Cleavable linker compositions and methods

Abstract

To provide an isolated polypeptide comprising a cleavable linker for minimizing off-target effects of a protein-based therapy in healthy tissue while maintaining activity of the protein-based therapy in disease tissue, and a complex comprising the polypeptide.SOLUTION: Provided herein are cleavable linkers, pharmaceutical compositions thereof, as well as nucleic acids, and methods for making and discovering the same. The cleavable linkers described herein have improved efficacy and safety.SELECTED DRAWING: Figure 3A

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 064,268, filed August 11, 2020, which is incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on August 5, 2021, is titled 52426_720_601_SL.txt, and is 58,325 bytes in size. Summary of the Invention

[0003] In certain embodiments herein, an isolated polypeptide is disclosed comprising a cleavable linker with 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 the peptide to the antigen-binding domain that binds to the target antigen or to the cytokine or cytokine fragment that binds to the cytokine receptor in an arrangement according to Formula I: A1-L1-P1, where A1 comprises the antigen-binding domain that binds to the target antigen or the cytokine or cytokine fragment that binds to the cytokine receptor, L1 comprises the cleavable linker, and P1 comprises a peptide that disrupts binding of the antigen-binding domain to the target antigen or disrupts binding of the cytokine to the cytokine receptor. In some embodiments, P1 is connected to the cleavable linker at the N-terminus and A1 is connected to the cleavable linker at the C-terminus. In some embodiments, P1 is connected to the cleavable linker at the C-terminus and A1 is connected to the cleavable linker at the N-terminus. In some embodiments, P1 is connected to A1 through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bond interactions, or a combination thereof. In some embodiments, P1 has less than 70% sequence homology to the target antigen or cytokine receptor. In some embodiments, P1 comprises a peptide sequence at least 10 amino acids in length. In some embodiments, P1 comprises a peptide sequence at least 10 amino acids in length and no more than 20 amino acids in length. In some embodiments, P1 comprises a peptide sequence at least 16 amino acids in length. In some embodiments, P1 comprises a peptide sequence no more than 40 amino acids in length. In some embodiments, P1 comprises a cyclic peptide or a linear peptide. In some embodiments, P1 comprises a cyclic peptide. In some embodiments, P1 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, A1 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 of a camelid-derived single domain antibody (VHH), 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, A1 comprises a Fab light chain polypeptide or a Fab heavy chain polypeptide. In some embodiments, A1 comprises an epidermal growth factor receptor (EGFR) binding domain. In some embodiments, the target antigen comprises an effector cell antigen. In some embodiments, A1 comprises an scFv. In some embodiments, the scFv comprises an anti-CD3e single chain variable fragment. In some embodiments, A1 comprises a 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 a 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 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 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 an arrangement according to Formula II: A2-L2-P2, where A2 comprises a second antigen-binding domain or a second cytokine, L2 comprises a second cleavable linker, and P2 comprises a second peptide that inhibits binding of the second antigen-binding domain to a second target antigen or inhibits 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, P2 is connected to the second cleavable linker at the N-terminus and A2 is connected to the second cleavable linker at the C-terminus. In some embodiments, P2 is connected to the second cleavable linker at the C-terminus and A2 is connected to the second cleavable linker at the N-terminus. In some embodiments, P2 is bound to A2 through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bonding interactions, or a combination thereof. In some embodiments, P2 has less than 70% sequence homology to the second target antigen or the second cytokine receptor. In some embodiments, P2 comprises a peptide sequence at least 10 amino acids in length. In some embodiments, P2 comprises a peptide sequence at least 10 amino acids in length and no more than 20 amino acids in length. In some embodiments, P2 comprises a peptide sequence at least 16 amino acids in length. In some embodiments, P2 comprises a peptide sequence no more than 40 amino acids in length. In some embodiments, P2 comprises a cyclic peptide or a linear peptide. In some embodiments, P2 comprises a cyclic peptide. In some embodiments, A2 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 of a camelid-derived single domain antibody (VHH), 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, A2 comprises a Fab light chain polypeptide or a Fab heavy chain polypeptide. In some embodiments, A2 comprises an epidermal growth factor receptor (EGFR) binding domain. In some embodiments, the second target antigen comprises an effector cell antigen. In some embodiments, A2 comprises an scFv. In some embodiments, the scFv comprises an anti-CD3e single-chain variable fragment. In some embodiments, A2 comprises a 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 a 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, a GM-CSF receptor, an IL-2 receptor, an IL-4 receptor, an IL-6 receptor, an IL-7 receptor, an IL-10 receptor, an IL-12 receptor, an IL-15 receptor, an IL-21 receptor, or a TGF-β receptor. In some embodiments, the second cytokine or second cytokine fragment comprises 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 IL-2, IL-12, IL-6, IL-4, IL-10, or TGFβ.

[0004] Disclosed herein is a pharmaceutical composition comprising an isolated polypeptide comprising a cleavable linker according to any of the above embodiments and a pharmaceutically acceptable excipient.

[0005] Disclosed herein is an isolated recombinant nucleic acid molecule encoding an isolated polypeptide comprising a cleavable linker according to any of the above embodiments.

[0006] Disclosed herein is a vector comprising an isolated recombinant nucleic acid molecule according to the above embodiments.

[0007] Disclosed herein is a method for producing an isolated polypeptide comprising a cleavable linker, the method comprising culturing a cell comprising the vector of the above embodiments under conditions that result in expression of the polypeptide.

[0008] Disclosed herein is a method for producing an isolated polypeptide comprising a cleavable linker, the method comprising: (a) culturing a cell comprising the isolated recombinant nucleic acid molecule of the above embodiments under conditions resulting in expression of the polypeptide; and (b) isolating the polypeptide. [Brief explanation of the drawings]

[0009] The novel features of the present disclosure are set forth with particularity in the appended claims. The features and advantages of the present disclosure will be better understood 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.

[0010] [Figure 1A] FIG. 1 illustrates the binding of polypeptide complexes PC-1, PC-2, PC-3, PC-4, and PC-5 containing EGFR masking. [Figure 1B] FIG. 1 illustrates the binding of polypeptide complexes PC-1, PC-2, PC-3, PC-4, and PC-5 after cleavage by the tumor protease MTSP1. [Figure 2A] FIG. 1 illustrates the binding of polypeptide complexes PC-1, PC-2, PC-3, PC-4, and PC-5, which contain CD3ε masking. [Figure 2B] FIG. 1 illustrates the binding of polypeptide complexes PC-1, PC-2, PC-3, PC-4, and PC-5 after cleavage by the tumor protease MTSP1. [Figure 3A]FIG. 1 illustrates the binding of polypeptide complexes PC-2, PC-3, PC-4, PC-5, PC-1, and PC-6 to EGFR-biotin as measured by ELISA. [Figure 3B] FIG. 1 illustrates binding to CD3ε-biotin as measured by ELISA. [Figure 4A] FIG. 1 illustrates the cytotoxicity of polypeptide complexes PC-2, PC-3, PC-4, PC-5, PC-1, and PC-6 against tumor target cells HCT116. [Figure 4B] FIG. 1 illustrates the cytotoxicity of polypeptide complexes PC-2, PC-3, PC-4, PC-5, PC-1, and PC-6 against tumor target cells HCT116. [Figure 4C] FIG. 1 illustrates the cytotoxicity of polypeptide complexes PC-2, PC-3, PC-4, PC-5, PC-1, and PC-6 against tumor target cells HCT116. [Figure 4D] FIG. 1 illustrates the cytotoxicity of polypeptide complexes PC-2, PC-3, PC-4, PC-5, PC-1, and PC-6 against tumor target cells HCT116. [Figure 4E] FIG. 1 illustrates the cytotoxicity of polypeptide complexes PC-2, PC-3, PC-4, PC-5, PC-1, and PC-6 against tumor target cells HCT116. [Figure 5A] FIG. 1 illustrates the pharmacokinetics of polypeptides PC-1, PC-2, PC-3, PC-7, PC-4, and PC-5 in cynomolgus monkeys. [Figure 5B] FIG. 1 illustrates the pharmacokinetics of polypeptides PC-1, PC-2, PC-3, PC-7, PC-4, and PC-5 in cynomolgus monkeys. [Figure 5C] FIG. 1 illustrates the pharmacokinetics of polypeptides PC-1, PC-2, PC-3, PC-7, PC-4, and PC-5 in cynomolgus monkeys. [Figure 5D]FIG. 1 illustrates the pharmacokinetics of polypeptides PC-1, PC-2, PC-3, PC-7, PC-4, and PC-5 in cynomolgus monkeys. [Figure 6A] FIG. 1 illustrates cytokine release of polypeptide complexes PC-1, PC-2, PC-3, PC-7, PC-4, and PC-5 in cynomolgus monkeys. [Figure 6B] FIG. 1 illustrates cytokine release of polypeptide complexes PC-1, PC-2, PC-3, PC-7, PC-4, and PC-5 in cynomolgus monkeys. [Figure 6C] FIG. 1 illustrates cytokine release of polypeptide complexes PC-1, PC-2, PC-3, PC-7, PC-4, and PC-5 in cynomolgus monkeys. [Figure 6D] FIG. 1 illustrates cytokine release of polypeptide complexes PC-1, PC-2, PC-3, PC-7, PC-4, and PC-5 in cynomolgus monkeys. [Figure 7A] FIG. 1 illustrates a graph of AST and ALT values ​​for polypeptide complexes PC-1, PC-2, PC-3, PC-4, and PC-5 in cynomolgus monkeys. [Figure 7B] FIG. 1 illustrates a graph of AST and ALT values ​​for polypeptide complexes PC-1, PC-2, PC-3, PC-4, and PC-5 in cynomolgus monkeys. DETAILED DESCRIPTION OF THE INVENTION

[0011] Protein therapeutics, such as antibodies, T cell receptor (TCR), and cytokine therapeutics, have proven effective against a variety of diseases and disorders. As with any therapeutic approach, it is necessary to maintain the activity of the protein therapeutic in diseased tissue while minimizing off-target effects in healthy tissue. One such strategy is to create an inactive form of the protein therapeutic, in which the necessary binding site of the protein therapeutic is blocked with a peptide associated with the protein therapeutic, thereby preventing the protein therapeutic from binding to or interacting with its cognate receptor or target antigen in healthy tissue. To activate the protein therapeutic in a desired disease microenvironment, a peptide is linked to the protein therapeutic using a linker that is cleavable by a protease specific to the disease microenvironment. The peptide is then released from the protein therapeutic when in the disease microenvironment.

[0012] Thus, disclosed herein are cleavable linkers applicable to various formats of protein therapeutics that are used to maintain the activity of protein therapeutics in diseased tissues while reducing the off-target effects of protein therapeutics in healthy tissues. The cleavable linkers disclosed herein have desirable properties, including, but not limited to, an increased rate of proteolysis by tumor proteases compared to control linkers, and being cleavable by an expanded panel of tumor proteases while also possessing a comparable safety profile.

[0013] Specific Definitions The terminology used herein is for the purpose of describing specific instances 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, the terms "including," "includes," "having," "has," "with," or variations thereof, when used in either the detailed description and / or claims, are intended to be inclusive, as is the term "comprising."

[0014] The term "about" or "approximately" means within an acceptable error range for a 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 one or more standard deviations per practice for a given value. When particular values ​​are described in this application and claims, unless otherwise specified, the term "about" should be assumed to mean an acceptable error range for the particular value.

[0015] "Fragment," as used herein, refers to a peptide or polypeptide that comprises less than the full-length amino acid sequence.

[0016] "Peptide," "P1," or "P2," as used herein, refers to an amino acid sequence of less than 50 amino acids, specifically excluding cytokine ligand binding domains, fragments, or muteins thereof, cytokine receptors, fragments, or muteins thereof, and any antibodies or antibody-binding fragments (e.g., single domain antibodies, Fab, scFv) that bind to a cytokine or to a cognate cytokine receptor.

[0017] Disclosed herein in some embodiments is an isolated polypeptide comprising a cleavable linker according to the amino acid sequence of SEQ ID NO:1 (LSGRSDAG).

[0018] 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).

[0019] 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).

[0020] In some embodiments, the cleavable linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 4, 5, and 6.

[0021] In some embodiments, the isolated polypeptide comprises a cleavable linker with the amino acid sequence of linker 1 of SEQ ID NO: 3 (ISSGLLSGRSDAG), linker 2 of SEQ ID NO: 4 (AAGLLAPPGGLSGRSDAG), linker 3 of SEQ ID NO: 5 (SPLGLSGRSDAG), or linker 4 of SEQ ID NO: 6 (LSGRSDAGSPLGLAG), or an isolated polypeptide comprising a cleavable linker with one, two, or three amino acid substitutions, additions, or deletions relative to the amino acid sequence of linker 1, linker 2, linker 3, or linker 4.

[0022] In some embodiments, the cleavable linker comprises the amino acid sequence of Linker1. In some embodiments, the cleavable linker consists of the amino acid sequence of Linker1. In some embodiments, the cleavable linker has one amino acid substitution, addition, or deletion relative to the amino acid sequence of Linker1. In some embodiments, the cleavable linker has two amino acid substitutions, additions, or deletions relative to the amino acid sequence of Linker1. In some embodiments, the cleavable linker has three amino acid substitutions, additions, or deletions relative to the amino acid sequence of Linker1.

[0023] 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 one amino acid substitution, addition, or deletion relative to the amino acid sequence of Linker 2. In some embodiments, the cleavable linker has two amino acid substitutions, additions, or deletions relative to the amino acid sequence of Linker 2. In some embodiments, the cleavable linker has three amino acid substitutions, additions, or deletions relative to the amino acid sequence of Linker 2.

[0024] 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 one amino acid substitution, addition, or deletion relative to the amino acid sequence of Linker 3. In some embodiments, the cleavable linker has two amino acid substitutions, additions, or deletions relative to the amino acid sequence of Linker 3. In some embodiments, the cleavable linker has three amino acid substitutions, additions, or deletions relative to the amino acid sequence of Linker 3.

[0025] 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 one amino acid substitution, addition, or deletion relative to the amino acid sequence of linker 4. In some embodiments, the cleavable linker has two amino acid substitutions, additions, or deletions relative to the amino acid sequence of linker 4. In some embodiments, the cleavable linker has three amino acid substitutions, additions, or deletions relative to the amino acid sequence of linker 4.

[0026] In some embodiments, the amino acid substitutions, additions, or deletions result 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 substitutions are conservative amino acid substitutions. Among common amino acids, for example, "conservative amino acid substitutions" are exemplified by substitutions between amino acids within each of: (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.

[0027] In some embodiments, the cleavable linker comprises a modified amino acid or unnatural amino acid, or a modified unnatural amino acid, or a combination thereof. In some embodiments, the modified amino acid or unnatural 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 a 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 a phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carbosylation, 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 may be made anywhere on the peptide backbone, cleavable linkers or amino acid side chains.

[0028] In some embodiments, the cleavable linker is cleavable by a protease. In some embodiments, the protease is present at elevated levels in a disease-state microenvironment compared to levels in healthy tissue or a non-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 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 different proteases. In some embodiments, the cleavable linker is cleaved by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more than 20 different proteases.

