Cleavable linker compositions and methods

Abstract

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.

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 Aug. 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 named 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 that comprises a cleavable linker according to the amino acid sequence of SEQ ID NO:1 (LSGRSDAG). In some embodiments, the cleavable linker comprises the amino acid sequence of SEQ ID NO:3 (ISSGLLSGRSDAG). In some embodiments, the cleavable linker comprises the amino acid sequence of SEQ ID NO:26 (AGLLAPPGGLSGRSDAG). In some embodiments, the cleavable linker comprises the amino acid sequence of SEQ ID NO:4 (AAGLLAPPGGLSGRSDAG). In some embodiments, the cleavable linker comprises the amino acid sequence of SEQ ID NO:5 (SPLGLSGRSDAG). In some embodiments, the cleavable linker comprises the amino acid sequence of SEQ ID NO:6 (LSGRSDAGSPLGLAG). In some embodiments, the cleavable linker is cleavable by a protease. In some embodiments, the protease comprises a tumor-specific protease. In some embodiments, the protease comprises a matrix metalloprotease (MMP) or a serine protease. In some embodiments, the matrix metalloprotease comprises MMP2, MMP7, MMP9, MMP13, or MMP14. In some embodiments, the serine protease comprises matriptase, urokinase, or hepsin. In some embodiments, the isolated polypeptide further comprises an antigen binding domain that binds to a target antigen. In some embodiments, the antigen binding domain is C-terminal to the cleavable linker. In some embodiments, the isolated polypeptide further comprises a cytokine or cytokine fragment that binds to a cytokine receptor. In some embodiments, the cytokine or cytokine fragment is C-terminal to the cleavable linker. In some embodiments, the cleavable linker is represented by 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: 1comprises an antigen-binding domain that binds to a target antigen, or a cytokine or cytokine fragment that binds to a cytokine receptor, 1 comprises a cleavable linker, P 1 comprises a peptide that impairs binding of an antigen-binding domain to a target antigen or that impairs binding of a cytokine to a cytokine receptor. 1 is connected at its N-terminus to a cleavable linker, 1 is connected at the C-terminus to a cleavable linker. 1 is connected at its C-terminus to a cleavable linker, 1 is connected to a cleavable linker at the N-terminus. 1 A is connected to A through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bonding interactions, or a combination thereof. 1 In some embodiments, P 1 has less than 70% sequence homology to the target antigen or cytokine receptor. 1 comprises a peptide sequence of at least 10 amino acids in length. 1 In some embodiments, P comprises a peptide sequence at least 10 amino acids in length and no more than 20 amino acids in length. 1 comprises a peptide sequence of at least 16 amino acids in length. 1 comprises a peptide sequence of 40 amino acids or less in length. 1 comprises a cyclic or linear peptide. In some embodiments, P 1 In some embodiments, P comprises a cyclic peptide. 1 is further linked to a half-life extending moiety. In some embodiments, the half-life extending moiety is a single domain antibody. In some embodiments, the single domain antibody comprises 10G. In some embodiments, A 1comprises 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, the A 1 comprises a Fab light chain polypeptide or a Fab heavy chain polypeptide. 1 In some embodiments, the target antigen comprises an effector cell antigen. 1 In some embodiments, the scFv comprises an anti-CD3e single chain variable fragment. 1 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, 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 cytokine or cytokine fragment comprises an interferon, a GM-CSF, an IL-2, an IL-7, an IL-12, an IL-15, or an IL-21. In some embodiments, the cytokine or cytokine fragment comprises an IL-2, an IL-12, an IL-6, an IL-4, an IL-10, or a TGFβ. In some embodiments, the isolated polypeptide forms a complex with a second isolated polypeptide comprising a second antigen-binding domain or a second cytokine or a second cytokine fragment. In some embodiments, the second isolated polypeptide has formula II:A 2 -L 2 -P 2In the formula, A 2 comprises a second antigen-binding domain or a second cytokine, and L 2 comprises a second cleavable linker, and P 2 comprises a second peptide that inhibits binding of the second antigen-binding domain to a second target antigen or inhibits binding of a second cytokine or a second cytokine fragment to a second cytokine receptor. In some embodiments, the second cleavable linker comprises the amino acid sequence of SEQ ID NO:1 (LSGRSDAG). In some embodiments, the second cleavable linker comprises the amino acid sequence of SEQ ID NO:3 (ISSGLLSGRSDAG). In some embodiments, the second cleavable linker comprises the amino acid sequence of SEQ ID NO:26 (AGLLAPPGGLSGRSDAG). In some embodiments, the second cleavable linker comprises the amino acid sequence of SEQ ID NO:4 (AAGLLAPPGGLSGRSDAG). In some embodiments, the second cleavable linker comprises the amino acid sequence of SEQ ID NO:5 (SPLGLSGRSDAG). In some embodiments, the second cleavable linker comprises the amino acid sequence of SEQ ID NO:6 (LSGRSDAGSPLGLAG). In some embodiments, P 2 is connected at its N-terminus to a second cleavable linker, and A 2 is connected at the C-terminus to a second cleavable linker. 2 is connected at its C-terminus to a second cleavable linker, 2 is connected at the N-terminus to a second cleavable linker. 2 A is connected to A through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bonding interactions, or a combination thereof. 2 In some embodiments, P 2 has less than 70% sequence homology to a second target antigen or a second cytokine receptor. 2 comprises a peptide sequence of at least 10 amino acids in length. 2In some embodiments, P comprises a peptide sequence at least 10 amino acids in length and no more than 20 amino acids in length. 2 comprises a peptide sequence of at least 16 amino acids in length. 2 comprises a peptide sequence of 40 amino acids or less in length. 2 comprises a cyclic or linear peptide. In some embodiments, P 2 In some embodiments, A comprises a cyclic peptide. 2 comprises an antibody, a single chain variable fragment (scFv), a heavy chain variable domain (VH domain), a light chain variable domain (VL domain), a variable domain 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, the A 2 comprises a Fab light chain polypeptide or a Fab heavy chain polypeptide. 2 In some embodiments, the second target antigen comprises an effector cell antigen. 2 In some embodiments, the scFv comprises an anti-CD3e single chain variable fragment. 2comprises a second cytokine. In some embodiments, the second cytokine or the second cytokine fragment is a wild-type cytokine. In some embodiments, the second cytokine or the 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 the second cytokine fragment comprises an interferon, a GM-CSF, an IL-2, an IL-7, an IL-12, an IL-15, or an IL-21. In some embodiments, the second cytokine or the second cytokine fragment comprises an IL-2, an IL-12, an IL-6, an IL-4, an IL-10, or a 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 pharma- ceutically 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 the 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, comprising culturing a cell comprising the vector of the above embodiments under conditions resulting in expression of the polypeptide.

[0008] Disclosed herein is a method for producing an isolated polypeptide comprising a cleavable linker, the method comprising the steps of: (a) culturing a cell comprising an isolated recombinant nucleic acid molecule of the above embodiments under conditions resulting in expression of the polypeptide; and (b) isolating the polypeptide. [Brief description 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 ​​of 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 ​​of polypeptide complexes PC-1, PC-2, PC-3, PC-4, and PC-5 in cynomolgus monkeys. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[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, there is a need to maintain the activity of the protein therapeutic in diseased tissues while minimizing the off-target effects of the protein therapeutic in healthy tissues. One such strategy is to create an inactive form of the protein therapeutic, where 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 when in healthy tissue. To activate the protein therapeutic in the desired disease state microenvironment, a peptide is linked to the protein therapeutic using a linker that is cleavable by a protease specific to the disease state microenvironment. The peptide is then released from the protein therapeutic when in the disease state microenvironment.

[0012] Accordingly, disclosed herein are cleavable linkers applicable to various forms of protein-based therapeutic agents that are used to maintain the activity of protein-based therapeutic agents in diseased tissues while reducing the off-target effects of protein-based therapeutic agents in healthy tissues. The cleavable linkers disclosed herein have desired properties, such as, but not limited to, an increased rate of proteolysis by tumor proteases compared to a control linker and being cleavable by an extended panel of tumor proteases while having an equivalent safety profile.

[0013] Specific Definitions The terms used herein are for the purpose of describing only specific examples and are 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 dictates otherwise. Further, the terms such as "including", "includes", "having", "has", "with", or variations thereof are intended to be included in the same manner as the term "comprising" as long as they are used in any of the detailed description and / or claims.

[0014] The term "about" or "approximately" means within an acceptable error range of a particular value as determined by one of ordinary skill in the art and will depend in part on how the value is measured or determined, e.g., the limitations of the measuring system. For example, "about" can mean within one or more standard deviations of a given value as it is implemented each time. When a particular value is set forth in the present application and claims, unless otherwise stated, the term "about" should be assumed to mean an acceptable error range for that particular value.

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

[0016] "Peptide", "P 1 " or "P 2 " 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 comprises 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.

[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 substitution, addition, or deletion results in an amino acid sequence that is at least 75% identical, such as 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 of the proteins described herein. In some embodiments, the amino acid substitution is a conservative amino acid substitution. 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 a non-natural amino acid, or a combination thereof. In some embodiments, the modified amino acid or the non-natural amino acid comprises a post-translational modification. In some embodiments, the cleavable linker comprises a modification including, but not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or a nucleotide derivative, covalent attachment of a lipid or a 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 may be made anywhere on the peptide backbone, on cleavable linkers or on amino acid side chains.

