments, the cleavable linker has improved stability in human serum that is at least 90× higher than the stability in human serum for linkers without the cleavable linker sequences. In some embodiments, the cleavable linker has improved stability in human serum that is at least 100× higher than the stability in human serum for linkers without the cleavable linker sequences. In some embodiments, the cleavable linker has improved stability in human serum that is at least 120× higher than the stability in human serum for linkers without the cleavable linker sequences. 
     In some embodiments, the isolated polypeptide comprising the cleavable linker has increased rates of proteolysis as compared to an isolated polypeptide of the same amino acid sequence but comprising a cleavable linker according to the amino acid sequence of SEQ ID NO: 2. 
     In some embodiments, the isolated polypeptide comprising the cleavable linker has improved or equivalent serum stability as compared to an isolated polypeptide of the same amino acid sequence but comprising a cleavable linker according to the amino acid sequence of SEQ ID NO: 2. 
     In some embodiments, the isolated polypeptide comprising the cleavable linker has improved or equivalent in vitro tumor cell killing as compared to an isolated polypeptide of the same amino acid sequence but comprising a cleavable linker according to the amino acid sequence of SEQ ID NO: 2. 
     In some embodiments, the isolated polypeptide comprising the cleavable linker has improved or equivalent pharmacokinetic parameters in cynomolgus monkeys as compared to an isolated polypeptide of the same amino acid sequence but comprising a cleavable linker according to the amino acid sequence of SEQ ID NO: 2. 
     In some embodiments, the isolated polypeptide comprising the cleavable linker has improved or equivalent liver toxicity levels in cynomolgus monkeys as compared to an isolated polypeptide of the same amino acid sequence but comprising a cleavable linker according to the amino acid sequence of SEQ ID NO: 2. 
     In some embodiments, the isolated polypeptide further comprises an antigen binding domain that binds to a target antigen. In some embodiments, the antigen binding domain is C-terminal to the cleavable linker. In some embodiments, the isolated polypeptide further comprises a cytokine or cytokine fragment that binds to a cytokine receptor. In some embodiments, the cytokine or cytokine fragment is C-terminal to the cleavable linker. 
     In some embodiments, the cleavable linker connects a peptide to an antigen binding domain that binds to a target antigen or to a cytokine that binds to a cytokine receptor in a configuration according to Formula I: 
       A 1 -L 1 -P 1   (Formula I)
 
     wherein A 1  comprises the antigen binding domain that binds to the target antigen or the cytokine that binds to the cytokine receptor; L 1  comprises the cleavable linker; P 1  comprises a peptide that impairs binding of the antigen binding domain to the target antigen or impairs binding of the cytokine to the cytokine receptor. In some embodiments, P 1  is connected N-terminal to the cleavable linker and A 1  is connected C-terminal to the cleavable linker. In some embodiments, P 1  is connected C-terminal to the cleavable linker and A 1  is connected N-terminal to the cleavable linker. 
     In some embodiments, the isolated polypeptide is complexed with a second isolated polypeptide comprising a second antigen binding domain or a second cytokine. In some embodiments, the second isolated polypeptide is in a configuration according to Formula II: 
       A 2 -L 2 -P 2   (Formula II)
 
