Compositions and methods related to activatable therapeutic agents

Abstract

Described herein are methods for assessing likelihood of response of subjects to activatable therapeutic agents and compositions, kits, and methods of preparing and using activatable therapeutic agents. Also described herein are methods for assessing likelihood of response of subjects to activatable therapeutic agents. In some cases, the activatable therapeutic agents of the compositions, kits, and methods disclosed herein can comprise a mammalian protein-derived sequence.

Description

REFERENCE STATEMENT

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 054525 filed on July 21, 2020, entitled "COMPOSITIONS AND METHODS RELATED TO ACTI VAT ABLE THERAPEUTIC AGENTS" which is incorporated herein in their entireties. SEQUENCE LISTING STATEMENT:

[0002] A computer readable form of the Sequence Listing is filed with this application by electronic submission and is incorporated into this application by reference in its entirety. The Sequence Listing is contained in the file created on July 15, 2021 having the file name “791-601 20-1831-WO_ST25_FINAL.txt” and is 1700 kb in size. BACKGROUND

[0003] A key challenge in developing prodrug therapeutics is avoiding unwanted immunogenicity and nonspecific activation at biological sites in vivo other than the target site. Various release sites have been optimized in vitro and incorporated into prodrugs for programmed and targeted activation, for example, by protease(s) natively produced at or near diseased tissue(s). Such engineered release segments can form Tor B-cell epitopes that can elicit undesired immunogenicity in patients. Further, there is currently a lack of methods for adequately predicting in vivo responses of patients to prodrugs. In particular, with respect to protease-activated prodrugs, diseased tissues being targeted often contain a multitude of proteases with varying activities and specificities, which is difficult to reconstitute in vitro and complicates any prediction of in vivo prodrug activation. There remains a need for identifying new peptide segments that can be incorporated into a variety of prodrug therapeutic, diagnostic and prophylactic compositions for a more effective and reliable release mechanism. There also remains a need for developing more accurate and robust methods for predicting therapeutic responses and outcomes upon administration of prodrugs or other activatable compositions. SUMMARY

[0004] In certain aspects, the present disclosure provides a method for assessing a likelihood of a subject being responsive to a therapeutic agent that is activatable by a mammalian protease expressed in the subject, the method comprising: (a) determining, in a biological sample from the subject, a presence or an amount of (i) a polypeptide comprising at least five, at least six, at least seven, at least eight, at least nine, or at least ten consecutive amino acid residues shown in a sequence set forth in Column V of Table A (or a subset thereol); or (ii) a polypeptide comprising at least five, at least six, at least seven, at least eight, at least nine, or at least ten consecutive amino acids shown in a sequence set forth in Column IV of Table A (or a subset thereof); or (iii) a polypeptide comprising at least five, at least six, at least seven, at least eight, at least nine, or at least ten consecutive amino acids shown in a sequence set forth in Column VI of Table A (or a subset thereof); and (b) designating the subject as being likely to respond to the therapeutic agent when the polypeptide of (i), (ii) or (iii) is present and / or if its amount exceeds a threshold.

[0005] In some embodiments of the method for assessing the likelihood of the subject being responsive to the therapeutic agent, the therapeutic agent comprises a peptide substrate, which peptide substrate is susceptible to cleavage by the mammalian protease at a scissile bond. In some embodiments, 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 peptide substrate that is either N-terminal or C-terminal side of the scissile bond. In some embodiments, the peptide substrate is susceptible to cleavage by the mammalian protease at a scissile bond, and wherein the polypeptide of (i), (ii), or (iii) is a cleavage product of a reporter polypeptide comprising a substrate sequence that is susceptible to cleavage by the same mammalian protease at a scissile bond and where the reporter polypeptide comprises a sequence set forth in Column II or III of Table A (or a subset thereof), tn some embodiments, the peptide substrate is susceptible to cleavage by the mammalian protease at a scissile bond, and wherein the polypeptide of (i), (ii), or (iii) is a cleavage product of a human protein that comprises a portion containing at least five or six consecutive amino acid residues of the peptide substrate that includes the scissile bond.