[0028] In some embodiments, the cleavable linker is cleavable by a protease. In some embodiments, the protease is present at higher levels in a disease state microenvironment compared to levels in healthy tissue or in 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, or 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 that does not have a cleavable linker sequence. In some embodiments, the cleavable linker has an increased proteolysis rate of at least 5 times compared to the proteolysis rate of a linker that does not have a cleavable linker sequence. In some embodiments, the cleavable linker has an increased proteolysis rate of at least 8 times compared to the proteolysis rate of a linker that does not have a cleavable linker sequence. In some embodiments, the cleavable linker has an increased proteolysis rate of at least 10 times compared to the proteolysis rate of a linker that does not have a cleavable linker sequence. In some embodiments, the cleavable linker has an increased proteolysis rate of at least 15 times compared to the proteolysis rate of a linker that does not have a cleavable linker sequence. In some embodiments, the cleavable linker has an increased proteolysis rate of at least 20 times compared to the proteolysis rate of a linker that does not have a cleavable linker sequence. In some embodiments, the cleavable linker has an increased proteolysis rate of at least 25 times compared to the proteolysis rate of a linker that does not have a cleavable linker sequence. In some embodiments, the cleavable linker has an increased proteolysis rate of at least 30 times compared to the proteolysis rate of a linker that does not have a cleavable linker sequence. In some embodiments, the cleavable linker has an increased proteolysis rate of at least 40 times compared to the proteolysis rate of a linker that does not have a cleavable linker sequence. In some embodiments, the cleavable linker has an increased proteolysis rate of at least 50 times compared to the proteolysis rate of a linker that does not have a cleavable linker sequence. In some embodiments, the cleavable linker has an increased proteolysis rate of at least 60 times compared to the proteolysis rate of a linker that does not have a cleavable linker sequence. In some embodiments, the cleavable linker has an increased proteolysis rate of at least 70 times compared to the proteolysis rate of a linker that does not have 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 in human serum of a linker that does not have a cleavable linker sequence. In some embodiments, the cleavable linker has at least 30-fold improved stability in human serum compared to the stability in human serum of a linker that does not have a cleavable linker sequence. In some embodiments, the cleavable linker has at least 40-fold improved stability in human serum compared to the stability in human serum of a linker that does not have a cleavable linker sequence. In some embodiments, the cleavable linker has at least 50-fold improved stability in human serum compared to the stability in human serum of a linker that does not have a cleavable linker sequence. In some embodiments, the cleavable linker has at least 60-fold improved stability in human serum compared to the stability in human serum of a linker that does not have a cleavable linker sequence. In some embodiments, the cleavable linker has at least 70-fold improved stability in human serum compared to the stability in human serum of a linker that does not have 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 human serum. 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 human serum. 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 human serum. 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 human serum. 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 in human serum.

[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 a 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 with the amino acid sequence of SEQ ID NO:2.

[0034] In some embodiments, the isolated polypeptide comprising a 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 with the amino acid sequence of SEQ ID NO:2.

[0035] In some embodiments, the isolated polypeptide comprising the cleavable linker has improved or comparable hepatotoxicity values ​​in cynomolgus monkeys compared to an isolated polypeptide of the same amino acid sequence but comprising a cleavable linker with 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: A 1 -L 1 -P 1 (Formula I) and connecting the peptide 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 the invention. In the formula, A 1 The L comprises an antigen-binding domain that binds to a target antigen, or a cytokine that binds to a cytokine receptor. 1 comprises a cleavable linker, P 1 comprises a peptide that impairs binding of an antigen-binding domain to a target antigen or that impairs binding of a cytokine to a cytokine receptor. 1 is connected at its N-terminus to a cleavable linker, 1is connected at the C-terminus to a cleavable linker. 1 is connected at its C-terminus to a cleavable linker, 1 is connected at its N-terminus to a cleavable linker.

[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 Formula II: A 2 -L 2 -P 2 (Formula II) In the arrangement by In the formula, A 2 comprises a second antigen-binding domain or a second cytokine, 2 comprises a second cleavable linker, and P 2 comprises a second peptide that impairs binding of the second antigen-binding domain to a second target antigen or impairs binding of a second cytokine to a second cytokine receptor.

[0039] In some embodiments, P 2 is connected at its N-terminus to a second cleavable linker, and A 2 is connected at the C-terminus to a second cleavable linker. 2 is connected at its C-terminus to a second cleavable linker, 2 is connected at the N-terminus to a second cleavable linker.

[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, L 1 or L 2 is at least 8 amino acids in length. 1 or L 2 is at least 10 amino acids in length but not more than 50 amino acids in length. 1 or L 2 is at least 10 amino acids in length but not more than 30 amino acids in length. 1 or L 2 is at least 18 amino acids in length. 1 or L 2 is at least 26 amino acids in length. 1 or L 2 is at least 30 amino acids in length. 1 or L 2 is at least 40 amino acids in length. 1 or L 2 is at least 50 amino acids in length.

[0042] Peptide (P 1 or P 2) In some embodiments, P 1 comprises a peptide that impairs binding of the antigen-binding domain to a target antigen. In some embodiments, P 1 In some embodiments, P comprises a peptide that impairs the binding of a cytokine to a cytokine receptor. 1 A is connected to A through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bonding interactions, or a combination thereof. 1 In some embodiments, P 1 A binds to the cytokine receptor at or near the binding site. 1 In some embodiments, P 1 A is located at or near the antigen-binding site. 1 In some embodiments, P 1 L 1 is cleaved by protease to form P 1 When exposed to a target antigen or cytokine receptor, A 1 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 P 1 A is a stimulator of the A receptor for target antigens or cytokine receptors by non-steric blocking. 1 In some embodiments, P 1 A binds to a target antigen or cytokine receptor through a covalent interaction. 1 In some embodiments, P 1 is not a cytokine, a cytokine binding fragment, a cytokine mutein, or a combination of the cognate receptors of those cytokines.1 is not an antibody or fragment thereof that binds to a cytokine receptor.

[0043] In some embodiments, P 2 comprises a peptide that impairs binding of the second antigen-binding domain to a second target antigen. In some embodiments, P 2 comprises a peptide that impairs binding of a second cytokine to a second cytokine receptor. 2 A is connected to A through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bonding interactions, or a combination thereof. 2 In some embodiments, P 2 A binds to the cytokine receptor at or near the binding site. 2 In some embodiments, P 2 A is located at or near the antigen-binding site. 2 In some embodiments, P 2 L 2 is cleaved by protease to form P 2 When exposed to a target antigen or cytokine receptor, A 2 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 P 2 A is a cytochrome P450 receptor agonist that binds to target antigens or cytokine receptors by non-steric blocking. 2 In some embodiments, P 2 A binds to a target antigen or cytokine receptor through a covalent interaction. 2 In some embodiments, P 2is not a cytokine, a cytokine binding fragment, a cytokine mutein, or a combination of the cognate receptors of those cytokines. 2 is not an antibody or fragment thereof that binds to a cytokine receptor.

[0044] In some embodiments, P 1 has less than 70% sequence homology to the target antigen. 1 has less than 75% sequence homology to the target antigen. 1 has less than 80% sequence homology to the target antigen. 1 has less than 85% sequence homology to the target antigen. 1 has less than 90% sequence homology to the target antigen. 1 has less than 95% sequence homology to the target antigen. 1 has less than 98% sequence homology to the target antigen. 1 has less than 99% sequence homology to the target antigen.

[0045] In some embodiments, P 1 has less than 70% sequence homology to a cytokine receptor. 1 has less than 75% sequence homology to a cytokine receptor. 1 has less than 80% sequence homology to a cytokine receptor. 1 has less than 85% sequence homology to a cytokine receptor. 1 has less than 90% sequence homology to a cytokine receptor. 1 has less than 95% sequence identity to a cytokine receptor. 1has less than 98% sequence identity to a cytokine receptor. 1 has less than 99% sequence homology to cytokine receptors.

[0046] In some embodiments, P 2 has less than 70% sequence homology to the second target antigen. 2 has less than 75% sequence homology to the second target antigen. 2 has less than 80% sequence homology to the second target antigen. 2 has less than 85% sequence homology to the second target antigen. 2 has less than 90% sequence homology to the second target antigen. 2 has less than 95% sequence homology to the second target antigen. 2 has less than 98% sequence identity to the second target antigen. 2 has less than 99% sequence homology to the second target antigen.

[0047] In some embodiments, P 2 has less than 70% sequence homology to the second cytokine receptor. 2 has less than 75% sequence homology to a second cytokine receptor. 2 has less than 80% sequence homology to the second cytokine receptor. 2 has less than 85% sequence homology to a second cytokine receptor. 2 has less than 90% sequence homology to the second cytokine receptor. 2 has less than 95% sequence homology to the second cytokine receptor. 2has less than 98% sequence identity to the second cytokine receptor. 2 has less than 99% sequence homology to a second cytokine receptor.

[0048] In some embodiments, P 1 or P 2 comprises a de novo amino acid sequence that shares less than 50% sequence homology to a cytokine, a cytokine receptor, or an antibody or fragment thereof that binds to a cytokine or cytokine receptor. 1 or P 2 comprises a de novo amino acid sequence that shares less than 40% sequence homology to a cytokine, a cytokine receptor, or an antibody or fragment thereof that binds to a cytokine or cytokine receptor. 1 or P 2 comprises a de novo amino acid sequence that shares less than 30% sequence homology to a cytokine, a cytokine receptor, or an antibody or fragment thereof that binds to a cytokine or cytokine receptor. 1 or P 2 comprises a de novo amino acid sequence that shares less than 20% sequence homology to a cytokine, a cytokine receptor, or an antibody or fragment thereof that binds to a cytokine or cytokine receptor. 1 or P 2 comprises a de novo amino acid sequence that shares less than 10% sequence homology to a cytokine, a cytokine receptor, or an antibody or fragment thereof that binds to a cytokine or cytokine receptor. 1 or P 2 are identified from a peptide library containing random amino acid sequences.