     wherein A 2  comprises the second antigen binding domain or the second cytokine; L 2  comprises a second cleavable linker; and P 2  comprises a second peptide that impairs binding of the second antigen binding domain to a second target antigen or impairs binding of the second cytokine to a second cytokine receptor. 
     In some embodiments, P 2  is connected N-terminal to the second cleavable linker and A 2  is connected C-terminal to the second cleavable linker. In some embodiments, P 2  is connected C-terminal to the second cleavable linker and A 2  is connected N-terminal to the second cleavable linker. 
     In some embodiments, the second cleavable linker comprises the amino acid sequence of SEQ ID NO: 1 (LSGRSDAG). In some embodiments, the second cleavable linker comprises the amino acid sequence of SEQ ID NO: 3 (ISSGLLSGRSDAG). In some embodiments, the second cleavable linker comprises the amino acid sequence of SEQ ID NO: 26 (AGLLAPPGGLSGRSDAG). In some embodiments, the second cleavable linker comprises the amino acid sequence of SEQ ID NO: 4 (AAGLLAPPGGLSGRSDAG). In some embodiments, the second cleavable linker comprises the amino acid sequence of SEQ ID NO: 5 (SPLGLSGRSDAG). In some embodiments, the second cleavable linker comprises the amino acid sequence of SEQ ID NO: 6 (LSGRSDAGSPLGLAG). 
     In some embodiments, L 1  or L 2  is at least 8 amino acids in length. In some embodiments, L 1  or L 2  is at least 10 amino acids in length but no more than 50 amino acids in length. In some embodiments, L 1  or L 2  is at least 10 amino acids in length but no more than 30 amino acids in length. In some embodiments, L 1  or L 2  is at least 18 amino acids in length. In some embodiments, L 1  or L 2  is at least 26 amino acids in length. In some embodiments, L 1  or L 2  is at least 30 amino acids in length. In some embodiments, L 1  or L 2  is at least 40 amino acids in length. In some embodiments, L 1  or L 2  is at least 50 amino acids in length. 
     Peptide (P 1  or P 2 ) 
     In some embodiments, P 1  comprises a peptide that impairs binding of the antigen binding domain to the target antigen. In some embodiments, P 1  comprises a peptide that impairs binding of the cytokine to the cytokine receptor. In some embodiments, P 1  is bound to A 1  through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bonding interactions, or a combination thereof. In some embodiments, P 1  is bound to A 1  at or near a cytokine receptor binding site. In some embodiments, P 1  is bound to A 1  at or near an antigen binding site. In some embodiments, P 1  becomes unbound from A 1  when L 1  is cleaved by the protease thereby exposing P 1  to the target antigen or cytokine receptor. In some embodiments, the protease comprises a tumor specific protease. In some embodiments, the protease comprises a matrix metalloprotease (MMP) or a serine protease. In some embodiments, the matrix metalloprotease comprises MMP2, MMP7, MMP9, MMP13, or MMP14. In some embodiments, the serine protease comprises matriptase, urokinase, or hepsin. In some embodiments, P 1  impairs binding of A 1  to the target antigen or cytokine receptor by non-steric blocking. In some embodiments, P 1  impairs binding of A 1  to the target antigen or cytokine receptor through covalent interactions. In some embodiments, P 1  is not a cytokine, cytokine binding fragment, cytokine mutein, or combinations thereof of the cognate receptor of the cytokine. In some embodiments, A 1  is not an antibody or fragment thereof that binds to the cytokine receptor. 
     In some embodiments, P 2  comprises a peptide that impairs binding of the second antigen binding domain to the second target antigen. In some embodiments, P 2  comprises a peptide that impairs binding of the second cytokine to the second cytokine receptor. In some embodiments, 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 a cytokine receptor binding site. In some embodiments, P 2  is bound to A 2  at or near an antigen binding site. In some embodiments, P 2  becomes unbound from A 2  when L 2  is cleaved by the protease thereby exposing P 2  to the second target antigen or second cytokine receptor. In some embodiments, the protease comprises a tumor specific protease. 
     In some embodiments, the protease comprises a matrix metalloprotease (MMP) or a serine protease. In some embodiments, the matrix metalloprotease comprises MMP2, MMP7, MMP9, MMP13, or MMP14. In some embodiments, the serine protease comprises matriptase, urokinase, or hepsin. In some embodiments, P 2  impairs binding of A 2  to the second target antigen or second cytokine receptor by non-steric blocking. In some embodiments, P 2  impairs binding of A 2  to the second target antigen or second cytokine receptor through covalent interactions. In some embodiments, P 2  is not a cytokine, cytokine binding fragment, cytokine mutein, or combinations thereof of the cognate receptor of the cytokine. In some embodiments, A 2  is not an antibody or fragment thereof that binds to the cytokine receptor. 
     In some embodiments, P 1  has less than 70% sequence homology to the target antigen. In some embodiments, P 1  has less than 75% sequence homology to the target antigen. In some embodiments, P 1  has less than 80% sequence homology to the target antigen. In some embodiments, P 1  has less than 85% sequence homology to the target antigen. In some embodiments, P 1  has less than 90% sequence homology to the target antigen. In some embodiments, P 1  has less than 95% sequence homology to the target antigen. In some embodiments, P 1  has less than 98% sequence homology to the target antigen. In some embodiments, P 1  has less than 99% sequence homology to the target antigen. 
     In some embodiments, P 1  has less than 70% sequence homology to the cytokine receptor. In some embodiments, P 1  has less than 75% sequence homology to the cytokine receptor. In some embodiments, P 1  has less than 80% sequence homology to the cytokine receptor. In some embodiments, P 1  has less than 85% sequence homology to the cytokine receptor. In some embodiments, P 1  has less than 90% sequence homology to the cytokine receptor. In some embodiments, P 1  has less than 95% sequence homology to the cytokine receptor. In some embodiments, P 1  has less than 98% sequence homology to the cytokine receptor. In some embodiments, P 1  has less than 99% sequence homology to the cytokine receptor. 
     In some embodiments, P 2  has less than 70% sequence homology to the second target antigen. In some embodiments, P 2  has less than 75% sequence homology to the second target antigen. In some embodiments, P 2  has less than 80% sequence homology to the second target antigen. In some embodiments, P 2  has less than 85% sequence homology to the second target antigen. In some embodiments, P 2  has less than 90% sequence homology to the second target antigen. In some embodiments, P 2  has less than 95% sequence homology to the second target antigen. In some embodiments, P 2  has less than 98% sequence homology to the second target antigen. In some embodiments, P 2  has less than 99% sequence homology to the second target antigen. 
     In some embodiments, P 2  has less than 70% sequence homology to the second cytokine receptor. In some embodiments, P 2  has less than 75% sequence homology to the second cytokine receptor. In some embodiments, P 2  has less than 80% sequence homology to the second cytokine receptor. In some embodiments, P 2  has less than 85% sequence homology to the second cytokine receptor. In some embodiments, P 2  has less than 90% sequence homology to the second cytokine receptor. In some embodiments, P 2  has less than 95% sequence homology to the second cytokine receptor. In some embodiments, P 2  has less than 98% sequence homology to the second cytokine receptor. In some embodiments, P 2  has less than 99% sequence homology to the second cytokine receptor. 
     In some embodiments, P 1  or P 2  comprises a de novo amino acid sequence that shares less than 50% sequence homology to a cytokine, cytokine receptor, or antibody or fragments thereof that bind to the cytokine or cytokine receptor. In some embodiments, P 1  or P 2  comprises a de novo amino acid sequence that shares less than 40% sequence homology to a cytokine, cytokine receptor, or antibody or fragments thereof that bind to the cytokine or cytokine receptor. In some embodiments, P 1  or P 2  comprises a de novo amino acid sequence that shares less than 30% sequence homology to a cytokine, cytokine receptor, or antibody or fragments thereof that bind to the cytokine or cytokine receptor. In some embodiments, P 1  or P 2  comprises a de novo amino acid sequence that shares less than 20% sequence homology to a cytokine, cytokine receptor, or antibody or fragments thereof that bind to the cytokine or cytokine receptor. In some embodiments, P 1  or P 2  comprises a de novo amino acid sequence that shares less than 10% sequence homology to a cytokine, cytokine receptor, or antibody or fragments thereof that bind to the cytokine or cytokine receptor. In some embodiments, P 1  or P 2  is identified from a peptide library that contains random amino acid sequences. 
     In some embodiments, P 1  or P 2  comprises a de novo amino acid sequence that shares less than 50% sequence homology to the target antigen. In some embodiments, P 1  or P 2  comprises a de novo amino acid sequence that shares less than 40% sequence homology to the target antigen. In some embodiments, P 1  or P 2  comprises a de novo amino acid sequence that shares less than 30% sequence homology to the target antigen. In some embodiments, P 1  or P 2  comprises a de novo amino acid sequence that shares less than 20% sequence homology to the target antigen. In some embodiments, P 1  or P 2  comprises a de novo amino acid sequence that shares less than 10% sequence homology to the target antigen. In some embodiments, P 1  or P 2  is identified from a peptide library that contains random amino acid sequences. 
     In some embodiments, P 1  or P 2  comprises a peptide sequence of at least 5 amino acids in length. In some embodiments, P 1  or P 2  comprises a peptide sequence of at least 6 amino acids in length. In some embodiments, P 1  or P 2  comprises a peptide sequence of at least 10 amino acids in length. In some embodiments, P 1  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, P 1  or P 2  comprises a peptide sequence of at least 16 amino acids in length. In some embodiments, P 1  or P 2  comprises a peptide sequence of no more than 40 amino acids in length. In some embodiments, P 1  or P 2  comprises at least two cysteine amino acid residues. In some embodiments, P 1  or P 2  comprises a cyclic peptide or a linear peptide. In some embodiments, P 1  or P 2  comprises a cyclic peptide. In some embodiments, P 1  or P 2  comprises a linear peptide. 
     In some embodiments, P 1  or P 2  comprise a modified amino acid or non-natural amino acid, or a modified non-natural amino acid, or a combination thereof. In some embodiments, the modified amino acid or a modified non-natural amino acid comprises a post-translational modification. In some embodiments P 1  or P 2  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 ubiquitination. Modifications are made anywhere to P 1  or P 2  including the peptide backbone, the amino acid side chains, and the terminus. 
     In some embodiments, P 1  or P 2  does not comprise albumin or an albumin fragment. In some embodiments, P 1  or P 2  does not comprise an albumin binding domain. 
     A 1  and A 2    
     In some embodiments, A 1  or A 2  is an antigen recognizing molecule. In some embodiments, the antigen recognizing molecule is an antibody or an antibody fragment. In some embodiments, the antibody or the antibody fragment thereof comprises a single chain variable fragment, a single domain antibody, Fab, Fab′. In some embodiments, the antibody or antibody fragment thereof comprises a single chain variable fragment (scFv), a heavy chain variable domain (VH domain), a light chain variable domain (VL domain), or a variable domain (VHH) of a camelid derived single domain antibody. In some embodiments, the antibody or antibody fragment thereof comprises a single-chain variable fragment. In some embodiments, the antibody or antibody fragment thereof is humanized or human. 
     In some embodiments, A 1  or A 2  is a Fab. In some embodiments, the Fab comprises (a) a Fab light chain polypeptide; and (b) a Fab heavy chain polypeptide. In some embodiments, L 1  or L 2  is bound to N-terminus of the Fab light chain polypeptide. In some embodiments L 1  or L 2  is bound to N-terminus of the Fab heavy chain polypeptide. In some embodiments, L 1  or L 2  is bound to C-terminus of the Fab light chain polypeptide. In some embodiments, L 1  or L 2  is bound to C-terminus of the Fab heavy chain polypeptide. 
     In some embodiments, A 1  or A 2  is a single chain variable fragment (scFv). In some embodiments, L 1  or L 2  is bound to N-terminus of the scFv. In some embodiments, L 1  or L 2  is bound to C-terminus of the scFv. In some embodiments, the scFv comprises a light chain variable domain and a heavy chain variable domain. In some embodiments, L 1  or L 2  is bound to a N-terminus of the light chain variable domain of the single chain variable fragment (scFv). In some embodiments, L 1  or L 2  is bound to a N-terminus of the heavy chain variable domain of the single chain variable fragment (scFv). 
     In some embodiments, the antibody or antibody fragment thereof comprises an epidermal growth factor receptor (EGFR) binding domain. In some embodiments, the antibody or antibody fragment thereof comprises a cluster of differentiation 3 (CD3) binding domain. In some embodiments, the antibody or antibody fragment thereof comprises a cluster of differentiation 3 epsilon (CD3e) binding domain. In some embodiments, the target antigen comprises EGFR. In some embodiments, the target antigen comprises CD3. In some embodiments, the target antigen comprises CD3ε. 
     In some embodiments, A 1  or A 2  binds to a polypeptide that is part of a TCR-CD3 complex on the effector cell. In some embodiments, the target antigen is an anti-CD3 effector cell antigen. In some embodiments, the polypeptide that is part of the TCR-CD3 complex is human CD3ε. In some embodiments, A 1  or A 2  comprises an anti-CD3e single-chain variable fragment. In some embodiments, A 1  or A 2  comprises an anti-CD3e single-chain variable fragment that has a K D  binding of 1 μM or less to CD3 on CD3 expressing cells. In some embodiments, A t  or A 2  comprises a variable light chain and variable heavy chain each of which is capable of specifically binding to human CD3. In some embodiments, A 1  or A 2  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, F111-409, CLB-T3.4.2, TR-66, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII-87, 12F6, T3/RW2-8C8, T3/RW2-4B6, OKT3D, M-T301, SMC2, F101.01, UCHT-1, WT-31, 15865, 15865v12, 15865v16, and 15865v19. 
     In some embodiments, A 1  or A 2  is a soluble T cell receptor (TCR). Native TCRs are transmembrane receptors expressed on the surface of T cells that recognize antigens bound to major histocompatibility complex molecules (MHC). Native TCRs are heterodimeric and comprise an alpha polypeptide chain and a beta polypeptide chain linked through a disulfide bond. The alpha polypeptide chain and the beta polypeptide chain are expressed as part of a complex with accessory proteins which include, for example, two CD3 epsilon polypeptides, one CD3 gamma polypeptide, one CD3 delta polypeptide, and two CD3 zeta polypeptides. When a TCR engages with a target antigen and MHC, the T cell is activated resulting in a series of signaling events mediated by associated enzymes, co-receptors, adapter molecules, and activated or released transcription factors. 
     In native TCRs, the alpha polypeptide chain and the beta polypeptide chain comprise an extracellular domain, a transmembrane domain, and a cytoplasmic domain. Each extracellular domain comprises a variable region (V), a joining region (J), and a constant region (C). The constant region is N-terminal to the transmembrane domain, and the transmembrane domain is N-terminal to the cytoplasmic domain. The variable regions of both the alpha polypeptide chain and the beta polypeptide chain comprise three hypervariable or complementarity determining regions (CDRs). The beta polypeptide chain usually contains a short diversity region between the variable and joining regions. The three CDRs are embedded into a framework sequence, with one CDR being the hypervariable region named CDR3. The alpha chain variable region (Vα) and the beta chain variable region (Vβ) are of several types that are distinguished by their framework sequences, CDR1 and CDR2 sequences, and a partly defined CDR3 sequence. 
     TCRs are described using the International Immunogenetics (IMGT) TCR nomenclature. The Vα in IMGT nomenclature is referred to by a unique “TRAV” number. In the same way, Vβ is referred to by a unique “TRBV” number. The corresponding joining and constant regions are referred to as TRAJ and TRAC, respectively for the α joining and constant regions, and TRBJ and TRBC, respectively for the β joining and constant regions. The sequences defined by the IMGT nomenclature are known in the art and are contained within the online IMGT public database. 
     In some embodiments, the soluble TCR is a single chain TCR comprising a variable region of a TCR alpha extracellular domain, or fragment thereof, and a variable region of a TCR beta extracellular domain, or fragment thereof. In some embodiments, the soluble TCR comprises an alpha TCR polypeptide comprising a TCR alpha extracellular domain and a beta TCR polypeptide comprising a TCR beta extracellular domain. 
     In some embodiments, the soluble TCR is a single chain TCR comprising a variable region of a TCR alpha extracellular domain, or fragment thereof, and a variable region of a TCR beta extracellular domain, or fragment thereof. In some embodiments, the soluble TCR comprises an alpha TCR polypeptide comprising a TCR alpha extracellular domain and a beta TCR polypeptide comprising a TCR beta extracellular domain. In some embodiments, L 1  is bound to N-terminus of the alpha TCR polypeptide. In some embodiments, L 1  is bound to N-terminus of the beta TCR polypeptide. In some embodiments, A 2  is bound to C-terminus of the alpha TCR polypeptide. In some embodiments, A 2  is bound to N-terminus of the alpha TCR polypeptide. In some embodiments, A 2  is bound to C-terminus of the beta TCR polypeptide. In some embodiments, A 2  is bound to N-terminus of the beta TCR polypeptide. In some embodiments, L 1  is bound to N-terminus of the alpha TCR polypeptide and A 2  is bound to N-terminus of the beta TCR polypeptide. In some embodiments, L 1  is bound to N-terminus of the alpha TCR polypeptide and A 2  is bound to C-terminus of the beta TCR polypeptide. In some embodiments, L 1  is bound to N-terminus of the alpha TCR polypeptide and A 2  is bound to C-terminus of the alpha TCR polypeptide. In some embodiments, L 1  is bound to N-terminus of the beta TCR polypeptide and A 2  is bound to N-terminus of the alpha TCR polypeptide. In some embodiments, L 1  is bound to N-terminus of the beta TCR polypeptide and A 2  is bound to C-terminus of the beta TCR polypeptide. In some embodiments, L 1  is bound to N-terminus of the beta TCR polypeptide and A 2  is bound to C-terminus of the alpha TCR polypeptide. 
     In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the target antigen or second target antigen as compared to the binding affinity for the target antigen or second target antigen of a polypeptide or polypeptide complex that does not have P 1  or P 2  or L 1  or L 2 . In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the target antigen or second target antigen that is at least 5× higher than the binding affinity for the target antigen of a form of the polypeptide or polypeptide complex that does not have P 1  or P 2  or L 1  or L 2 . In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the target antigen or second target antigen that is at least 8× higher than the binding affinity for the target antigen or second target antigen of a form of the polypeptide or polypeptide complex that does not have P 1  or P 2  or L 1  or L 2 . In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the target antigen or second target antigen that is at least 10× higher than the binding affinity for the target antigen or second target antigen of a form of the polypeptide or polypeptide complex that does not have P 1  or P 2  or L 1  or L 2 . In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the target antigen or second target antigen that is at least 20× higher than the binding affinity for the target antigen or second target antigen of a form of the polypeptide or polypeptide complex that does not have P 1  or P 2  or L 1  or L 2 . In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the target antigen or second target antigen that is at least 25× higher than the binding affinity for the target antigen or second target antigen of a form of the polypeptide or polypeptide complex that does not have P 1  or P 2  or L 1  or L 2 . In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the target antigen or second target antigen that is at least 30× higher than the binding affinity for the target antigen or second target antigen of a form of the polypeptide or polypeptide complex that does not have P 1  or P 2  or L 1  or L 2 . In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the target antigen or second target antigen that is at least 40× higher than the binding affinity for the target antigen or second target antigen of a form of the polypeptide or polypeptide complex that does not have P 1  or P 2  or L 1  or L 2 . In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the target antigen or second target antigen that is at least 50× higher than the binding affinity for the target antigen or second target antigen of a form of the polypeptide or polypeptide complex that does not have P 1  or P 2  or L 1  or L 2 . In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the target antigen or second target antigen that is at least 60× higher than the binding affinity for the target antigen or second target antigen of a form of the polypeptide or polypeptide complex that does not have P 1  or P 2  or L 1  or L 2 . In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the target antigen or second target antigen that is at least 70× higher than the binding affinity for the target antigen or second target antigen of a form of the polypeptide or polypeptide complex that does not have P 1  or P 2  or L 1  or L 2 . In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the target antigen or second target antigen that is at least 75× higher than the binding affinity for the target antigen or second target antigen of a form of the polypeptide or polypeptide complex that does not have P 1  or P 2  or L 1  or L 2 . In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the target antigen or second target antigen that is at least 80× higher than the binding affinity for the target antigen or second target antigen of a form of the polypeptide or polypeptide complex that does not have P 1  or P 2  or L 1  or L 2 . In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the target antigen or second target antigen that is at least 90× higher than the binding affinity for the target antigen or second target antigen of a form of the polypeptide or polypeptide complex that does not have P 1  or P 2  or L 1  or L 2 . In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the target antigen or second target antigen that is at least 100× higher than the binding affinity for the target antigen or second target antigen of a form of the polypeptide or polypeptide complex that does not have P 1  or P 2  or L 1  or L 2 . In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the target antigen or second target antigen that is at least 120× higher than the binding affinity for the target antigen or second target antigen of a form of the polypeptide or polypeptide complex that does not have P 1  or P 2  or L 1  or L 2 . 
     In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the target antigen or second target antigen as compared to the binding affinity for the target antigen or second target antigen of the polypeptide or polypeptide complex in which L 1  or L 2  has been cleaved. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the target antigen or second target antigen that is at least 5× higher than the binding affinity for the target antigen or second target antigen of the polypeptide or polypeptide complex in which L 1  or L 2  has been cleaved. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the target antigen or second target antigen that is at least 8× higher than the binding affinity for the target antigen or second target antigen of the polypeptide or polypeptide complex in which L 1  or L 2  has been cleaved. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the target antigen or second target antigen that is at least 10× higher than the binding affinity for the target antigen or second target antigen of the polypeptide or polypeptide complex in which L 1  or L 2  has been cleaved. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the target antigen or second target antigen that is at least 20× higher than the binding affinity for the target antigen or second target antigen of the polypeptide or polypeptide complex in which L 1  or L 2  has been cleaved. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the target antigen or second target antigen that is at least 25× higher than the binding affinity for the target antigen or second target antigen of the polypeptide or polypeptide complex in which L 1  or L 2  has been cleaved. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the target antigen or second target antigen that is at least 30× higher than the binding affinity for the target antigen or second target antigen of the polypeptide or polypeptide complex in which L 1  or L 2  has been cleaved. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the target antigen or second target antigen that is at least 40× higher than the binding affinity for the target antigen or second target antigen of the polypeptide or polypeptide complex in which L 1  or L 2  has been cleaved. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the target antigen or second target antigen that is at least 50× higher than the binding affinity for the target antigen or second target antigen of the polypeptide or polypeptide complex in which L 1  or L 2  has been cleaved. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the target antigen or second target antigen that is at least 60× higher than the binding affinity for the target antigen or second target antigen of the polypeptide or polypeptide complex in which L 1  or L 2  has been cleaved. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the target antigen or second target antigen that is at least 70× higher than the binding affinity for the target antigen or second target antigen of the polypeptide or polypeptide complex in which L 1  or L 2  has been cleaved. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the target antigen or second target antigen that is at least 75× higher than the binding affinity for the target antigen or second target antigen of the polypeptide or polypeptide complex in which L 1  or L 2  has been cleaved. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the target antigen or second target antigen that is at least 80× higher than the binding affinity for the target antigen or second target antigen of the polypeptide or polypeptide complex in which L 1  or L 2  has been cleaved. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the target antigen or second target antigen that is at least 90× higher than the binding affinity for the target antigen or second target antigen of the polypeptide or polypeptide complex in which L 1  or L 2  has been cleaved. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the target antigen or second target antigen that is at least 100× higher than the binding affinity for the target antigen or second target antigen of the polypeptide or polypeptide complex in which L 1  or L 2  has been cleaved. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the target antigen or second target antigen that is at least 120× higher than the binding affinity for the target antigen or second target antigen of the polypeptide or polypeptide complex in which L 1  or L 2  has been cleaved. In some embodiments, L 1  or L 2  is cleaved by a protease. In some embodiments, the protease comprises a tumor specific protease. In some embodiments, the protease comprises a matrix metalloprotease (MMP) or a serine protease. In some embodiments, the matrix metalloprotease comprises MMP2, MMP7, MMP9, MMP13, or MMP14. In some embodiments, the serine protease comprises matriptase, urokinase, or hepsin. 
     In some embodiments, A 1  or A 2  is a cytokine or cytokine fragment. In some embodiments, A 1  or A 2  is a mutein of the cytokine or the cytokine fragment. In some embodiments, the cytokine or the cytokine fragment is a mutein of the cytokine or the cytokine fragment. 
     Cytokines are a diverse group of small peptides, including chemokines, interferons, interleukins, lymphokines, adipokines, mesenchymal growth factors, and tumor necrosis factors, which are involved in intercellular signaling in a variety of biological pathways. They are particularly important in immune and inflammatory responses. Signaling occurs following recognition of the cytokine by a corresponding cytokine receptor, which are transmembrane receptors comprising an extracellular domain for ligand binding and an intracellular domain that allows signal transduction. 
     The diversity of cytokines comes with a corresponding diversity in cytokine receptors, which can comprise a single chain or subunit or dimeric/multimeric domains. Cytokine receptors include Type I cytokine receptors, exemplified by interleukin receptors, and Type II cytokine receptors, exemplified by interferon receptors, both of which comprise a cytokine receptor homology domain (CHD). The CHD of Type I cytokine receptors share a common amino acid motif (WSXWS (SEQ ID NO: 27)), while Type II cytokine receptors lack this motif. Cytokine receptors can include an alpha subunit, beta subunit, gamma subunit, or dimeric, or trimeric combinations thereof. In one example, a high affinity receptor for IL-2 comprises an IL-2Rα subunit, IL-2Rβ subunit, and IL-2Rγ subunit, an intermediate affinity receptor for IL-2 comprises only the IL-2Rβ subunit and IL-2Rγ subunit, and low affinity receptor for IL-2 comprises only the IL-2Rα subunit. 
     In some embodiments, the cytokine is a chemokine, an interferon, an interleukin, a lymphokine, an adipokine, a growth factor, or a tumor necrosis factor. In some embodiments, the interferon (IFN) is IFNα, IFNβ, IFNγ, or a fragment thereof. In some embodiments, the interleukin (IL) is IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-15, IL-21, or a fragment thereof. In some embodiments, the growth factor is granulocyte-macrophage colony-stimulating factor (GM-CSF) or a fragment thereof. In some embodiments the cytokine is TGF-γ. 
     In some embodiments, a cytokine mutein is a variant of a wild-type cytokine. In some embodiments, a cytokine mutein is a mutant of a wild-type cytokine. In some embodiments, the cytokine mutein comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or more than 50 amino acid substitutions relative to a wild-type cytokine. In some embodiments, the cytokine mutein comprises no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or more than 50 amino acid substitutions relative to a wild-type cytokine. In some embodiments, the cytokine mutein is a non-naturally occurring cytokine. In some embodiments, the cytokine mutein comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or more than 50 amino acid substitutions relative to a naturally occurring cytokine. In some embodiments, the cytokine mutein comprises no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or more than 50 amino acid substitutions relative to a naturally occurring cytokine. 
     In some embodiments, the cytokine or cytokine fragment binds to a cytokine receptor. In some embodiments, the cytokine receptor is a receptor for a chemokine, an interferon, an interleukin, a lymphokine, an adipokine, a growth factor, or a tumor necrosis factor. In some embodiments, the cytokine receptor is a type I cytokine receptor or a type II cytokine receptor. In some embodiments, the cytokine receptor is a dimer or a trimer. In some embodiments, the cytokine receptor comprises an alpha subunit, a beta subunit, a gamma subunit, or any combination thereof. For example, in some embodiments, the cytokine receptor comprises an alpha subunit, a beta subunit, and a gamma subunit. In another example, in some embodiments, the cytokine receptor comprises a beta subunit and a gamma subunit. In some embodiments, the cytokine receptor comprises an alpha subunit and a beta subunit. 
     In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity for its cytokine receptor as compared to the binding affinity for the cytokine receptor of a polypeptide or polypeptide complex that does not have P 1  or P 2  or L 1  or L 2 . In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its cytokine receptor that is at least 5× weaker than the binding affinity for the cytokine receptor of a form of the polypeptide or polypeptide complex that does not have P 1  or P 2  or L 1  or L 2 . In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its cytokine receptor that is at least 8× weaker than the binding affinity for the cytokine receptor of a form of the polypeptide or polypeptide complex that does not have P 1  or P 2  or L 1  or L 2 . In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its cytokine receptor that is at least 10× weaker than the binding affinity for the cytokine receptor of a form of the polypeptide or polypeptide complex that does not have P 1  or P 2  or L 1  or L 2 . In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its cytokine receptor that is at least 20× weaker than the binding affinity for the cytokine receptor of a form of the polypeptide or polypeptide complex that does not have P 1  or P 2  or L 1  or L 2 . In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its cytokine receptor that is at least 25× weaker than the binding affinity for the cytokine receptor of a form of the polypeptide or polypeptide complex that does not have P 1  or P 2  or L 1  or L 2 . In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its cytokine receptor that is at least 30× weaker than the binding affinity for the cytokine receptor of a form of the polypeptide or polypeptide complex that does not have P 1  or P 2  or L 1  or L 2 . In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its cytokine receptor that is at least 40× weaker than the binding affinity for the cytokine receptor of a form of the polypeptide or polypeptide complex that does not have P 1  or P 2  or L 1  or L 2 . In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its cytokine receptor that is at least 50× weaker than the binding affinity for the cytokine receptor of a form of the polypeptide or polypeptide complex that does not have P 1  or P 2  or L 1  or L 2 In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its cytokine receptor that is at least 60× weaker than the binding affinity for the cytokine receptor of a form of the polypeptide or polypeptide complex that does not have P 1  or P 2  or L 1  or L 2 In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its cytokine receptor that is at least 70× weaker than the binding affinity for the cytokine receptor of a form of the polypeptide or polypeptide complex that does not have P 1  or P 2  or L 1  or L 2 . In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its cytokine receptor that is at least 75× weaker than the binding affinity for the cytokine receptor of a form of the polypeptide or polypeptide complex that does not have P 1  or P 2  or L 1  or L 2 . In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its cytokine receptor that is at least 80× weaker than the binding affinity for the cytokine receptor of a form of the polypeptide or polypeptide complex that does not have P 1  or P 2  or L 1  or L 2 . In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its cytokine receptor that is at least 90× weaker than the binding affinity for the cytokine receptor of a form of the polypeptide or polypeptide complex that does not have P 1  or P 2  or L 1  or L 2 . In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its cytokine receptor that is at least 100× weaker than the binding affinity for the cytokine receptor of a form of the polypeptide or polypeptide complex that does not have P 1  or P 2  or L 1  or L 2 . In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its cytokine receptor that is at least 120× weaker than the binding affinity for the cytokine receptor of a form of the polypeptide or polypeptide complex that does not have P 1  or P 2  or L 1  or L 2 . In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its cytokine receptor that is at least 150× weaker than the binding affinity for the cytokine receptor of a form of the polypeptide or polypeptide complex that does not have P 1  or P 2  or L 1  or L 2 . In some embodiments, the cytokine or cytokine fragment comprises an interferon, GM-CSF, IL-2, IL-7, IL-12, IL-15, or IL-21. In some embodiments, the cytokine or cytokine fragment comprises IL-2, IL-12, IL-6, IL-4, IL-10, or TGF-3. In some embodiments, the cytokine receptor comprises an interferon receptor, GM-CSF receptor, IL-2 receptor, IL-7 receptor, IL-12 receptor, IL-15 receptor, or IL-21 receptor. In some embodiments, the cytokine receptor comprises IL-2 receptor, IL-12 receptor, IL-6 receptor, IL-4 receptor, IL-10 receptor, or TGF-β receptor. 
     In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its cytokine receptor as compared to the binding affinity for the cytokine receptor of the polypeptide or polypeptide complex in which L 1  or L 2  has been cleaved. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its cytokine receptor that is at least 5× weaker than the binding affinity for the cytokine receptor of the polypeptide or polypeptide complex in which L 1  or L 2  has been cleaved. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its cytokine receptor that is at least 8× weaker than the binding affinity for the cytokine receptor of the polypeptide or polypeptide complex in which L 1  or L 2  has been cleaved. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its cytokine receptor that is at least 10× weaker than the binding affinity for the cytokine receptor of the polypeptide or polypeptide complex in which L 1  or L 2  has been cleaved. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its cytokine receptor that is at least 15× weaker than the binding affinity for the cytokine receptor of the polypeptide or polypeptide complex in which L 1  or L 2  has been cleaved. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its cytokine receptor that is at least 20× weaker than the binding affinity for the cytokine receptor of the polypeptide or polypeptide complex in which L 1  or L 2  has been cleaved. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its cytokine receptor that is at least 25× weaker than the binding affinity for the cytokine receptor of the polypeptide or polypeptide complex in which L 1  or L 2  has been cleaved. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its cytokine receptor that is at least 30× weaker than the binding affinity for the cytokine receptor of the polypeptide or polypeptide complex in which L 1  or L 2  has been cleaved. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its cytokine receptor that is at least 40× weaker than the binding affinity for the cytokine receptor of the polypeptide or polypeptide complex in which L 1  or L 2  has been cleaved. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its cytokine receptor that is at least 50× weaker than the binding affinity for the cytokine receptor of the polypeptide or polypeptide complex in which L 1  or L 2  has been cleaved. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its cytokine receptor that is at least 60× weaker than the binding affinity for the cytokine receptor of the polypeptide or polypeptide complex in which L 1  or L 2  has been cleaved. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its cytokine receptor that is at least 70× weaker than the binding affinity for the cytokine receptor of the polypeptide or polypeptide complex in which L 1  or L 2  has been cleaved. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its cytokine receptor that is at least 75× weaker than the binding affinity for the cytokine receptor of the polypeptide or polypeptide complex in which L 1  or L 2  has been cleaved. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its cytokine receptor that is at least 80× weaker than the binding affinity for the cytokine receptor of the polypeptide or polypeptide complex in which L 1  or L 2  has been cleaved. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its cytokine receptor that is at least 90× weaker than the binding affinity for the cytokine receptor of the polypeptide or polypeptide complex in which L 1  or L 2  has been cleaved. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its cytokine receptor that is at least 100× weaker than the binding affinity for the cytokine receptor of the polypeptide or polypeptide complex in which L 1  or L 2  has been cleaved. In some embodiments, the cytokine or cytokine fragment comprises an interferon, GM-CSF, IL-2, IL-7, IL-12, IL-15, or IL-21. In some embodiments, the cytokine receptor comprises an interferon receptor, GM-CSF receptor, IL-2 receptor, IL-7 receptor, IL-12 receptor, IL-15 receptor, or IL-21 receptor. In some embodiments, L 1  or L 2  is cleaved by a protease. In some embodiments, the protease comprises a tumor specific protease. In some embodiments, the protease comprises a matrix metalloprotease (MMP) or a serine protease. In some embodiments, the matrix metalloprotease comprises MMP2, MMP7, MMP9, MMP13, or MMP14. In some embodiments, the serine protease comprises matriptase, urokinase, or hepsin. 
     Half-Life Extending Moiety 
     In some embodiments, P 1  is further linked to a half-life extending moiety. In some embodiments, P 1  is further linked to a half-life extending moiety in a configuration according to Formula Ia 
       A 1 -L 1 -P 1 -L 3 -H 1   (Formula Ia)
 