[0006] In some embodiments of the method for assessing the likelihood of the subject being responsive to the 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 shown in a sequence set forth in Column V of Table A (or a subset thereof), tn 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 shown in a sequence set forth in Column IV of Table A (or a subset thereof), tn 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 shown in a sequence set forth in Column VI of Table A (or a subset thereof).

[0007] tn some embodiments of the method for assessing the likelihood of the subject being responsive to the therapeutic agent, (a) comprises determining the presence or the amount of any two of (i)-(iii). In some embodiments, (a) comprises determining the presence or the amount of all three of (i)-(iii).

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

[0009] In some embodiments of the method for assessing the likelihood of the subject being responsive to the therapeutic agent, the mammalian protease is a serine protease, a cysteine protease, an aspartate protease, a threonine protease, or a metalloproteinase. In some embodiments, the mammalian protease is selected from the group consisting of 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 domaincontaining protein 17 (ADAM17), disintegrin and metalloproteinase domain-containing protein 9 (ADAM9), disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), Cathepsin B, Cathepsin D, Cathepsin E, Cathepsin K, cathepsin L, cathepsin S, Fibroblast activation protein alpha, Hepsin, kallikrein-2, kallikrein-4, kallikrein-3, Prostate-specific antigen (PSA), kallikrein-13, Legumain, 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 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 type serine protease 1 (MT-SP1), matriptase, 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), legumain, and matriptase. In some embodiments, the mammalian protease is preferentially expressed or activated in a target tissue or cell.

[0010] In some embodiments of the method for assessing the likelihood of the subject being responsive to the therapeutic agent, the target tissue or cell is a tumor. In some embodiments, the target tissue or cell produces or is co-localized with the mammalian protease.

[0011] In some embodiments of the method for assessing the likelihood of the subject being responsive to the therapeutic agent, the target tissue or cell contains therein or thereon, or is associated with in proximity thereto, a reporter polypeptide. In some embodiments, the reporter polypeptide is a polypeptide selected from the group consisting of 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, epididymis secretory sperm binding protein, secretogranin-2, angiotensinogen, transgelin-2, pancreatic prohormone, neurosecretory protein VGF, ceruloplasmin, PDZ and LIM domain protein 1, multimerin-1, inter-alpha-trypsin inhibitor heavy chain H2, N-acetylmuramoyl-L-alanine amidase, histone Hl.4, adhesion G-protein coupled receptor G6, mannan-binding lectin serine protease 2, prothrombin, deleted in malignant brain tumors 1 protein, desmoglein-3, calsyntenin-1, alpha-2-macroglobulin, myosin-9, sodium / potassium-transporting ATPase subunit gamma, oncoprotein-induced transcript 3 protein, serglycin, histidine-rich glycoprotein, inter-alpha-trypsin inhibitor heavy chain H5, integrin alpha-IIb, membrane-associated progesterone receptor component 1, histone Hl.2, rho GDP-dissociation inhibitor 2, zinc-alpha-2-gly coprotein, talin-1, secretogranin-1, neutrophil defensin 3, cytochrome P450 2E1, gastric inhibitory polypeptide, transcription initiation factor TFIID subunit 1, integral membrane protein 2B, pigment epithelium-derived factor, voltage-dependent N-type calcium channel subunit alpha-IB, ras GTPase-activating protein nGAP, type I cytoskeletal 17, sulfhydryl oxidase 1, homeobox protein Hox-B2, transcription factor SOX-10, E3 ubiquitin-protein ligase SIAH2, decorin, secreted protein acidic and rich in cysteine (SPARC), laminin gamma 1 chain, vimentin, and nidogen-1 (NIDI). In some embodiments, the reporter polypeptide is a polypeptide selected from the group consisting of 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-l 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, epididymis secretory sperm binding protein, zyxin, apolipoprotein C-III, secretogranin-2, angiotensinogen, c-reactive protein, serum albumin, transgelin-2, pancreatic prohormone, neurosecretory protein VGF, ceruloplasmin, PDZ and LIM domain protein 1, tubulin alpha-4 A chain, multimerin-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 Hl.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, probable non-functional immunoglobulin kappa variable 2D-24, prothrombin, coagulation factor IX, apolipoprotein LI, deleted in malignant brain tumors 1 protein, desmoglein-3, calsyntenin-1, immunoglobulin lambda constant 3, complement C5, alpha-2-macroglobulin, myosin-9, sodium / potassium-transporting ATPase subunit gamma, immunoglobulin kappa variable 2-28, oncoprotein-induced transcript 3 protein, serglycin, coagulation factor XII, coagulation factor XIIIA chain, insulin, histidine-rich glycoprotein, immunoglobulin kappa variable 3-11, immunoglobulin kappa variable 1-39, collagen alpha-l(I) chain, inter-alpha-trypsin inhibitor heavy chain H5, latent-transforming growth factor beta-binding protein 2, integrin alpha-IIb, membrane-associated progesterone receptor component 1, immunoglobulin lambda variable 6-57, immunoglobulin kappa variable 3-15, complement Clr subcomponent-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 inhibitory polypeptide, immunoglobulin heavy variable 3-15, immunoglobulin lambda variable 2-11, transcription initiation factor TFIID subunit 1, collagen alpha-l(VII) chain, integral membrane protein 2B, pigment epithelium-derived factor, voltage-dependent N-type calcium channel subunit alpha-lB, immunoglobulin lambda variable 3-27, ras GTPase-activating protein nGAP, keratin, type I cytoskeletal 17, tubulin beta chain, sulfhydryl oxidase 1, immunoglobulin kappa variable 4-1, complement Clr subcomponent, 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 comprises a sequence set forth in Columns II-VI of Table A (or a subset thereol). In some embodiments, the reporter polypeptide is selected from the group set forth in Column I of Table A (or a subset thereol).