[0049] In some embodiments, P 1 or P 2comprises a de novo amino acid sequence that shares less than 50% sequence homology to the target antigen. 1 or P 2 comprises a de novo amino acid sequence that shares less than 40% sequence homology to the target antigen. 1 or P 2 comprises a de novo amino acid sequence that shares less than 30% sequence homology to the target antigen. 1 or P 2 comprises a de novo amino acid sequence that shares less than 20% sequence homology to the target antigen. 1 or P 2 comprises a de novo amino acid sequence that shares less than 10% sequence homology to the target antigen. 1 or P 2 are identified from a peptide library containing random amino acid sequences.

[0050] In some embodiments, P 1 or P 2 comprises a peptide sequence of at least 5 amino acids in length. 1 or P 2 comprises a peptide sequence of at least 6 amino acids in length. 1 or P 2 comprises a peptide sequence of at least 10 amino acids in length. 1 or P 2 In some embodiments, P comprises a peptide sequence at least 10 amino acids in length and no more than 20 amino acids in length. 1 or P 2 comprises a peptide sequence of at least 16 amino acids in length. 1 or P 2 comprises a peptide sequence of 40 amino acids or less in length. 1 or P 2 contains at least two cysteine ​​amino acid residues.1 or P 2 comprises a cyclic or linear peptide. In some embodiments, P 1 or P 2 In some embodiments, P comprises a cyclic peptide. 1 or P 2 comprises a linear peptide.

[0051] In some embodiments, P 1 or P 2 comprises modified amino acids or unnatural amino acids, or modified unnatural amino acids, or combinations thereof. In some embodiments, the modified amino acids or modified unnatural amino acids comprise post-translational modifications. In some embodiments, P 1 or P 2 Modifications include, but are 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 modified to modify the peptide backbone, amino acid side chains, and termini, including PAGEs. 1 or P 2 This is done everywhere.

[0052] In some embodiments, P 1 or P 2 does not contain albumin or albumin fragments. 1 or P 2 does not contain an albumin binding domain.

[0053] A 1 and A2 In some embodiments, A 1 Or A 2 is an antigen recognition molecule. In some embodiments, the antigen recognition 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, 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, A 1 Or A 2 In some embodiments, a Fab comprises (a) a Fab light chain polypeptide and (b) a Fab heavy chain polypeptide. In some embodiments, L 1 or L 2 is attached to the N-terminus of the Fab light chain polypeptide. 1 or L 2 is attached to the N-terminus of the Fab heavy chain polypeptide. 1 or L 2 is attached to the C-terminus of the Fab light chain polypeptide. 1 or L 2 is attached to the C-terminus of the Fab heavy chain polypeptide.

[0055] In some embodiments, A 1 Or A 2 is a single chain variable fragment (scFv). In some embodiments, L 1 or L 2 is attached to the N-terminus of the scFv. 1 or L 2is 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. 1 or L 2 is attached to the N-terminus of the light chain variable domain of the single chain variable fragment (scFv). 1 or L 2 is attached to the N-terminus of the heavy chain variable domain of a 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, A 1 Or A 2 A 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, A 1 Or A 2 comprises an anti-CD3e single chain variable fragment. 1 Or A 2 is the K of binding to CD3 on CD3-expressing cells D In some embodiments, the anti-CD3e single chain variable fragment has an A of 1 μM or less. 1 Or A 2 In some embodiments, A comprises a variable light chain and a variable heavy chain, each of which is capable of specifically binding to human CD3.1 Or A 2 are muromonab-CD3 (OKT3), otelixizumab (TRX4), teplizumab (MGA031), visilizumab (Nuvion), SP34, X35, VIT3, BMA030 (BW264 / 56), CLB-T3 / 3, CRIS7, YTH12.5, F111-409, CLB-T3.4.2, TR-66, WT32, SPv-T3b, 1 The complementarity determining region (CDR) is selected from the group consisting of 1D8, 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, A 1 Or A 2 CD3 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 disulfide bonds. 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. Upon engagement of the TCR with a target antigen and MHC, the T cell is activated, resulting in a series of signaling events mediated by associated enzymes, co-receptors, adapter molecules, and activated or released transcription factors.

[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 the β chain variable region (Vβ) are several types of regions that are 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 referred to as 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 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. In some embodiments, the soluble TCR comprises an alpha TCR polypeptide comprising a TCR alpha extracellular domain, and a beta TCR polypeptide comprising a TCR beta extracellular domain.

[0062] In some embodiments, the soluble TCR is a single chain TCR comprising the variable region of the TCR alpha extracellular domain or a fragment thereof, and the variable region of the TCR beta extracellular domain or a fragment thereof. In some embodiments, the soluble TCR comprises an alpha TCR polypeptide comprising the TCR alpha extracellular domain, and a beta TCR polypeptide comprising the TCR beta extracellular domain. In some embodiments, the L 1 is attached to the N-terminus of the αTCR polypeptide. 1 is attached to the N-terminus of the βTCR polypeptide. 2 is attached to the C-terminus of the αTCR polypeptide. 2 is attached to the N-terminus of the αTCR polypeptide. 2 is attached to the C-terminus of the βTCR polypeptide. 2 is attached to the N-terminus of the βTCR polypeptide. 1 is attached to the N-terminus of the αTCR polypeptide and 2 is attached to the N-terminus of the βTCR polypeptide. 1 is attached to the N-terminus of the αTCR polypeptide and 2 is attached to the C-terminus of the βTCR polypeptide. 1 is attached to the N-terminus of the αTCR polypeptide and 2 is attached to the C-terminus of the αTCR polypeptide. 1 is attached to the N-terminus of the βTCR polypeptide and 2 is attached to the N-terminus of the αTCR polypeptide. 1 is attached to the N-terminus of the βTCR polypeptide and 2 is attached to the C-terminus of the βTCR polypeptide. 1 is attached to the N-terminus of the βTCR polypeptide and 2 is attached to the C-terminus of the αTCR polypeptide.

[0063] In some embodiments, the polypeptide or polypeptide complex is 1 Or P 2 or L 1 Or L 2 In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity for the target antigen or the second target antigen compared to the binding affinity for the target antigen or the second target antigen of a polypeptide or polypeptide complex not having P. 1 Or P 2 or L 1 Or L 2 In some embodiments, the polypeptide or polypeptide complex has a binding affinity for a target antigen or a second target antigen that is at least 5 times weaker than the binding affinity for the target antigen of a form of the polypeptide or polypeptide complex that does not have P. 1 Or P 2 or L 1 Or L 2 In some embodiments, the polypeptide or polypeptide complex has 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 a polypeptide or polypeptide complex that does not have P. 1 Or P 2 or L 1 Or L 2 In some embodiments, the polypeptide or polypeptide complex has 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 a polypeptide or polypeptide complex that does not have P. 1 Or P 2 or L 1 Or L 2 In some embodiments, the polypeptide or polypeptide complex has a binding affinity for the target antigen or second target antigen that is at least 20-fold weaker than the binding affinity for the target antigen or second target antigen of a polypeptide or polypeptide complex that does not have P. 1 Or P2 or L 1 Or L 2 In some embodiments, the polypeptide or polypeptide complex has a binding affinity for the target antigen or second target antigen that is at least 25 times weaker than the binding affinity for the target antigen or second target antigen of a polypeptide or polypeptide complex that does not have P. 1 Or P 2 or L 1 Or L 2 In some embodiments, the polypeptide or polypeptide complex has a binding affinity for the target antigen or second target antigen that is at least 30-fold weaker than the binding affinity for the target antigen or second target antigen of a polypeptide or polypeptide complex that does not have P. 1 Or P 2 or L 1 Or L 2 In some embodiments, the polypeptide or polypeptide complex has a binding affinity for the target antigen or second target antigen that is at least 40-fold weaker than the binding affinity for the target antigen or second target antigen of a polypeptide or polypeptide complex that does not have P. 1 Or P 2 or L 1 Or L 2 In some embodiments, the polypeptide or polypeptide complex has a binding affinity for the target antigen or second target antigen that is at least 50-fold weaker than the binding affinity for the target antigen or second target antigen of a polypeptide or polypeptide complex that does not have P. 1 Or P 2 or L 1 Or L 2 In some embodiments, the polypeptide or polypeptide complex has a binding affinity for the target antigen or second target antigen that is at least 60-fold weaker than the binding affinity for the target antigen or second target antigen of a polypeptide or polypeptide complex that does not have P. 1 Or P 2 or L 1 Or L 2In some embodiments, the polypeptide or polypeptide complex has a binding affinity for the target antigen or second target antigen that is at least 70 times weaker than the binding affinity for the target antigen or second target antigen of a polypeptide or polypeptide complex that does not have P. 1 Or P 2 or L 1 Or L 2 In some embodiments, the polypeptide or polypeptide complex has a binding affinity for the target antigen or second target antigen that is at least 75 times weaker than the binding affinity for the target antigen or second target antigen of a polypeptide or polypeptide complex that does not have P. 1 Or P 2 or L 1 Or L 2 In some embodiments, the polypeptide or polypeptide complex has a binding affinity for the target antigen or second target antigen that is at least 80 times weaker than the binding affinity for the target antigen or second target antigen of a polypeptide or polypeptide complex that does not have P. 1 Or P 2 or L 1 Or L 2 In some embodiments, the polypeptide or polypeptide complex has a binding affinity for the target antigen or second target antigen that is at least 90 times weaker than the binding affinity for the target antigen or second target antigen of a polypeptide or polypeptide complex that does not have P. 1 Or P 2 or L 1 Or L 2 In some embodiments, the polypeptide or polypeptide complex has a binding affinity for the target antigen or second target antigen that is at least 100 times weaker than the binding affinity for the target antigen or second target antigen of a polypeptide or polypeptide complex that does not have P. 1 Or P 2 or L 1 Or L 2The polypeptide or polypeptide complex has a binding affinity for the target antigen or second target antigen that is at least 120-fold weaker than the binding affinity for the target antigen or second target antigen of the polypeptide or polypeptide complex not having the