     wherein H 1  is the half-life extending moiety and L 3  is a linker that connects H 1  to P 1 . In some embodiments, L 3  is a non-cleavable linker. In some embodiments, the half-life extending moiety (H 1 ) does not block A 1  binding to the target antigen. In some embodiments, the half-life extending moiety (H 1 ) does not have binding affinity to A 1 . In some embodiments, the half-life extending moiety (H 1 ) does not have binding affinity to the target antigen. In some embodiments, the half-life extending moiety (H 1 ) does not shield A 1  from the target antigen. In some embodiments, the half-life extending moiety (H 1 ) is not directly linked to A 1 . 
     In some embodiments, the half-life extending moiety (H 1 ) does not block A 1  binding to the cytokine receptor. In some embodiments, the half-life extending moiety (H 1 ) does not have binding affinity to the cytokine or cytokine receptor. In some embodiments, the half-life extending moiety (H 1 ) does not shield the cytokine or cytokine fragment from the cytokine receptor. In some embodiments, the half-life extending moiety (H 1 ) is not directly linked to the cytokine or cytokine fragment. 
     In some embodiments, H 1  comprises an amino acid sequence that has repetitive sequence motifs. In some embodiments, H 1  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 H 1  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. 
     In some embodiments, H 1  comprises a polymer. In some embodiments, the polymer is polyethylene glycol (PEG). In some embodiments, H 1  comprises albumin. In some embodiments, H 1  comprises a Fc domain. In some embodiments, the albumin is serum albumin. In some embodiments, the albumin is human serum albumin. In some embodiments, H 1  comprises a polypeptide, a ligand, or a small molecule. In some embodiments, the polypeptide, the ligand or the small molecule binds serum protein or a fragment thereof, a circulating immunoglobulin or a fragment thereof, or CD35/CR1. In some embodiments, the serum protein comprises a thyroxine-binding protein, a transthyretin, a 1-acid glycoprotein, a transferrin, transferrin receptor or a transferrin-binding portion thereof, a fibrinogen, or an albumin. In some embodiments, the circulating immunoglobulin molecule comprises IgG1, IgG2, IgG3, IgG4, s1gA, IgM or IgD. In some embodiments, the serum protein is albumin. In some embodiments, the polypeptide is an antibody. In some embodiments, the antibody comprises a single domain antibody, a single chain variable fragment or a Fab. In some embodiments, the antibody comprises a single domain antibody. In some embodiments, the antibody comprises a single domain antibody that binds to albumin. In some embodiments, the antibody comprises a single domain antibody that binds to human serum albumin. In some embodiments, the antibody is a human or humanized antibody. In some embodiments, the single domain antibody is selected from the group consisting of 645gH1gL1, 645dsgH5gL4, 23-13-A01-sc02, A10m3 or a fragment thereof, DOM7r-31, DOM7h-11-15, Alb-1, Alb-8, Alb-23, 10G, 10GE, and SA21. 
     In some embodiments, H 1  comprises a single domain antibody. In some embodiments, H 1  comprises a single domain antibody that binds to albumin. In some embodiments, H 1  comprises a single domain antibody that binds to human serum albumin. 
     In some embodiments, H 1  comprise a modified amino acid or non-natural amino acid, or a modified non-natural amino acid, or a combination thereof. In some embodiments, the modified amino acid or a modified non-natural amino acid comprises a post-translational modification. In some embodiments, H 1  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 ubiquitination. Modifications are made anywhere to H 1  including the peptide backbone, the amino acid side chains, and the terminus. 
     Polynucleotides Encoding Polypeptides or Polypeptide Complexes 
     Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes as disclosed herein. Described herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding polypeptides comprising a cleavable linker. 
     As disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding polypeptides comprising a cleavable linker according to the amino acid sequence of SEQ ID NO: 1 (LSGRSDAG). 
     In some embodiments, the cleavable linker comprises the amino acid sequence of SEQ ID NO: 3 (ISSGLLSGRSDAG). In some embodiments, the cleavable linker comprises the amino acid sequence of SEQ ID NO: 26 (AGLLAPPGGLSGRSDAG). In some embodiments, the cleavable linker comprises the amino acid sequence of SEQ ID NO: 4 (AAGLLAPPGGLSGRSDAG). In some embodiments, the cleavable linker comprises the amino acid sequence of SEQ ID NO: 5 (SPLGLSGRSDAG). In some embodiments, the cleavable linker comprises the amino acid sequence of SEQ ID NO: 6 (LSGRSDAGSPLGLAG). 
     Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding an isolated polypeptide comprising a cleavable linker according to the amino acid sequence of Linker 1 (ISSGLLSGRSDAG) (SEQ ID NO: 3), Linker 2 (AAGLLAPPGGLSGRSDAG) (SEQ ID NO: 4), Linker 3 (SPLGLSGRSDAG) (SEQ ID NO: 5), or Linker 4 (LSGRSDAGSPLGLAG) (SEQ ID NO: 6), or an isolated polypeptide comprising a cleavable linker that has 1, 2, or 3 amino acid substitutions, additions, or deletions relative to the amino acid sequence of Linker 1, Linker 2, Linker 3, or Linker 4. Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding an isolated polypeptide comprising a cleavable linker according to the amino acid sequence of Linker 1 (ISSGLLSGRSDAG) (SEQ ID NO: 3). Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding an isolated polypeptide comprising a cleavable linker according to the amino acid sequence of Linker 2 (AAGLLAPPGGLSGRSDAG) (SEQ ID NO: 4). Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding an isolated polypeptide comprising a cleavable linker according to the amino acid sequence of Linker 3 (SPLGLSGRSDAG) (SEQ ID NO: 5). Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding an isolated polypeptide comprising a cleavable linker according to the amino acid sequence of Linker 4 (LSGRSDAGSPLGLAG) (SEQ ID NO: 6). 
     Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding an isolated polypeptide comprising a cleavable linker according to the amino acid sequence LSGRSDAG (SEQ ID NO: 1). 
     Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes according to Formula I: 
       A 1 -L 1 -P 1   (Formula I)
 
     wherein A 1  comprises the antigen binding domain that binds to the target antigen or the cytokine that binds to the cytokine receptor; L 1  comprises the cleavable linker; and P 1  comprises a peptide that impairs binding of the antigen binding domain to the target antigen or impairs binding of the cytokine to the cytokine receptor. 
     Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes comprising Formula I: 
       A 1 -L 1 -P 1   (Formula I)
 
     wherein A 1  comprises the antigen binding domain that binds to the target antigen or the cytokine that binds to the cytokine receptor; L 1  comprises the cleavable linker; and P 1  comprises a peptide that impairs binding of the antigen binding domain to the target antigen or impairs binding of the cytokine to the cytokine receptor. Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes according to Formula I: 
       A 1 -L 1 -P 1   (Formula I)
 
     wherein A 1  is the antigen binding domain that binds to the target antigen or the cytokine that binds to the cytokine receptor; L 1  is the cleavable linker; and P 1  is a peptide that impairs binding of the antigen binding domain to the target antigen or impairs binding of the cytokine to the cytokine receptor. Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes comprising Formula I: 
       A 1 -L 1 -P 1   (Formula I)
 
     wherein A 1  is the antigen binding domain that binds to the target antigen or the cytokine that binds to the cytokine receptor; L 1  is the cleavable linker; and P 1  is a peptide that impairs binding of the antigen binding domain to the target antigen or impairs binding of the cytokine to the cytokine receptor. 
     Disclosed herein, in some embodiments, are polypeptides or polypeptide complexes, wherein the isolated polypeptide is complexed with a second isolated polypeptide comprising a second antigen binding domain or a second cytokine. Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes according to Formula II: 
       A 2 -L 2 -P 2   (Formula II)
 
     wherein A 2  comprises the second antigen binding domain or the second cytokine; L 2  comprises a second cleavable linker; and P 2  comprises a second peptide that impairs binding of the second antigen binding domain to a second target antigen or impairs binding of the second cytokine to a second cytokine receptor. Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes comprising Formula II: 
       A 2 -L 2 -P 2   (Formula II)
 
     wherein A 2  comprises the second antigen binding domain or the second cytokine; L 2  comprises a second cleavable linker; and P 2  comprises a second peptide that impairs binding of the second antigen binding domain to a second target antigen or impairs binding of the second cytokine to a second cytokine receptor. Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes according to Formula II: 
       A 2 -L 2 -P 2   (Formula II)
 
     wherein A 2  is the second antigen binding domain or the second cytokine; L 2  is a second cleavable linker; and P 2  is a second peptide that impairs binding of the second antigen binding domain to a second target antigen or impairs binding of the second cytokine to a second cytokine receptor. Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes comprising Formula II: 
       A 2 -L 2 -P 2   (Formula II)
 
     wherein A 2  is the second antigen binding domain or the second cytokine; L 2  is a second cleavable linker; and P 2  is a second peptide that impairs binding of the second antigen binding domain to a second target antigen or impairs binding of the second cytokine to a second cytokine receptor. 
     Pharmaceutical Compositions 
     Disclosed herein, in some embodiments, are pharmaceutical compositions comprising: (a) the polypeptides or polypeptide complexes as disclosed herein; and (b) a pharmaceutically acceptable excipient. 
     In some embodiments, the pharmaceutical composition comprises (a) the polypeptides or polypeptide complexes comprising a cleavable linker according to the amino acid sequence of SEQ ID NO: 1 (LSGRSDAG) and (b) a pharmacetically acceptable excipient. 
     In some embodiments, the pharmaceutical composition comprises (a) the polypeptides or polypeptide complexes comprising a cleavable linker according to the amino acid sequence of SEQ ID NO: 3 (ISSGLLSGRSDAG) and (b) a pharmacetically acceptable excipient. 
     In some embodiments, the pharmaceutical composition comprises (a) the polypeptides or polypeptide complexes comprising a cleavable linker according to the amino acid sequence of SEQ ID NO: 26 (AGLLAPPGGLSGRSDAG) and (b) a pharmacetically acceptable excipient. 
     In some embodiments, the pharmaceutical composition comprises (a) the polypeptides or polypeptide complexes comprising a cleavable linker according to the amino acid sequence of SEQ ID NO: 4 (AAGLLAPPGGLSGRSDAG) and (b) a pharmacetically acceptable excipient. 
     In some embodiments, the pharmaceutical composition comprises (a) the polypeptides or polypeptide complexes comprising a cleavable linker according to the amino acid sequence of SEQ ID NO: 5 (SPLGLSGRSDAG) and (b) a pharmacetically acceptable excipient. 
     In some embodiments, the pharmaceutical composition comprises (a) the polypeptides or polypeptide complexes comprising a cleavable linker according to the amino acid sequence of SEQ ID NO: 6 (LSGRSDAGSPLGLAG) and (b) a pharmacetically acceptable excipient. 
     In some embodiments, the pharmaceutical composition comprises (a) the polypeptides or polypeptide complexes comprising a cleavable linker according to the amino acid sequence of Linker 1 (ISSGLLSGRSDAG) (SEQ ID NO: 3), Linker 2 (AAGLLAPPGGLSGRSDAG) (SEQ ID NO: 4), Linker 3 (SPLGLSGRSDAG) (SEQ ID NO: 5), or Linker 4 (LSGRSDAGSPLGLAG) (SEQ ID NO: 6), or the polypeptides or polypeptide complexes comprising a cleavable linker that has 1, 2, or 3 amino acid substitutions, additions, or deletions relative to the amino acid sequence of Linker 1, Linker 2, Linker 3, or Linker 4 and (b) a pharmacetically acceptable excipient. In some embodiments, the pharmaceutical composition comprises (a) the polypeptides or polypeptide complexes comprising a cleavable linker according to the amino acid sequence of Linker 1 (ISSGLLSGRSDAG) (SEQ ID NO: 3) and (b) a pharmacetically acceptable excipient. In some embodiments, the pharmaceutical composition comprises (a) the polypeptides or polypeptide complexes comprising a cleavable linker according to the amino acid sequence of Linker 2 (AAGLLAPPGGLSGRSDAG) (SEQ ID NO: 4) and (b) a pharmacetically acceptable excipient. In some embodiments, the pharmaceutical composition comprises (a the polypeptides or polypeptide complexes comprising a cleavable linker according to the amino acid sequence of Linker 3 (SPLGLSGRSDAG) (SEQ ID NO: 5) and (b) a pharmacetically acceptable excipient. In some embodiments, the pharmaceutical composition comprises (a) the polypeptides or polypeptide complexes comprising a cleavable linker according to the amino acid sequence of Linker 4 (LSGRSDAGSPLGLAG) (SEQ ID NO: 6) and (b) a pharmacetically acceptable excipient. In some embodiments, the pharmaceutical composition comprises (a) the polypeptides or polypeptide complexes comprising a cleavable linker according to the the amino acid sequence LSGRSDAG (SEQ ID NO: 1) and (b) a pharmacetically acceptable excipient. 
     In some embodiments, the pharmaceutical composition comprises (a) isolated polypeptide polypeptides or polypeptide complexes according to Formula I: 
       A 1 -L 1 -P 1   (Formula I)
 
     wherein A 1  comprises the antigen binding domain that binds to the target antigen or the cytokine that binds to the cytokine receptor; L 1  comprises the cleavable linker; and P 1  comprises a peptide that impairs binding of the antigen binding domain to the target antigen or impairs binding of the cytokine to the cytokine receptor; and (b) a pharmacetically acceptable excipient. In some embodiments, the pharmaceutical composition comprises (a) isolated polypeptide polypeptides or polypeptide complexes comprising Formula I: 
       A 1 -L 1 -P 1   (Formula I)
 