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

[0013] In some embodiments of the method for assessing the likelihood of the subject being responsive to the therapeutic agent, the disease or condition is a cancer or an inflammatory or autoimmune disease. In some embodiments, the disease or condition is selected from the group consisting of carcinoma, Hodgkin’s lymphoma, and non-Hodgkin’s lymphoma, diffuse large B cell lymphoma, follicular lymphoma, mantle cell lymphoma, blastoma, breast cancer, ER / PR+ breast cancer, Her2+ breast cancer, triple-negative breast cancer, colon cancer, colon cancer with malignant ascites, mucinous tumors, prostate cancer, head and neck cancer, skin cancer, melanoma, genito-urinary tract cancer, ovarian cancer, ovarian cancer with malignant ascites, peritoneal carcinomatosis, uterine serous carcinoma, endometrial cancer, cervix cancer, colorectal, uterine cancer, mesothelioma in the peritoneum, kidney cancer, Wilm’s tumor, lung cancer, small-cell lung cancer, non-small cell lung cancer, gastric cancer, stomach cancer, small intestine cancer, liver cancer, hepatocarcinoma, hepatoblastoma, liposarcoma, pancreatic cancer, gall bladder cancer, cancers of the bile duct, esophageal cancer, salivary gland carcinoma, thyroid cancer, epithelial cancer, arrhenoblastoma, adenocarcinoma, sarcoma, and B-cell derived chronic lymphatic leukemia. In some embodiments, the disease or condition is selected from the group consisting of ankylosing spondylitis (AS), arthritis (for example, and 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-Barre syndrome (GBS), Hashimoto’s disease, suppurative scab, Kawasaki disease, IgA nephropathy, idiopathic thrombocytopenic purpura, inflammatory bowel disease (IBD) (for example, and not limited to, Crohn’s disease (CD), clonal disease, ulcerative colitis, collagen colitis, lymphocytic colitis, ischemic colitis, empty colitis, Behcet’s syndrome, infectious colitis, indeterminate colitis, interstitial Cystitis), lupus (for example, and not limited to, systemic lupus erythematosus, discoid lupus, subacute cutaneous lupus erythematosus, cutaneous lupus erythematosus (such as chilblain lupus erythematosus), drug-induced lupus, neonatal lupus, lupus nephritis), mixed connective tissue disease, morphea, multiple sclerosis (MS), severe muscle Force disorder, narcolepsy, neuromuscular angina, pemphigus vulgaris, pernicious anemia, psoriasis, psoriatic arthritis, polymyositis, primary biliary cirrhosis, relapsing polychondritis, schizophrenia, scleroderma, Sjogren’s syndrome, systemic stiffness syndrome, temporal arteritis (also known as giant cell arteritis), vasculitis, vitiligo, Wegener’s granulomatosis, transplant rejection-associated immune reaction(s) (for example, and not limited to, renal transplant rejection, lung transplant rejection, liver transplant rejection), psoriasis, Wiskott-Aldrich syndrome, autoimmune lymphoproliferative syndrome, myasthenia gravis, inflammatory chronic rhinosinusitis, colitis, celiac disease, Barrett’s esophagus, inflammatory gastritis, autoimmune nephritis, autoimmune hepatitis, autoimmune carditis, autoimmune encephalitis, autoimmune mediated hematological disease, asthma, atopic dermatitis, atopy, allergy, allergic rhinitis, scleroderma, bronchitis, pericarditis, 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, systemic inflammatory response syndrome.