[0064] In some embodiments, the polypeptide or polypeptide complex comprises L 1 or L 2 In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity for the target antigen or second target antigen compared to the binding affinity for the target antigen or second target antigen of a polypeptide or polypeptide complex in which the L 1 or L 2 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 a polypeptide or polypeptide complex in which L is truncated. 1 or L 2 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 a polypeptide or polypeptide complex in which L is truncated. 1 or L 2 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 a polypeptide or polypeptide complex in which L is truncated. 1 or L 2 In some embodiments, the polypeptide or polypeptide complex has a binding affinity for the target antigen or second target antigen that is at least 20-fold weaker than the binding affinity for the target antigen or second target antigen of a polypeptide or polypeptide complex in which L is truncated. 1 or L 2In some embodiments, the polypeptide or polypeptide complex has a binding affinity for the target antigen or second target antigen that is at least 25-fold weaker than the binding affinity for the target antigen or second target antigen of a polypeptide or polypeptide complex in which L is truncated. 1 or L 2 In some embodiments, the polypeptide or polypeptide complex has a binding affinity for the target antigen or second target antigen that is at least 30-fold weaker than the binding affinity for the target antigen or second target antigen of a polypeptide or polypeptide complex in which L is truncated. 1 or L 2 In some embodiments, the polypeptide or polypeptide complex has a binding affinity for the target antigen or second target antigen that is at least 40-fold weaker than the binding affinity for the target antigen or second target antigen of a polypeptide or polypeptide complex in which L is truncated. 1 or L 2 In some embodiments, the polypeptide or polypeptide complex has a binding affinity for the target antigen or second target antigen that is at least 50-fold weaker than the binding affinity for the target antigen or second target antigen of a polypeptide or polypeptide complex in which L is truncated. 1 or L 2 In some embodiments, the polypeptide or polypeptide complex has a binding affinity for the target antigen or second target antigen that is at least 60-fold weaker than the binding affinity for the target antigen or second target antigen of a polypeptide or polypeptide complex in which L is truncated. 1 or L 2 In some embodiments, the polypeptide or polypeptide complex has a binding affinity for the target antigen or second target antigen that is at least 70-fold weaker than the binding affinity for the target antigen or second target antigen of a polypeptide or polypeptide complex in which L is truncated. 1 or L 2In some embodiments, the polypeptide or polypeptide complex has a binding affinity for the target antigen or second target antigen that is at least 75 times weaker than the binding affinity for the target antigen or second target antigen of a polypeptide or polypeptide complex in which L is truncated. 1 or L 2 In some embodiments, the polypeptide or polypeptide complex has a binding affinity for the target antigen or second target antigen that is at least 80-fold weaker than the binding affinity for the target antigen or second target antigen of a polypeptide or polypeptide complex in which L is truncated. 1 or L 2 In some embodiments, the polypeptide or polypeptide complex has a binding affinity for the target antigen or second target antigen that is at least 90-fold weaker than the binding affinity for the target antigen or second target antigen of a polypeptide or polypeptide complex in which L is truncated. 1 or L 2 In some embodiments, the polypeptide or polypeptide complex has a binding affinity for the target antigen or second target antigen that is at least 100-fold weaker than the binding affinity for the target antigen or second target antigen of a polypeptide or polypeptide complex in which L is truncated. 1 or L 2 In some embodiments, the L has a binding affinity for the target antigen or the second target antigen that is at least 120-fold weaker than the binding affinity for the target antigen or the second target antigen of a polypeptide or polypeptide complex in which the L is truncated. 1 or L 2 is cleaved by a protease. In some embodiments, the protease comprises a tumor-specific protease. In some embodiments, the protease comprises a matrix metalloprotease (MMP) or a serine protease. In some embodiments, the matrix metalloprotease comprises MMP2, MMP7, MMP9, MMP13, or MMP14. In some embodiments, the serine protease comprises matriptase, urokinase, or hepsin.

[0065] In some embodiments, A 1 Or A 2 is a cytokine or a cytokine fragment. In some embodiments, A 1 Or A 2 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 the corresponding cytokine receptor, a transmembrane receptor that contains an extracellular domain for ligand binding and an intracellular domain that allows signal transduction.

[0067] The diversity of cytokines is accompanied by a corresponding diversity of cytokine receptors, which can include single chains or subunits or dimer / multimer domains. Examples of cytokine receptors include type I cytokine receptors exemplified by interleukin receptors, and type II cytokine receptors exemplified by interferon receptors, both of which contain cytokine receptor homology domains (CHDs). The CHD of type I cytokine receptors share a common amino acid motif (WSXWS (SEQ ID NO: 27)), while type II cytokine receptors lack this motif. Cytokine receptors can include α subunits, β subunits, γ subunits, or combinations of their dimers or trimers. In one example, the high-affinity receptor for IL-2 includes the IL-2Rα subunit, the IL-2Rβ subunit, and the IL-2Rγ subunit, the intermediate-affinity receptor for IL-2 includes only the IL-2Rβ subunit and the IL-2Rγ subunit, and the low-affinity receptor for IL-2 includes only the 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 the 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, an interferon, an interleukin, a lymphokine, an adipokine, a growth factor, or a tumor necrosis factor. In some embodiments, the cytokine receptor is a type I cytokine receptor or a type II cytokine receptor. In some embodiments, the cytokine receptor is a dimer or a trimer. In some embodiments, the cytokine receptor comprises an alpha subunit, a beta subunit, a gamma subunit, or any combination thereof. For example, in some embodiments, the cytokine receptor comprises an alpha subunit, a beta subunit, and a gamma subunit. In another example, in some embodiments, the cytokine receptor comprises a beta subunit and a gamma subunit. In some embodiments, the cytokine receptor comprises an alpha subunit and a beta subunit.