[0029] In some embodiments, the cleavable linker has an increased proteolysis rate compared to the proteolysis rate of a linker without a cleavable linker sequence. In some embodiments, the cleavable linker has an increased proteolysis rate of at least 5-fold compared to the proteolysis rate of a linker without a cleavable linker sequence. In some embodiments, the cleavable linker has an increased proteolysis rate of at least 8-fold compared to the proteolysis rate of a linker without a cleavable linker sequence. In some embodiments, the cleavable linker has an increased proteolysis rate of at least 10-fold compared to the proteolysis rate of a linker without a cleavable linker sequence. In some embodiments, the cleavable linker has an increased proteolysis rate of at least 15-fold compared to the proteolysis rate of a linker without a cleavable linker sequence. In some embodiments, the cleavable linker has an increased proteolysis rate of at least 20-fold compared to the proteolysis rate of a linker without a cleavable linker sequence. In some embodiments, the cleavable linker has an increased proteolysis rate of at least 25-fold compared to the proteolysis rate of a linker without a cleavable linker sequence. In some embodiments, the cleavable linker has an at least 30-fold increase in proteolysis rate compared to the proteolysis rate of a linker without a cleavable linker sequence. In some embodiments, the cleavable linker has an at least 40-fold increase in proteolysis rate compared to the proteolysis rate of a linker without a cleavable linker sequence. In some embodiments, the cleavable linker has an at least 50-fold increase in proteolysis rate compared to the proteolysis rate of a linker without a cleavable linker sequence. In some embodiments, the cleavable linker has an at least 60-fold increase in proteolysis rate compared to the proteolysis rate of a linker without a cleavable linker sequence. In some embodiments, the cleavable linker has an at least 70-fold increase in proteolysis rate compared to the proteolysis rate of a linker without a cleavable linker sequence.In some embodiments, the cleavable linker has at least a 75-fold increase in proteolysis rate compared to the proteolysis rate of a linker without a cleavable linker sequence. In some embodiments, the cleavable linker has at least an 80-fold increase in proteolysis rate compared to the proteolysis rate of a linker without a cleavable linker sequence. In some embodiments, the cleavable linker has at least a 90-fold increase in proteolysis rate compared to the proteolysis rate of a linker without a cleavable linker sequence. In some embodiments, the cleavable linker has at least a 100-fold increase in proteolysis rate compared to the proteolysis rate of a linker without a cleavable linker sequence. In some embodiments, the cleavable linker has at least a 120-fold increase in proteolysis rate compared to the proteolysis rate of a linker without a cleavable linker sequence. 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 MMP 14. In some embodiments, the serine protease comprises matriptase, urokinase, or hepsin.

[0030] In some embodiments, the cleavable linker has improved stability in human serum compared to the stability in human serum of a linker without a cleavable linker sequence. In some embodiments, the cleavable linker has at least 5-fold improved stability in human serum compared to the stability in human serum of a linker without a cleavable linker sequence. In some embodiments, the cleavable linker has at least 8-fold improved stability in human serum compared to the stability in human serum of a linker without a cleavable linker sequence. In some embodiments, the cleavable linker has at least 10-fold improved stability in human serum compared to the stability in human serum of a linker without a cleavable linker sequence. In some embodiments, the cleavable linker has at least 15-fold improved stability in human serum compared to the stability in human serum of a linker without a cleavable linker sequence. In some embodiments, the cleavable linker has at least 20-fold improved stability in human serum compared to the stability in human serum of a linker without a cleavable linker sequence. In some embodiments, the cleavable linker has at least 25-fold improved stability in human serum compared to the stability of a linker without a cleavable linker sequence. In some embodiments, the cleavable linker has at least 30-fold improved stability in human serum compared to the stability of a linker without a cleavable linker sequence. In some embodiments, the cleavable linker has at least 40-fold improved stability in human serum compared to the stability of a linker without a cleavable linker sequence. In some embodiments, the cleavable linker has at least 50-fold improved stability in human serum compared to the stability of a linker without a cleavable linker sequence. In some embodiments, the cleavable linker has at least 60-fold improved stability in human serum compared to the stability of a linker without a cleavable linker sequence. In some embodiments, the cleavable linker has at least 70-fold improved stability in human serum compared to the stability of a linker without a cleavable linker sequence.In some embodiments, the cleavable linker has at least 75-fold improved stability in human serum compared to the stability of a linker without a cleavable linker sequence. In some embodiments, the cleavable linker has at least 80-fold improved stability in human serum compared to the stability of a linker without a cleavable linker sequence. In some embodiments, the cleavable linker has at least 90-fold improved stability in human serum compared to the stability of a linker without a cleavable linker sequence. In some embodiments, the cleavable linker has at least 100-fold improved stability in human serum compared to the stability of a linker without a cleavable linker sequence. In some embodiments, the cleavable linker has at least 120-fold improved stability in human serum compared to the stability of a linker without a cleavable linker sequence.

[0031] In some embodiments, the isolated polypeptide comprising a cleavable linker has an increased rate of proteolysis 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.

[0032] In some embodiments, the isolated polypeptide comprising a cleavable linker has improved or equivalent serum stability 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.

[0033] In some embodiments, the isolated polypeptide comprising the cleavable linker exhibits improved or equivalent in vitro tumor cell killing 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.

[0034] In some embodiments, the isolated polypeptide comprising the cleavable linker has improved or comparable pharmacokinetic parameters in cynomolgus monkeys 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.

[0035] In some embodiments, the isolated polypeptide comprising the cleavable linker has improved or equivalent hepatotoxicity values ​​in cynomolgus monkeys 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.

[0036] 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.

[0037] In some embodiments, the cleavable linker has Formula I: A1-L1-P1 (Formula I) the peptide is connected to an antigen-binding domain that binds to a target antigen or to a cytokine that binds to a cytokine receptor in an arrangement according to wherein A1 comprises an antigen-binding domain that binds to a target antigen or a cytokine that binds to a cytokine receptor, L1 comprises a cleavable linker, and P1 comprises a peptide that disrupts binding of the antigen-binding domain to the target antigen or disrupts binding of the cytokine to a cytokine receptor. In some embodiments, P1 is connected to the cleavable linker at the N-terminus, and A1 is connected to the cleavable linker at the C-terminus. In some embodiments, P1 is connected to the cleavable linker at the C-terminus, and A1 is connected to the cleavable linker at the N-terminus.

[0038] 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 has a structure represented by Formula II: A2-L2-P2 (Formula II) In the arrangement by wherein A2 comprises a second antigen-binding domain or a second cytokine, L2 comprises a second cleavable linker, and P2 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.

[0039] In some embodiments, P2 is connected to the second cleavable linker at the N-terminus and A2 is connected to the second cleavable linker at the C-terminus. In some embodiments, P2 is connected to the second cleavable linker at the C-terminus and A2 is connected to the second cleavable linker at the N-terminus.

[0040] 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).

[0041] In some embodiments, L1 or L2 is at least 8 amino acids in length. In some embodiments, L1 or L2 is at least 10 amino acids in length but not more than 50 amino acids in length. In some embodiments, L1 or L2 is at least 10 amino acids in length but not more than 30 amino acids in length. In some embodiments, L1 or L2 is at least 18 amino acids in length. In some embodiments, L1 or L2 is at least 26 amino acids in length. In some embodiments, L1 or L2 is at least 30 amino acids in length. In some embodiments, L1 or L2 is at least 40 amino acids in length. In some embodiments, L1 or L2 is at least 50 amino acids in length.

[0042] Peptide (P1 or P2) In some embodiments, P1 comprises a peptide that impairs binding of the antigen-binding domain to a target antigen. In some embodiments, P1 comprises a peptide that impairs binding of a cytokine to a cytokine receptor. In some embodiments, P1 is bound to A1 via ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bonding interactions, or a combination thereof. In some embodiments, P1 is bound to A1 at or near the cytokine receptor binding site. In some embodiments, P1 is bound to A1 at or near the antigen binding site. In some embodiments, P1 becomes unbound to A1 when L1 is cleaved by a protease, exposing P1 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, P1 impairs binding of A1 to a target antigen or cytokine receptor by non-steric blocking. In some embodiments, P1 impairs binding of A1 to a target antigen or cytokine receptor by a covalent interaction. In some embodiments, P1 is not a cytokine, a cytokine-binding fragment, a cytokine mutein, or a combination of the cytokines' cognate receptors. In some embodiments, A1 is not an antibody or fragment thereof that binds to a cytokine receptor.

[0043] In some embodiments, P2 comprises a peptide that impairs binding of a second antigen-binding domain to a second target antigen. In some embodiments, P2 comprises a peptide that impairs binding of a second cytokine to a second cytokine receptor. In some embodiments, P2 is bound to A2 via ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bonding interactions, or a combination thereof. In some embodiments, P2 is bound to A2 at or near the cytokine receptor binding site. In some embodiments, P2 is bound to A2 at or near the antigen binding site. In some embodiments, P2 becomes unbound to A2 when L2 is cleaved by a protease, exposing P2 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, P2 impairs binding of A2 to a target antigen or cytokine receptor by non-steric blocking. In some embodiments, P2 impairs binding of A2 to a target antigen or cytokine receptor by a covalent interaction. In some embodiments, P2 is not a cytokine, a cytokine-binding fragment, a cytokine mutein, or a combination of the cytokine's cognate receptors. In some embodiments, A2 is not an antibody or fragment thereof that binds to a cytokine receptor.

[0044] In some embodiments, P1 has less than 70% sequence homology to the target antigen. In some embodiments, P1 has less than 75% sequence homology to the target antigen. In some embodiments, P1 has less than 80% sequence homology to the target antigen. In some embodiments, P1 has less than 85% sequence homology to the target antigen. In some embodiments, P1 has less than 90% sequence homology to the target antigen. In some embodiments, P1 has less than 95% sequence homology to the target antigen. In some embodiments, P1 has less than 98% sequence homology to the target antigen. In some embodiments, P1 has less than 99% sequence homology to the target antigen.

[0045] In some embodiments, P1 has less than 70% sequence homology to a cytokine receptor. In some embodiments, P1 has less than 75% sequence homology to a cytokine receptor. In some embodiments, P1 has less than 80% sequence homology to a cytokine receptor. In some embodiments, P1 has less than 85% sequence homology to a cytokine receptor. In some embodiments, P1 has less than 90% sequence homology to a cytokine receptor. In some embodiments, P1 has less than 95% sequence homology to a cytokine receptor. In some embodiments, P1 has less than 98% sequence homology to a cytokine receptor. In some embodiments, P1 has less than 99% sequence homology to a cytokine receptor.

[0046] In some embodiments, P2 has less than 70% sequence homology to the second target antigen. In some embodiments, P2 has less than 75% sequence homology to the second target antigen. In some embodiments, P2 has less than 80% sequence homology to the second target antigen. In some embodiments, P2 has less than 85% sequence homology to the second target antigen. In some embodiments, P2 has less than 90% sequence homology to the second target antigen. In some embodiments, P2 has less than 95% sequence homology to the second target antigen. In some embodiments, P2 has less than 98% sequence homology to the second target antigen. In some embodiments, P2 has less than 99% sequence homology to the second target antigen.

[0047] In some embodiments, P2 has less than 70% sequence homology to the second cytokine receptor. In some embodiments, P2 has less than 75% sequence homology to the second cytokine receptor. In some embodiments, P2 has less than 80% sequence homology to the second cytokine receptor. In some embodiments, P2 has less than 85% sequence homology to the second cytokine receptor. In some embodiments, P2 has less than 90% sequence homology to the second cytokine receptor. In some embodiments, P2 has less than 95% sequence homology to the second cytokine receptor. In some embodiments, P2 has less than 98% sequence homology to the second cytokine receptor. In some embodiments, P2 has less than 99% sequence homology to the second cytokine receptor.

[0048] In some embodiments, P1 or P2 comprises a de novo amino acid sequence that shares less than 50% sequence homology with a cytokine, cytokine receptor, or antibody or fragment thereof that binds to a cytokine or cytokine receptor. In some embodiments, P1 or P2 comprises a de novo amino acid sequence that shares less than 40% sequence homology with a cytokine, cytokine receptor, or antibody or fragment thereof that binds to a cytokine or cytokine receptor. In some embodiments, P1 or P2 comprises a de novo amino acid sequence that shares less than 30% sequence homology with a cytokine, cytokine receptor, or antibody or fragment thereof that binds to a cytokine or cytokine receptor. In some embodiments, P1 or P2 comprises a de novo amino acid sequence that shares less than 20% sequence homology with a cytokine, cytokine receptor, or antibody or fragment thereof that binds to a cytokine or cytokine receptor. In some embodiments, P1 or P2 comprises a de novo amino acid sequence that shares less than 10% sequence homology with a cytokine, cytokine receptor, or antibody or fragment thereof that binds to a cytokine or cytokine receptor. In some embodiments, P1 or P2 is identified from a peptide library containing random amino acid sequences.

[0049] In some embodiments, P1 or P2 comprises a de novo amino acid sequence that shares less than 50% sequence homology with the target antigen. In some embodiments, P1 or P2 comprises a de novo amino acid sequence that shares less than 40% sequence homology with the target antigen. In some embodiments, P1 or P2 comprises a de novo amino acid sequence that shares less than 30% sequence homology with the target antigen. In some embodiments, P1 or P2 comprises a de novo amino acid sequence that shares less than 20% sequence homology with the target antigen. In some embodiments, P1 or P2 comprises a de novo amino acid sequence that shares less than 10% sequence homology with the target antigen. In some embodiments, P1 or P2 is identified from a peptide library containing random amino acid sequences.

[0050] In some embodiments, P1 or P2 comprises a peptide sequence at least 5 amino acids in length. In some embodiments, P1 or P2 comprises a peptide sequence at least 6 amino acids in length. In some embodiments, P1 or P2 comprises a peptide sequence at least 10 amino acids in length. In some embodiments, P1 or P2 comprises a peptide sequence at least 10 amino acids in length and no more than 20 amino acids in length. In some embodiments, P1 or P2 comprises a peptide sequence at least 16 amino acids in length. In some embodiments, P1 or P2 comprises a peptide sequence no more than 40 amino acids in length. In some embodiments, P1 or P2 comprises at least two cysteine ​​amino acid residues. In some embodiments, P1 or P2 comprises a cyclic peptide or a linear peptide. In some embodiments, P1 or P2 comprises a cyclic peptide. In some embodiments, P1 or P2 comprises a linear peptide.

[0051] In some embodiments, P1 or P2 comprises a modified amino acid or unnatural amino acid, or a modified unnatural amino acid, or a combination thereof. In some embodiments, the modified amino acid or unnatural amino acid comprises a post-translational modification. In some embodiments, P1 or P2 comprises a modification including, but not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of a 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 a phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carbosylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA-mediated addition of amino acids to proteins such as arginylation, and ubiquitination. Modifications can be made anywhere on P1 or P2, including the peptide backbone, amino acid side chains, and termini.

[0052] In some embodiments, P1 or P2 does not comprise albumin or an albumin fragment, hi some embodiments, P1 or P2 does not comprise an albumin binding domain.

[0053] A1 and A2 In some embodiments, A1 or A2 is an antigen-recognizing molecule. In some embodiments, the antigen-recognizing molecule is an antibody or antibody fragment. In some embodiments, the antibody or antibody fragment comprises a single-chain variable fragment, a single-domain antibody, Fab, or Fab'. In some embodiments, the antibody or antibody fragment comprises a single-chain variable fragment (scFv), a heavy-chain variable domain (VH domain), a light-chain variable domain (VL domain), or a variable domain (VHH) of a camelid-derived single-domain antibody. In some embodiments, the antibody or antibody fragment comprises a single-chain variable fragment. In some embodiments, the antibody or antibody fragment is humanized or human.

