Compositions and methods relating to activatable therapeutic agents

Abstract

This invention provides a novel peptide segment that can be incorporated into various therapeutic, diagnostic, and prophylactic prodrug compositions for a more effective and reliable release mechanism. It also provides a more accurate and robust method for predicting therapeutic responses and outcomes upon administration of prodrugs or other activatable compositions. [Solution] Methods for evaluating the potential response of a subject to an activatable therapeutic agent, as well as compositions, kits, and methods for preparing and using an activatable therapeutic agent, are provided. Methods for evaluating the potential response of a subject to an activatable therapeutic agent are also provided. In some cases, the activatable therapeutic agents of the compositions, kits, and methods provided herein may include sequences derived from mammalian proteins. In certain embodiments, methods are provided for evaluating the potential responsiveness of a subject to a therapeutic agent that can be activated by a mammalian protease expressed in the subject.

Description

[Technical Field]

[0001] References This application claims priority to U.S. Provisional Patent Application No. 63 / 054525, filed on 21 July 2020, titled "Compositions and Methods Related to Activatable Therapeutic Agents," which is incorporated herein by reference in its entirety.

[0002] Sequence listing description: A computer-readable sequence listing is filed electronically together with this application. The sequence listing is incorporated herein by reference in its entirety. The sequence listing is contained in a file created on July 15, 2021, with the filename "791-601_20-1831-WO_ST25_FINAL.txt", and is 1700kb in size. [Background technology]

[0003] background A major challenge in the development of prodrug therapeutics is avoiding undesirable immunogenicity and nonspecific activation at non-target sites in vivo. Various release sites have been optimized in vitro and incorporated into prodrugs for programmed and targeted activation, for example, by proteases naturally produced in or near diseased tissue. Such engineered release segments may form T-cell or B-cell epitopes, which can induce undesirable immunogenicity in patients. Furthermore, there is currently a lack of methods to adequately predict a patient's in vivo response to prodrugs. In particular, with respect to protease-activated prodrugs, the targeted diseased tissue often contains numerous proteases with varying activities and specificities, which are difficult to reconstitute in vitro and complicate any prediction of in vivo prodrug activation. The identification of novel peptide segments that can be incorporated into various therapeutic, diagnostic, and prophylactic prodrug compositions for more effective and reliable release mechanisms remains necessary. There is still a need to develop more accurate and robust methods for predicting therapeutic responses and outcomes when prodrugs or other activatable compositions are administered. [Overview of the Initiative] [Means for solving the problem]

[0004] overview In certain embodiments, the present disclosure is a method for evaluating the likelihood that a subject is responsive to a therapeutic agent that can be activated by a mammalian protease expressed in the subject, (a) In biological samples from the subject, (i) A polypeptide comprising at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 consecutive amino acid residues as shown in the sequence in column V of Table A (or a subset thereof), or (ii) A polypeptide comprising at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 consecutive amino acids as shown in the sequence in column IV (or a subset thereof) of Table A, or (iii) The step of determining the presence or amount of a polypeptide containing at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 consecutive amino acids as shown in the sequence in column VI of Table A; and (b) If polypeptides (i), (ii), or (iii) are present and / or if their quantity exceeds a threshold, the step of designating the subject as likely to respond to the therapeutic agent. This provides a method that includes [something].

[0005] In some embodiments of a method for evaluating the potential responsiveness of a subject to a therapeutic agent, the therapeutic agent comprises a peptide substrate, which is sensitive to cleavage by a mammalian protease at an easily cleavable linkage. In some embodiments, the polypeptide of (i), (ii), or (iii) comprises a portion of the peptide substrate containing at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten consecutive amino acid residues at either the N-terminal or C-terminal end of the easily cleavable linkage. In some embodiments, the sequence of the peptide substrate is sensitive to cleavage by a mammalian protease at an easily cleavable linkage, and the polypeptide of (i), (ii), or (iii) is a cleavage product of a reporter polypeptide containing a substrate sequence sensitive to cleavage by the same mammalian protease at an easily cleavable linkage, wherein the reporter polypeptide comprises a sequence (or a subset thereof) listed in column II or III of Table A. In some embodiments, the peptide substrate sequence is sensitive to cleavage by mammalian proteases at the easily cleaved linkage, and the polypeptide of (i), (ii), or (iii) is a cleavage product of a human protein comprising a portion containing at least five or six consecutive amino acid residues of the peptide substrate containing the easily cleaved linkage.

[0006] In some embodiments of the method for evaluating the likelihood that a subject is responsive to a therapeutic agent, the polypeptide of (i) comprises at least six, at least seven, at least eight, at least nine, or at least ten consecutive amino acid residues as shown in the sequence listed in column V (or a subset thereof) of Table A. In some embodiments, the polypeptide of (ii) comprises at least six, at least seven, at least eight, at least nine, or at least ten consecutive amino acids as shown in the sequence listed in column IV (or a subset thereof) of Table A. In some embodiments, the polypeptide of (iii) comprises at least six, at least seven, at least eight, at least nine, or at least ten consecutive amino acids as shown in the sequence listed in column VI (or a subset thereof) of Table A.

[0007] In some embodiments of a method for evaluating the likelihood that a subject is responsive to a therapeutic agent, (a) includes determining the presence or amount of any two of (i) to (iii). In some embodiments, (a) includes determining the presence or amount of all three of (i) to (iii).

[0008] In some embodiments of the method for evaluating the likelihood that a subject is responsive to a therapeutic agent, the threshold is zero or a nominal value. In some embodiments, the biological sample includes a serum or plasma sample. In some embodiments, the biological sample includes a serum sample. In some embodiments, the biological sample includes a plasma sample.

[0009] In some embodiments of a method for evaluating the likelihood that a subject is responsive to a therapeutic agent, the mammalian protease is a serine protease, cysteine ​​protease, aspartate protease, threonine protease, or metalloproteinase.In some embodiments, the mammalian protease is a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), a disintegrin and metalloproteinase domain-containing protein 12 (ADAM12), a disintegrin and metalloproteinase domain-containing protein 15 (ADAM15), a disintegrin and metalloproteinase domain-containing protein 17 (ADAM17), and a disintegrin and metalloproteinase domain-containing protein Kallikrein-9 (ADAM9), disintegrin and metalloproteinase with thrombospondin motif 5 (ADAMTS5), cathepsin B, cathepsin D, cathepsin E, cathepsin K, cathepsin L, cathepsin S, fibroblast-activating protein alpha, hepsin, kallikrein-2, kallikrein-4, kallikrein-3, prostate-specific antigen (PSA), kallikrein-13, regmine, matrix metallopeptidase 1 (MMP-1), matrix metallopeptidase 10 (MMP-10) ), Matrix metallopeptidase 11 (MMP-11), Matrix metallopeptidase 12 (MMP-12), Matrix metallopeptidase 13 (MMP-13), Matrix metallopeptidase 14 (MMP-14), Matrix metallopeptidase 16 (MMP-16), Matrix metallopeptidase 2 (MMP-2), Matrix metallopeptidase 3 (MMP-3), Matrix metallopeptidase 7 (MMP-7), Matrix metallopeptidase 8 (MMP-8), Mat The following are selected from the group consisting of rix metallopeptidase 9 (MMP-9), matrix metallopeptidase 4 (MMP-4), matrix metallopeptidase 5 (MMP-5), matrix metallopeptidase 6 (MMP-6), matrix metallopeptidase 15 (MMP-15), neutrophil elastase, protease-activated receptor 2 (PAR2), plasmin, prostasin, PSMA-FOLH1, membrane-bound serine protease 1 (MT-SP1), matryptase, and u-plasminogen.In some embodiments, the mammalian protease is selected from the group consisting of matrix metallopeptidase 1 (MMP1), matrix metallopeptidase 2 (MMP2), matrix metallopeptidase 7 (MMP7), matrix metallopeptidase 9 (MMP9), matrix metallopeptidase 11 (MMP11), matrix metallopeptidase 14 (MMP14), urokinase-type plasminogen activator (uPA), regmine, and matryptase. In some embodiments, the mammalian protease is preferentially expressed or activated in the target tissue or cell.

[0010] In some embodiments of the method for evaluating the likelihood that a target is responsive to a therapeutic agent, the target tissue or cells are tumors. In some embodiments, the target tissue or cells produce or co-localize with mammalian proteases.

[0011] In some embodiments of a method for evaluating the potential responsiveness of a target to a therapeutic agent, the target tissue or cells contain a reporter polypeptide within or on them, or are associated with a reporter polypeptide in their vicinity. In some embodiments, the reporter polypeptide is a coagulation factor, complement component, tubulin, immunoglobulin, apolipoprotein, serum amyloid, insulin, growth factor, fibrinogen, PDZ domain protein, LIM domain protein, c-reactive protein, serum albumin, versican, collagen, elastin, keratin, kininogen-1, alpha-2-antiplasmin, clusterin, biglycan, alpha-1-antitrypsin, transthyretin, alpha-1-antichymotrypsin, glucagon, hepcidin, thymosin beta-4, haptoglobin, hemoglobin subunit alpha, caveolae-associated protein 2, alpha-2-HS-glycoprotein, chromogranin-A, vitronectin, hemopexin, epididymal sperm-binding protein, secretogranin-2, angiothex Insinogen, transgerin-2, pancreatic prohormone, neurosecretory protein VGF, ceruloplasmin, PDZ and LIM domain protein 1, multimelin-1, inter-alpha-trypsin inhibitor heavy chain H2, N-acetylmuramoyl-L-alanine amidase, histone H1.4, adhesion G protein-coupled receptor G6, mannan-binding lectin serine protease 2, prothrombin, malignant brain tumor deletion 1 protein, desmoglein-3, calcin-tenin-1, alpha-2-macroglobulin, myosin-9, sodium / potassium transport ATPase subunit gamma, oncoprotein-inducible transcript 3 protein, cerglycine, histidine-rich glycoprotein, inter-alpha-trypsin inhibitor heavy chain H5, integrin alpha-IIb, membrane-bound progesterone receptor component 1, histone H1.2. A polypeptide selected from the group consisting of rho GDP dissociation inhibitor 2, zinc-alpha-2-glycoprotein, talin-1, secretogranin-1, neutrophil defensin 3, cytochrome P450 2E1, gastric suppressor polypeptide, transcription initiation factor TFIID subunit 1, membrane-bound protein 2B, pigment epithelium-derived factor, voltage-gated N-type calcium channel subunit alpha-1B, ras GTPase-activating protein nGAP, type I cytoskeleton 17, sulfhydryl oxidase 1, homeobox protein Hox-B2, transcription factor SOX-10, E3 ubiquitin-protein ligase SIAH2, decorin, acidic, cysteine-rich secretory protein (SPARC), laminin gamma 1 chain, vimentin, and nidogen-1 (NID1). In some embodiments, the reporter polypeptide is versican, type II collagen alpha-1 chain, kininogen-1, complement C4-A, complement C4-B, complement C3, alpha-2-antiplasmin, clusterin, biglycan, elastin, fibrinogen alpha chain, alpha-1-antitrypsin, fibrinogen beta chain, type III collagen alpha-1 chain, serum amyloid A-1 protein, transthyretin, apolipoprotein AI, apolipoprotein AI isoform 1, alpha-1-antichymotrypsin, glucagon, hepcidin, serum amyloid A-2 protein, thymosin beta-4, haptoglobin, hemoglobin subunit alpha, caveo R-related protein 2, alpha-2-HS-glycoprotein, chromogranin-A, vitronectin, hemopexin, epididymal sperm-binding protein, zyxin, apolipoprotein C-III, secretogranin-2, angiotensinogen, c-reactive protein, serum albumin, transgerin-2, pancreatic prohormones, neurosecretory protein VGF, ceruloplasmin, PDZ and LIM domain proteins 1, tubulin alpha-4A chain, multimelin-1, inter-alpha-trypsin inhibitor heavy chain H2, apolipoprotein CI, fibrinogen gamma chain, N-acetylmuramoyl-L-alanine amidase, immunoglobulin lambda variable 3-21, histone H1.4, Adhesion G protein-coupled receptor G6, Immunoglobulin lambda variable 3-25, Immunoglobulin lambda variable 1-51, Immunoglobulin lambda variable 1-36, Mannan-binding lectin serine protease 2, Immunoglobulin kappa variable 3-20, Immunoglobulin kappa variable 2-30, Insulin-like growth factor II, Apolipoprotein A-II, Immunoglobulin kappa variable 2D-24 (likely non-functional), Prothrombin, Coagulation factor IX, Apolipoprotein L1, Malignant brain tumor deletion 1 protein, Desmoglein-3, Calcinetenin-1, Immunoglobulin lambda constant 3, Complement C5, Alpha-2-macroglobulin, Myosin-9, Sodium / potassium transport ATPase subunit gamma, Immunoglobulin kappa variable 2-28, Oncoprotein-inducible transcript 3 protein, Cerglycine, Coagulation factor XII, Coagulation factor XIII A chain, insulin, histidine-rich glycoprotein, immunoglobulin kappa variable 3-11, immunoglobulin kappa variable 1-39, collagen alpha-1(I) chain, inter-alpha-trypsin inhibitor heavy chain H5, latent transforming growth factor beta-binding protein 2, integrin alpha-IIb, membrane-bound progesterone receptor component 1, immunoglobulin lambda variable 6-57, immunoglobulin kappa variable 3-15, complement C1r small component-like protein, histone H1.2, Rho GDP dissociation inhibitor 2, latent transforming growth factor beta-binding protein 4, collagen alpha-1 (XVIII) chain, immunoglobulin lambda variable 2-18, zinc-alpha-2-glycoprotein, talin-1, secretogranin-1, neutrophil defensin 3, cytochrome P450 2E1, gastric suppressor polypeptide, immunoglobulin heavy chain variable 3-15, immunoglobulin lambda variable 2-11, transcription initiation factor TFIID subunit 1, collagen alpha-1 (VII) chain, membrane endogenous protein 2B, pigment epithelium-derived factor, voltage-gated N-type calcium channel subunit alpha-1B, immunoglobulin lambda variable 3-27, ras The reporter polypeptide is selected from the group consisting of GTPase-activated protein nGAP, keratin, type I cytoskeleton 17, tubulin beta chain, sulfhydryl oxidase 1, immunoglobulin kappa variable 4-1, complement C1r minor component, homeobox protein Hox-B2, transcription factor SOX-10, E3 ubiquitin-protein ligase SIAH2, decorin, SPARC, type I collagen alpha-1 chain, type IV collagen alpha-1 chain, laminin gamma 1 chain, vimentin, type III collagen, type IV collagen alpha-3 chain, type VII collagen alpha-1 chain, type VI collagen alpha-1 chain, type V collagen alpha-1 chain, nidogen-1, and type VI collagen alpha-3 chain. In some embodiments, the reporter polypeptide includes the sequences listed in columns II-VI (or subsets thereof) of Table A. In some embodiments, the reporter polypeptide is selected from the group listed in column I (or subset thereof) of Table A.

[0012] In some embodiments of a method for evaluating the likelihood of a subject being responsive to a therapeutic agent, the target tissue or cells are characterized by an increased amount or activity of mammalian proteases in the vicinity of the target tissue or cells compared to non-target tissue or cells in the subject. In some embodiments, the subject is suffering from or suspected of suffering from a disease or condition characterized by increased expression or activity of mammalian proteases in the vicinity of the target tissue or cells compared to the corresponding non-target tissue or cells in the subject.

[0013] In some embodiments of the method for evaluating the likelihood of a subject being responsive to a therapeutic agent, the disease or condition is cancer, inflammatory disease, or autoimmune disease. In some embodiments, the disease or condition is carcinoma, Hodgkin lymphoma, and non-Hodgkin lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, blastoma, breast cancer, ER / PR + Breast cancer, Her2 +The group consists of breast cancer, triple-negative breast cancer, colon cancer, colon cancer with malignant ascites, mucinous neoplasms, prostate cancer, head and neck cancer, skin cancer, melanoma, genitourinary cancer, ovarian cancer, ovarian cancer with malignant ascites, peritoneal carcinomatosis, serous uterine carcinoma, endometrial cancer, cervical cancer, colorectal cancer, uterine cancer, peritoneal mesothelioma, kidney cancer, Wilms' tumor, lung cancer, small cell lung cancer, non-small cell lung cancer, gastric cancer, stomach cancer, small intestine cancer, liver cancer, hepatocellular carcinoma, hepatoblastoma, liposarcoma, pancreatic cancer, gallbladder cancer, bile duct cancer, esophageal cancer, salivary gland cancer, thyroid cancer, epithelial cancer, masculinizing neoplasm, adenocarcinoma, sarcoma, and B-cell chronic lymphocytic leukemia.In some embodiments, the disease or condition is ankylosing spondylitis (AS), arthritis (e.g., rheumatoid arthritis (RA), juvenile idiopathic arthritis (JIA), osteoarthritis (OA), psoriatic arthritis (PsA), gout, chronic arthritis), Chagas disease, chronic obstructive pulmonary disease (COPD), dermatomyositis, type 1 diabetes mellitus, endometriosis, Goodpasture syndrome, Graves' disease, Guillain-Barré syndrome (GBS), Hashimoto's disease, suppurative crus, Kawasaki disease, IgA nephropathy, idiopathic thrombocytopenic purpura, inflammatory bowel disease (IBD) (e.g., not limited to these) Although not limited to these, Crohn's disease (CD), Crohn's disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischemic colitis, vacant colitis, Behçet's syndrome, infectious colitis, indeterminate colitis, interstitial cystitis), lupus (for example, systemic lupus erythematosus, discoid lupus, subacute cutaneous lupus erythematosus, cutaneous lupus erythematosus (e.g., frostbite-like lupus erythematosus), drug-induced lupus, neonatal lupus, lupus nephritis), mixed connective tissue disease, Morphea, multiple sclerosis (MS), myocardial infarction, narcolepsy Lepsy, neuromuscular anguina, pemphigus vulgaris, pernicious anemia, psoriasis, psoriatic arthritis, polymyositis, primary biliary cirrhosis, relapsing polychondritis, schizophrenia, scleroderma, Sjögren's syndrome, generalized rigidity syndrome, temporal arteritis (also known as giant cell arteritis), vasculitis, vitiligo, Wegener's granulomatosis, transplant rejection-associated immune reactions (e.g., kidney transplant rejection, lung transplant rejection, liver transplant rejection, etc., though not limited to these), psoriasis, Wiscott-Aldrich syndrome, autoimmune lymphoproliferative syndrome, myasthenia gravis, inflammatory chronic sinusitis, colitis, celiac disease, Barrett's esophagus, The group is selected from inflammatory gastritis, autoimmune nephritis, autoimmune hepatitis, autoimmune carditis, autoimmune encephalitis, autoimmune-mediated hematological disorders, asthma, atopic dermatitis, atopy, allergies, allergic rhinitis, scleroderma, bronchitis, and pericarditis, and the inflammatory diseases are Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, inflammatory lung disease, inflammatory skin disease, atherosclerosis, myocardial infarction, stroke, Gram-positive shock, Gram-negative shock, sepsis, septic shock, hemorrhagic shock, anaphylactic shock, and systemic inflammatory response syndrome.

[0014] In some embodiments of the method for evaluating the potential responsiveness of a subject to a therapeutic agent, the therapeutic agent is an anticancer agent. In some embodiments, the therapeutic agent is an activatable therapeutic agent. In some embodiments, the therapeutic agent is an activatable therapeutic agent or a non-natural activatable therapeutic agent as described herein.

[0015] In some embodiments of the method for evaluating the potential responsiveness of a subject to a therapeutic agent, the therapeutic agent further comprises a masking moiety (MM). In some embodiments of the method for evaluating the potential responsiveness of a subject to a therapeutic agent, the masking moiety (MM) may be released from the therapeutic agent upon cleavage of the peptide substrate by a mammalian protease. In some embodiments, the masking moiety (MM), in its uncleaved state, interferes with the interaction between the therapeutic agent and the target tissue or cell. In some embodiments, the bioactivity of the therapeutic agent may be enhanced upon cleavage of the peptide substrate by a mammalian protease. In some embodiments, the masking moiety (MM) is an elongated recombinant polypeptide (XTEN). In some embodiments, XTEN is characterized by (i) containing at least 100 amino acids; (ii) at least 90% of its amino acid residues being selected from glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), and proline (P); and (iii) containing at least four different amino acids selected from G, A, S, T, E, and P.

[0016] Some embodiments of the method for assessing the likelihood that a subject is responsive to a therapeutic agent further include the step of sending a designation to the healthcare provider and / or the subject.

[0017] Some embodiments of the method for evaluating the likelihood that a subject is responsive to a therapeutic agent further include, after (b), a step of contacting the therapeutic agent with a mammalian protease.

[0018] Some embodiments of a method for evaluating the likelihood that a subject is responsive to a therapeutic agent further include, after (b), a step of administering an effective amount of the therapeutic agent to the subject based on the designation in step (b).

[0019] In some embodiments of a method for evaluating the likelihood that a subject is responsive to a therapeutic agent, (a) includes detecting the polypeptide of (i), (ii), or (iii) by immunoassay. In some embodiments, the immunoassay utilizes an antibody that specifically binds to the polypeptide of (i), (ii), or (iii) or its epitope.

[0020] In some embodiments of a method for evaluating the likelihood that a subject is responsive to a therapeutic agent, (a) includes detecting the polypeptide (including its derivatives (fragments)) of (i), (ii), or (iii) by using a mass spectrometer (MS).

[0021] Some embodiments of the method involve the use of diagnostic reagents to assess the likelihood that a subject with a disease or disorder is responsive to a therapeutic agent that can be activated by a mammalian protease expressed in the subject.

[0022] In a particular embodiment, the diagnostic reagent is used to assess the likelihood that a subject with a disease or disorder is responsive to a therapeutic agent that can be activated by a mammalian protease expressed in the subject.

[0023] Some embodiments include a kit for performing a method to assess the likelihood that a subject with a disease or disorder is responsive to a therapeutic agent that can be activated by a mammalian protease expressed in the subject, comprising a reagent for detecting the presence or amount of proteolytic peptide products generated by the action of a mammalian protease.

[0024] In certain embodiments, the present disclosure relates to a method for treating a subject that requires a therapeutic agent that can be activated by a mammalian protease expressed in the subject, The step includes administering an effective amount of the therapeutic agent to the subject, and the subject then, in a biological sample from the subject, (i) A polypeptide comprising at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 consecutive amino acid residues as shown in the sequence in column V (or a subset thereof) of Table A, or (ii) A polypeptide comprising at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 consecutive amino acids as shown in the sequence in column IV (or a subset thereof) of Table A, or (iii) A polypeptide comprising at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 consecutive amino acids as shown in the sequence in column VI of Table A (or a subset thereof). It has been shown to express, or (iv) Provide a method for which the expression level of polypeptide (i), (ii), or (iii) exceeds a threshold.

[0025] In some embodiments for treating a subject with a therapeutic agent, polypeptide (i) contains at least 6, at least 7, at least 8, at least 9, or at least 10 consecutive amino acid residues as shown in the sequence listed in column V (or a subset thereof) of Table A. In some embodiments, polypeptide (ii) contains at least 6, at least 7, at least 8, at least 9, or at least 10 consecutive amino acids as shown in the sequence listed in column IV (or a subset thereof) of Table A. In some embodiments, polypeptide (iii) contains at least 6, at least 7, at least 8, at least 9, or at least 10 consecutive amino acids as shown in the sequence listed in column VI (or a subset thereof) of Table A. In some embodiments, the subject is shown to express any two of (i) to (iii) in a biological sample. In some embodiments, the subject is shown to express all three of (i) to (iii) in a biological sample.

[0026] In some embodiments for treating a target with a therapeutic agent, the therapeutic agent comprises a peptide substrate sensitive to cleavage by mammalian proteases. In some embodiments, the peptide substrate sequence is sensitive to cleavage by mammalian proteases at a cleavable linkage, and the polypeptide of (i), (ii), or (iii) comprises a portion containing at least four consecutive amino acid residues of the peptide substrate at either the N-terminal or C-terminal side of the cleavable linkage. In some embodiments, the portion of the peptide substrate at the N-terminal side of the cleavable linkage has up to three or two amino acid substitutions or up to one amino acid substitution to the C-terminal sequence containing 4 to 10 amino acid residues of the sequences (or subsets thereof) listed in column IV or V of Table A, and none of the amino acid substitutions are in a position corresponding to an amino acid residue directly adjacent to the corresponding cleavable linkage. In some embodiments, the peptide substrate portion at the N-terminus of the easily cleavable bond has up to three or two amino acid substitutions or up to one amino acid substitution to a C-terminal sequence containing 4 to 10 amino acid residues of the sequence described in column IV (or a subset thereof) of Table A, and none of the amino acid substitutions are located in positions corresponding to amino acid residues directly adjacent to the corresponding easily cleavable bond. In some embodiments, the peptide substrate portion at the N-terminus of the easily cleavable bond has up to three or two amino acid substitutions or up to one amino acid substitution to a C-terminal sequence containing 4 to 10 amino acid residues of the sequence described in column V (or a subset thereof) of Table A, and none of the amino acid substitutions are located in positions corresponding to amino acid residues directly adjacent to the corresponding easily cleavable bond. In some embodiments, the peptide substrate portion at the N-terminus of the easily cleavable bond includes a C-terminal sequence containing 4 to 10 amino acid residues of the sequence described in column IV or V (or a subset thereof) of Table A. In some embodiments, the peptide substrate portion at the N-terminus of the easily cleavable bond includes a C-terminal sequence containing 4 to 10 amino acid residues of the sequence described in column IV (or a subset thereof) of Table A. In some embodiments, the peptide substrate portion at the N-terminus of the easily cleavable bond includes a C-terminal sequence containing 4 to 10 amino acid residues of the sequence described in column V (or a subset thereof) of Table A.In some embodiments, the peptide substrate portion at the C-terminus of the easily cleavable bond has up to three or two amino acid substitutions or up to one amino acid substitution to the N-terminal sequence containing 4 to 10 amino acid residues of the sequence described in column V or VI (or a subset thereof) of Table A, and none of the amino acid substitutions are located in positions corresponding to amino acid residues directly adjacent to the corresponding easily cleavable bond. In some embodiments, the peptide substrate portion at the C-terminus of the easily cleavable bond has up to three or two amino acid substitutions or up to one amino acid substitution to the N-terminal sequence containing 4 to 10 amino acid residues of the sequence described in column V (or a subset thereof) of Table A, and none of the amino acid substitutions are located in positions corresponding to amino acid residues directly adjacent to the corresponding easily cleavable bond. In some embodiments, the peptide substrate portion at the C-terminus of the easily cleavable bond has up to three or two amino acid substitutions or up to one amino acid substitution to the N-terminal sequence containing 4 to 10 amino acid residues of the sequence described in column VI (or a subset thereof) of Table A, and none of the amino acid substitutions are located in positions corresponding to amino acid residues directly adjacent to the corresponding easily cleavable bond. In some embodiments, the peptide substrate portion at the C-terminus of the easily cleavable bond includes an N-terminal sequence containing 4 to 10 amino acid residues of the sequence described in column V or VI (or a subset thereof) of Table A. In some embodiments, the peptide substrate portion at the C-terminus of the easily cleavable bond includes an N-terminal sequence containing 4 to 10 amino acid residues of the sequence described in column V (or a subset thereof) of Table A. In some embodiments, the peptide substrate portion at the C-terminus of the easily cleavable bond includes an N-terminal sequence containing 4 to 10 amino acid residues of the sequence described in column VI (or a subset thereof) of Table A.

[0027] In some embodiments for treating a target with a therapeutic agent, the threshold is zero or a nominal value. In some embodiments, the biological sample includes a serum or plasma sample. In some embodiments, the biological sample includes a serum sample. In some embodiments, the biological sample includes a plasma sample.

[0028] In some embodiments for treating the target with a therapeutic agent, the mammalian protease is a serine protease, cysteine ​​protease, aspartate protease, threonine protease, or metalloproteinase.In some embodiments, the mammalian protease is a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), a disintegrin and metalloproteinase domain-containing protein 12 (ADAM12), a disintegrin and metalloproteinase domain-containing protein 15 (ADAM15), a disintegrin and metalloproteinase domain-containing protein 17 (ADAM17), and a disintegrin and metalloproteinase domain-containing protein Kallikrein-9 (ADAM9), disintegrin and metalloproteinase with thrombospondin motif 5 (ADAMTS5), cathepsin B, cathepsin D, cathepsin E, cathepsin K, cathepsin L, cathepsin S, fibroblast-activating protein alpha, hepsin, kallikrein-2, kallikrein-4, kallikrein-3, prostate-specific antigen (PSA), kallikrein-13, regmine, matrix metallopeptidase 1 (MMP-1), matrix metallopeptidase 10 (MMP-10) ), Matrix metallopeptidase 11 (MMP-11), Matrix metallopeptidase 12 (MMP-12), Matrix metallopeptidase 13 (MMP-13), Matrix metallopeptidase 14 (MMP-14), Matrix metallopeptidase 16 (MMP-16), Matrix metallopeptidase 2 (MMP-2), Matrix metallopeptidase 3 (MMP-3), Matrix metallopeptidase 7 (MMP-7), Matrix metallopeptidase 8 (MMP-8), Mat The following are selected from the group consisting of rix metallopeptidase 9 (MMP-9), matrix metallopeptidase 4 (MMP-4), matrix metallopeptidase 5 (MMP-5), matrix metallopeptidase 6 (MMP-6), matrix metallopeptidase 15 (MMP-15), neutrophil elastase, protease-activated receptor 2 (PAR2), plasmin, prostasin, PSMA-FOLH1, membrane-bound serine protease 1 (MT-SP1), matryptase, and u-plasminogen.In some embodiments, the mammalian protease is selected from the group consisting of matrix metallopeptidase 1 (MMP1), matrix metallopeptidase 2 (MMP2), matrix metallopeptidase 7 (MMP7), matrix metallopeptidase 9 (MMP9), matrix metallopeptidase 11 (MMP11), matrix metallopeptidase 14 (MMP14), urokinase-type plasminogen activator (uPA), regmine, and matryptase. In some embodiments, the mammalian protease is preferentially expressed or activated in a target tissue or cell. In some embodiments, the target tissue or cell is a tumor. In some embodiments, the target tissue or cell produces the mammalian protease or co-localizes with the mammalian protease.

[0029] In some embodiments for treating a target with a therapeutic agent, the target tissue or cells contain or associate with a reporter polypeptide in or near them. In some embodiments, the reporter polypeptide is a coagulation factor, complement component, tubulin, immunoglobulin, apolipoprotein, serum amyloid, insulin, growth factor, fibrinogen, PDZ domain protein, LIM domain protein, c-reactive protein, serum albumin, versican, collagen, elastin, keratin, kininogen-1, alpha-2-antiplasmin, clusterin, biglycan, alpha-1-antitrypsin, transthyretin, alpha-1-antichymotrypsin, glucagon, hepcidin, thymosin beta-4, haptoglobin, hemoglobin subunit alpha, caveolae-associated protein 2, alpha-2-HS-glycoprotein, chromogranin-A, vitronectin, hemopexin, epididymal sperm-binding protein, secretogranin-2, angiothene Insinogen, transgerin-2, pancreatic prohormone, neurosecretory protein VGF, ceruloplasmin, PDZ and LIM domain protein 1, multimelin-1, inter-alpha-trypsin inhibitor heavy chain H2, N-acetylmuramoyl-L-alanine amidase, histone H1.4, adhesion G protein-coupled receptor G6, mannan-binding lectin serine protease 2, prothrombin, malignant brain tumor deletion 1 protein, desmoglein-3, calcin-tenin-1, alpha-2-macroglobulin, myosin-9, sodium / potassium transport ATPase subunit gamma, oncoprotein-inducible transcript 3 protein, cerglycine, histidine-rich glycoprotein, inter-alpha-trypsin inhibitor heavy chain H5, integrin alpha-IIb, membrane-bound progesterone receptor component 1, histone H1.2. A polypeptide selected from the group consisting of rho GDP dissociation inhibitor 2, zinc-alpha-2-glycoprotein, talin-1, secretogranin-1, neutrophil defensin 3, cytochrome P450 2E1, gastric suppressor polypeptide, transcription initiation factor TFIID subunit 1, membrane-bound protein 2B, pigment epithelium-derived factor, voltage-gated N-type calcium channel subunit alpha-1B, ras GTPase-activating protein nGAP, type I cytoskeleton 17, sulfhydryl oxidase 1, homeobox protein Hox-B2, transcription factor SOX-10, E3 ubiquitin-protein ligase SIAH2, decorin, acidic, cysteine-rich secretory protein (SPARC), laminin gamma 1 chain, vimentin, and nidogen-1 (NID1). In some embodiments, the reporter polypeptide is versican, type II collagen alpha-1 chain, kininogen-1, complement C4-A, complement C4-B, complement C3, alpha-2-antiplasmin, clusterin, biglycan, elastin, fibrinogen alpha chain, alpha-1-antitrypsin, fibrinogen beta chain, type III collagen alpha-1 chain, serum amyloid A-1 protein, transthyretin, apolipoprotein AI, apolipoprotein AI isoform 1, alpha-1-antichymotrypsin, glucagon, hepcidin, serum amyloid A-2 protein, thymosin beta-4, haptoglobin, hemoglobin subunit alpha, caveo R-related protein 2, alpha-2-HS-glycoprotein, chromogranin-A, vitronectin, hemopexin, epididymal sperm-binding protein, zyxin, apolipoprotein C-III, secretogranin-2, angiotensinogen, c-reactive protein, serum albumin, transgerin-2, pancreatic prohormones, neurosecretory protein VGF, ceruloplasmin, PDZ and LIM domain proteins 1, tubulin alpha-4A chain, multimelin-1, inter-alpha-trypsin inhibitor heavy chain H2, apolipoprotein CI, fibrinogen gamma chain, N-acetylmuramoyl-L-alanine amidase, immunoglobulin lambda variable 3-21, histone H1.4, Adhesion G protein-coupled receptor G6, Immunoglobulin lambda variable 3-25, Immunoglobulin lambda variable 1-51, Immunoglobulin lambda variable 1-36, Mannan-binding lectin serine protease 2, Immunoglobulin kappa variable 3-20, Immunoglobulin kappa variable 2-30, Insulin-like growth factor II, Apolipoprotein A-II, Immunoglobulin kappa variable 2D-24 (likely non-functional), Prothrombin, Coagulation factor IX, Apolipoprotein L1, Malignant brain tumor deletion 1 protein, Desmoglein-3, Calcinetenin-1, Immunoglobulin lambda constant 3, Complement C5, Alpha-2-macroglobulin, Myosin-9, Sodium / potassium transport ATPase subunit gamma, Immunoglobulin kappa variable 2-28, Oncoprotein-inducible transcript 3 protein, Cerglycine, Coagulation factor XII, Coagulation factor XIII A chain, insulin, histidine-rich glycoprotein, immunoglobulin kappa variable 3-11, immunoglobulin kappa variable 1-39, collagen alpha-1(I) chain, inter-alpha-trypsin inhibitor heavy chain H5, latent transforming growth factor beta-binding protein 2, integrin alpha-IIb, membrane-bound progesterone receptor component 1, immunoglobulin lambda variable 6-57, immunoglobulin kappa variable 3-15, complement C1r small component-like protein, histone H1.2, Rho GDP dissociation inhibitor 2, latent transforming growth factor beta-binding protein 4, collagen alpha-1 (XVIII) chain, immunoglobulin lambda variable 2-18, zinc-alpha-2-glycoprotein, talin-1, secretogranin-1, neutrophil defensin 3, cytochrome P450 2E1, gastric suppressor polypeptide, immunoglobulin heavy chain variable 3-15, immunoglobulin lambda variable 2-11, transcription initiation factor TFIID subunit 1, collagen alpha-1 (VII) chain, membrane endogenous protein 2B, pigment epithelium-derived factor, voltage-gated N-type calcium channel subunit alpha-1B, immunoglobulin lambda variable 3-27, ras The reporter polypeptide is selected from the group consisting of GTPase-activated protein nGAP, keratin, type I cytoskeleton 17, tubulin beta chain, sulfhydryl oxidase 1, immunoglobulin kappa variable 4-1, complement C1r minor component, homeobox protein Hox-B2, transcription factor SOX-10, E3 ubiquitin-protein ligase SIAH2, decorin, SPARC, type I collagen alpha-1 chain, type IV collagen alpha-1 chain, laminin gamma 1 chain, vimentin, type III collagen, type IV collagen alpha-3 chain, type VII collagen alpha-1 chain, type VI collagen alpha-1 chain, type V collagen alpha-1 chain, nidogen-1, and type VI collagen alpha-3 chain. In some embodiments, the reporter polypeptide includes the sequences listed in columns II-VI (or subsets thereof) of Table A. In some embodiments, the reporter polypeptide is selected from the group listed in column I (or subset thereof) of Table A.