[0071] In some embodiments, the polypeptide or polypeptide complex is 1 Or P 2 or L 1 Or L 2 In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity to a cytokine receptor compared to the binding affinity to the cytokine receptor of a polypeptide or polypeptide complex not having P. 1 Or P 2 or L 1 Or L 2 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 the cytokine receptor of a form of the polypeptide or polypeptide complex that does not have P. 1 Or P 2 or L 1 Or L 2In 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 to the cytokine receptor of a form of the polypeptide or polypeptide complex that does not have P. 1 Or P 2 or L 1 Or L 2 In some embodiments, the polypeptide or polypeptide complex has a binding affinity for a cytokine receptor that is at least 10-fold weaker than the binding affinity for the cytokine receptor of a form of the polypeptide or polypeptide complex that does not have P. 1 Or P 2 or L 1 Or L 2 In some embodiments, the polypeptide or polypeptide complex has a binding affinity for a cytokine receptor that is at least 20-fold weaker than the binding affinity for the cytokine receptor of a form of the polypeptide or polypeptide complex that does not have P. 1 Or P 2 or L 1 Or L 2 In some embodiments, the polypeptide or polypeptide complex has a binding affinity for a cytokine receptor that is at least 25 times weaker than the binding affinity for the cytokine receptor of a form of the polypeptide or polypeptide complex that does not have P. 1 Or P 2 or L 1 Or L 2 In some embodiments, the polypeptide or polypeptide complex has a binding affinity for a cytokine receptor that is at least 30-fold weaker than the binding affinity for the cytokine receptor of a form of the polypeptide or polypeptide complex that does not have P. 1 Or P 2 or L 1 Or L 2In some embodiments, the polypeptide or polypeptide complex has a binding affinity for a cytokine receptor that is at least 40 times weaker than the binding affinity for the cytokine receptor of a form of the polypeptide or polypeptide complex that does not have P. 1 Or P 2 or L 1 Or L 2 In some embodiments, the polypeptide or polypeptide complex has a binding affinity for a cytokine receptor that is at least 50-fold weaker than the binding affinity for the cytokine receptor of a form of the polypeptide or polypeptide complex that does not have P. 1 Or P 2 or L 1 Or L 2 In some embodiments, the polypeptide or polypeptide complex has a binding affinity for a cytokine receptor that is at least 60 times weaker than the binding affinity for the cytokine receptor of a form of the polypeptide or polypeptide complex that does not have P. 1 Or P 2 or L 1 Or L 2 In some embodiments, the polypeptide or polypeptide complex has a binding affinity for a cytokine receptor that is at least 70 times weaker than the binding affinity for the cytokine receptor of a form of the polypeptide or polypeptide complex that does not have P. 1 Or P 2 or L 1 Or L 2 In some embodiments, the polypeptide or polypeptide complex has a binding affinity for a cytokine receptor that is at least 75 times weaker than the binding affinity for the cytokine receptor of a form of the polypeptide or polypeptide complex that does not have P. 1 Or P 2 or L 1 Or L 2In some embodiments, the polypeptide or polypeptide complex has a binding affinity for a cytokine receptor that is at least 80 times weaker than the binding affinity for the cytokine receptor of a form of the polypeptide or polypeptide complex that does not have P. 1 Or P 2 or L 1 Or L 2 In some embodiments, the polypeptide or polypeptide complex has a binding affinity for a cytokine receptor that is at least 90 times weaker than the binding affinity for the cytokine receptor of a form of the polypeptide or polypeptide complex that does not have P. 1 Or P 2 or L 1 Or L 2 In some embodiments, the polypeptide or polypeptide complex has a binding affinity for a cytokine receptor that is at least 100 times weaker than the binding affinity for the cytokine receptor of a form of the polypeptide or polypeptide complex that does not have P. 1 Or P 2 or L 1 Or L 2 In some embodiments, the polypeptide or polypeptide complex has a binding affinity for a cytokine receptor that is at least 120 times weaker than the binding affinity for the cytokine receptor of a form of the polypeptide or polypeptide complex that does not have P. 1 Or P 2 or L 1 Or L 2The cytokine receptor has at least 150-fold weaker binding affinity to the cytokine receptor compared to the binding affinity to the cytokine receptor of a form of the polypeptide or polypeptide complex that does not have the above structure. In some embodiments, the cytokine or cytokine fragment comprises an interferon, GM-CSF, IL-2, IL-7, IL-12, IL-15, or IL-21. In some embodiments, the cytokine or cytokine fragment comprises an IL-2, IL-12, IL-6, IL-4, IL-10, or 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 comprises L 1 or L 2 In some embodiments, the polypeptide or polypeptide complex has a weaker binding affinity to a cytokine receptor compared to the binding affinity to the cytokine receptor of a polypeptide or polypeptide complex in which L is truncated. 1 or L 2 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 the cytokine receptor of a polypeptide or polypeptide complex in which L is truncated. 1 or L 2 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 the cytokine receptor of a polypeptide or polypeptide complex in which L is truncated. 1 or L 2In 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 the cytokine receptor of a polypeptide or polypeptide complex in which L is truncated. 1 or L 2 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 the cytokine receptor of a polypeptide or polypeptide complex in which L is truncated. 1 or L 2 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 the cytokine receptor of a polypeptide or polypeptide complex in which L is truncated. 1 or L 2 In some embodiments, the polypeptide or polypeptide complex has a binding affinity for a cytokine receptor that is at least 25-fold weaker than the binding affinity for the cytokine receptor of a polypeptide or polypeptide complex in which L is truncated. 1 or L 2 In some embodiments, the polypeptide or polypeptide complex has a binding affinity for a cytokine receptor that is at least 30-fold weaker than the binding affinity for the cytokine receptor of a polypeptide or polypeptide complex in which L is truncated. 1 or L 2 In some embodiments, the polypeptide or polypeptide complex has a binding affinity for a cytokine receptor that is at least 40-fold weaker than the binding affinity for the cytokine receptor of a polypeptide or polypeptide complex in which L is truncated. 1 or L 2 In some embodiments, the polypeptide or polypeptide complex has a binding affinity for a cytokine receptor that is at least 50-fold weaker than the binding affinity for the cytokine receptor of a polypeptide or polypeptide complex in which the L 1 or L2 In some embodiments, the polypeptide or polypeptide complex has a binding affinity for a cytokine receptor that is at least 60-fold weaker than the binding affinity for the cytokine receptor of a polypeptide or polypeptide complex in which L is truncated. 1 or L 2 In some embodiments, the polypeptide or polypeptide complex has a binding affinity for a cytokine receptor that is at least 70-fold weaker than the binding affinity for the cytokine receptor of a polypeptide or polypeptide complex in which L is truncated. 1 or L 2 In some embodiments, the polypeptide or polypeptide complex has a binding affinity for a cytokine receptor that is at least 75-fold weaker than the binding affinity for the cytokine receptor of a polypeptide or polypeptide complex in which L is truncated. 1 or L 2 In some embodiments, the polypeptide or polypeptide complex has a binding affinity for a cytokine receptor that is at least 80-fold weaker than the binding affinity for the cytokine receptor of a polypeptide or polypeptide complex in which L is truncated. 1 or L 2 In some embodiments, the polypeptide or polypeptide complex has a binding affinity for a cytokine receptor that is at least 90-fold weaker than the binding affinity for the cytokine receptor of a polypeptide or polypeptide complex in which L is truncated. 1 or L 2In some embodiments, the cytokine or cytokine fragment comprises an interferon, GM-CSF, IL-2, IL-7, IL-12, IL-15, or IL-21. In some embodiments, the cytokine receptor comprises an interferon receptor, 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 L 1 or L 2 is cleaved by a protease. In some embodiments, the protease comprises a tumor-specific protease. In some embodiments, the protease comprises a matrix metalloprotease (MMP) or a serine protease. In some embodiments, the matrix metalloprotease comprises MMP2, MMP7, MMP9, MMP13, or MMP14. In some embodiments, the serine protease comprises matriptase, urokinase, or hepsin.

[0073] Half-life extension moiety In some embodiments, P 1 is further linked to a half-life extending moiety. 1 is represented by formula Ia: A 1 -L 1 -P 1 -L 3 -H 1 (Formula Ia) is linked to the half-life extending moiety in an arrangement according to In the formula, H 1 is the half-life extending moiety, and L 3 H 1 P 1 In some embodiments, L 3 is a non-cleavable linker. In some embodiments, the half-life extending moiety (H 1 ) is A 1In some embodiments, the half-life extending moiety (H 1 ) is A 1 In some embodiments, the half-life extending moiety (H 1 ) has no binding affinity for the target antigen. In some embodiments, the half-life extending moiety (H 1 ) is A 1 In some embodiments, the half-life extending moiety (H 1 ) is A 1 Do not connect directly to

[0074] In some embodiments, the half-life extending moiety (H 1 ) is A 1 In some embodiments, the half-life extending moiety (H 1 ) has no binding affinity for a cytokine or cytokine receptor. In some embodiments, the half-life extending moiety (H 1 ) does not protect the cytokine or cytokine fragment from the cytokine receptor. In some embodiments, a half-life extending moiety (H 1 ) is not directly linked to the cytokine or cytokine fragment.

[0075] In some embodiments, H 1 comprises an amino acid sequence having a repeat sequence motif. 1 comprises an amino acid sequence having a highly ordered secondary structure. "Highly ordered secondary structure" as used in this context means 1 By "secondary structure," we mean that at least about 50%, about 70%, about 80%, or about 90% of the amino acid residues in the protein contribute to the secondary structure as measured by means including, but not limited to, spectrophotometric methods (e.g., circular dichroism spectroscopy in the "far-UV" region of the spectrum (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, H 1 comprises a polymer. In some embodiments, the polymer is polyethylene glycol (PEG). In some embodiments, H 1 In some embodiments, H 1 In some embodiments, the albumin is serum albumin. In some embodiments, the albumin is human serum albumin. In some embodiments, the H 1 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 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 albumin. In some embodiments, the antibody comprises a single domain antibody that binds 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, H 1comprises a single domain antibody. In some embodiments, H 1 comprises a single domain antibody that binds to albumin. In some embodiments, H 1 comprises a single domain antibody that binds to human serum albumin.

[0078] In some embodiments, H 1 comprises modified amino acids or unnatural amino acids, or modified unnatural amino acids, or combinations thereof. In some embodiments, the modified amino acids or modified unnatural amino acids comprise post-translational modifications. In some embodiments, H 1 These include, but are not limited to, modifications such as 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 modified to modify the peptide backbone, amino acid side chains, and termini, including H-terminus. 1 This is done everywhere.

[0079] Polynucleotides encoding polypeptides or polypeptide complexes In some embodiments herein, an isolated recombinant nucleic acid molecule is disclosed that encodes a polypeptide or polypeptide complex as disclosed herein. In some embodiments herein, an isolated recombinant nucleic acid molecule is disclosed 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] In some embodiments herein, an isolated recombinant nucleic acid molecule is disclosed 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. In some embodiments herein, an isolated recombinant nucleic acid molecule is disclosed that encodes an isolated polypeptide comprising a cleavable linker according to the amino acid sequence of linker 1 (ISSGLLSGRSDAG) (SEQ ID NO: 3). In some embodiments herein, an isolated recombinant nucleic acid molecule is disclosed that encodes an isolated polypeptide comprising a cleavable linker according to the amino acid sequence of linker 2 (AAGLLAPPGGLSGRSDAG) (SEQ ID NO: 4). In some embodiments herein, an isolated recombinant nucleic acid molecule is disclosed that encodes an isolated polypeptide comprising a cleavable linker according to the amino acid sequence of linker 3 (SPLGLSGRSDAG) (SEQ ID NO:5). In some embodiments herein, an isolated recombinant nucleic acid molecule is disclosed that encodes an isolated polypeptide comprising a cleavable linker according to the amino acid sequence of linker 4 (LSGRSDAGSPLGLAG) (SEQ ID NO:6). In some embodiments herein, an isolated recombinant nucleic acid molecule is disclosed that encodes an isolated polypeptide comprising a cleavable linker according to the amino acid sequence of LSGRSDAG (SEQ ID NO:1).

[0083] In some embodiments herein, the compound of formula I: A 1 -L 1 -P 1 (Formula I) Disclosed is an isolated recombinant nucleic acid molecule encoding a polypeptide or polypeptide complex according to In the formula, A 1The L comprises an antigen-binding domain that binds to a target antigen, or a cytokine that binds to a cytokine receptor. 1 comprises a cleavable linker, P 1 In some embodiments herein, the peptide of formula I: A 1 -L 1 -P 1 (Formula I) Disclosed is an isolated, recombinant nucleic acid molecule encoding a polypeptide or polypeptide complex comprising:

[0084] In the formula, A 1 The L comprises an antigen-binding domain that binds to a target antigen, or a cytokine that binds to a cytokine receptor. 1 comprises a cleavable linker, P 1 In some embodiments herein, the peptide of formula I: A 1 -L 1 -P 1 (Formula I) Disclosed is an isolated recombinant nucleic acid molecule encoding a polypeptide or polypeptide complex according to In the formula, A 1 is an antigen-binding domain that binds to a target antigen, or a cytokine that binds to a cytokine receptor, and L 1 is a cleavable linker, P 1 is a peptide that impairs binding of an antigen-binding domain to a target antigen or impairs binding of a cytokine to a cytokine receptor. In some embodiments herein, a peptide represented by Formula I: A 1 -L 1 -P 1 (Formula I) Disclosed is an isolated, recombinant nucleic acid molecule encoding a polypeptide or polypeptide complex comprising: In the formula, A 1 is an antigen-binding domain that binds to a target antigen, or a cytokine that binds to a cytokine receptor, and L 1 is a cleavable linker, P 1 is a peptide that impairs binding of an antigen-binding domain to a target antigen or that impairs binding of a cytokine to a cytokine receptor.