     wherein A 1  comprises the antigen binding domain that binds to the target antigen or the cytokine that binds to the cytokine receptor; L 1  comprises the cleavable linker; and P 1  comprises a peptide that impairs binding of the antigen binding domain to the target antigen or impairs binding of the cytokine to the cytokine receptor; and (b) a pharmacetically acceptable excipient. In some embodiments, the pharmaceutical composition comprises (a) isolated polypeptide polypeptides or polypeptide complexes according to Formula I: 
       A 1 -L 1 -P 1   (Formula I)
 
     wherein A 1  is the antigen binding domain that binds to the target antigen or the cytokine that binds to the cytokine receptor; L 1  is the cleavable linker; and P 1  is a peptide that impairs binding of the antigen binding domain to the target antigen or impairs binding of the cytokine to the cytokine receptor; and (b) a pharmacetically acceptable excipient. In some embodiments, the pharmaceutical composition comprises (a) isolated polypeptide polypeptides or polypeptide complexes comprising Formula I: 
       A 1 -L 1 -P 1   (Formula I)
 
     wherein A 1  is the antigen binding domain that binds to the target antigen or the cytokine that binds to the cytokine receptor; L 1  is the cleavable linker; and P 1  is a peptide that impairs binding of the antigen binding domain to the target antigen or impairs binding of the cytokine to the cytokine receptor; and (b) a pharmacetically acceptable excipient. 
     Disclosed herein, in some embodiments, are polypeptides or polypeptide complexes, wherein the isolated polypeptide is complexed with a second isolated polypeptide comprising a second antigen binding domain or a second cytokine. In some embodiments, the pharmaceutical composition comprises (a) isolated polypeptide polypeptides or polypeptide complexes according to Formula II: 
       A 2 -L 2 -P 2   (Formula II)
 
     wherein A 2  comprises the second antigen binding domain or the second cytokine; L 2  comprises a second cleavable linker; and P 2  comprises a second peptide that impairs binding of the second antigen binding domain to a second target antigen or impairs binding of the second cytokine to a second cytokine receptor; and (b) a pharmacetically acceptable excipient. In some embodiments, the pharmaceutical composition comprises (a) isolated polypeptide polypeptides or polypeptide complexes comprising Formula II: 
       A 2 -L 2 -P 2   (Formula II)
 
     wherein A 2  comprises the second antigen binding domain or the second cytokine; L 2  comprises a second cleavable linker; and P 2  comprises a second peptide that impairs binding of the second antigen binding domain to a second target antigen or impairs binding of the second cytokine to a second cytokine receptor; and (b) a pharmacetically acceptable excipient. In some embodiments, the pharmaceutical composition comprises (a) isolated polypeptide polypeptides or polypeptide complexes according to Formula II: 
       A 2 -L 2 -P 2   (Formula II)
 
     wherein A 2  is the second antigen binding domain or the second cytokine; L 2  is a second cleavable linker; and P 2  is a second peptide that impairs binding of the second antigen binding domain to a second target antigen or impairs binding of the second cytokine to a second cytokine receptor; and (b) a pharmacetically acceptable excipient. In some embodiments, the pharmaceutical composition comprises (a) isolated polypeptide polypeptides or polypeptide complexes comprising Formula II: 
       A 2 -L 2 -P 2   (Formula II)
 
     wherein A 2  is the second antigen binding domain or the second cytokine; L 2  is a second cleavable linker; and P 2  is a second peptide that impairs binding of the second antigen binding domain to a second target antigen or impairs binding of the second cytokine to a second cytokine receptor; and (b) a pharmacetically acceptable excipient. 
     In some embodiments, the polypeptide or polypeptide complex further comprises a detectable label, a therapeutic agent, or a pharmacokinetic modifying moiety. In some embodiments, the detectable label comprises a fluorescent label, a radiolabel, an enzyme, a nucleic acid probe, or a contrast agent. 
     For administration to a subject, the polypeptide or polypeptide complex as disclosed herein, may be provided in a pharmaceutical composition together with one or more pharmaceutically acceptable carriers or excipients. The term “pharmaceutically acceptable carrier” includes, but is not limited to, any carrier that does not interfere with the effectiveness of the biological activity of the ingredients and that is not toxic to the patient to whom it is administered. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Such carriers can be formulated by conventional methods and can be administered to the subject at a suitable dose. Preferably, the compositions are sterile. These compositions may also contain adjuvants such as preservative, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents. 
     The pharmaceutical composition may be in any suitable form, (depending upon the desired method of administration). It may be provided in unit dosage form, may be provided in a sealed container and may be provided as part of a kit. Such a kit may include instructions for use. It may include a plurality of said unit dosage forms. 
     The pharmaceutical composition may be adapted for administration by any appropriate route, including a parenteral (e.g., subcutaneous, intramuscular, or intravenous) route. Such compositions may be prepared by any method known in the art of pharmacy, for example by mixing the active ingredient with the carrier(s) or excipient(s) under sterile conditions. 
     Dosages of the substances of the present disclosure can vary between wide limits, depending upon the disease or disorder to be treated, the age and condition of the individual to be treated, etc. and a physician will ultimately determine appropriate dosages to be used. 
     Table 1 provides the amino acid sequences of constructs described herein. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Summary of Amino Acid Sequences 
               
            
           
           
               
               
               
               
            
               
                 Construct 
                 Construct 
                 Amino Acid Sequence 
                 SEQ ID 
               
               
                 ID 
                 Description 
                 (N to C) 
                 NO: 
               
               
                   
               
            
           
           
               
            
               
                 LINKER SEQUENCES 
               
            
           
           
               
               
               
               
            
               
                 Linker-0 
                 Cleavable linker 
                 ISSGLLSGRSDNH 
                  2 
               
               
                   
                 (control) 
                   
                   
               
               
                   
               
               
                 Linker-1 
                 Cleavable linker 
                 ISSGLLSGRSDAG 
                  3 
               
               
                   
               
               
                 Linker-2 
                 Cleavable linker 
                 AAGLLAPPGGLSGRSDAG 
                  4 
               
               
                   
               
               
                 Linker-3 
                 Cleavable linker 
                 SPLGLSGRSDAG 
                  5 
               
               
                   
               
               
                 Linker-4 
                 Cleavable linker 
                 LSGRSDAGSPLGLAG 
                  6 
               
               
                   
               
               
                 Linker-5 
                 Cleavable linker 
                 LSGRSDAG 
                  1 
               
               
                   
               
               
                 Linker-6 
                 Cleavable linker 
                 AGLLAPPGGLSGRSDAG 
                 26 
               
               
                   
               
            
           
           
               
            
               
                 PEPTIDE MASK SEQUENCES 
               
            
           
           
               
               
               
               
            
               
                 Peptide-1 
                 anti-EGFR peptide 
                 QGQSGQLSCEGWAMNREQCRA 
                  7 
               
               
                   
                 mask 
                   
                   
               
               
                   
               
               
                 Peptide-2 
                 anti-EGFR peptide 
                 GGPCRSHIDVAKPICV 
                  8 
               
               
                   
                 mask 
                   
                   
               
               
                   
               
               
                 Peptide-3 
                 anti-CD3 peptide 
                 QGQSGQGYLWGCEWNCGGITT 
                  9 
               
               
                   
                 mask 
                   
                   
               
               
                   
               
               
                 Peptide-4 
                 anti-CD3 peptide 
                 QGQSGSGYLWGCEWNCAGITT 
                 10 
               
               
                   
                 mask 
                   
                   
               
               
                   
               
            
           
           
               
            
               
                 HALF-LIFE EXTENDING MOIETIES 
               
            
           
           
               
               
               
               
            
               
                 HE-1 
                 10G single domain 
                 EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVR 
                 11 
               
               
                   
                 antibody 
                 QAPGKGLEWVSSISGSGRDTLYADSVKGRFTISRDNAKT 
                   
               
               
                   
                   
                 TLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSS 
                   
               
               
                   
               
            
           
           
               
            
               
                 FULL LENGTH CONSTRUCTS 
               
            
           
           
               
               
               
               
            
               
                 PC-1 
                 Light Chain 
                 QGQSGQLSCEGWAMNREQCRAGSSGGSGGSGGSGISSG 
                 12 
               
               
                   
                 Sequence: N- 
                 LLSGRSDNHGSSGTDILLTQSPVILSVSPGERVSFSCRASQ 
                   
               
               
                   
                 [Peptide-1]-[Linker- 
                 SIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGT 
                   
               
               
                   
                 0]-[anti-EGFR Fab 
                 DFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKR 
                   
               
               
                   
                 light chain]-C 
                 TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ 
                   
               
               
                   
                 (control) 
                 WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY 
                   
               
               
                   
                   
                 EKHKVYACEVTHQGLSSPVTKSFNRGEC 
                   
               
               
                   
               
               
                 PC-1 
                 Heavy Chain 
                 EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVR 
                 13 
               
               
                   
                 Sequence: N-[10G 
                 QAPGKGLEWVSSISGSGRDTLYADSVKGRFTISRDNAKT 
                   
               
               
                   
                 SDA]-[Peptide-3]- 
                 TLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSG 
                   
               
               
                   
                 [Linker-0]-[anti-CD3 
                 GGGSGGGSQGQSGQGYLWGCEW 
                   
               
               
                   
                 scFv (light chain- 
                 NCGGITTGSSGGSGGSGGISSGLLSGRSDNHGGGSQTVVT 
                   
               
               
                   
                 heavy chain)]-[anti- 
                 QEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQ 
                   
               
               
                   
                 EGFR Fab heavy 
                 APRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPE 
                   
               
               
                   
                 chain]-C 
                 DEAEYYCALWYSNLWVFGGGTKLTVLGGGGSGGGGSG 
                   
               
               
                   
                 (control) 
                 GGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAM 
                   
               
               
                   
                   
                 NWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI 
                   
               
               
                   
                   
                 SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYV 
                   
               
               
                   
                   
                 SWFAYWGQGTLVTVSSGGGGSQVQLKQSGPGLVQPSQS 
                   
               
               
                   
                   
                 LSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGN 
                   
               
               
                   
                   
                 TDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCA 
                   
               
               
                   
                   
                 RALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSK 
                   
               
               
                   
                   
                 STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA 
                   
               
               
                   
                   
                 VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD 
                   
               
               
                   
                   
                 KKVEPKSCGGHHHHHHHHGGGLNDIFEAQKIEWHE 
                   
               
               
                   
               
               
                 PC-2 
                 Light Chain 
                 GGPCRSHIDVAKPICVGGGGSGGSISSGLLSGRSDAGGGG 
                 14 
               
               
                   
                 Sequence: N- 
                 SDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRT 
                   
               
               
                   
                 [Peptide-2]-[Linker- 
                 NGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDI 
                   
               
               
                   
                 1]-[anti-EGFR Fab 
                 ADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSD 
                   
               
               
                   
                 light chain]-C 
                 EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ 
                   
               
               
                   
                   
                 ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ 
                   
               
               
                   
                   
                 GLSSPVTKSFNRGEC 
                   
               
               
                   
               
               
                 PC-2 
                 Heavy Chain 
                 EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVR 
                 15 
               
               
                   
                 Sequence: N-[10G 
                 QAPGKGLEWVSSISGSGRDTLYADSVKGRFTISRDNAKT 
                   
               
               
                   
                 SDA]-[Peptide-4]- 
                 TLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSG 
                   
               
               
                   
                 [Linker-1]-[anti-CD3 
                 GGGSGGGSQGQSGQGYLWGCEWNCAGITTGSSGGSGGS 
                   
               
               
                   
                 scFv (light chain- 
                 GGISSGLLSGRSDAGGGGSQTVVTQEPSLTVSPGGTVTLT 
                   
               
               
                   
                 heavy chain)]-[anti- 
                 CRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPG 
                   
               
               
                   
                 EGFR Fab heavy 
                 TPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNL 
                   
               
               
                   
                 chain]-C 
                 WVFGGGTKLTVLGGGGSGGGGSGGGGSEVQLVESGGG 
                   
               
               
                   
                   
                 LVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEW 
                   
               
               
                   
                   
                 VARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQM 
                   
               
               
                   
                   
                 NNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVT 
                   
               
               
                   
                   
                 VSSGGGGSQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNY 
                   
               
               
                   
                   
                 GVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSIN 
                   
               
               
                   
                   
                 KDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAY 
                   
               
               
                   
                   
                 WGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCL 
                   
               
               
                   
                   
                 VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS 
                   
               
               
                   
                   
                 VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCAAA 
                   
               
               
                   
                   
                 HHHHHHHH 
                   
               
               
                   
               
               
                 PC-3 
                 Light Chain 
                 GGPCRSHIDVAKPICVGGGGSSGGSAAGLLAPPGGLSGRS 
                 16 
               
               
                   
                 Sequence: N- 
                 DAGGGGSDILLTQSPVILSVSPGERVSFSCRASQSIGTNIH 
                   
               
               
                   
                 [Peptide-2]-[Linker- 
                 WYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSIN 
                   
               
               
                   
                 2]-[anti-EGFR Fab 
                 SVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSV 
                   
               
               
                   
                 light chain]-C 
                 FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL 
                   
               
               
                   
                   
                 QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA 
                   
               
               
                   
                   
                 CEVTHQGLSSPVTKSFNRGEC 
                   
               
               
                   
               
               
                 PC-3 
                 Heavy Chain 
                 EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVR 
                 17 
               
               
                   
                 Sequence: N-[10G 
                 QAPGKGLEWVSSISGSGRDTLYADSVKGRFTISRDNAKT 
                   
               
               
                   
                 SDA]-[Peptide-4]- 
                 TLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSG 
                   
               
               
                   
                 [linker Linker-2]- 
                 GGGSGGGSQGQSGQGYLWGCEWNCAGITTGSSGGSAA 
                   
               
               
                   
                 [anti-CD3 scFv (light 
                 GLLAPPGGLSGRSDAGGGGSQTVVTQEPSLTVSPGGTVT 
                   
               
               
                   
                 chain-heavy chain)]- 
                 LTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRA 
                   
               
               
                   
                 [anti-EGFR Fab 
                 PGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSN 
                   
               
               
                   
                 heavy chain]-C 
                 LWVFGGGTKLTVLGGGGSGGGGSGGGGSEVQLVESGG 
                   
               
               
                   
                   
                 GLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLE 
                   
               
               
                   
                   
                 WVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQ 
                   
               
               
                   
                   
                 MNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLV 
                   
               
               
                   
                   
                 TVSSGGGGSQVQLKQSGPGLVQPSQSLSITCTVSGFSLTN 
                   
               
               
                   
                   
                 YGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSI 
                   
               
               
                   
                   
                 NKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFA 
                   
               
               
                   
                   
                 YWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGC 
                   
               
               
                   
                   
                 LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS 
                   
               
               
                   
                   
                 SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCAA 
                   
               
               
                   
                   
                 AHHHHHHHH 
                   
               
               
                   
               
               
                 PC-4 
                 Light Chain 
                 GGPCRSHIDVAKPICVGGGGSGGGGSPLGLSGRSDAGGG 
                 18 
               
               
                   
                 Sequence: N- 
                 GSDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQR 
                   
               
               
                   
                 [Peptide-2]-[Linker- 
                 TNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESED 
                   
               
               
                   
                 3]-[anti-EGFR Fab 
                 IADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPS 
                   
               
               
                   
                 light chain]-C 
                 DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS 
                   
               
               
                   
                   
                 QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH 
                   
               
               
                   
                   
                 QGLSSPVTKSFNRGEC 
                   
               
               
                   
               
               
                 PC-4 
                 Heavy Chain 
                 EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVR 
                 19 
               
               
                   
                 Sequence: N-[10G 
                 QAPGKGLEWVSSISGSGRDTLYADSVKGRFTISRDNAKT 
                   
               
               
                   
                 SDA]-[Peptide-4]- 
                 TLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSG 
                   
               
               
                   
                 [Linker-3]-[anti-CD3 
                 GGGSGGGSQGQSGQGYLWGCEWNCAGITTGSSGGSGG 
                   
               
               
                   
                 scFv (light chain- 
                 GSGGSPLGLSGRSDAGGGGSQTVVTQEPSLTVSPGGTVT 
                   
               
               
                   
                 heavy chain)]-[anti- 
                 LTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRA 
                   
               
               
                   
                 EGFR Fab heavy 
                 PGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSN 
                   
               
               
                   
                 chain]-C 
                 LWVFGGGTKLTVLGGGGSGGGGSGGGGSEVQLVESGG 
                   
               
               
                   
                   
                 GLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLE 
                   
               
               
                   
                   
                 WVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQ 
                   
               
               
                   
                   
                 MNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLV 
                   
               
               
                   
                   
                 TVSSGGGGSQVQLKQSGPGLVQPSQSLSITCTVSGFSLTN 
                   
               
               
                   
                   
                 YGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSI 
                   
               
               
                   
                   
                 NKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFA 
                   
               
               
                   
                   
                 YWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGC 
                   
               
               
                   
                   
                 LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS 
                   
               
               
                   
                   
                 SVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCAA 
                   
               
               
                   
                   
                 AHHHHHHHH 
                   
               
               
                   
               