[0030] In some embodiments for treating a subject with a therapeutic agent, the target tissue or cells are characterized by an increased amount or activity of mammalian proteases in the vicinity of the target tissue or cells compared to non-target tissue or cells in the subject. In some embodiments, the subject is suffering from or suspected of suffering from a disease or condition characterized by increased expression or activity of mammalian proteases in the vicinity of the target tissue or cells compared to the corresponding non-target tissue or cells in the subject. In some embodiments, the disease or condition is cancer, an inflammatory disease, or an autoimmune disease.In some embodiments, the disease or condition is ankylosing spondylitis (AS), arthritis (e.g., rheumatoid arthritis (RA), juvenile idiopathic arthritis (JIA), osteoarthritis (OA), psoriatic arthritis (PsA), gout, chronic arthritis), Chagas disease, chronic obstructive pulmonary disease (COPD), dermatomyositis, type 1 diabetes mellitus, endometriosis, Goodpasture syndrome, Graves' disease, Guillain-Barré syndrome (GBS), Hashimoto's disease, suppurative crus, Kawasaki disease, IgA nephropathy, idiopathic thrombocytopenic purpura, inflammatory bowel disease (IBD) (e.g., not limited to these) Although not limited to these, Crohn's disease (CD), Crohn's disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischemic colitis, vacant colitis, Behçet's syndrome, infectious colitis, indeterminate colitis, interstitial cystitis), lupus (for example, systemic lupus erythematosus, discoid lupus, subacute cutaneous lupus erythematosus, cutaneous lupus erythematosus (e.g., frostbite-like lupus erythematosus), drug-induced lupus, neonatal lupus, lupus nephritis), mixed connective tissue disease, Morphea, multiple sclerosis (MS), myocardial infarction, narcolepsy Lepsy, neuromuscular anguina, pemphigus vulgaris, pernicious anemia, psoriasis, psoriatic arthritis, polymyositis, primary biliary cirrhosis, relapsing polychondritis, schizophrenia, scleroderma, Sjögren's syndrome, generalized rigidity syndrome, temporal arteritis (also known as giant cell arteritis), vasculitis, vitiligo, Wegener's granulomatosis, transplant rejection-associated immune reactions (e.g., kidney transplant rejection, lung transplant rejection, liver transplant rejection, etc., though not limited to these), psoriasis, Wiscott-Aldrich syndrome, autoimmune lymphoproliferative syndrome, myasthenia gravis, inflammatory chronic sinusitis, colitis, celiac disease, Barrett's esophagus, The group is selected from inflammatory gastritis, autoimmune nephritis, autoimmune hepatitis, autoimmune carditis, autoimmune encephalitis, autoimmune-mediated hematological disorders, asthma, atopic dermatitis, atopy, allergies, allergic rhinitis, scleroderma, bronchitis, and pericarditis, and the inflammatory diseases are Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, inflammatory lung disease, inflammatory skin disease, atherosclerosis, myocardial infarction, stroke, Gram-positive shock, Gram-negative shock, sepsis, septic shock, hemorrhagic shock, anaphylactic shock, and systemic inflammatory response syndrome.In some embodiments, the disease or condition is carcinoma, Hodgkin lymphoma, and non-Hodgkin lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, blastoma, breast cancer, ER / PR. + Breast cancer, Her2 + The therapeutic agent is selected from the group consisting of breast cancer, triple-negative breast cancer, colon cancer, colon cancer with malignant ascites, mucinous neoplasm, prostate cancer, head and neck cancer, skin cancer, melanoma, genitourinary cancer, ovarian cancer, ovarian cancer with malignant ascites, peritoneal carcinomatosis, serous uterine carcinoma, endometrial cancer, cervical cancer, colorectal cancer, uterine cancer, peritoneal mesothelioma, kidney cancer, Wilms' tumor, lung cancer, small cell lung cancer, non-small cell lung cancer, gastric cancer, stomach cancer, small intestine cancer, liver cancer, hepatocellular carcinoma, hepatoblastoma, liposarcoma, pancreatic cancer, gallbladder cancer, bile duct cancer, esophageal cancer, salivary gland cancer, thyroid cancer, epithelial cancer, masculinizing neoplasm, adenocarcinoma, sarcoma, and B-cell chronic lymphocytic leukemia. In some embodiments, the therapeutic agent is an anticancer agent. In some embodiments, the therapeutic agent is an activatable therapeutic agent. In some embodiments, the therapeutic agent is a non-natural, activatable therapeutic agent described herein.

[0031] In some embodiments for treating a target with a therapeutic agent, the therapeutic agent includes a masking moiety (MM). In some embodiments, the masking moiety (MM) may be released from the therapeutic agent upon cleavage of the peptide substrate by a mammalian protease. In some embodiments, the masking moiety (MM), in its uncleaved state, interferes with the interaction between the therapeutic agent and the target tissue or cell. In some embodiments, the bioactivity of the therapeutic agent may be enhanced upon cleavage of the peptide substrate by a mammalian protease. In some embodiments, the masking moiety (MM) is an elongated recombinant polypeptide (XTEN). In some embodiments, the XTEN is characterized by (i) containing at least 100 amino acids; (ii) at least 90% of its amino acid residues being selected from glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), and proline (P); and (iii) containing at least four different amino acids selected from G, A, S, T, E, and P.

[0032] In some embodiments for treating a subject with a therapeutic agent, the subject is determined to be potentially responsive to the therapeutic agent by the method described herein.

[0033] In certain embodiments, the Disclosure provides a method for treating a disease or condition in a subject, comprising the step of administering one or more therapeutically effective doses of the therapeutic agents or pharmaceutical compositions described herein to a subject in need thereof.

[0034] In some embodiments of a method for treating a disease or condition in a subject, the subject is selected from the group consisting of mice, rats, monkeys, and humans. In some embodiments, the subject is human. In some embodiments, the subject is determined to be potentially responsive to a therapeutic agent or pharmaceutical composition. In some embodiments, the likelihood of response is 50% or higher. In some embodiments, the likelihood of response is determined by the method described herein.

[0035] In some embodiments of methods for treating a disease or condition in a subject, the disease or condition is cancer, an inflammatory disease, or an autoimmune disease.In some embodiments, the disease or condition is ankylosing spondylitis (AS), arthritis (e.g., rheumatoid arthritis (RA), juvenile idiopathic arthritis (JIA), osteoarthritis (OA), psoriatic arthritis (PsA), gout, chronic arthritis), Chagas disease, chronic obstructive pulmonary disease (COPD), dermatomyositis, type 1 diabetes mellitus, endometriosis, Goodpasture syndrome, Graves' disease, Guillain-Barré syndrome (GBS), Hashimoto's disease, suppurative crus, Kawasaki disease, IgA nephropathy, idiopathic thrombocytopenic purpura, inflammatory bowel disease (IBD) (e.g., not limited to these) Although not limited to these, Crohn's disease (CD), Crohn's disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischemic colitis, vacant colitis, Behçet's syndrome, infectious colitis, indeterminate colitis, interstitial cystitis), lupus (for example, systemic lupus erythematosus, discoid lupus, subacute cutaneous lupus erythematosus, cutaneous lupus erythematosus (e.g., frostbite-like lupus erythematosus), drug-induced lupus, neonatal lupus, lupus nephritis), mixed connective tissue disease, Morphea, multiple sclerosis (MS), myocardial infarction, narcolepsy Lepsy, neuromuscular anguina, pemphigus vulgaris, pernicious anemia, psoriasis, psoriatic arthritis, polymyositis, primary biliary cirrhosis, relapsing polychondritis, schizophrenia, scleroderma, Sjögren's syndrome, generalized rigidity syndrome, temporal arteritis (also known as giant cell arteritis), vasculitis, vitiligo, Wegener's granulomatosis, transplant rejection-associated immune reactions (e.g., kidney transplant rejection, lung transplant rejection, liver transplant rejection, etc., though not limited to these), psoriasis, Wiscott-Aldrich syndrome, autoimmune lymphoproliferative syndrome, myasthenia gravis, inflammatory chronic sinusitis, colitis, celiac disease, Barrett's esophagus, The group is selected from inflammatory gastritis, autoimmune nephritis, autoimmune hepatitis, autoimmune carditis, autoimmune encephalitis, autoimmune-mediated hematological disorders, asthma, atopic dermatitis, atopy, allergies, allergic rhinitis, scleroderma, bronchitis, and pericarditis, and the inflammatory diseases are Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, inflammatory lung disease, inflammatory skin disease, atherosclerosis, myocardial infarction, stroke, Gram-positive shock, Gram-negative shock, sepsis, septic shock, hemorrhagic shock, anaphylactic shock, and systemic inflammatory response syndrome.In some embodiments, the disease or condition is carcinoma, Hodgkin lymphoma, and non-Hodgkin lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, blastoma, breast cancer, ER / PR. + Breast cancer, Her2 + The group consists of breast cancer, triple-negative breast cancer, colon cancer, colon cancer with malignant ascites, mucinous neoplasms, prostate cancer, head and neck cancer, skin cancer, melanoma, genitourinary cancer, ovarian cancer, ovarian cancer with malignant ascites, peritoneal carcinomatosis, serous uterine carcinoma, endometrial cancer, cervical cancer, colorectal cancer, uterine cancer, peritoneal mesothelioma, kidney cancer, Wilms' tumor, lung cancer, small cell lung cancer, non-small cell lung cancer, gastric cancer, stomach cancer, small intestine cancer, liver cancer, hepatocellular carcinoma, hepatoblastoma, liposarcoma, pancreatic cancer, gallbladder cancer, bile duct cancer, esophageal cancer, salivary gland cancer, thyroid cancer, epithelial cancer, masculinizing neoplasm, adenocarcinoma, sarcoma, and B-cell chronic lymphocytic leukemia.

[0036] In certain embodiments, this disclosure provides the use of the therapeutic agents described herein in the preparation of a medicament for the treatment of a disease or condition in a subject.

[0037] In certain embodiments, this disclosure provides the use of the pharmaceutical compositions described herein in the preparation of a medicament for the treatment of a disease or condition in a subject.

[0038] In some embodiments of use, the subject is selected from the group consisting of mice, rats, monkeys, and humans. In some embodiments, the subject is human. In some embodiments, the subject is determined to be potentially responsive to the therapeutic agent or pharmaceutical composition. In some embodiments, the likelihood of response is 50% or higher. In some embodiments, the likelihood of response is determined by the method described herein.

[0039] In some embodiments of use, the disease or condition is cancer, inflammatory disease, or autoimmune disease. In some embodiments, the disease or condition is carcinoma, Hodgkin lymphoma, and non-Hodgkin lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, blastoma, breast cancer, ER / PR + Breast cancer, Her2 +The group consists of breast cancer, triple-negative breast cancer, colon cancer, colon cancer with malignant ascites, mucinous neoplasms, prostate cancer, head and neck cancer, skin cancer, melanoma, genitourinary cancer, ovarian cancer, ovarian cancer with malignant ascites, peritoneal carcinomatosis, serous uterine carcinoma, endometrial cancer, cervical cancer, colorectal cancer, uterine cancer, peritoneal mesothelioma, kidney cancer, Wilms' tumor, lung cancer, small cell lung cancer, non-small cell lung cancer, gastric cancer, stomach cancer, small intestine cancer, liver cancer, hepatocellular carcinoma, hepatoblastoma, liposarcoma, pancreatic cancer, gallbladder cancer, bile duct cancer, esophageal cancer, salivary gland cancer, thyroid cancer, epithelial cancer, masculinizing neoplasm, adenocarcinoma, sarcoma, and B-cell chronic lymphocytic leukemia.In some embodiments, the disease or condition is ankylosing spondylitis (AS), arthritis (e.g., rheumatoid arthritis (RA), juvenile idiopathic arthritis (JIA), osteoarthritis (OA), psoriatic arthritis (PsA), gout, chronic arthritis), Chagas disease, chronic obstructive pulmonary disease (COPD), dermatomyositis, type 1 diabetes mellitus, endometriosis, Goodpasture syndrome, Graves' disease, Guillain-Barré syndrome (GBS), Hashimoto's disease, suppurative crus, Kawasaki disease, IgA nephropathy, idiopathic thrombocytopenic purpura, inflammatory bowel disease (IBD) (e.g., not limited to these) Although not limited to these, Crohn's disease (CD), Crohn's disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischemic colitis, vacant colitis, Behçet's syndrome, infectious colitis, indeterminate colitis, interstitial cystitis), lupus (for example, systemic lupus erythematosus, discoid lupus, subacute cutaneous lupus erythematosus, cutaneous lupus erythematosus (e.g., frostbite-like lupus erythematosus), drug-induced lupus, neonatal lupus, lupus nephritis), mixed connective tissue disease, Morphea, multiple sclerosis (MS), myocardial infarction, narcolepsy Lepsy, neuromuscular anguina, pemphigus vulgaris, pernicious anemia, psoriasis, psoriatic arthritis, polymyositis, primary biliary cirrhosis, relapsing polychondritis, schizophrenia, scleroderma, Sjögren's syndrome, generalized rigidity syndrome, temporal arteritis (also known as giant cell arteritis), vasculitis, vitiligo, Wegener's granulomatosis, transplant rejection-associated immune reactions (e.g., kidney transplant rejection, lung transplant rejection, liver transplant rejection, etc., though not limited to these), psoriasis, Wiscott-Aldrich syndrome, autoimmune lymphoproliferative syndrome, myasthenia gravis, inflammatory chronic sinusitis, colitis, celiac disease, Barrett's esophagus, The group is selected from inflammatory gastritis, autoimmune nephritis, autoimmune hepatitis, autoimmune carditis, autoimmune encephalitis, autoimmune-mediated hematological disorders, asthma, atopic dermatitis, atopy, allergies, allergic rhinitis, scleroderma, bronchitis, and pericarditis, and the inflammatory diseases are Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, inflammatory lung disease, inflammatory skin disease, atherosclerosis, myocardial infarction, stroke, Gram-positive shock, Gram-negative shock, sepsis, septic shock, hemorrhagic shock, anaphylactic shock, and systemic inflammatory response syndrome.

[0040] In some embodiments, the Disclosure provides a therapeutic agent (e.g., an activatable therapeutic agent, or a non-natural activatable therapeutic agent) comprising a release segment (RS) directly or indirectly linked to a bioactive moiety (BM), wherein the RS comprises a peptide substrate having an amino acid sequence sensitive to cleavage by a mammalian protease at an easily cleavable bond, and the peptide substrate comprises an amino acid sequence having up to three amino acid substitutions (or up to two amino acid substitutions, or up to one amino acid substitution) to the sequences listed in column II or III (or a subset thereof) of Table A.

[0041] In some embodiments, the Disclosure relates to a therapeutic agent (e.g., an activatable therapeutic agent, or a non-natural activatable therapeutic agent) comprising a release segment (RS) directly or indirectly linked to a bioactive moiety (BM), wherein the RS comprises a peptide substrate having an amino acid sequence sensitive to cleavage by mammalian proteases at an easily cleavable bond, and the therapeutic agent is configured to be activated in or near a target tissue or cell in a subject. The target tissue or cell contains, or is associated with, a reporter sequence that can be cleaved by a mammalian protease at the cleavage sequence, either within or on the tissue or cell. The present invention provides a therapeutic agent in which the peptide substrate contains an amino acid sequence having up to three amino acid substitutions (or up to two amino acid substitutions, or up to one amino acid substitution) relative to the cleavage sequence of a reporter polypeptide.

[0042] In some embodiments of the therapeutic agent, the reporter polypeptide is a coagulation factor, complement component, tubulin, immunoglobulin, apolipoprotein, serum amyloid, insulin, growth factor, fibrinogen, PDZ domain protein, LIM domain protein, c-reactive protein, serum albumin, versican, collagen, elastin, keratin, kininogen-1, alpha-2-antiplasmin, clusterin, biglycan, alpha-1-antitrypsin, transthyretin, alpha-1-antichymotrypsin, glucagon, hepcidin, thymosin beta-4, haptoglobin, hemoglobin subunit alpha, caveolae-associated protein 2, alpha-2-HS-glycoprotein, chromogranin-A, vitronectin, hemopexin, epididymal secretory sperm-binding protein, secretogranin-2, and andipose tissue. Otensinogen, transgerin-2, pancreatic prohormone, neurosecretory protein VGF, ceruloplasmin, PDZ and LIM domain protein 1, multimelin-1, inter-alpha-trypsin inhibitor heavy chain H2, N-acetylmuramoyl-L-alanine amidase, histone H1.4, adhesion G protein-coupled receptor G6, mannan-binding lectin serine protease 2, prothrombin, malignant brain tumor deletion 1 protein, desmoglein-3, calcinthenin-1, alpha-2-macroglobulin, myosin-9, sodium / potassium transport ATPase subunit gamma, oncoprotein-inducible transcript 3 protein, cerglycine, histidine-rich glycoprotein, inter-alpha-trypsin inhibitor heavy chain H5, integrin alpha-IIb, membrane-bound progesterone receptor component 1, histone H1.The group selected consists of 2, rho GDP dissociation inhibitor 2, zinc-alpha-2-glycoprotein, talin-1, secretogranin-1, neutrophil defensin 3, cytochrome P450 2E1, gastric suppressor polypeptide, transcription initiation factor TFIID subunit 1, membrane-bound protein 2B, pigment epithelial-derived factor, voltage-gated N-type calcium channel subunit alpha-1B, ras GTPase-activating protein nGAP, type I cytoskeleton 17, sulfhydryl oxidase 1, homeobox protein Hox-B2, transcription factor SOX-10, E3 ubiquitin-protein ligase SIAH2, decorin, acidic, cysteine-rich secretory protein (SPARC), laminin gamma 1 chain, vimentin, and nidogen-1 (NID1).

[0043] In some embodiments of the therapeutic agent, the reporter polypeptide is versican, type II collagen alpha-1 chain, kininogen-1, complement C4-A, complement C4-B, complement C3, alpha-2-antiplasmin, clusterin, biglycan, elastin, fibrinogen alpha chain, alpha-1-antitrypsin, fibrinogen beta chain, type III collagen alpha-1 chain, serum amyloid A-1 protein, transthyretin, apolipoprotein AI, apolipoprotein AI isoform 1, alpha-1-antichymotrypsin, glucagon, hepcidin, serum amyloid A-2 protein, thymosin beta-4, haptoglobin, hemoglobin subunit alpha, and Beola-related protein 2, alpha-2-HS-glycoprotein, chromogranin-A, vitronectin, hemopexin, epididymal sperm-binding protein, zyxin, apolipoprotein C-III, secretogranin-2, angiotensinogen, c-reactive protein, serum albumin, transgerin-2, pancreatic prohormones, neurosecretory protein VGF, ceruloplasmin, PDZ and LIM domain proteins 1, tubulin alpha-4A chain, multimelin-1, inter-alpha-trypsin inhibitor heavy chain H2, apolipoprotein CI, fibrinogen gamma chain, N-acetylmuramoyl-L-alanine amidase, immunoglobulin lambda variable 3-21, histone H1.4, Adhesion G protein-coupled receptor G6, Immunoglobulin lambda variable 3-25, Immunoglobulin lambda variable 1-51, Immunoglobulin lambda variable 1-36, Mannan-binding lectin serine protease 2, Immunoglobulin kappa variable 3-20, Immunoglobulin kappa variable 2-30, Insulin-like growth factor II, Apolipoprotein A-II, Immunoglobulin kappa variable 2D-24 (likely non-functional), Prothrombin, Coagulation factor IX, Apolipoprotein L1, Malignant brain tumor deletion 1 protein, Desmoglein-3, Calcinetenin-1, Immunoglobulin lambda constant 3, Complement C5, Alpha-2-macroglobulin, Myosin-9, Sodium / potassium transport ATPase subunit gamma, Immunoglobulin kappa variable 2-28, Oncoprotein-inducible transcript 3 protein, Cerglycine, Coagulation factor XII, Coagulation factor XIII A chain, insulin, histidine-rich glycoprotein, immunoglobulin kappa variable 3-11, immunoglobulin kappa variable 1-39, collagen alpha-1(I) chain, inter-alpha-trypsin inhibitor heavy chain H5, latent transforming growth factor beta-binding protein 2, integrin alpha-IIb, membrane-bound progesterone receptor component 1, immunoglobulin lambda variable 6-57, immunoglobulin kappa variable 3-15, complement C1r small component-like protein, histone H1.2, Rho GDP dissociation inhibitor 2, latent transforming growth factor beta-binding protein 4, collagen alpha-1 (XVIII) chain, immunoglobulin lambda variable 2-18, zinc-alpha-2-glycoprotein, talin-1, secretogranin-1, neutrophil defensin 3, cytochrome P450 2E1, gastric suppressor polypeptide, immunoglobulin heavy chain variable 3-15, immunoglobulin lambda variable 2-11, transcription initiation factor TFIID subunit 1, collagen alpha-1 (VII) chain, membrane endogenous protein 2B, pigment epithelium-derived factor, voltage-gated N-type calcium channel subunit alpha-1B, immunoglobulin lambda variable 3-27, ras This polypeptide is selected from the group consisting of GTPase-activated protein nGAP, keratin, type I cytoskeleton 17, tubulin beta chain, sulfhydryl oxidase 1, immunoglobulin kappa variable 4-1, complement C1r minor component, homeobox protein Hox-B2, transcription factor SOX-10, E3 ubiquitin-protein ligase SIAH2, decorin, SPARC, type I collagen alpha-1 chain, type IV collagen alpha-1 chain, laminin gamma 1 chain, vimentin, type III collagen, type IV collagen alpha-3 chain, type VII collagen alpha-1 chain, type VI collagen alpha-1 chain, type V collagen alpha-1 chain, nidogen-1, and type VI collagen alpha-3 chain.

[0044] In some embodiments of the therapeutic agent, the cleavage sequence of the reporter polypeptide is a cleavage sequence described in column II or III (or a subset thereof) of Table A. In some embodiments, the cleavage sequence does not contain a methionine residue immediately N-terminal to the easily cleavable bond (contained therein) if methionine is the first residue at the N-terminus of the reporter polypeptide. In some embodiments, the target tissue or cell is characterized by an increased amount or activity of mammalian protease in the vicinity of the target tissue or cell compared to non-target tissue or cells in the subject. In some embodiments, the mammalian protease is produced in the target tissue or cell. In some embodiments, the peptide substrate contains an amino acid sequence having up to three amino acid substitutions, up to two amino acid substitutions, or up to one amino acid substitution relative to the sequences described in column II or III (or a subset thereof) of Table A. In some embodiments, the peptide substrate contains an amino acid sequence having up to three amino acid substitutions relative to the sequences described in column II or III (or a subset thereof) of Table A. In some embodiments, the easily cleavable bond is not immediately C-terminal to the methionine residue.

[0045] In some embodiments of the therapeutic agent, the peptide substrate contains 6 to 25 or 6 to 20 amino acid residues. In some embodiments of the therapeutic agent, the peptide substrate contains 6 to 25 amino acid residues. In some embodiments of the therapeutic agent, the peptide substrate contains 6 to 20 amino acid residues. In some embodiments, the peptide substrate contains 7 to 12 amino acid residues. In some embodiments, the peptide substrate contains an amino acid sequence having up to two amino acid substitutions relative to the sequence described in column II or III (or a subset thereof) of Table A. In some embodiments, the peptide substrate contains an amino acid sequence having up to one amino acid substitution relative to the sequence described in column II or III (or a subset thereof) of Table A. In some embodiments, up to three amino acid substitutions, up to two amino acid substitutions, or up to one amino acid substitution are not located in positions corresponding to amino acid residues directly adjacent to the corresponding easily cleavable bonds of the corresponding sequences shown in column II or III (or a subset thereof) of Table A. In some embodiments, the peptide substrate contains an amino acid sequence identical to the sequence described in column II or III (or a subset thereof) of Table A. In some embodiments, the peptide substrate does not contain a methionine residue immediately to the N-terminus of the easily cleavable bond (which is contained therein). In some embodiments, the peptide substrate does not contain an amino acid sequence selected from the group consisting of #279, #280, #282, #283, #298, #299, #302, #303, #305, #307, #308, #349, #396, #397, #416, #417, #418, #458, #459, #460, #466, #481, and #482 (or any combination thereof) in column II of Table A. In some embodiments, the peptide substrate contains two or three sequences listed in column II or III (or a subset thereof) of Table A. In some embodiments where the peptide substrate contains two sequences listed in column II or III (or a subset thereof) of Table A, the two sequences partially overlap each other. In some embodiments, where the peptide substrate includes two sequences listed in column II or III (or a subset thereof) of Table A, the two sequences do not overlap.In some embodiments, where the peptide substrate includes three sequences listed in column II or III (or a subset thereof) of Table A, two or all of the three sequences do not overlap with each other. In some embodiments, where the peptide substrate includes three sequences listed in column II or III (or a subset thereof) of Table A, one of the three sequences partially overlaps with another sequence or both of the other sequences. In some embodiments, where the peptide substrate includes three sequences listed in column II or III (or a subset thereof) of Table A, two of the three sequences partially overlap with each other. In some embodiments, where the peptide substrate includes three sequences listed in column II or III (or a subset thereof) of Table A, each of the two sequences partially overlaps with each other. In some embodiments, where the peptide substrate includes three sequences listed in column II or III (or a subset thereof) of Table A, all three sequences partially overlap with each other. In some embodiments, the peptide substrate, which is sensitive to cleavage by mammalian proteases, is sensitive to cleavage by multiple mammalian proteases, including mammalian proteases. In some embodiments, peptide substrates sensitive to cleavage by multiple mammalian proteases have up to three amino acid substitutions, or up to two amino acid substitutions, or up to one amino acid substitution relative to the sequences listed in Table 1(j). In some embodiments, peptide substrates sensitive to cleavage by multiple mammalian proteases have up to three amino acid substitutions relative to the sequences listed in Table 1(j). In some embodiments, peptide substrates sensitive to cleavage by multiple mammalian proteases have up to two amino acid substitutions relative to the sequences listed in Table 1(j). In some embodiments, peptide substrates sensitive to cleavage by multiple mammalian proteases have up to one amino acid substitution relative to the sequences listed in Table 1(j). In some embodiments, none of the up to three amino acid substitutions, up to two amino acid substitutions, or up to one amino acid substitution are located at positions corresponding to amino acid residues directly adjacent to the corresponding easily cleavable bonds in the corresponding sequences listed in Table 1(j).In some embodiments, the peptide substrate, which is sensitive to cleavage by multiple mammalian proteases, includes the sequences listed in Table 1(j).

[0046] In some embodiments of the therapeutic agent, the release segment (RS) may be cleaved when it is near a target tissue or cell, and the target tissue or cell produces a mammalian protease that uses the RS as a peptide substrate. In some embodiments, the mammalian protease for cleaving the release segment (RS) is a serine protease, cysteine ​​protease, aspartate protease, threonine protease, or metalloproteinase.In some embodiments, the mammalian proteases for cleaving the release segment (RS) are disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), disintegrin and metalloproteinase domain-containing protein 12 (ADAM12), disintegrin and metalloproteinase domain-containing protein 15 (ADAM15), disintegrin and metalloproteinase domain-containing protein 17 (ADAM17), disintegrin and metalloproteinase domain-containing protein Inase domain-containing protein 9 (ADAM9), disintegrin and metalloproteinase with thrombospondin motif 5 (ADAMTS5), cathepsin B, cathepsin D, cathepsin E, cathepsin K, cathepsin L, cathepsin S, fibroblast-activating protein alpha, hepsin, kallikrein-2, kallikrein-4, kallikrein-3, prostate-specific antigen (PSA), kallikrein-13, regmine, matrix metallopeptidase 1 (MMP-1), matrix metallopeptidase 10 (MMP-10), Matrix metallopeptidase 11 (MMP-11), Matrix metallopeptidase 12 (MMP-12), Matrix metallopeptidase 13 (MMP-13), Matrix metallopeptidase 14 (MMP-14), Matrix metallopeptidase 16 (MMP-16), Matrix metallopeptidase 2 (MMP-2), Matrix metallopeptidase 3 (MMP-3), Matrix metallopeptidase 7 (MMP-7), Matrix metallopeptidase 8 (MMP- 8) Selected from the group consisting of matrix metallopeptidase 9 (MMP-9), matrix metallopeptidase 4 (MMP-4), matrix metallopeptidase 5 (MMP-5), matrix metallopeptidase 6 (MMP-6), matrix metallopeptidase 15 (MMP-15), neutrophil elastase, protease-activated receptor 2 (PAR2), plasmin, prostasin, PSMA-FOLH1, membrane-bound serine protease 1 (MT-SP1), matryptase, and u-plasminogen.In some embodiments, the mammalian protease for cleavage of the release segment (RS) is selected from the group consisting of matrix metallopeptidase 1 (MMP1), matrix metallopeptidase 2 (MMP2), matrix metallopeptidase 7 (MMP7), matrix metallopeptidase 9 (MMP9), matrix metallopeptidase 11 (MMP11), matrix metallopeptidase 14 (MMP14), urokinase-type plasminogen activator (uPA), legumain, and matriptase.

[0047] In some embodiments of the therapeutic agent, the therapeutic agent further comprises a masking moiety (MM) directly or indirectly linked to the release segment (RS). In some embodiments, the therapeutic agent in the uncleaved state has a structural arrangement of BM-RS-MM or MM-RS-BM from the N-terminus to the C-terminus. In some embodiments of the therapeutic agent, upon cleavage of the release segment (RS), the masking moiety (MM) is released from the therapeutic agent. In some embodiments, the masking moiety (MM) comprises an extended recombinant polypeptide (XTEN). In some embodiments, XTEN is characterized by (i) comprising at least 100 amino acids; (ii) at least 90% of its amino acid residues being selected from glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E) and proline (P); and (iii) comprising at least 4 different amino acids selected from G, A, S, T, E and P. In some embodiments, the extended recombinant polypeptide (XTEN) comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequences described in Tables 2b - 2c. In some embodiments, when the masking moiety (MM) is linked to the therapeutic agent, it interferes with the interaction between the bioactive moiety (BM) and the target tissue or cell, and thus the dissociation constant (K d ) between the target cell marker of the target tissue or cell and the BM of the therapeutic agent is, when the therapeutic agent is in the uncleaved state, the dissociation constant (K d) is larger compared to the corresponding bioactive moiety. In some embodiments, the therapeutic agent provides a broader therapeutic area compared to the corresponding bioactive moiety by delivering BM to target tissue or cells. In some embodiments, the therapeutic agent has a longer terminal phase half-life compared to that of the corresponding bioactive moiety. In some embodiments, the therapeutic agent is less immunogenic compared to the corresponding bioactive moiety. In some embodiments, immunogenicity is confirmed by measuring the production of IgG antibodies that selectively bind to the bioactive moiety after administration of an equivalent dose to the subject. In some embodiments, the therapeutic agent has a larger apparent molecular weight coefficient compared to the corresponding bioactive moiety under physiological conditions.

[0048] In some embodiments of the therapeutic agent, the release segment (RS) is a first release segment (RS1), the easily cleavable bond is the first easily cleavable bond, and the therapeutic agent further comprises a second release segment (RS2) directly or indirectly linked to the bioactive moiety (BM), wherein RS2 comprises a second peptide substrate or comprises cleavage by a mammalian protease at the second easily cleavable bond. In some embodiments, the mammalian protease for cleavage of RS2 is the same as the mammalian protease for cleavage of RS1. In some embodiments, the mammalian protease for cleavage of RS2 is different from the mammalian protease for cleavage of RS1. In some embodiments, RS2 has the same amino acid sequence as RS1. In some embodiments, RS2 has a different amino acid sequence than RS1.In some embodiments, RS1 and RS2 each contain disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), disintegrin and metalloproteinase domain-containing protein 12 (ADAM12), disintegrin and metalloproteinase domain-containing protein 15 (ADAM15), disintegrin and metalloproteinase domain-containing protein 17 (ADAM17), and disintegrin and metalloproteinase domain-containing protein 9 ( ADAM9), disintegrin and metalloproteinase with thrombospondin motif 5 (ADAMTS5), cathepsin B, cathepsin D, cathepsin E, cathepsin K, cathepsin L, cathepsin S, fibroblast-activating protein alpha, hepsin, kallikrein-2, kallikrein-4, kallikrein-3, prostate-specific antigen (PSA), kallikrein-13, regmine, matrix metallopeptidase 1 (MMP-1), matrix metallopeptidase 10 (MMP-10), matrix metallo Peptidase 11 (MMP-11), Matrix Metallopeptidase 12 (MMP-12), Matrix Metallopeptidase 13 (MMP-13), Matrix Metallopeptidase 14 (MMP-14), Matrix Metallopeptidase 16 (MMP-16), Matrix Metallopeptidase 2 (MMP-2), Matrix Metallopeptidase 3 (MMP-3), Matrix Metallopeptidase 7 (MMP-7), Matrix Metallopeptidase 8 (MMP-8), Matrix Metallopeptidase 9 (MMP -9) The peptide substrates include different mammalian proteases selected from the group consisting of matrix metallopeptidase 4 (MMP-4), matrix metallopeptidase 5 (MMP-5), matrix metallopeptidase 6 (MMP-6), matrix metallopeptidase 15 (MMP-15), neutrophil elastase, protease-activated receptor 2 (PAR2), plasmin, prostasin, PSMA-FOLH1, membrane-bound serine protease 1 (MT-SP1), matryptase, and u-plasminogen.In some embodiments, RS1 and RS2 each contain a peptide substrate for a different mammalian protease selected from the group consisting of matrix metallopeptidase 1 (MMP1), matrix metallopeptidase 2 (MMP2), matrix metallopeptidase 7 (MMP7), matrix metallopeptidase 9 (MMP9), matrix metallopeptidase 11 (MMP11), matrix metallopeptidase 14 (MMP14), urokinase-type plasminogen activator (uPA), regmine, and matryptase. In some embodiments, the second easily cleaved bond is not located immediately C-terminal to the methionine residue.

[0049] In some embodiments of the therapeutic agent, the second peptide substrate contains 6 to 25 or 6 to 20 amino acid residues. In some embodiments of the therapeutic agent, the second peptide substrate contains 6 to 25 amino acid residues. In some embodiments of the therapeutic agent, the second peptide substrate contains 6 to 20 amino acid residues. In some embodiments, the second peptide substrate contains 7 to 12 amino acid residues. In some embodiments, the second peptide substrate includes an amino acid sequence having up to 3 amino acid substitutions, up to 2 amino acid substitutions, or up to 1 amino acid substitution relative to the sequence described in column II or III (or a subset thereof) of Table A. In some embodiments, the second peptide substrate includes an amino acid sequence having up to 3 amino acid substitutions relative to the sequence described in column II or III (or a subset thereof) of Table A. In some embodiments, the second peptide substrate includes an amino acid sequence having up to 2 amino acid substitutions relative to the sequence described in column II or III (or a subset thereof) of Table A. In some embodiments, the second peptide substrate includes an amino acid sequence having up to 1 amino acid substitution relative to the sequence described in column II or III (or a subset thereof) of Table A. In some embodiments, up to three amino acid substitutions, up to two amino acid substitutions, or up to one amino acid substitution (of the second peptide substrate) are not located in positions corresponding to amino acid residues directly adjacent to the corresponding cleavage-promoting bonds in the corresponding sequences shown in column II or III (or a subset thereof) of Table A. In some embodiments, the second peptide substrate contains the same amino acid sequence as the sequences shown in column II or III (or a subset thereof) of Table A. In some embodiments, the second peptide substrate does not contain a methionine residue immediately N-terminal to the cleavage-promoting bond (which is contained therein). In some embodiments, the second peptide substrate does not contain an amino acid sequence selected from the group consisting of #279, #280, #282, #283, #298, #299, #302, #303, #305, #307, #308, #349, #396, #397, #416, #417, #418, #458, #459, #460, #466, #481, and #482 (or any combination thereof) in column II of Table A.In some embodiments, the second peptide substrate includes two or three sequences listed in column II or III (or a subset thereof) of Table A. In some embodiments where the second peptide substrate includes two sequences listed in column II or III (or a subset thereof) of Table A, the two sequences (of the second peptide substrate) partially overlap each other. In some embodiments where the second peptide substrate includes two sequences listed in column II or III (or a subset thereof) of Table A, the two sequences (of the second peptide substrate) do not overlap each other. In some embodiments where the second peptide substrate includes three sequences listed in column II or III (or a subset thereof) of Table A, two or all of the three sequences (of the second peptide substrate) do not overlap each other. In some embodiments where the second peptide substrate includes three sequences listed in column II or III (or a subset thereof) of Table A, one of the three sequences (of the second peptide substrate) partially overlaps with another sequence or both of the other sequences of the three sequences. In some embodiments, where the second peptide substrate includes three sequences listed in column II or III (or a subset thereof) of Table A, two of the three sequences (of the second peptide substrate) partially overlap each other. In some embodiments, where the second peptide substrate includes three sequences listed in column II or III (or a subset thereof) of Table A, each of the two sequences (of the second peptide substrate) partially overlaps each other. In some embodiments, where the second peptide substrate includes three sequences listed in column II or III (or a subset thereof) of Table A, all three sequences (of the second peptide substrate) partially overlap each other. In some embodiments, the second peptide substrate, which is sensitive to cleavage by mammalian proteases, is sensitive to cleavage by multiple mammalian proteases, including mammalian proteases. In some embodiments, the second peptide substrate, which is sensitive to cleavage by multiple mammalian proteases, has up to three amino acid substitutions, or up to two amino acid substitutions, or up to one amino acid substitution relative to the sequences listed in Table 1(j).In some embodiments, the second peptide substrate, which is sensitive to cleavage by multiple mammalian proteases, has up to three amino acid substitutions relative to the sequence listed in Table 1(j). In some embodiments, the second peptide substrate, which is sensitive to cleavage by multiple mammalian proteases, has up to two amino acid substitutions relative to the sequence listed in Table 1(j). In some embodiments, the second peptide substrate, which is sensitive to cleavage by multiple mammalian proteases, has up to one amino acid substitution relative to the sequence listed in Table 1(j). In some embodiments, none of the up to three amino acid substitutions, up to two amino acid substitutions, or up to one amino acid substitution (of the second peptide substrate) are located at positions corresponding to amino acid residues directly adjacent to the corresponding easily cleavable bonds in the corresponding sequences listed in Table 1(j). In some embodiments, the second peptide substrate, which is sensitive to cleavage by multiple mammalian proteases, includes the sequence listed in Table 1(j).