[0085] In some embodiments herein, a polypeptide or polypeptide complex is disclosed, wherein the isolated polypeptide is complexed with a second isolated polypeptide comprising a second antigen-binding domain or a second cytokine. In some embodiments herein, a polypeptide or polypeptide complex is disclosed, wherein the isolated polypeptide is complexed with a second isolated polypeptide comprising a second antigen-binding domain or a second cytokine. A 2 -L 2 -P 2 (Formula II) Disclosed is an isolated recombinant nucleic acid molecule encoding a polypeptide or polypeptide complex according to In the formula, A 2 comprises a second antigen-binding domain or a second cytokine, 2 comprises a second cleavable linker, and P 2 comprises a second peptide that impairs binding of the second antigen-binding domain to a second target antigen or impairs binding of a second cytokine to a second cytokine receptor. In some embodiments herein, a peptide represented by Formula II: A 2 -L 2 -P 2 (Formula II) Disclosed is an isolated, recombinant nucleic acid molecule encoding a polypeptide or polypeptide complex comprising: In the formula, A 2 comprises a second antigen-binding domain or a second cytokine, 2 comprises a second cleavable linker, and P 2comprises a second peptide that impairs binding of the second antigen-binding domain to a second target antigen or impairs binding of a second cytokine to a second cytokine receptor. In some embodiments herein, a peptide represented by Formula II: A 2 -L 2 -P 2 (Formula II) Disclosed is an isolated recombinant nucleic acid molecule encoding a polypeptide or polypeptide complex according to In the formula, A 2 is a second antigen-binding domain or a second cytokine, and L 2 is a second cleavable linker, and P 2 is a second peptide that impairs binding of a second antigen-binding domain to a second target antigen or impairs binding of a second cytokine to a second cytokine receptor. In some embodiments herein, a peptide represented by Formula II: A 2 -L 2 -P 2 (Formula II) Disclosed is an isolated recombinant nucleic acid molecule encoding a polypeptide or polypeptide complex comprising: In the formula, A 2 is a second antigen-binding domain or a second cytokine, and L 2 is a second cleavable linker, and P 2 is a second peptide that impairs binding of a second antigen-binding domain to a second target antigen or impairs binding of a second cytokine to a second cytokine receptor.

[0086] Pharmaceutical Compositions Disclosed herein in some embodiments is a pharmaceutical composition comprising (a) a polypeptide or polypeptide complex disclosed herein; and (b) a pharma- ceutically acceptable excipient.

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

[0088] In some embodiments, a 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 pharma- ceutically 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 pharma- ceutically 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 pharma- ceutically acceptable excipient.

[0091] In some embodiments, a 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 pharma- ceutically acceptable excipient.

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

[0094] In some embodiments, the pharmaceutical composition comprises (a) a compound of formula I: A 1 -L 1 -P 1 (Formula I) 2. An isolated polypeptide or polypeptide complex according to claim 1, In the formula, A 1 The L comprises an antigen-binding domain that binds to a target antigen, or a cytokine that binds to a cytokine receptor. 1 comprises a cleavable linker, P 1 comprises an isolated polypeptide or polypeptide complex comprising a peptide that impairs binding of an antigen-binding domain to a target antigen or that impairs binding of a cytokine to a cytokine receptor, and (b) a pharma- ceutical acceptable excipient. In some embodiments, the pharmaceutical composition comprises (a) a polypeptide comprising a peptide of Formula I: A 1 -L 1 -P 1 (Formula I) 1. An isolated polypeptide or polypeptide complex comprising: In the formula, A 1 The L comprises an antigen-binding domain that binds to a target antigen, or a cytokine that binds to a cytokine receptor. 1 comprises a cleavable linker, P 1 comprises an isolated polypeptide or polypeptide complex comprising a peptide that impairs binding of an antigen-binding domain to a target antigen or that impairs binding of a cytokine to a cytokine receptor, and (b) a pharma- ceutical acceptable excipient. In some embodiments, the pharmaceutical composition comprises (a) a polypeptide comprising a peptide of Formula I: A 1 -L 1 -P 1 (Formula I) 2. An isolated polypeptide or polypeptide complex according to claim 1, In the formula, A 1 is an antigen-binding domain that binds to a target antigen, or a cytokine that binds to a cytokine receptor, and L 1 is a cleavable linker, P 1In some embodiments, the pharmaceutical composition comprises an isolated polypeptide or polypeptide complex that is a peptide that impairs binding of an antigen-binding domain to a target antigen or that impairs binding of a cytokine to a cytokine receptor, and (b) a pharma- ceutical acceptable excipient. A 1 -L 1 -P 1 (Formula I) 1. An isolated polypeptide or polypeptide complex comprising: In the formula, A 1 is an antigen-binding domain that binds to a target antigen, or a cytokine that binds to a cytokine receptor, and L 1 is a cleavable linker, P 1 comprises an isolated polypeptide or polypeptide complex, which is a peptide that impairs binding of an antigen-binding domain to a target antigen or that impairs binding of a cytokine to a cytokine receptor, and (b) a pharma- ceutically acceptable excipient.

[0095] In some embodiments herein, a polypeptide or polypeptide complex is disclosed, 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 having Formula II: A 2 -L 2 -P 2 (Formula II) 2. An isolated polypeptide or polypeptide complex according to claim 1, In the formula, A 2 comprises a second antigen-binding domain or a second cytokine, 2 comprises a second cleavable linker, and P 2comprises an isolated polypeptide or polypeptide complex comprising a second peptide that impairs binding of a second antigen-binding domain to a second target antigen or impairs binding of a second cytokine to a second cytokine receptor, and (b) a pharma- ceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises: (a) a polypeptide comprising a polypeptide of Formula II: A 2 -L 2 -P 2 (Formula II) 1. An isolated polypeptide or polypeptide complex comprising: In the formula, A 2 comprises a second antigen-binding domain or a second cytokine, 2 comprises a second cleavable linker, and P 2 comprises an isolated polypeptide or polypeptide complex comprising a second peptide that impairs binding of a second antigen-binding domain to a second target antigen or impairs binding of a second cytokine to a second cytokine receptor, and (b) a pharma- ceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises: (a) a polypeptide comprising a polypeptide of Formula II: A 2 -L 2 -P 2 (Formula II) 2. An isolated polypeptide or polypeptide complex according to claim 1, In the formula, A 2 is a second antigen-binding domain or a second cytokine, and L 2 is a second cleavable linker, and P 2 comprises an isolated polypeptide or polypeptide complex that is a second peptide that impairs binding of a second antigen-binding domain to a second target antigen or impairs binding of a second cytokine to a second cytokine receptor, and (b) a pharma- ceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises: (a) a polypeptide comprising a polypeptide of Formula II: A 2 -L 2 -P 2 (Formula II) 1. An isolated polypeptide or polypeptide complex comprising: In the formula, A 2 is a second antigen-binding domain or a second cytokine, and L 2 is a second cleavable linker, and P 2 comprises an isolated polypeptide or polypeptide complex, which is a second peptide that impairs binding of a second antigen-binding domain to a second target antigen or impairs binding of a second cytokine to a second cytokine receptor, and (b) a pharma- ceutically 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 polypeptide or polypeptide complex disclosed herein may be provided in a pharmaceutical composition together with one or more pharma- ceutically 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, and the like. Such carriers can be formulated by conventional methods and administered to a subject in a suitable dosage. Preferably, the pharmaceutical composition is sterile. These compositions may further contain auxiliary agents such as preservatives, emulsifiers, dispersing agents, and the like. 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 a 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 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 will ultimately be determined by the physician as to 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 having at least or about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, or more than 400 amino acids of SEQ ID NO:1, 2, 4, 5, 7, 8, 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25.

[0107] 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. It is possible to determine appropriate parameters for aligning sequences, including the algorithm required to achieve maximum alignment over the entire length of the sequences being compared. However, for the purposes herein, amino acid sequence identity percent values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program is the author Genentech, Inc., and the source code has been submitted with user documentation to the United States Copyright Office, Washington, DC, 20559, and is registered under United States 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 (which may alternatively be phrased as a given amino acid sequence A having or containing a particular % amino acid sequence identity to a 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 an alignment of programs 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 stated, 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 (e.g., antibodies and binding fragments thereof) described herein are produced by any method known in the art and useful for the synthesis of polypeptides (e.g., antibodies), in particular, chemical synthesis or recombinant expression, and preferably are produced 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 expressing 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 produce polyclonal antibodies, or more preferably, by producing monoclonal antibodies, e.g., as described in Kohler and Milstein (1975, Nature 256:495-497), or as described in 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 in Huse et al., 1989, Science 246:1275-1281) for clones of Fab fragments that bind a specific antigen, 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).

[0113] In some embodiments, techniques developed to produce "chimeric antibodies" by splicing genes from a human antibody molecule of appropriate biological activity together with an antibody 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 mouse 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 to produce a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli are also optionally used (Skerra et al., 1988, Science 242:1038-1041).