[0054] In some embodiments, A1 or A2 is a Fab. In some embodiments, a Fab comprises (a) a Fab light chain polypeptide and (b) a Fab heavy chain polypeptide. In some embodiments, L1 or L2 is attached to the N-terminus of the Fab light chain polypeptide. In some embodiments, L1 or L2 is attached to the N-terminus of the Fab heavy chain polypeptide. In some embodiments, L1 or L2 is attached to the C-terminus of the Fab light chain polypeptide. In some embodiments, L1 or L2 is attached to the C-terminus of the Fab heavy chain polypeptide.

[0055] In some embodiments, A1 or A2 is a single-chain variable fragment (scFv). In some embodiments, L1 or L2 is attached to the N-terminus of the scFv. In some embodiments, L1 or L2 is attached to the 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, L1 or L2 is attached to the N-terminus of the light chain variable domain of the single-chain variable fragment (scFv). In some embodiments, L1 or L2 is attached to the N-terminus of the heavy chain variable domain of the single-chain variable fragment (scFv).

[0056] 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 (CD3ε) 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.

[0057] In some embodiments, A1 or A2 binds to a polypeptide that is part of the TCR-CD3 complex on an 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, A1 or A2 comprises an anti-CD3e single-chain variable fragment. In some embodiments, A1 or A2 binds to a polypeptide that is part of the TCR-CD3 complex on an effector cell ... DIn some embodiments, A1 or A2 comprises an anti-CD3e single-chain variable fragment having a variable light chain and a variable heavy chain capable of specifically binding to human CD3, respectively. In some embodiments, A1 or A2 comprises an anti-CD3e single-chain variable fragment having a variable light chain and a variable heavy chain capable of specifically binding to human CD3, respectively. In some embodiments, A1 or A2 comprises an anti-CD3e single-chain variable fragment 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, Complementarity determining regions (CDRs) selected from the group consisting of 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.

[0058] In some embodiments, A1 or A2 is a soluble T cell receptor (TCR). Natural TCRs are transmembrane receptors expressed on the surface of T cells that recognize antigens bound to major histocompatibility complex molecules (MHC). Natural TCRs are heterodimers, comprising an α polypeptide chain and a β polypeptide chain linked via a disulfide bond. The α and β polypeptide chains are expressed as part of a complex with accessory proteins, including, for example, two CD3ε polypeptides, one CD3γ polypeptide, one CD3δ polypeptide, and two CD3ζ polypeptides. Engagement of the TCR with a target antigen and MHC activates the T cell, resulting in a series of signaling events mediated by associated enzymes, co-receptors, adapter molecules, and activated or released transcription factors.

[0059] In natural TCRs, the α and β polypeptide chains contain an extracellular domain, a transmembrane domain, and a cytoplasmic domain. Each extracellular domain contains a variable region (V), a joining region (J), and a constant region (C). The constant region is N-terminal to the transmembrane domain, which is N-terminal to the cytoplasmic domain. The variable regions of both the α and β polypeptide chains contain three hypervariable or complementarity-determining regions (CDRs). The β polypeptide chain usually contains a short diversity region between the variable region and the joining region. The three CDRs are embedded in framework sequences, and one CDR is a hypervariable region named CDR3. The α chain variable region (Vα) and β chain variable region (Vβ) are several types of regions distinguished by their framework sequences, CDR1 and CDR2 sequences, and partially defined CDR3 sequences.

[0060] TCRs are described using the International Immunogenetics (IMGT) TCR nomenclature. In the IMGT nomenclature, Vα is referenced by a unique "TRAV" number. Similarly, Vβ is referenced by a unique "TRBV" number. The corresponding binding and constant regions are designated TRAJ and TRAC for the α binding and constant regions, respectively, and TRBJ and TRBC for the β binding and constant regions, respectively. Sequences defined by the IMGT nomenclature are known in the art and are available in the online IMGT public database.

[0061] In some embodiments, the soluble TCR is a single-chain TCR comprising the variable region of a TCR alpha extracellular domain or a fragment thereof, and the variable region of a TCR beta extracellular domain or a fragment thereof, hi 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.

[0062] In some embodiments, the soluble TCR is a single-chain TCR comprising the variable region of a TCR alpha extracellular domain or a fragment thereof and the variable region of a TCR beta extracellular domain or a fragment thereof. In some embodiments, the soluble TCR comprises an αTCR polypeptide comprising a TCR alpha extracellular domain and a βTCR polypeptide comprising a TCR beta extracellular domain. In some embodiments, L1 is attached to the N-terminus of the αTCR polypeptide. In some embodiments, L1 is attached to the N-terminus of the βTCR polypeptide. In some embodiments, A2 is attached to the C-terminus of the αTCR polypeptide. In some embodiments, A2 is attached to the N-terminus of the αTCR polypeptide. In some embodiments, A2 is attached to the C-terminus of the βTCR polypeptide. In some embodiments, L1 is attached to the N-terminus of the αTCR polypeptide and A2 is attached to the N-terminus of the βTCR polypeptide. In some embodiments, L1 is attached to the N-terminus of the αTCR polypeptide and A2 is attached to the N-terminus of the βTCR polypeptide. In some embodiments, L1 is attached to the N-terminus of the αTCR polypeptide and A2 is attached to the C-terminus of the βTCR polypeptide. In some embodiments, L1 is attached to the N-terminus of the αTCR polypeptide and A2 is attached to the C-terminus of the αTCR polypeptide. In some embodiments, L1 is attached to the N-terminus of the βTCR polypeptide and A2 is attached to the N-terminus of the αTCR polypeptide. In some embodiments, L1 is attached to the N-terminus of the βTCR polypeptide and A2 is attached to the C-terminus of the βTCR polypeptide. In some embodiments, L1 is attached to the N-terminus of the βTCR polypeptide and A2 is attached to the C-terminus of the αTCR polypeptide.

[0063] In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity for the target antigen or second target antigen than the binding affinity of the polypeptide or polypeptide complex without P1 or P2 or L1 or L2. In some embodiments, the polypeptide or polypeptide complex has a binding affinity for the target antigen or second target antigen that is at least 5-fold weaker than the binding affinity of a form of the polypeptide or polypeptide complex without P1 or P2 or L1 or L2. In some embodiments, the polypeptide or polypeptide complex has a binding affinity for the target antigen or second target antigen that is at least 8-fold weaker than the binding affinity of the polypeptide or polypeptide complex without P1 or P2 or L1 or L2. In some embodiments, the polypeptide or polypeptide complex has a binding affinity for the target antigen or second target antigen that is at least 10-fold weaker than the binding affinity of the polypeptide or polypeptide complex without P1 or P2 or L1 or L2. In some embodiments, the polypeptide or polypeptide complex has at least 20-fold weaker binding affinity for the target antigen or second target antigen than the binding affinity for the target antigen or second target antigen of the polypeptide or polypeptide complex without P1 or P2 or L1 or L2. In some embodiments, the polypeptide or polypeptide complex has at least 25-fold weaker binding affinity for the target antigen or second target antigen than the binding affinity for the target antigen or second target antigen of the polypeptide or polypeptide complex without P1 or P2 or L1 or L2. In some embodiments, the polypeptide or polypeptide complex has at least 30-fold weaker binding affinity for the target antigen or second target antigen than the binding affinity for the target antigen or second target antigen of the polypeptide or polypeptide complex without P1 or P2 or L1 or L2.In some embodiments, the polypeptide or polypeptide complex has at least 40-fold weaker binding affinity for the target antigen or second target antigen than the binding affinity of the polypeptide or polypeptide complex without P1 or P2 or L1 or L2. In some embodiments, the polypeptide or polypeptide complex has at least 50-fold weaker binding affinity for the target antigen or second target antigen than the binding affinity of the polypeptide or polypeptide complex without P1 or P2 or L1 or L2. In some embodiments, the polypeptide or polypeptide complex has at least 60-fold weaker binding affinity for the target antigen or second target antigen than the binding affinity of the polypeptide or polypeptide complex without P1 or P2 or L1 or L2. In some embodiments, the polypeptide or polypeptide complex has at least 70-fold weaker binding affinity for the target antigen or second target antigen than the binding affinity of the polypeptide or polypeptide complex without P1 or P2 or L1 or L2. In some embodiments, the polypeptide or polypeptide complex has at least 75-fold weaker binding affinity for the target antigen or second target antigen than the binding affinity of the polypeptide or polypeptide complex without P1 or P2 or L1 or L2 for the target antigen or second target antigen. In some embodiments, the polypeptide or polypeptide complex has at least 80-fold weaker binding affinity for the target antigen or second target antigen than the binding affinity of the polypeptide or polypeptide complex without P1 or P2 or L1 or L2 for the target antigen or second target antigen. In some embodiments, the polypeptide or polypeptide complex has at least 90-fold weaker binding affinity for the target antigen or second target antigen than the binding affinity of the polypeptide or polypeptide complex without P1 or P2 or L1 or L2 for the target antigen or second target antigen.In some embodiments, the polypeptide or polypeptide complex has at least 100-fold weaker binding affinity for the target antigen or second target antigen than the binding affinity for the target antigen or second target antigen of the polypeptide or polypeptide complex without P1 or P2 or L1 or L2. In some embodiments, the polypeptide or polypeptide complex has at least 120-fold weaker binding affinity for the target antigen or second target antigen than the binding affinity for the target antigen or second target antigen of the polypeptide or polypeptide complex without P1 or P2 or L1 or L2.

[0064] In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity for the target antigen or second target antigen than the binding affinity for the target antigen or second target antigen of the polypeptide or polypeptide complex in which L1 or L2 is truncated. In some embodiments, the polypeptide or polypeptide complex has a binding affinity for the target antigen or second target antigen that is at least 5-fold weaker than the binding affinity for the target antigen or second target antigen of the polypeptide or polypeptide complex in which L1 or L2 is truncated. In some embodiments, the polypeptide or polypeptide complex has a binding affinity for the target antigen or second target antigen that is at least 8-fold weaker than the binding affinity for the target antigen or second target antigen of the polypeptide or polypeptide complex in which L1 or L2 is truncated. In some embodiments, the polypeptide or polypeptide complex has a binding affinity for the target antigen or second target antigen that is at least 10-fold weaker than the binding affinity for the target antigen or second target antigen of the polypeptide or polypeptide complex in which L1 or L2 is truncated. In some embodiments, the polypeptide or polypeptide complex has at least 20-fold weaker binding affinity for the target antigen or second target antigen than the binding affinity for the target antigen or second target antigen of the polypeptide or polypeptide complex in which L1 or L2 is truncated. In some embodiments, the polypeptide or polypeptide complex has at least 25-fold weaker binding affinity for the target antigen or second target antigen than the binding affinity for the target antigen or second target antigen of the polypeptide or polypeptide complex in which L1 or L2 is truncated. In some embodiments, the polypeptide or polypeptide complex has at least 30-fold weaker binding affinity for the target antigen or second target antigen than the binding affinity for the target antigen or second target antigen of the polypeptide or polypeptide complex in which L1 or L2 is truncated.In some embodiments, the polypeptide or polypeptide complex has at least 40-fold weaker binding affinity for the target antigen or second target antigen than the binding affinity for the target antigen or second target antigen of the polypeptide or polypeptide complex in which L1 or L2 is truncated. In some embodiments, the polypeptide or polypeptide complex has at least 50-fold weaker binding affinity for the target antigen or second target antigen than the binding affinity for the target antigen or second target antigen of the polypeptide or polypeptide complex in which L1 or L2 is truncated. In some embodiments, the polypeptide or polypeptide complex has at least 60-fold weaker binding affinity for the target antigen or second target antigen than the binding affinity for the target antigen or second target antigen of the polypeptide or polypeptide complex in which L1 or L2 is truncated. In some embodiments, the polypeptide or polypeptide complex has at least 70-fold weaker binding affinity for the target antigen or second target antigen than the binding affinity for the target antigen or second target antigen of the polypeptide or polypeptide complex in which L1 or L2 is truncated. In some embodiments, the polypeptide or polypeptide complex has at least 75-fold weaker binding affinity for the target antigen or second target antigen than the binding affinity for the target antigen or second target antigen of the polypeptide or polypeptide complex in which L1 or L2 is truncated. In some embodiments, the polypeptide or polypeptide complex has at least 80-fold weaker binding affinity for the target antigen or second target antigen than the binding affinity for the target antigen or second target antigen of the polypeptide or polypeptide complex in which L1 or L2 is truncated. In some embodiments, the polypeptide or polypeptide complex has at least 90-fold weaker binding affinity for the target antigen or second target antigen than the binding affinity for the target antigen or second target antigen of the polypeptide or polypeptide complex in which L1 or L2 is truncated.In some embodiments, the polypeptide or polypeptide complex has at least 100-fold weaker binding affinity for the target antigen or second target antigen than the binding affinity for the target antigen or second target antigen of a polypeptide or polypeptide complex in which L1 or L2 is truncated. In some embodiments, the polypeptide or polypeptide complex has at least 120-fold weaker binding affinity for the target antigen or second target antigen than the binding affinity for the target antigen or second target antigen of a polypeptide or polypeptide complex in which L1 or L2 is truncated. In some embodiments, L1 or L2 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.

[0065] In some embodiments, A1 or A2 is a cytokine or cytokine fragment. In some embodiments, A1 or A2 is a mutein of a cytokine or cytokine fragment. In some embodiments, the cytokine or cytokine fragment is a mutein of a cytokine or cytokine fragment.

[0066] Cytokines are a diverse group of small peptides, including chemokines, interferons, interleukins, lymphokines, adipokines, mesenchymal growth factors, and tumor necrosis factors, that are involved in intercellular signaling in various biological pathways. Cytokines are particularly important in immune and inflammatory responses. Signaling occurs following recognition of the cytokine by its corresponding cytokine receptor, a transmembrane receptor that contains an extracellular domain for ligand binding and an intracellular domain that enables signal transduction.

[0067] The diversity of cytokines is accompanied by a corresponding diversity of cytokine receptors, which can include single chains, subunits, or dimer / multimer domains. Cytokine receptors include type I cytokine receptors, exemplified by interleukin receptors, and type II cytokine receptors, exemplified by interferon receptors, both of which contain a cytokine receptor homology domain (CHD). The CHDs 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 α subunits, β subunits, γ subunits, or dimeric or trimeric combinations thereof. In one example, a high-affinity receptor for IL-2 contains an IL-2Rα subunit, an IL-2Rβ subunit, and an IL-2Rγ subunit; an intermediate-affinity receptor for IL-2 contains only an IL-2Rβ subunit and an IL-2Rγ subunit; and a low-affinity receptor for IL-2 contains only an IL-2Rα subunit.

[0068] In some embodiments, the cytokine is a chemokine, interferon, interleukin, lymphokine, adipokine, growth factor, or 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-β.

[0069] In some embodiments, the cytokine mutein is a variant of a wild-type cytokine. In some embodiments, the cytokine mutein is a mutant of a wild-type cytokine. In some embodiments, the cytokine mutein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or more than 50 amino acid substitutions compared to the wild-type cytokine. In some embodiments, the cytokine mutein comprises at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or more than 50 amino acid substitutions compared to the 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 compared to a naturally occurring cytokine. In some embodiments, the cytokine mutein contains at most 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.