[0050] In some embodiments of the therapeutic agent, the second release segment (RS2) may be cleaved when it is near a target tissue or cell, and the target tissue or cell produces a mammalian protease that uses RS2 as a peptide substrate. This mammalian protease includes proteases produced by the tumor and proteases produced by the tumor environment (melieu). In some embodiments, the mammalian protease for cleaving the second release segment (RS2) is a serine protease, cysteine ​​protease, aspartate protease, threonine protease, or metalloproteinase.In some embodiments, the mammalian proteases for cleaving the release segment (RS) are disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), disintegrin and metalloproteinase domain-containing protein 12 (ADAM12), disintegrin and metalloproteinase domain-containing protein 15 (ADAM15), disintegrin and metalloproteinase domain-containing protein 17 (ADAM17), disintegrin and metalloproteinase domain-containing protein Inase domain-containing protein 9 (ADAM9), disintegrin and metalloproteinase with thrombospondin motif 5 (ADAMTS5), cathepsin B, cathepsin D, cathepsin E, cathepsin K, cathepsin L, cathepsin S, fibroblast-activating protein alpha, hepsin, kallikrein-2, kallikrein-4, kallikrein-3, prostate-specific antigen (PSA), kallikrein-13, regmine, matrix metallopeptidase 1 (MMP-1), matrix metallopeptidase 10 (MMP-10), Matrix metallopeptidase 11 (MMP-11), Matrix metallopeptidase 12 (MMP-12), Matrix metallopeptidase 13 (MMP-13), Matrix metallopeptidase 14 (MMP-14), Matrix metallopeptidase 16 (MMP-16), Matrix metallopeptidase 2 (MMP-2), Matrix metallopeptidase 3 (MMP-3), Matrix metallopeptidase 7 (MMP-7), Matrix metallopeptidase 8 (MMP- 8) Selected from the group consisting of matrix metallopeptidase 9 (MMP-9), matrix metallopeptidase 4 (MMP-4), matrix metallopeptidase 5 (MMP-5), matrix metallopeptidase 6 (MMP-6), matrix metallopeptidase 15 (MMP-15), neutrophil elastase, protease-activated receptor 2 (PAR2), plasmin, prostasin, PSMA-FOLH1, membrane-bound serine protease 1 (MT-SP1), matryptase, and u-plasminogen.In some embodiments, the mammalian protease for cleaving the second release segment (RS2) is selected from the group consisting of matrix metallopeptidase 1 (MMP1), matrix metallopeptidase 2 (MMP2), matrix metallopeptidase 7 (MMP7), matrix metallopeptidase 9 (MMP9), matrix metallopeptidase 11 (MMP11), matrix metallopeptidase 14 (MMP14), urokinase-type plasminogen activator (uPA), regmine, and matryptase.

[0051] In some embodiments of the therapeutic agent, the masking portion (MM) is a first masking portion (MM1), and the therapeutic agent further comprises a second masking portion (MM2) directly or indirectly linked to a second release segment (RS2). In some embodiments, the uncleaved therapeutic agent has a structural configuration of MM1-RS1-BM-RS2-MM2, MM1-RS2-BM-RS1-MM2, MM2-RS1-BM-RS2-MM1, or MM2-RS2-BM-RS1-MM1 from N-terminus to C-terminus. In some embodiments of the therapeutic agent, the second masking portion (MM2) is released from the therapeutic agent upon cleavage of the second release segment (RS2). In some embodiments, the second masking portion (MM2) comprises a second elongated recombinant polypeptide (XTEN2). In some embodiments, XTEN2 is characterized by (i) containing at least 100 amino acids; (ii) at least 90% of its amino acid residues being selected from glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), and proline (P); and (iii) containing at least four different amino acids selected from G, A, S, T, E, and P. In some embodiments, XTEN2 contains an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from the group of sequences listed in Tables 2b to 2c. In some embodiments, the first masking portion (MM1) and the second masking portion (MM2), when both are linked in the therapeutic agent, interfere with the interaction between the bioactive portion (BM) and the target tissue or cells, and therefore the dissociation constant (K) of the BM of the therapeutic agent with the target cell marker present in the target tissue or cells. d ) is the dissociation constant (K) of the corresponding bioactive moiety when the therapeutic agent is in an uncleaved state. d) is larger compared to the corresponding bioactive moiety. In some embodiments, therapeutic agents in which the bioactive moiety (BM) is directly or indirectly linked to one or both of the first masking moiety (MM1) and the second masking moiety (MM2) provide a broader therapeutic range compared to the corresponding bioactive moiety by delivering the BM to the target tissue or cells. In some embodiments, therapeutic agents in which the bioactive moiety (BM) is directly or indirectly linked to one or both of the first masking moiety (MM1) and the second masking moiety (MM2) have a longer terminal phase half-life compared to that of the corresponding bioactive moiety. In some embodiments, therapeutic agents in which the bioactive moiety (BM) is directly or indirectly linked to one or both of the first masking moiety (MM1) and the second masking moiety (MM2) have lower immunogenicity compared to the corresponding bioactive moiety. In some embodiments of therapeutic agents, immunogenicity is confirmed by measuring the production of IgG antibodies that selectively bind to the bioactive moiety after administration of an equivalent dose to the subject. In some embodiments, therapeutic agents in which a bioactive moiety (BM) is directly or indirectly linked to one or both of a first masking moiety (MM1) and a second masking moiety (MM2) have a larger apparent molecular weight coefficient under physiological conditions compared to the corresponding bioactive moiety. In some embodiments, the therapeutic agent comprises a fusion polypeptide or conjugate.

[0052] In some embodiments of the therapeutic agent, the bioactive moiety (BM) comprises a bioactive peptide (BP). In some embodiments, the BP comprises an antibody, a cytokine, a cell receptor, or a fragment thereof.

[0053] In some embodiments, the therapeutic agent comprises a recombinant polypeptide. In some embodiments, the recombinant polypeptide comprises a bioactive peptide (BP) and a release segment (RS). In some embodiments, the recombinant polypeptide comprises a bioactive peptide (BP), a release segment (RS), and a masking moiety (MM). In some embodiments, the uncleaved recombinant polypeptide has a structural configuration of BP-RS-MM or MM-RS-BP from the N-terminus to the C-terminus. In some embodiments, the recombinant polypeptide comprises a bioactive peptide (BP), a first release segment (RS1), and a second release segment (RS2). In some embodiments, the recombinant polypeptide comprises a bioactive peptide (BP), a first release segment (RS1), a second release segment (RS2), a first masking moiety (MM1), and a second masking moiety (MM2). In some embodiments, the uncleaved recombinant polypeptide has a structural configuration from N-terminus to C-terminus of MM1-RS1-BP-RS2-MM2, MM1-RS2-BP-RS1-MM2, MM2-RS1-BP-RS2-MM1, or MM2-RS2-BP-RS1-MM1. In some embodiments, the recombinant polypeptide comprises a bioactive peptide (BP), a first release segment (RS1), a second release segment (RS2), a first extended recombinant polypeptide (XTEN1), and a second extended recombinant polypeptide (XTEN2). In some embodiments, the uncleaved recombinant polypeptide has a structural configuration from N-terminus to C-terminus of XTEN1-RS1-BP-RS2-XTEN2, XTEN1-RS2-BP-RS1-XTEN2, XTEN2-RS1-BP-RS2-XTEN1, or XTEN2-RS2-BP-RS1-XTEN1.

[0054] In some embodiments of the therapeutic agent, the bioactive polypeptide (BP) includes a binding moiety having binding affinity to a target cell marker on a target tissue or cell. In some embodiments, the target cell marker is an effector cell antigen expressed on the surface of effector cells. In some embodiments, the binding moiety is an antibody. In some embodiments, the binding moiety is Fv, Fab, Fab', Fab'-SH, or a nanobody (single-domain antibody or V). HH The antibody is selected from the group consisting of Fv, Fab, Fab', Fab'-SH, nanobody (single-domain antibody or V). In some embodiments, the binding site is a first binding site, the target cell marker is a first target cell marker, and the bioactive polypeptide (BP) further comprises a second binding site directly or indirectly linked to the first binding site, the second binding site having binding affinity to the second target cell marker on a target tissue or cell. In some embodiments, the second target cell marker is a marker on tumor cells or cancer cells. In some embodiments, the second binding site is an antibody. In some embodiments, the second binding site is Fv, Fab, Fab', Fab'-SH, nanobody (single-domain antibody or V HH The antibody is selected from the group consisting of (also known as) linear antibodies and single-chain variable fragments (scFv).

[0055] Certain aspects of this disclosure provide isolated nucleic acids, which include (a) polynucleotides encoding recombinant polypeptides as described herein, or (b) reverse complements of the polynucleotides of (a).

[0056] Certain aspects of this disclosure provide an expression vector comprising a polynucleotide sequence described herein and a recombinant regulatory sequence operably linked to the polynucleotide sequence.

[0057] Certain embodiments of this disclosure provide isolated host cells, which contain expression vectors described herein. In some embodiments, the host cells are prokaryotes. In some embodiments, the host cells are E. coli or mammalian cells. In some embodiments, the host cells are E. coli. In some embodiments, the host cells are mammalian cells.

[0058] Some aspects of this disclosure provide pharmaceutical compositions comprising therapeutic agents described herein and one or more pharmaceutically suitable excipients. In some embodiments, the pharmaceutical compositions are formulated for oral, intradermal, subcutaneous, intravenous, intra-arterial, intra-abdominal, intraperitoneal, subarachnoid, or intramuscular administration. In some embodiments, the pharmaceutical compositions are in liquid or frozen form. In some embodiments, the pharmaceutical compositions are contained in pre-filled syringes for single-dose injection. In some embodiments, the pharmaceutical compositions are formulated as lyophilized powders that are restored before administration.

[0059] Some aspects of this disclosure provide a kit which includes the pharmaceutical composition, container, and label or accompanying information on or attached to the container as described herein.

[0060] In certain embodiments, the Disclosure provides methods for preparing therapeutic agents provided herein (e.g., activatable therapeutic agents, or non-natural activatable therapeutic agents).

[0061] In certain embodiments, the present disclosure relates to a method for preparing a therapeutic agent (e.g., an activatable therapeutic agent, or a non-natural activatable therapeutic agent), (a) A step of culturing host cells containing a nucleic acid construct encoding a recombinant polypeptide under conditions sufficient to express the recombinant polypeptide in the host cells, wherein the recombinant polypeptide comprises a bioactive polypeptide (BP), a release segment (RS), and a masking moiety (MM), RS comprises a peptide substrate that is sensitive to cleavage by mammalian proteases in its easily cleavable bond, and the peptide substrate comprises an amino acid sequence having up to three or two amino acid substitutions (or up to one amino acid substitution) to the sequence described in column II or III (or a subset thereof) of Table A. The recombinant polypeptide has a structural configuration of BP-RS-MM or MM-RS-BP from the N-terminus to the C-terminus; and (b) A step of recovering the therapeutic agent containing the recombinant polypeptide (e.g., an activatable therapeutic agent, or a non-natural activatable therapeutic agent). This provides a method that includes this.

[0062] In some embodiments of the method for preparing a therapeutic agent, the peptide substrate sensitive to cleavage by mammalian proteases is sensitive to cleavage by multiple mammalian proteases, including mammalian proteases. In some embodiments, the peptide substrate sensitive to cleavage by multiple mammalian proteases has up to three amino acid substitutions, or up to two amino acid substitutions, or up to one amino acid substitution relative to the sequence listed in Table 1(j). In some embodiments, the peptide substrate sensitive to cleavage by multiple mammalian proteases contains the sequence listed in Table 1(j). In some embodiments, the peptide substrate does not contain SEQ ID NO: 1. In some embodiments, the peptide substrate does not contain SEQ ID NO: 2. In some embodiments, the peptide substrate does not contain SEQ ID NO: 3. In some embodiments, the peptide substrate does not contain SEQ ID NO: 4. In some embodiments, the peptide substrate does not contain SEQ ID NO: 5. In some embodiments, the peptide substrate does not contain SEQ ID NO: 6. In some embodiments, the peptide substrate does not contain SEQ ID NO: 7. In some embodiments, the peptide substrate does not contain SEQ ID NO: 8. In some embodiments, the masking moiety (MM) contains an extended recombinant polypeptide (XTEN).

[0063] In some embodiments of the method for preparing a therapeutic agent, the release segment (RS) is a first release segment (RS1), the peptide substrate is a first peptide substrate, the easily cleavable bond is a first easily cleavable bond, the masking moiety (MM) is a first masking moiety (MM1), and the recombinant polypeptide further comprises a second release segment (RS2) and a second masking moiety (MM2), wherein RS2 is sensitive to cleavage by mammalian proteases at the second easily cleavable bond. The recombinant polypeptide comprises a second peptide substrate, the second peptide substrate comprising an amino acid sequence having up to three amino acid substitutions, up to two amino acid substitutions, or up to one amino acid substitution relative to the sequence described in column II or III (or a subset thereof) of Table A, and the recombinant polypeptide has a structural configuration of MM1-RS1-BP-RS2-MM2, MM1-RS2-BP-RS1-MM2, MM2-RS1-BP-RS2-MM1, or MM2-RS2-BP-RS1-MM1 from the N-terminus to the C-terminus.

[0064] In some embodiments of the method for preparing a therapeutic agent, the second peptide substrate, which is sensitive to cleavage by mammalian proteases, is sensitive to cleavage by a plurality of mammalian proteases, including mammalian proteases. In some embodiments, the second peptide substrate, which is sensitive to cleavage by a plurality of mammalian proteases, has up to three amino acid substitutions, or up to two amino acid substitutions, or up to one amino acid substitution relative to the sequence listed in Table 1(j). In some embodiments, the second peptide substrate, which is sensitive to cleavage by a plurality of mammalian proteases, contains the sequence listed in Table 1(j). In some embodiments, the second peptide substrate does not contain SEQ ID NO: 1. In some embodiments, the second peptide substrate does not contain SEQ ID NO: 2. In some embodiments, the second peptide substrate does not contain SEQ ID NO: 3. In some embodiments, the second peptide substrate does not contain SEQ ID NO: 4. In some embodiments, the second peptide substrate does not contain SEQ ID NO: 5. In some embodiments, the second peptide substrate does not contain SEQ ID NO: 6. In some embodiments, the second peptide substrate does not contain SEQ ID NO: 7. In some embodiments, the second peptide substrate does not contain SEQ ID NO: 8. In some embodiments, one of the first masking moiety (MM1) and the second masking moiety (MM2) comprises an elongated recombinant polypeptide (XTEN). In some embodiments, the elongated recombinant polypeptide (XTEN) is characterized by (i) comprising at least 100 amino acids; (ii) at least 90% of its amino acid residues being selected from glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), and proline (P); and (iii) comprising at least four different amino acids selected from G, A, S, T, E, and P. In some embodiments, the elongated recombinant polypeptide (XTEN) comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to a sequence selected from the group listed in Tables 2b-2c.In some embodiments, the elongated recombinant polypeptide (XTEN) is a first elongated recombinant polypeptide (XTEN1), and the other of the first masking moiety (MM1) and the second masking moiety (MM2) comprises a second elongated recombinant polypeptide (XTEN2). In some embodiments, the second elongated recombinant polypeptide (XTEN2) is characterized by (i) containing at least 100 amino acids; (ii) at least 90% of its amino acid residues being selected from glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), and proline (P); and (iii) containing at least four different amino acids selected from G, A, S, T, E, and P. In some embodiments, XTEN1 and XTEN2 each contain an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from the group of sequences listed in Tables 2b to 2c.

[0065] In some embodiments of the method for preparing therapeutic agents, the masking portion (MM), when linked to the recombinant polypeptide, interferes with the interaction between the BP and the target tissue or cell, and therefore the dissociation constant (K) of the BP of the recombinant polypeptide with the target cell marker present in the target tissue or cell. d The dissociation constant (K) of the corresponding bioactive peptide is measured in an in vitro assay at equivalent molar concentration when the recombinant polypeptide is in an uncleaved state. d It is larger compared to ). In some embodiments, the first masking portion (MM1) and the second masking portion (MM2), when both are linked in the recombinant polypeptide, interfere with the interaction between BP and the target tissue or cell, and therefore the dissociation constant (K) of the BP of the recombinant polypeptide between the target cell marker present in the target tissue or cell and the target cell marker present in the target tissue or cell. d The dissociation constant (K) of the corresponding bioactive peptide is measured in an in vitro assay at equivalent molar concentration when the recombinant polypeptide is in an uncleaved state. dIt is larger compared to ). In some embodiments, in vitro assays include cell membrane integrity assays, mixed cell culture assays, cell-based competitive binding assays, FACS-based propidium iodide assays, trypan blue influx assays, photometric enzyme release assays, and radiometric assays. 51 A Cr release assay, a fluorescence-based europium release assay, a calcein AM release assay, a photometric MTT assay, an XTT assay, a WST-1 assay, an Alamer Blue assay, a radiometric 3H-Thd integration assay, a cloning assay measuring cell division activity, a fluorescence-based rhodamine 123 assay measuring mitochondrial transmembrane gradient, an apoptosis assay monitored by phosphatidylserine exposure based on FACS, an ELISA-based TUNEL assay, a sandwich ELISA, a caspase activity assay, a cell-based LDH release assay, and a cell morphology assay, or any combination thereof, may be selected. In some embodiments, the activatable therapeutic agent is an activatable therapeutic agent described herein or a non-natural activatable therapeutic agent.

[0066] Further aspects and advantages of the Disclosure will be readily apparent to those skilled in the art from the following detailed description, which shows and describes only exemplary embodiments of the Disclosure. As you may understand, other different embodiments of the Disclosure are possible, and some of its details can be modified in various obvious ways, all without departing from the Disclosure. Therefore, the drawings and description should be considered as aiding to understanding and not as limiting. Inclusion by reference

[0067] All published documents, patents, and patent applications referenced herein are incorporated by reference to the same extent as each individual published document, patent, or patent application is specifically and individually indicated as being incorporated by reference. If any of the incorporated published documents, patents, or patent applications conflict with the disclosures contained herein, this specification is intended to supersede and / or prevail over any such conflicts.

[0068] Novel features of the present invention are described in particular in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by referring to the following detailed description illustrating exemplary embodiments in which the principles of the present invention are utilized, and to the following accompanying drawings (also known as "Figure" and "FIG." in this specification). [Brief explanation of the drawing]

[0069] [Figure 1]Figure 1 shows the nomenclature for peptide biomarker sequences (e.g., any of those listed in Table A) within a reporter polypeptide (e.g., a protein in or adjacent to a target tissue or cell from which the biomarker sequence is generated). An illustrative reporter polypeptide sequence contains two cleavage sequences, both of which can be recognized and cleaved by mammalian enzymes (e.g., mammalian proteases). For example, in some cases, the first and second cleavage sequences can be recognized and cleaved by the same enzyme or the same set of enzymes. As an alternative example, in some cases, the first and second cleavage sequences can be recognized and cleaved by different enzymes or different sets of enzymes. The first cleavage sequence contains a first easily cleavable linkage; the second cleavage sequence is C-terminal to the first cleavage sequence and contains a second easily cleavable linkage. The first and second easily cleavable links (e.g., indicated by hyphens (-) in Table A) divide the illustrative reporter polypeptide into three parts. An illustrative reporter polypeptide can be cleaved with corresponding enzymes whose substrates are the first and second cleavage sequences to obtain an N-terminal fragment (N-terminus relative to the first easily cleavable bond), a central fragment (between the first and second easily cleavable bonds), and a C-terminal fragment (C-terminal side relative to the second easily cleavable bond). The N-terminal, central, or C-terminal fragments (if present) (e.g., any of those listed in Table A), or their derivatives, can function as peptide biomarker sequences. The first or second cleavage sequence (e.g., any of those listed in Table A) can be incorporated into a release segment of an activatable therapeutic agent (e.g., any of those listed herein).

[0070] [Figure 2]Figure 2 shows the nomenclature for peptide substrates and their cleavage bonds for cleavage. The exemplary peptide substrate contains eight consecutive amino acid residues, of which four amino acid residues (with side chain groups R4, R3, R2, and R1 in order from N-terminus to C-terminus) are located immediately N-terminally to the cleavage bond, and four amino acid residues (with side chain groups R'1, R'2, R'3, and R'4 in order from N-terminus to C-terminus) are located immediately C-terminally to the cleavage bond. For example, mammalian proteases can recognize up to four residues on either side of the cleavage bond. Upon cleavage, the exemplary peptide substrate separates into an N-terminal proteolytic fragment and a C-terminal proteolytic fragment. The four amino acid residues immediately N-terminally to the cleavage bond of the exemplary peptide substrate form the C-terminus of the N-terminal proteolytic fragment, and the four amino acid residues immediately C-terminally to the cleavage bond of the exemplary peptide substrate form the N-terminus of the C-terminal proteolytic fragment.

[0071] [Figure 3] Figure 3 shows the structural arrangement of an exemplary activatable antibody (AA) composition comprising the antibody or a fragment thereof, a masking moiety (MM), and a release segment (RS).

[0072] [Figure 4] Figure 4 shows the structural arrangement of an exemplary activatable antibody complex (AAC) composition in which cross-masking occurs, such that target binding by both antibodies or fragments decays in their uncleaved state, and target binding increases upon cleavage of the release segment (RS), which allows for the degradation of the complex. In this figure, the two antibodies or fragments are referred to as antibody domain 1 (ABD1) and antibody domain 2 (ABD2), respectively.

[0073] [Figure 5] Figure 5 shows the structural arrangement of an exemplary activatable antibody-acid (AAC) composition comprising two antibodies or their fragments, a masking moiety (MM), and a release segment (RS).

[0074] [Figure 6] Figure 6 shows the structural arrangement of an exemplary activatable antibody complex (AAC) composition comprising four antibodies or their fragments, two masking moieties (MMs), and three release segments (RSs).

[0075] [Figure 7] Figure 7 shows the structural arrangement of an exemplary activatable antibody composition (AA) comprising one antibody or antibody fragment (AB), two masking moieties (MM), and two release segments (RS).

[0076] [Figure 8] Figure 8 shows the structural arrangement of XTEN-modified protease-activated T cell engagers (XPATs). Each illustrative XPAT contains two binding sites, each linked to XTEN by a release segment.

[0077] [Figure 9] Figure 9 shows the results of cleavage by mammalian proteases of release segments having sequences similar to those found in collagen I. Cleavage sites are identified by stars (★), and parts with sequences identical to collagen are underlined. Sequences that have been manipulated so as not to be recognized or cleaved by proteases that recognize collagen-derived cleavage sites are described as 818-NonClv(RSR-3058), and amino acids different from the collagen sequence are shown in black. [Modes for carrying out the invention]

[0078] Detailed explanation In various cancer treatment modalities, drugs that can be conditionally activated in the tumor microenvironment have been developed. However, there is still a need to develop more accurate and robust methods to predict whether the administration of these therapies will actually lead to a therapeutic response and outcome when administered as a prodrug or other activatable composition. It is recognized that there is a cascade of events leading to metastatic growth of cancer cells. The central factor in these events is the interaction between cancer cells and their microenvironment, through which tumor cells proliferate, build new blood vessels, leave the primary tumor bed, and ultimately invade and persist in secondary sites of metastatic tumor growth. The extracellular matrix (ECM) of the tumor microenvironment consists of various macromolecules, including collagen and glycoproteins. The basement membrane of the ECM is formed primarily by type IV collagen, although type I and III collagens are the most abundant proteins in the basal stromal matrix. In healthy tissue, the ECM is constantly remodeled, which is mainly mediated by matrix metalloproteinases (MMPs), and matrix degradation is balanced by protein formation. This controlled remodeling of ECM is disrupted during cancer development and progression.

[0079] During the MMP-mediated degradation of the ECM, small fragments of ECM turnover products are generated and released into the bloodstream. Several studies have shown that serum levels of collagen degradation fragments are elevated in cancer patients compared to healthy controls. Bager et al. found that MMP-degraded type I, III, and IV collagen levels (i.e., C1M, C3M, and C4M, respectively; Cancer Biomark. 2015;15:783-788) were 1.5 to 6 times higher in ovarian and breast cancer patients than in controls. In this invention, it is demonstrated that cleavage of the ECM by MMP results in cleavage products that are very similar to the MMP cleavage sites of the protease-cleavable linker in XPAT. The data presented herein demonstrate that the protease-cleavable linker used in the XPAT of this invention is cleaved more efficiently by purified MMP than ECM. Therefore, the presence of ECM peptides in cancer patients can serve as an indicator that the patient's tumor has a microenvironment with adequate protease (e.g., MMP) activity capable of cleaving the protease-cleavable linker in XPAT. Thus, the presence of ECM peptides in a cancer patient's sample predicts whether a given patient or tumor can cleave XPAT, and therefore whether the tumor can be treated. This enables a personalized approach to determine whether XPAT is cleaved in a given tumor type by determining whether plasma levels of certain cleavage products derived from the extracellular matrix are elevated in subjects having that tumor type.

[0080] Before reading further about the embodiments of this disclosure, it should be understood that such embodiments are provided merely as examples, and that various alternative forms of the embodiments of this disclosure described herein may be used in practicing the invention. A great many variations, modifications, and substitutions will readily come to mind for those skilled in the art without departing from the invention.

[0081] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art in which the invention pertains. Methods and materials similar to or equivalent to those described herein may be used in carrying out or testing the invention, but preferred methods and materials are described below. In case of any inconsistency, this specification shall prevail, including definitions. Furthermore, materials, methods, and examples are provided for illustrative purposes only and are not intended to limit the scope. A great many variations, alterations, and substitutions will readily come to mind for those skilled in the art without departing from the invention. definition

[0082] In relation to this application, the following terms have the meanings of those terms unless otherwise specified.

[0083] As used herein and in the claims, the terms “a,” “an,” and “the” are used generally to mean “at least one,” “at least first,” “one or more,” or “more,” the components or steps referred to, unless an upper limit is specifically stated after them. For example, “cleavage sequence,” as used herein, means “at least first cleavage sequence,” but includes multiple cleavage sequences. The operable limits and parameters for combinations, as well as the amounts of any single component, will be apparent to those skilled in the art in view of this disclosure.

[0084] When used herein in relation to therapeutic agents, the term "activatable" generally means that the activity or biological activity of a therapeutic agent can be enhanced upon activation, for example, by physical, chemical, or physiological processes (e.g., enzymatic and metabolic processes).

[0085] As used herein, the term “activatable therapeutic agent” generally refers to a therapeutic agent whose activity or biological activity can be enhanced upon activation, for example, by physical, chemical, or physiological processes (e.g., enzymatic and metabolic processes). For example, the term “activatable therapeutic agent” may refer to a therapeutic agent that is inactive (or less active) (at least in one aspect inactive) and configured (at least in an aspect in which it is inactive before activation) to be activated (i.e., in vitro, in vivo, or ex vivo) to an active (or more active) state. Another example is that the term “activatable therapeutic agent” may refer to an active therapeutic agent (at least in one aspect active) whose activity or biological activity can be further enhanced (i.e., in vitro, in vivo, or ex vivo). Non-limiting examples of activatable therapeutic agents include prodrugs, probodies, and pro-parts.

[0086] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to generally refer to polymers of amino acids of any length. The polymers may be linear or branched, may contain modified amino acids, or may have non-amino acid intercalations. These terms also encompass amino acid polymers modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other operation, such as conjugation with a labeling component.

[0087] As used herein with respect to the structure of polypeptides, “N-terminus” (or “amino-terminus”) and “C-terminus” (or “carboxyl-terminus”) generally refer to the amino and carboxyl ends, respectively, of the polypeptide.

[0088] When used herein in relation to a polypeptide or polynucleotide sequence of interest, the term "N-terminal sequence" generally means the N-terminus of the polypeptide or polynucleotide sequence of interest that is not preceded by any other amino acid residues or nucleotide residues. When used herein in relation to a polypeptide or polynucleotide sequence of interest, the term "C-terminal sequence" generally means the C-terminus of the polypeptide or polynucleotide sequence of interest that is not followed by any other amino acid residues or nucleotide residues.

[0089] The terms “not naturally occurring” and “unnatural” are used interchangeably herein. When used herein in reference to therapeutic agents, “not naturally occurring” or “unnatural” generally means that the agent is not of biological origin in mammals (including but not limited to humans). When applied to sequences, and as used herein, “not naturally occurring” or “unnatural” means a polypeptide or polynucleotide sequence that does not have a wild-type or naturally occurring sequence found in mammals, is not complementary to such a sequence, or does not have a high degree of homology to such a sequence. For example, a polypeptide or fragment not naturally occurring may, when suitably aligned, share up to 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50%, or even lower amino acid sequence identity compared to a natural sequence.

[0090] As used herein, the term “antibody” generally refers to an immunoglobulin molecule or any fragment thereof that is immunoreactive with an antigen of interest. For example, an antibody fragment may retain the ability to bind to its ligand but have a smaller molecular size and may be in a single-chain form. The term “antibody” is used herein in its broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments provided they exhibit the desired antigen-binding activity. Full-length antibodies may be, for example, monoclonal, recombinant, chimeric, deimmunized, humanized, and human antibodies.

[0091] When applied to bioactive proteins, a “variant” is a protein that has sequence homology to a native bioactive protein, retaining at least a portion of the therapeutic activity and / or bioactivity of the bioactive protein. For example, a variant protein may share at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of amino acid sequence identity with respect to a reference bioactive protein. As used herein, the term “bioactive protein variant” includes proteins that have been intentionally modified, such as by site-directed mutagenesis, synthesis or insertion of the encoding gene, or proteins that have been accidentally modified by mutation, and that retain activity.

[0092] The term “sequence variant” means a polypeptide that has been modified compared to its native or original sequence by one or more amino acid insertions, deletions, or substitutions. Insertions may be located at either or both ends of a protein and / or within an internal region of the amino acid sequence. A non-limiting example is the substitution of an amino acid in XTEN with a different amino acid. In the case of a deletion variant, one or more amino acid residues are removed from the polypeptide described herein. Thus, a deletion variant includes all fragments of the polypeptide sequence described. In the case of a substitution variant, one or more amino acid residues are removed from the polypeptide and replaced with substitute residues. In one embodiment, the substitution is conserved in nature, and this type of conserved substitution is well known in the art. With respect to antibodies or bioactive polypeptides, a sequence variant will retain at least a portion of the binding affinity or bioactivity of the unmodified polypeptide, respectively.

[0093] The term “moiety” means a component of a larger composition, or a protein-like moiety intended to be incorporated into a larger composition, such as a continuous or discontinuous sequence bound to a larger polypeptide. Moieties of a larger composition can confer desired functionality. For example, an antibody fragment may retain its ability to bind to its ligand but have a smaller molecular size and be in a single-chain form. Masking moieties (including, but not limited to, extended recombinant polypeptides (XTENs)) can confer functionality that increases the molecular weight and / or extends the half-life of the resulting larger composition to which the masking moieties associate.

[0094] The terms “binding domain” and “binding moiety” are used interchangeably herein and refer, respectively, to a portion that has specific binding affinity to an antigen (e.g., an effector cell antigen, or a tumor-specific marker or antigen on a target cell).

[0095] As used herein, “release segment” or “RS” generally refers to a peptide having one or more cleavage sites in its sequence that can be recognized and cleaved by one or more mammalian enzymes (e.g., one or more proteases).

[0096] As used herein, “peptide substrate” generally refers to an amino acid sequence that is recognized by an enzyme (e.g., a mammalian protease) and is cleaved by this enzyme at a peptide bond (or its peptide bond) within the peptide substrate, resulting in the separation of two consecutive amino acid residues that were previously connected by a peptide bond (or cleavable bond) upon cleavage. As used herein, “cleavable bond” generally refers to a peptide bond that connects consecutive amino acids by an amide linkage that can (or is cleaved) by an enzyme (e.g., a mammalian protease). For example, with respect to a peptide substrate, a cleavable bond divides the peptide substrate into a C-terminal proteolytic fragment (or C-terminal fragment) and an N-terminal proteolytic fragment (or N-terminal fragment), the C-terminal proteolytic fragment (or C-terminal fragment) being on the N-terminal side of the cleavable bond in the peptide substrate, and the N-terminal proteolytic fragment (or N-terminal fragment) being on the C-terminal side of the cleavable bond in the peptide substrate. For example, the (presumed) easily cleavable connections of each cleavage sequence listed in Table A are indicated by a hyphen (-).

[0097] As used herein, the term “easily cleavable bond” generally refers to a peptide bond between two amino acids that can be cleaved by one or more proteases.

[0098] As used herein, the term “mammalian protease” generally means a protease that is normally present in body fluids, cells, and tissues, but which may be found at higher levels in certain target tissues or cells of mammals, for example, in diseased tissue (e.g., tumors).

[0099] The term “inside” when referring to a first polypeptide that will be ligated into a second polypeptide includes not only the ligation or fusion of additional components that connect the N-terminus of the first or second polypeptide to the C-terminus of the second or first polypeptide, respectively, but also the insertion of the first polypeptide into the sequence of the second polypeptide. For example, when an RS component is ligated “inside” a recombinant polypeptide, RS may be N-terminally ligated, C-terminally ligated, or inserted between any two amino acids of the XTEN polypeptide.

[0100] The term “directly linked,” as used herein in reference to therapeutic agents, generally refers to a structure in which one part is connected to or bonded to another part without an intervening tether. The term “indirectly linked,” as used herein in reference to therapeutic agents, generally refers to a structure in which one part of a therapeutic agent is connected to or bonded to another part of the therapeutic agent via an intervening tether. The terms “link,” “linked,” and “linking,” as used herein in reference to therapeutic agents, generally include both covalent and noncovalent bonding of one part of a therapeutic agent to another part of the therapeutic agent.

[0101] When applied to the forms of compositions provided herein, “activity” (e.g., “biological activity”) generally refers to action or effect, whether measured by in vitro, ex vivo, or in vivo assays or by clinical efficacy, including but not limited to receptor binding, antagonist activity, agonist activity, cellular or physiological response, cell lysis, cell death, or effects generally known in the art with respect to the effector components of the composition.

[0102] "Effector cells," as used herein, include any eukaryotic cells capable of producing an effect on target cells. For example, effector cells may induce loss of membrane integrity, nuclear condensation, nuclear disintegration, apoptosis, lysis, and / or death of target cells. In another example, effector cells may induce division, growth, differentiation, or alteration of signaling pathways in target cells.

[0103] "Effector cell antigens" refer to molecules expressed by effector cells, including but not limited to cell surface molecules such as proteins, glycoproteins, or lipoproteins. Effector cell antigens can function as binding counterparts to the binding site of a target recombinant polypeptide.

[0104] As used herein, the term "ELISA" refers to enzyme-linked immunosorbent assays described herein or otherwise known in the art.

[0105] "Host cells" generally include individual cells or cell cultures that may or may have been recipients of a target vector into which an exogenous nucleic acid has been introduced, such as those described herein. Host cells include offspring of a single host cell. Offspring may not necessarily be completely identical (in terms of morphology or total DNA complement genomics) to the original parent cell due to spontaneous, accidental, or intentional mutations. Host cells include cells to which the vectors of this disclosure have been transfected in vivo.

[0106] When used to describe the various polypeptides disclosed herein, the term “isolated” generally means polypeptides identified, separated, and / or recovered from components of their natural environment or from more complex mixtures (e.g., during protein purification). Contaminating components of their natural environment are substances that would generally interfere with the diagnostic or therapeutic use of the polypeptide and may include enzymes, hormones, and other protein-like or non-protein-like solutes. As will be apparent to those skilled in the art, polynucleotides, peptides, polypeptides, proteins, antibodies, or fragments thereof that do not exist in nature do not require “isolation” to distinguish them from their naturally occurring counterparts. In addition, “concentrated,” “isolated,” or “diluted” polynucleotides, peptides, polypeptides, proteins, antibodies, or fragments thereof are distinguishable from their naturally occurring counterparts because the concentration or number of molecules per unit volume is generally higher than that of their naturally occurring counterparts. Generally, polypeptides produced by recombinant means and expressed in host cells are considered “isolated.”

[0107] An "isolated nucleic acid" is a nucleic acid molecule that has been isolated from at least one contaminating nucleic acid molecule that is identified and normally associated with the polypeptide-coding nucleic acid in its natural source. For example, an isolated polypeptide-coding nucleic acid molecule is in a form or setting other than those found in nature. Therefore, an isolated polypeptide-coding nucleic acid molecule is distinguished from its specific polypeptide-coding nucleic acid molecule as it would be in a native cell. However, an isolated polypeptide-coding nucleic acid molecule may include polypeptide-coding nucleic acid molecules contained in cells that normally express polypeptides, for example, if the nucleic acid molecule is in a different chromosomal or extrachromosomal location than that in a native cell.

[0108] A "chimeric" protein or polypeptide contains at least one fusion polypeptide that includes at least one region in a position different from its naturally occurring position within the sequence. These regions may normally exist in separate proteins and be brought together into the fusion peptide, or they may normally exist in the same protein but be positioned in a new configuration within the fusion polypeptide. Chimeric proteins may be produced, for example, by chemical synthesis, or by recombination and translation of polynucleotides in which the peptide regions encode in a desired relationship.

[0109] The terms “fused” and “fusion” are used interchangeably herein and refer to the linking of two or more peptide or polypeptide sequences by recombinant means. A “fusion protein” or “chimeric protein” contains a first amino acid sequence linked to a second amino acid sequence that is not inherently linked in nature.

[0110] "Uncleaved" and "uncleaved state" are used interchangeably herein and refer to a polypeptide that has not been cleaved or digested by a protease, and therefore remains intact.

[0111] The term "XTEN-modified" is used to describe a peptide or polypeptide that has been modified by linking or fusing one or more XTEN polypeptides (described below) to the peptide or polypeptide, whether by recombinant means or chemical crosslinking means.

[0112] "Crosslinking" and "conjugating" are used interchangeably herein and refer to the covalent bonding of two different molecules through a chemical reaction. Crosslinking can be carried out by one or more chemical reactions, as is known in the art.