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

[0116] In some embodiments, a variety of 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, when transformed or transfected with the appropriate nucleotide coding sequences, express the antibodies or binding fragments thereof in situ. These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli or Bacillus subtilis) transformed with a recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vector containing the coding sequence for the antibody or binding fragment thereof; yeast (e.g., Saccharomyces Pichia) transformed with a recombinant yeast expression vector containing the coding sequence for the antibody or binding fragment thereof; insect cell systems infected with a recombinant viral expression vector (e.g., baculovirus) containing the coding sequence for the antibody or binding fragment thereof; plant cell systems infected with a recombinant viral expression vector (e.g., cauliflower mosaic virus (CaMV) or tobacco mosaic virus (TMV)) or a recombinant plasmid expression vector (e.g., Ti plasmid) containing the coding sequence for the antibody or binding fragment thereof; or mammalian cell systems (e.g., COS, CHO, BH, 293, 293T, 3T3 cells) harboring a recombinant expression construct containing a promoter derived from a mammalian cell genome (e.g., metallothionein promoter) or a mammalian virus (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 the antibody 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 medium for 1-2 days and switched to selective medium. The selectable marker in the recombinant plasmid confers resistance to the selection, allowing cells to stably integrate the plasmid into their chromosomes and grow to give rise to foci that are cloned and expanded into cell lines. This method may be conveniently used to engineer cell lines that express the antibody or binding fragments thereof.

[0118] In some examples, a number of selection systems may be 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) are utilized in tk cells, hgprt cells, or aprt cells, respectively. Antimetabolite resistance is also 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 12:131-134). 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 can be used are described in Ausubel et al. (eds., Current Protocols in Molecular Biology, 1993, John Wiley & Sons, NY; Gene Transfer and Expression, A Laboratory Manual, 1990, by Kriegler, Stockton Press, NY; and Chapters 12 and 13 of Current Protocols in Human Genetics, 1994, by Dracopoli et al. (eds.), John Wiley & Sons, NY; J. Mol. Biol. 150:1, 1981, by Colberre-Garapin et al.).

[0119] In some cases, the expression level of an antibody is increased by vector amplification (see, for example, 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) for consideration). When the marker of the vector system expressing the antibody is amplifiable, the level of inhibitors present during the culture of the host cells increases, resulting in an increase in the copy number of the marker gene. Since the amplified region is associated with the nucleotide sequence of the antibody, the production of the antibody will also increase (Mol. Cell Biol. 3:257, 1983, by Crouse et al.).

[0120] Optionally, any method known in the art for the purification of an antibody is used, such as chromatography (e.g., ion exchange, affinity, specifically affinity for a specific antigen after 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 eukaryotic or prokaryotic sources. Optionally, the vector is obtained from bacterial sources (e.g., E. coli), insect sources, yeast sources (e.g., Pichia pastoris), algae sources, or mammalian sources. Exemplary bacterial vectors include pACYC177, pASK75, pBAD vector series, pBADM vector series, pET vector series, pETM vector series, pGEX vector series, pHAT, pHAT2, pMal-c2, pMal-p2, pQE vector series, pRSET A, pRSET B, pRSET C, pTrcHis2 series, pZA31-Luc, pZE21-MCS-1, pFLAG ATS, pFLAG CTS, pFLAG MAC, pFLAG Shift-12c, pTAC-MAT-1, pFLAG CTC, or pTAC-MAT-2.

[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-MAT 2, 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 can 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 examples, the cell-free system is a mixture of cytoplasmic and / or nuclear components from cells and is used for in vitro nucleic acid synthesis. In some cases, the cell-free system utilizes either prokaryotic or eukaryotic components. Sometimes nucleic acid synthesis is obtained 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, E.coli S30 Extract system, 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, the gram-positive bacteria include Actinomycetes, Firmicutes, and Tenericutes. In some cases, the gram-negative bacteria include Aquifex, Deinococcus Thermus, Fibrobacter-Chlorobium / Bacteroides (FCB group), Fusobacterium, Gemmatimonas, Nitrospira, Planctomyces-Vercomicrobium / Chlamydia (PVC group), Proteobacteria, Spirochetes, or Synergistes. Other bacteria can be Acidobacteria, Chloroflexi, Chrysiogenes, Cyanobacteria, Deferibacter, Dictyoglomi, Thermodesulfobacteria, or Thermotoga. The bacterial cell can 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, basidiomycetes, and zygomycota. In some instances, yeasts include the Ascomycota or Basidiomycota phylum. In some instances, the Ascomycota phylum includes the Saccharomycetes (true yeasts, e.g., saccharomyces cerevisiae (baker's yeast)) and the Taphrina (e.g., Schizosaccharomycetes (fission yeast)). In some instances, the Basidiomycota phylum includes the Agaricota (e.g., Tremellomycetes) and the Urobacterium (e.g., Microbotryomycetes).

[0132] Exemplary yeasts or filamentous fungi include, for example, those of the genera 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 brussels, 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 includes a cell obtained from a mollusc, an arthropod, an annelid, or a sponge.Optionally, the additional animal cell is a mammalian cell, such as a primate, an ape, a horse, a cow, a pig, a dog, a cat, or a rodent.Optionally, the rodent includes 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 293A cell line, 293FT cell line, 293F cells, 293H cells, CHO DG44 cells, CHO-S cells, CHO-K1 cells, 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 has the ability to express 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 does not integrate the genetic material of interest into its genome and does not have the ability to express 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, 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 In another aspect of the invention, an article of manufacture is provided that contains materials useful for the treatment, prevention, and / or diagnosis of the disorders described above. 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 that is pierceable by a hypodermic needle). At least one active agent in the composition is a bispecific antibody that includes 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 pharma- ceutically 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 In some embodiments, the isolated polypeptides comprising the cleavable linker described herein are used in a method of treating cancer. In some embodiments, the cancer has cells expressing EGFR. In some embodiments, the polypeptides or polypeptide complexes described herein are used in a method of treating colorectal cancer (CRC), squamous cell carcinoma of the head and neck (SCCHN), non-small cell lung cancer (NSCLC), prostate cancer, breast cancer, 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, the polypeptides or polypeptide complexes described herein are used in a method of treating a subject resistant to treatment with an EGFR inhibitor. In some embodiments, the polypeptides or polypeptide complexes described herein are used in a method of treating a subject with a KRAS mutation that is resistant to treatment with an EGFR inhibitor. In some embodiments, the polypeptides or polypeptide complexes described herein are 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 is 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 with 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 includes 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 includes 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 includes the isolated polypeptide of embodiment 12, wherein the antigen-binding domain is C-terminal to the cleavable linker.

[0157] Embodiment 14 includes 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 is a method for preparing a cleavable linker comprising the steps of: 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 a target antigen, or a cytokine or cytokine fragment that binds to a cytokine receptor, 1 comprises a cleavable linker, P 1The polypeptide of any one of embodiments 1 to 15 comprises a peptide that impairs binding of an antigen-binding domain to a target antigen or that impairs binding of a cytokine to a cytokine receptor.

[0160] Embodiment 17 is P 1 is connected at its N-terminus to a cleavable linker, and A 1 is connected at its C-terminus to a cleavable linker.

[0161] In embodiment 18, P 1 is connected at its C-terminus to a cleavable linker, and A 1 is connected at its N-terminus to a cleavable linker.

[0162] Embodiment 19 is a method for producing a medicament for use in a pharmaceutical composition comprising the steps of: 1 through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bonding interactions, or a combination thereof. 1 19. The method of claim 17, further comprising administering to said patient an isolated polypeptide comprising the steps of:

[0163] In the twentieth embodiment, P 1 has less than 70% sequence homology to the target antigen or cytokine receptor.

[0164] Embodiment 21 is P 1 21. The isolated polypeptide of any one of embodiments 16 to 20, wherein the isolated polypeptide comprises a peptide sequence of at least 10 amino acids in length.

[0165] In embodiment 22, P 1 22. The isolated polypeptide of any one of embodiments 16 to 21, comprising a peptide sequence of at least 10 amino acids in length and no more than 20 amino acids in length.

[0166] Embodiment 23 is P 123. The isolated polypeptide of any one of embodiments 16 to 22, wherein the isolated polypeptide comprises a peptide sequence of at least 16 amino acids in length.

[0167] Embodiment 24 is P 1 24. The isolated polypeptide of any one of embodiments 16 to 23, wherein the isolated polypeptide comprises a peptide sequence of 40 amino acids or less in length.

[0168] In embodiment 25, P 1 25. The isolated polypeptide of any one of embodiments 16 to 24, wherein said polypeptide comprises a cyclic or linear peptide.

[0169] In embodiment 26, P 1 26. The isolated polypeptide of any one of embodiments 16 to 25, wherein

[0170] In embodiment 27, P 1 27. The isolated polypeptide of any one of embodiments 16 to 26, wherein said polypeptide 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] In embodiment 30, A 1 30. The isolated polypeptide of any one of embodiments 16 to 29, 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.

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

[0175] In embodiment 32, A 1 32. The isolated polypeptide of any one of embodiments 30 to 31, wherein said isolated polypeptide comprises a Fab light chain polypeptide or a Fab heavy chain polypeptide.

[0176] Embodiment 33 is A 1 comprises an epidermal growth factor receptor (EGFR) binding domain.

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

[0178] In embodiment 35, A 1 The isolated polypeptide of embodiment 34, wherein said polypeptide 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] In embodiment 37, A 1 The isolated polypeptide of any one of embodiments 16 to 29, wherein the isolated polypeptide 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 includes 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 includes 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 includes the isolated polypeptide of any one of embodiments 37 to 41, wherein the cytokine or cytokine fragment comprises an interferon, GM-CSF, IL-2, IL-7, IL-12, IL-15, or IL-21.

[0186] Embodiment 43 includes 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 includes 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 is a method for treating a cancer, comprising administering to a patient a cancer treatment ... 2 -L 2 -P 2 In the formula, A 2 comprises a second antigen-binding domain or a second cytokine, and L 2 comprises a second cleavable linker, and P 2 comprises the isolated polypeptide of embodiment 44, which comprises a second peptide that inhibits binding of the second antigen-binding domain to a second target antigen or inhibits binding of a second cytokine or a second cytokine fragment to a second cytokine receptor.