[0014] In some embodiments of the method for assessing the likelihood of the subject being responsive to the therapeutic agent, the therapeutic agent is an anti-cancer agent. In some embodiments, the therapeutic agent is an activatable therapeutic agent. In some embodiments, the therapeutic agent is an activatable therapeutic agent, or non-natural, activatable therapeutic agent as described herein.

[0015] In some embodiments of the method for assessing the likelihood of the subject being responsive to the therapeutic agent, the therapeutic agent further comprises a masking moiety (MM). In some embodiments of the method for assessing the likelihood of the subject being responsive to the therapeutic agent, the masking moiety (MM) is capable of being released from the therapeutic agent upon cleavage of the peptide substrate by the mammalian protease. In some embodiments, the masking moiety (MM) interferes with an interaction of the therapeutic agent, in an uncleaved state, to a target tissue or cell. In some embodiments, a bioactivity of the therapeutic agent is capable of being enhanced upon cleavage of the peptide substrate by the mammalian protease. In some embodiments, the masking moiety (MM) is an extended recombinant polypeptide (XTEN). In some embodiments, the XTEN is characterized in that: (i) it comprises at least 100 amino acids; (ii) at least 90% of the amino acid residues of it are selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P); and (iii) it comprises at least 4 different types of amino acids selected from G, A, S, T, E, and P.

[0016] In some embodiments of the method for assessing the likelihood of the subject being responsive to the therapeutic agent, further comprises transmitting the designation to a healthcare provider and / or the subject.

[0017] In some embodiments of the method for assessing the likelihood of the subject being responsive to the therapeutic agent, further comprises, subsequent to (b), contacting the therapeutic agent with the mammalian protease.

[0018] In some embodiments of the method for assessing the likelihood of the subject being responsive to the therapeutic agent, further comprises, subsequent to (b), administering to the subject an effective amount of the therapeutic agent based on the designation of step (b).

[0019] In some embodiments of the method for assessing the likelihood of the subject being responsive to the therapeutic agent, (a) comprises detecting the polypeptide of (i), (ii) or (iii) in an immuno-assay. In some embodiments, the immuno-assay utilizes an antibody that specifically binds to the polypeptide of (i), (ii) or (iii), or an epitope thereof.

[0020] In some embodiments of the method for assessing the likelihood of the subject being responsive to the therapeutic agent, (a) comprises detecting the polypeptide of (i), (ii) or (iii) (or a derivative (including fragment(s)) thereol) by using a mass spectrometer (MS)

[0021] In some embodiment of the method is use of a diagnostic reagent for assessing a likelihood of a subject being responsive to a therapeutic agent that is activatable by a mammalian protease expressed in said subject having a disease or disorder.

[0022] In certain aspects the diagnostic reagent is used for assessing a likelihood of a subject being responsive to a therapeutic agent that is activatable by a mammalian protease expressed in said subject having a disease or disorder.

[0023] In some embodiments is a kit for the practice of a method for assessing a likelihood of a subject being responsive to a therapeutic agent that is activatable by a mammalian protease expressed in said subject having a disease or disorder comprising a reagent for detecting the presence or amount of a proteolytic peptide product produced by action of said mammalian protease.