[0113] With respect to polypeptides, a "linear sequence" or "sequence" is the order of amino acids in a polypeptide from the amino terminus to the carboxyl terminus (N-terminus to C-terminus), where adjacent residues in the sequence are consecutive in the polypeptide's primary structure. A "partial sequence" is a linear sequence of a portion of a polypeptide that is known to contain additional residues in one or both directions.

[0114] The term "heterogeneous" means that it originates from an entity that is genotype-clearly different from the rest of the entity being compared. For example, a glycine-rich sequence that has been removed from its natural coding sequence and operably ligated to a coding sequence other than the natural sequence is a heterogeneous glycine-rich sequence. When the term "heterogeneous" is applied to polynucleotides and polypeptides, it means that the polynucleotide or polypeptide originates from an entity that is genotype-clearly different from the rest of the entity being compared.

[0115] The terms “polynucleotide,” “nucleic acid,” “nucleotide,” and “oligonucleotide” are used interchangeably. These terms refer to nucleotides of any length, encompassing single nucleic acids as well as multiple nucleic acids, whether deoxyribonucleotides, ribonucleotides, or their analogues. Polynucleotides may have any three-dimensional structure and may perform any known or unknown function. The following are non-exclusive examples of polynucleotides: coding or non-coding regions of genes or gene fragments from which a single locus (or multiple loci) has been defined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may include modified nucleotides, such as methylated nucleotides and nucleotide analogs. Modifications to the nucleotide structure, where present, may be conferred before or after the polymer is assembled. Non-nucleotide components may also be interspersed in the nucleotide sequence. Polynucleotides may be further modified after polymerization, such as through conjugation with labeling components.

[0116] As used herein, the term “reporter polypeptide” refers to a human polypeptide or protein that, under certain circumstances, can be acted upon to produce a detectable signal (e.g., one that can be enzymatically digested to produce a detectable peptide sequence) that can be identified and characterized extracellularly, extraorganically, extratissueally, or extracorporeally. For example, a “reporter polypeptide” may be a human protein that can be cleaved by a protease that can also cleave an activatable therapeutic agent containing a peptide substrate (e.g., as described below herein). Non-limiting examples of peptide substrates include those described in the “Release Segment (RS)” section below herein.

[0117] The term "complementary polynucleotide" refers to a polynucleotide molecule that has a complementary base sequence and reverse orientation compared to a reference sequence, and therefore can hybridize with the reference sequence with perfect fidelity.

[0118] When applied to polynucleotides, "recombinant" means that the polynucleotide is the product of a recombination step, which may include cloning, restriction, and / or ligation steps, and other steps that result in the expression of a recombinant protein in a host cell.

[0119] The terms “gene” and “gene fragment” are used interchangeably herein. These terms refer to a polynucleotide containing at least one open reading frame capable of encoding a particular protein after transcription and translation. A gene or gene fragment may be genomic or cDNA, provided that its polynucleotide contains at least one open reading frame capable of covering the entire coding region or a segment thereof. A “fusion gene” is a gene composed of at least two heterogeneous polynucleotides that are linked together.

[0120] The terms “homologousness,” “homologous,” or “identity” are interchangeable and refer to sequence similarity between two or more polynucleotide sequences or between two or more polypeptide sequences. When using a program such as BestFit to determine sequence identity, similarity, or homology between two different amino acid sequences, default settings can be used, or an appropriate scoring matrix such as bloomum45 or bloomum80 can be selected to optimize the identity, similarity, or homology score. Preferably, homologous polynucleotides are those that hybridize under stringent conditions as defined herein and, when optimally aligned, have sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, more preferably 95%, more preferably 97%, more preferably 98%, and even more preferably 99% compared to their respective sequences. Homologous polypeptides, when optimally aligned over sequences of equivalent length, preferably have sequence identity that is at least 70%, preferably at least 80%, more preferably at least 90%, and even more preferably at least 95-99% identical.

[0121] The terms “identity percentage,” “sequence identity percentage,” and “identity %” refer, when applied to polynucleotide sequences, to the percentage of residue matching between at least two polynucleotide sequences aligned using a standard algorithm. Such algorithms can optimize the alignment between two sequences by inserting gaps in a standardized and reproducible manner into the sequences to be compared, thereby achieving a more meaningful comparison of the two sequences. The identity percentage may be measured over the length of the entire defined polynucleotide sequence, or over a shorter length, for example, over the length of a fragment taken from a larger defined polynucleotide sequence, e.g., a fragment of at least 45, at least 60, at least 90, at least 120, at least 150, at least 210, or at least 450 residues consecutively. It is understood that such lengths are illustrative only, and any fragment length supported by the sequences shown in the tables, figures, or sequence listings herein may be used to describe the length over which the identity percentage can be measured. The percentage of sequence identity is calculated by comparing two optimally aligned sequences across a comparison window, determining the number of matching positions (positions where the same residue exists in both polypeptide sequences), dividing the number of matching positions by the total number of positions within the comparison window (e.g., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity. When sequences of different lengths are compared, the length of the comparison window is defined by the shortest sequence. Conservative substitutions are not considered when calculating sequence identity.

[0122] The “sequence identity percentage (%)” and “identity percentage (%)” for polypeptide sequences identified herein are defined as the percentage of amino acid residues in a query sequence that are identical to amino acid residues of a second reference polypeptide sequence or a portion thereof of equivalent length, after achieving optimal alignment by aligning the sequences, introducing gaps as necessary to obtain the maximum sequence identity percentage, and not considering any conservative substitutions as part of the sequence identity. Alignment for determining amino acid sequence identity percentage can be achieved in various ways within the scope of the skills in the art, for example, using commonly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve optimal alignment over the entire length of the sequences to be compared. The identity percentage may be measured over the entire length of a defined polypeptide sequence, or over a shorter length, for example, over a fragment taken from a larger defined polypeptide sequence, e.g., a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70, or at least 150 consecutive residues. It is understood that such lengths are illustrative only, and any fragment length supported by the sequences shown in the tables, figures, or sequence listings herein may be used to describe the length over which the identity percentage can be measured.

[0123] As used herein, the term “expression” refers to the process by which a polynucleotide produces a gene product, such as RNA or polypeptide. This term includes, but is not limited to, the transcription of a polynucleotide into messenger RNA (mRNA), transfer RNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA), or any other RNA product, and the translation of mRNA into polypeptides. Expression produces a “gene product.” As used herein, a gene product may be either a nucleic acid, such as messenger RNA produced by the transcription of a gene, or a polypeptide translated from the transcript. Gene products described herein further include nucleic acids with post-transcriptional modifications, such as polyadenylation or splicing, or polypeptides with post-translational modifications, such as methylation, glycosylation, lipid addition, association with other protein subunits, or protein cleavage.

[0124] The terms “vector” or “expression vector” refer interchangeably to nucleic acid molecules that transfer inserted nucleic acid molecules into and / or between host cells, preferably nucleic acid molecules that self-replicate in a suitable host. The term includes vectors that primarily function for the insertion of DNA or RNA into cells, vector replication vectors that primarily function for the replication of DNA or RNA, and expression vectors that function for the transcription and / or translation of DNA or RNA. Vectors that provide more than one of the above functions are also included. An “expression vector” is a polynucleotide that can be transcribed and translated into polypeptides when introduced into a suitable host cell. “Expression system” usually implies a suitable host cell containing an expression vector capable of functioning to produce a desired expression product.

[0125] The term "t" 1 / 2 "Half-life," "Terminal phase half-life," "Elimination half-life," and "Cyclical half-life" are used interchangeably in this specification, and when used herein, ln(2) / K el This generally refers to the terminal phase half-life calculated as follows: K elθ is the terminal phase elimination rate constant calculated by linear regression of the terminal linear portion of the logarithmic concentration-time curve. Half-life generally refers to the time required for half of the administered substance accumulated in a living organism to be metabolized or eliminated by normal biological processes. When the clearance curve of a given polypeptide is constructed as a function of time, the curve is usually biphasic, having a rapid α phase and a longer beta phase. The typical beta phase half-life of a human antibody in humans is 21 days. Half-life can be measured using timed samples from any body fluid, but is most typically measured in serum or plasma samples.

[0126] The term "molecular weight" generally refers to the sum of the atomic weights of the constituent atoms in a molecule. Theoretically, molecular weight can be determined by summing the atomic masses of the constituent atoms in a molecule. When applied to polypeptides, molecular weight is calculated based on the amino acid composition, by adding the molecular weights of each type of amino acid in that composition, or by estimation from comparison with molecular weight standards on an SDS electrophoresis gel. The calculated molecular weight of a molecule may differ from the apparent molecular weight of the molecule, which generally refers to the molecular weight of the molecule determined by one or more analytical techniques. "Apparent molecular weight coefficient" and "apparent molecular weight" are related terms, and when used in relation to polypeptides, these terms refer to a measure of the relative increase or decrease in apparent molecular weight indicated by a particular amino acid or polypeptide sequence. Apparent molecular weight can be determined, for example, by comparison with a globular protein standard measured in "apparent kD" units using size exclusion chromatography (SEC) or a similar method. The apparent molecular weight coefficient is the ratio of apparent molecular weight to "molecular weight," the latter calculated by addition based on amino acid composition as described above, or by estimation from comparison with molecular weight standards on an SDS electrophoresis gel. The determination of apparent molecular weight and apparent molecular weight coefficient is described in U.S. Patent No. 8,673,860, among many other methods.

[0127] The term "hydrodynamic radius" or "Stokes radius" refers to the effective radius (R in nm) of a molecule in solution. h) is the effective radius, measured by assuming that the molecule is an object that moves through a solution and is subject to resistance due to the viscosity of the solution. In embodiments of this disclosure, the hydrodynamic radius measurement of the XTEN polypeptide correlates with the “apparent molecular weight coefficient,” a more intuitive measure. The “hydrodynamic radius” of a protein affects not only its diffusion rate in aqueous solutions but also its electrophoretic ability in polymer gels. The hydrodynamic radius of a protein is determined by its molecular weight and also by its structure, including its shape and density. Methods for determining the hydrodynamic radius, such as the use of size exclusion chromatography (SEC), as described in U.S. Patents 6,406,632 and 7,294,513, are well known in the art. Most proteins have a spherical structure, which is the densest three-dimensional structure a protein can have, and in which the hydrodynamic radius is smallest. Some proteins adopt random, open, unstructured, or "linear" higher-order structures, resulting in a much larger hydrodynamic radius compared to typical globular proteins of similar molecular weight.

[0128] "Physiological conditions" refer to a set of conditions in a viable host, as well as in vitro conditions, including temperature, salt concentration, and pH, that mimic those conditions in the target organism. Physiologically appropriate host conditions for use in in vitro assays have been established. Generally, physiological buffers contain physiologically appropriate salt concentrations and are adjusted to a neutral pH in the range of about 6.5 to about 7.8, and preferably about 7.0 to about 7.5. Various physiological buffers are described in Sambrook et al. (2001). Physiologically appropriate temperatures are in the range of about 25°C to about 38°C, and preferably about 35°C to about 37°C.

[0129] The term “binding site” is used herein in its broadest sense and is intended to include, specifically, categories of cytokines, cell receptors, antibodies, or antibody fragments that have a specific affinity for an antigen or ligand, such as a cell surface receptor, a target cell marker, or an antigen or glycoprotein, oligonucleotide, enzyme substrate, antigenic determinant, or a binding site that may be present in or on the surface of a tissue or cell.

[0130] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, for example, in which the individual antibodies constituting the population are identical and / or bind to the same epitope, except for a presumed variant antibody, which may contain, for example, a spontaneous mutation or one that arises during the production of the monoclonal antibody preparation, and such variants are generally present in small amounts. In contrast to polyclonal antibody preparations, which generally contain different antibodies against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is an antibody against a single determinant on an antigen. Therefore, the modifier "monoclonal" indicates the characteristic of an antibody obtained from a substantially homogeneous population of antibodies, and this modifier should not be interpreted as requiring the production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention can be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of a human immunoglobulin locus. Such methods and other exemplary methods for producing monoclonal antibodies are known in the art or are described herein.

[0131] As used herein, "antibody fragment" generally refers to a molecule other than an intact antibody that contains a portion of an intact antibody and binds to an antigen to which the intact antibody binds. Examples of antibody fragments include Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, single-chain diabodies, linear antibodies, nanobodies (single-domain antibodies (including single-domain camelid antibodies) or V HH Examples include, but are not limited to, single-chain variable fragment (scFv) antibody molecules (also known as), and polyspecific antibodies formed from antibody fragments.

[0132] The terms "scFv" or "single-chain variable fragment" are used interchangeably herein to refer to antibody fragment forms that include regions of a variable heavy ("VH") chain and a variable light ("VL") chain, or regions of two copies of either a VH chain or a VL chain, linked by a short, flexible peptide linker. While scFv is not actually an antibody fragment, it is a fusion protein of the heavy-chain variable region (VH) and light-chain variable region (VL) of an immunoglobulin, and can be readily expressed in functional form in E. coli or mammalian cells in either a VL-VH or VH-VL orientation from the N-terminus to the C-terminus.

[0133] The terms “antigen,” “target cell marker,” and “ligand” are used interchangeably herein to refer to a binding site, an antibody, an antibody fragment, or a molecule based on an antibody fragment to which it binds or which has binding specificity, and are either a binding site or a binding determinant.

[0134] The term "epitope" refers to a specific site on an antigen molecule to which an antibody, antibody fragment, or binding site binds. An epitope is the ligand for an antibody, antibody fragment, or binding site.

[0135] The term “diagnostic reagent” is used herein to refer to any reagent used in vivo or in vitro for the detection or screening of a particular disease. This detection or screening includes, but is not limited to, assays, antibodies, tests, nucleic acid-based tests including RT-PCR, etc.

[0136] As used herein, “CD3” or “surface antigen classification 3” means the T cell surface antigen CD3 complex, comprising all known CD3 subunits, e.g., CD3 epsilon, CD3 delta, CD3 gamma, CD3 zeta, CD3 alpha, and CD3 beta, either individually or independently combined. The extracellular domains of CD3 epsilon, gamma, and delta contain immunoglobulin-like domains and are therefore considered part of the immunoglobulin superfamily.

[0137] The terms “specific binding,” “specifically binding,” or “binding properties” are used interchangeably herein to refer to the high binding affinity of a binding moiety to its corresponding target. Typically, specific binding, when measured by one or more of the assays disclosed herein, is approximately 10 -6 Less than M (for example, 10 -7 M~10 -12 M) Dissociation constant or K d It will be a tightening.

[0138] The term "affinity," as used herein, generally refers to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated herein, "binding affinity" refers to the intrinsic binding affinity representing the 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y is generally expressed by the dissociation constant (K). d ) can be expressed by. As used herein, “greater binding affinity” or “increased binding affinity” means lower K d It means a value, for example, 1 × 10 -9 M is 1 × 10 -8 A binding affinity greater than M, while a "smaller binding affinity" is associated with a higher K d It means a value, for example, 1 × 10 -7 M is 1 × 10 -8 It has a binding affinity smaller than M.

[0139] "Inhibition constant" or "K i The term "1" is used interchangeably and refers to the dissociation constant of an enzyme-inhibitor complex, or the reciprocal of the binding affinity of the inhibitor to the enzyme.

[0140] "Dissociation constant" or "K" d The term "L" is used interchangeably and refers to the affinity between ligand "L" and protein "P," for example, how strongly the ligand binds to a particular protein. This is represented by formula K. d It can be calculated using the formula =[L][P] / [LP], where [P], [L], and [LP] represent the molar concentrations of the protein, ligand, and complex, respectively. on When used herein, the term "k" is intended to refer to the association rate constant for the association of an antibody with an antigen to form an antibody / antigen complex, as is known in the art. offWhen used herein, is intended to refer to the dissociation rate constant for the dissociation of an antibody from an antibody / antigen complex as is known in the art. Binding events can be detected using techniques such as flow cytometry or surface plasmon resonance. Assays may include soluble antigens or receptor molecules, or the assay may determine binding to receptors expressed by cells. Such assays may include cell-based assays, including assays for proliferation, cell death, apoptosis, and cell migration. The binding affinity of a composition of interest to a target ligand can be assayed using binding or competitive binding assays, e.g., Biacore assays using a chip-bound receptor or binding protein, as described in U.S. Patent No. 5,534,617, or ELISA assays; assays described in the examples herein; radioreceptor assays; reporter gene activity assays; or other assays known in the art. For example, exemplary reporter gene activity assays can be obtained based on genetically engineered cells, generated by stably introducing appropriate genes into the target receptor and signaling pathway, such that binding to the engineered receptor triggers a signaling cascade leading to activation of the engineered gene pathway and subsequent production of a signature polypeptide (e.g., an enzyme). The binding affinity constant can then be determined using standard methods, e.g., scatchard analysis as described by van Zoelen, et al., Trends Pharmacol Sciences (1998) 19)12):487, or other methods known in the art.

[0141] "Target cell markers" refer to molecules expressed by target cells; antigenic determinants; or binding sites that may be present in or on the surface of target tissues or cells, and can function as ligands for the binding site. Non-limiting examples of target cell markers include the target markers listed in Table 6.

[0142] The term “target tissue” generally refers to tissue that is the cause or part of a disease, including but not limited to cancer or an inflammatory condition. Sources of diseased target tissue include organs within the body, tumors, cancerous cells or populations of cancerous cells, or cells found forming a matrix or associated with populations of cancerous cells, bone, skin, and cells that produce cytokines or factors that contribute to the disease.

[0143] The term “target cell” generally refers to a cell having a ligand for the binding portion of the target composition, an antibody, or an antibody fragment, and which is associated with or causes a disease or pathological condition, including cancer cells, tumor cells, and inflammatory cells. Ligands of target cells are referred herein as “target cell markers” or “target cell antigens” and include, but are not limited to, cell surface receptors or antigens, cytokines, cytokine receptors, MHC proteins, and exogenously presented cytosolic proteins or peptides. As used herein, “target cell” does not include effector cells.

[0144] As used herein, “immunoassay” generally refers to a biochemical test that measures the presence or concentration of a substance in a sample, such as a biological sample, by using the reaction of an antibody (or a fragment thereof) to its alloantigen, for example, the specific binding of an antibody to a protein. Both the presence of an antigen or the amount of an antigen present can be measured.

[0145] As used herein, “mass spectrometer (MS)” generally refers to an instrument that includes means for ionizing molecules to detect charged molecules. The mass spectrum produced by the mass spectrometer can be used to identify a molecule of interest based on its molar mass. Non-limiting examples of “mass spectrometer (MS)” include any combination with liquid chromatography (LC), such as liquid chromatography and mass spectrometry (LC-MS), and liquid chromatography and tandem mass spectrometry (LC-MS / MS).

[0146] Where used herein, the terms “treatment,” “to treat,” “to alleviate,” and “to improve” are interchangeable herein. These terms generally refer to an approach to obtain beneficial or desired outcomes, including but not limited to therapeutic and / or preventive benefits. Therapeutic benefit means the elimination or improvement of the underlying disorder to be treated. Therapeutic benefit may also be obtained by the elimination or improvement of one or more physiological symptoms, or by improvement in one or more clinical parameters related to the underlying disorder, such that improvement is observed in the subject even though the subject may still be suffering from the underlying disorder. For preventive benefit, a composition may be administered to a subject at risk of developing a particular disease, or to a subject reporting one or more physiological symptoms of the disease, even if the subject has not been diagnosed with the disease.

[0147] When used herein, “therapeutic effect” or “therapeutic benefit” generally refers to physiological effects resulting from the administration of the polypeptides of this disclosure, other than the ability to induce the production of antibodies against an antigen epitope present on a bioactive protein, including, but not limited to, the reduction, improvement, or prevention of disease in humans or other animals, or the improvement of one or more clinical parameters related to an underlying disorder, or the enhancement of the physical or mental health of humans or animals. For preventive benefits, the compositions may be administered to subjects at risk of developing a particular disease, a recurrence of a past disease, a disease state or symptom, or to subjects reporting one or more physiological symptoms of the disease, even if the disease has not been diagnosed.

[0148] The terms “therapeutic dose” and “therapeutic load” as used herein generally refer to the amount of a drug or bioactive protein, either alone or as part of a polypeptide composition, that, when administered to a subject in a single dose or repeated dose, can produce any detectable beneficial effect on any symptom, aspect, measurable parameter or characteristic of a disease or condition. Such effect does not necessarily have to be beneficial. Determining the therapeutic dose is well within the capabilities of those skilled in the art, in particular in light of the detailed disclosures provided herein.

[0149] The term "equomolar dose" generally means that the amount of a substance administered to a subject is equimolar, based on the molecular weight of the substance used in that dose.

[0150] The term “therapeutically effective non-toxic dose,” as used herein, generally refers to a tolerable dose of a composition as defined herein that is high enough to cause depletion of tumor or cancer cells, tumor removal, tumor reduction, or stabilization of the disease without serious toxic effects in the subject or without inherent toxic effects. Such a therapeutically effective non-toxic dose can be determined by dose-escalation studies described in the Art, and such a dose should be less than the dose that induces serious adverse side effects.

[0151] The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals typically characterized by unregulated cell growth / proliferation. composition Therapeutic drugs

[0152] In some embodiments, therapeutic agents (or activatable therapeutic agents, or unnatural activatable therapeutic agents) are provided herein, comprising a release segment (RS) (e.g., described in the release segment below or elsewhere in this specification) directly or indirectly linked to a bioactive moiety (BM) (e.g., described in the bioactive moiety section below or elsewhere in this specification). The bioactive moiety (BM) may be a bioactive peptide (BP) (e.g., described in the bioactive moiety section below or elsewhere in this specification). The release segment (RS) may comprise a peptide substrate (e.g., described in the release segment below or elsewhere in this specification) that is sensitive to cleavage by a mammalian protease (e.g., described in the release segment section below or elsewhere in this specification) at an easily cleavable bond. The therapeutic agent may further include a masking moiety (MM) directly or indirectly linked to the release segment (RS) (e.g., as described in the section on masking moieties below or elsewhere in this specification). The biological activity of the therapeutic agent may be enhanced upon cleavage of the peptide substrate by a mammalian protease (thereby releasing the masking moiety). In its uncleaved state, the therapeutic agent may have a structural configuration of BM-RS-MM or MM-RS-BM from the N-terminus to the C-terminus. Upon cleavage of the release segment (RS), the masking moiety (MM) may be released from the therapeutic agent. The masking moiety (MM) may include an extended recombinant polypeptide (XTEN). In its uncleaved state, the therapeutic agent may have a structural configuration of BM-RS-XTEN or XTEN-RS-BM from the N-terminus to the C-terminus.

[0153] In some embodiments of a therapeutic agent (or activatable therapeutic agent, or unnatural activatable therapeutic agent), the therapeutic agent may further include a second release segment (RS2) (e.g., as described in the section on release segments below or elsewhere in this specification) directly or indirectly linked to the bioactive moiety (BM). The second release segment (RS2) may include a second peptide substrate (e.g., as described in the section on release segments below or elsewhere in this specification) cleaved at the second cleavable linkage by a mammalian protease (e.g., as described in the section on release segments below or elsewhere in this specification). The biological activity of the therapeutic agent may be enhanced when one or both of the first and second peptide substrates are cleaved by a mammalian protease (thereby releasing one or both of the first and second masking moieties). The mammalian protease for cleaving the second release segment (RS2) may be the same as the mammalian protease for cleaving the first release segment (RS1). The mammalian protease for cleaving the second release segment (RS2) may be different from the mammalian protease for cleaving the first release segment (RS1). The second release segment (RS2) may have the same amino acid sequence as the first release segment (RS1). The second release segment (RS2) may have a different amino acid sequence than the first release segment (RS1). In some embodiments, the easily cleavable bond (or first easily cleavable bond, or second easily cleavable bond) is not located immediately C-terminal to the methionine residue. In some embodiments, the first easily cleavable bond is not located immediately C-terminus of the methionine residue. In some embodiments, the second easily cleavable bond is not located immediately C-terminus of the methionine residue.

[0154] In some embodiments of a therapeutic agent (or activatable therapeutic agent, or non-natural activatable therapeutic agent), where the masking portion (MM) may be a first masking portion (MM1), the therapeutic agent may further include a second masking portion (MM2) (e.g., as described in the following section on masking portions in this specification or elsewhere) directly or indirectly linked to a second release segment (RS2). The uncleaved therapeutic agent may have a structural configuration of MM1-RS1-BM-RS2-MM2, MM1-RS2-BM-RS1-MM2, MM2-RS1-BM-RS2-MM1, or MM2-RS2-BM-RS1-MM1 from N-terminus to C-terminus. Upon cleavage of the second release segment (RS2), the second masking portion (MM2) may be released from the therapeutic agent. The first masking portion (MM1) may include a first elongated recombinant polypeptide (XTEN1). The second masking region (MM2) may contain a second extended recombinant polypeptide (XTEN2). The uncleaved therapeutic agent may have a structural configuration from N-terminus to C-terminus of XTEN1-RS1-BP-RS2-XTEN2, XTEN1-RS2-BP-RS1-XTEN2, XTEN2-RS1-BP-RS2-XTEN1, or XTEN2-RS2-BP-RS1-XTEN1.

[0155] In some embodiments of a therapeutic agent (or an activatable therapeutic agent, or a non-natural activatable therapeutic agent), the therapeutic agent may comprise a fusion polypeptide (e.g., a recombinant fusion protein) or a conjugate (e.g., linked by chemical conjugation). In some embodiments, the therapeutic agent may be configured to activate in or near a target tissue or cell in a subject (e.g., as described in the section on target tissues or cells below in this specification or elsewhere in this specification). The therapeutic agent may be an anticancer agent (e.g., an activatable anticancer agent, or a non-natural activatable anticancer agent). The therapeutic agent may be configured to be activated by one or more mammalian proteases (e.g., one of those described herein or any combination thereof).

[0156] In some embodiments of a therapeutic agent (or an activatable therapeutic agent, or a non-natural activatable therapeutic agent), the therapeutic agent may comprise a recombinant polypeptide. The recombinant polypeptide may comprise a bioactive peptide (BP) and a release segment (RS). The recombinant polypeptide may comprise a bioactive peptide (BP), a release segment (RS), and a masking moiety (MM). The uncleaved recombinant polypeptide may have a structural configuration of BP-RS-MM or MM-RS-BP from the N-terminus to the C-terminus. The recombinant polypeptide may comprise a bioactive peptide (BP), a first release segment (RS1), and a second release segment (RS2). The recombinant polypeptide may comprise a bioactive peptide (BP), a first release segment (RS1), a second release segment (RS2), a first masking moiety (MM1), and a second masking moiety (MM2). Uncleaved recombinant polypeptides may have the structural configuration MM1-RS1-BP-RS2-MM2, MM1-RS2-BP-RS1-MM2, MM2-RS1-BP-RS2-MM1, or MM2-RS2-BP-RS1-MM1 from the N-terminus to the C-terminus. Recombinant polypeptides may contain a bioactive peptide (BP), a first release segment (RS1), a second release segment (RS2), a first extended recombinant polypeptide (XTEN1), and a second extended recombinant polypeptide (XTEN2). Uncleaved recombinant polypeptides may have the structural configuration XTEN1-RS1-BP-RS2-XTEN2, XTEN1-RS2-BP-RS1-XTEN2, XTEN2-RS1-BP-RS2-XTEN1, or XTEN2-RS2-BP-RS1-XTEN1 from the N-terminus to the C-terminus. Release segment (RS)

[0157] In some embodiments of a therapeutic agent (or an activatable therapeutic agent, or a non-natural activatable therapeutic agent), the release segment (RS) (or a first release segment (RS1), or a second release segment (RS2)) may each independently contain a peptide substrate that is sensitive to cleavage by a mammalian protease at an easily cleavable bond. If a target tissue or cell can produce a mammalian protease (e.g., one described in the section on target tissues or cells below or anywhere else in this specification) for which the release segment (RS) (or a first release segment (RS1), or a second release segment (RS2)) is a peptide substrate of that mammalian protease, then the release segment (RS) (or a first release segment (RS1), or a second release segment (RS2)) may each independently cleave when it is in the vicinity of the target tissue or cell (e.g., one described in the section on target tissues or cells below or anywhere else in this specification).

[0158] In some embodiments of the therapeutic agent (or activatable therapeutic agent, or non-natural activatable therapeutic agent), the peptide substrate (or first peptide substrate, or second peptide substrate) may have up to four, up to three, up to two, or up to one amino acid substitutions with respect to the cleavage sequence (e.g., those described in Tables 1(a)-1(j) or Table A) of the reporter polypeptide (e.g., those described in the section on target tissues or cells below or elsewhere in this specification). The peptide substrate (or first peptide substrate, or second peptide substrate) may have up to four, up to three, up to two, or up to one amino acid substitutions with respect to the cleavage sequence of the reporter polypeptide (e.g., those described in Tables 1(a)-1(j) or Table A). The peptide substrate (or first peptide substrate, or second peptide substrate) may contain the same amino acid sequence as the cleavage sequence of the reporter polypeptide (e.g., those described in Tables 1(a)-1(j) or Table A). In some embodiments of the therapeutic agent (or activatable therapeutic agent, or unnatural activatable therapeutic agent), the peptide substrate (or first peptide substrate, or second peptide substrate) may include an amino acid sequence having up to four, up to three, up to two, or up to one amino acid substitution with respect to the sequences listed in column II or III of Table A (or a subset thereof) and / or the groups listed in Tables 1(a) to 1(j) (or any subset thereof). The peptide substrate (or the first peptide substrate, or the second peptide substrate) may contain the same amino acid sequence as those listed in column II or III of Table A (or a subset thereof) and / or the groups listed in Tables 1(a) to 1(j) (or any subset thereof).In some embodiments, the peptide substrate (or first peptide substrate, or second peptide substrate) includes two or three sequences listed in column II or III (or a subset thereof) of Table A. In some embodiments where the peptide substrate (or first peptide substrate, or second peptide substrate) includes two sequences listed in column II or III (or a subset thereof) of Table A, the two sequences partially overlap each other. In some embodiments where the peptide substrate (or first peptide substrate, or second peptide substrate) includes two sequences listed in column II or III (or a subset thereof) of Table A, the two sequences do not overlap each other. In some embodiments where the peptide substrate (or first peptide substrate, or second peptide substrate) includes three sequences listed in column II or III (or a subset thereof) of Table A, two or all of the three sequences do not overlap each other. In some embodiments, where the peptide substrate (or the first peptide substrate, or the second peptide substrate) includes three sequences listed in column II or III (or a subset thereof) of Table A, one of the three sequences partially overlaps with another sequence or both of the other sequences. In some embodiments, where the peptide substrate (or the first peptide substrate, or the second peptide substrate) includes three sequences listed in column II or III (or a subset thereof) of Table A, two of the three sequences partially overlap each other. In some embodiments, where the peptide substrate (or the first peptide substrate, or the second peptide substrate) includes three sequences listed in column II or III (or a subset thereof) of Table A, each of the three sequences partially overlaps each other. In some embodiments, where the peptide substrate (or the first peptide substrate, or the second peptide substrate) includes three sequences listed in column II or III (or a subset thereof) of Table A, all three sequences partially overlap each other.In some embodiments, up to four, up to three, up to two, or up to one amino acid substitutions are not located in positions corresponding to amino acid residues directly adjacent to easily cleaved links in the sequences listed in column II or III (or a subset thereof) of Table A. In some embodiments, up to four, up to three, up to two, or up to one amino acid substitutions are not located in positions corresponding to amino acid residues directly adjacent to easily cleaved links in the corresponding sequences selected from the group listed in Tables 1(a) to 1(i) (or any subset thereof). In some embodiments, up to four, up to three, up to two, or up to one amino acid substitutions are not located in positions corresponding to amino acid residues directly adjacent to easily cleaved links in the corresponding sequences selected from the group listed in Table 1(j) (or any subset thereof). The peptide substrate (or the first peptide substrate, or the second peptide substrate) may contain 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid residues, or a range of any two of the aforementioned values. The peptide substrate may contain 6 to 25 or 6 to 20 amino acid residues. The peptide substrate may contain 6 to 25 amino acid residues. The peptide substrate may contain 6 to 20 amino acid residues. In some embodiments, the peptide substrate contains 7 to 12 amino acid residues. The peptide substrate may include amino acid sequence fragments listed in column II or III of Table A (or a subset thereof) and / or groups listed in Tables 1(a) to 1(j) (or any subset thereof). A peptide substrate fragment may contain at least four amino acid residues and a corresponding cleavable linkage (e.g., those listed in Tables 1(a) to 1(j) or Table A). A peptide substrate fragment may contain at least five, at least six, at least seven, at least eight, at least nine, or at least ten amino acid residues. In some cases, the peptide substrate portion at the N-terminus of the cleavable linkage may have up to four, or up to three, or up to two, or up to one amino acid substitutions relative to the C-terminal sequence containing four to ten amino acid residues of the sequence listed in column IV or V (or a subset thereof) of Table A.The peptide substrate portion at the N-terminus of the easily cleavable bond may contain a C-terminal sequence containing 4 to 10 amino acid residues of the sequence described in column IV or V (or a subset thereof) of Table A. In some cases, the peptide substrate portion at the N-terminus of the easily cleavable bond may have up to 4, or up to 3, or up to 2, or up to 1 amino acid substitutions to the C-terminal sequence containing 4 to 10 amino acid residues of the sequence described in column IV (or a subset thereof) of Table A. The peptide substrate portion at the N-terminus of the easily cleavable bond may contain a C-terminal sequence containing 4 to 10 amino acid residues of the sequence described in column IV (or a subset thereof) of Table A. In some cases, the peptide substrate portion at the N-terminus of the easily cleavable bond may have up to 4, or up to 3, or up to 2, or up to 1 amino acid substitutions to the C-terminal sequence containing 4 to 10 amino acid residues of the sequence described in column V (or a subset thereof) of Table A. The peptide substrate portion at the N-terminus of the easily cleavable bond may contain a C-terminal sequence containing 4 to 10 amino acid residues of the sequence described in column V (or a subset thereof) of Table A. In some cases, the peptide substrate portion at the C-terminus of the easily cleavable bond may have up to 4, or up to 3, or up to 2, or up to 1 amino acid substitutions to the N-terminal sequence containing 4 to 10 amino acid residues of the sequence described in column V or VI (or a subset thereof) of Table A. The peptide substrate portion at the C-terminus of the easily cleavable bond may __ the N-terminal sequence containing 4 to 10 amino acid residues of the sequence described in column V or VI (or a subset thereof) of Table A. In some cases, the peptide substrate portion at the C-terminus of the easily cleavable bond may have up to 4, or up to 3, or up to 2, or up to 1 amino acid substitutions to the N-terminal sequence containing 4 to 10 amino acid residues of the sequence described in column V (or a subset thereof) of Table A. The peptide substrate portion at the C-terminus of the easily cleavable bond can have an N-terminal sequence containing 4 to 10 amino acid residues of the sequence listed in column V (or a subset thereof) of Table A.In some cases, the portion of the peptide substrate at the C-terminus of the easily cleavable linkage may have up to four, or up to three, or up to two, or up to one amino acid substitutions to the N-terminal sequence containing 4 to 10 amino acid residues of the sequence described in column VI (or a subset thereof) of Table A. The portion of the peptide substrate at the C-terminus of the easily cleavable linkage may __ the N-terminal sequence containing 4 to 10 amino acid residues of the sequence described in column VI (or a subset thereof) of Table A. In some embodiments in which the peptide substrate contains an easily cleavable linkage (for cleavage by one or more mammalian proteases), the peptide substrate does not contain a methionine residue immediately to the N-terminus of the easily cleavable linkage. In some embodiments in which the peptide substrate contains multiple easily cleavable links, the peptide substrate does not contain a methionine residue immediately to the N-terminus of at least one of the multiple easily cleavable links. In some embodiments in which the peptide substrate contains multiple easily cleavable links, the peptide substrate does not contain a methionine residue immediately to the N-terminus of each of the multiple easily cleavable links. In some embodiments, the peptide substrate does not contain an amino acid sequence selected from the group consisting of #279, #280, #282, #283, #298, #299, #302, #303, #305, #307, #308, #349, #396, #397, #416, #417, #418, #458, #459, #460, #466, #481, and #482 (or any combination thereof) in column II of Table A.