               
                 PC-5 
                 Light Chain 
                 GGPCRSHIDVAKPICVGGGGSGGLSGRSDAGSPLGLAGS 
                 20 
               
               
                   
                 Sequence: N- 
                 GGSDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQ 
                   
               
               
                   
                 [Peptide-2]-[Linker- 
                 RTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESE 
                   
               
               
                   
                 4]-[anti-EGFR Fab 
                 DIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPP 
                   
               
               
                   
                 light chain]-C 
                 SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN 
                   
               
               
                   
                   
                 SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT 
                   
               
               
                   
                   
                 HQGLSSPVTKSFNRGEC 
                   
               
               
                   
               
               
                 PC-5 
                 Heavy Chain 
                 EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVR 
                 21 
               
               
                   
                 Sequence: N-[10G 
                 QAPGKGLEWVSSISGSGRDTLYADSVKGRFTISRDNAKT 
                   
               
               
                   
                 SDA]-[Peptide-4]- 
                 TLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSG 
                   
               
               
                   
                 [Linker-4]-[anti-CD3 
                 GGGSGGGSQGQSGQGYLWGCEWNCAGITTGSSGGSGGL 
                   
               
               
                   
                 scFv (light chain- 
                 SGRSDAGSPLGLAGSGGGSQTVVTQEPSLTVSPGGTVTL 
                   
               
               
                   
                 heavy chain)]-[anti- 
                 TCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAP 
                   
               
               
                   
                 EGFR Fab heavy 
                 GTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNL 
                   
               
               
                   
                 chain]-C 
                 WVFGGGTKLTVLGGGGSGGGGSGGGGSEVQLVESGGG 
                   
               
               
                   
                   
                 LVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEW 
                   
               
               
                   
                   
                 VARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQM 
                   
               
               
                   
                   
                 NNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVT 
                   
               
               
                   
                   
                 VSSGGGGSQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNY 
                   
               
               
                   
                   
                 GVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSIN 
                   
               
               
                   
                   
                 KDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAY 
                   
               
               
                   
                   
                 WGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCL 
                   
               
               
                   
                   
                 VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS 
                   
               
               
                   
                   
                 VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCAAA 
                   
               
               
                   
                   
                 HHHHHHHH 
                   
               
               
                   
               
               
                 PC-6 
                 N-[anti-EGFR Fab 
                 QILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTN 
                 22 
               
               
                   
                 heavy chain]-C 
                 GSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIA 
                   
               
               
                   
                   
                 DYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDE 
                   
               
               
                   
                   
                 QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE 
                   
               
               
                   
                   
                 SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG 
                   
               
               
                   
                   
                 LSSPVTKSFNRGEC 
                   
               
               
                   
               
               
                 PC-6 
                 N-[anti-CD3 scFv 
                 QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQ 
                 23 
               
               
                   
                 (light chain-heavy 
                 QKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLS 
                   
               
               
                   
                 chain)]-[anti-EGFR 
                 GVQPEDEAEYYCALWYSNLWVFGGGTKLTVLGGGGSG 
                   
               
               
                   
                 Fab heavy chain]-C 
                 GGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFN 
                   
               
               
                   
                   
                 TYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVK 
                   
               
               
                   
                   
                 DRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFG 
                   
               
               
                   
                   
                 NSYVSWFAYWGQGTLVTVSSGGGGSQVQLKQSGPGLV 
                   
               
               
                   
                   
                 QPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVI 
                   
               
               
                   
                   
                 WSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDT 
                   
               
               
                   
                   
                 AIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVF 
                   
               
               
                   
                   
                 PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS 
                   
               
               
                   
                   
                 GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK 
                   
               
               
                   
                   
                 PSNTKVDKKVEPKSCGGHHHHHHHHGGGLNDIFEAQKI 
                   
               
               
                   
                   
                 EWHE 
                   
               
               
                   
               
               
                 PC-7 
                 N-[anti-EGFR Fab 
                 DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTN 
                 24 
               
               
                   
                 heavy chain]-C 
                 GSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIA 
                   
               
               
                   
                   
                 DYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDE 
                   
               
               
                   
                   
                 QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE 
                   
               
               
                   
                   
                 SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG 
                   
               
               
                   
                   
                 LSSPVTKSFNRGEC 
                   
               
               
                   
               
               
                 PC-7 
                 N-[anti-CD3 scFv 
                 QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQ 
                 25 
               
               
                   
                 (light chain-heavy 
                 QKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLS 
                   
               
               
                   
                 chain)]-[anti-EGFR 
                 GVQPEDEAEYYCALWYSNLWVFGGGTKLTVLGGGGSG 
                   
               
               
                   
                 Fab heavy chain]-C 
                 GGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFN 
                   
               
               
                   
                   
                 TYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVK 
                   
               
               
                   
                   
                 DRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFG 
                   
               
               
                   
                   
                 NSYVSWFAYWGQGTLVTVSSGGGGSQVQLKQSGPGLV 
                   
               
               
                   
                   
                 QPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVI 
                   
               
               
                   
                   
                 WSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDT 
                   
               
               
                   
                   
                 AIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVF 
                   
               
               
                   
                   
                 PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS 
                   
               
               
                   
                   
                 GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK 
                   
               
               
                   
                   
                 PSNTKVDKKVEPKSCGGHHHHHHHHGGGLNDIFEAQKI 
                   
               
               
                   
                   
                 EWHE 
               
               
                   
               
            
           
         
       
     