[0070] 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, interferon, interleukin, lymphokine, adipokine, growth factor, or 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 trimer. In some embodiments, the cytokine receptor comprises an α subunit, a β subunit, a γ subunit, or any combination thereof. For example, in some embodiments, the cytokine receptor comprises an α subunit, a β subunit, and a γ subunit. In another example, in some embodiments, the cytokine receptor comprises a β subunit and a γ subunit. In some embodiments, the cytokine receptor comprises an α subunit and a β subunit.

[0071] In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity to a cytokine receptor than the binding affinity of a polypeptide or polypeptide complex that does not have P1 or P2 or L1 or L2. In some embodiments, the polypeptide or polypeptide complex has a binding affinity to a cytokine receptor that is at least 5-fold weaker than the binding affinity of a form of the polypeptide or polypeptide complex that does not have P1 or P2 or L1 or L2. In some embodiments, the polypeptide or polypeptide complex has a binding affinity to a cytokine receptor that is at least 8-fold weaker than the binding affinity of a form of the polypeptide or polypeptide complex that does not have P1 or P2 or L1 or L2. In some embodiments, the polypeptide or polypeptide complex has a binding affinity to a cytokine receptor that is at least 10-fold weaker than the binding affinity of a form of the polypeptide or polypeptide complex that does not have P1 or P2 or L1 or L2. In some embodiments, the polypeptide or polypeptide complex has a binding affinity to a cytokine receptor that is at least 20-fold weaker than the binding affinity of a form of the polypeptide or polypeptide complex that does not have P1 or P2 or L1 or L2. In some embodiments, the polypeptide or polypeptide complex has at least 25-fold weaker binding affinity to a cytokine receptor than a form of the polypeptide or polypeptide complex that does not have P1 or P2 or L1 or L2. In some embodiments, the polypeptide or polypeptide complex has at least 30-fold weaker binding affinity to a cytokine receptor than a form of the polypeptide or polypeptide complex that does not have P1 or P2 or L1 or L2.In some embodiments, the polypeptide or polypeptide complex has at least 40-fold weaker binding affinity to a cytokine receptor than a form of the polypeptide or polypeptide complex that does not have P1 or P2 or L1 or L2. In some embodiments, the polypeptide or polypeptide complex has at least 50-fold weaker binding affinity to a cytokine receptor than a form of the polypeptide or polypeptide complex that does not have P1 or P2 or L1 or L2. In some embodiments, the polypeptide or polypeptide complex has at least 60-fold weaker binding affinity to a cytokine receptor than a form of the polypeptide or polypeptide complex that does not have P1 or P2 or L1 or L2. In some embodiments, the polypeptide or polypeptide complex has at least 70-fold weaker binding affinity to a cytokine receptor than a form of the polypeptide or polypeptide complex that does not have P1 or P2 or L1 or L2. In some embodiments, the polypeptide or polypeptide complex has at least 75-fold weaker binding affinity to a cytokine receptor than a form of the polypeptide or polypeptide complex that does not have P1 or P2 or L1 or L2. In some embodiments, the polypeptide or polypeptide complex has at least 80-fold weaker binding affinity to a cytokine receptor than the binding affinity of a form of the polypeptide or polypeptide complex that does not have P1 or P2 or L1 or L2 to the cytokine receptor. In some embodiments, the polypeptide or polypeptide complex has at least 90-fold weaker binding affinity to a cytokine receptor than the binding affinity of a form of the polypeptide or polypeptide complex that does not have P1 or P2 or L1 or L2 to the cytokine receptor.In some embodiments, the polypeptide or polypeptide complex has at least 100-fold weaker binding affinity to a cytokine receptor than a form of the polypeptide or polypeptide complex that does not have P1 or P2 or L1 or L2. In some embodiments, the polypeptide or polypeptide complex has at least 120-fold weaker binding affinity to a cytokine receptor than a form of the polypeptide or polypeptide complex that does not have P1 or P2 or L1 or L2. In some embodiments, the polypeptide or polypeptide complex has at least 150-fold weaker binding affinity to a cytokine receptor than a form of the polypeptide or polypeptide complex that does not have P1 or P2 or L1 or L2. 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-β. In some embodiments, the cytokine receptor comprises an interferon receptor, a GM-CSF receptor, an IL-2 receptor, an IL-7 receptor, an IL-12 receptor, an IL-15 receptor, or an IL-21 receptor. In some embodiments, the cytokine receptor comprises an IL-2 receptor, an IL-12 receptor, an IL-6 receptor, an IL-4 receptor, an IL-10 receptor, or a TGF-β receptor.

[0072] In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity for a cytokine receptor than the binding affinity for a polypeptide or polypeptide complex in which L1 or L2 is truncated. In some embodiments, the polypeptide or polypeptide complex has a binding affinity for a cytokine receptor that is at least 5-fold weaker than the binding affinity for a cytokine receptor for a polypeptide or polypeptide complex in which L1 or L2 is truncated. In some embodiments, the polypeptide or polypeptide complex has a binding affinity for a cytokine receptor that is at least 8-fold weaker than the binding affinity for a cytokine receptor for a polypeptide or polypeptide complex in which L1 or L2 is truncated. In some embodiments, the polypeptide or polypeptide complex has a binding affinity for a cytokine receptor that is at least 10-fold weaker than the binding affinity for a cytokine receptor for a polypeptide or polypeptide complex in which L1 or L2 is truncated. In some embodiments, the polypeptide or polypeptide complex has a binding affinity for a cytokine receptor that is at least 15-fold weaker than the binding affinity for a cytokine receptor for a polypeptide or polypeptide complex in which L1 or L2 is truncated. In some embodiments, the polypeptide or polypeptide complex has a binding affinity for a cytokine receptor that is at least 20-fold weaker than the binding affinity for a cytokine receptor for a polypeptide or polypeptide complex in which L1 or L2 is truncated. In some embodiments, the polypeptide or polypeptide complex has at least 25-fold weaker binding affinity for a cytokine receptor than the binding affinity for a polypeptide or polypeptide complex in which L1 or L2 is truncated, hi some embodiments, the polypeptide or polypeptide complex has at least 30-fold weaker binding affinity for a cytokine receptor than the binding affinity for a polypeptide or polypeptide complex in which L1 or L2 is truncated.In some embodiments, the polypeptide or polypeptide complex has at least 40-fold weaker binding affinity to a cytokine receptor than the binding affinity of a polypeptide or polypeptide complex in which L1 or L2 is truncated. In some embodiments, the polypeptide or polypeptide complex has at least 50-fold weaker binding affinity to a cytokine receptor than the binding affinity of a polypeptide or polypeptide complex in which L1 or L2 is truncated. In some embodiments, the polypeptide or polypeptide complex has at least 60-fold weaker binding affinity to a cytokine receptor than the binding affinity of a polypeptide or polypeptide complex in which L1 or L2 is truncated. In some embodiments, the polypeptide or polypeptide complex has at least 70-fold weaker binding affinity to a cytokine receptor than the binding affinity of a polypeptide or polypeptide complex in which L1 or L2 is truncated. In some embodiments, the polypeptide or polypeptide complex has at least 75-fold weaker binding affinity to a cytokine receptor than the binding affinity of a polypeptide or polypeptide complex in which L1 or L2 is truncated. In some embodiments, the polypeptide or polypeptide complex has at least 80-fold weaker binding affinity to a cytokine receptor than the binding affinity of a polypeptide or polypeptide complex in which L1 or L2 is truncated. In some embodiments, the polypeptide or polypeptide complex has at least 90-fold weaker binding affinity for a cytokine receptor compared to the binding affinity for a polypeptide or polypeptide complex in which L1 or L2 is truncated, hi some embodiments, the polypeptide or polypeptide complex has at least 100-fold weaker binding affinity for a cytokine receptor compared to the binding affinity for a polypeptide or polypeptide complex in which L1 or L2 is truncated.In some embodiments, the cytokine or cytokine fragment comprises 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, L1 or L2 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.

[0073] Half-life extension moiety In some embodiments, P1 is further linked to a half-life extending moiety. In some embodiments, P1 is a compound of formula Ia: A1-L1-P1-L3-H1 (Formula Ia) is linked to the half-life extending moiety in an arrangement according to wherein H1 is a half-life extending moiety and L3 is a linker connecting H1 to P1. In some embodiments, L3 is a non-cleavable linker. In some embodiments, the half-life extending moiety (H1) does not prevent A1 from binding to the target antigen. In some embodiments, the half-life extending moiety (H1) has no binding affinity for A1. In some embodiments, the half-life extending moiety (H1) has no binding affinity for the target antigen. In some embodiments, the half-life extending moiety (H1) does not protect A1 from the target antigen. In some embodiments, the half-life extending moiety (H1) is not directly linked to A1.

[0074] In some embodiments, the half-life extending moiety (H1) does not prevent A1 from binding to the cytokine receptor. In some embodiments, the half-life extending moiety (H1) has no binding affinity for the cytokine or cytokine receptor. In some embodiments, the half-life extending moiety (H1) does not protect the cytokine or cytokine fragment from the cytokine receptor. In some embodiments, the half-life extending moiety (H1) is not directly linked to the cytokine or cytokine fragment.

[0075] In some embodiments, H1 comprises an amino acid sequence having a repeat sequence motif. In some embodiments, H1 comprises an amino acid sequence having a highly ordered secondary structure. "Highly ordered secondary structure," as used in this context, means that at least about 50%, about 70%, about 80%, or about 90% of the amino acid residues of H1 contribute to secondary structure as measured by means including, but not limited to, spectrophotometric methods (e.g., circular dichroism spectroscopy in the "far-UV" spectral region (190-250 nm) and computer programs or algorithms such as the Chou-Fasman algorithm or the Garnier-Osguthorpe-Robson ("GOR") algorithm).

[0076] In some embodiments, H1 comprises a polymer. In some embodiments, the polymer is polyethylene glycol (PEG). In some embodiments, H1 comprises albumin. In some embodiments, H1 comprises an Fc domain. In some embodiments, the albumin is serum albumin. In some embodiments, the albumin is human serum albumin. In some embodiments, H1 comprises a polypeptide, a ligand, or a small molecule. In some embodiments, the polypeptide, ligand, or small molecule binds to a serum protein or fragment thereof, a circulating immunoglobulin or fragment thereof, or CD35 / CR1. In some embodiments, the serum protein comprises thyroxine-binding protein, transthyretin, 1-acid glycoprotein, transferrin, transferrin receptor or a transferrin-binding portion thereof, fibrinogen, or albumin. In some embodiments, the circulating immunoglobulin molecule comprises IgG1, IgG2, IgG3, IgG4, slgA, IgM, or IgD. In some embodiments, the serum protein is albumin. In some embodiments, the polypeptide is an antibody. In some embodiments, the antibody comprises a single domain antibody, a single chain variable fragment, or a Fab. In some embodiments, the 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 antibody or a humanized antibody. In some embodiments, the single domain antibody is selected from the group consisting of 645gH1gL1, 645dsgH5gL4, 23-13-A01-sc02, A10m3, or fragments thereof, DOM7r-31, DOM7h-11-15, Alb-1, Alb-8, Alb-23, 10G, 10GE, and SA21.

[0077] In some embodiments, H1 comprises a single domain antibody. In some embodiments, H1 comprises a single domain antibody that binds to albumin. In some embodiments, H1 comprises a single domain antibody that binds to human serum albumin.

[0078] In some embodiments, H1 comprises a modified amino acid or unnatural amino acid, or a modified unnatural amino acid, or a combination thereof. In some embodiments, the modified amino acid or unnatural amino acid comprises a post-translational modification. In some embodiments, H1 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 cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carbosylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA-mediated addition of amino acids to proteins such as arginylation, and ubiquitination. Modifications can be made anywhere on H1, including the peptide backbone, amino acid side chains, and termini.

[0079] Polynucleotides encoding polypeptides or polypeptide complexes Disclosed herein in some embodiments is an isolated recombinant nucleic acid molecule that encodes a polypeptide or polypeptide complex disclosed herein. Disclosed herein in some embodiments is an isolated recombinant nucleic acid molecule that encodes a polypeptide that includes a cleavable linker.

[0080] Disclosed herein in some embodiments is an isolated recombinant nucleic acid molecule that encodes a polypeptide comprising a cleavable linker according to the amino acid sequence of SEQ ID NO:1 (LSGRSDAG).

[0081] 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).

[0082] Disclosed herein in some embodiments is an isolated recombinant nucleic acid molecule that encodes 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 having one, two, or three 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 is an isolated recombinant nucleic acid molecule that encodes 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 is an isolated recombinant nucleic acid molecule that encodes 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 is an isolated recombinant nucleic acid molecule 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 is an isolated recombinant nucleic acid molecule 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 is an isolated recombinant nucleic acid molecule encoding an isolated polypeptide comprising a cleavable linker according to the amino acid sequence of LSGRSDAG (SEQ ID NO: 1).

[0083] In some embodiments herein, a compound of Formula I: A1-L1-P1 (Formula I) Disclosed is an isolated recombinant nucleic acid molecule encoding a polypeptide or polypeptide complex according to wherein A1 comprises an antigen-binding domain that binds to a target antigen or a cytokine that binds to a cytokine receptor, L1 comprises a cleavable linker, and P1 comprises a peptide that impairs binding of the antigen-binding domain to the target antigen or impairs binding of the cytokine to a cytokine receptor. In some embodiments herein, a compound represented by Formula I: A1-L1-P1 (Formula I) Disclosed is an isolated recombinant nucleic acid molecule encoding a polypeptide or polypeptide complex comprising:

[0084] wherein A1 comprises an antigen-binding domain that binds to a target antigen or a cytokine that binds to a cytokine receptor, L1 comprises a cleavable linker, and P1 comprises a peptide that impairs binding of the antigen-binding domain to the target antigen or impairs binding of the cytokine to a cytokine receptor. In some embodiments herein, a compound represented by Formula I: A1-L1-P1 (Formula I) Disclosed is an isolated recombinant nucleic acid molecule encoding a polypeptide or polypeptide complex according to wherein A1 is an antigen-binding domain that binds to a target antigen or a cytokine that binds to a cytokine receptor, L1 is a cleavable linker, and P1 is a peptide that impairs binding of the antigen-binding domain to the target antigen or impairs binding of the cytokine to a cytokine receptor. In some embodiments herein, a compound represented by Formula I: A1-L1-P1 (Formula I) Disclosed is an isolated recombinant nucleic acid molecule encoding a polypeptide or polypeptide complex comprising: wherein A1 is an antigen-binding domain that binds to a target antigen or a cytokine that binds to a cytokine receptor, L1 is a cleavable linker, and P1 is a peptide that impairs binding of the antigen-binding domain to the target antigen or impairs binding of the cytokine to a cytokine receptor.