[0189] Embodiment 46 includes 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 includes 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 includes 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 includes 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 includes 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 is P 2 is connected at its N-terminus to a second cleavable linker, and A 2 is connected at its C-terminus to a second cleavable linker.

[0196] Embodiment 53 is P 2 is connected at its C-terminus to a second cleavable linker, and A 2is connected at its N-terminus to a second cleavable linker.

[0197] Embodiment 54 is P 2 through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bonding interactions, or a combination thereof. 2 The invention further comprises an isolated polypeptide according to any one of embodiments 45 to 53, which is linked to

[0198] Embodiment 55 is a method for producing a medicament for use in a pharmaceutical composition comprising the steps of: 2 has less than 70% sequence homology to the second target antigen or the second cytokine receptor.

[0199] Embodiment 56 is a method for producing a 56-well plate comprising the steps of: 2 56. The isolated polypeptide of any one of embodiments 45 to 55, wherein the isolated polypeptide comprises a peptide sequence of at least 10 amino acids in length.

[0200] In embodiment 57, P 2 57. The isolated polypeptide of any one of embodiments 45 to 56, comprising a peptide sequence at least 10 amino acids in length and no more than 20 amino acids in length.

[0201] Embodiment 58 is P 2 58. The isolated polypeptide of any one of embodiments 45 to 57, wherein the isolated polypeptide comprises a peptide sequence of at least 16 amino acids in length.

[0202] Embodiment 59 is a method for producing a 2 57. The isolated polypeptide of any one of embodiments 45 to 56, wherein said isolated polypeptide comprises a peptide sequence of 40 or less amino acids in length.

[0203] Embodiment 60 is a method for manufacturing a 2 60. The isolated polypeptide of any one of embodiments 45 to 59, wherein said polypeptide comprises a cyclic or linear peptide.

[0204] Embodiment 61 is P 2 The isolated polypeptide of any one of embodiments 45 to 60, wherein

[0205] In embodiment 62, A 2 62. The isolated polypeptide of any one of embodiments 45 to 61, 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.

[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 is A 2 63. The isolated polypeptide of embodiment 62, wherein said polypeptide comprises a Fab light chain polypeptide or a Fab heavy chain polypeptide.

[0208] In embodiment 65, A 2 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] In embodiment 67, A 2 63. The isolated polypeptide of embodiment 62, wherein said polypeptide 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 is A 2comprises a second cytokine.

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

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

[0215] Embodiment 72 includes 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 includes 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 an interferon, GM-CSF, IL-2, IL-7, IL-12, IL-15, or IL-21.

[0218] Embodiment 75 includes 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 includes a pharmaceutical composition comprising an isolated polypeptide comprising a cleavable linker according to any one of the above embodiments, and a pharma- ceutically 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 above 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 above embodiments, comprising culturing a cell comprising the vector according to embodiment 78 under conditions resulting 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. EXAMPLES

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

[0225] Briefly, polypeptide complexes PC-1, PC-2, PC-3, PC-4, and PC-5 were generated that contain peptide masks genetically fused to the polypeptide complexes with 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 assessed for EGFR and CD3ε binding.

[0231] Briefly, the binding of polypeptide complexes PC-1, PC-2, PC-3, PC-4, and PC-5 containing EGFR masking was determined. 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 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, the 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 seen 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 seen 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, culture medium and non-adherent cells were removed and replaced with fresh medium containing the polypeptide complexes titrated 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 complex was evaluated for its pharmacokinetics and safety in cynomolgus monkeys.

[0251] Cynomolgus monkeys

[0252] Young male naive cynomolgus monkeys were housed in pairs by group and identified by tattoos unique to their bodies. All animals were acclimated to the housing conditions for 3 days before the start of the test. Before the start, all animals underwent a physical examination by a test veterinarian. Only animals that were healthy and met the criteria in other respects according to the judgment of the test veterinarian were registered for the test. Food was withheld overnight before administration. Purina 5049 was given daily in an amount appropriate for the size of the animal. Tap water was made freely available through an automatic watering device.

[0253] Pharmacokinetics

[0254] The pharmacokinetics of the polypeptide complexes PC-1, PC-2, PC-3, PC-4, and PC-5 were determined in male naive cynomolgus monkeys weighing 2 - 3 kg. Briefly, two groups of housed monkeys were used for each dosing group and acclimated to the surroundings before dosing. Before dosing and bleeding, the animals were sedated with ketamine HCL 10 - 20 mg / kg IM. The concentrated test substance was diluted with sterile phosphate-buffered saline and administered to the animals in an amount relative to the mass of the animal in kg. The dose of each test substance was administered intravenously at a dose of 1 mL / kg. For administration, the left and right limbs were clamped and prepped with alcohol. The saphenous vein was identified and a standard catheter was placed for IV bolus injection (either in the left or right limb). The test substance administration solution was flowed into the catheter by syringe (attached), and bolus injection was performed by manually compressing the syringe.

[0255] For blood collection, animals were sedated with ketamine, the femoral deltoid muscle was prepared, and blood was collected from the femoral vein using a 22G 1.5 inch needle, vacutainer sheath, and collection tube. After venipuncture, manual compression of the vein was maintained until hemostasis was achieved. Blood was collected based on the animal's weight and did not exceed the IACUC-specified AGI maximum blood loss. Blood was collected into EDTA tubes and processed to plasma. Blood samples were chilled and centrifuged at 3000xg for 10 minutes to separate cells from plasma. 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 antibody was coated directly onto the ELISA plate. Standard dilutions of the polypeptide complex in cynomolgus monkey serum were used to generate a standard curve to which the 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 samples 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 concentration was used to calculate the undisclosed 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 the polypeptide complexes 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] From these data, it was found that the polypeptide complex containing a cleavable linker extended the serum half-life in cynomolgus monkeys.

[0262] Example 5. Cytokine Release in Cynomolgus Monkeys In vivo Cytokine release in cynomolgus monkeys was measured.

[0263] Cytokines present in the plasma after treatment were measured using an assay kit (catalog number 557800) for non-human primate Th1 / Th2 cytometric bead array manufactured by BD Biosciences according to the manufacturer's instructions. The data are shown in FIGS. 6A to 6D and Table 21.

[0264]

Table 21

[0265] Example 6. Liver ALT / AST in Cynomolgus Monkeys In vivo ALT / AST values were measured. As seen in FIGS. 7A and 7B and Table 22, the polypeptide complex 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 is 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 according to the amino acid sequence of SEQ ID NO:6 (LSGRSDAGSPLGLAG), said isolated polypeptide further comprising an antigen binding domain that binds to a target antigen, said cleavable linker connecting a peptide to said antigen binding domain that binds to said target antigen in an arrangement according to formula I: A 1 -L 1 -P 1 , where A 1 comprises the antigen binding domain that binds to the target antigen, L 1 comprises the cleavable linker, and P 1 comprises a peptide that disrupts binding of the antigen binding domain to the target antigen.

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

3. The isolated polypeptide of claim 2 , wherein the protease comprises a tumor-specific protease.

4. The isolated polypeptide of claim 2 , wherein the protease comprises a matrix metalloprotease (MMP) or a serine protease.

5. The isolated polypeptide of claim 4, wherein the matrix metalloprotease comprises MMP2, MMP7, MMP9, MMP13, or MMP14.

6. 5. The isolated polypeptide of claim 4, wherein the serine protease comprises matriptase, urokinase, or hepsin.

7. 2. The isolated polypeptide of claim 1, wherein the antigen-binding domain is C-terminal to the cleavable linker.

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

9. P １ The isolated polypeptide of claim 1 , wherein said polypeptide has less than 70% sequence identity to said target antigen.

10. P １ 2. The isolated polypeptide of claim 1, comprising a peptide sequence at least 10 amino acids in length and no more than 20 amino acids in length.

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

12. The isolated polypeptide of claim 11, wherein the half-life extending moiety is a single domain antibody.

13. A １ 2. The isolated polypeptide of claim 1, 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.

14. A １ The isolated polypeptide of claim 13, comprising said scFv.

15. The isolated polypeptide of claim 14, wherein the scFv comprises an anti-CD3e single chain variable fragment.

16. A complex comprising the isolated polypeptide of claim 1 and a second isolated polypeptide comprising a second antigen-binding domain.

17. The second isolated polypeptide has the formula II:A ２ -L ２ -P ２ In the formula: ２ comprises the second antigen-binding domain, and L ２ comprises a second cleavable linker, P ２ comprises a second peptide that impairs binding of the second antigen-binding domain to a second target antigen, the second antigen-binding domain comprising a Fab light chain polypeptide or a Fab heavy chain polypeptide, and the second target antigen comprises a tumor antigen.

18. 18. The conjugate of claim 17, wherein the second cleavable linker comprises the amino acid sequence of SEQ ID NO:1 (LSGRSDAG).

19. 18. The conjugate of claim 17, wherein the second cleavable linker comprises the amino acid sequence of SEQ ID NO:3 (ISSGLLSGRSDAG).

20. 18. The conjugate of claim 17, wherein the second cleavable linker comprises the amino acid sequence of SEQ ID NO:26 (AGLLAPPGGLSGRSDAG).

21. 18. The conjugate of claim 17, wherein the second cleavable linker comprises the amino acid sequence of SEQ ID NO: 4 (AAGLLAPPGGLSGRSDAG).

22. 18. The conjugate of claim 17, wherein the second cleavable linker comprises the amino acid sequence of SEQ ID NO:5 (SPLGLSGRSDAG).

23. 18. The conjugate of claim 17, wherein the second cleavable linker comprises the amino acid sequence of SEQ ID NO:6 (LSGRSDAGSPLGLAG).

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