[0159] In some embodiments of a therapeutic agent (or activatable therapeutic agent, or unnatural activatable therapeutic agent) comprising (1) a first release segment (RS1) containing a first peptide substrate and (2) a second release segment (RS2) containing a second peptide substrate, the second peptide substrate may contain 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid residues, or a range of any two of the aforementioned values. The second peptide substrate may contain 6 to 25 or 6 to 20 amino acid residues. The second peptide substrate may contain 6 to 25 amino acid residues. The second peptide substrate may contain 6 to 20 amino acid residues. The second peptide substrate may contain 7 to 12 amino acid residues. The second peptide substrate may include an amino acid sequence having up to four, up to three, up to two, or up to one amino acid substitution with respect to the sequences listed in column II or III (or a subset thereof) of Table A and / or the groups listed in Tables 1(a) to 1(j) (or any subset thereof). The second peptide substrate may include an amino acid sequence having up to four, up to three, up to two, or up to one amino acid substitution with respect to the sequences listed in column II or III (or a subset thereof) of Table A and / or the groups listed in Tables 1(a) to 1(j) (or any subset thereof). The second peptide substrate may include an amino acid sequence identical to the sequences listed in column II or III (or a subset thereof) of Table A and / or the groups listed in Tables 1(a) to 1(j) (or any subset thereof). In some embodiments, the second peptide substrate includes two or three sequences listed in column II or III (or a subset thereof) of Table A. In some embodiments, where the second peptide substrate includes two sequences listed in column II or III (or a subset thereof) of Table A, the two sequences (of the second peptide substrate) partially overlap each other. In some embodiments, where the second peptide substrate includes two sequences listed in column II or III (or a subset thereof) of Table A, the two sequences (of the second peptide substrate) do not overlap each other.In some embodiments, where the second peptide substrate includes three sequences listed in column II or III (or a subset thereof) of Table A, two or all of the three sequences (of the second peptide substrate) do not overlap with each other. In some embodiments, where the second peptide substrate includes three sequences listed in column II or III (or a subset thereof) of Table A, one of the three sequences (of the second peptide substrate) partially overlaps with another sequence (of the second peptide substrate) or both of the other sequences (of the second peptide substrate). In some embodiments, where the second peptide substrate includes three sequences listed in column II or III (or a subset thereof) of Table A, two of the three sequences (of the second peptide substrate) partially overlap with each other. In some embodiments, where the second peptide substrate includes three sequences listed in column II or III (or a subset thereof) of Table A, each of the three sequences (of the second peptide substrate) partially overlaps with each other. In some embodiments, where the second peptide substrate includes three sequences listed in column II or III (or a subset thereof) of Table A, all three sequences (of the second peptide substrate) partially overlap each other. In some embodiments, where the second peptide substrate includes a cleavable linkage (for cleavage by one or more mammalian proteases), the second peptide substrate does not contain a methionine residue immediately to the N-terminus of the cleavable linkage. In some embodiments, where the second peptide substrate includes multiple cleavable links, the second peptide substrate does not contain a methionine residue immediately to the N-terminus of at least one of the multiple cleavable links. In some embodiments, where the second peptide substrate includes multiple cleavable links, the second peptide substrate does not contain a methionine residue immediately to the N-terminus of each of the multiple cleavable links.In some embodiments, the second peptide substrate does not contain an amino acid sequence selected from the group consisting of #279, #280, #282, #283, #298, #299, #302, #303, #305, #307, #308, #349, #396, #397, #416, #417, #418, #458, #459, #460, #466, #481, and #482 (or any combination thereof) in column II of Table A.

[0160] In some embodiments of this disclosure, the peptide substrate (or first peptide substrate, or second peptide substrate) does not contain a sequence selected from SEQ ID NOs: 1 to 8. In some embodiments, the peptide substrate (or first peptide substrate, or second peptide substrate) does not contain the sequence of SEQ ID NO: 1. In some embodiments, the peptide substrate (or first peptide substrate, or second peptide substrate) does not contain the sequence of SEQ ID NO: 2. In some embodiments, the peptide substrate (or first peptide substrate, or second peptide substrate) does not contain the sequence of SEQ ID NO: 3. In some embodiments, the peptide substrate (or first peptide substrate, or second peptide substrate) does not contain the sequence of SEQ ID NO: 4. In some embodiments, the peptide substrate (or first peptide substrate, or second peptide substrate) does not contain the sequence of SEQ ID NO: 5. In some embodiments, the peptide substrate (or first peptide substrate, or second peptide substrate) does not contain the sequence of SEQ ID NO: 6. In some embodiments, the peptide substrate (or first peptide substrate, or second peptide substrate) does not contain the sequence of SEQ ID NO: 7. In some embodiments, the peptide substrate (or the first peptide substrate, or the second peptide substrate) does not contain the sequence of Sequence ID No. 8. In some embodiments, the peptide substrate (or the first peptide substrate, or the second peptide substrate) does not contain a methionine residue immediately N-terminal to one or more easily cleavable bonds (contained in the peptide substrate) (for cleavage by one or more mammalian proteases). In some embodiments, the peptide substrate (or the first peptide substrate, or the second peptide substrate) does not contain a methionine residue immediately N-terminal to one or more easily cleavable bonds (contained in the peptide substrate). In some embodiments, the peptide substrate (or the first peptide substrate, or the second peptide substrate) does not contain a methionine residue immediately N-terminal to any of the easily cleavable bonds (contained in the peptide substrate).In some embodiments, the peptide substrate (or the first peptide substrate or the second peptide substrate) does not contain an amino acid sequence selected from the group consisting of #279, #280, #282, #283, #298, #299, #302, #303, #305, #307, #308, #349, #396, #397, #416, #417, #418, #458, #459, #460, #466, #481 and #482 (or any combination thereof) in column II of Table A.

[0161] In some embodiments of the therapeutic agent (or activatable therapeutic agent, or unnatural activatable therapeutic agent), the sequence of 6 to 10 consecutive amino acids of the peptide substrate (e.g., first peptide substrate, second peptide substrate, etc.) contains up to 4, up to 3, up to 2, or up to 1 amino acid substitutions with respect to the corresponding sequence of 6 to 10 consecutive amino acids of the sequence listed in column II or III (or a subset thereof) of Table A. In some embodiments, the sequence of 6 to 10 consecutive amino acids of the peptide substrate (e.g., first peptide substrate, second peptide substrate, etc.) is identical to the corresponding sequence of 6 to 10 consecutive amino acids of the sequence listed in column II or III (or a subset thereof) of Table A. In some embodiments, the sequence of 8 to 10 consecutive amino acids of the peptide substrate (e.g., first peptide substrate, second peptide substrate, etc.) contains up to 3, up to 2, or up to 1 amino acid substitutions with respect to the corresponding sequence of 8 to 10 consecutive amino acids of the sequence listed in column II or III (or a subset thereof) of Table A. In some embodiments, the sequence of 8 to 10 consecutive amino acids of the peptide substrate (e.g., the first peptide substrate, the second peptide substrate, etc.) is identical to the corresponding sequence of 8 to 10 consecutive amino acids of the sequence described in column II or III (or a subset thereof) of Table A. In some embodiments, the sequence of 8 consecutive amino acids of the peptide substrate (e.g., the first peptide substrate, the second peptide substrate, etc.) contains up to 3, up to 2, or up to 1 amino acid substitutions relative to the corresponding sequence of 8 consecutive amino acids of the sequence described in column II or III (or a subset thereof) of Table A. In some embodiments, the sequence of 8 consecutive amino acids of the peptide substrate (e.g., the first peptide substrate or the second peptide substrate, etc.) is identical to the corresponding sequence of 8 consecutive amino acids of the sequence described in column II or III (or a subset thereof) of Table A. In some embodiments, the nine consecutive amino acid sequences of the peptide substrate (e.g., the first peptide substrate or the second peptide substrate) include up to three, up to two, or up to one amino acid substitutions to the corresponding nine consecutive amino acid sequences of the sequences listed in column II or III (or a subset thereof) of Table A.In some embodiments, the nine consecutive amino acid sequences of the peptide substrate (e.g., the first peptide substrate, the second peptide substrate, etc.) are identical to the corresponding nine consecutive amino acid sequences of the sequences listed in column II or III (or a subset thereof) of Table A. In some embodiments, the ten consecutive amino acid sequences of the peptide substrate (e.g., the first peptide substrate, the second peptide substrate, etc.) contain up to three, up to two, or up to one amino acid substitutions compared to the corresponding ten consecutive amino acid sequences of the sequences listed in column II or III (or a subset thereof) of Table A. In some embodiments, the ten consecutive amino acid sequences of the peptide substrate (e.g., the first peptide substrate, or the second peptide substrate, etc.) are identical to the corresponding ten consecutive amino acid sequences of the sequences listed in column II or III (or a subset thereof) of Table A.

[0162] In some embodiments of a therapeutic agent (or an activatable therapeutic agent, or a non-natural activatable therapeutic agent), the release segment (RS) (or a first release segment (RS1), or a second release segment (RS2)) may (each independently) contain a peptide substrate (or a first peptide substrate, or a second peptide substrate) cleaved by a mammalian protease, such as a serine protease, cysteine ​​protease, aspartate protease, threonine protease, or metalloproteinase. The release segment (RS) (or the first release segment (RS1), or the second release segment (RS2)) contains disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), disintegrin and metalloproteinase domain-containing protein 12 (ADAM12), disintegrin and metalloproteinase domain-containing protein 15 (ADAM15), disintegrin and metalloproteinase domain-containing protein 17 (ADAM17), disintegrin and metalloproteinase domain-containing protein 9 (ADAM9), disintegrin and metalloproteinase with thrombospondin motif 5 (ADAMTS5), cathepsin B, cathepsin D, cathepsin E, cathepsin K, cathepsin L, cathepsin S, fibroblast-activating protein alpha, hep Syn, Kallikrein-2, Kallikrein-4, Kallikrein-3, Prostate-Specific Antigen (PSA), Kallikrein-13, Regmine, Matrix Metallopeptidase 1 (MMP-1), Matrix Metallopeptidase 10 (MMP-10), Matrix Metallopeptidase 11 (MMP-11), Matrix Metallopeptidase 12 (MMP-12), Matrix Metallopeptidase 13 (MMP-13), Matrix Matrices metallopeptidase 14 (MMP-14), matrix metallopeptidase 16 (MMP-16), matrix metallopeptidase 2 (MMP-2), matrix metallopeptidase 3 (MMP-3), matrix metallopeptidase 7 (MMP-7), matrix metallopeptidase 8 (MMP-8), matrix metallopeptidase 9 (MMP-9), matrix metallopeptidase 4 (MMP-4),It may (independently) contain a peptide substrate (or a first peptide substrate or a second peptide substrate) cleaved by a mammalian protease selected from the group consisting of matrix metallopeptidase 5 (MMP-5), matrix metallopeptidase 6 (MMP-6), matrix metallopeptidase 15 (MMP-15), neutrophil elastase, protease-activated receptor 2 (PAR2), plasmin, prostasin, PSMA-FOLH1, membrane-bound serine protease 1 (MT-SP1), matryptase, and u-plasminogen. The release segment (RS) (or the first release segment (RS1), or the second release segment (RS2)) is matrix metallopeptidase 1 (MMP1) (the sequence listed in Table 1(a) is, for example, but not limited to, the substrate sequence), matrix metallopeptidase 2 (MMP2) (the sequence listed in Table 1(b) is, for example, but not limited to, the substrate sequence), matrix metallopeptidase 7 (MMP7) (the sequence listed in Table 1(c) is, for example, but not limited to, the substrate sequence), matrix metallopeptidase 9 (MMP9) (the sequence listed in Table 1(d) is, for example, but not limited to, the substrate sequence), matrix metallopeptidase 11 (MMP11) (Table 1(e) The peptide substrate (or first peptide substrate, or second peptide substrate) cleaved by a mammalian protease selected from the group consisting of: (for example, but not limited to) the sequences listed, which are substrate sequences; matrix metallopeptidase 14 (MMP14) (for example, but not limited to) the sequences listed in Table 1(f), which are substrate sequences; urokinase-type plasminogen activator (uPA) (for example, but not limited to) the sequences listed in Table 1(g), which are substrate sequences; regmine (for example, but not limited to) the sequences listed in Table 1(h), which are substrate sequences; and matryptase (for example, but not limited to) the sequences listed in Table 1(i), which are substrate sequences;A peptide substrate (or a first peptide substrate, or a second peptide substrate) that is cleaved by multiple mammalian proteases may (independently) contain multiple peptide substrates (or a first peptide substrate, or a second peptide substrate) that are sensitive to cleavage by mammalian proteases may be sensitive to cleavage by multiple mammalian proteases, including mammalian proteases. A peptide substrate (or a first peptide substrate, or a second peptide substrate) that is sensitive to cleavage by multiple mammalian proteases may have up to four, or up to three, or up to two, or up to one amino acid substitutions relative to the sequences listed in Table 1(j). A peptide substrate (or a first peptide substrate, or a second peptide substrate) that is sensitive to cleavage by multiple mammalian proteases may have up to four, or up to three, or up to two, or up to one amino acid substitutions relative to the sequences listed in Table 1(j). A peptide substrate (or the first peptide substrate, or the second peptide substrate) that is sensitive to cleavage by multiple mammalian proteases may have up to four, three, two, or one amino acid substitutions relative to the sequences listed in Table 1(j). A peptide substrate (or the first peptide substrate, or the second peptide substrate) that is sensitive to cleavage by multiple mammalian proteases may contain the sequences listed in Table 1(j).

[0163] In some embodiments of a therapeutic agent (or activatable therapeutic agent, or non-natural activatable therapeutic agent) comprising a set of release segments, each release segment in the set may (independently) contain a peptide substrate cleaved by a mammalian protease, e.g., serine protease, cysteine ​​protease, aspartate protease, threonine protease, or metalloproteinase.Each release segment in the set is: disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), disintegrin and metalloproteinase domain-containing protein 12 (ADAM12), disintegrin and metalloproteinase domain-containing protein 15 (ADAM15), disintegrin and metalloproteinase domain-containing protein 17 (ADAM17), disintegrin and metalloproteinase domain-containing protein 9 (ADAM9), thrombos Disintegrins and metalloproteinases with Pondin motif 5 (ADAMTS5), cathepsin B, cathepsin D, cathepsin E, cathepsin K, cathepsin L, cathepsin S, fibroblast-activating protein alpha, hepsin, kallikrein-2, kallikrein-4, kallikrein-3, prostate-specific antigen (PSA), kallikrein-13, regmine, matrix metallopeptidase 1 (MMP-1), matrix metallopeptidase 10 (MMP-10), matrix metallopeptidase 11 (MMP- 11) Matrix metallopeptidase 12 (MMP-12), Matrix metallopeptidase 13 (MMP-13), Matrix metallopeptidase 14 (MMP-14), Matrix metallopeptidase 16 (MMP-16), Matrix metallopeptidase 2 (MMP-2), Matrix metallopeptidase 3 (MMP-3), Matrix metallopeptidase 7 (MMP-7), Matrix metallopeptidase 8 (MMP-8), Matrix metallopeptidase 9 (MMP-9), Matrix metallop It may (independently) contain peptide substrates for different mammalian proteases (independently) selected from the group consisting of plutidase 4 (MMP-4), matrix metallopeptidase 5 (MMP-5), matrix metallopeptidase 6 (MMP-6), matrix metallopeptidase 15 (MMP-15), neutrophil elastase, protease-activated receptor 2 (PAR2), plasmin, prostasin, PSMA-FOLH1, membrane-bound serine protease 1 (MT-SP1), matryptase, and u-plasminogen.Each release segment in the set is matrix metallopeptidase 1 (MMP1) (the sequence listed in Table 1(a) is the substrate sequence, for example, but is not limited to this), matrix metallopeptidase 2 (MMP2) (the sequence listed in Table 1(b) is the substrate sequence, for example, but is not limited to this), matrix metallopeptidase 7 (MMP7) (the sequence listed in Table 1(c) is the substrate sequence, for example, but is not limited to this), matrix metallopeptidase 9 (MMP9) (the sequence listed in Table 1(d) is the substrate sequence, for example, but is not limited to this), matrix metallopeptidase 11 (MMP11) (the sequence listed in Table 1(e) However, the release segments may (independently) include peptide substrates for different mammalian proteases selected (independently) from the group consisting of, for example but not limited to, the following: matrix metallopeptidase 14 (MMP14) (the sequences listed in Table 1(f) are substrate sequences, for example but not limited to, the following): urokinase-type plasminogen activator (uPA) (the sequences listed in Table 1(g) are substrate sequences, for example but not limited to, the following): regmine (the sequences listed in Table 1(h) are substrate sequences, for example but not limited to, the following): and matryptase (the sequences listed in Table 1(i) are substrate sequences, for example but not limited to, the following): in some cases, at least one release segment (RS) of the set of release segments may (independently) include peptide substrates for cleavage by multiple mammalian proteases. Peptide substrates sensitive to cleavage by multiple mammalian proteases may have up to four, three, two, or one amino acid substitutions relative to the sequences listed in Table 1(j). Peptide substrates sensitive to cleavage by multiple mammalian proteases may have up to four, three, two, or one amino acid substitutions relative to the sequences listed in Table 1(j). Peptide substrates sensitive to cleavage by multiple mammalian proteases may have up to four, three, two, or one amino acid substitutions relative to the sequences listed in Table 1(j).Peptide substrates that are sensitive to cleavage by multiple mammalian proteases may contain the sequences listed in Table 1(j). It will be understood by those skilled in the art that the sequences listed in Tables 1(a) to 1(j) may be alternatively or further cleaved by one or more other proteases having substrate specificities similar to those of the corresponding proteases identified as capable of cleaving the sequences in the corresponding tables.

Table 1a

Table 1b

Table 1c

Table 1d

Table 1e

Table 1f

Table 1g

Table 1h

Table 1i

Table 1j

[0164] The masking moieties (MMs) of this disclosure can mask a bioactive moiety (BM) (or any component or fragment thereof) of an activatable therapeutic composition (e.g., those described herein) by specifically or nonspecificly interacting with the BM, thereby inhibiting or reducing the BM's ability to bind to a designated target (at least in certain cases). In some cases, the masking moiety (MM) can interfere with and / or inhibit the binding of the BM to its designated target (e.g., an antigen target) by specifically binding to the bioactive moiety (e.g., an antibody or antibody fragment) or having a specific affinity for it. In some cases, the masking moiety does not have a significant affinity for the bioactive moiety but exerts its masking effect due to nonspecific steric hindrance.

[0165] In some embodiments of a therapeutic agent (or an activatable therapeutic agent, or a non-natural activatable therapeutic agent), a masking moiety (MM) (or a first masking moiety (MM1), or a second masking moiety (MM2)) when linked to the corresponding therapeutic agent may interfere (independently, individually, or collectively) with the interaction between the bioactive moiety (BM) and target tissue or cells (e.g., those described in the section on target tissue or cells below or elsewhere in this specification) and the dissociation constant (K) of the BM of the therapeutic agent. d ) is the dissociation constant (K) of the bioactive portion corresponding to the target cell marker (such that the release segment (RS) is cleaved and MM remains after release) when the therapeutic agent is in an uncleaved state. d ) can be larger compared to the target cell marker and the dissociation constant (K) of the bioactive moiety (BM) of the therapeutic agent when the therapeutic agent is in an uncleaved state. d ) is the dissociation constant (K) of the target cell marker and the corresponding bioactive moiety. d) can be at least (approximately) twice as large, at least (approximately) five times as large, at least (approximately) ten times as large, at least (approximately) fifty times as large, at least (approximately) one hundred times as large, at least (approximately) two hundred times as large, at least (approximately) three hundred times as large, at least (approximately) four hundred times as large, at least (approximately) five hundred times as large, at least (approximately) six hundred times as large, at least (approximately) seven hundred times as large, at least (approximately) eight hundred times as large, at least (approximately) nine hundred times as large, or at least (approximately) one thousand times as large. Dissociation constant (K d ) can be measured under equimolar concentrations in in vitro assays. In vitro assays include cell membrane integrity assays, mixed cell culture assays, cell-based competitive binding assays, FACS-based propidium iodide assays, trypan blue influx assays, photometric enzyme release assays, and radiometric assays. 51 The following assays may be selected from Cr release assays, fluorescence-based europium release assays, calcein AM release assays, photometric MTT assays, XTT assays, WST-1 assays, Alamer Blue assays, radiometric 3H-Thd integration assays, cloning assays measuring cell division activity, fluorescence-based rhodamine 123 assays measuring mitochondrial transmembrane gradients, apoptosis assays monitored by phosphatidylserine exposure based on FACS, ELISA-based TUNEL assays, sandwich ELISAs, caspase activity assays, cell-based LDH release assays, and cell morphology assays, reporter gene activity assays, or any combination thereof.

[0166] In some embodiments of a therapeutic agent (or an activatable therapeutic agent, or a non-natural activatable therapeutic agent), the therapeutic agent can deliver BM to a target tissue or cell (e.g., as described in the section below on target tissues or cells in this specification or elsewhere) to enhance the safety profile compared to the corresponding bioactive moiety (such as the release segment (RS) being cleaved and MM being released and remaining), for example, to improve the maximum tolerable exposure level (MTEL) and / or reduce side effects (e.g., cytotoxicity). A therapeutic agent in which a bioactive moiety (BM) is linked (directly or indirectly) to a masking moiety (MM) (or a first masking moiety (MM1) or a second masking moiety (MM2)) can deliver the BM to target tissue or cells at least 2 times, at least 5 times, at least 10 times, at least 50 times, at least 100 times, at least 200 times, at least 300 times, at least 400 times, or at least 500 times greater than the corresponding bioactive moiety, resulting in an enhanced safety profile, for example, by improving the maximum tolerable exposure level (MTEL) and / or reducing side effects (e.g., cytotoxicity).

[0167] In some embodiments of a therapeutic agent (or an activatable therapeutic agent, or a non-natural activatable therapeutic agent), the therapeutic agent may have a longer terminal phase half-life compared to that of the corresponding bioactive moiety. A therapeutic agent in which a bioactive moiety (BM) is linked (directly or indirectly) to a masking moiety (MM) (or a first masking moiety (MM1), or a second masking moiety (MM2)) may have a terminal phase half-life that is at least (approximately) twice, at least (approximately) five times, at least (approximately) ten times, at least (approximately) fifteen times, at least (approximately) twenty times, at least (approximately) fifty times, or at least (approximately) 100 times longer than that of the corresponding bioactive moiety.

[0168] In some embodiments, the therapeutic agent may have lower immunogenicity compared to the corresponding bioactive moiety. A therapeutic agent in which the bioactive moiety (BM) is (directly or indirectly) linked to a masking moiety (MM) (or a first masking moiety (MM1) or a second masking moiety (MM2)) may have up to approximately half, up to approximately one-fifth, or up to approximately one-tenth the immunogenicity of the corresponding bioactive moiety. Immunogenicity can be confirmed by measuring the production of IgG antibodies that selectively bind to the bioactive moiety after administration of an equivalent dose to the subject.

[0169] In some embodiments, the therapeutic agent may have a larger apparent molecular weight coefficient under physiological conditions compared to the corresponding bioactive moiety. A therapeutic agent in which a bioactive moiety (BM) is linked (directly or indirectly) to a masking moiety (MM) (or a first masking moiety (MM1) or a second masking moiety (MM2)) may have an apparent molecular weight coefficient under physiological conditions that is at least (approximately) 1.5 times, at least (approximately) 2 times, at least (approximately) 5 times, at least (approximately) 8 times, at least (approximately) 10 times, at least (approximately) 12 times, at least (approximately) 15 times, at least (approximately) 18 times, or at least (approximately) 20 times larger than that of the corresponding bioactive moiety.

[0170] In some embodiments of a therapeutic agent (or activatable therapeutic agent, or non-natural activatable therapeutic agent) comprising a first masking portion (MM1) and a second masking portion (MM2), MM1 and MM2, when both linked in the therapeutic agent, may interfere (independently, individually or collectively) with the interaction between the bioactive portion (BM) and target tissue or cells (e.g., those described in the section on target tissue or cells below or elsewhere in this specification) and the dissociation constant (K) of the bioactive portion (BM) of the therapeutic agent with respect to target cell markers (e.g., those described in the section on target tissue or cells below or elsewhere in this specification) present in the target tissue or cells. d) is the dissociation constant (K) of the corresponding bioactive peptide (such that when the therapeutic agent is in an uncleaved state, one or both of the first release segment (RS1) and the second release segment (RS2) are cleaved, and one or both of MM1 and MM2 remain after release). d ) can be larger compared to the target cell marker and the dissociation constant (K) of the bioactive moiety (BM) of the therapeutic agent when the therapeutic agent is in an uncleaved state. d ) is the dissociation constant (K) of the corresponding bioactive peptide. d ) can be at least (approximately) twice as large, at least (approximately) five times as large, at least (approximately) ten times as large, at least (approximately) fifty times as large, at least (approximately) one hundred times as large, at least (approximately) two hundred times as large, at least (approximately) three hundred times as large, at least (approximately) four hundred times as large, at least (approximately) five hundred times as large, at least (approximately) six hundred times as large, at least (approximately) seven hundred times as large, at least (approximately) eight hundred times as large, at least (approximately) nine hundred times as large, or at least (approximately) one thousand times as large. Dissociation constant (K d ) can be measured under equimolar concentrations in in vitro assays. In vitro assays include cell membrane integrity assays, mixed cell culture assays, cell-based competitive binding assays, FACS-based propidium iodide assays, trypan blue influx assays, photometric enzyme release assays, and radiometric assays. 51 The following assays may be selected from: Cr release assays, fluorescence-based europium release assays, calcein AM release assays, photometric MTT assays, XTT assays, WST-1 assays, Alamer Blue assays, radiometric 3H-Thd integration assays, cloning assays measuring cell division activity, fluorescence-based rhodamine 123 assays measuring mitochondrial transmembrane gradients, apoptosis assays monitored by phosphatidylserine exposure based on FACS, ELISA-based TUNEL assays, sandwich ELISAs, caspase activity assays, cell-based LDH release assays, reporter gene activity assays, and cell morphology assays, or any combination thereof.

[0171] In some embodiments of a therapeutic agent (or activatable therapeutic agent, or non-natural activatable therapeutic agent) comprising a first masking moiety (MM1) and a second masking moiety (MM2), a therapeutic agent in which a bioactive moiety (BM) is directly or indirectly linked to one or both of MM1 and MM2 can deliver the bioactive moiety (BM) to a target tissue or cell, thereby resulting in an enhanced safety profile compared to a corresponding bioactive moiety (such as one or both of the first release segment (RS1) and the second release segment (RS2) being cleaved and one or both of MM1 and MM2 being released and remaining), for example, an improved maximum tolerable exposure level (MTEL) and / or reduced side effects (e.g., cytotoxicity). A therapeutic agent in which a bioactive moiety (BM) is linked (directly or indirectly) to one or both of MM1 and MM2 can deliver the BM to target tissue or cells at least 2 times, at least 5 times, at least 10 times, at least 50 times, at least 100 times, at least 200 times, at least 300 times, at least 400 times, or at least 500 times greater than the corresponding bioactive moiety, resulting in an enhanced safety profile, for example, an improved maximum tolerable exposure level (MTEL), and / or a reduction in side effects (e.g., cytotoxicity).

[0172] In some embodiments of a therapeutic agent (or activatable therapeutic agent, or unnatural activatable therapeutic agent) comprising a first masking moiety (MM1) and a second masking moiety (MM2), a therapeutic agent in which a bioactive moiety (BM) is directly or indirectly linked to one or both of MM1 and MM2 may have a longer terminal phase half-life compared to that of the corresponding bioactive moiety (such as one or both of the first release segment (RS1) and the second release segment (RS2) being cleaved and one or both of MM1 and MM2 being released). A therapeutic agent in which a bioactive moiety (BM) is linked (directly or indirectly) to one or both of MM1 and MM2 may have a terminal phase half-life at least (approximately) twice, at least (approximately) five times, at least (approximately) ten times, at least (approximately) fifteen times, at least (approximately) twenty times, at least (approximately) fifty times, or at least (approximately) 100 times longer than the terminal phase half-life of the corresponding bioactive moiety.

[0173] In some embodiments of a therapeutic agent (or activatable therapeutic agent, or non-natural activatable therapeutic agent) comprising a first masking moiety (MM1) and a second masking moiety (MM2), a therapeutic agent in which a bioactive moiety (BM) is directly or indirectly linked to one or both of MM1 and MM2 may have lower immunogenicity compared to the corresponding bioactive moiety (such as one or both of the first release segment (RS1) and the second release segment (RS2) being cleaved and one or both of MM1 and MM2 being released). A therapeutic agent in which a bioactive moiety (BM) is linked (directly or indirectly) to one or both of MM1 and MM2 may have up to approximately half, up to approximately one-fifth, or up to approximately one-tenth the immunogenicity of the corresponding bioactive moiety. Immunogenicity can be confirmed by measuring the production of IgG antibodies that selectively bind to the bioactive moiety after administration of an equivalent dose to the subject.

[0174] In some embodiments of a therapeutic agent (or activatable therapeutic agent, or unnatural activatable therapeutic agent) comprising a first masking moiety (MM1) and a second masking moiety (MM2), a therapeutic agent in which a bioactive moiety (BM) is directly or indirectly linked to one or both of MM1 and MM2 may have a larger apparent molecular weight coefficient under physiological conditions compared to the corresponding bioactive moiety. A therapeutic agent in which a bioactive moiety (BM) is linked (directly or indirectly) to one or both of MM1 and MM2 may have an apparent molecular weight coefficient under physiological conditions that is at least (approximately) 1.5 times, at least (approximately) 2 times, at least (approximately) 5 times, at least (approximately) 8 times, at least (approximately) 10 times, at least (approximately) 12 times, at least (approximately) 15 times, at least (approximately) 18 times, or at least (approximately) 20 times larger than the corresponding bioactive moiety.

[0175] In some embodiments of a therapeutic agent (or an activatable therapeutic agent, or a non-natural activatable therapeutic agent), the masking moiety (MM) (or a first masking moiety (MM1), or a second masking moiety (MM2)) may (each independently) comprise an extended recombinant polypeptide (XTEN). The XTEN may be characterized by (i) comprising at least 100 amino acids; (ii) having at least 90% of its amino acid residues selected from glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), and proline (P); and (iii) comprising at least four different amino acids selected from G, A, S, T, E, and P. XTEN may be characterized by (i) containing at least 150 amino acids; (ii) at least 90% of its amino acid residues being selected from glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), and proline (P); and (iii) containing at least four different amino acids selected from G, A, S, T, E, and P. The extended recombinant polypeptide (XTEN) may contain (each independently) amino acid sequences having at least (approximately) 90%, at least (approximately) 91%, at least (approximately) 92%, at least (approximately) 93%, at least (approximately) 94%, at least (approximately) 95%, at least (approximately) 96%, at least (approximately) 97%, at least (approximately) 98%, at least (approximately) 99%, or 100% sequence identity with respect to the sequences listed in Tables 2b-2c or any subset thereof.

[0176] In some embodiments of a therapeutic agent (or activatable therapeutic agent, or non-natural activatable therapeutic agent) comprising (1) a first masking moiety (MM1) comprising a first elongated recombinant polypeptide (XTEN1) and (2) a second masking moiety (MM2) comprising a second elongated recombinant polypeptide (XTEN2), XTEN2 may be characterized by (i) comprising at least 100 amino acids; (ii) at least 90% of its amino acid residues being selected from glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), and proline (P); and (iii) comprising at least four different amino acids selected from G, A, S, T, E, and P. XTEN2 may be characterized by (i) containing at least 150 amino acids; (ii) at least 90% of its amino acid residues being selected from glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), and proline (P); and (iii) containing at least four different amino acids selected from G, A, S, T, E, and P. XTEN2 may contain amino acid sequences having at least (approximately) 90%, at least (approximately) 91%, at least (approximately) 92%, at least (approximately) 93%, at least (approximately) 94%, at least (approximately) 95%, at least (approximately) 96%, at least (approximately) 97%, at least (approximately) 98%, at least (approximately) 99%, or 100% sequence identity with sequences selected from the group of sequences listed in Tables 2b-2c or any subset thereof.

[0177] In some embodiments, an XTEN (or XTEN1, or XTEN2) may contain or be formed from a plurality of non-overlapping sequence motifs (each independently). At least one of the non-overlapping sequence motifs may be repeated (or repeated at least twice in the corresponding XTEN). At least one of the non-overlapping sequence motifs may not be repeated (or appear only once in the corresponding XTEN). The plurality of non-overlapping sequence motifs may include (i) a set of (repeating) non-overlapping sequence motifs, where each motif in the set is repeated at least twice in the corresponding XTEN; and (ii) a non-overlapping (non-repeating) sequence motif that appears only once (or appears only once) in the corresponding XTEN. Each non-overlapping sequence motif may be 9–14 (or 10–14, or 11–13) amino acids in length. Each non-overlapping sequence motif may be 12 amino acids in length. Multiple non-overlapping sequence motifs may include a set of non-overlapping (repeating) sequence motifs, each of which (1) may be repeated at least twice in the corresponding XTEN, and (2) may be between 9 and 14 amino acids in length. A set of (repeating) non-overlapping sequence motifs may include 12-mer sequence motifs selected from the group listed in Table 2a. A set of (repeating) non-overlapping sequence motifs may include 12-mer sequence motifs selected from the group listed in Table 2a. A set of (repeating) non-overlapping sequence motifs may include at least two, at least three, or all four of the 12-mer sequence motifs from the group listed in Table 2a. [Table 2a] *This shows individual motif sequences that form a "family sequence" when used together in various permutations. [Table 2b-1] [Table 2b-2] Table 2b-3 Table 2b-4 Table 2b-5 Table 2b-6 Table 2b-7 Table 2b-8 Table 2b-9 Table 2b-10 Table 2b-11 Table 2b-12 Table 2c-1 Table 2c-2 Table 2c-3 Table 2c-4 Table 2c-5 Table 2c-6

[0178] Additional examples of XTEN sequences that may be used in accordance with this disclosure are U.S. Patent Applications Publications 2010 / 0239554A1, 2010 / 0323956A1, 2011 / 0046060A1, 2011 / 0046061A1, 2011 / 0077199A1, 2011 / 0172146A1, 2018 / 0244736A1, 2018 / 0346952A1, and 2019 / 0153115A1; U.S. Patents 8,673,860, 9,371,369, and 9,926,351. Disclosed in Patent Nos. 9,249,211 and 9,976,166; and in International Patent Publication Nos. WO2010 / 091122A1, WO2010 / 144502A2, WO2010 / 144508A1, WO2011 / 028228A1, WO2011 / 028229A1, WO2011 / 028344A2, WO2014 / 011819A2, WO2015 / 023891, WO2016 / 077505A2, WO2017 / 040344A2, and WO2019 / 126576A1.

[0179] In general, XTENs are polypeptides having substantially non-repeating sequences that do not exist in nature, possessing low levels of secondary or tertiary structure under physiological conditions, or having neither secondary nor tertiary structure, and also possessing additional properties as described in subsequent paragraphs. XTENs may have at least (about) 100, at least (about) 150, at least (about) 200, at least (about) 300, at least (about) 400, at least (about) 500, at least (about) 600, at least (about) 700, at least (about) 800, at least (about) 900, at least (about) 1,000 amino acids, or a range between any of the above. As used herein, XTENs specifically exclude whole antibodies or antibody fragments (e.g., single-chain antibodies and Fc fragments). XTEN polypeptides are useful as fusion partners because they play a variety of roles in conferring certain desirable properties when linked to compositions, for example, one or more bioactive moieties (e.g., those described herein). The resulting compositions have enhanced properties compared to the corresponding one or more bioactive moieties not linked to XTEN, such as enhanced pharmacokinetic, physicochemical, and pharmacological properties, as well as improved toxicological and pharmaceutical properties, which makes these compositions useful in treating certain conditions in which one or more bioactive moieties are known in the art.

[0180] The unstructured and physicochemical properties of XTEN are partly attributable to its total amino acid composition, which is biased towards 4-6 hydrophilic amino acids, its quantifiable, substantially non-repeating amino acid sequence design, and the resulting XTEN polypeptide length. With respect to advantageous properties common to XTEN but rarely found in natural polypeptides, the properties of XTEN disclosed herein can be independent of the absolute primary amino acid sequence, as demonstrated by the diversity of exemplary sequences in Tables 2b-2c, which exhibit similar properties across various length ranges and impart enhanced properties to compositions in which they are linked, many of which are demonstrated in these embodiments. In fact, the compositions of this disclosure are not limited to the XTENs specifically listed in Table 8 or 10, but rather embodiments are particularly intended to include at least sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequences of Tables 2b-2c when optimally aligned, as they exhibit the properties of the XTENs described herein. It has been confirmed that the properties of such XTENs are more similar to those of non-proteinoid hydrophilic polymers (e.g., polyethylene glycol, or "PEG") than to those of proteins. The XTENs of this disclosure exhibit one or more of the following advantageous properties: defined uniform length (for a given sequence), conformational mobility, reduced or absent secondary structures, high degree of random coil formation, high water solubility, high protease resistance, low immunogenicity, low binding to mammalian receptors, defined charge, and increased hydrodynamic (or Stokes) radius; similar properties to certain hydrophilic polymers (e.g., polyethylene glycol) that make the XTENs of this disclosure particularly useful as fusion partners.

[0181] The XTENs described herein are designed to behave like denatured peptide sequences under physiological conditions, despite their extended polymer length. "Denatured" refers to a state of peptide in solution characterized by a large degree of freedom in the three-dimensional structure of the peptide backbone. Most peptides and proteins adopt denatured structures in the presence of high concentrations of denaturants or at high temperatures. Peptides in denatured structures are characterized, for example, by a characteristic circular dichroism (CD) spectrum and the absence of long-range interactions as determined by NMR. "Denatured structure" and "unstructured structure" are used synonymously herein. In some embodiments, this disclosure provides compositions comprising XTEN sequences that, under physiological conditions, resemble denatured sequences with substantially no secondary structure. "Substantially no" as used in this context means that, as measured or determined by methods described herein, including algorithms or spectrophotometric assays, at least about 80%, or about 90%, or about 95%, or about 97%, or at least about 99% of the XTEN amino acid residues of the XTEN sequence do not contribute to the secondary structure.