[0085] In some embodiments disclosed herein, a polypeptide or polypeptide complex is provided, wherein the isolated polypeptide is complexed with a second isolated polypeptide comprising a second antigen-binding domain or a second cytokine. In some embodiments disclosed herein, a polypeptide or polypeptide complex is provided, wherein the isolated polypeptide is complexed with a second isolated polypeptide comprising a second antigen-binding domain or a second cytokine. A2-L2-P2 (Formula II) Disclosed is an isolated recombinant nucleic acid molecule encoding a polypeptide or polypeptide complex according to wherein A2 comprises a second antigen-binding domain or a second cytokine, L2 comprises a second cleavable linker, and P2 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 herein, a compound represented by Formula II: A2-L2-P2 (Formula II) Disclosed is an isolated recombinant nucleic acid molecule encoding a polypeptide or polypeptide complex comprising: wherein A2 comprises a second antigen-binding domain or a second cytokine, L2 comprises a second cleavable linker, and P2 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 herein, a compound represented by Formula II: A2-L2-P2 (Formula II) Disclosed is an isolated recombinant nucleic acid molecule encoding a polypeptide or polypeptide complex according to wherein A2 is a second antigen-binding domain or a second cytokine, L2 is a second cleavable linker, and P2 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. In some embodiments herein, a compound represented by Formula II: A2-L2-P2 (Formula II) Disclosed is an isolated recombinant nucleic acid molecule encoding a polypeptide or polypeptide complex comprising: wherein A2 is a second antigen-binding domain or a second cytokine, L2 is a second cleavable linker, and P2 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.

[0086] Pharmaceutical Composition Disclosed herein in some embodiments are pharmaceutical compositions comprising (a) a polypeptide or polypeptide complex disclosed herein, and (b) a pharmaceutically acceptable excipient.

[0087] In some embodiments, the pharmaceutical composition comprises (a) a polypeptide or polypeptide complex comprising a cleavable linker with the amino acid sequence of SEQ ID NO: 1 (LSGRSDAG), and (b) a pharmaceutically acceptable excipient.

[0088] In some embodiments, the pharmaceutical composition comprises (a) a polypeptide or polypeptide complex comprising a cleavable linker according to the amino acid sequence of SEQ ID NO: 3 (ISSGLLSGRSDAG), and (b) a pharmaceutically acceptable excipient.

[0089] In some embodiments, the pharmaceutical composition comprises (a) a polypeptide or polypeptide complex comprising a cleavable linker with the amino acid sequence of SEQ ID NO: 26 (AGLLAPPGGLSGRSDAG), and (b) a pharmaceutically acceptable excipient.

[0090] In some embodiments, the pharmaceutical composition comprises (a) a polypeptide or polypeptide complex comprising a cleavable linker with the amino acid sequence of SEQ ID NO: 4 (AAGLLAPPGGLSGRSDAG), and (b) a pharmaceutically acceptable excipient.

[0091] In some embodiments, the pharmaceutical composition comprises (a) a polypeptide or polypeptide complex comprising a cleavable linker with the amino acid sequence of SEQ ID NO: 5 (SPLGLSGRSDAG), and (b) a pharmaceutically acceptable excipient.

[0092] In some embodiments, the pharmaceutical composition comprises (a) a polypeptide or polypeptide complex comprising a cleavable linker with the amino acid sequence of SEQ ID NO: 6 (LSGRSDAGSPLGLAG), and (b) a pharmaceutically acceptable excipient.

[0093] In some embodiments, a pharmaceutical composition comprises (a) a polypeptide or polypeptide complex 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 a polypeptide or polypeptide complex comprising a cleavable linker having one, two, or three 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 pharmaceutically acceptable excipient. In some embodiments, a pharmaceutical composition comprises (a) a polypeptide or polypeptide complex comprising a cleavable linker according to the amino acid sequence of linker 1 (ISSGLLSGRSDAG) (SEQ ID NO: 3); and (b) a pharmaceutically acceptable excipient. In some embodiments, a pharmaceutical composition comprises (a) a polypeptide or polypeptide complex comprising a cleavable linker according to the amino acid sequence of linker 2 (AAGLLAPPGGLSGRSDAG) (SEQ ID NO: 4), and (b) a pharmaceutically acceptable excipient. In some embodiments, a pharmaceutical composition comprises (a) a polypeptide or polypeptide complex comprising a cleavable linker according to the amino acid sequence of linker 3 (SPLGLSGRSDAG) (SEQ ID NO: 5), and (b) a pharmaceutically acceptable excipient. In some embodiments, a pharmaceutical composition comprises (a) a polypeptide or polypeptide complex comprising a cleavable linker according to the amino acid sequence of linker 4 (LSGRSDAGSPLGLAG) (SEQ ID NO: 6), and (b) a pharmaceutically acceptable excipient. In some embodiments, a pharmaceutical composition comprises (a) a polypeptide or polypeptide complex comprising a cleavable linker according to the amino acid sequence of LSGRSDAG (SEQ ID NO: 1), and (b) a pharmaceutically acceptable excipient.

[0094] In some embodiments, the pharmaceutical composition comprises (a) a compound of Formula I: A1-L1-P1 (Formula I) 1. An isolated polypeptide or polypeptide complex according to wherein A1 comprises an antigen-binding domain that binds to a target antigen or a cytokine that binds to a cytokine receptor, L1 comprises a cleavable linker, and P1 comprises a peptide that impairs binding of the antigen-binding domain to the target antigen or impairs binding of the cytokine to a cytokine receptor; and (b) a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises: (a) an isolated polypeptide or polypeptide complex represented by Formula I: A1-L1-P1 (Formula I) 1. An isolated polypeptide or polypeptide complex comprising: wherein A1 comprises an antigen-binding domain that binds to a target antigen or a cytokine that binds to a cytokine receptor, L1 comprises a cleavable linker, and P1 comprises a peptide that impairs binding of the antigen-binding domain to the target antigen or impairs binding of the cytokine to a cytokine receptor; and (b) a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises: (a) an isolated polypeptide or polypeptide complex represented by Formula I: A1-L1-P1 (Formula I) 1. An isolated polypeptide or polypeptide complex according to wherein A1 is an antigen-binding domain that binds to a target antigen or a cytokine that binds to a cytokine receptor, L1 is a cleavable linker, and P1 is a peptide that impairs binding of the antigen-binding domain to a target antigen or impairs binding of the cytokine to a cytokine receptor; and (b) a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises: (a) an isolated polypeptide or polypeptide complex represented by Formula I: A1-L1-P1 (Formula I) 1. An isolated polypeptide or polypeptide complex comprising: wherein A1 is an antigen-binding domain that binds to a target antigen or a cytokine that binds to a cytokine receptor, L1 is a cleavable linker, and P1 is a peptide that impairs binding of the antigen-binding domain to the target antigen or impairs binding of the cytokine to a cytokine receptor; and (b) a pharmaceutically acceptable excipient.

[0095] Disclosed herein in some embodiments is a polypeptide or polypeptide complex, 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) a polypeptide of Formula II: A2-L2-P2 (Formula II) 1. An isolated polypeptide or polypeptide complex according to wherein A2 comprises a second antigen-binding domain or a second cytokine, L2 comprises a second cleavable linker, and P2 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 pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises: (a) a polypeptide or polypeptide complex represented by Formula II: A2-L2-P2 (Formula II) 1. An isolated polypeptide or polypeptide complex comprising: wherein A2 comprises a second antigen-binding domain or a second cytokine, L2 comprises a second cleavable linker, and P2 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 pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises: (a) a polypeptide or polypeptide complex represented by Formula II: A2-L2-P2 (Formula II) 1. An isolated polypeptide or polypeptide complex according to wherein A2 is a second antigen-binding domain or a second cytokine, L2 is a second cleavable linker, and P2 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 pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises: (a) an isolated polypeptide or polypeptide complex of Formula II: A2-L2-P2 (Formula II) 1. An isolated polypeptide or polypeptide complex comprising: wherein A2 is a second antigen-binding domain or a second cytokine, L2 is a second cleavable linker, and P2 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 pharmaceutically acceptable excipient.

[0096] In some embodiments, the polypeptide or polypeptide complex further comprises a detectable label, a therapeutic agent, or a pharmacokinetic modifying moiety, hi some embodiments, the detectable label comprises a fluorescent label, a radioactive label, an enzyme, a nucleic acid probe, or an imaging agent.

[0097] For administration to a subject, the polypeptides or polypeptide complexes 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 component and is not toxic to the patient to whom it is administered. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate-buffered saline, water, emulsions such as oil / water emulsions, various types of wetting agents, sterile solutions, etc. Such carriers can be formulated using conventional methods and administered to a subject in an appropriate dosage. Preferably, the pharmaceutical composition is sterile. These compositions may further contain auxiliary substances such as preservatives, emulsifiers, dispersing agents, etc. Prevention of microbial action may be ensured by including various antibacterial and antifungal agents.

[0098] The pharmaceutical composition may be in any suitable form (depending on the desired method of administration). The pharmaceutical composition may be provided in unit dosage form, provided in a hermetically sealed container, and provided as part of a kit. Such a kit may also include instructions for use. It may also contain a plurality of the unit dosage forms described above.

[0099] The pharmaceutical compositions may be adapted for administration by any suitable route, including parenteral (e.g., subcutaneous, intramuscular, or intravenous) routes. Such compositions may be prepared by methods known in the art of pharmacy, for example, by mixing the active ingredient with the carrier or excipient under sterile conditions.

[0100] The dosage of the substances of the present disclosure can vary within a wide range depending on the disease or disorder being treated, the age and condition of the individual being treated, etc., and ultimately the physician will determine the appropriate dosage to be used.

[0101] Table 1 provides the amino acid sequences of the constructs described herein.

[0102] [Table 1-1]

[0103]

Table 1-2

[0104]

Table 1-3

[0105]

Table 1-4

[0106] In some embodiments, the polypeptide or polypeptide complex comprises 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 NO: 1, 2, 4, 5, 7, 8, 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In some embodiments, the sequence comprises at least or about 95% homology to SEQ ID NO: 1, 2, 4, 5, 7, 8, 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In some embodiments, the sequence comprises at least or about 97% homology to SEQ ID NO: 1, 2, 4, 5, 7, 8, 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In some embodiments, the sequence comprises at least or about 99% homology to SEQ ID NO: 1, 2, 4, 5, 7, 8, 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In some embodiments, the sequence comprises at least or about 100% homology to SEQ ID NO: 1, 2, 4, 5, 7, 8, 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In some examples, the sequence includes at least a portion of SEQ ID NO: 1, 2, 4, 5, 7, 8, 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 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.

[0107] The percent sequence identity (%) with respect to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are identical to 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, but not allowing any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in a variety of known ways, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software, etc. Appropriate parameters for aligning sequences can be determined, including the algorithm required to achieve maximum alignment across the entire length of the sequences being compared. However, for purposes herein, percent amino acid sequence identity values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program is owned by Genentech, Inc., and its source code has been submitted, along with user documentation, to the U.S. Copyright Office, Washington, DC 20559, and 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 must be compiled for use on UNIX operating systems, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0108] In situations where ALIGN-2 is utilized to compare amino acid sequences, the percent amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (alternatively, the phrase "given amino acid sequence A having or containing a particular % amino acid sequence identity to given amino acid sequence B") is calculated as 100 x 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 the program's alignment of A and B, and Y is the total number of amino acid residues in B. It should be recognized that the length of amino acid sequence A is equal to the length of amino acid sequence B, and the % amino acid sequence identity of A to B is not equal to the % amino acid sequence identity of B to A. Unless otherwise noted, all % amino acid sequence identity values ​​used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

[0109] Production of polypeptides containing cleavable linkers In some embodiments, the polypeptides described herein (e.g., antibodies and binding fragments thereof) are produced by any method known in the art and useful for synthesizing polypeptides (e.g., antibodies), specifically, chemical synthesis or recombinant expression, preferably by recombinant expression techniques.

[0110] In some examples, antibodies or binding fragments thereof are recombinantly expressed, and nucleic acids encoding the antibodies or binding fragments thereof are assembled from chemically synthesized oligonucleotides (e.g., those described in Kutmeier et al., 1994, BioTechniques 17:242), which involve synthesis of overlapping oligonucleotides containing portions of the antibody-encoding sequence, annealing and ligation of those oligonucleotides, and PCR amplification of the ligated oligonucleotides.

[0111] Alternatively, nucleic acid molecules encoding antibodies are produced, optionally from a suitable source (e.g., an antibody cDNA library or a cDNA library generated from any tissue or cell that expresses immunoglobulins), by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence, or by cloning using oligonucleotide probes specific for the particular gene sequence.

[0112] In some embodiments, the antibody or antigen-binding fragment thereof is produced, optionally by immunizing an animal 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 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, clones encoding at least the Fab portion of the antibody are optionally obtained by screening a Fab expression library (e.g., as described by Huse et al., 1989 Science 246:1275-1281) or by screening an antibody library (see, e.g., Clackson et al., 1991 Nature 352:624; Hane et al., 1997 Proc. Natl. Acad. Sci. USA 94:4937) for clones of Fab fragments that bind a specific antigen.

[0113] In some embodiments, techniques developed to produce "chimeric antibodies" by splicing genes from a human antibody molecule of appropriate biological activity together with antibodies from a mouse antibody molecule of appropriate antigen specificity (Morrison et al., 1984, Proc. Natl. Acad. Sci. 81:851-855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454) are used. Chimeric antibodies are molecules in which different portions are derived from different animal species, such as those having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region.

[0114] 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, 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward, 1989, Nature 334:544-54) are suitable for producing 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 assembling functional Fv fragments in E. coli are also optionally used (Skerra et al., 1988, Science 242:1038-1041).

[0115] In some embodiments, an expression vector containing an antibody nucleotide sequence, or the antibody nucleotide sequence, is introduced into host cells by conventional techniques (e.g., electroporation, liposome transfection, calcium phosphate precipitation), and the transfected cells are then cultured by conventional techniques to produce the antibody. In certain embodiments, antibody expression is regulated by a constitutive, inducible, or tissue-specific promoter.

[0116] In some embodiments, various host-expression vector systems are utilized to express the antibodies or binding fragments thereof described herein. Such host-expression systems not only represent vehicles for producing and purifying the antibody coding sequence, but also cells that express the antibody or binding fragment thereof in situ when transformed or transfected with the appropriate nucleotide coding sequence. These include, but are not limited to, microorganisms such as bacteria (e.g., Escherichia coli or Bacillus subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the coding sequence for an antibody or binding fragment thereof; yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing the coding sequence for an antibody or binding fragment thereof; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing the coding sequence for an antibody or binding fragment thereof; plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus (CaMV) or tobacco mosaic virus (TMV)) or recombinant plasmid expression vectors (e.g., Ti plasmid) containing the coding sequence for an antibody or binding fragment thereof; or mammalian cell systems (e.g., COS, CHO, BH, 293, 293T, 3T3 cells) harboring recombinant expression constructs containing promoters derived from mammalian cell genomes (e.g., metallothionein promoter) or mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter).

[0117] For long-term, high-yield production of recombinant proteins, stable expression is preferred. In some instances, cell lines that stably express antibodies are optionally engineered. Rather than using expression vectors containing viral origins of replication, host cells are transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.) and a selectable marker. After introducing the foreign DNA, engineered cells are grown in enriched media for 1-2 days and then switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to selection, allowing cells to stably integrate the plasmid into their chromosomes and grow to generate foci that are cloned and expanded into cell lines. This method can be conveniently used to engineer cell lines that express antibodies or binding fragments thereof.