[0024] In certain aspects, the present disclosure provides a method for treating a subject in need of a therapeutic agent that is activatable by a mammalian protease expressed in the subject, the method comprising: administering an effective amount of the therapeutic agent to the subject, wherein the subject has been shown to express in a biological sample from the subject: (i) a polypeptide comprising at least five, at least six, at least seven, at least eight, at least nine, or at least ten consecutive amino acid residues shown in a sequence set forth in Column V of Table A (or a subset thereol); or (ii) a polypeptide comprising at least five, at least six, at least seven, at least eight, at least nine, or at least ten consecutive amino acids shown in a sequence set forth in Column IV of Table A (or a subset thereol); or (iii) a polypeptide comprising at least five, at least six, at least seven, at least eight, at least nine, or at least ten consecutive amino acids shown in a sequence set forth in Column VI of Table A (or a subset thereof); or (iv) expression level of polypeptide (i), (ii) or (iii) exceeds a threshold.

[0025] In some embodiments for treating the subject with the 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 shown in a sequence set forth in Column V of Table A (or a subset thereof), tn 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 shown in a sequence set forth in Column IV of Table A (or a subset thereof), tn 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 shown in a sequence set forth in Column VI of Table A (or a subset thereof), tn some embodiments, the subject has been shown to express in the biological sample any two of (i)-(iii). In some embodiments, the subject has been shown to express in the biological sample all three of (i)-(iii).

[0026] In some embodiments for treating the subject with the therapeutic agent, the therapeutic agent comprises a peptide substrate susceptible to cleavage by the mammalian protease. In some embodiments, the peptide substrate is susceptible to cleavage by the mammalian protease at a scissile bond, and wherein the polypeptide of (i), (ii), or (iii) comprises a portion containing at least four consecutive amino acid residues of the peptide substrate that is either N-terminal or C-terminal of the scissile bond. In some embodiments, a portion of the peptide substrate that is N-terminal of the scissile bond has at most three or two amino acid substitutions or at most one amino acid substitution with respect to a C-terminal end sequence containing from four to ten amino acid residues of a sequence set forth in Column IV or V of Table A (or a subset thereof), wherein none of the amino acid substitution is at a position corresponding to an amino acid residue immediately adjacent to a corresponding scissile bond. In some embodiments, a portion of the peptide substrate that is N-terminal of the scissile bond has at most three or two amino acid substitutions or at most one amino acid substitution with respect to a C-terminal end sequence containing from four to ten amino acid residues of a sequence set forth in Column IV of Table A (or a subset thereof), wherein none of the amino acid substitution is at a position corresponding to an amino acid residue immediately adjacent to a corresponding scissile bond. In some embodiments, a portion of the peptide substrate that is N-terminal of the scissile bond has at most three or two amino acid substitutions or at most one amino acid substitution with respect to a C-terminal end sequence containing from four to ten amino acid residues of a sequence set forth in Column V of Table A (or a subset thereof), wherein none of the amino acid substitution is at a position corresponding to an amino acid residue immediately adjacent to a corresponding scissile bond. In some embodiments, the portion of the peptide substrate that is N-terminal of the scissile bond comprises a C-terminal end sequence containing from four to ten amino acid residues of a sequence set forth in Column IV or V of Table A (or a subset thereof), tn some embodiments, the portion of the peptide substrate that is N-terminal of the scissile bond comprises a C-terminal end sequence containing from four to ten amino acid residues of a sequence set forth in Column IV of Table A (or a subset thereof). In some embodiments, the portion of the peptide substrate that is N-terminal of the scissile bond comprises a C-terminal end sequence containing from four to ten amino acid residues of a sequence set forth in Column V of Table A (or a subset thereof), tn some embodiments, a portion of the peptide substrate that is C-terminal of the scissile bond has at most three or two amino acid substitutions or at most one amino acid substitution with respect to an N-terminal end sequence containing from four to ten amino acid residues of a sequence set forth in Column V or VI of Table A (or a subset thereof), wherein none of the amino acid substitution is at a position corresponding to an amino acid residue immediately adjacent to a corresponding scissile bond. In some embodiments, a portion of the peptide substrate that is C-terminal of the scissile bond has at most three or two amino acid substitutions or at most one amino acid substitution with respect to an N-terminal end sequence containing from four to ten amino acid residues of a sequence set forth in Column V of Table A (or a subset thereof), wherein none of the amino acid substitution is at a position corresponding to an amino acid residue immediately adjacent to a corresponding scissile bond. In some embodiments, a portion of the peptide substrate that is C-terminal of the scissile bond has at most three or two amino acid substitutions or at most one amino acid substitution with respect to an N-terminal end sequence containing from four to ten amino acid residues of a sequence set forth in Column VI of Table A (or a subset thereof), wherein none of the amino acid substitution is at a position corresponding to an amino acid residue immediately adjacent to a corresponding scissile bond. In some embodiments, the portion of the peptide substrate that is C-terminal of the scissile bond comprises an N-terminal end sequence containing from four to ten amino acid residues of a sequence set forth in Column V or VI of Table A (or a subset thereof), tn some embodiments, the portion of the peptide substrate that is C-terminal of the scissile bond comprises an N-terminal end sequence containing from four to ten amino acid residues of a sequence set forth in Column V of Table A (or a subset thereof), tn some embodiments, the portion of the peptide substrate that is C-terminal of the scissile bond comprises an N-terminal end sequence containing from four to ten amino acid residues of a sequence set forth in Column VI of Table A (or a subset thereof).