[0182] Various well-established methods and assays are known in the art for determining and confirming the physicochemical properties of target XTENs and target polypeptide compositions in which they are incorporated. Such properties include, but are not limited to, secondary or tertiary structure, solubility, protein aggregation, stability, absolute and apparent molecular weight, purity and homogeneity, melting properties, contaminants, and water content. Methods for measuring such properties include analytical centrifugation, EPR, HPLC-ion exchange, HPLC-size exclusion chromatography (SEC), HPLC-reverse phase, light scattering, capillary electrophoresis, circular dichroism, differential scanning calorimetry, fluorescence, HPLC-ion exchange, HPLC-size exclusion, IR, NMR, Raman spectroscopy, refractive index measurement, and UV / visible spectroscopy. In particular, secondary structure can be measured by spectrophotometric methods, for example, by circular dichroism spectroscopy in the "far UV" spectral region (190-250 nm). Secondary structural elements, such as alpha-helices and beta-sheets, each give rise to characteristic shapes and sizes in the CD spectrum, and the absence of these structural elements also gives rise to characteristic shapes and sizes in the CD spectrum. Secondary structures can also be predicted for polypeptide sequences using certain computer programs or algorithms, such as the well-known Chou-Fasman algorithm (Chou, PY, et al. (1974) Biochemistry, 13: 222-45) as described in U.S. Patent Application Publication No. 20030228309A1 and the Garnier-Osguthorpe-Robson algorithm ("GOR IV algorithm") (Garnier J, Gibrat JF, Robson B. (1996), GOR method for predicting protein secondary structure from amino acid sequence. Methods Enzymol 266:540-553).For a given sequence, the algorithm predicts whether secondary structure is present to some extent or not at all, and can express this as, for example, the total number and / or percentage of residues in sequences that form alpha-helices or beta-sheets, or as the percentage of residues in sequences that are predicted to result in the formation of random coils (lacking secondary structure). Polypeptide sequences can be analyzed using the Chou-Fasman algorithm, for example, using the site fasta.bioch.virginia.edu / fasta_www2 / fasta_www.cgi?rm=misc1 on the World Wide Web, and the GOR IV algorithm at npsa-pbil.ibcp.fr / cgi-bin / npsa_automat.pl?page=npsa_gor4.html (both accessed on December 8, 2017). The properties of random coils can be determined by various methods, including a method using intrinsic viscosity measurement that increases or decreases with chain length in a structure-dependent manner (Tanford, C., Kawahara, K. & Lapanje, S. (1966) J. Biol. Chem. 241, 1921-1923), and a method using size exclusion chromatography (Squire, PG, Calculation of hydrodynamic parameters of random coil polymers from size exclusion chromatography and comparison with parameters by conventional methods. Journal of Chromatography, 1981, 5, 433-442). Further methods are disclosed in Arnau, et al., Prot Expr and Purif (2006) 48, 1-13.

[0183] In some embodiments of this disclosure, the activatable therapeutic agent is an activatable antibody (AA) composition, where the masking moiety (MM) refers to an amino acid sequence attached to the antibody or antibody fragment (AB) and is arranged to reduce the ability of AB to bind to its designated binding target by specifically binding to the antigen-binding domain of AB (such as the complementarity-determining region (CDR)). Such binding may be non-covalent. In some embodiments, the activatable antibody composition may be prevented from binding to its designated binding target by binding the MM to the N-terminus or C-terminus of the activatable antibody composition.

[0184] Alternatively, the masking moiety (MM) may not specifically bind to AB, but rather interfere with AB-target binding through nonspecific interactions such as steric hindrance. For example, the MM may be positioned in a non-cleaved, activatable antibody composition such that, depending on the tertiary or quaternary structure of the activatable antibody, the MM can mask AB through charge-based interactions, thereby retaining the MM in place and interfering with target access to AB. The masking moiety (MM) can allosterically or sterically interfere with and / or inhibit the binding of the antibody or antibody fragment (AB) to its target.

[0185] When an antibody or antibody fragment (AB) is modified with MM and present in the presence of a target, the specific binding of AB to that target may be reduced or inhibited compared to the specific binding of unmodified AB to the target. The dissociation constant (K) of MM-modified AB to the target of AB d ) is generally the corresponding K for AB targets that are not modified with MM. d It may be greater than this. Conversely, the binding affinity of MM-modified AB to a target may generally be lower than the binding affinity of MM-unmodified AB to a target. In some embodiments, the masking portion (MM) of an activatable antibody composition is greater than the equilibrium dissociation constant (K) for binding to the antibody or fragment, which is greater than the equilibrium dissociation constant (K) for binding the antibody or fragment to its designated binding target (near or at the site of the disease in the target). d ) may have.

[0186] If an antibody or antibody fragment (AB) is modified with a release segment (RS) and a masking moiety (MM), and in the presence of a target but in the absence of sufficient protease or protease activity to cleave the RS, the specific binding of the modified AB to the target may generally be reduced or inhibited compared to the specific binding of the RS and MM-modified AB in the presence of sufficient protease or protease activity to cleave the target and the RS. For example, if the modified antibody is an activatable antibody composition and contains a release segment (RS), the AB may not be masked at the time of cleavage of the RS in the presence of a protease, preferably a disease-specific protease. Thus, the MM results in masking of AB from target binding when the activatable antibody composition is uncleaved, but does not substantially or significantly interfere with or compete with the binding of the target to AB when the activatable antibody composition is a cleaved configuration. A schematic diagram of an exemplary activatable antibody (AA) composition is provided in Figure 3. As illustrated, the release segment (RS) is configured such that, in a cleaved (or relatively active) state and in the presence of the target, the antibody or antibody fragment (AB) binds to the target, but in an uncleaved (or relatively inactive) state and in the presence of the target, the specific binding of AB to that target is reduced or inhibited. The specific binding of the antibody or antibody fragment (AB) to its target may be reduced due to the inhibition or masking of the ability of AB to specifically bind to that target by the masking portion (MM).

[0187] In some embodiments of activatable antibody compositions in which an antibody or antibody fragment (AB) is capable of specifically binding to its designated binding target, the attachment of a masking moiety (MM) to the antibody or antibody fragment (AB) may reduce the ability of AB to bind to its designated binding target compared to the ability of AB not attached to the MM (for example, when assayed in vitro using a target substitution assay). Such attachment of the MM to the AB may result in a persistent reduction in the ability of AB to bind to its designated binding target.

[0188] The masking moiety (MM) may be provided in various different forms. In certain embodiments, the MM may be selected to be a known binding partner of an antibody or antibody fragment (AB), provided that, after cleavage of the release segment (RS), it binds AB with lower affinity and / or avidity than the target protein to which AB is designed to bind, thereby reducing the interference of the MM in target-AB binding. In other words, as discussed above, the MM masks AB from target binding when the activatable antibody composition is uncleaved, but does not substantially or significantly interfere with or compete with target binding when the activatable antibody composition is in a cleaved configuration. In specific embodiments, AB and MM do not contain the amino acid sequences of a naturally occurring binding partner pair, such that at least one of AB and MM does not have the amino acid sequences of members of the naturally occurring binding partner. The masking moiety (MM) may not contain more than 50% amino acid sequence identity with respect to the natural binding partner of the antibody or antibody fragment (AB). The masking portion (MM) may contain a consensus sequence specific to the binding of an antibody class to a designated binding target (e.g., an affected target). The MM may be a polypeptide with an amino acid length of 40 or less (e.g., 2 to 40). The MM may be attached to an antibody composition that can be activated by covalent bond.

[0189] In some embodiments, the Disclosure provides an activatable antibody complex (AAC) composition (illustrated in Figure 4) comprising: (1) two antibodies or antibody fragments (AB1 and AB2) each capable of specifically binding to their designated binding targets; (2) at least one masking portion (MM) attached to either AB1 or AB2, capable of inhibiting the specific binding of AB1 and AB2 to their designated binding targets; and (3) at least one release segment (RS) attached to either AB1 or AB2, capable of being specifically cleaved by a protease, thereby activating the AAC composition. In some embodiments, if the AAC is in an uncleaved state, the MM can inhibit the specific binding of AB1 and AB2 to their designated binding targets; if the AAC is in a cleaved state, the MM does not inhibit the specific binding of AB1 and AB2 to their designated binding targets. The two ABs can bind to different targets or different epitopes on the same target.

[0190] In some embodiments, MM does not inhibit the cell entry of the activatable antibody composition.

[0191] In some embodiments, the masking portion (MM) may include an antialbumin domain, such as a single-domain antibody (sdAb) antialbumin domain. In some embodiments, the antialbumin domain may include a non-CDR loop, a CDR loop, or any combination thereof. In some embodiments, the antialbumin domain may include both a non-CDR loop and a CDR loop. A non-CDR loop may be able to mask AB by binding to one or more antibodies or antibody fragments (AB) (e.g., a CDR of AB) in an activatable antibody (AA) composition, thereby inhibiting or reducing AB's ability to bind to its designated target (at least in some cases). A CDR loop may be able to bind albumin (e.g., human serum albumin), thereby (at least in some cases) masking AB in an activatable antibody (AA) composition from binding to its designated target by steric or allosteric impairment and / or conferring an extension of the half-life to the AA composition. In some embodiments, the non-CDR loop may be manipulated at different positions on the antialbumin sdAb domain. In some embodiments, MM may inhibit or reduce the ability of AB to bind to its designated target by (1) (1a) specific binding to the target recognition region of AB and / or (1b) stereomasking the target recognition region of AB, and / or MM may confer an extension of the half-life of AA containing AB by (2) binding to albumin. MM may be (directly or indirectly) attached to an antibody composition that can be activated by covalent bond.

[0192] As illustrated in the schematic diagram shown in Figure 5, an exemplary activatable antibody complex (AAC) composition may include: (1) at least two antibodies or antibody fragments (AB1 and AB2) each capable of specifically binding to their designated binding targets; (2) at least one masking portion (MM) attached to AB1 or AB2 capable of inhibiting the specific binding of AB1 or AB2 to their designated binding targets; and (3) at least one release segment (RS) attached to AB1 or AB2 capable of being specifically cleaved by a protease, thereby activating the activatable antibody complex (AAC) composition. In some embodiments, if the AA is in an uncleaved state, the MM can inhibit the specific binding of AB1 or AB2 to their designated binding targets, and if the activatable antibody complex (AAC) composition is in a cleaved state, the MM does not inhibit the specific binding of AB1 or AB2 to their designated binding targets. In some embodiments, the masking portion (MM) can be attached to both AB1 and AB2 by two separate release segments (RS). In other words, MM is positioned between AB1 and AB2 and may be connected to either the C-terminus of AB1 or the N-terminus of AB2, or to the N-terminus of AB1 and the C-terminus of AB2.

[0193] In some embodiments of this disclosure, the activatable therapeutic agent is an activatable antibody (AA) composition, where the masking moiety (MM) refers to an amino acid sequence attached to an antibody or antibody fragment (AB) (e.g., but not limited to scFv, sdAb, or fragments thereof) and is arranged to reduce the ability of AB to dimerize with another antibody or antibody fragment, thereby preventing the formation of an antibody or antibody fragment capable of binding to a target. Such binding may be non-covalent. In some embodiments, the activatable antibody composition may be prevented from binding to a designated binding target by the MM binding to the N-terminus or C-terminus of the activatable antibody composition.

[0194] When an antibody or antibody fragment (AB) is modified with MM and present in the presence of a target, the specific binding of AB to its dimerization partner may be reduced or inhibited compared to the specific binding of unmodified AB to its dimerization partner. The dissociation constant (K) of MM-modified AB to its dimerization partner. d ) is generally the corresponding K for the dimerization partner of AB that is not modified with MM. d It may be greater than this. Conversely, the binding affinity of MM-modified AB to its dimerization partner may generally be lower than the binding affinity of unMM-modified AB to its dimerization partner. In some embodiments, the masking moiety (MM) of the activatable antibody composition is greater than the equilibrium dissociation constant (K) for binding to the antibody or fragment with respect to the binding of the antibody or fragment to its designated dimerization partner. d ) may have.

[0195] When an antibody or antibody fragment (AB) is modified with a release segment (RS) and a masking moiety (MM), and in the presence of a target but in the absence of a protease or protease activity sufficient to cleave the RS, the specific ability of the modified AB to dimerize with another antibody or antibody fragment and the resulting ability of the dimer to bind to its designated binding target may generally be reduced or inhibited compared to the specific dimerization ability of the RS and MM-modified AB, and the subsequent ability of the dimer to bind to its designated binding target, in the presence of a protease or protease activity sufficient to cleave the RS and the target. For example, if the modified antibody is an activatable antibody composition and contains a release segment (RS), the AB may not be masked at the time of cleavage of the RS in the presence of a protease, preferably a disease-specific protease. Therefore, if the activatable antibody composition is uncleaved, MM results in masking from the dimerization of AB with another AB and a reduction or inhibition of the binding of the resulting dimer to its designated binding target; however, if the activatable antibody composition is in a cleaved configuration, MM does not substantially or significantly interfere with or compete with the reduction or inhibition of dimerization to another AB and the binding of the resulting dimer to its designated binding target.

[0196] The masking portion can be provided in various forms. In some embodiments, the masking domain may be an inhibitory antibody or antibody fragment (IAB; e.g., a VL or VH domain, but not limited to these) provided that the MM reduces the interference of the MM in AB-AB dimerization by binding AB with lower affinity and / or avidity than the dimerization partner designed for AB to dimerize after cleavage of the release segment (RS). In other words, as discussed above, the MM masks AB from dimerization to another AB when the activatable antibody composition is uncleaved, but does not substantially or significantly interfere with or compete with dimerization with another AB when the activatable antibody composition is a cleaved configuration. The MM may be attached to the activatable antibody composition by covalent bond.

[0197] In some embodiments, the Disclosure provides an activatable antibody complex (AAC) composition (illustrated in Figure 6) comprising: (1) two antibodies or antibody fragments (AB1 and AB2); (2) two masking moieties (MMs) attached to one of AB1 and AB2, respectively, which reduce or inhibit the specific dimerization of AB1 and AB2, thereby enabling the subsequent binding of the AB1-AB2 complex to its designated binding target; (3) at least three release segments (RSs) attached to AB1, AB2 and MM, which are specifically cleaved by a protease and thereby activate the AAC composition; and (4) at least one additional antibody or antibody fragment (AB3 and / or AB4; e.g., scFv or sdAb, but not limited to these) attached to AB1 and / or AB2. In some embodiments, if the AAC is in an uncleaved state, the MM can inhibit or reduce the specific dimerization of AB1 and AB2, and subsequently inhibit or reduce the binding of the resulting AB1-AB2 dimer to its designated binding target; if the AAC is in a cleaved state, the MM neither reduces nor inhibits the specific dimerization of AB1 and AB2, nor reduces nor inhibits the subsequent binding of the AB1-AB2 dimer to its designated binding target. If two or more additional ABs are attached to AB1 and / or AB2, the additional ABs may bind to the same target or to different targets.

[0198] In some embodiments, MM may include coiled-coil domains, for example, but not limited to, (1) a high-affinity parallel heterodimer leucine zipper coiled-coil domain containing or lacking cysteine, (2) a low-affinity parallel heterodimer coiled-coil leucine zipper domain containing or lacking cysteine, (3) a disulfide-linked covalent coiled-coil domain, (4) an anti-parallel heterodimer leucine zipper coiled-coil domain, or (5) a helix-turn-helix homodimer leucine zipper coiled-coil domain. MM can be (directly or indirectly) attached to an antibody composition that can be activated by covalent bonding. In some embodiments, MM may reduce or inhibit the binding of AB to its intended target by steric or allosteric interference.

[0199] In some embodiments, the Disclosure provides an activatable antibody complex (AAC) composition (illustrated in Figure 7) comprising: (1) at least one antibody or antibody fragment (AB); (2) at least one masking portion (MM) attached to AB capable of inhibiting the specific binding of AB to its designated binding target; and (3) at least one release segment (RS) attached to AB capable of being specifically cleaved by a protease, thereby activating the AAC composition. In some embodiments, when the AAC is in an uncleaved state, the MM can reduce or inhibit the specific binding of AB to its designated binding target, and when the AAC is in a cleaved state, the MM neither reduces nor inhibits the specific binding of AB to its designated binding target.

[0200] In some embodiments, the activatable therapeutic agent may incorporate the cleavage sequences described herein and / or may be administered to patients identified as potential responders to the therapeutic agent based on the identification of peptide biomarkers (further described herein) in a biological sample derived from the subject. Biologically active moiety (BM)

[0201] In some embodiments of a therapeutic agent (or an activatable therapeutic agent, or a non-natural activatable therapeutic agent), the bioactive moiety (BM) may comprise a bioactive peptide (BP). The bioactive peptide (BP) may comprise an antibody, cytokine, cell receptor, or a fragment thereof. The bioactive polypeptide (BP) may comprise a binding moiety having binding affinity to a target cell marker on a target tissue or cell. The target cell marker may be an effector cell antigen expressed on the surface of effector cells. The binding moiety may be an antibody. The antibody may be Fv, Fab, Fab', Fab'-SH, or a nanobody (single-domain antibody or V). HH It can be selected from the group consisting of (also known as) linear antibodies and single-chain variable fragments (scFv).

[0202] In some embodiments of a therapeutic agent (or activatable therapeutic agent, or non-natural activatable therapeutic agent), where the binding site may be a first binding site and the target cell marker may be the first target cell marker, the bioactive polypeptide (BP) may further include a second binding site directly or indirectly linked to the first binding site. The second binding site may have binding affinity to a second target cell marker on a target tissue or cell. The second target cell marker may be a marker on tumor cells or cancer cells. The second binding site may be an antibody. The second binding site may be Fv, Fab, Fab', Fab'-SH, or a nanobody (single-domain antibody or V). HH The antibody may be selected from the group consisting of (also known as) linear antibodies and single-chain variable fragments (scFv).

[0203] In some embodiments disclosed herein, a bioactive moiety (BM) or bioactive peptide (BP) can exhibit binding specificity to a given target (or a given number of targets) and / or other desired biological properties when used in vivo or in in vitro assays. For example, a BM or BP may be an agonist, receptor, ligand, antagonist, enzyme, antibody (e.g., monospecific or bispecific), or hormone. BMs or BPs that are used for or are known to be useful for diseases or disorders in which natural BMs or BPs have a relatively short terminal phase half-life, and where improved pharmacokinetic parameters (which may be released as needed from the conjugated or fusion polypeptide by cleavage of spacer sequences) allow for lower dosing or enhanced pharmacological effects. Minimum effective dose or blood concentration (C min ) and maximum tolerated dose or blood concentration (C max BMs or BPs with a relatively narrow therapeutic range between ) are also of interest. In such cases, linking of BMs or BPs within a conjugate or fusion polypeptide containing a selected masking moiety such as XTEN can result in improvements to these properties, and as a result, they may be more useful as therapeutic or prophylactic agents compared to BMs or BPs that are not linked to a masking moiety such as XTEN. BMs or BPs encompassed by the compositions of the present invention described herein may be useful in the treatment of a variety of therapeutic or disease categories, including but not limited to glucose and insulin disorders, metabolic disorders, cardiovascular diseases, coagulation and hemorrhagic disorders, growth disorders or conditions, endocrine disorders, eye diseases, kidney diseases, liver diseases, tumorigenic conditions, inflammatory conditions, autoimmune conditions, and the like.

[0204] In some embodiments of the compositions disclosed herein, where the bioactive portion is a bioactive peptide (BP), the BP is adapted to the amino acid sequence of a glucose-regulating peptide or glucagon-like peptide (natural or synthetic analog) listed in Tables 3a-3c (for example, as described in more detail in the section on glucose-regulating peptides below), or to the amino acid sequence of a protein associated with metabolic disorders and cardiology listed in Table 3d (for example, as described in more detail in the section on metabolic disorders and cardiovascular proteins below), or to the amino acid sequence of a growth hormone listed in Table 3f (for example, as described in more detail in the section on growth hormone proteins below), or to the cytokine listed in Table 3g (for example, as described in more detail in the section on cytokines below). The peptide sequence may contain at least approximately 80% sequence identity (e.g., at least approximately 81%, at least approximately 82%, at least approximately 83%, at least approximately 84%, at least approximately 85%, at least approximately 86%, at least approximately 87%, at least approximately 88%, at least approximately 89%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or 100% sequence identity) to the amino acid sequence of the transduction domain in Table 3h (e.g., at least approximately 83%, at least approximately 84%, at least approximately 85%, at least approximately 86%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or 100% sequence identity). In some embodiments of the compositions of this disclosure, the BP sequence may include one or more substitutions shown in Table 4 (for example, those described in more detail below herein).

[0205] In some embodiments of the compositions disclosed herein, where the bioactive portion is a bioactive peptide (BP), the BP may include antibodies (e.g., monospecific, dispecific, trispecific, or polyspecific antibodies) (as defined herein above, the term “antibody” includes, among other things, antibody fragments) (as described in more detail below in the section on antibodies). The antibody may include a binding domain (or binding portion) having binding affinity to an effector cell antigen. The effector cell antigen may be expressed on the surface of effector cells selected from plasma cells, T cells, B cells, cytokine-induced killer cells (CIK cells), mast cells, dendritic cells, regulatory T cells (RegT cells), helper T cells, myeloid cells, and NK cells. The effector cell antigen may be expressed on or within effector cells. The effector cell antigen may be expressed on T cells, e.g., CD4 + CD8 +Effector cell antigens may be expressed on T cells or natural killer (NK) cells. Effector cell antigens may be expressed on the surface of T cells. Effector cell antigens may be expressed on B cells, pluripotent stem cells, dendritic cells, or myeloid cells. The binding domain (or binding moiety) may include VH and VL regions derived from a monoclonal antibody capable of binding to human CD3. In some embodiments in which the binding domain (or binding moiety) has binding affinity to CD3, the binding domain (or binding moiety) may have binding affinity to members of the CD3 complex, including all known CD3 subunits of the CD3 complex in individual or independently combined forms, e.g., CD3 epsilon, CD3 delta, CD3 gamma, CD3 zeta, CD3 alpha, and CD3 beta. A binding domain (or binding moiety) having binding affinity to CD3 may have binding affinity to CD3 epsilon, CD3 delta, CD3 gamma, CD3 zeta, CD3 alpha, or CD3 beta. In some embodiments of the compositions of this disclosure, the binding domain (or binding moiety) that binds to human CD3 may be derived from an anti-CD3 antibody selected from the group of antibodies listed in Tables 5a to 5e. The binding domain (or binding moiety) that binds to human CD3 may include VH and VL regions, each of which exhibits at least (about) 90%, at least (about) 91%, at least (about) 92%, at least (about) 93%, at least (about) 94%, at least (about) 95%, at least (about) 96%, at least (about) 97%, at least (about) 98%, at least (about) 99%, or 100% sequence identity with respect to the VL and VH sequences of a pair of anti-CD3 antibodies selected from those listed in Table 5a or 5d.The binding domain (or binding portion) that binds to human CD3 may include VH and VL regions, each of which exhibits at least (approximately) 90%, at least (approximately) 91%, at least (approximately) 92%, at least (approximately) 93%, at least (approximately) 94%, at least (approximately) 95%, at least (approximately) 96%, at least (approximately) 97%, at least (approximately) 98%, at least (approximately) 99%, or 100% sequence identity with respect to the paired VL and VH sequences of the huUCHT1 anti-CD3 antibody in Table 5a. The binding domain (or binding portion) that binds to human CD3 may include CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-H3 regions, each of which may originate from a monoclonal antibody selected from the group of antibodies listed in Tables 5a-5b or Table 5d. The binding domain (or binding portion) that binds to human CD3 may contain FRs that independently exhibit at least (approximately) 90%, at least (approximately) 91%, at least (approximately) 92%, at least (approximately) 93%, at least (approximately) 94%, at least (approximately) 95%, at least (approximately) 96%, at least (approximately) 97%, at least (approximately) 98%, at least (approximately) 99%, or 100% sequence identity with respect to the corresponding FRs listed in Table 5c. The binding domain (or binding moiety) that binds to human CD3 may contain a single-chain variable fragment (scFv) sequence exhibiting at least (approximately) 90%, at least (approximately) 91%, at least (approximately) 92%, at least (approximately) 93%, at least (approximately) 94%, at least (approximately) 95%, at least (approximately) 96%, at least (approximately) 97%, at least (approximately) 98%, at least (approximately) 99%, or 100% sequence identity with respect to the anti-CD3 scFv sequences listed in Table 5e. In the embodiments described above, the VH and / or VL domains may consist of an scFv, a diabody, a single-domain antibody, or a single-domain camelid antibody. The antibody may contain a binding domain (or binding moiety) that has a specific binding affinity to a tumor-specific marker or to an antigen (or target antigen) on a target cell.Tumor-specific markers, or antigens of target cells, include alpha-4 integrin, Ang2, B7-H3, B7-H6 (e.g., its natural ligand Nkp30 rather than an antibody fragment), CEACAM5, cMET, CTLA4, FOLR1, EpCAM (epithelial cell adhesion molecule), CCR5, CD19, HER2, and HER2. neu, HER3, HER4, HER1 (EGFR), PD-L1, PSMA, CEA, TROP-2, MUC1 (mucin), MUC-2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, MUC16, βhCG, Lewis-Y, CD20, CD33, CD38, CD30, CD56 (NCAM), CD133, ganglioside GD3, 9-O-acetyl-GD3, GM2, Globo H, fucosyl GM1, GD2, carbonic anhydrase IX, CD44v6, Nectin-4, Sonic Hedgehog (Shh), Wue-1, plasma cell antigen 1 (PC-1), melanoma chondroitin sulfate proteoglycan (MCSP), CCR8, prostate 6 transmembrane antigen (STEAP), mesothelin, A33 antigen, prostate stem cell antigen (PSCA), Ly-6, desmoglein 4, fetal acetylcholine receptor (fnAChR), CD25, cancer antigen 19-9 (CA19-9), cancer antigen 125 (CA-125), Müllerian inhibitor receptor type II (MISIIR), sialylated Tn antigen (sTN), fibroblast-activating antigen (FAP), endosialin (CD248), epidermal growth factor receptor variant II The group may be selected from the following: I (EGFRvIII), tumor-associated antigen L6 (TAL6), SAS, CD63, TAG72, Thomsen-Friedenreich antigen (TF-antigen), insulin-like growth factor I receptor (IGF-IR), Cora antigen, CD7, CD22, CD70 (e.g., its natural ligand rather than an antibody fragment, CD27), CD79a, CD79b, G250, MT-MMP, fibroblast-activating antigen (FAP), alpha-fetoprotein (AFP), VEGFR1, VEGFR2, DLK1, SP17, ROR1, EphA2, ENPP3, glucan 3 (GPC3), and TPBG / 5T4 (trophotrophic membrane glycoprotein).Tumor-specific markers, or antigens of target cells, may be selected from alpha-4 integrin, Ang2, CEACAM5, cMET, CTLA4, FOLR1, EpCAM (epithelial cell adhesion molecule), CD19, HER2, HER2 neu, HER3, HER4, HER1 (EGFR), PD-L1, PSMA, CEA, TROP-2, MUC1 (mucin), Lewis-Y, CD20, CD33, CD38, mesothelin, CD70 (e.g., its natural ligand rather than an antibody fragment, CD27), VEGFR1, VEGFR2, ROR1, EphA2, ENPP3, glypican 3 (GPC3), and TPBG / 5T4 (trophotrophic glycoprotein). Tumor-specific markers, or antigens of target cells, may be any one of those listed in the “Target” column of Table 6. A binding domain (or binding region) having binding affinity to a tumor-specific marker or to an antigen on a target cell may include VH and VL regions such that each VH and VL region can exhibit at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or 100% sequence identity with any one of the paired VL and VH sequences listed in the “VH sequence” and “VL sequence” columns of Table 6.Without limiting the scope, further exemplary tumor antigen targets include FGFR2, LIV1, TRK, RET, BCMA, CD71, CD166, SSTR2, cKIT, VISTA, GPNMB, DLL3, CD123, LAMP1, P-cadherin, Ephrin-A4, PTK7, NaPi2b, GCC, C4.4a, Mucin 17, FLT3, NKG2D ligand, SLAMF7, IL13a2R, CLL-1 / CLEC12A, CD66e, IL3Ra, CD5, ULBP1, B7H4, CSPG4, SDC1, IL1RAP, Survivin, CD138, CD74, TIM1, SLITRK6, CD37, CD142, AXL, ETBR, Cadherin 6, FGFR3, CA6, CanAg(Muc The group may be selected from one novel glycophorin, integrin alpha V, crypto 1 (TDGF1), CD352, and NOTCH3.

[0206] The biological activity of the BP embodiments described herein can be evaluated using the assays or measurement / determination parameters described herein, and sequences that retain at least (about) 40%, at least (about) 50%, at least (about) 55%, at least (about) 60%, at least (about) 70%, at least (about) 80%, at least (about) 90%, at least (about) 95%, or higher activity compared to the corresponding natural BP sequence will be considered suitable for inclusion in the compositions of this disclosure. Glucose-regulating peptides

[0207] Endocrine and obesity-related diseases or disorders are prevalent in most developed countries and represent a significant and ever-increasing healthcare burden in most developed countries. They encompass a wide variety of conditions affecting the body's organs, tissues, and circulatory system. Of particular concern are the following endocrine and obesity-related diseases or disorders: Diabetes is the most important of these and is one of the leading causes of death in the United States. Diabetes is divided into two main subclasses: Type 1, also known as juvenile diabetes mellitus or insulin-dependent diabetes mellitus (IDDM), and Type 2, also known as adult-onset diabetes mellitus or non-insulin-dependent diabetes mellitus (NIDDM). Type 1 diabetes mellitus is a form of autoimmune disease that completely or partially destroys the insulin-producing cells of the pancreas in such individuals, requiring the use of exogenous insulin throughout their lives. Even in well-controlled individuals, sudden complications can occur, some of which are fatal.

[0208] In patients with type 2 diabetes, the rise in blood glucose levels after meals does not adequately stimulate insulin production by the pancreas. In addition, peripheral tissues generally exhibit resistance to the effects of insulin, and such individuals often have higher plasma insulin levels (hyperinsulinemia) than normal as the body attempts to overcome this insulin resistance. In advanced stages of the disease, insulin secretion is also impaired.

[0209] Insulin resistance and hyperinsulinemia are also linked to two other metabolic disorders that pose significant health risks: impaired glucose tolerance and metabolic obesity. Impaired glucose tolerance is characterized by normal pre-meal glucose levels that tend to rise after meals (hyperglycemia). Individuals with these conditions are considered to be at higher risk of diabetes and coronary artery disease. Obesity is also a risk factor for a group of conditions called insulin resistance syndromes, or "X syndromes," which include hypertension, coronary artery disease (atherosclerosis), and lactic acidosis and related conditions. While the pathogenesis of obesity is thought to be multifactorial, the underlying problem is that in obese individuals, nutrient utilization and energy expenditure are only balanced when there is an excess of adipose tissue. Other related conditions or disorders include, but are not limited to, gestational diabetes, juvenile diabetes, obesity, hyperappetite, insufficient satiety, metabolic disorders, glucagonoma, retinal neurodegenerative processes, and the "honeymoon phase" of type 1 diabetes.

[0210] Dyslipidemia is common among diabetic patients and is generally characterized by elevated plasma triglycerides, low HDL (high-density lipoprotein) cholesterol, normal to high LDL (low-density lipoprotein) cholesterol levels, and elevated levels of small, high-density LDL particles in the blood. Dyslipidemia is a major contributing factor to the increased incidence of coronary events and mortality among diabetic patients.

[0211] Most metabolic processes in glucose homeostasis and insulin response are regulated by multiple peptides and hormones, and many such peptides and hormones, as well as their analogues, have proven useful in the treatment of metabolic diseases and disorders. Many of these peptides tend to be highly homologous to one another, even when they have opposing biological functions. Glucose-increasing peptides are exemplified by the peptide hormone glucagon, while glucose-decreasing peptides include exendin-4, glucagon-like peptide 1, and amylin. However, the use of therapeutic peptides and / or hormones, even when enhanced by the use of small molecule drugs, has yielded only limited success in the management of such diseases and disorders. Dose optimization, in particular, is crucial for drugs and biologics used to treat metabolic diseases, especially those with narrow therapeutic ranges. Hormones, and peptides involved in glucose homeostasis in general, often have narrow therapeutic ranges. Narrow therapeutic ranges, coupled with the fact that such hormones and peptides generally have short half-lives requiring frequent dosing to achieve clinical benefit, make the management of such patients difficult. Chemical modifications of therapeutic proteins, such as PEGylation, can alter their in vivo clearance rate and subsequent serum half-life, but require additional manufacturing steps and result in heterogeneous final products. Furthermore, unacceptable side effects from chronic administration have been reported. Alternatively, genetic modification by fusion of the Fc domain with a therapeutic protein or peptide increases the size of the therapeutic protein, resulting in decreased renal clearance and promoting recirculation from lysosomes via the FcRn receptor. Surprisingly, the Fc domain does not fold efficiently during recombinant expression and tends to form insoluble precipitates known as inclusion bodies. These inclusion bodies must be made soluble, and the functional protein restored, which is a time-consuming, inefficient, and costly process.

[0212] In some embodiments of the compositions of this disclosure, the bioactive peptides (BPs) may include peptides involved in glucose homeostasis, insulin resistance, and obesity (collectively, “glucose-regulating peptides”), and these compositions may be useful in the treatment of glucose, insulin, and obesity disorders, diseases, and related conditions. Glucose-regulating peptides may include any protein of biological, therapeutic, or prophylactic interest, or of biological, therapeutic, or prophylactic function, which may be useful in the prevention, treatment, mediation, or alleviation of diseases, disorders, or conditions of glucose homeostasis, insulin resistance, or obesity. Suitable glucose-regulating peptides that can be linked to a masking moiety (e.g., XTEN) may include any bioactive polypeptide that increases glucose-dependent secretion of insulin by pancreatic beta cells or enhances the action of insulin. Glucose-regulating peptides may also include any bioactive polypeptide that stimulates proinsulin gene transcription in pancreatic beta cells. Furthermore, glucose-regulating peptides may also include any bioactive polypeptide that delays gastric emptying time and reduces food intake. Glucose-regulating peptides may also include any bioactive polypeptide that inhibits glucagon release from alpha cells of the islets of Langerhans. Table 3a provides a non-limiting list of glucose-regulating peptide sequences that may be included in the compositions of the present disclosure. In some embodiments of the compositions disclosed herein, where the bioactive portion may be a bioactive peptide (BP), the BP may include peptide sequences exhibiting at least (about) 80% sequence identity to the amino acid sequences of the glucose-regulating peptides listed in Table 3a (e.g., at least (about) 81%, at least (about) 82%, at least (about) 83%, at least (about) 84%, at least (about) 85%, at least (about) 86%, at least (about) 87%, at least (about) 88%, at least (about) 89%, at least (about) 90%, at least (about) 91%, at least (about) 92%, at least (about) 93%, at least (about) 94%, at least (about) 95%, at least (about) 96%, at least (about) 97%, at least (about) 98%, at least (about) 99%, or 100% sequence identity). [Table 3a-1] [Table 3a-2]

[0213] "Adrenomedullin" or "ADM" refers to the human adrenomedullin peptide hormone, as well as species and sequence variants thereof that possess at least a portion of the bioactivity of mature ADM. ADM is produced from a 185-amino acid preprohormone by sequential enzymatic cleavage and amidation, resulting in a 52-amino acid bioactive peptide with a measurable plasma half-life of 22 minutes. The ADM-containing fusion protein of the present invention may be particularly used in diabetes mellitus for its stimulating effect on insulin secretion from islet cells for glucose regulation, or in subjects with persistent hypotension. The complete genomic substructure of human AM has been reported (Ishimitsu, et al., Biochem. Biophys. Res. Commun 203:631-639 (1994)), and analogs of the ADM peptide have been cloned, as described in U.S. Patent No. 6,320,022.

[0214] "Amylin" refers to a human peptide hormone called amylin, plumrintide, and a variant thereof having at least a portion of the biological activity of mature amylin, as described in U.S. Patent No. 5,234,906. Amylin is a 37-amino acid polypeptide hormone co-secreted with insulin by pancreatic beta cells in response to nutrient intake (Koda et al., Lancet 339:1179-1180. 1992), and has been reported to modulate several vital pathways of carbohydrate metabolism, including the incorporation of glucose into glycogen. The amylin-containing fusion protein of the present invention may be particularly used in diabetes and obesity to influence the rate of glucose appearance in the bloodstream by regulating gastric emptying and suppressing glucagon secretion and food intake. Thus, the fusion protein can complement the action of insulin, which regulates the rate of glucose elimination from the bloodstream and its uptake by peripheral tissues. Amylin analogs have been cloned as described in U.S. Patents No. 5,686,411 and No. 7,271,238. Amyrin mimetic compounds that retain biological activity can be created. For example, plumrintide has the sequence KCNTATCATNRLANFLVHSSNNFGPILPPTNVGSNTY (SEQ ID NO: 271), in which amino acids from the rat amyrin sequence are substituted for amino acids from the human amyrin sequence. In one embodiment, the present invention envisions a fusion protein containing an amyrin mimetic of the sequence KCNTATCATX1RLANFLVHSSNNFGX2ILX2X2TNVGSNTY (SEQ ID NO: 275) (wherein X1 is independently N or Q, and X2 is independently S, P, or G). In one embodiment, the amyrin mimetic incorporated into the composition of the present disclosure may have the sequence KCNTATCATNRLANFLVHSSNNFGGILGGTNVGSNTY (SEQ ID NO: 276). In another embodiment in which the amylin mimetic is used at the C-terminus of the composition, the mimetic may have the sequence KCNTATCATNRLANFLVHSSNNFGGILGGTNVGSNTY(NH2) (SEQ ID NO: 276).

[0215] Calcitonin (CT) refers to the human calcitonin protein, as well as its species and sequence variants, including salmon calcitonin ("sCT"), which possesses at least a portion of the bioactivity of mature CT. CT is a 32-amino acid peptide cleaved from the thyroid's larger prohormones, and while this peptide appears to function in the nervous and vascular systems, it has also been reported to be a potent hormone-mediated factor in the satiety reflex. The name CT derives from its secretion in response to induced hypercalcemia and its rapid calcium-lowering effect. CT is produced and secreted by neuroendocrine cells of the thyroid called C cells. CT affects osteoclasts, and inhibition of osteoclast function by CT leads to reduced bone resorption. In vitro effects of CT include rapid loss of wavy margins and reduced release of lysosomal enzymes. The primary functions of CT(1-32) are to cope with acute hypercalcemia in emergencies and / or to protect the skeleton during periods of "calcium stress," such as growth, pregnancy, and lactation. (See Becker, JCEM, 89(4): 1512-1525 (2004) and Sexton, Current Medicinal Chemistry 6: 1067-1093 (1999)). The calcitonin-containing fusion protein of the present invention may be used in particular for the treatment of osteoporosis and as a therapeutic agent for Paget's disease of bone. Synthetic calcitonin peptides have been produced as described in U.S. Patents 5,175,146 and 5,364,840.