     Polypeptides or polypeptide complexes, in some embodiments, comprise a sequence set forth in Table 1. In some embodiments, the sequence comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 1, 2, 4, 5, 7, 8, 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In some instances, the sequence comprises at least or about 95% homology to SEQ ID NOs: 1, 2, 4, 5, 7, 8, 9, 12, 13, 14, 15, 16, 17, 18, 1 9 , 20, 21, 22, 23, 24, or 25. In some instances, the sequence comprises at least or about 97% homology to SEQ ID NOs: 1, 2, 4, 5, 7, 8, 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In some instances, the sequence comprises at least or about 99% homology to SEQ ID NOs: 1, 2, 4, 5, 7, 8, 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In some instances, the sequence comprises at least or about 100% homology to SEQ ID NOs: 1, 2, 4, 5, 7, 8, 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In some instances, the sequence comprises at least a portion having at least or about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, or more than 400 amino acids of SEQ ID NOs: 1, 2, 4, 5, 7, 8, 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. 
     Percent (%) sequence identity with respect to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide 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 known for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Appropriate parameters for aligning sequences are able to be determined, including algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or may be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary. 
     In situations where ALIGN-2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X/Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program&#39;s alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program. 
     Production of Polypeptides Comprising Cleavable Linkers 
     In some embodiments, polypeptides described herein (e.g., antibodies and its binding fragments) are produced using any method known in the art to be useful for the synthesis of polypeptides (e.g., antibodies), in particular, by chemical synthesis or by recombinant expression, and are preferably produced by recombinant expression techniques. 
     In some instances, an antibody or its binding fragment thereof is expressed recombinantly, and the nucleic acid encoding the antibody or its binding fragment is assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., 1994, BioTechniques 17:242), which involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligation of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR. 
     Alternatively, a nucleic acid molecule encoding an antibody is optionally generated from a suitable source (e.g., an antibody cDNA library, or cDNA library generated from any tissue or cells expressing the immunoglobulin) by PCR amplification using synthetic primers hybridizable to the 3′ and 5′ ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence. 
     In some instances, an antibody or its binding is optionally generated by immunizing an animal, such as a mouse, to generate polyclonal antibodies or, more preferably, by generating monoclonal antibodies, e.g., as described by Kohler and Milstein (1975, Nature 256:495-497) or, as described by Kozbor et al. (1983, Immunology Today 4:72) or Cole et al. (1985 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Alternatively, a clone encoding at least the Fab portion of the antibody is optionally obtained by screening Fab expression libraries (e.g., as described in Huse et al., 1989, Science 246:1275-1281) for clones of Fab fragments that bind the specific antigen or by screening antibody libraries (See, e.g., Clackson et al., 1991, Nature 352:624; Hane et al., 1997 Proc. Natl. Acad. Sci. USA 94:4937). 
     In some embodiments, techniques developed for the production of “chimeric antibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci. 81:851-855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454) by splicing genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity are used. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region. 
     In some embodiments, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,694,778; Bird, 1988, Science 242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989, Nature 334:544-54) are adapted to produce single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Techniques for the assembly of functional Fv fragments in  E. coli  are also optionally used (Skerra et al., 1988, Science 242:1038-1041). 
     In some embodiments, an expression vector comprising the nucleotide sequence of an antibody or the nucleotide sequence of an antibody is transferred to a host cell by conventional techniques (e.g., electroporation, liposomal transfection, and calcium phosphate precipitation), and the transfected cells are then cultured by conventional techniques to produce the antibody. In specific embodiments, the expression of the antibody is regulated by a constitutive, an inducible or a tissue, specific promoter. 
     In some embodiments, a variety of host-expression vector systems is utilized to express an antibody, or its binding fragment described herein. Such host-expression systems represent vehicles by which the coding sequences of the antibody is produced and subsequently purified, but also represent cells that are, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody or its binding fragment in situ. These include, but are not limited to, microorganisms such as bacteria (e.g.,  E. coli  and  B. subtilis ) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing an antibody or its binding fragment coding sequences; yeast (e.g.,  Saccharomyces Pichia ) transformed with recombinant yeast expression vectors containing an antibody or its binding fragment coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing an antibody or its binding fragment coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV)) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing an antibody or its binding fragment coding sequences; or mammalian cell systems (e.g., COS, CHO, BH, 293, 293T, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g. the adenovirus late promoter; the vaccinia virus 7.5K promoter). 
     For long-term, high-yield production of recombinant proteins, stable expression is preferred. In some instances, cell lines that stably express an antibody are optionally engineered. Rather than using expression vectors that contain viral origins of replication, host cells are transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells are then allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci that in turn are cloned and expanded into cell lines. This method can advantageously be used to engineer cell lines which express the antibody or its binding fragments. 
     In some instances, a number of selection systems are used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &amp; Szybalski, 192, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes are employed in tk-, hgprt- or aprt- cells, respectively. Also, antimetabolite resistance are used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Proc. Natl. Acad. Sci. USA 77:357; O&#39;Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan &amp; Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May 1993, TIB TECH 11(5):155-215) and hygro, which confers resistance to hygromycin (Santerre et al., 1984, Gene 30:147). Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds., 1993, Current Protocols in Molecular Biology, John Wiley &amp; Sons, NY; Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY; and in Chapters 12 and 13, Dracopoli et al. (eds), 1994, Current Protocols in Human Genetics, John Wiley &amp; Sons, NY.; Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1). 
     In some instances, the expression levels of an antibody are increased by vector amplification (for a review, see Bebbington and Hentschel, the use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York, 1987)). When a marker in the vector system expressing an antibody is amplifiable, an increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the nucleotide sequence of the antibody, production of the antibody will also increase (Crouse et al., 1983, Mol. Cell Biol. 3:257). 
     In some instances, any method known in the art for purification of an antibody is used, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. 
     Expression Vectors 
     In some embodiments, vectors include any suitable vectors derived from either a eukaryotic or prokaryotic sources. In some cases, vectors are obtained from bacteria (e.g.  E. coli ), insects, yeast (e.g.  Pichia pastoris ), algae, or mammalian sources. Exemplary bacterial vectors include pACYC177, pASK75, pBAD vector series, pBADM vector series, pET vector series, pETM vector series, pGEX vector series, pHAT, pHAT2, pMal-c2, pMal-p2, pQE vector series, pRSET A, pRSET B, pRSET C, pTrcHis2 series, pZA31-Luc, pZE21-MCS-1, pFLAG ATS, pFLAG CTS, pFLAG MAC, pFLAG Shift-12c, pTAC-MAT-1, pFLAG CTC, or pTAC-MAT-2. 
     Exemplary insect vectors include pFastBac1, pFastBac DUAL, pFastBac ET, pFastBac HTa, pFastBac HTb, pFastBac HTc, pFastBac M30a, pFastBact M30b, pFastBac, M30c, pVL1392, pVL1393, pVL1393 M10, pVL1393 M11, pVL1393 M12, FLAG vectors such as pPolh-FLAG1 or pPolh-MAT 2, or MAT vectors such as pPolh-MAT1, or pPolh-MAT2. 
     In some cases, yeast vectors include Gateway® pDEST™ 14 vector, Gateway® pDEST™ 15 vector, Gateway® pDEST™ 17 vector, Gateway® pDEST™ 24 vector, Gateway® pYES-DEST52 vector, pBAD-DEST49 Gateway® destination vector, pAO815  Pichia  vector, pFLD1 Pichi  pastoris  vector, pGAPZA,B, &amp; C  Pichia pastoris  vector, pPIC3.5K  Pichia  vector, pPIC6 A, B, &amp; C  Pichia  vector, pPIC9K  Pichia  vector, pTEF1/Zeo, pYES2 yeast vector, pYES2/CT yeast vector, pYES2/NT A, B, &amp; C yeast vector, or pYES3/CT yeast vector. 
     Exemplary algae vectors include pChlamy-4 vector or MCS vector. 
     Examples of mammalian vectors include transient expression vectors or stable expression vectors. Mammalian transient expression vectors may include pRK5, p3xFLAG-CMV 8, pFLAG-Myc-CMV 19, pFLAG-Myc-CMV 23, pFLAG-CMV 2, pFLAG-CMV 6a, b, c, pFLAG-CMV 5.1, pFLAG-CMV 5a, b, c, p3xFLAG-CMV 7.1, pFLAG-CMV 20, p3xFLAG-Myc-CMV 24, pCMV-FLAG-MAT1, pCMV-FLAG-MAT2, pBICEP-CMV 3, or pBICEP-CMV 4. Mammalian stable expression vector may include pFLAG-CMV 3, p3xFLAG-CMV 9, p3xFLAG-CMV 13, pFLAG-Myc-CMV 21, p3xFLAG-Myc-CMV 25, pFLAG-CMV 4, p3xFLAG-CMV 10, p3xFLAG-CMV 14, pFLAG-Myc-CMV 22, p3xFLAG-Myc-CMV 26, pBICEP-CMV 1, or pBICEP-CMV 2. 
     In some instances, a cell-free system is a mixture of cytoplasmic and/or nuclear components from a cell and is used for in vitro nucleic acid synthesis. In some cases, a cell-free system utilizes either prokaryotic cell components or eukaryotic cell components. Sometimes, a nucleic acid synthesis is obtained in a cell-free system based on for example  Drosophila  cell,  Xenopus  egg, or HeLa cells. Exemplary cell-free systems include, but are not limited to,  E. coli  S30 Extract system,  E. coli  T7 S30 system, or PURExpress®. 
     Host Cells 
     In some embodiments, a host cell includes any suitable cell such as a naturally derived cell or a genetically modified cell. In some instances, a host cell is a production host cell. In some instances, a host cell is a eukaryotic cell. In other instances, a host cell is a prokaryotic cell. In some cases, a eukaryotic cell includes fungi (e.g., yeast cells), animal cell or plant cell. In some cases, a prokaryotic cell is a bacterial cell. Examples of bacterial cell include gram-positive bacteria or gram-negative bacteria. Sometimes the gram-negative bacteria is anaerobic, rod-shaped, or both. 
     In some instances, gram-positive bacteria include Actinobacteria, Firmicutes or Tenericutes. In some cases, gram-negative bacteria include Aquificae, Deinococcus-Thermus, Fibrobacteres-Chlorobi/Bacteroidetes (FCB group), Fusobacteria, Gemmatimonadetes, Nitrospirae, Planctomycetes-Verrucomicrobia/Chlamydiae (PVC group), Proteobacteria, Spirochaetes or Synergistetes. Other bacteria can be Acidobacteria, Chloroflexi, Chrysiogenetes, Cyanobacteria, Deferribacteres, Dictyoglomi, Thermodesulfobacteria or Thermotogae. A bacterial cell can be  Escherichia coli, Clostridium botulinum , or  Coli  bacilli. 
     Exemplary prokaryotic host cells include, but are not limited to, BL21, Machl™, DH10B™, TOP10, DH5α, DH10Bac™, OmniMax™, MegaX™, DH12S™, INV110, TOP10F′, INVαF, TOP10/P3, ccdB Survival, PIR1, PIR2, Stbl2™, Stbl3™, or Stbl4™. 
     In some instances, animal cells include a cell from a vertebrate or from an invertebrate. In some cases, an animal cell includes a cell from a marine invertebrate, fish, insects, amphibian, reptile, or mammal. In some cases, a fungus cell includes a yeast cell, such as brewer&#39;s yeast, baker&#39;s yeast, or wine yeast. 
     Fungi include ascomycetes such as yeast, mold, filamentous fungi, basidiomycetes, or zygomycetes. In some instances, yeast includes Ascomycota or Basidiomycota. In some cases, Ascomycota includes Saccharomycotina (true yeasts, e.g.  Saccharomyces cerevisiae  (baker&#39;s yeast)) or Taphrinomycotina (e.g. Schizosaccharomycetes (fission yeasts)). In some cases, Basidiomycota includes Agaricomycotina (e.g. Tremellomycetes) or Pucciniomycotina (e.g. Microbotryomycetes). 
     Exemplary yeast or filamentous fungi include, for example, the genus:  Saccharomyces, Schizosaccharomyces, Candida, Pichia, Hansenula, Kluyveromyces, Zygosaccharomyces, Yarrowia, Trichosporon, Rhodosporidi, Aspergillus, Fusarium , or  Trichoderma . Exemplary yeast or filamentous fungi include, for example, the species:  Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida utilis, Candida boidini, Candida albicans, Candida tropicalis, Candida stellatoidea, Candida glabrata, Candida krusei, Candida parapsilosis, Candida guilliermondii, Candida viswanathii, Candida lusitaniae, Rhodotorula mucilaginosa, Pichia metanolica, Pichia angusta, Pichia pastoris, Pichia anomala, Hansenula polymorpha, Kluyveromyces lactis, Zygosaccharomyces rouxii, Yarrowia lipolytica, Trichosporon pullulans, Rhodosporidium toru - Aspergillus niger, Aspergillus nidulans, Aspergillus awamori, Aspergillus oryzae, Trichoderma reesei, Yarrowia lipolytica, Brettanomyces bruxellensis, Candida stellata, Schizosaccharomyces pombe, Torulaspora delbrueckii, Zygosaccharomyces bailii, Cryptococcus neoformans, Cryptococcus gattii , or  Saccharomyces boulardii.    
     Exemplary yeast host cells include, but are not limited to,  Pichia pastoris  yeast strains such as GS115, KM71H 1 , SMD1168, SMD1 168H, and X-33; and  Saccharomyces cerevisiae  yeast strain such as INVSc1. 
     In some instances, additional animal cells include cells obtained from a mollusk, arthropod, annelid or sponge. In some cases, an additional animal cell is a mammalian cell, e.g., from a primate, ape, equine, bovine, porcine, canine, feline or rodent. In some cases, a rodent includes mouse, rat, hamster, gerbil, hamster, chinchilla, fancy rat, or guinea pig. 
     Exemplary mammalian host cells include, but are not limited to, 293A cell line, 293FT cell line, 293F cells, 293 H cells, CHO DG44 cells, CHO-S cells, CHO-K1 cells, FUT8 KO CHOK1, Expi293F™ cells, F1p-In™ T-REx™ 293 cell line, F1p-In™-293 cell line, F1p-In™-3T3 cell line, F1p-In™-BHK cell line, F1p-In™-CHO cell line, F1p-In™-CV-1 cell line, F1p-In™-Jurkat cell line, FreeStyle™ 293-F cells, FreeStyle™ CHO-S cells, GripTite™ 293 MSR cell line, GS-CHO cell line, HepaRG™ cells, T-REx™ Jurkat cell line, Per.C6 cells, T-REx™-293 cell line, T-REx™-CHO cell line, and T-REx™-HeLa cell line. 
     In some instances, a mammalian host cell is a stable cell line, or a cell line that has incorporated a genetic material of interest into its own genome and has the capability to express the product of the genetic material after many generations of cell division. In some cases, a mammalian host cell is a transient cell line, or a cell line that has not incorporated a genetic material of interest into its own genome and does not have the capability to express the product of the genetic material after many generations of cell division. 
     Exemplary insect host cells include, but are not limited to,  Drosophila  S2 cells, Sf9 cells, Sf21 cells, High Five™ cells, and expresSF+® cells. 
     In some instances, plant cells include a cell from algae. Exemplary insect cell lines include, but are not limited to, strains from  Chlamydomonas reinhardtii  137c, or Synechococcus  elongatus  PPC 7942. 
     Articles of Manufacture 
     In another aspect of the invention, an article of manufacture containing materials useful for the treatment, prevention and/or diagnosis of the disorders described above is provided. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper that is pierceable by a hypodermic injection needle). At least one active agent in the composition is a bispecific antibody comprising a first antigen-binding site that specifically binds to CD3 and a second antigen-binding site that specifically binds to a tumor antigen. 
     The label or package insert indicates that the composition is used for treating the condition of choice. Moreover, the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises the bispecific antibody of the invention; and (b) a second container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherwise therapeutic agent. The article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the compositions can be used to treat a particular condition. 
     Alternatively, or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer&#39;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. 
     Methods of Treatment 
     In some embodiments, the isolated polypeptide comprising the cleavable linkers described herein are used in a method of treating cancer. In some embodiments, the cancer has cells that express EGFR. In some embodiments, the polypeptides or polypeptide complexes described herein are used in a method of treating colorectal cancer (CRC), squamous cell carcinoma of the head and Neck (SCCHN), non-small cell lung cancer (NSCLC), prostate cancer, breast cancer, colon/rectum cancer, head and neck cancer, esophagogastric cancer, liver cancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, kidney cancer, or pancreatic cancer. In some embodiments, the polypeptides or polypeptide complexes described herein are used in a method of treating subjects who are resistant to EGFR inhibitor treatment. In some embodiments, the polypeptides or polypeptide complexes described herein are used in a method of treating subjects who harbor KRAS mutations. In some embodiments, the polypeptides or polypeptide complexes described herein are used in a method of treating subjects who are resistant to EGFR inhibitor treatment and harbor KRAS mutations. 
     While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby. 
     EMBODIMENTS 
     Embodiment 1 comprises an isolated polypeptide comprising a cleavable linker according to the amino acid sequence of SEQ ID NO: 1 (LSGRSDAG). 
     Embodiment 2 comprises an isolated polypeptide of embodiment 1, wherein the cleavable linker comprises the amino acid sequence of SEQ ID NO: 3 (ISSGLLSGRSDAG). 
     Embodiment 3 comprises an isolated polypeptide of any one of embodiments 1-2, wherein the cleavable linker comprises the amino acid sequence of SEQ ID NO: 26 (AGLLAPPGGLSGRSDAG). 
     Embodiment 4 comprises an isolated polypeptide of any one of embodiments 1-3, wherein the cleavable linker comprises the amino acid sequence of SEQ ID NO: 4 (AAGLLAPPGGLSGRSDAG). 
     Embodiment 5 comprises an isolated polypeptide of any one of embodiments 1-4, wherein the cleavable linker comprises the amino acid sequence of SEQ ID NO: 5 (SPLGLSGRSDAG). 
     Embodiment 6 comprises an isolated polypeptide of any one of embodiments 1-5, wherein the cleavable linker comprises the amino acid sequence of SEQ ID NO: 6 (LSGRSDAGSPLGLAG). 
     Embodiment 7 comprises an isolated polypeptide of any one of embodiments 1-6, wherein the cleavable linker is cleavable by a protease. 
     Embodiment 8 comprises an isolated polypeptide of embodiment 7, wherein the protease comprises a tumor specific protease. 
     Embodiment 9 comprises an isolated polypeptide of any one of embodiments 7-8, wherein the protease comprises a matrix metalloprotease (MMP) or a serine protease. 
     Embodiment 10 comprises an isolated polypeptide of embodiment 9, wherein the matrix metalloprotease comprises MMP2, MMP7, MMP9, MMP13, or MMP14. 
     Embodiment 11 comprises an isolated polypeptide of embodiment 9, wherein the serine protease comprises matriptase, urokinase, or hepsin. 
     Embodiment 12 comprises an isolated polypeptide of any one of embodiments 1-11, wherein the isolated polypeptide further comprises an antigen binding domain that binds to a target antigen. 
     Embodiment 13 comprises an isolated polypeptide of embodiment 12, wherein the antigen binding domain is C-terminal to the cleavable linker. 
     Embodiment 14 comprises an isolated polypeptide of any one of embodiments 1-11, wherein the isolated polypeptide further comprises a cytokine or cytokine fragment that binds to a cytokine receptor. 
     Embodiment 15 comprises an isolated polypeptide of embodiment 14, wherein the cytokine or cytokine fragment is C-terminal to the cleavable linker. 
     Embodiment 16 comprises an isolated polypeptide of any one of embodiments 1-15, wherein the cleavable linker connects a peptide to an antigen binding domain that binds to a target antigen or to a cytokine or cytokine fragment that binds to a cytokine receptor in a configuration according to Formula I: A 1 -L 1 -P 1  wherein A 1  comprises the antigen binding domain that binds to the target antigen or the cytokine or cytokine fragment that binds to the cytokine receptor; L 1  comprises the cleavable linker; P 1  comprises a peptide that impairs binding of the antigen binding domain to the target antigen or impairs binding of the cytokine to the cytokine receptor. 
     Embodiment 17 comprises an isolated polypeptide of embodiment 16, wherein P 1  is connected N-terminal to the cleavable linker and A 1  is connected C-terminal to the cleavable linker. 
     Embodiment 18 comprises an isolated polypeptide of embodiment 16, wherein P 1  is connected C-terminal to the cleavable linker and A 1  is connected N-terminal to the cleavable linker. 
     Embodiment 19 comprises an isolated polypeptide of any one of embodiments 16-18, wherein P 1  is bound to A 1  through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bonding interactions, or a combination thereof. 
     Embodiment 20 comprises an isolated polypeptide of any one of embodiments 16-19, wherein P 1  has less than 70% sequence homology to the target antigen or the cytokine receptor. 
     Embodiment 21 comprises an isolated polypeptide of any one of embodiments 16-20, wherein P 1  comprises a peptide sequence of at least 10 amino acids in length. 
     Embodiment 22 comprises an isolated polypeptide of any one of embodiments 16-21, wherein P 1  comprises a peptide sequence of at least 10 amino acids in length and no more than 20 amino acids in length. 
     Embodiment 23 comprises an isolated polypeptide of any one of embodiments 16-22, wherein P 1  comprises a peptide sequence of at least 16 amino acids in length. 
     Embodiment 24 comprises an isolated polypeptide of any one of embodiments 16-23, wherein P 1  comprises a peptide sequence of no more than 40 amino acids in length. 
     Embodiment 25 comprises an isolated polypeptide of any one of embodiments 16-24, wherein P 1  comprises a cyclic peptide or a linear peptide. 
     Embodiment 26 comprises an isolated polypeptide of any one of embodiments 16-25, wherein P 1  comprises a cyclic peptide. 
     Embodiment 27 comprises an isolated polypeptide of any one of embodiments 16-26, wherein P 1  is further linked to a half-life extending moiety. 
     Embodiment 28 comprises an isolated polypeptide of embodiment 27, wherein the half-life extending moiety is a single-domain antibody. 
     Embodiment 29 comprises an isolated polypeptide of embodiment 28, wherein the single domain antibody comprises 10G. 
     Embodiment 30 comprises an isolated polypeptide of any one of embodiments 16-29, wherein A 1  comprises an antibody, 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, a Fab, a Fab′, a Fab light chain polypeptide, or a Fab heavy chain polypeptide. 
     Embodiment 31 comprises an isolated polypeptide of any one of embodiments 16-30, wherein the target antigen comprises a tumor antigen. 
     Embodiment 32 comprises an isolated polypeptide of any one of embodiments 30-31, wherein A 1  comprises the Fab light chain polypeptide or the Fab heavy chain polypeptide. 
     Embodiment 33 comprises an isolated polypeptide of any one of embodiments 16-32, wherein A 1  comprises an epidermal growth factor receptor (EGFR) binding domain. 
     Embodiment 34 comprises an isolated polypeptide of any one of embodiments 16-30, wherein the target antigen comprises an effector cell antigen. 
     Embodiment 35 comprises an isolated polypeptide of embodiment 34, wherein A 1  comprises the scFv. 
     Embodiment 36 comprises an isolated polypeptide of embodiment 35, wherein the scFv comprises an anti-CD3e single chain variable fragment. 
     Embodiment 37 comprises an isolated polypeptide of any one of embodiments 16-29, wherein A 1  comprises the cytokine. 
     Embodiment 38 comprises an isolated polypeptide of embodiment 37, wherein the cytokine or cytokine fragment is a wild-type cytokine. 
     Embodiment 39 comprises an isolated polypeptide of embodiment 37, wherein the cytokine or cytokine fragment is a mutein of the cytokine. 
     Embodiment 40 comprises an isolated polypeptide of any one of embodiments 37-39, wherein the cytokine receptor is an interferon receptor or an interleukin receptor. 
     Embodiment 41 comprises an isolated polypeptide of any one of embodiments 37-40, wherein the cytokine receptor comprises an interferon receptor, GM-CSF receptor, IL-2 receptor, IL-4 receptor, IL-6 receptor, IL-7 receptor, IL-10 receptor, IL-12 receptor, IL-15 receptor, IL-21 receptor, or TGF-β receptor. 
     Embodiment 42 comprises an isolated polypeptide of any one of embodiments 37-41, wherein the cytokine or cytokine fragment comprises an interferon, GM-CSF, IL-2, IL-7, IL-12, IL-15, or IL-21. 
     Embodiment 43 comprises an isolated polypeptide of any one of embodiments 37-42, wherein the cytokine or cytokine fragment comprises an IL-2, IL-12, IL-6, IL-4, IL-10, or TGFβ. 
     Embodiment 44 comprises an isolated polypeptide of any one of embodiments 1-43, wherein the isolated polypeptide is complexed with a second isolated polypeptide comprising a second antigen binding domain or a second cytokine or second cytokine fragment. 
     Embodiment 45 comprises an isolated polypeptide of embodiment 44, wherein the second isolated polypeptide is in a configuration according to Formula II: A 2 -L 2 -P 2  wherein A 2  comprises the second antigen binding domain or the second cytokine; L 2  comprises a second cleavable linker; P 2  comprises a second peptide that impairs binding of the second antigen binding domain to a second target antigen or impairs binding of the second cytokine or second cytokine fragment to a second cytokine receptor. 
     Embodiment 46 comprises an isolated polypeptide of embodiment 45, wherein the second cleavable linker comprises the amino acid sequence of SEQ ID NO: 1 (LSGRSDAG). 
     Embodiment 47 comprises an isolated polypeptide of any one of embodiments 45-46, wherein the second cleavable linker comprises the amino acid sequence of SEQ ID NO: 3 (ISSGLLSGRSDAG). 
     Embodiment 48 comprises an isolated polypeptide of any one of embodiments 45-47, wherein the second cleavable linker comprises the amino acid sequence of SEQ ID NO: 26 (AGLLAPPGGLSGRSDAG). 
     Embodiment 49 comprises an isolated polypeptide of any one of embodiments 45-48, wherein the second cleavable linker comprises the amino acid sequence of SEQ ID NO: 4 (AAGLLAPPGGLSGRSDAG). 
     Embodiment 50 comprises an isolated polypeptide of any one of embodiments 45-49, wherein the second cleavable linker comprises the amino acid sequence of SEQ ID NO: 5 (SPLGLSGRSDAG). 
     Embodiment 51 comprises an isolated polypeptide of any one of embodiments 45-50, wherein the second cleavable linker comprises the amino acid sequence of SEQ ID NO: 6 (LSGRSDAGSPLGLAG). 
     Embodiment 52 comprises an isolated polypeptide of any one of embodiments 45-51, wherein P 2  is connected N-terminal to the second cleavable linker and A 2  is connected C-terminal to the second cleavable linker. 
     Embodiment 53 comprises an isolated polypeptide of any one of embodiments 45-51, wherein P 2  is connected C-terminal to the second cleavable linker and A 2  is connected N-terminal to the second cleavable linker. 
     Embodiment 54 comprises an isolated polypeptide of any one of embodiments 45-53, wherein P 2  is bound to A 2  through ionic interactions, electrostatic interactions, hydrophobic interactions, P 1 -stacking interactions, and H-bonding interactions, or a combination thereof. 
     Embodiment 55 comprises an isolated polypeptide of any one of embodiments 45-54, wherein P 2  has less than 70% sequence homology to the second target antigen or the second cytokine receptor. 
     Embodiment 56 comprises an isolated polypeptide of any one of embodiments 45-55, wherein P 2  comprises a peptide sequence of at least 10 amino acids in length. 
     Embodiment 57 comprises an isolated polypeptide of any one of embodiments 45-56, wherein P 2  comprises a peptide sequence of at least 10 amino acids in length and no more than 20 amino acids in length. 
     Embodiment 58 comprises an isolated polypeptide of any one of embodiments 45-57, wherein P 2  comprises a peptide sequence of at least 16 amino acids in length. 
     Embodiment 59 comprises an isolated polypeptide of any one of embodiments 45-56, wherein P 2  comprises a peptide sequence of no more than 40 amino acids in length. 
     Embodiment 60 comprises an isolated polypeptide of any one of embodiments 45-59, wherein P 2  comprises a cyclic peptide or a linear peptide. 
     Embodiment 61 comprises an isolated polypeptide of any one of embodiments 45-60, wherein P 2  comprises a cyclic peptide. 
     Embodiment 62 comprises an isolated polypeptide of any one of embodiments 45-61, wherein A 2  comprises an antibody, 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, a Fab, a Fab′, a Fab light chain polypeptide, or a Fab heavy chain polypeptide. 
     Embodiment 63 comprises an isolated polypeptide of any one of embodiments 45-62, wherein the second target antigen comprises a tumor antigen. 
     Embodiment 64 comprises an isolated polypeptide of embodiment 62, wherein A 2  comprises the Fab light chain polypeptide or the Fab heavy chain polypeptide. 
     Embodiment 65 comprises an isolated polypeptide of any one of embodiments 45-64, wherein A 2  comprises an epidermal growth factor receptor (EGFR) binding domain. 
     Embodiment 66 comprises an isolated polypeptide of any one of embodiments 45-62, wherein the second target antigen comprises an effector cell antigen. 
     Embodiment 67 comprises an isolated polypeptide of embodiment 62, wherein A 2  comprises the scFv. 
     Embodiment 68 comprises an isolated polypeptide of any one of embodiments 66-67, wherein the scFv comprises an anti-CD3e single chain variable fragment. 
     Embodiment 69 comprises an isolated polypeptide of any one of embodiments 45-61, wherein A 2  comprises the second cytokine. 
     Embodiment 70 comprises an isolated polypeptide of embodiment 69, wherein the second cytokine or second cytokine fragment is a wild-type cytokine. 
     Embodiment 71 comprises an isolated polypeptide of embodiment 69, wherein the second cytokine or second cytokine fragment is a mutein of the cytokine. 
     Embodiment 72 comprises an isolated polypeptide of any one of embodiments 69-71, wherein the second cytokine receptor is an interferon receptor or an interleukin receptor. 
     Embodiment 73 comprises an isolated polypeptide of any one of embodiments 69-72, wherein the second cytokine receptor comprises an interferon receptor, GM-CSF receptor, IL-2 receptor, IL-4 receptor, IL-6 receptor, IL-7 receptor, IL-10 receptor, IL-12 receptor, IL-15 receptor, IL-21 receptor, or TGF-β receptor. 
     Embodiment 74 comprises an isolated polypeptide of any one of embodiments 69-73, wherein the second cytokine or second cytokine fragment comprises an interferon, GM-CSF, IL-2, IL-7, IL-12, IL-15, or IL-21. 
     Embodiment 75 comprises an isolated polypeptide of any one of embodiments 69-74, wherein the second cytokine or second cytokine fragment comprises an IL-2, IL-12, IL-6, IL-4, IL-10, or TGFβ. 
     Embodiment 76 comprises a pharmaceutical composition comprising: the isolated polypeptide comprising a cleavable linker of any one of the above embodiments; and a pharmaceutically acceptable excipient. 
     Embodiment 77 comprises an isolated recombinant nucleic acid molecule encoding the isolated polypeptide comprising a cleavable linker of any one of the above embodiments. 
     Embodiment 78 comprises a vector comprising the isolated recombinant nucleic acid molecule according to Embodiment 77. 
     Embodiment 79 comprises a method of producing an isolated polypeptide comprising a cleavable linker according to any of the above embodiments comprising culturing a cell under conditions that lead to expression of the polypeptide, wherein the cell comprises the vector of embodiment 78. 
     Embodiment 80 comprises a method of manufacturing an isolated polypeptide comprising a cleavable linker, the method comprising: (a) culturing a cell comprising the recombinant nucleic acid molecule of embodiment 77 under conditions that lead to expression of the polypeptide, and (b) isolating the polypeptide. 
     Examples 
     Example 1. Proteolysis Rates and Serum Stability 
     The polypeptide complexes were evaluated for tumor and serum protease activity. 
     Briefly, polypeptide complexes PC-1, PC-2, PC-3, PC-4, and PC-5 were generated comprising peptide masks genetically fused to the polypeptide complexes using cleavable linkers recognized by various tumor proteases. The polypeptide complexes were exposed to various tumor proteases. Cleavage rate was determined when the polypeptide complexes were exposed to MMP2, MMP7, MMP9, MMP13, MMP14, uPa, MTSP1, and Hepsin. The data for apparent cleavage rate and relative serum stability are seen in Tables 2-4. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Apparent Cleavage Rate Comparisons 
               