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

[0028] In some embodiments for treating the subject with the therapeutic agent, the mammalian protease is a serine protease, a cysteine protease, an aspartate protease, a threonine protease, or a metalloproteinase. In some embodiments, the mammalian protease is selected from the group consisting of disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), disintegrin and metalloproteinase domain-containing protein 12 (ADAM12), disintegrin and metalloproteinase domaincontaining protein 15 (ADAM 15), disintegrin and metalloproteinase domain-containing protein 17 (ADAM17), disintegrin and metalloproteinase domain-containing protein 9 (ADAM9), disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), Cathepsin B, Cathepsin D, Cathepsin E, Cathepsin K, cathepsin L, cathepsin S, Fibroblast activation protein alpha, Hepsin, kallikrein-2, kallikrein-4, kallikrein-3, Prostate-specific antigen (PSA), kallikrein-13, Legumain, 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 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 type serine protease 1 (MT-SP1), matriptase, 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), legumain, and matriptase. 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 or is co-localized with the mammalian protease.

[0029] In some embodiments for treating the subject with the therapeutic agent, the target tissue or cell contains therein or thereon, or is associated with in proximity thereto, a reporter polypeptide. In some embodiments, the reporter polypeptide is a polypeptide selected from the group consisting of 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, epididymis secretory sperm binding protein, secretogranin-2, angiotensinogen, transgelin-2, pancreatic prohormone, neurosecretory protein VGF, ceruloplasmin, PDZ and LIM domain protein 1, multimerin-1, inter-alpha-trypsin inhibitor heavy chain H2, N-acetylmuramoyl-L-alanine amidase, histone Hl.4, adhesion G-protein coupled receptor G6, mannan-binding lectin serine protease 2, prothrombin, deleted in malignant brain tumors 1 protein, desmoglein-3, calsyntenin-1, alpha-2-macroglobulin, myosin-9, sodium / potassium-transporting ATPase subunit gamma, oncoprotein-induced transcript 3 protein, serglycin, histidine-rich glycoprotein, inter-alpha-trypsin inhibitor heavy chain H5, integrin alpha-IIb, membrane-associated progesterone receptor component 1, histone Hl.2, rho GDP-dissociation inhibitor 2, zinc-alpha-2-glycoprotein, talin-1, secretogranin-1, neutrophil defensin 3, cytochrome P450 2E1, gastric inhibitory polypeptide, transcription initiation factor TFIID subunit 1, integral membrane protein 2B, pigment epithelium-derived factor, voltage-dependent N-type calcium channel subunit alpha-lB, ras GTPase-activating protein nGAP, type I cytoskeletal 17, sulfhydryl oxidase 1, homeobox protein Hox-B2, transcription factor SOX-10, E3 ubiquitin-protein ligase SIAH2, decorin, secreted protein acidic and rich in cysteine (SPARC), laminin gamma 1 chain, vimentin, and nidogen-1 (NIDI). In some embodiments, the reporter polypeptide is a polypeptide selected from the group consisting of 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-l 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, epididymis secretory sperm binding protein, zyxin, apolipoprotein C-III, secretogranin-2, angiotensinogen, c-reactive protein, serum albumin, transgelin-2, pancreatic prohormone, neurosecretory protein VGF, ceruloplasmin, PDZ and LIM domain protein 1, tubulin alpha-4 A chain, multimerin-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 Hl.