[0118] In some examples, a number of selection systems are used, including but not limited to, the genes for herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska and Szybalski, 192, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817), in tk cells, hgprt cells, or aprt cells, respectively. Additionally, antimetabolite resistance is used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980 Proc. Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981 Proc. Natl. Acad. Sci. USA 78:1527), gpt, which confers resistance to mycophenolic acid (Mulligan and Berg, 1981 Proc. Natl. Acad. Sci. USA 78:2072), and neo, which confers resistance to the aminoglycoside G-418 (Clinical Pharmacy 12:488-505; Wu and Wu, 1991 Biotherapy 12:488-505). 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), as well as hygro, which confers resistance to hygromycin (Santerre et al. 1984 Gene 30:147).Methods known in the art of recombinant DNA technology that may be used are described in Ausubel et al. (eds., 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY; and Chapters 12 and 13 of Dracopoli et al. (eds.), 1994, Current Protocols in Human Genetics, John Wiley & Sons, NY; Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1).

[0119] In some instances, antibody expression levels can be increased by vector amplification (for a review, see Bebbington and Hentschel, "The Use of Vectors Based on Gene Amplification for the Expression of Cloned Genes in Mammalian Cells in DNA Cloning," Vol. 3, Academic Press, New York, 1987). If the antibody expression vector-based marker is amplifiable, increasing the level of inhibitor present in the host cell culture will result in an increase in marker gene copy number. Since the amplified region is associated with the antibody sequence, antibody production will also increase (Crouse et al., 1983, Mol. Cell Biol. 3:257).

[0120] Optionally, any method known in the art for purifying antibodies is used, such as chromatography (e.g., ion exchange, affinity, particularly affinity for specific antigens followed by Protein A, and sizing column chromatography), centrifugation, differential solubility, or any other standard technique for protein purification.

[0121] Expression vector In some embodiments, the vector comprises any suitable vector derived from either a eukaryotic or prokaryotic source. Optionally, the vector is obtained from a bacterial source (e.g., E. coli), an insect source, a yeast source (e.g., Pichia pastoris), an algae source, or a mammalian source. 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.

[0122] 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 and pPolh-MAT2, or MAT vectors such as pPolh-MAT1 and pPolh-MAT2.

[0123] Optionally, the yeast vector includes a Gateway® pDEST™ 14 vector, a Gateway® pDEST™ 15 vector, a Gateway® pDEST™ 17 vector, a Gateway® pDEST™ 24 vector, a Gateway® pYES-DEST52 vector, a pBAD-DEST49 Gateway® destination vector, a pAO815 Pichia vector, a pFLD1 Pichi pastoris vector, a pGAPZA,B,&C Pichia pastoris vector, a pPIC3.5K Pichia vector, a pPIC6 A,B,&C Pichia vector, a pPIC9K Pichia vector, pTEF1 / Zeo, a pYES2 yeast vector, a pYES2 / CT yeast vector, a pYES2 / NT A,B,&C yeast vector, or a pYES3 / CT yeast vector.

[0124] Exemplary algal vectors include the pChlamy-4 vector and the MCS vector.

[0125] Examples of mammalian vectors include transient expression vectors and 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 vectors 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.

[0126] In some instances, cell-free systems are mixtures of cytoplasmic and / or nuclear components from cells and are used for in vitro nucleic acid synthesis. In some cases, cell-free systems utilize either prokaryotic or eukaryotic components. Sometimes nucleic acid synthesis is achieved in cell-free systems based on, for example, Drosophila cells, Xenopus eggs, or HeLa cells. Exemplary cell-free systems include, but are not limited to, the E. coli S30 Extract system, the E. coli T7 S30 system, or PURExpress®.

[0127] host cell In some embodiments, the host cell includes any suitable cell, such as a naturally occurring cell or a genetically modified cell. In some examples, the host cell is a production host cell. In some examples, the host cell is a eukaryotic cell. In other examples, the host cell is a prokaryotic cell. Optionally, the eukaryotic cell includes a fungus (e.g., a yeast cell), an animal cell, or a plant cell. Optionally, the prokaryotic cell is a bacterial cell. Examples of bacterial cells include gram-positive and gram-negative bacteria. Sometimes, the gram-negative bacteria are anaerobes, bacilli, or both.

[0128] In some examples, Gram-positive bacteria include Actinomycetes, Firmicutes, and Tenericutes. In some cases, Gram-negative bacteria include Aquifex, Deinococcus Thermus, Fibrobacter-Chlorobium / Bacteroides (FCB group), Fusobacterium, Gemmatimonas, Nitrospira, Planctomycetes-Verrucomicrobium / Chlamydia (PVC group), Proteobacteria, Spirochetes, or Synergists. Other bacteria may be Acidobacteria, Chloroflexi, Chrysiogenes, Cyanobacteria, Deferibacter, Dictyoglomi, Thermodesulfobacteria, or Thermotoga. Bacterial cells may be Escherichia coli, Clostridium botulinum, or Coli bacilli.

[0129] Exemplary prokaryotic host cells include, but are not limited to, BL21, Mach1™, DH10B™, TOP10, DH5α, DH10Bac™, OmniMax™, MegaX™, DH12S™, INV110, TOP10F′, InvαF, TOP10 / P3, ccdB Survival, PIR1, PIR2, Stbl2™, Stbl3™, or Stbl4™.

[0130] In some examples, the animal cell comprises a cell derived from a vertebrate or invertebrate. Optionally, the animal cell comprises a cell derived from a marine invertebrate, a fish, an insect, an amphibian, a reptile, or a mammal. Optionally, the fungal cell comprises a yeast cell, such as a brewer's yeast, baker's yeast, or wine yeast.

[0131] Fungi include ascomycota, such as yeasts, molds, filamentous fungi, basidiomycota, and zygomycota. In some instances, yeasts include the Ascomycota or Basidiomycota phylum. Optionally, the Ascomycota includes the subphylum Saccharomycetes (true yeasts, e.g., Saccharomyces cerevisiae (baker's yeast)) and the subphylum Taphrina (e.g., Schizosaccharomycetes (fission yeast)). Optionally, the phylum Basidiomycota includes the subphylum Agaricomycetes (e.g., Tremellomycetes) and the subphylum Urobacterium (e.g., Microbotryomycetes).

[0132] Exemplary yeasts or filamentous fungi include, for example, Saccharomyces, Schizosaccharomyces, Candida, Pichia, Hansenula, Kluyveromyces, Zygosaccharomyces, Yarrowia, Trichosporon, Rhodosporidi, Aspergillus, Fusarium, or Trichoderma. Exemplary yeasts or filamentous fungi include, for example, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida utilis, Candida boidini, Candida albicans, Candida tropicalis, Candida stellatoidea, Candida glabrata, Candida krusei, Candida parapsilosis, Candida guilliermondii, Candida viswanathi, Candida lusitaniae, Rhodotorula mutilaginosa, Pichia methanolica, 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 brusselsii, Candida stellata, Schizosaccharomyces pombe, Torulaspora delbrueckii, Zygosaccharomyces bailii, Cryptococcus neoformans, Cryptococcus gattii, or Saccharomyces boulardii.

[0133] Exemplary yeast host cells include, but are not limited to, Pichia pastoris yeast strains such as GS115, KM71H, SMD1168, SMD1168H, X-33, and Saccharomyces cerevisiae yeast strains such as INVSc1.

[0134] In some examples, the additional animal cell comprises a cell obtained from a mollusk, an arthropod, an annelid, or a sponge.Optionally, the additional animal cell is a mammalian cell derived from, for example, a primate, an ape, a horse, a cow, a pig, a dog, a cat, or a rodent.Optionally, the rodent comprises a mouse, a rat, a hamster, a gerbil, a hamster, a chinchilla, a fancy rat, or a guinea pig.

[0135] Exemplary mammalian host cells include the 293A cell line, the 293FT cell line, 293F cells, 293H cells, CHO DG44 cells, CHO-S cells, CHO-K1 cells, and FUT8 KO cells. CHOK1, Expi293F(TM) cells, Flp-In(TM) T-REx(TM) 293 cell line, Flp-In(TM)-293 cell line, Flp-In(TM)-3T3 cell line, Flp -In(TM)-BHK Cell Line, Flp-In(TM)-CHO Cell Line, Flp-In(TM)-CV-1 Cell Line, Flp-In(TM)-Jurkat Cell Line, FreeStyle(TM) 293-F cells, FreeStyle(TM) CHO-S cells, GripTite(TM) 293MSR cell line, GS-CHO cell line, HepaRG(TM) cells, T-REx(TM) Jur Examples include, but are not limited to, kat cell line, Per.C6 cell, T-REx™-293 cell line, T-REx™-CHO cell line, T-REx™-HeLa cell line.

[0136] In some instances, the mammalian host cell is a stable cell line or a cell line that has integrated the genetic material of interest into its genome and is capable of expressing the product of the genetic material after many generations of cell division. Optionally, the mammalian host cell is a transient cell line or a cell line that has not integrated the genetic material of interest into its genome and is not capable of expressing the product of the genetic material after many generations of cell division.

[0137] Exemplary insect host cells include, but are not limited to, Drosophila S2 cells, Sf9 cells, Sf21 cells, High Five™ cells, and expresSF+® cells.

[0138] In some examples, plant cells include cells derived from algae. Exemplary insect cell lines include, but are not limited to, strains derived from Chlamydomonas reinhardtii 137c and Synechococcus elongatus PPC7942.

[0139] manufactured goods Another aspect of the present invention provides an article of manufacture containing materials useful for the treatment, prevention, and / or diagnosis of the aforementioned disorders. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container may be formed from a variety of materials, such as glass or plastic. The container holds a composition that is effective, by itself or in combination with another composition, to treat, prevent, and / or diagnose a disease and may have a sterile access port (e.g., the solution may be an IV bag or vial with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is a bispecific antibody comprising a first antigen-binding site that specifically binds to CD3 and a second antigen-binding site that specifically binds to a tumor antigen.

[0140] The label or package insert indicates that the composition is used to treat a disease of choice. Additionally, the article of manufacture may comprise (a) a first container containing a composition comprising a bispecific antibody of the invention, and (b) a second container containing a composition comprising an additional cytotoxic or other therapeutic agent. The article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the composition can be used to treat a particular disease.

[0141] Alternatively, or additionally, the article of manufacture may further comprise a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, dextrose solution, etc. It may further comprise other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0142] Treatment method In some embodiments, an isolated polypeptide comprising a cleavable linker described herein is used in a method of treating cancer. In some embodiments, the cancer has cells that express EGFR. In some embodiments, a polypeptide or polypeptide complex described herein is 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, breast / rectal cancer, head and neck cancer, esophageal cancer, liver cancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, kidney cancer, or pancreatic cancer. In some embodiments, a polypeptide or polypeptide complex described herein is used in a method of treating a subject resistant to treatment with an EGFR inhibitor. In some embodiments, a polypeptide or polypeptide complex described herein is used in a method of treating a subject with a KRAS mutation. In some embodiments, a polypeptide or polypeptide complex described herein is used in a method of treating a subject with a KRAS mutation that is resistant to treatment with an EGFR inhibitor.

[0143] While preferred embodiments of the present invention have been shown and described herein, it will be apparent 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 will be understood that various alternatives to the embodiments of the invention described herein may be used in practicing the invention. The following claims define the scope of the invention, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0144] Embodiment Embodiment 1 includes an isolated polypeptide comprising a cleavable linker according to the amino acid sequence of SEQ ID NO:1 (LSGRSDAG).

[0145] Embodiment 2 includes the isolated polypeptide of embodiment 1, wherein the cleavable linker comprises the amino acid sequence of SEQ ID NO:3 (ISSGLLSGRSDAG).

[0146] Embodiment 3 comprises the isolated polypeptide of any one of embodiments 1 to 2, wherein the cleavable linker comprises the amino acid sequence of SEQ ID NO:26 (AGLLAPPGGLSGRSDAG).

[0147] Embodiment 4 comprises the isolated polypeptide of any one of embodiments 1 to 3, wherein the cleavable linker comprises the amino acid sequence of SEQ ID NO: 4 (AAGLLAPPGGLSGRSDAG).

[0148] Embodiment 5 comprises the isolated polypeptide of any one of embodiments 1 to 4, wherein the cleavable linker comprises the amino acid sequence of SEQ ID NO:5 (SPLGLSGRSDAG).

[0149] Embodiment 6 comprises the isolated polypeptide of any one of embodiments 1 to 5, wherein the cleavable linker comprises the amino acid sequence of SEQ ID NO: 6 (LSGRSDAGSPLGLAG).

[0150] Embodiment 7 comprises the isolated polypeptide of any one of embodiments 1 to 6, wherein the cleavable linker is cleavable by a protease.

[0151] Embodiment 8 includes the isolated polypeptide of embodiment 7, wherein the protease comprises a tumor-specific protease.

[0152] Embodiment 9 comprises the isolated polypeptide of any one of embodiments 7 to 8, wherein the protease comprises a matrix metalloproteinase (MMP) or a serine protease.

[0153] Embodiment 10 includes the isolated polypeptide of embodiment 9, wherein the matrix metalloprotease includes MMP2, MMP7, MMP9, MMP13, or MMP14.

[0154] Embodiment 11 includes the isolated polypeptide of embodiment 9, wherein the serine protease comprises matriptase, urokinase, or hepsin.

[0155] Embodiment 12 comprises the isolated polypeptide of any one of embodiments 1 to 11, further comprising an antigen-binding domain that binds to a target antigen.

[0156] Embodiment 13 comprises the isolated polypeptide of embodiment 12, wherein the antigen-binding domain is C-terminal to the cleavable linker.

[0157] Embodiment 14 comprises the isolated polypeptide of any one of embodiments 1 to 11, further comprising a cytokine or cytokine fragment that binds to a cytokine receptor.

[0158] Embodiment 15 includes the isolated polypeptide of embodiment 14, wherein the cytokine or cytokine fragment is C-terminal to a cleavable linker.

[0159] Embodiment 16 includes the isolated polypeptide of any one of embodiments 1 to 15, wherein the cleavable linker connects the peptide to the antigen-binding domain that binds to the target antigen, or to the cytokine or cytokine fragment that binds to the cytokine receptor, in an arrangement according to Formula I: A1-L1-P1, where A1 comprises the antigen-binding domain that binds to the target antigen, or the cytokine or cytokine fragment that binds to the cytokine receptor, L1 comprises the cleavable linker, and P1 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.

[0160] Embodiment 17 includes the isolated polypeptide of embodiment 16, wherein P1 is connected to a cleavable linker at the N-terminus and A1 is connected to a cleavable linker at the C-terminus.

[0161] Embodiment 18 includes the isolated polypeptide of embodiment 16, wherein P1 is connected to a cleavable linker at the C-terminus and A1 is connected to a cleavable linker at the N-terminus.

[0162] Embodiment 19 includes the isolated polypeptide of any one of embodiments 16 to 18, wherein P1 is bound to A1 through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bonding interactions, or a combination thereof.

[0163] Embodiment 20 comprises the isolated polypeptide of any one of embodiments 16 to 19, wherein P1 has less than 70% sequence homology to the target antigen or cytokine receptor.

[0164] Embodiment 21 comprises the isolated polypeptide of any one of embodiments 16 to 20, wherein P1 comprises a peptide sequence at least 10 amino acids in length.

[0165] Embodiment 22 comprises the isolated polypeptide of any one of embodiments 16 to 21, wherein P1 comprises a peptide sequence at least 10 amino acids in length and no more than 20 amino acids in length.

[0166] Embodiment 23 comprises the isolated polypeptide of any one of embodiments 16 to 22, wherein P1 comprises a peptide sequence at least 16 amino acids in length.