            
           
           
               
               
               
               
               
               
            
               
                 Tumor 
                   
                   
                   
                   
                   
               
               
                 Proteases 
                  PC-1 
                 PC-2 
                 PC-3 
                 PC-4 
                 PC-5 
               
               
                   
               
               
                 MMP2 
                  45 
                  45 
                  5 
                  5 
                  5 
               
               
                   
                  10 &lt; r &lt; 10 
                  10 &lt; r &lt; 10 
                 &gt;10 
                 &gt;10 
                 &gt;10 
               
               
                 MMP7 
                  &lt;2.5 × 10 
                  &lt;2.5 × 10 3   
                  45 
                  5 
                  5 
               
               
                   
                   
                   
                  10 &lt; r &lt; 10 
                 &gt;10 
                 &gt;10 
               
               
                 MMP9 
                  &lt;2.5 ×10 
                  &lt;2.5 × 10 3   
                  45 
                  5 
                  5 
               
               
                   
                   
                   
                  10 &lt;r&lt;10 
                 &gt;10 
                 &gt;10 
               
               
                 MMP13 
                  45 
                  45 
                  5 
                  5 
                  5 
               
               
                   
                  10 &lt; r &lt; 10 
                  10 &lt; r &lt; 10 
                 &gt;10 
                 &gt;10 
                 &gt;10 
               
               
                 MMP14 
                  45 
                  45 
                  45 
                  45 
                  45 
               
               
                   
                  10 &lt; r &lt; 10 
                  10 &lt; r &lt; 10 
                  10 &lt; r &lt; 10 
                  10 &lt; r &lt; 10 
                  10 &lt; r &lt; 10 
               
               
                 uPa 
                  2.5 × 10 3  &lt; r &lt; 10 4   
                  2.5 × 10 3  &lt; r &lt; 10 
                  2.5 × 10 3  &lt; r &lt; 10 4   
                  2.5 × 10 3  &lt; r &lt; 10 4   
                  2.5 × 10 3  &lt; r &lt; 10 4   
               
               
                 MTSP1 
                  2.5 × 10 3  &lt; r &lt; 10 4   
                  2.5 × 10 3  &lt; r &lt; 10 4   
                  45 
                  45 
                  45 
               
               
                   
                   
                   
                  10 &lt; r &lt; 10 
                  10 &lt; r &lt; 10 
                  10 &lt; r &lt; 10 
               
               
                 Hepsin 
                  5 
                  5 
                  45 
                  45 
                  45 
               
               
                   
                 &gt;10 
                 &gt;10 
                  10 &lt; r &lt; 10 
                  10 &lt; r &lt;10 
                  10 &lt; r &lt; 10 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Apparent Cleavage Rate Constants 
               
               
                 Apparent Cleavage Rate Constants (M −1  s −1 ) (EGFR side) 
               
            
           
           
               
               
               
               
               
               
            
               
                 Tumor  
                   
                   
                   
                   
                   
               
               
                 Protease 
                 PC-1 
                 PC-2 
                 PC-3 
                 PC-4 
                 PC-5 
               
               
                   
               
               
                 MMP2 
                 1.99E+04 
                 2.47E+04 
                 3.63E+05 
                 3.29E+05 
                 3.48E+05 
               
               
                 MMP7 
                 1.50E+02 
                 1.57E+02 
                 1.34E+04 
                 1.76E+05 
                 9.15E+04 
               
               
                 MMP9 
                 2.35E+03 
                 5.00E+01 
                 4.10E+04 
                 2.36E+05 
                 2.82E+05 
               
               
                 MMP13 
                 1.89E+04 
                 1.20E+04 
                 1.56E+05 
                 2.75E+05 
                 3.80E+05 
               
               
                 MMP14 
                 1.01E+04 
                 1.07E+04 
                 2.78E+04 
                 1.42E+04 
                 1.95E+04 
               
               
                 uPa 
                 8.39E+03 
                 4.32E+03 
                 3.41E+03 
                 4.03E+03 
                 5.88E+03 
               
               
                 MTSP1 
                 9.96E+03 
                 8.63E+03 
                 3.83E+04 
                 3.92E+04 
                 5.65E+04 
               
               
                 Hepsin 
                 2.53E+05 
                 2.53E+05 
                 7.00E+04 
                 4.26E+04 
                 8.28E+04 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Relative Serum Stability 
               
               
                 Relative serum stability (% cleavage per day)  
               
               
                 (EGFR side/CD3e side) 
               
            
           
           
               
               
               
               
               
               
            
               
                 Serum 
                 PC-1 
                 PC-2 
                 PC-3 
                 PC-4 
                 PC-5 
               
               
                   
               
               
                 Human 
                 0.51%/ 
                 0.52%/  
                 0.44%/ 
                 0.44%/ 
                 0.72%/ 
               
               
                   
                 0.56% 
                 0.65% 
                 0.63% 
                 0.82% 
                 0.71% 
               
               
                   
               
            
           
         
       
     
     The data shows that serum proteolytic activity is greater than blood. The data also shows the cleavable linker sequences have increased rates of proteolysis while retaining stability in human serum. 
     Example 2. Confirmation of Comparable Masking with Cleavable Linkers 
     The polypeptide complexes were evaluated for EGFR and CD36 binding. 
     Briefly, the binding of polypeptide complexes PC-1, PC-2, PC-3, PC-4, and PC-5 comprising EGFR masking was determined. As seen in  FIG.  1 A , EGFR masking blocks binding for the various polypeptide complexes. Following cleavage by the tumor protease MTSP1, the polypeptide complexes are able to bind ( FIG.  1 B ). 
     Details of the EGFR binding shifts are seen in Tables 5-8. 
     
       
         
           
               
               
               
               
             
               
                   
                 TABLE 5 
               
               
                   
                   
               
               
                   
                 Step 
                 Time 
                 pH 
               
               
                   
                   
               
             
            
               
                   
                 Baseline: Octet buffer 
                  60 sec 
                 pH 7.4 
               
               
                   
                 Load: 30 nM EGFR-biotin  
                 300 sec 
                 pH 7.4 
               
               
                   
                 (2.169 ug/mL) 
                   
                   
               
               
                   
                 Biocytin quench (100 uM) 
                 300 sec 
                 pH 7.4 
               
               
                   
                 Baseline: Octet buffer 
                  90 sec 
                 pH 7.4 
               
               
                   
                 Association: 
                 300 sec 
                 pH 7.4 
               
               
                   
                 25 nM PC-2 
                   
                   
               
               
                   
                 25 nM PC-3 
                   
                   
               
               
                   
                 25 nM PC-4 
                   
                   
               
               
                   
                 25 nM PC-5 
                   
                   
               
               
                   
                 25 nM PC-2 + MTSP1 
                   
                   
               
               
                   
                 25 nMPC-3 + MTSP1 
                   
                   
               
               
                   
                 25 nM PC-4 + MTSP1 
                   
                   
               
               
                   
                 25 nM PC-5 + MTSP1 
                   
                   
               
               
                   
                 Dissociation: Octet buffer 
                 600 sec 
                 pH 7.4 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
               
               
               
               
               
             
               
                 TABLE 6 
               
               
                   
               
               
                   
                 Loading 
                 Conc. 
                   
                 KD 
                   
                   
                   
                   
                   
               
               
                 Sample ID 
                 Sample ID 
                 (nM) 
                 KD (M) 
                 Error 
                 kon(1/Ms) 
                 kdis(1/s) 
                 Full R{circumflex over ( )}2 
                 Full X{circumflex over ( )}2 
                 Response 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                 PC-2 
                 EGFR 
                 25 
                 No significant binding 
               
               
                 PC-3 
                 EGFR 
                 25 
                 No significant binding 
               
               
                 PC-4 
                 EGFR 
                 25 
                 No significant binding 
               
               
                 PC-5 
                 EGFR 
                 25 
                 No significant binding 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 PC-2 + MTSP1 
                 EGFR 
                 25 
                 1.34E−09 
                 2.87E−11 
                 3.48E+05 
                 4.66E−04 
                 0.9735 
                 2.3843 
                 1.5573 
               
               
                 PC-3 + MTSP1 
                 EGFR 
                 25 
                 9.46E−10 
                 1.99E−11 
                 4.72E+05 
                 4.46E−04 
                 0.9684 
                 2.3105 
                 1.5016 
               
               
                 PC-4 + MTSP1 
                 EGFR 
                 25 
                 1.19E−09 
                 2.56E−11 
                 3.82E+05 
                 4.54E−04 
                 0.9715 
                 2.45   
                 1.553  
               
               
                 PC-5 + MTSP1 
                 EGFR 
                 25 
                 1.45E−09 
                 2.98E−11 
                 3.50E+05 
                 5.07E−04 
                 0.9728 
                 1.9973 
                 1.4156 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
             
               
                   
                 TABLE 7 
               
               
                   
                   
               
               
                   
                 Step 
                 Time 
                 pH 
               
               
                   
                   
               
             
            
               
                   
                 Baseline: Octet buffer 
                  60 sec 
                 pH 7.4 
               
               
                   
                 Load: 30 nM EGFR-biotin 
                 300 sec 
                 pH 7.4 
               
               
                   
                 (2.169 ug/mL) 
                   
                   
               
               
                   
                 Biocytin quench (100 uM) 
                 300 sec 
                 pH 7.4 
               
               
                   
                 Baseline: 5% human serum 
                 300 sec 
                 pH 7.4 
               
               
                   
                 Association: 
                 300 sec 
                 pH 7.4 
               
               
                   
                 30 nM PC-1, 
                   
                   
               
               
                   
                 30 nM PC-1 + MTSP1 
                   
                   
               
               
                   
                 30 nM PC-6, 
                   
                   
               
               
                   
                 buffer 
                   
                   
               
               
                   
                 Dissociation: 5% human  
                 600 sec 
                 pH 7.4 
               
               
                   
                 serum 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
               
               
               
               
               
             
               
                 TABLE 8 
               
               
                   
               
               
                   
                 Loading 
                 Conc. 
                   
                 KD 
                   
                   
                   
                   
                   
               
               
                 Sample ID 
                 Sample ID 
                 (nM) 
                 KD (M) 
                 Error 
                 kon(1/Ms) 
                 kdis(1/s) 
                 Full R{circumflex over ( )}2 
                 Full X{circumflex over ( )}2 
                 Response 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                 PC-1 
                 EGFR- 
                 50 
                 No significant binding 
               
               
                   
                 biotin 
                   
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 PC-1 + MTSP1 
                 EGFR- 
                 50 
                 1.91E−09 
                 3.85E−11 
                 2.60E+05 
                 4.98E−04 
                 0.965  
                 1.7318 
                 1.2733 
               
               
                   
                 biotin 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 PC-6 
                 EGFR- 
                 50 
                 1.77E−09 
                 3.19E−11 
                 4.93E+05 
                 8.72E−04 
                 0.9535 
                 1.4956 
                 1.0938 
               
               
                   
                 biotin 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 buffer 
                 EGFR- 
                 50 
                 &lt;1.0E−12 
                 6.00E−09 
                 1.29E+04 
                 &lt;1.0E−07 
                 0.5559 
                 0.1511 
                 0.0151 
               
               
                   
                 biotin 
               
               
                   
               
            
           
         
       
     
     The polypeptide complexes were also evaluated for CD3E binding. Briefly, the binding of the polypeptide complexes PC-1, PC-2, PC-3, PC-4, and PC-5 comprising CD3ε masking was determined. As seen in  FIG.  2 A , the masks block binding for the various polypeptide complexes. Following cleavage by the tumor protease MTSP1, the polypeptide complexes are able to bind ( FIG.  2 B ). 
     Details of the CD3ε binding shifts are seen in Tables 9-12. 
     
       
         
           
               
               
               
               
             
               
                   
                 TABLE 9 
               
               
                   
                   
               
               
                   
                 Step 
                 Time 
                 pH 
               
               
                   
                   
               
             
            
               
                   
                 Baseline: Octet buffer 
                  60 sec 
                 pH 7.4 
               
               
                   
                 Load: 30 nM CD3e-biotin  
                 300 sec 
                 pH 7.4 
               
               
                   
                 (0.465 ug/mL) 
                   
                   
               
               
                   
                 Biocytin quench (100 uM) 
                 300 sec 
                 pH 7.4 
               
               
                   
                 Baseline: Octet buffer 
                  90 sec 
                 pH 7.4 
               
               
                   
                 Association: 
                 300 sec 
                 pH 7.4 
               
               
                   
                 25 nM PC-2 
                   
                   
               
               
                   
                 25 nM PC-3 
                   
                   
               
               
                   
                 25 nM PC-4 
                   
                   
               
               
                   
                 25 nM PC-5 
                   
                   
               
               
                   
                 25 nM PC-2 + MTSP1 
                   
                   
               
               
                   
                 25 nM PC-3 + MTSP1 
                   
                   
               
               
                   
                 25 nM PC-4 + MTSP1 
                   
                   
               
               
                   
                 25 nM PC-5 + MTSP1 
                   
                   
               
               
                   
                 Dissociation: Octet buffer 
                 600 sec 
                 pH 7.4 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
               
               
               
               
               
             
               
                 TABLE 10 
               
               
                   
               
               
                   
                 Loading 
                 Conc. 
                   