[0216] "Calcitonin gene-related peptide" or "CGRP" means human CGRP peptide, as well as species and sequence variants thereof that have at least a portion of the biological activity of mature CGRP. Calcitonin gene-related peptide is a member of the calcitonin family of peptides and exists in humans in two forms: α-CGRP (a 37-amino acid peptide) and β-CGRP. CGRP has 43-46% sequence identity with human amyrin. The CGRP-containing fusion protein of the present invention may be particularly used to reduce diabetes-related morbidity, alleviate hyperglycemia and insulin deficiency, inhibit lymphocyte infiltration into pancreatic islets, and protect beta cells from autoimmune destruction. Methods for producing synthetic and recombinant CGRP are described in U.S. Patent No. 5,374,618.

[0217] "Cholecystokinin" or "CCK" refers to the human CCK peptide, as well as its species and sequence variants having at least a portion of the biological activity of mature CCK. CCK-58 is the mature sequence, but the CCK-33 amino acid sequence, first identified in humans, is the primary circulating form of this peptide. The CCK family also includes pentagastrin or CCK-5, which is an 8-amino acid in vivo C-terminal fragment ("CCK-8"); the C-terminal peptide CCK(29-33); and CCK-4, which is the C-terminal tetrapeptide CCK(30-33). CCK is a digestive system peptide hormone involved in stimulating the digestion of fats and proteins. The CCK-33 and CCK-8-containing fusion proteins of the present invention may be used in particular to reduce the increase in circulating glucose and promote the increase in circulating insulin after dietary digestion. Analogues of CCK-8 have been prepared as described in U.S. Patent No. 5,631,230.

[0218] "Excendin-3" refers to a glucose-regulating peptide isolated from Heloderma horridum, and its sequence variant having at least a portion of the biological activity of mature excendin-3. Excendin-3 amide is a specific excendin receptor antagonist that mediates the increase of pancreatic cAMP and the release of insulin and amylase. The excendin-3-containing fusion protein of the present invention may be particularly used in the treatment of diabetes and impaired insulin resistance. The sequence and methods for assaying it are described in U.S. Patent No. 5,4242,86.

[0219] "Excendin-4" refers to the glucose-regulating peptide found in the saliva of the Gila monster Heloderma suspectum, as well as its species and sequence variants, including the natural 39-amino acid sequence His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser and its homologous sequences and peptide mimes and variants; including natural sequences, e.g., from primates, and non-natural sequences, having at least a portion of the bioactivity of mature excendin-4. Exendin-4 is an incretin polypeptide hormone that reduces blood glucose, increases insulin secretion, slows gastric emptying, and improves satiety, resulting in a significant improvement in postprandial hyperglycemia. Exendin has some sequence similarity to members of the glucagon-like peptide family and is most identical to GLP-1 (Goke, et al., J. Biol. Chem., 268:19650-55 (1993)). Various homologous sequences may be functionally equivalent to natural exendin-4 and GLP-1. Conservation of GLP-1 sequences from different species is presented in Regulatory Peptides 2001 98 p.1-12. Table 3b shows sequences from a wide variety of species, while Table 3c lists synthetic GLP-1 analogs, all of which are intended for use in the compositions described herein. Exendin-4 binds to the GLP-1 receptor on insulin-secreting βTC1 cells and also stimulates somatostatin release and inhibits gastrin release in isolated stomachs (Goke, et al., J. Biol. Chem. 268:19650-55, 1993). As a GLP-1 mimic, exendin-4 exhibits a similar broad range of biological activity but has a longer half-life than GLP-1, with an average terminal phase half-life of 2.4 hours. Exenatide, marketed as Byetta, is a synthetic version of exendin-4.However, due to its short half-life, exenatide is currently administered twice daily, which limits its usefulness. The exendin-4-containing fusion protein of the present invention may be particularly useful in the treatment of diabetes and impaired insulin resistance.

[0220] "Fibroblast growth factor 21," or "FGF-21," refers to the human protein encoded by the FGF21 gene, or species and sequence variants thereof that possess at least a portion of the biological activity of mature FGF21. FGF-21 stimulates glucose uptake in adipocytes but not in other cell types, and its effect is additive to insulin activity. Injection of FGF-21 in ob / ob mice results in increased Glut1 in adipose tissue. FGF21 also prevents diet-induced obesity in transgenic mice when overexpressed and lowers blood glucose and triglyceride levels when administered to diabetic rodents (Kharitonenkov A, et al., (2005). "FGF-21 as a novel metabolic regulator". J. Clin. Invest. 115: 1627-35). The FGF-21-containing fusion protein of the present invention may be particularly used in the treatment of diabetes, including by producing increased energy expenditure, fat utilization rate, and lipid excretion. FGF-21 has been cloned as disclosed in U.S. Patent No. 6,716,626.

[0221] "FGF-19" or "fibroblast growth factor 19" refers to the human protein encoded by the FGF19 gene, or species and sequence variants thereof that have at least a portion of the biological activity of mature FGF-19. FGF-19 is a protein member of the fibroblast growth factor (FGF) family. Members of the FGF family have a wide range of mitogen and cell survival activities and are involved in various biological processes. FGF-19 increases the hepatic expression of leptin receptors, increases metabolic rate, stimulates glucose uptake in adipocytes, and leads to weight loss in obese mouse models (Fu, L, et al.). The FGF-19-containing fusion protein of the present invention may be particularly used to increase metabolic rate and in the improvement of diet-induced and leptin-deficient diabetes. FGF-19 has been cloned and expressed as described in U.S. Patent Application Publication No. 20020042367.

[0222] "Gastrin" refers to human gastrin peptide, a shortened version thereof having at least a portion of the bioactivity of mature gastrin, and species and sequence variants. Gastrin is a linear peptide hormone produced by G cells in the duodenum and in the pyloric sinuses of the stomach and secreted into the bloodstream. Gastrin is found primarily in three forms: gastrin-34 ("major gastrin"), gastrin-17 ("minor gastrin"), and gastrin-14 ("mini-gastrin"). Gastrin shares sequence homology with CCK. The gastrin-containing fusion proteins of the present invention may be used in particular for glucose regulation in the treatment of obesity and diabetes. Gastrin is synthesized as described in U.S. Patent No. 5,843,446.

[0223] "Ghrelin" refers to the human hormone that induces satiety, or its species and sequence variants, including naturally occurring, processed 27 or 28 amino acid sequences and homologous sequences. Ghrelin is primarily produced by P / D1 cells covering the fundus of the human stomach and by pancreatic epsilon cells that stimulate hunger, and is considered the counterpart hormone to leptin. Ghrelin levels increase before meals and decrease after meals, which can lead to increased food intake and, through its effects exerted at the hypothalamic level, can increase fat mass. Ghrelin also stimulates the release of growth hormone. Ghrelin is acylated by n-octanoic acid at a serine residue, and this acylation is crucial for binding to the GHS1a receptor and for ghrelin's GH-releasing ability. The ghrelin-containing fusion protein of the present invention can be used in particular as an agonist, for example, to selectively stimulate GI duct motility in gastrointestinal motility disorders, to accelerate gastric emptying, or to stimulate the release of growth hormone. Ghrelin analogs with sequence substitutions, or truncated variants, such as those described in U.S. Patent No. 7,385,026, may be particularly used as fusion partners for XTEN for use as antagonists for improving glucose homeostasis, treating insulin resistance, and treating obesity. The isolation and characterization of ghrelin have been reported (Kojima M, et al., Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature. 1999;402(6762):656-660.), and synthetic analogs have been prepared by peptide synthesis, as described in U.S. Patent No. 6,967,237.

[0224] "Glucagon" means human glucagon glucose-regulating peptide, or its species and sequence variants, including the natural 29-amino acid sequence and homologous sequence; natural sequence variants, e.g., from primates, and non-natural sequence variants, having at least a portion of the bioactivity of mature glucagon. The term "glucagon," as used herein, also includes peptide mimes of glucagon. Natural glucagon is produced by the pancreas and released when blood glucose levels begin to drop too low, causing the liver to convert stored glycogen into glucose and release it into the bloodstream. The action of glucagon is the opposite of insulin, which signals the body's cells to take up glucose from the blood; however, glucagon also stimulates the release of insulin, so that newly available glucose in the bloodstream can be taken up and used by insulin-dependent tissues. The glucagon-containing fusion proteins of the present invention may be used in particular to raise blood glucose levels in individuals with existing hepatic glycogen stores and to maintain glucose homeostasis in diabetes. Glucagon has been cloned as disclosed in U.S. Patent No. 4,826,763.

[0225] "GLP-1" refers to human glucagon-like peptide-1 and its sequence variants having at least a portion of the biological activity of mature GLP-1. The term "GLP-1" includes human GLP-1(1-37), GLP-1(7-37), and GLP-1(7-36) amides. GLP-1 stimulates insulin secretion, but only during periods of hyperglycemia. The safety of GLP-1 compared to insulin is enhanced by this property and by the observation that the amount of insulin secreted is proportional to the degree of hyperglycemia. The biological half-life of GLP-1(7-37)OH is only 3-5 minutes (U.S. Patent No. 5,118,666). The GLP-1-containing fusion protein of the present invention may be used in particular for glucose regulation in the treatment of diabetes and impaired insulin resistance. As described in U.S. Patent No. 5,118,666, GLP-1 has been cloned and derivatives have been prepared. Tables 3b-3c show non-limiting examples of glucagon-like peptide sequences and their synthetic analogs from a wide variety of species. In some embodiments of the compositions disclosed herein, where the bioactive portion may be a bioactive peptide (BP), the BP may comprise a peptide sequence exhibiting at least approximately 80% sequence identity (e.g., at least approximately 81%, at least approximately 82%, at least approximately 83%, at least approximately 84%, at least approximately 85%, at least approximately 86%, at least approximately 87%, at least approximately 88%, at least approximately 89%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or 100% sequence identity) to the amino acid sequence of a glucagon-like peptide (natural or synthetic analog) listed in Tables 3b-3c. [Table 3b-1] [Table 3b-2] [Table 3c-1] [Table 3c-2] [Table 3c-3]

[0226] The natural GLP sequence can be described by several sequence motifs presented below. The letters in parentheses represent acceptable amino acids at each sequence position: [HVY][AGISTV][DEHQ][AG][ILMPSTV][FLY][DINST][ADEKNST][ADENSTV][LMVY][ANRSTY][EHIKNQRST][AHILMQVY][LMRT][ADEGKQS][ADEGKNQSY][AEIKLMQR][AKQRSVY][AILMQSTV][GKQR][DEKLQR][FHLVWY][ILV][ADEGHIKNQRST][ADEGNRSTW][GILVW][AIKLMQSV][ADGIKNQRST][GKRSY]. In addition, synthetic analogs of GLP-1 may be useful as fusion partners for masking moieties (e.g., XTEN) to create fusion compositions with useful bioactivity in treating glucose-related disorders. Further sequences homologous to exendin-4 or GLP-1 can be found using standard homology search techniques.

[0227] "GLP-2" refers to human glucagon-like peptide-2 and its sequence variants that possess at least a portion of the biological activity of mature GLP-2. More specifically, GLP-2 is a 33-amino acid peptide that is co-secreted together with GLP-1 from enteroendocrine cells in the small and large intestines.

[0228] "IGF-1" or "insulin-like growth factor 1" refers to the human IGF-1 protein, as well as species and sequence variants thereof that possess at least a portion of the biological activity of mature IGF-1. Formerly known as somatomedin C, IGF-1 is a polypeptide anabolic hormone that is similar to insulin in molecular structure and modulates the action of growth hormone. IGF-1 consists of 70 amino acids and is primarily produced by the liver as an endocrine hormone, and is also produced in target tissues in a paracrine / autocrine manner. The IGF-1-containing fusion protein of the present invention may be particularly used for glucose regulation in the treatment of diabetes and impaired insulin resistance. IGF-1 has been cloned and expressed in E. coli and yeast, as described in U.S. Patent Application Publication No. 5,324,639.

[0229] "IGF-2" or "insulin-like growth factor 2" refers to the human IGF-2 protein, as well as species and sequence variants of that protein that possess at least a portion of the biological activity of mature IGF-2. IGF-2 is a polypeptide protein hormone that is similar to insulin in terms of molecular structure and primarily plays a role as a growth-promoting hormone during pregnancy. IGF-2 has been cloned, as described by Bell GI, et al. The isolation of the human insulin-like growth factor gene, insulin-like growth factor II, and the insulin gene is ongoing. Proc Natl Acad Sci US A. 1985. 82(19):6450-4.

[0230] "INGAP," or "pancreatic islet neogenesis-associated protein," or "pancreatic beta-cell growth factor" refers to the human INGAP peptide, as well as species and sequence variants thereof that possess at least a portion of the biological activity of mature INGAP. INGAP can initiate pancreatic ductal cell proliferation, which is a prerequisite for islet neogenesis. The INGAP-containing fusion protein of the present invention may be particularly used in the treatment and prevention of diabetes mellitus and impaired insulin resistance. INGAP has been cloned and expressed as described in R Rafaeloff R, et al., Cloning and sequencing of the pancreatic islet neogenesis associated protein (INGAP) gene and its expression in islet neogenesis in hamsters. J Clin Invest. 1997. 99(9): 2100-2109.

[0231] "Intermedin" or "AFP-6" refers to human intermedin peptides, as well as species and sequence variants thereof that possess at least a portion of the biological activity of mature intermedin. Intermedin is a ligand for calcitonin receptor-like receptors. Intermedin treatment results in blood pressure reduction and suppression of gastric emptying activity in both normal and hypertensive subjects, and is involved in glucose homeostasis. The intermedin-containing fusion proteins of the present invention may be particularly used in the treatment of diabetes, insulin resistance disorders, and obesity. Intermedin peptides and variants have been cloned as described in U.S. Patent No. 6,965,013.

[0232] "Leptin" means leptin naturally occurring from any species, as well as its bioactive D isoform, or fragments and sequence variants. Leptin plays a vital role in regulating energy intake and energy expenditure, including appetite and metabolism. The leptin-containing fusion protein of the present invention may be used in particular for glucose regulation in the treatment of diabetes, in the treatment of impaired insulin resistance, and in the treatment of obesity. Leptin is a polypeptide product of the ob gene, as described in International Patent Publication No. WO96 / 05309. Leptin has been cloned as described in U.S. Patent No. 7,112,659, and leptin analogs and fragments have been cloned as described in U.S. Patents No. 5,521,283, U.S. Patent No. 5,532,336, PCT / US96 / 22308 and PCT / US96 / 01471.

[0233] "Neuromedin" refers to the neuromedin family of peptides, including neuromedin U and S peptides, as well as their sequence variants. The naturally active human neuromedin U peptide hormone is neuromedin-U25, particularly its amide form. These processed active peptide hormones, as well as their analogs, derivatives, and fragments, are of particular interest. The neuromedin U family includes various truncated or spliced ​​variants, e.g., FLFHYSKTQKLGKSNVVEELQSPFASQSRGYFLFRPRN (SEQ ID NO: 409). A typical example of the neuromedin S family is human neuromedin S having the sequence ILQRGSGTAAVDFTKKDHTATWGRPFFLFRPRN (SEQ ID NO: 267), particularly its amide form. The neuromedin fusion proteins of the present invention may be used in particular to treat obesity, treat diabetes, reduce food intake, and in other related conditions and disorders described herein. Neuromedin modules combined with amylin family peptides, exendin peptide family, or GLP I peptide family modules are of particular interest.

[0234] "Oxintmodulin," or "OXM," refers to human oxintmodulin, as well as species and sequence variants thereof having at least a portion of the biological activity of mature OXM. OXM is a 37-amino acid peptide produced in the colon, containing an 8-amino acid carboxy-terminal extension following a 29-amino acid sequence of glucagon. OXM is known to suppress appetite. The OXM-containing fusion protein of the present invention may be used in particular for glucose regulation in the treatment of diabetes, in the treatment of impaired insulin resistance, and in the treatment of obesity, and may be used as a treatment for weight loss.

[0235] "PYY" refers to the human peptide YY polypeptide, as well as species and sequence variants thereof that possess at least a portion of the biological activity of mature PYY. PYY is a human full-length, 36-amino acid peptide, PYY 1-36 and PYY 3-36 It contains both, and these have a PP folded structure motif. PYY inhibits gastric motility and increases water and electrolyte absorption in the colon. PYY can also suppress pancreatic juice secretion. The PPY-containing fusion proteins of the present invention may be used in particular for glucose regulation in the treatment of diabetes, in the treatment of impaired insulin resistance, and in the treatment of obesity. Analogues of PYY have been prepared as described in U.S. Patents 5,604,203, 5,574,010 and 7,166,575.

[0236] "Urocortin" refers to the human urocortin peptide hormone and its sequence variants having at least a portion of the bioactivity of mature urocortin. There are three human urocortins: Ucn-1, Ucn-2, and Ucn-3. Further urocortins and analogs are described in U.S. Patent No. 6,214,797. Urocortins Ucn-2 and Ucn-3 have food intake suppression, antihypertensive, cardioprotective, and inotropic properties. Ucn-2 and Ucn-3 have the ability to suppress chronic HPA activation after stress stimuli such as diet / fasting, are specific to the CRF type 2 receptor, and do not activate CRF-R1, which mediates ACTH release. Therapeutic agents containing urocortin, e.g., Ucn-2 or Ucn-3, may be useful for vasodilation and therefore may be useful for cardiovascular applications, e.g., chronic heart failure. The urocortin-containing fusion protein of the present invention may also be particularly used in the treatment or prevention of conditions associated with ACTH release stimulation, hypertension due to vasodilatory effects, inflammation mediated by means other than elevated ACTH, hyperthermia, loss of appetite, congestive heart failure, stress, anxiety, and psoriasis. The urocortin-containing fusion protein can also be combined with natriuretic peptide modules, the amylin family, and the exendin family, or the GLP1 family module to provide enhanced cardiovascular benefits, such as in the treatment of CHF, by resulting in beneficial vasodilatory effects. Metabolic diseases and cardiovascular proteins

[0237] Metabolic and cardiovascular diseases represent a significant healthcare burden in most developed countries, with cardiovascular disease remaining the leading cause of death and disability in the United States and most European countries. Metabolic diseases and disorders encompass a wide variety of conditions affecting the body's organs, tissues, and circulatory system. Diabetes is the most important of these, and is one of the leading causes of death in the United States, as it results in both lesions and metabolic dysfunction in the vascular system, central nervous system, major organs, and peripheral tissues. Insulin resistance and hyperinsulinemia are also linked to two other metabolic disorders that pose significant health risks: impaired glucose tolerance and metabolic obesity. Impaired glucose tolerance is characterized by normal pre-meal glucose levels that tend to rise after meals (hyperglycemia). Individuals with these conditions are considered to be at higher risk of diabetes and coronary artery disease. Obesity is also a risk factor for a group of conditions known as insulin resistance syndrome, or "X syndrome," including hypertension, coronary artery disease (arteriosclerosis), lactic acidosis, and related conditions. While the pathogenesis of obesity is thought to be multifactorial, the fundamental problem is that in obese individuals, nutrient utilization and energy expenditure only become balanced when there is an excess of adipose tissue.

[0238] Dyslipidemia is more common in patients with diabetes and those with cardiovascular disease and is generally characterized by parameters such as elevated plasma triglycerides, low HDL (high-density lipoprotein) cholesterol, normal to high LDL (low-density lipoprotein) cholesterol levels, and elevated levels of small, high-density LDL particles in the blood. Dyslipidemia and hypertension are major contributors to the increased incidence of coronary events, renal disease, and mortality in individuals with metabolic diseases such as diabetes and cardiovascular disease.

[0239] Cardiovascular diseases, among many others, can manifest throughout the body through numerous disorders, symptoms, and changes in clinical parameters affecting the heart, vascular system, and organ systems, including aneurysms, angina pectoris, atherosclerosis, cerebrovascular disorders (stroke), cerebrovascular diseases, congestive heart failure, coronary artery disease, myocardial infarction, decreased cardiac output and peripheral vascular disease, hypertension, hypotension, and blood markers (e.g., C-reactive protein, BNP, and enzymes such as CPK, LDH, SGPT, SGOT).

[0240] Most metabolic processes and many cardiovascular parameters are regulated by multiple peptides and hormones ("metabolic proteins"), and many such peptides and hormones, as well as their analogues, have proven useful in treating such diseases and disorders. However, the use of therapeutic peptides and / or hormones, even when enhanced by the use of small molecule drugs, has yielded only limited success in managing such diseases and disorders. Dose optimization, in particular, is crucial for drugs and biologics used to treat metabolic diseases, especially those with narrow therapeutic ranges. Hormones, and peptides involved in glucose homeostasis in general, often have narrow therapeutic ranges. A narrow therapeutic range, coupled with the fact that such hormones and peptides generally have short half-lives requiring frequent dosing to achieve clinical benefit, makes the management of such patients difficult. Therefore, there is still a need for therapeutic agents with broader therapeutic ranges and increased efficacy and safety in the treatment of metabolic diseases.

[0241] In some embodiments of the compositions disclosed herein, the bioactive peptides (BPs) may include bioactive metabolite proteins, and the compositions may be useful for the treatment of metabolic and cardiovascular diseases and disorders. The metabolite proteins may include any proteins of biological, therapeutic or preventive interest, or of biological, therapeutic or preventive function, that are useful for the prevention, treatment, mediation or alleviation of metabolic or cardiovascular diseases, disorders or conditions. Table 3d provides a non-limiting list of such sequences of metabolite BPs that may be included in the compositions of the present invention (e.g., therapeutic agents). In some embodiments of the compositions disclosed herein, where the bioactive portion is a bioactive peptide (BP), the BP may comprise a peptide sequence exhibiting at least approximately 80% sequence identity to the amino acid sequences of the metabolite proteins listed in Table 3d (e.g., at least approximately 81%, at least approximately 82%, at least approximately 83%, at least approximately 84%, at least approximately 85%, at least approximately 86%, at least approximately 87%, at least approximately 88%, at least approximately 89%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or 100% sequence identity). [Table 3d-1] [Table 3d-2]

[0242] "Anti-CD3" refers to monoclonal antibodies, species, and sequence variants against the T cell surface protein CD3, including OKT3 (also known as muromonab) and the humanized anti-CD3 monoclonal antibody (hOKT31(Ala-Ala)) (KC Herold et al., New England Journal of Medicine 346:1692-1698. 2002), as well as fragments thereof. Anti-CD3 prevents T cell activation and proliferation by binding to the T cell receptor complex present on all differentiated T cells. The anti-CD3-containing fusion proteins of the present invention may be used in particular to delay the onset of type 1 diabetes, including the use of anti-CD3 as a therapeutic effector as well as the use of anti-CD3 as a targeted portion for a second therapeutic BP in the compositions of this disclosure. The sequences of the variable region of anti-CD3 and the creation of anti-CD3 are described in U.S. Patents No. 5,885,573 and No. 6,491,916.

[0243] "IL-1ra" refers to the human IL-1 receptor antagonist protein, as well as its species and sequence variants that possess at least a portion of the biological activity of mature IL-1ra, including the sequence variant anakinra (Kineret®). Human IL-1ra is a mature glycoprotein consisting of 152 amino acid residues. The inhibitory effect of IL-1ra arises from its binding to the type I IL-1 receptor. The protein has a native molecular weight of 25 kDa, and the molecule exhibits limited sequence homology with IL-1α (19%) and IL-1β (26%). Anakinra is an unglycosylated recombinant human IL-1ra and differs from endogenous human IL-1ra in that it has a methionine tag at its N-terminus. The commercialized version of anakinra is marketed as Kineret®. It binds to the IL-1 receptor with the same avidity as native IL-1ra and IL-1b, but does not result in receptor activation (signal transduction). This is due to the fact that IL-1α and IL-1β have two receptor-binding motifs, while IL-1ra has only one. Anakinra has 153 amino acids and a size of 17.3 kD, with a reported half-life of approximately 4-6 hours.

[0244] Increased IL-1 production has been reported in patients with various viral, bacterial, fungal, and parasitic infections; intravascular coagulation; high-dose IL-2 therapy; solid tumors; leukemia; Alzheimer's disease; HIV-1 infection; autoimmune disorders; trauma (surgery); hemodialysis; ischemic disease (myocardial infarction); non-infectious hepatitis; asthma; UV irradiation; closed head trauma; pancreatitis; peritonitis; graft-versus-host disease; transplant rejection; and healthy subjects after strenuous exercise. There is a correlation between increased IL-1b production in patients with Alzheimer's disease and the possible role of IL-1 in the release of amyloid precursor protein. IL-1 is associated with diseases such as type 2 diabetes, obesity, hyperglycemia, hyperinsulinemia, type 1 diabetes, insulin resistance, retinal neurodegenerative processes, conditions and states characterized by insulin resistance, acute myocardial infarction (AMI), acute coronary syndrome (ACS), atherosclerosis, chronic inflammatory disorders, rheumatoid arthritis, intervertebral disc degeneration, sarcoidosis, Crohn's disease, ulcerative colitis, gestational diabetes, increased appetite, insufficient satiety, metabolic disorders, glucagonoma, airway secretion disorders, osteoporosis, central nervous system diseases, restenosis, neurodegenerative diseases, renal failure, congestive heart failure, nephrotic syndrome, and liver cirrhosis. It is also associated with conditions such as pulmonary edema, hypertension, disorders requiring reduced food intake, irritable bowel syndrome, myocardial infarction, stroke, postoperative catabolic changes, hibernating myocardium, diabetic cardiomyopathy, urinary sodium excretion deficiency, excessive urinary potassium concentration, conditions or disorders accompanied by toxic polycystic cysts, polycystic ovary syndrome, respiratory distress, chronic skin ulcers, nephropathy, left ventricular systolic dysfunction, gastrointestinal diarrhea, postoperative dumping syndrome, irritable bowel syndrome, severe polyneuropathy (CIPN), systemic inflammatory response syndrome (SIRS), dyslipidemia, post-ischemia-reperfusion injury, and coronary heart disease risk factor (CHDRF) syndrome. The IL-1ra-containing fusion protein of the present invention may be particularly used in the treatment of any of the aforementioned diseases and disorders. IL-1ra has been cloned as described in U.S. Patents No. 5,075,222 and No. 6,858,409.

[0245] "Natriuretic peptides" refers to atrial natriuretic peptides (ANP), brain natriuretic peptides (BNP or type B natriuretic peptide), and type C natriuretic peptides (CNP); and their human and non-human species and sequence variants having at least a portion of the biological activity of the mature corresponding natriuretic peptides. Alpha atrial natriuretic peptide (aANP) or (ANP), brain natriuretic peptide (BNP), and type C natriuretic peptide (CNP) are homologous polypeptide hormones involved in the regulation of fluid and electrolyte homeostasis. Sequences of useful forms of natriuretic peptides are disclosed in U.S. Patent Application Publication No. 20010027181. Examples of ANPs include human ANP (Kangawa et al., BBRC 118:131 (1984)), or ANPs from various species, including pig and rat ANPs (Kangawa et al., BBRC 121:585 (1984)). Sequence analysis reveals that preproBNP consists of 134 residues and is cleaved into 108-amino acid proBNP. Cleavage of the 32-amino acid sequence from the C-terminus of proBNP yields the circulating physiologically active form, human BNP (77-108). The 32-amino acid human BNP is involved in the formation of disulfide bonds (Sudoh et al., BBRC 159:1420 (1989), and U.S. Patents 5,114,923, 5,674,710, 5,674,710 and 5,948,761). Compositions containing one or more natriuretic functions may be useful in the treatment of hypertension, diuretic induction, natriuretic induction, vasoconduct dilation or relaxation, natriuretic peptide receptor (e.g., NPR-A) binding, inhibition of 108-apida secretion from the kidneys, inhibition of aldosterone secretion from the adrenal glands, treatment of cardiovascular diseases and disorders, reduction, cessation or reversal of cardiac remodeling after cardiac events or as a result of congestive heart failure, treatment of renal diseases and disorders; treatment or prevention of ischemic stroke; and treatment of asthma.

[0246] "FGF-2" or "heparin-binding growth factor 2" refers to the human FGF-2 protein, as well as species and sequence variants of its mature counterpart that possess at least a portion of its biological activity. FGF-2 has been shown to stimulate the proliferation of neural stem cells differentiated into striatal-like neurons and protect striatal neurons in a toxin-induced model of Huntington's disease, and may also be useful in the treatment of cardiac reperfusion injury, potentially promoting endothelial cell growth, anti-angiogenic and tumor-suppressing properties, wound healing, and bone fracture healing. FGF-2 has been cloned as described in Burgess, WH and Maciag, T., Ann. Rev. Biochem., 58:575-606 (1989); Coulier, F., et al., 1994, Prog. Growth Factor Res. 5:1; and PCT Publication WO87 / 01728.

[0247] "TNF receptor" refers to the human receptor for TNF, as well as its species and sequence variants that possess at least a portion of the bioreceptor activity of mature TNFR. The p75 TNF receptor molecule is the extracellular domain of the p75 TNF receptor, which is from a family of structurally homologous...

Claims

1. A method for evaluating the likelihood that a subject with a disease or disorder is responsive to a therapeutic agent that can be activated by a mammalian protease expressed in the subject, a. A step of determining the presence or amount of proteolytic peptide products produced by the action of the mammalian protease in a biological sample from the subject suffering from the disease or disorder, wherein the peptide is i. Consists of at least five or six consecutive amino acid residues as shown in the sequence in column V of Table A, or ii. Containing at least five or six consecutive amino acids as shown in the sequence in column IV of Table A, or iii. A step comprising at least five or six consecutive amino acids as shown in the sequence in column VI of Table A; and b. The step of designating the subject as likely to respond to the therapeutic agent if the peptide (i), (ii), or (iii) is present and / or if its amount exceeds a threshold. Methods that include...

2. The method according to claim 1, wherein the therapeutic agent comprises a peptide substrate having an amino acid sequence that is sensitive to cleavage by the mammalian protease in an easily cleavable bond.

3. The method according to claim 2, wherein the polypeptide of (i), (ii), or (iii) comprises a portion containing at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten consecutive amino acid residues of the sequence of the peptide substrate, either at the N-terminal or C-terminal end of the easily cleavable bond.

4. The method according to claim 1 or 2, wherein the sequence of the peptide substrate is sensitive to cleavage by the mammalian protease at an easily cleavable bond, the polypeptide of (i), (ii), or (iii) is a cleavage product of a reporter polypeptide containing a substrate sequence sensitive to cleavage by the same mammalian protease at an easily cleavable bond, and the reporter polypeptide contains the sequence described in column II or III of Table A.

5. The method according to claim 1 or 2, wherein the sequence of the peptide substrate is sensitive to cleavage by the mammalian protease at an easily cleavable bond, and the polypeptide of (i), (ii), or (iii) is a cleavage product of a human protein comprising a portion containing at least five or six consecutive amino acid residues of the peptide substrate sequence including the easily cleavable bond.

6. The method according to any one of claims 1 to 5, wherein the polypeptide in (i) comprises at least seven, at least eight, at least nine, or at least ten consecutive amino acid residues as shown in the sequence in column V of Table A.

7. The method according to any one of claims 1 to 6, wherein the polypeptide of (ii) comprises at least seven, at least eight, at least nine, or at least ten consecutive amino acids as shown in the sequence in column IV of Table A.

8. The method according to any one of claims 1 to 7, wherein the polypeptide of (iii) comprises at least seven, at least eight, at least nine, or at least ten consecutive amino acids as shown in the sequence in column VI of Table A.

9. The method according to any one of claims 1 to 8, wherein step (a) comprises determining the presence or quantity of any two of (i) to (iii).

10. The method according to any one of claims 1 to 9, wherein the threshold is zero or a nominal value.

11. The method according to any one of claims 1 to 10, wherein the biological sample comprises a serum or plasma sample.

12. The method according to any one of claims 1 to 11, wherein the mammalian protease is a serine protease, a cysteine ​​protease, an aspartate protease, a threonine protease, or a metalloproteinase.

13. The mammalian proteases are: disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), disintegrin and metalloproteinase domain-containing protein 12 (ADAM12), disintegrin and metalloproteinase domain-containing protein 15 (ADAM15), disintegrin and metalloproteinase domain-containing protein 17 (ADAM17), and disintegrin and metalloproteinase domain-containing protein 9 (ADAM9). ), disintegrin and metalloproteinase having thrombospondin motif 5 (ADAMTS5), cathepsin B, cathepsin D, cathepsin E, cathepsin K, cathepsin L, cathepsin S, fibroblast-activating protein alpha, hepsin, kallikrein-2, kallikrein-4, kallikrein-3, prostate-specific antigen (PSA), kallikrein-13, regmine, matrix metallopeptidase 1 (MMP-1), matrix metallopeptidase 10 (MMP-10), matrix metal Matrix metallopeptidase 11 (MMP-11), Matrix metallopeptidase 12 (MMP-12), Matrix metallopeptidase 13 (MMP-13), Matrix metallopeptidase 14 (MMP-14), Matrix metallopeptidase 16 (MMP-16), Matrix metallopeptidase 2 (MMP-2), Matrix metallopeptidase 3 (MMP-3), Matrix metallopeptidase 7 (MMP-7), Matrix metallopeptidase 8 (MMP-8), Matrix metallopeptidase The method according to claim 12, wherein a selection is made from the group consisting of -ase 9 (MMP-9), matrix metallopeptidase 4 (MMP-4), matrix metallopeptidase 5 (MMP-5), matrix metallopeptidase 6 (MMP-6), matrix metallopeptidase 15 (MMP-15), neutrophil elastase, protease-activated receptor 2 (PAR2), plasmin, prostasin, PSMA-FOLH1, membrane-bound serine protease 1 (MT-SP1), matryptase, and u-plasminogen.

14. The method according to claim 12, wherein the mammalian protease is selected from the group consisting of matrix metallopeptidase 1 (MMP1), matrix metallopeptidase 2 (MMP2), matrix metallopeptidase 7 (MMP7), matrix metallopeptidase 9 (MMP9), matrix metallopeptidase 11 (MMP11), matrix metallopeptidase 14 (MMP14), urokinase-type plasminogen activator (uPA), regmine, and matryptase.

15. The method according to any one of claims 1 to 14, wherein the mammalian protease is preferentially expressed or activated in the target tissue or cell.

16. The method according to claim 15, wherein the target tissue or cells are a tumor.

17. The method according to claim 15 or 16, wherein the target tissue or cell produces the mammalian protease or co-localizes with the mammalian protease.

18. The method according to any one of claims 15 to 17, wherein the target tissue or cell contains a reporter polypeptide in or on it, or is associated with a reporter polypeptide in its vicinity.

19. The reporter polypeptide is a coagulation factor, complement component, tubulin, immunoglobulin, apolipoprotein, serum amyloid, insulin, growth factor, fibrinogen, PDZ domain protein, LIM domain protein, c-reactive protein, serum albumin, versican, collagen, elastin, keratin, kininogen-1, alpha-2-antiplasmin, clusterin, biglycan, alpha-1-antitrypsin, transthyretin, alpha-1-antichymotrypsin, glucagon, hepcidin, thymosin beta-4, haptoglobin, hemoglobin subunit alpha, caveolae-associated protein 2, alpha-2-HS glycoprotein, chromogranin-A, vitronectin, hemopexin, epididymal sperm-binding protein, secretogranin-2, angiotensinogen, Transgerin-2, pancreatic prohormone, neurosecretory protein VGF, ceruloplasmin, PDZ and LIM domain protein 1, multimelin-1, inter-alpha-trypsin inhibitor heavy chain H2, N-acetylmuramoyl-L-alanine amidase, histone H1.4, adhesion G protein-coupled receptor G6, mannan-binding lectin serine protease 2, prothrombin, malignant brain tumor deletion 1 protein, desmoglein-3, calcin-tenin-1, alpha-2-macroglobulin, myosin-9, sodium / potassium transport ATPase subunit gamma, oncoprotein-inducible transcript 3 protein, cerglycine, histidine-rich glycoprotein, inter-alpha-trypsin inhibitor heavy chain H5, integrin alpha-IIb, membrane-bound progesterone receptor component 1, histone H1.2, rh GDP dissociation inhibitor 2, zinc-alpha-2-glycoprotein, talin-1, secretogranin-1, neutrophil defensin 3, cytochrome P450 2E1, gastric suppressor polypeptide, transcription initiation factor TFIID subunit 1, membrane endogenous protein 2B, pigment epithelium-derived factor, voltage-gated N-type calcium channel subunit alpha-1B, rasThe method according to claim 4 or 18, wherein the polypeptide is selected from the group consisting of GTPase-activating protein nGAP, type I cytoskeleton 17, sulfhydryl oxidase 1, homeobox protein Hox-B2, transcription factor SOX-10, E3 ubiquitin-protein ligase SIAH2, decorin, acidic, cysteine-rich secretory protein (SPARC), laminin gamma 1 chain, vimentin, and nidogen-1 (NID1).