[0167] Embodiment 24 comprises the isolated polypeptide of any one of embodiments 16 to 23, wherein P1 comprises a peptide sequence of 40 or fewer amino acids in length.

[0168] Embodiment 25 comprises the isolated polypeptide of any one of embodiments 16 to 24, wherein P1 comprises a cyclic or linear peptide.

[0169] Embodiment 26 comprises the isolated polypeptide of any one of embodiments 16 to 25, wherein P1 comprises a cyclic peptide.

[0170] Embodiment 27 comprises the isolated polypeptide of any one of embodiments 16 to 26, wherein P1 is further linked to a half-life extending moiety.

[0171] Embodiment 28 comprises the isolated polypeptide of embodiment 27, wherein the half-life extending moiety is a single domain antibody.

[0172] Embodiment 29 comprises the isolated polypeptide of embodiment 28, wherein the single domain antibody comprises 10G.

[0173] Embodiment 30 comprises the isolated polypeptide of any one of embodiments 16 to 29, wherein A1 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 of a camelid-derived single domain antibody (VHH), a Fab, a Fab', a Fab light chain polypeptide, or a Fab heavy chain polypeptide.

[0174] Embodiment 31 comprises the isolated polypeptide of any one of embodiments 16 to 30, wherein the target antigen comprises a tumor antigen.

[0175] Embodiment 32 comprises the isolated polypeptide of any one of embodiments 30 to 31, wherein A1 comprises a Fab light chain polypeptide or a Fab heavy chain polypeptide.

[0176] Embodiment 33 comprises the isolated polypeptide of any one of embodiments 16 to 32, wherein A1 comprises an epidermal growth factor receptor (EGFR) binding domain.

[0177] Embodiment 34 comprises the isolated polypeptide of any one of embodiments 16 to 30, wherein the target antigen comprises an effector cell antigen.

[0178] Embodiment 35 comprises the isolated polypeptide of embodiment 34, wherein A1 comprises an scfv.

[0179] Embodiment 36 comprises the isolated polypeptide of embodiment 35, wherein the scFv comprises an anti-CD3e single chain variable fragment.

[0180] Embodiment 37 comprises the isolated polypeptide of any one of embodiments 16 to 29, wherein A1 comprises a cytokine.

[0181] Embodiment 38 includes the isolated polypeptide of embodiment 37, wherein the cytokine or cytokine fragment is a wild-type cytokine.

[0182] Embodiment 38 includes the isolated polypeptide of embodiment 37, wherein the cytokine or cytokine fragment is a mutein of a cytokine.

[0183] Embodiment 40 comprises the isolated polypeptide of any one of embodiments 37 to 39, wherein the cytokine receptor is an interferon receptor or an interleukin receptor.

[0184] Embodiment 41 comprises the isolated polypeptide of any one of Embodiments 37 to 40, wherein the cytokine receptor comprises an interferon receptor, a GM-CSF receptor, an IL-2 receptor, an IL-4 receptor, an IL-6 receptor, an IL-7 receptor, an IL-10 receptor, an IL-12 receptor, an IL-15 receptor, an IL-21 receptor, or a TGF-β receptor.

[0185] Embodiment 42 comprises the isolated polypeptide of any one of embodiments 37 to 41, wherein the cytokine or cytokine fragment comprises interferon, GM-CSF, IL-2, IL-7, IL-12, IL-15, or IL-21.

[0186] Embodiment 43 comprises the isolated polypeptide of any one of embodiments 37 to 42, wherein the cytokine or cytokine fragment comprises IL-2, IL-12, IL-6, IL-4, IL-10, or TGFβ.

[0187] Embodiment 44 comprises the isolated polypeptide of any one of embodiments 1 to 43 complexed with a second isolated polypeptide comprising a second antigen-binding domain or a second cytokine or a second cytokine fragment.

[0188] Embodiment 45 includes the isolated polypeptide of embodiment 44, wherein the second isolated polypeptide is in an arrangement according to Formula II: A2-L2-P2, where A2 comprises a second antigen-binding domain or a second cytokine, L2 comprises a second cleavable linker, and P2 comprises a second peptide that inhibits binding of the second antigen-binding domain to a second target antigen or inhibits binding of the second cytokine or second cytokine fragment to a second cytokine receptor.

[0189] Embodiment 46 comprises the isolated polypeptide of embodiment 45, wherein the second cleavable linker comprises the amino acid sequence of SEQ ID NO:1 (LSGRSDAG).

[0190] Embodiment 47 comprises the isolated polypeptide of any one of embodiments 45 to 46, wherein the second cleavable linker comprises the amino acid sequence of SEQ ID NO: 3 (ISSGLLSGRSDAG).

[0191] Embodiment 48 comprises the isolated polypeptide of any one of embodiments 45 to 47, wherein the second cleavable linker comprises the amino acid sequence of SEQ ID NO: 26 (AGLLAPPGGLSGRSDAG).

[0192] Embodiment 49 comprises the isolated polypeptide of any one of embodiments 45 to 48, wherein the second cleavable linker comprises the amino acid sequence of SEQ ID NO: 4 (AAGLLAPPGGLSGRSDAG).

[0193] Embodiment 50 comprises the isolated polypeptide of any one of embodiments 45 to 49, wherein the second cleavable linker comprises the amino acid sequence of SEQ ID NO: 5 (SPLGLSGRSDAG).

[0194] Embodiment 51 comprises the isolated polypeptide of any one of embodiments 45 to 50, wherein the second cleavable linker comprises the amino acid sequence of SEQ ID NO: 6 (LSGRSDAGSPLGLAG).

[0195] Embodiment 52 comprises the isolated polypeptide of any one of embodiments 45 to 51, wherein P2 is connected at the N-terminus to a second cleavable linker and A2 is connected at the C-terminus to a second cleavable linker.

[0196] Embodiment 53 comprises the isolated polypeptide of any one of embodiments 45 to 51, wherein P2 is connected to the second cleavable linker at the C-terminus and A2 is connected to the second cleavable linker at the N-terminus.

[0197] Embodiment 54 includes the isolated polypeptide of any one of embodiments 45 to 53, wherein P2 is bound to A2 via ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bonding interactions, or a combination thereof.

[0198] Embodiment 55 comprises the isolated polypeptide of any one of embodiments 45 to 54, wherein P2 has less than 70% sequence homology to the second target antigen or the second cytokine receptor.

[0199] Embodiment 56 comprises the isolated polypeptide of any one of embodiments 45 to 55, wherein P2 comprises a peptide sequence at least 10 amino acids in length.

[0200] Embodiment 57 comprises the isolated polypeptide of any one of embodiments 45 to 56, wherein P2 comprises a peptide sequence at least 10 amino acids in length and no more than 20 amino acids in length.

[0201] Embodiment 58 comprises the isolated polypeptide of any one of embodiments 45 to 57, wherein P2 comprises a peptide sequence at least 16 amino acids in length.

[0202] Embodiment 59 comprises the isolated polypeptide of any one of embodiments 45 to 56, wherein P2 comprises a peptide sequence of 40 or fewer amino acids in length.

[0203] Embodiment 60 comprises the isolated polypeptide of any one of embodiments 45 to 59, wherein P2 comprises a cyclic or linear peptide.

[0204] Embodiment 61 comprises the isolated polypeptide of any one of embodiments 45 to 60, wherein P2 comprises a cyclic peptide.

[0205] Embodiment 62 comprises the isolated polypeptide of any one of embodiments 45 to 61, wherein A2 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 of a camelid-derived single domain antibody (VHH), a Fab, a Fab', a Fab light chain polypeptide, or a Fab heavy chain polypeptide.

[0206] Embodiment 63 comprises the isolated polypeptide of any one of embodiments 45 to 62, wherein the second target antigen comprises a tumor antigen.

[0207] Embodiment 64 comprises the isolated polypeptide of embodiment 62, wherein A2 comprises a Fab light chain polypeptide or a Fab heavy chain polypeptide.

[0208] Embodiment 65 comprises the isolated polypeptide of any one of embodiments 45 to 64, wherein A2 comprises an epidermal growth factor receptor (EGFR) binding domain.

[0209] Embodiment 66 comprises the isolated polypeptide of any one of embodiments 45 to 62, wherein the second target antigen comprises an effector cell antigen.

[0210] Embodiment 67 comprises the isolated polypeptide of embodiment 62, wherein A2 comprises an scfv.

[0211] Embodiment 68 comprises the isolated polypeptide of any one of embodiments 66 to 67, wherein the scFv comprises an anti-CD3e single chain variable fragment.

[0212] Embodiment 69 comprises the isolated polypeptide of any one of embodiments 45 to 61, wherein A2 comprises a second cytokine.

[0213] Embodiment 70 includes the isolated polypeptide of embodiment 69, wherein the second cytokine or second cytokine fragment is a wild-type cytokine.

[0214] Embodiment 71 includes the isolated polypeptide of embodiment 69, wherein the second cytokine or second cytokine fragment is a mutein of a cytokine.

[0215] Embodiment 72 comprises the isolated polypeptide of any one of embodiments 69 to 71, wherein the second cytokine receptor is an interferon receptor or an interleukin receptor.

[0216] Embodiment 73 comprises the isolated polypeptide of any one of embodiments 69 to 72, wherein the second cytokine receptor comprises an interferon receptor, a GM-CSF receptor, an IL-2 receptor, an IL-4 receptor, an IL-6 receptor, an IL-7 receptor, an IL-10 receptor, an IL-12 receptor, an IL-15 receptor, an IL-21 receptor, or a TGF-β receptor.

[0217] Embodiment 74 includes the isolated polypeptide of any one of embodiments 69 to 73, wherein the second cytokine or second cytokine fragment comprises interferon, GM-CSF, IL-2, IL-7, IL-12, IL-15, or IL-21.

[0218] Embodiment 75 comprises the isolated polypeptide of any one of embodiments 69 to 74, wherein the second cytokine or second cytokine fragment comprises IL-2, IL-12, IL-6, IL-4, IL-10, or TGFβ.

[0219] Embodiment 76 comprises a pharmaceutical composition comprising an isolated polypeptide comprising a cleavable linker according to any one of the preceding embodiments and a pharmaceutically acceptable excipient.

[0220] Embodiment 77 includes an isolated, recombinant nucleic acid molecule encoding an isolated polypeptide comprising a cleavable linker according to any one of the preceding embodiments.

[0221] Embodiment 78 includes a vector comprising the isolated recombinant nucleic acid molecule of embodiment 77.

[0222] Embodiment 79 includes a method for producing an isolated polypeptide comprising a cleavable linker according to any one of the preceding embodiments, the method comprising culturing a cell comprising the vector according to embodiment 78 under conditions that result in expression of the polypeptide.

[0223] Embodiment 80 includes a method of producing an isolated polypeptide comprising a cleavable linker, the method comprising the steps of (a) culturing a cell comprising the isolated recombinant nucleic acid molecule of embodiment 77 under conditions resulting in expression of the polypeptide, and (b) isolating the polypeptide. [Example]

[0224] Example 1. Proteolysis rate and serum stability The polypeptide complexes were evaluated for tumor and serum protease activity.

[0225] Briefly, we generated polypeptide complexes PC-1, PC-2, PC-3, PC-4, and PC-5, which contain 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 rates were determined when the polypeptide complexes were exposed to MMP2, MMP7, MMP9, MMP13, MMP14, uPa, MTSP1, and hepsin. Data on apparent cleavage rates and relative serum stability can be found in Tables 2–4.

[0226] [Table 2]

[0227] [Table 3]

[0228] [Table 4]

[0229] The data show that serum proteolytic activity is higher than blood. The data also show that the cleavable linker sequence increased the proteolytic rate while maintaining stability in human serum.

[0230] Example 2. Confirmation of equivalent masking with cleavable linkers The polypeptide complexes were evaluated for EGFR and CD3ε binding.

[0231] Briefly, we determined the binding of polypeptide complexes PC-1, PC-2, PC-3, PC-4, and PC-5 containing EGFR masking. As seen in Figure 1A, EGFR masking blocks binding to the various polypeptide complexes. After cleavage by the tumor protease MTSP1, the polypeptide complexes become available for binding (Figure 1B).

[0232] Details of the EGFR binding shifts can be seen in Tables 5-8.

[0233] [Table 5]

[0234] [Table 6]

[0235] [Table 7]

[0236] [Table 8]

[0237] The polypeptide complexes were further evaluated for CD3ε binding. Briefly, binding of polypeptide complexes PC-1, PC-2, PC-3, PC-4, and PC-5, which contain CD3ε masking, was determined. As seen in Figure 2A, this mask blocks binding to the various polypeptide complexes. After cleavage by the tumor protease MTSP1, the polypeptide complexes become available for binding (Figure 2B).

[0238] Details of the CD3ε binding shifts can be found in Tables 9-12.

[0239] [Table 9]

[0240] [Table 10]

[0241] [Table 11]

[0242] [Table 12]

[0243] Binding of the polypeptide complexes was assessed using an enzyme-linked immunosorbent assay (ELISA). Biotinylated peptides were captured on neutravidin-coated plates. A secondary antibody was used to detect bound polypeptide complexes. Data for PC-2, PC-3, PC-4, PC-5, PC-1, and PC-6, both with and after mask cleavage, are shown in Figures 3A-B, and EC50 binding data are summarized in Tables 13-14.

[0244] [Table 13]

[0245] [Table 14]

[0246] Example 3. Confirmation of equivalent T cell shift with cleavable linkers The polypeptide complexes were then evaluated in functional in vitro tumor cell killing and associated T cell activation assays.

[0247] Briefly, HCT116 cells were plated onto 96-well tissue-culture-treated flat-bottom plates and allowed to adhere overnight. The following day, the culture medium and non-adherent cells were removed and replaced with fresh medium containing titrated polypeptide complexes at the indicated concentrations. Data for PC-2, PC-3, PC-4, PC-5, PC-1, and PC-6 can be seen in Figure 14 and Tables 15-16.

[0248] [Table 15]

[0249] [Table 16]

[0250] Example 4. In vivo PK comparison in cynomolgus monkeys The polypeptide conjugates were evaluated for pharmacokinetics and safety in cynomolgus monkeys.

[0251] Cynomolgus monkeys

[0252] Young, male, naive cynomolgus monkeys were housed in pairs and group-housed and identified with unique body tattoos. All animals were acclimated to the housing conditions for three days prior to the start of the study. Prior to initiation, all animals underwent a physical examination by the study veterinarian. Only animals that were healthy in the opinion of the study veterinarian and otherwise met the criteria were enrolled in the study. Food was withheld overnight prior to dosing. Purina 5049 was provided daily in amounts appropriate for the animal's size. Tap water was available ad libitum via an automatic watering system.

[0253] Pharmacokinetics

[0254] The pharmacokinetics of polypeptide conjugates PC-1, PC-2, PC-3, PC-4, and PC-5 were determined in naive male cynomolgus monkeys weighing 2–3 kg. Briefly, two housed groups of monkeys were used per treatment group and allowed to acclimate to their surroundings before administration. Prior to dosing and bleeding, animals were sedated with ketamine HCl at 10–20 mg / kg IM. Concentrated test substances were diluted in sterile phosphate-buffered saline and administered to animals at a dose equivalent to the animal's mass in kg. Each test substance dose was administered intravenously at a volume of 1 mL / kg. For administration, the left and right limbs were clipped and prepped with alcohol. The saphenous vein was identified and a standard catheter was placed to allow for IV bolus injections (in either the left or right limb). The test substance dosing solution was drawn into the catheter via a syringe, and the bolus injection was administered by manually compressing the syringe.