                   
                   
                   
                   
                   
                   
               
               
                 Sample ID 
                 Sample ID 
                 (nM) 
                 KD (M) 
                 KD Error 
                 kon(1/Ms) 
                 kdis(1/s) 
                 Full R{circumflex over ( )}2 
                 Full X{circumflex over ( )}2 
                 Response 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                 PC-2 
                 CD3e 
                 25 
                 No significant binding 
               
               
                 PC-3 
                 CD3e 
                 25 
                 No significant binding 
               
               
                 PC-4 
                 CD3e 
                 25 
                 No significant binding 
               
               
                 PC-5 
                 CD3e 
                 25 
                 No significant binding 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 PC-2 + MTSP1 
                 CD3e 
                 25 
                 1.41E−08 
                 3.66E−10 
                 2.37E+05 
                 3.33E−03 
                 0.9829 
                 1.2588 
                 1.0342 
               
               
                 PC-3 + MTSP1 
                 CD3e 
                 25 
                 1.00E−08 
                 2.60E−10 
                 3.37E+05 
                 3.37E−03 
                 0.976  
                 2.078  
                 1.1036 
               
               
                 PC-4 + MTSP1 
                 CD3e 
                 25 
                 1.19E−08 
                 3.14E−10 
                 2.78E+05 
                 3.30E−03 
                 0.9786 
                 1.5272 
                 1.0227 
               
               
                 PC-5 + MTSP1 
                 CD3e 
                 25 
                 1.32E−08 
                 3.33E−10 
                 2.91E+05 
                 3.84E−03 
                 0.9832 
                 1.452  
                 1.0526 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
             
               
                   
                 TABLE 11 
               
               
                   
                   
               
               
                   
                 Step 
                 Time 
                 pH 
               
               
                   
                   
               
             
            
               
                   
                 Baseline: Octet buffer 
                  60 sec 
                 pH 7.4 
               
               
                   
                 Load: 30nM CD3e-biotin 
                 300 sec 
                 pH 7.4 
               
               
                   
                 (0.46ug/mL) 
                   
                   
               
               
                   
                 Biocytin quench (IOOuM) 
                 300 sec 
                 pH 7.4 
               
               
                   
                 Baseline: 5% human serum 
                 300 sec 
                 pH 7.4 
               
               
                   
                 Association: 
                 300 sec 
                 pH 7.4 
               
               
                   
                 30nMPC-l, 
                   
                   
               
               
                   
                 30nM PC-1 + MTSP1 
                   
                   
               
               
                   
                 30nM PC-6, 
                   
                   
               
               
                   
                 buffer 
                   
                   
               
               
                   
                 Dissociation: 5% human serum 
                 600 sec 
                 pH 7.4 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
               
               
               
               
               
             
               
                 TABLE 12 
               
               
                   
               
               
                   
                 Loading 
                 Conc. 
                   
                 KD 
                   
                   
                   
                   
                   
               
               
                 Sample ID 
                 Sample ID 
                 (nM) 
                 KD (M) 
                 Error 
                 kon(1/Ms) 
                 kdis(1/s) 
                 Full R{circumflex over ( )}2 
                 Full X{circumflex over ( )}2 
                 Response 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                 PC-1 
                 CD3e- 
                 50 
                 No significant binding 
               
               
                   
                 biotin 
                   
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 PC-1 + MTSP1 
                 CD3e- 
                 50 
                 3.15E−08 
                 1.01E−09 
                 1.25E+05 
                 3.95E−03 
                 0.9774 
                 2.6058 
                 1.2556 
               
               
                   
                 biotin 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 PC-6 
                 CD3e- 
                 50 
                 2.26E−08 
                 6.81E−10 
                 1.33E+05 
                 3.02E−03 
                 0.9702 
                 4.4245 
                 1.5652 
               
               
                   
                 biotin 
                   
                   
                   
                   
                   
                   
                   
                   
               
            
           
           
               
               
               
               
            
               
                 buffer 
                 CD3e- 
                 50 
                 No significant binding 
               
               
                   
                 biotin 
               
               
                   
               
            
           
         
       
     
     The polypeptide complexes binding was evaluated using enzyme linked immunosorbent assays (ELISAs). Biotinylated peptides were captured on neutravidin coated plates. A secondary antibody was used to detect bound polypeptide complex. Data for PC-2, PC-3, PC-4, PC-5, PC-1, and PC-6 comprising the mask and following cleavage of the mask is seen in  FIGS.  3 A- 3 B  and EC50 binding data is summarized in Tables 13-14. 
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 13 
               
               
                   
                   
               
               
                   
                 EC50 nM 
                 Masked 
                 Cleaved 
                 Shift 
               
               
                   
                   
               
             
            
               
                   
                 PC-2 
                 21.29 
                 0.13 
                 169.5 
               
               
                   
                 PC-3 
                 23.47 
                 0.12 
                 198.2 
               
               
                   
                 PC-4 
                 20.59 
                 0.15 
                 139.3 
               
               
                   
                 PC-5 
                 18.28 
                 0.13 
                 143.5 
               
               
                   
                 PC-1 
                 44.79 
                 — 
                 282.8 
               
               
                   
                 PC-6 
                 — 
                 0.16 
                 — 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 14 
               
               
                   
                   
               
               
                   
                 EC50 nM 
                 Masked 
                 Cleaved 
                 Shift 
               
               
                   
                   
               
             
            
               
                   
                 PC-2 
                 2.082 
                 0.101 
                 20.6x 
               
               
                   
                 PC-3 
                 1.792 
                 0.1007 
                 17.8x 
               
               
                   
                 PC-4 
                 2.439 
                 0.119 
                 20.5x 
               
               
                   
                 PC-5 
                 2.665 
                 0.121 
                 22.0x 
               
               
                   
                 PC-1 
                 4.598 
                 0.1383 
                 33.2x 
               
               
                   
                 PC-6 
                 — 
                 0.069 
                 — 
               
               
                   
                   
               
            
           
         
       
     
     Example 3. Confirmation of Comparable T-Cell Shifts with Cleavable Linkers 
     The polypeptide complexes were next evaluated in functional in vitro tumor cell killing and related T cell activation studies. 
     Briefly, HCT116 cells were seeded onto 96 well tissue culture treated flat bottom plates and allowed to adhere overnight. The following day, culture medium and nonadherent cells were removed and replaced with fresh medium containing titrated the polypeptide complexes at concentrations indicated. The data for PC-2, PC-3, PC-4, PC-5, PC-1, and PC-6 is seen in  FIGS.  4 A- 4 E  and Tables 15-16. 
     
       
         
           
               
             
               
                 TABLE 15 
               
             
            
               
                   
               
               
                 Tumor cell viability, IC50 (pM) 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 Cytotox 
                   
                   
                   
                   
                   
                   
                 PC-2 + 
                 PC-3 + 
                 PC-4 + 
                 PC-5 + 
               
               
                 time 
                 PC-6 
                 PC-1 
                 PC-2 
                 PC-3 
                 PC-4 
                 PC-5 
                 MTSP1 
                 MTSP1 
                 MTSP1 
                 MTSP1 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                  24 hr 
                 1.13 
                 555.6 
                 326.6 
                 578.3 
                 539.9 
                 869.4 
                 0.2377 
                 0.2811 
                 0.3557 
                 0.2294 
               
               
                  48 hr 
                 0.4675 
                 460.9 
                 301.8 
                 389.1 
                 419.7 
                 499.6 
                 0.1998 
                 0.1843 
                 0.1596 
                 0.2197 
               
               
                  72 hr 
                 0.5507 
                 763.5 
                 389.3 
                 444 
                 522.6 
                 588.6 
                 0.2632 
                 0.2402 
                 0.218 
                 0.3065 
               
               
                  96 hr 
                 0.7548 
                 1023 
                 475.3 
                 511.8 
                 659.1 
                 712.9 
                 0.3296 
                 0.3099 
                 0.2809 
                 0.394 
               
               
                 120 hr 
                   
                   
                 577.5 
                 589.6 
                 773.2 
                 824.2 
                 0.3901 
                 0.3679 
                 0.3351 
                 0.4737 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 16 
               
             
            
               
                   
               
               
                 Tumor cell viability, Functional Shift 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 Cytotox 
                   
                   
                   
                   
                   
                   
                 PC-2 + 
                 PC-3 + 
                 PC-4 + 
                 PC-5 + 
               
               
                 time 
                 PC-6 
                 PC-1 
                 PC-2 
                 PC-3 
                 PC-4 
                 PC-5 
                 MTSP1 
                 MTSP1 
                 MTSP1 
                 MTSP1 
               
               
                   
               
               
                  24 hr 
                 1x 
                  491.7x 
                   289x 
                 511.8x 
                  477.8x 
                  769.4x 
                 0.2x 
                 0.2x 
                 0.3x 
                 0.2x 
               
               
                  48 hr 
                 1x 
                  985.9x 
                 645.6x 
                 832.3x 
                  897.8x 
                 1068.7x 
                 0.4x 
                 0.4x 
                 0.3x 
                 0.5x 
               
               
                  72 hr 
                 1x 
                 1386.4x 
                 706.9x 
                 806.2x 
                    949x 
                 1068.8x 
                 0.5x 
                 0.4x 
                 0.4x 
                 0.6x 
               
               
                  96 hr 
                 1x 
                 1355.3x 
                 629.7x 
                 678.1x 
                  873.2x 
                  944.5x 
                 0.4x 
                 0.4x 
                 0.4x 
                 0.5x 
               
               
                 120 hr 
                   
                   
                 765.1x 
                 781.1x 
                 1024.4x 
                 1091.9x 
                 0.5x 
                 0.5x 
                 0.4x 
                 0.6x 
               
               
                   
               
            
           
         
       
     
     Example 4. In Vivo Cynomolgus Monkey PK Comparison 
     The polypeptide complexes were assessed for pharmacokinetic and safety in cynomolgus monkey. 
     Cynomolgus Monkeys 
     Young naïve male cynomolgus monkeys were paired housed by group and identified by unique body tattoo. All animals were acclimated to housing conditions for 3 days prior to the start of the study. Prior to initiation all animals had undergone a physical examination by the study veterinarian. Only animals that, in the opinion of the study veterinarian, were healthy and otherwise met the criteria were admitted to the study. Food was withheld overnight prior to dosing. Purina 5049 was provided daily in amounts appropriate for the size of the animal. Tap water was provided ad libitum via automatic watering device. 
     Pharmacokinetics 
     Polypeptide complex pharmacokinetics for polypeptide complexes PC-1, PC-2, PC-3, PC-4, and PC-5 were determined in naïve male cynomolgus monkeys weighing 2-3 kg. Briefly, two group housed monkeys were used per dosing group and allowed to acclimate to their surroundings prior to dosing. Animals were sedated with Ketamine HCL 10-20 mg/kg IM prior to dosing and bleeding. Concentrated test articles were diluted in sterile phosphate buffered saline and administered to animals at a quantity relative to the animals&#39; mass in kg. The dose for each test article was administered intravenously at 1 mL/kg dosing volume. For dosing, the left and right limbs were clipped and prepped with alcohol. The saphenous vein was identified, and a standard catheter was placed for IV bolus infusion (in either the left or right limb). The test article dosing solution was attached to the catheter via syringe and the bolus infusion occurred via manual compression of the syringe. 
     For blood collections, animals were sedated using ketamine, the femoral triangle was prepared, and blood was collected from the femoral vein using a 22G 1.5 inch needle, vacutainer sheath, and collection tube. Following venipuncture, manual compression of the vein was maintained until hemostasis was achieved. Blood collections were based on weight of the animals and did not exceed AGI maximum bleeds as set forth by IACUC. Blood was collected in EDTA tubes and processed to plasma. The blood samples were centrifuged cold at 3000×g for 10 min to separate cells from plasma. The plasma supernatant was harvested and stored frozen prior to analysis. 
     The concentration of the polypeptide complexes in cyno plasma samples was determined by ELISA. Briefly, anti-histag capture antibody was coated directly on ELISA plates. Standard dilutions of polypeptide complex in cyno serum were used to generate a calibration curve to which animal PK test samples could be compared. Standards and test samples were added to the plate and incubated cold overnight. Several different dilutions of test samples were used to make sure signals landed within appropriate dynamic range of the standard curve. Plates were washed and incubated with an anti-human HRP detection antibody for a brief time. Plates were washed, developed, and stopped using standard ELISA techniques. Standard curves plotting absorbance at 450 nm versus known polypeptide complex concentration were used to calculate the concentration of unknown test articles in each mouse PK plasma sample. Concentration of polypeptide complexes were plotted versus time and fit to a standard two stage distribution and elimination pharmacokinetic model. The calculated pharmacokinetic and parameters for polypeptide complexes PC-1, PC-7, PC-4, and PC-5 from cynomolgus monkey are shown in  FIGS.  5 A- 5 D  and Tables 17-20. 
     
       
         
           
               
               
               
               
               
               
               
             
               
                 TABLE 17 
               
               
                   
               
               
                   
                 Dose 
                 C max   
                 T 1/2   
                 Vd 
                 CL 
                 AUC (0→d7)    
               
               
                 Construct 
                 (ug/kg) 
                 (nM) 
                 (hr) 
                 (L) 
                 (ml/h/kg) 
                 (nM · min) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 PC-1 
                 100 
                 51 
                 100 
                 0.06 
                 0.14 
                 165,895 
               
               
                 PC-2 
                 100 
                   
                   
                   
                   
                   
               
               
                 PC-3 
                 100 
                   
                   
                   
                   
                   
               
               
                 PC-4 
                 100 
                 48 
                 91 
                 0.07 
                 0.16 
                 221,875 
               
               
                 PC-5 
                 100 
                 61 
                 97 
                 0.05 
                 0.12 
                 244,051 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 18 
               
             
            
               
                   
               
               
                 Cyno PK Parameters 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                 CL 
               
               
                   
                   
                 Dose 
                 C max   
                 T 1/2   
                 Vd 
                 (ml/ 
               
               
                   
                 Construct 
                 (ug/kg) 
                 (nM) 
                 (hr) 
                 (L) 
                 h/kg) 
               
               
                   
                   
               
               
                   
                 PC-1 
                 300 
                 153 
                 109 
                 0.06 
                  0.13 
               
               
                   
                 PC-1 
                 100 
                  51 
                 100 
                 0.06 
                  0.14 
               
               
                   
                 PC-7 
                  10 
                  1.7 
                  1.2 
                 0.24 
                  47 
               
               
                   
                 PC-7 
                  3 
                  0.17 
                  0.3 
                 0.68 
                 491 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
               
               
             
               
                   
                 TABLE 19 
               
               
                   
                   
               
               
                   
                   
                 Dose 
                 C max   
                 T 1/2   
                 Vd 
                 CL 
               
               
                   
                 Construct 
                 (ug/kg) 
                 (nM) 
                 (hr) 
                 (L) 
                 (ml/h/kg) 
               
               
                   
                   
               
             
            
               
                   
                 PC-1 
                 100 
                 51 
                 100 
                 0.06 
                 0.14 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 20 
               
               
                   
                   
               
               
                   
                 Parameter 
                 PC-2 100 ug/kg 
                 PC-3 100 ug/kg 
                 Units 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 CMAX 
                 40.37 
                 53.78 
                 nM 
               
               
                   
                 t1/2 
                 69.44 
                 104.57 
                 hr 
               
               
                   
                 Vd 
                 0.08 
                 0.06 
                 L 
               
               
                   
                 VSS 
                 0.06 
                 0.11 
                 L 
               
               
                   
                 CL 
                 0.26 
                 0.13 
                 mL/hr/kg 
               
               
                   
                 BW 
                 3.00 
                 3.00 
                 kg 
               
               
                   
                 7 day AUC 
                 207834 
                 305952 
                 nM · min 
               
               
                   
                   
               
            
           
         
       
     
     Example 5. In Vivo Cynomolgus Monkey Cytokine Release 
     Cytokine release was measured in cynomolgus monkeys. 
     Cytokines present in plasma post treatment were measured using the non-human primate Th1/Th2 cytometric bead array assay kit from BD Biosciences (Cat no. 557800) according to the manufacturer&#39;s instructions. Data is shown in  FIGS.  6 A- 61   ) and Table 21. 
     
       
         
           
               
               
               
               
               
               
               
               
             
               
                 TABLE 21 
               
               
                   
               
             
            
               
                 Property 
                 PC-7 
                 PC-7 
                 PC-1 
                 PC-2 
                 PC-3 
                 PC-4 
                 PC-5 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                   
                 3 ug/kg 
                 10 ug/kg 
                 100 ug/kg 
               
               
                   
               
            
           
           
               
            
               
                 Plasma Cytokine Levels (Cmax pg/ml) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 IL-6 
                 1,932 
                 5,352 
                 BQL 
                 TBD 
                 TBD 
                 234 
                 493 
               
               
                 TNFα 
                 1,267 
                 2,497 
                 BQL 
                 TBD 
                 TBD 
                 BQL 
                 BQL 
               
               
                 IFNγ 
                 BQL 
                 109 
                 BQL 
                 TBD 
                 TBD 
                 BQL 
                 BQL 
               
               
                 IL-2 
                 87 
                 412 
                 BQL 
                 TBD 
                 TBD 
                 BQL 
                 BQL 
               
               
                   
               
            
           
         
       
     
     Example 6. In Vivo Cynomolgus Monkey Liver ALT/AST 
     ALT/AST levels were measured. As seen in  FIGS.  7 A and  7 B  and Table 22, polypeptide complexes prevented liver toxicity in cynomolgus monkeys. 
     
       
         
           
               
               
               
               
               
               
             
               
                 TABLE 22 
               
             
            
               
                   
               
               
                   
                 PC-1 
                 PC-2 
                 PC-3 
                 PC-4 
                 PC-5 
               
            
           
           
               
               
            
               
                 Property 
                 100 ug/kg 
               
            
           
           
               
            
               
                 Plasma Levels (U/L) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 ALT 
                 18 
                 TBD 
                 TBD 
                 40 
                 41 
               
               
                 AST 
                  9 
                 TBD 
                 TBD 
                 40 
                 51 
               
               
                   
               
            
           
         
       
     
     While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.