20. The reporter polypeptide is versican, type II collagen alpha-1 chain, kininogen-1, complement C4-A, complement C4-B, complement C3, alpha-2-antiplasmin, clusterin, biglycan, elastin, fibrinogen alpha chain, alpha-1-antitrypsin, fibrinogen beta chain, type III collagen alpha-1 chain, serum amyloid A-1 protein, transthyretin, apolipoprotein A-I, apolipoprotein A-I isoform 1, alpha-1-antichymotrypsin, glucagon, hepcidin , serum amyloid A-2 protein, thymosin beta-4, haptoglobin, hemoglobin subunit alpha, caveolae-associated protein 2, alpha-2-HS glycoprotein, chromogranin-A, vitronectin, hemopexin, epididymal sperm-binding protein, zyxin, apolipoprotein C-III, secretogranin-2, angiotensinogen, c-reactive protein, serum albumin, transgerin-2, pancreatic prohormones, neurosecretory protein VGF, ceruloplasmin, PDZ and LIM domain protein 1, Iubrin alpha-4A chain, multimelin-1, inter-alpha-trypsin inhibitor heavy chain H2, apolipoprotein C-I, fibrinogen gamma chain, N-acetylmuramoyl-L-alanine amidase, immunoglobulin lambda variable 3-21, histone H1.4, adhesion G protein-coupled receptor G6, immunoglobulin lambda variable 3-25, immunoglobulin lambda variable 1-51, immunoglobulin lambda variable 1-36, mannan-binding lectin serine protease 2, immunoglobulin kappa variable 3-20, immunoglobulin kappa variable 2-30, insulin Phosphorus-like growth factor II, apolipoprotein A-II, highly non-functional immunoglobulin kappa variable 2D-24, prothrombin, coagulation factor IX, apolipoprotein L1, malignant brain tumor deletion 1 protein, desmoglein-3, calcin-tenin-1, immunoglobulin lambda constant 3, complement C5, alpha-2-macroglobulin, myosin-9, sodium / potassium transport ATPase subunit gamma, immunoglobulin kappa variable 2-28, oncoprotein-inducible transcript 3 protein, cerglycine, coagulation factor XII, coagulation factor XIIIA chain, insulin, histidine-rich glycoprotein, immunoglobulin kappa variable 3-11, immunoglobulin kappa variable 1-39, collagen alpha-1(I) chain, inter-alpha-trypsin inhibitor heavy chain H5, latent transforming growth factor beta-binding protein 2, integrin alpha-IIb, membrane-bound progesterone receptor component 1, immunoglobulin lambda variable 6-57, immunoglobulin kappa variable 3-15, complement C1r small component-like protein, histone H1.2, rhogenetic GDP dissociation inhibitor 2, latent transforming growth factor beta-binding protein 4, collagen alpha-1(XVIII) chain, immunoglobulin lambda variable 2-18, zinc-alpha-2-glycoprotein, talin-1, secretogranin-1, neutrophil defensin 3, cytochrome P450 2E1, gastric suppressor polypeptide, immunoglobulin heavy chain variable 3-15, immunoglobulin lambda variable 2-11, transcription initiation factor TFIID subunit 1, collagen alpha-1(VII) chain, membrane endogenous protein 2B, pigment epithelium-derived factor, voltage-gated N-type calcium channel subunit alpha-1B, immunoglobulin lambda variable 3-27, ras The method according to claim 4 or 18, wherein the polypeptide is selected from the group consisting of GTPase-activated protein nGAP, keratin, type I cytoskeleton 17, tubulin beta chain, sulfhydryl oxidase 1, immunoglobulin kappa variable 4-1, complement C1r minor component, homeobox protein Hox-B2, transcription factor SOX-10, E3 ubiquitin-protein ligase SIAH2, decorin, SPARC, type I collagen alpha-1 chain, type IV collagen alpha-1 chain, laminin gamma 1 chain, vimentin, type III collagen, type IV collagen alpha-3 chain, type VII collagen alpha-1 chain, type VI collagen alpha-1 chain, type V collagen alpha-1 chain, nidogen-1, and type VI collagen alpha-3 chain.

21. The method according to any one of claims 18 to 20, wherein the reporter polypeptide comprises the sequence described in columns II to VI of Table A.

22. The method according to any one of claims 15 to 18, wherein the target tissue or cells are characterized by an increase in the amount or activity of the mammalian protease in the vicinity of the target tissue or cells compared to non-target tissue or cells in the subject.

23. The method according to any one of claims 1 to 22, wherein the subject is suffering from or suspected of suffering from a disease or condition characterized by increased expression or activity of the mammalian protease in the vicinity of the target tissue or cells compared to the corresponding non-target tissue or cells in the subject.

24. The method according to claim 23, wherein the disease or condition is cancer, an inflammatory disease, or an autoimmune disease.

25. The aforementioned disease or condition is carcinoma, Hodgkin lymphoma, and non-Hodgkin lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, blastoma, breast cancer, ER / PR ＋ Breast cancer, HER2 ＋ The method according to claim 24, selected from the group consisting of breast cancer, triple-negative breast cancer, colon cancer, colon cancer with malignant ascites, mucinous neoplasm, prostate cancer, head and neck cancer, skin cancer, melanoma, genitourinary cancer, ovarian cancer, ovarian cancer with malignant ascites, peritoneal carcinomatosis, serous uterine carcinoma, endometrial cancer, cervical cancer, colorectal cancer, uterine cancer, mesothelioma of the peritoneum, kidney cancer, Wilms' tumor, lung cancer, small cell lung cancer, non-small cell lung cancer, gastric cancer, stomach cancer, small intestine cancer, liver cancer, hepatocellular carcinoma, hepatoblastoma, liposarcoma, pancreatic cancer, gallbladder cancer, bile duct cancer, esophageal cancer, salivary gland cancer, thyroid cancer, epithelial cancer, masculinizing neoplasm, adenocarcinoma, sarcoma, and B-cell chronic lymphocytic leukemia.

26. The aforementioned diseases or conditions include ankylosing spondylitis (AS), arthritis (for example, but not limited to rheumatoid arthritis (RA), juvenile idiopathic arthritis (JIA), osteoarthritis (OA), psoriatic arthritis (PsA), gout, chronic arthritis), Chagas disease, chronic obstructive pulmonary disease (COPD), dermatomyositis, type 1 diabetes, endometriosis, Goodpasture syndrome, Graves' disease, Guillain-Barré syndrome (GBS), Hashimoto's disease, suppurative crus, Kawasaki disease, IgA nephropathy, idiopathic thrombocytopenic purpura, inflammatory bowel disease (IBD) (for example, but not limited to these, Lone's disease (CD), Crohn's disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischemic colitis, sparse colitis, Behçet's syndrome, infectious colitis, indeterminate colitis, interstitial cystitis), lupus (e.g., systemic lupus erythematosus, discoid lupus, subacute cutaneous lupus erythematosus, cutaneous lupus erythematosus (e.g., frostbite-like lupus erythematosus), drug-induced lupus, neonatal lupus, lupus nephritis), mixed connective tissue disease, Morphea, multiple sclerosis (MS), myocardial infarction, narcolepsy, neuromuscular aorticoid nephritis Nginseng, pemphigus vulgaris, pernicious anemia, psoriasis, psoriatic arthritis, polymyositis, primary biliary cirrhosis, relapsing polychondritis, schizophrenia, scleroderma, Sjögren's syndrome, generalized rigidity syndrome, temporal arteritis (also known as giant cell arteritis), vasculitis, vitiligo, Wegener's granulomatosis, transplant rejection-associated immune reactions (e.g., kidney transplant rejection, lung transplant rejection, liver transplant rejection, etc., though not limited to these), psoriasis, Wiscott-Aldrich syndrome, autoimmune lymphoproliferative syndrome, myasthenia gravis, inflammatory chronic sinusitis, colitis, celiac disease, Barrett's esophagus, inflammatory gastritis, autoimmune The method according to claim 24, wherein the inflammatory disease is selected from the group consisting of nephritis, autoimmune hepatitis, autoimmune carditis, autoimmune encephalitis, autoimmune-mediated hematological disorders, asthma, atopic dermatitis, atopy, allergy, allergic rhinitis, scleroderma, bronchitis, and pericarditis, and the inflammatory disease is Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, inflammatory lung disease, inflammatory skin disease, atherosclerosis, myocardial infarction, stroke, Gram-positive shock, Gram-negative shock, sepsis, septic shock, hemorrhagic shock, anaphylactic shock, and systemic inflammatory response syndrome.

27. The method according to any one of claims 1 to 26, wherein the therapeutic agent is an anticancer agent.

28. The method according to any one of claims 1 to 27, wherein the therapeutic agent is an activatable therapeutic agent.

29. The method according to any one of claims 1 to 28, wherein the therapeutic agent further comprises a masking portion (MM).

30. The method according to claim 29, wherein the masking portion (MM) can be released from the therapeutic agent when the peptide substrate is cleaved by the mammalian protease.

31. The method according to claim 29 or 30, wherein the masking portion (MM) interferes with the interaction between the therapeutic agent and the target tissue or cells in an uncut state.

32. The method according to any one of claims 29 to 31, wherein the biological activity of the therapeutic agent can be enhanced when the peptide substrate is cleaved by the mammalian protease.

33. The method according to any one of claims 29 to 32, wherein the masking portion (MM) is an elongated recombinant polypeptide (XTEN).

34. The aforementioned XTEN is, (i) It must contain at least 100 amino acids; (ii) at least 90% of the amino acid residues are selected from glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), and proline (P); and (iii) Contains at least four different amino acids selected from G, A, S, T, E, and P. The method according to claim 33, characterized by the above.

35. The method according to any one of claims 1 to 34, further comprising the step of evaluating whether the subject is responsive to the therapeutic agent by contacting the therapeutic agent with the mammalian protease, after (b).

36. The method according to any one of claims 1 to 34, wherein (a) is to detect the polypeptide of (i), (ii), or (iii) by immunoassay.

37. The method according to claim 36, wherein the immunoassay utilizes an antibody that specifically binds to the polypeptide or epitope of (i), (ii), or (iii).

38. The method according to any one of claims 1 to 37, wherein (a) detects the polypeptide of (i), (ii), or (iii) by using a mass spectrometer (MS).

39. The method according to any one of claims 1 to 38, further comprising the step of administering an effective amount of the therapeutic agent to the subject based on the designation of step (b), following (b).

40. A method for treating a target with an activatable therapeutic agent, (a) Identifying the subject as potentially responsive to the activatable therapeutic agent based on the identification of peptide biomarkers in a biological sample from the subject, wherein the activatable therapeutic agent comprises a peptide substrate sequence that is sensitive to cleavage by mammalian proteases in an easily cleavable bond; and (b) A step of administering the activatable therapeutic agent to the subject based on the identification of the subject in (a). A method comprising the peptide biomarker comprising a portion identical to at least four consecutive amino acid residues of the peptide substrate sequence located either at the N-terminus or C-terminus of the easily cleavable bond.

41. The method according to claim 40, wherein the peptide biomarker is derived from a reporter polypeptide comprising the sequence described in columns II to VI of Table A.

42. The method according to claim 40 or 41, wherein the peptide biomarker has an amino acid sequence identical to the sequence of a reporter polypeptide containing the sequences described in columns II to VI of Table A.

43. The method according to any one of claims 40 to 42, wherein the peptide substrate sequence contains 6 to 25 or 6 to 20 amino acid residues.

44. The method according to claim 43, wherein the peptide substrate sequence contains 7 to 12 amino acid residues.

45. The method according to any one of claims 40 to 44, wherein the peptide substrate sequence includes an amino acid sequence having up to three amino acid substitutions, up to two amino acid substitutions, or up to one amino acid substitution with respect to the sequence described in column II or III of Table A, and none of the amino acid substitutions are in a position corresponding to an amino acid residue directly adjacent to the corresponding easily cleavable bond shown in Table A.

46. The method according to claim 45, wherein the peptide substrate sequence includes the amino acid sequence described in column II or III of Table A.

47. The method according to any one of claims 40 to 46, wherein the peptide substrate sequence, which is sensitive to cleavage by the mammalian protease, is sensitive to cleavage by a plurality of mammalian proteases, including the mammalian protease.

48. The method according to claim 47, wherein the peptide substrate sequence, which is sensitive to cleavage by the plurality of mammalian proteases, has a maximum of three amino acid substitutions, a maximum of two amino acid substitutions, or a maximum of one amino acid substitution relative to the sequence described in Table 1(j), and none of the amino acid substitutions are in a position corresponding to an amino acid residue directly adjacent to the corresponding easily cleavable bond.

49. The method according to claim 47 or 48, wherein the peptide substrate sequence sensitive to cleavage by the plurality of mammalian proteases includes the sequence listed in Table 1(j).

50. The method according to claim 45, wherein the peptide substrate sequence has the same amino acid sequence as the sequence fragment described in column II or III of Table A, and the fragment comprises at least four consecutive amino acid residues directly adjacent to the corresponding easily cleavable bond shown in Table A.

51. The method according to claim 50, wherein the fragment contains at least five, at least six, at least seven, at least eight, at least nine, or at least ten amino acid residues.

52. The method according to any one of claims 40 to 51, wherein the portion of the peptide substrate sequence located at the N-terminus of the easily cleavable bond has a maximum of three amino acid substitutions, a maximum of two amino acid substitutions, or a maximum of one amino acid substitution with respect to the C-terminal sequence containing 4 to 10 amino acid residues of the sequence described in column IV or V of Table A, and none of the amino acid substitutions are in a position corresponding to an amino acid residue directly adjacent to the corresponding easily cleavable bond.

53. The method according to claim 52, wherein the portion of the peptide substrate sequence located at the N-terminus of the easily cleavable bond includes a C-terminal sequence containing 4 to 10 amino acid residues of the sequence described in column IV or V of Table A.

54. The method according to any one of claims 40 to 52, wherein the portion of the peptide substrate sequence located on the C-terminal side of the easily cleavable bond has a maximum of three amino acid substitutions, a maximum of two amino acid substitutions, or a maximum of one amino acid substitution with respect to the N-terminal sequence containing 4 to 10 amino acid residues of the sequence described in column V or VI of Table A, and none of the amino acid substitutions are in a position corresponding to an amino acid residue directly adjacent to the corresponding easily cleavable bond.

55. The method according to claim 54, wherein the portion of the peptide substrate sequence located at the C-terminus of the easily cleavable bond comprises an N-terminal sequence containing 4 to 10 amino acid residues of the sequence described in column V or VI of Table A.

56. The method according to any one of claims 40 to 55, wherein the possibility of the response is determined by the method according to any one of claims 1 to 39.

57. A method for treating a subject that requires a therapeutic agent that can be activated by a mammalian protease expressed in the subject, The procedure includes the step of administering an effective amount of the therapeutic agent to the subject, wherein the subject has been shown to express the following in a biological sample from the subject: (i) A polypeptide comprising at least five or six consecutive amino acid residues as shown in the sequence in column V of Table A, or (ii) A polypeptide comprising at least five or six consecutive amino acids as shown in the sequence in column IV of Table A, or (iii) A polypeptide comprising at least five or six consecutive amino acids as shown in the sequence in column VI of Table A, or (iv) The expression level of polypeptide (i), (ii), or (iii) exceeds the threshold. method.

58. The method according to claim 57, wherein the polypeptide in (i) comprises at least seven, at least eight, at least nine, or at least ten consecutive amino acid residues as shown in the sequence in column V of Table A.

59. The method according to claim 57 or 58, wherein the polypeptide of (ii) comprises at least seven, at least eight, at least nine, or at least ten consecutive amino acids as shown in the sequence in column IV of Table A.

60. The method according to any one of claims 57 to 59, wherein the polypeptide of (iii) comprises at least seven, at least eight, at least nine, or at least ten consecutive amino acids as shown in the sequence in column VI of Table A.

61. The method according to any one of claims 57 to 60, wherein the subject is shown to express any two of (i) to (iii) in the biological sample.

62. The method according to any one of claims 57 to 61, wherein the therapeutic agent comprises a peptide substrate sequence that is sensitive to cleavage by the mammalian protease.

63. The method according to claim 62, wherein the peptide substrate sequence is sensitive to cleavage by the mammalian protease at an easily cleavable bond, and the polypeptide of (i), (ii), or (iii) comprises a portion containing at least four consecutive amino acid residues of the peptide substrate sequence located either at the N-terminal or C-terminal end of the easily cleavable bond.

64. The method according to claim 63, wherein the portion of the peptide substrate sequence located at the N-terminus of the easily cleavable bond has a maximum of three amino acid substitutions, a maximum of two amino acid substitutions, or a maximum of one amino acid substitution with respect to the C-terminal sequence containing 4 to 10 amino acid residues of the sequence described in column IV or V of Table A, and none of the amino acid substitutions are located in a position corresponding to an amino acid residue directly adjacent to the corresponding easily cleavable bond.

65. The method according to claim 63 or 64, wherein the portion of the peptide substrate sequence located at the N-terminus of the easily cleavable bond includes a C-terminal sequence containing 4 to 10 amino acid residues of the sequence described in column IV or V of Table A.

66. The method according to any one of claims 63 to 65, wherein the portion of the peptide substrate sequence located on the C-terminal side of the easily cleavable bond has a maximum of three amino acid substitutions, a maximum of two amino acid substitutions, or a maximum of one amino acid substitution with respect to the N-terminal sequence containing 4 to 10 amino acid residues of the sequence described in column V or VI of Table A, and none of the amino acid substitutions are located in a position corresponding to an amino acid residue directly adjacent to the corresponding easily cleavable bond.

67. The method according to any one of claims 63 to 66, wherein the portion of the peptide substrate sequence located on the C-terminal side of the easily cleavable bond comprises an N-terminal sequence containing 4 to 10 amino acid residues of the sequence described in column V or VI of Table A.

68. The method according to any one of claims 57 to 67, wherein the threshold is zero or a nominal value.

69. The method according to any one of claims 40 to 68, wherein the biological sample comprises a serum or plasma sample.

70. The method according to any one of claims 40 to 69, wherein the mammalian protease is a serine protease, a cysteine ​​protease, an aspartate protease, a threonine protease, or a metalloproteinase.

71. The mammalian proteases are: disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), disintegrin and metalloproteinase domain-containing protein 12 (ADAM12), disintegrin and metalloproteinase domain-containing protein 15 (ADAM15), disintegrin and metalloproteinase domain-containing protein 17 (ADAM17), and disintegrin and metalloproteinase domain-containing protein 9 (ADAM9). ), disintegrin and metalloproteinase having thrombospondin motif 5 (ADAMTS5), cathepsin B, cathepsin D, cathepsin E, cathepsin K, cathepsin L, cathepsin S, fibroblast-activating protein alpha, hepsin, kallikrein-2, kallikrein-4, kallikrein-3, prostate-specific antigen (PSA), kallikrein-13, regmine, matrix metallopeptidase 1 (MMP-1), matrix metallopeptidase 10 (MMP-10), matrix metal Matrix metallopeptidase 11 (MMP-11), Matrix metallopeptidase 12 (MMP-12), Matrix metallopeptidase 13 (MMP-13), Matrix metallopeptidase 14 (MMP-14), Matrix metallopeptidase 16 (MMP-16), Matrix metallopeptidase 2 (MMP-2), Matrix metallopeptidase 3 (MMP-3), Matrix metallopeptidase 7 (MMP-7), Matrix metallopeptidase 8 (MMP-8), Matrix metallopeptidase The method according to claim 70, comprising a selection from the group consisting of -ase 9 (MMP-9), matrix metallopeptidase 4 (MMP-4), matrix metallopeptidase 5 (MMP-5), matrix metallopeptidase 6 (MMP-6), matrix metallopeptidase 15 (MMP-15), neutrophil elastase, protease-activated receptor 2 (PAR2), plasmin, prostasin, PSMA-FOLH1, membrane-bound serine protease 1 (MT-SP1), matryptase, and u-plasminogen.

72. The method according to claim 70, wherein the mammalian protease is selected from the group consisting of matrix metallopeptidase 1 (MMP1), matrix metallopeptidase 2 (MMP2), matrix metallopeptidase 7 (MMP7), matrix metallopeptidase 9 (MMP9), matrix metallopeptidase 11 (MMP11), matrix metallopeptidase 14 (MMP14), urokinase-type plasminogen activator (uPA), regmine, and matryptase.

73. The method according to any one of claims 40 to 71, wherein the mammalian protease is preferentially expressed or activated in the target tissue or cell.

74. The method according to claim 73, wherein the target tissue or cells are a tumor.

75. The method according to claim 73 or 74, wherein the target tissue or cells produce the mammalian protease or co-localize with the mammalian protease.

76. The method according to any one of claims 73 to 75, wherein the target tissue or cell contains a reporter polypeptide in or on it, or is associated with a reporter polypeptide in its vicinity.

77. The reporter polypeptide is a coagulation factor, complement component, tubulin, immunoglobulin, apolipoprotein, serum amyloid, insulin, growth factor, fibrinogen, PDZ domain protein, LIM domain protein, c-reactive protein, serum albumin, versican, collagen, elastin, keratin, kininogen-1, alpha-2-antiplasmin, clusterin, biglycan, alpha-1-antitrypsin, transthyretin, alpha-1-antichymotrypsin, glucagon, hepcidin, thymosin beta-4, haptoglobin, hemoglobin subunit alpha, caveolae-associated protein 2, alpha-2-HS glycoprotein, chromogranin-A, vitronectin, hemopexin, epididymal sperm-binding protein, secretogranin-2, angiotensinogen, Transgerin-2, pancreatic prohormone, neurosecretory protein VGF, ceruloplasmin, PDZ and LIM domain protein 1, multimelin-1, inter-alpha-trypsin inhibitor heavy chain H2, N-acetylmuramoyl-L-alanine amidase, histone H1.4, adhesion G protein-coupled receptor G6, mannan-binding lectin serine protease 2, prothrombin, malignant brain tumor deletion 1 protein, desmoglein-3, calcin-tenin-1, alpha-2-macroglobulin, myosin-9, sodium / potassium transport ATPase subunit gamma, oncoprotein-inducible transcript 3 protein, cerglycine, histidine-rich glycoprotein, inter-alpha-trypsin inhibitor heavy chain H5, integrin alpha-IIb, membrane-bound progesterone receptor component 1, histone H1.2, rh GDP dissociation inhibitor 2, zinc-alpha-2-glycoprotein, talin-1, secretogranin-1, neutrophil defensin 3, cytochrome P450 2E1, gastric suppressor polypeptide, transcription initiation factor TFIID subunit 1, membrane endogenous protein 2B, pigment epithelium-derived factor, voltage-gated N-type calcium channel subunit alpha-1B, rasThe method according to claim 76, wherein the polypeptide is selected from the group consisting of GTPase-activating protein nGAP, type I cytoskeleton 17, sulfhydryl oxidase 1, homeobox protein Hox-B2, transcription factor SOX-10, E3 ubiquitin-protein ligase SIAH2, decorin, acidic, cysteine-rich secretory protein (SPARC), laminin gamma 1 chain, vimentin, and nidogen-1 (NID1).

78. The reporter polypeptide is versican, type II collagen alpha-1 chain, kininogen-1, complement C4-A, complement C4-B, complement C3, alpha-2-antiplasmin, clusterin, biglycan, elastin, fibrinogen alpha chain, alpha-1-antitrypsin, fibrinogen beta chain, type III collagen alpha-1 chain, serum amyloid A-1 protein, transthyretin, apolipoprotein A-I, apolipoprotein A-I isoform 1, alpha-1-antichymotrypsin, glucagon, hepcidin , serum amyloid A-2 protein, thymosin beta-4, haptoglobin, hemoglobin subunit alpha, caveolae-associated protein 2, alpha-2-HS glycoprotein, chromogranin-A, vitronectin, hemopexin, epididymal sperm-binding protein, zyxin, apolipoprotein C-III, secretogranin-2, angiotensinogen, c-reactive protein, serum albumin, transgerin-2, pancreatic prohormones, neurosecretory protein VGF, ceruloplasmin, PDZ and LIM domain protein 1, Iubrin alpha-4A chain, multimelin-1, inter-alpha-trypsin inhibitor heavy chain H2, apolipoprotein C-I, fibrinogen gamma chain, N-acetylmuramoyl-L-alanine amidase, immunoglobulin lambda variable 3-21, histone H1.4, adhesion G protein-coupled receptor G6, immunoglobulin lambda variable 3-25, immunoglobulin lambda variable 1-51, immunoglobulin lambda variable 1-36, mannan-binding lectin serine protease 2, immunoglobulin kappa variable 3-20, immunoglobulin kappa variable 2-30, insulin Phosphorus-like growth factor II, apolipoprotein A-II, highly non-functional immunoglobulin kappa variable 2D-24, prothrombin, coagulation factor IX, apolipoprotein L1, malignant brain tumor deletion 1 protein, desmoglein-3, calcin-tenin-1, immunoglobulin lambda constant 3, complement C5, alpha-2-macroglobulin, myosin-9, sodium / potassium transport ATPase subunit gamma, immunoglobulin kappa variable 2-28, oncoprotein-inducible transcript 3 protein, cerglycine, coagulation factor XII, coagulation factor XIIIA chain, insulin, histidine-rich glycoprotein, immunoglobulin kappa variable 3-11, immunoglobulin kappa variable 1-39, collagen alpha-1(I) chain, inter-alpha-trypsin inhibitor heavy chain H5, latent transforming growth factor beta-binding protein 2, integrin alpha-IIb, membrane-bound progesterone receptor component 1, immunoglobulin lambda variable 6-57, immunoglobulin kappa variable 3-15, complement C1r small component-like protein, histone H1.2, rhogenetic GDP dissociation inhibitor 2, latent transforming growth factor beta-binding protein 4, collagen alpha-1(XVIII) chain, immunoglobulin lambda variable 2-18, zinc-alpha-2-glycoprotein, talin-1, secretogranin-1, neutrophil defensin 3, cytochrome P450 2E1, gastric suppressor polypeptide, immunoglobulin heavy chain variable 3-15, immunoglobulin lambda variable 2-11, transcription initiation factor TFIID subunit 1, collagen alpha-1(VII) chain, membrane endogenous protein 2B, pigment epithelium-derived factor, voltage-gated N-type calcium channel subunit alpha-1B, immunoglobulin lambda variable 3-27, ras The method according to claim 77, wherein the polypeptide is selected from the group consisting of GTPase-activated protein nGAP, keratin, type I cytoskeleton 17, tubulin beta chain, sulfhydryl oxidase 1, immunoglobulin kappa variable 4-1, complement C1r minor component, homeobox protein Hox-B2, transcription factor SOX-10, E3 ubiquitin-protein ligase SIAH2, decorin, SPARC, type I collagen alpha-1 chain, type IV collagen alpha-1 chain, laminin gamma 1 chain, vimentin, type III collagen, type IV collagen alpha-3 chain, type VII collagen alpha-1 chain, type VI collagen alpha-1 chain, type V collagen alpha-1 chain, nidogen-1, and type VI collagen alpha-3 chain.

79. The method according to any one of claims 76 to 78, wherein the reporter polypeptide comprises the sequence described in columns II to VI of Table A.

80. The method according to any one of claims 73 to 79, wherein the target tissue or cells are characterized by an increase in the amount or activity of the mammalian protease in the vicinity of the target tissue or cells compared to non-target tissue or cells in the subject.

81. The method according to any one of claims 40 to 80, wherein the subject is suffering from or suspected to be suffering from a disease or condition characterized by increased expression or activity of the mammalian protease in the vicinity of the target tissue or cells compared to the corresponding non-target tissue or cells in the subject.

82. The method according to claim 81, wherein the disease or condition is cancer, an inflammatory disease, or an autoimmune disease.

83. The aforementioned diseases or conditions include ankylosing spondylitis (AS), arthritis (for example, but not limited to rheumatoid arthritis (RA), juvenile idiopathic arthritis (JIA), osteoarthritis (OA), psoriatic arthritis (PsA), gout, chronic arthritis), Chagas disease, chronic obstructive pulmonary disease (COPD), dermatomyositis, type 1 diabetes, endometriosis, Goodpasture syndrome, Graves' disease, Guillain-Barré syndrome (GBS), Hashimoto's disease, suppurative crus, Kawasaki disease, IgA nephropathy, idiopathic thrombocytopenic purpura, inflammatory bowel disease (IBD) (for example, but not limited to these, Lone's disease (CD), Crohn's disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischemic colitis, sparse colitis, Behçet's syndrome, infectious colitis, indeterminate colitis, interstitial cystitis), lupus (e.g., systemic lupus erythematosus, discoid lupus, subacute cutaneous lupus erythematosus, cutaneous lupus erythematosus (e.g., frostbite-like lupus erythematosus), drug-induced lupus, neonatal lupus, lupus nephritis), mixed connective tissue disease, Morphea, multiple sclerosis (MS), myocardial infarction, narcolepsy, neuromuscular aorticoid nephritis Nginseng, pemphigus vulgaris, pernicious anemia, psoriasis, psoriatic arthritis, polymyositis, primary biliary cirrhosis, relapsing polychondritis, schizophrenia, scleroderma, Sjögren's syndrome, generalized rigidity syndrome, temporal arteritis (also known as giant cell arteritis), vasculitis, vitiligo, Wegener's granulomatosis, transplant rejection-associated immune reactions (e.g., kidney transplant rejection, lung transplant rejection, liver transplant rejection, etc., though not limited to these), psoriasis, Wiscott-Aldrich syndrome, autoimmune lymphoproliferative syndrome, myasthenia gravis, inflammatory chronic sinusitis, colitis, celiac disease, Barrett's esophagus, inflammatory gastritis, autoimmune The method according to claim 82, wherein the inflammatory disease is selected from the group consisting of nephritis, autoimmune hepatitis, autoimmune carditis, autoimmune encephalitis, autoimmune-mediated hematological disorders, asthma, atopic dermatitis, atopy, allergy, allergic rhinitis, scleroderma, bronchitis, and pericarditis, and the inflammatory disease is Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, inflammatory lung disease, inflammatory skin disease, atherosclerosis, myocardial infarction, stroke, Gram-positive shock, Gram-negative shock, sepsis, septic shock, hemorrhagic shock, anaphylactic shock, and systemic inflammatory response syndrome.

84. The aforementioned disease or condition is carcinoma, Hodgkin lymphoma, and non-Hodgkin lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, blastoma, breast cancer, ER / PR ＋ Breast cancer, HER2 ＋ The method according to claim 83, selected from the group consisting of breast cancer, triple-negative breast cancer, colon cancer, colon cancer with malignant ascites, mucinous neoplasm, prostate cancer, head and neck cancer, skin cancer, melanoma, genitourinary cancer, ovarian cancer, ovarian cancer with malignant ascites, peritoneal carcinomatosis, serous uterine carcinoma, endometrial cancer, cervical cancer, colorectal cancer, uterine cancer, mesothelioma of the peritoneum, kidney cancer, Wilms' tumor, lung cancer, small cell lung cancer, non-small cell lung cancer, gastric cancer, stomach cancer, small intestine cancer, liver cancer, hepatocellular carcinoma, hepatoblastoma, liposarcoma, pancreatic cancer, gallbladder cancer, bile duct cancer, esophageal cancer, salivary gland cancer, thyroid cancer, epithelial cancer, masculinizing neoplasm, adenocarcinoma, sarcoma, and B-cell chronic lymphocytic leukemia.

85. The method according to any one of claims 40 to 84, wherein the therapeutic agent is an anticancer agent.

86. The method according to any one of claims 40 to 85, wherein the therapeutic agent is an activatable therapeutic agent.

87. The method according to claim 86, wherein the therapeutic agent is a non-natural, activatable therapeutic agent.

88. The method according to any one of claims 40 to 86, wherein the therapeutic agent further comprises a masking portion (MM).

89. The method according to claim 88, wherein the masking portion (MM) can be released from the therapeutic agent when the peptide substrate sequence is cleaved by the mammalian protease.

90. The method according to claim 88 or 89, wherein the masking portion (MM) interferes with the interaction between the therapeutic agent and the target tissue or cells in an uncut state.

91. The method according to any one of claims 88 to 90, wherein the biological activity of the therapeutic agent can be enhanced when the peptide substrate is cleaved by the mammalian protease.

92. The method according to any one of claims 88 to 90, wherein the masking portion (MM) is an elongated recombinant polypeptide (XTEN).

93. The aforementioned XTEN is, (i) It must contain at least 100 amino acids; (ii) at least 90% of the amino acid residues are selected from glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), and proline (P); and (iii) Contains at least four different amino acids selected from G, A, S, T, E, and P. The method according to claim 92, characterized by the above.

94. The method according to any one of claims 57 to 93, wherein the subject is determined to have the potential to respond to the therapeutic agent.

95. A method according to any one of claims 57 to 94 for treating a disease or condition in a subject, comprising the step of administering to the subject in need one or more therapeutically effective doses of the activatable therapeutic agent or a pharmaceutical composition comprising the activatable therapeutic agent.

96. The method according to claim 95, wherein the subject is selected from the group consisting of mice, rats, monkeys, and humans.

97. The method according to claim 95, wherein the subject is a human.

98. The method according to any one of claims 95 to 97, wherein the subject is determined to have the potential to respond to the therapeutic agent or the pharmaceutical composition.

99. The method according to claim 98, wherein the probability of the response is 50% or higher.

100. The method according to claim 99 or 100, wherein the possibility of the response is determined by the method according to any one of claims 1 to 39.

101. The method according to any one of claims 95 to 100, wherein the disease or condition is cancer, an inflammatory disease, or an autoimmune disease.

102. The aforementioned diseases or conditions include ankylosing spondylitis (AS), arthritis (for example, but not limited to rheumatoid arthritis (RA), juvenile idiopathic arthritis (JIA), osteoarthritis (OA), psoriatic arthritis (PsA), gout, chronic arthritis), Chagas disease, chronic obstructive pulmonary disease (COPD), dermatomyositis, type 1 diabetes, endometriosis, Goodpasture syndrome, Graves' disease, Guillain-Barré syndrome (GBS), Hashimoto's disease, suppurative crus, Kawasaki disease, IgA nephropathy, idiopathic thrombocytopenic purpura, inflammatory bowel disease (IBD) (for example, but not limited to cytoplasmic purpura), Crohn's disease (CD), Crohn's disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischemic colitis, sparse colitis, Behçet's syndrome, infectious colitis, indeterminate colitis, interstitial cystitis), lupus (e.g., systemic lupus erythematosus, discoid lupus, subacute cutaneous lupus erythematosus, cutaneous lupus erythematosus (e.g., frostbite-like lupus erythematosus), drug-induced lupus, neonatal lupus, lupus nephritis), mixed connective tissue disease, Morphea, multiple sclerosis (MS), myocardial infarction, narcolepsy, neuromuscular angioplasia Gina, pemphigus vulgaris, pernicious anemia, psoriasis, psoriatic arthritis, polymyositis, primary biliary cirrhosis, relapsing polychondritis, schizophrenia, scleroderma, Sjögren's syndrome, generalized rigidity syndrome, temporal arteritis (also known as giant cell arteritis), vasculitis, vitiligo, Wegener's granulomatosis, transplant rejection-associated immune reactions (e.g., kidney transplant rejection, lung transplant rejection, liver transplant rejection, etc., though not limited to these), psoriasis, Wiscott-Aldrich syndrome, autoimmune lymphoproliferative syndrome, myasthenia gravis, inflammatory chronic sinusitis, colitis, celiac disease, Barrett's esophagus, inflammatory gastritis, autoimmune kidney The method according to claim 101, wherein the inflammatory disease is selected from the group consisting of inflammation, autoimmune hepatitis, autoimmune carditis, autoimmune encephalitis, autoimmune-mediated hematological disorders, asthma, atopic dermatitis, atopy, allergy, allergic rhinitis, scleroderma, bronchitis, and pericarditis, and the inflammatory disease is Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, inflammatory lung disease, inflammatory skin disease, atherosclerosis, myocardial infarction, stroke, Gram-positive shock, Gram-negative shock, sepsis, septic shock, hemorrhagic shock, anaphylactic shock, and systemic inflammatory response syndrome.

103. The aforementioned disease or condition is carcinoma, Hodgkin lymphoma, and non-Hodgkin lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, blastoma, breast cancer, ER / PR ＋ Breast cancer, HER2 ＋ The method according to claim 101, selected from the group consisting of breast cancer, triple-negative breast cancer, colon cancer, colon cancer with malignant ascites, mucinous neoplasm, prostate cancer, head and neck cancer, skin cancer, melanoma, genitourinary cancer, ovarian cancer, ovarian cancer with malignant ascites, peritoneal carcinomatosis, serous uterine carcinoma, endometrial cancer, cervical cancer, colorectal cancer, uterine cancer, mesothelioma of the peritoneum, kidney cancer, Wilms' tumor, lung cancer, small cell lung cancer, non-small cell lung cancer, gastric cancer, stomach cancer, small intestine cancer, liver cancer, hepatocellular carcinoma, hepatoblastoma, liposarcoma, pancreatic cancer, gallbladder cancer, bile duct cancer, esophageal cancer, salivary gland cancer, thyroid cancer, epithelial cancer, masculinizing neoplasm, adenocarcinoma, sarcoma, and B-cell chronic lymphocytic leukemia.

104. Use of a diagnostic reagent in an execution of any one of claims 1 to 39 for evaluating the likelihood that a subject is responsive to a therapeutic agent that can be activated by a mammalian protease expressed in the subject having a disease or disorder.

105. Use of a diagnostic reagent in an execution of any one of claims 40 to 103 for evaluating the likelihood that a subject is responsive to a therapeutic agent that can be activated by a mammalian protease expressed in a subject having a disease or disorder.

106. A kit for carrying out the method of claims 1 to 103, comprising a reagent for detecting the presence or amount of proteolytic peptide products produced by the action of a mammalian protease, for evaluating the likelihood that a subject with a disease or disorder is responsive to a therapeutic agent that can be activated by the mammalian protease expressed in the subject.

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