[0255] For blood collection, animals were sedated with ketamine, the deltoid femoris muscle was prepared, and blood was collected from the femoral vein using a 22G 1.5-inch needle, a vacutainer sheath, and a collection tube. After venipuncture, manual compression of the vein was maintained until hemostasis was achieved. Blood collection was based on the animal's weight and did not exceed the AGI maximum blood loss limit specified by the IACUC. Blood was collected into EDTA tubes and processed to plasma. Blood samples were chilled and centrifuged at 3000 x g for 10 minutes to separate cells from the plasma. The plasma supernatant was collected and stored frozen prior to analysis.

[0256] Polypeptide complex concentrations in cynomolgus monkey plasma samples were determined by ELISA. Briefly, anti-His tag capture antibodies were coated directly onto ELISA plates. Standard dilutions of the polypeptide complex in cynomolgus monkey serum were used to generate a standard curve to which animal PK test samples could be compared. Standard and test samples were added to the plate and incubated overnight in the cold. Several different dilutions of the test sample were used to ensure that the signal was within the appropriate dynamic range of the standard curve. The plate was washed and briefly incubated with anti-human HRP detection antibody. The plate was washed, developed, and stopped using standard ELISA techniques. A standard curve plotting absorbance at 450 nm against known polypeptide complex concentrations was used to calculate the unspecified test article concentration for each mouse PK plasma sample. Polypeptide complex concentrations were plotted versus time and fit to a standard two-stage distribution and elimination pharmacokinetic model. The pharmacokinetics and parameters calculated for polypeptide conjugates PC-1, PC-7, PC-4, and PC-5 obtained from cynomolgus monkeys are shown in Figures 5A-5D and Tables 17-20.

[0257] [Table 17]

[0258] [Table 18]

[0259] [Table 19]

[0260] [Table 20]

[0261] These data demonstrate that polypeptide conjugates containing cleavable linkers have extended serum half-lives in cynomolgus monkeys.

[0262] Example 5. Cytokine release in cynomolgus monkeys in vivo Cytokine release was measured in cynomolgus monkeys.

[0263] Cytokines present in plasma after treatment were measured using a BD Biosciences non-human primate Th1 / Th2 cytometric bead array assay kit (catalog no. 557800) according to the manufacturer's instructions. The data are shown in Figures 6A-6D and Table 21.

[0264] [Table 21]

[0265] Example 6. In vivo liver ALT / AST in cynomolgus monkeys ALT / AST levels were measured. As can be seen in Figures 7A and 7B and Table 22, the polypeptide conjugate prevented hepatotoxicity in cynomolgus monkeys.

[0266] [Table 22]

[0267] While preferred embodiments of the present invention have been shown and described herein, it will be apparent 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 will be understood that various alternatives to the embodiments of the invention described herein may be used in practicing the invention. The following claims define the scope of the invention, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. An isolated polypeptide comprising a cleavable linker with the amino acid sequence of SEQ ID NO:1 (LSGRSDAG).

2. 2. The isolated polypeptide of claim 1, wherein the cleavable linker comprises the amino acid sequence of SEQ ID NO: 3 (ISSGLLSGRSDAG).

3. 2. The isolated polypeptide of claim 1, wherein the cleavable linker comprises the amino acid sequence of SEQ ID NO: 26 (AGLLAPPGGLSGRSDAG).

4. 2. The isolated polypeptide of claim 1, wherein the cleavable linker comprises the amino acid sequence of SEQ ID NO: 4 (AAGLLAPPGGLSGRSDAG).

5. 2. The isolated polypeptide of claim 1, wherein the cleavable linker comprises the amino acid sequence of SEQ ID NO:5 (SPLGLSGRSDAG).

6. 2. The isolated polypeptide of claim 1, wherein the cleavable linker comprises the amino acid sequence of SEQ ID NO: 6 (LSGRSDAGSPLGLAG).

7. The isolated polypeptide of claim 1, wherein the cleavable linker is cleavable by a protease.

8. The isolated polypeptide of claim 7 , wherein the protease comprises a tumor-specific protease.

9. 8. The isolated polypeptide of claim 7, wherein the protease comprises a matrix metalloprotease (MMP) or a serine protease.

10. 10. The isolated polypeptide of claim 9, wherein the matrix metalloprotease comprises MMP2, MMP7, MMP9, MMP13, or MMP14.

11. 10. The isolated polypeptide of claim 9, wherein the serine protease comprises matriptase, urokinase, or hepsin.

12. The isolated polypeptide of claim 1, further comprising an antigen-binding domain that binds to a target antigen.

13. 13. The isolated polypeptide of claim 12, wherein the antigen-binding domain is C-terminal to the cleavable linker.

14. 13. The isolated polypeptide of claim 12, further comprising a cytokine or cytokine fragment that binds to a cytokine receptor.

15. 15. The isolated polypeptide of claim 14, wherein the cytokine or cytokine fragment is C-terminal to the cleavable linker.

16. The cleavable linker has the formula I:A １ -L １ -P １ The peptide is connected 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 an arrangement according to the formula: １ comprises an antigen-binding domain that binds to the target antigen, or a cytokine or cytokine fragment that binds to the cytokine receptor, and L １ comprises the cleavable linker, and P １ The isolated polypeptide of claim 1 , wherein the polypeptide comprises a peptide that impairs binding of an antigen-binding domain to the target antigen or that impairs binding of a cytokine to the cytokine receptor.

17. P １ is connected at the N-terminus to the cleavable linker, and A １ 17. The isolated polypeptide of claim 16, wherein is connected to the cleavable linker at the C-terminus.

18. P １ is connected at the C-terminus to the cleavable linker, and A １ 17. The isolated polypeptide of claim 16, wherein is connected at the N-terminus to the cleavable linker.

19. P １ is connected to A through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bonding interactions, or a combination thereof. １ 17. The isolated polypeptide of claim 16, wherein the polypeptide is linked to

20. P １ 17. The isolated polypeptide of claim 16, wherein said polypeptide has less than 70% sequence homology to said target antigen or said cytokine receptor.

21. P １ 17. The isolated polypeptide of claim 16, wherein said polypeptide comprises a peptide sequence at least 10 amino acids in length.

22. P １ 17. The isolated polypeptide of claim 16, wherein said polypeptide comprises a peptide sequence at least 10 amino acids in length and no more than 20 amino acids in length.

23. P １ 23. The isolated polypeptide of claim 22, wherein said polypeptide comprises a peptide sequence at least 16 amino acids in length.

24. P １ 17. The isolated polypeptide of claim 16, wherein said polypeptide comprises a peptide sequence of 40 amino acids or less in length.

25. P １ 17. The isolated polypeptide of claim 16, wherein said polypeptide comprises a cyclic peptide or a linear peptide.

26. P １ 17. The isolated polypeptide of claim 16, wherein said polypeptide comprises a cyclic peptide.

27. P １ 17. The isolated polypeptide of claim 16, further linked to a half-life extending moiety.

28. 28. The isolated polypeptide of claim 27, wherein the half-life extending moiety is a single domain antibody.

29. 29. The isolated polypeptide of claim 28, wherein the single domain antibody comprises 10G.

30. A １ 17. The isolated polypeptide of claim 16, comprising 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 of a camelid-derived single domain antibody (VHH), a Fab, a Fab', a Fab light chain polypeptide, or a Fab heavy chain polypeptide.

31. 31. The isolated polypeptide of claim 30, wherein the target antigen comprises a tumor antigen.

32. A １ 32. The isolated polypeptide of claim 31 , wherein said Fab light chain polypeptide or said Fab heavy chain polypeptide is comprised of:

33. A １ 31. The isolated polypeptide of claim 30, wherein said polypeptide comprises an epidermal growth factor receptor (EGFR) binding domain.

34. 17. The isolated polypeptide of claim 16, wherein the target antigen comprises an effector cell antigen.

35. A １ 35. The isolated polypeptide of claim 34, wherein said scFv comprises

36. 36. The isolated polypeptide of claim 35, wherein the scFv comprises an anti-CD3e single-chain variable fragment.

37. A １ The isolated polypeptide of claim 16, wherein said polypeptide comprises said cytokine.

38. 38. The isolated polypeptide of claim 37, wherein the cytokine or cytokine fragment is a wild-type cytokine.

39. 38. The isolated polypeptide of claim 37, wherein the cytokine or cytokine fragment is a mutein of the cytokine.

40. 17. The isolated polypeptide of claim 16, wherein the cytokine receptor is an interferon receptor or an interleukin receptor.

41. 17. The isolated polypeptide of claim 16, wherein the cytokine receptor comprises an interferon receptor, a GM-CSF receptor, an IL-2 receptor, an IL-4 receptor, an IL-6 receptor, an IL-7 receptor, an IL-10 receptor, an IL-12 receptor, an IL-15 receptor, an IL-21 receptor, or a TGF-β receptor.

42. 17. The isolated polypeptide of claim 16, wherein the cytokine or cytokine fragment comprises an interferon, GM-CSF, IL-2, IL-7, IL-12, IL-15, or IL-21.

43. 17. The isolated polypeptide of claim 16, wherein the cytokine or cytokine fragment comprises IL-2, IL-12, IL-6, IL-4, IL-10, or TGFβ.

44. 17. The isolated polypeptide of claim 16, which forms a complex with a second isolated polypeptide comprising a second antigen-binding domain or a second cytokine or a second cytokine fragment.

45. The second isolated polypeptide has the formula II:A ２ -L ２ -P ２ In the formula, A ２ comprises the second antigen-binding domain or the second cytokine, and L ２ comprises a second cleavable linker, and P ２ 45. The isolated polypeptide of claim 44, wherein said second peptide inhibits binding of said second antigen-binding domain to a second target antigen or inhibits binding of said second cytokine or second cytokine fragment to a second cytokine receptor.

46. 46. ​​The isolated polypeptide of claim 45, wherein the second cleavable linker comprises the amino acid sequence of SEQ ID NO: 1 (LSGRSDAG).

47. 46. ​​The isolated polypeptide of claim 45, wherein the second cleavable linker comprises the amino acid sequence of SEQ ID NO: 3 (ISSGLLSGRSDAG).

48. 46. ​​The isolated polypeptide of claim 45, wherein the second cleavable linker comprises the amino acid sequence of SEQ ID NO: 26 (AGLLAPPGGLSGRSDAG).

49. 46. ​​The isolated polypeptide of claim 45, wherein the second cleavable linker comprises the amino acid sequence of SEQ ID NO: 4 (AAGLLAPPGGLSGRSDAG).

50. 46. ​​The isolated polypeptide of claim 45, wherein the second cleavable linker comprises the amino acid sequence of SEQ ID NO: 5 (SPLGLSGRSDAG).

51. 46. ​​The isolated polypeptide of claim 45, wherein the second cleavable linker comprises the amino acid sequence of SEQ ID NO: 6 (LSGRSDAGSPLGLAG).

52. P ２ is connected at the N-terminus to the second cleavable linker, and A ２ is connected at the C-terminus to the second cleavable linker.

53. P ２ is connected at the C-terminus to the second cleavable linker, and A ２ is connected at the N-terminus to the second cleavable linker.

54. P ２ is connected to A through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bonding interactions, or a combination thereof. ２ 46. ​​The isolated polypeptide of claim 45, wherein the polypeptide is linked to

55. P ２ 46. ​​The isolated polypeptide of claim 45, wherein said polypeptide has less than 70% sequence homology to said second target antigen or said second cytokine receptor.

56. P ２ 46. ​​The isolated polypeptide of claim 45, wherein said polypeptide comprises a peptide sequence at least 10 amino acids in length.

57. P ２ 46. ​​The isolated polypeptide of claim 45, wherein said polypeptide comprises a peptide sequence at least 10 amino acids in length and no more than 20 amino acids in length.

58. P ２ 58. The isolated polypeptide of claim 57, wherein said polypeptide comprises a peptide sequence at least 16 amino acids in length.

59. P ２ 46. ​​The isolated polypeptide of claim 45, wherein said polypeptide comprises a peptide sequence of 40 amino acids or less in length.

60. P ２ 46. ​​The isolated polypeptide of claim 45, wherein said polypeptide comprises a cyclic peptide or a linear peptide.

61. P ２ 46. ​​The isolated polypeptide of claim 45, wherein said polypeptide comprises a cyclic peptide.

62. A ２ 46. ​​The isolated polypeptide of claim 45, comprising 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 of a camelid-derived single domain antibody (VHH), a Fab, a Fab', a Fab light chain polypeptide, or a Fab heavy chain polypeptide.

63. 46. ​​The isolated polypeptide of claim 45, wherein the second target antigen comprises a tumor antigen.

64. A ２ 63. The isolated polypeptide of claim 62, wherein said Fab light chain polypeptide or said Fab heavy chain polypeptide is comprised of:

65. A ２ 63. The isolated polypeptide of claim 62, wherein said polypeptide comprises an epidermal growth factor receptor (EGFR) binding domain.

66. 46. ​​The isolated polypeptide of claim 45, wherein the second target antigen comprises an effector cell antigen.

67. A ２ 63. The isolated polypeptide of claim 62, wherein said scFv comprises

68. 68. The isolated polypeptide of claim 67, wherein the scFv comprises an anti-CD3e single-chain variable fragment.

69. A ２ 46. ​​The isolated polypeptide of claim 45, wherein said second cytokine comprises

70. 70. The isolated polypeptide of claim 69, wherein the second cytokine or second cytokine fragment is a wild-type cytokine.

71. 70. The isolated polypeptide of claim 69, wherein the second cytokine or second cytokine fragment is a mutein of the cytokine.

72. 70. The isolated polypeptide of claim 69, wherein the second cytokine receptor is an interferon receptor or an interleukin receptor.

73. 73. The isolated polypeptide of claim 72, wherein the second cytokine receptor comprises an interferon receptor, a GM-CSF receptor, an IL-2 receptor, an IL-4 receptor, an IL-6 receptor, an IL-7 receptor, an IL-10 receptor, an IL-12 receptor, an IL-15 receptor, an IL-21 receptor, or a TGF-β receptor.

74. 73. The isolated polypeptide of claim 72, wherein the second cytokine or second cytokine fragment comprises an interferon, GM-CSF, IL-2, IL-7, IL-12, IL-15, or IL-21.

75. 73. The isolated polypeptide of claim 72, wherein the second cytokine or second cytokine fragment comprises IL-2, IL-12, IL-6, IL-4, IL-10, or TGFβ.

76. A pharmaceutical composition comprising an isolated polypeptide comprising the cleavable linker of claim 1 and a pharmaceutically acceptable excipient.

77. 10. An isolated recombinant nucleic acid molecule encoding an isolated polypeptide comprising the cleavable linker of claim 1.

78. 78. A vector comprising the isolated recombinant nucleic acid molecule of claim 77.

79. 80. A method for producing an isolated polypeptide comprising a cleavable linker, the method comprising culturing a cell comprising the vector of claim 78 under conditions that result in expression of the polypeptide.

80. 78. A method for producing an isolated polypeptide comprising a cleavable linker, the method comprising: (a) culturing a cell comprising the isolated recombinant nucleic acid molecule of claim 77 under conditions that result in expression of the polypeptide; and (b) isolating the polypeptide.

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