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, probable non-functional immunoglobulin kappa variable 2D-24, prothrombin, coagulation factor IX, apolipoprotein LI, deleted in malignant brain tumors 1 protein, desmoglein-3, calsyntenin-1, immunoglobulin lambda constant 3, complement C5, alpha-2-macroglobulin, myosin-9, sodium / potassium-transporting ATPase subunit gamma, immunoglobulin kappa variable 2-28, oncoprotein-induced transcript 3 protein, serglycin, coagulation factor XII, coagulation factor XIIIA chain, insulin, histidine-rich glycoprotein, immunoglobulin kappa variable 3-11, immunoglobulin kappa variable 1-39, collagen alpha-l(I) chain, inter-alpha-trypsin inhibitor heavy chain H5, latent-transforming growth factor beta-binding protein 2, integrin alpha-IIb, membrane-associated progesterone receptor component 1, immunoglobulin lambda variable 6-57, immunoglobulin kappa variable 3-15, complement Clr subcomponent-like protein, histone Hl.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 inhibitory polypeptide, immunoglobulin heavy variable 3-15, immunoglobulin lambda variable 2-11, transcription initiation factor TFIID subunit 1, collagen alpha-l(VII) chain, integral membrane protein 2B, pigment epithelium-derived factor, voltage-dependent N-type calcium channel subunit alpha-IB, immunoglobulin lambda variable 3-27, ras GTPase-activating protein nGAP, keratin, type I cytoskeletal 17, tubulin beta chain, sulfhydryl oxidase 1, immunoglobulin kappa variable 4-1, complement Clr subcomponent, 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 comprises a sequence set forth in Columns II-VI of Table A (or a subset thereof). In some embodiments, the reporter polypeptide is selected from the group set forth in Column I of Table A (or a subset thereof).

[0030] tn some embodiments for treating the subject with the therapeutic agent, the target tissue or cell is characterized by an increased amount or activity of the mammalian protease in proximity to the target tissue or cell as compared to a non-target tissue or cell in the subject. In some embodiments, the subject is suffering from, or is suspected of suffering from, a disease or condition characterized by an increased expression or activity of the mammalian protease in proximity to a target tissue or cell as compared to a corresponding non-target tissue or cell in the subject. In some embodiments, the disease or condition is a cancer or an inflammatory or autoimmune disease, in some embodiments, the disease or condition is selected from the group consisting of ankylosing spondylitis (AS), arthritis (for example, and 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-Barre syndrome (GBS), Hashimoto’s disease, suppurative scab, Kawasaki disease, IgA nephropathy, idiopathic thrombocytopenic purpura, inflammatory bowel disease (IBD) (for example, and not limited to, Crohn’s disease (CD), clonal disease, ulcerative colitis, collagen colitis, lymphocytic colitis, ischemic colitis, empty colitis, Behcet’s syndrome, infectious colitis, indeterminate colitis, interstitial Cystitis), lupus (for example, and not limited to, systemic lupus erythematosus, discoid lupus, subacute cutaneous lupus erythematosus, cutaneous lupus erythematosus (such as chilblain lupus erythematosus), drug-induced lupus, neonatal lupus, lupus nephritis), mixed connective tissue disease, morphea, multiple sclerosis (MS), severe muscle Force disorder, narcolepsy, neuromuscular angina, pemphigus vulgaris, pernicious anemia, psoriasis, psoriatic arthritis, polymyositis, primary biliary cirrhosis, relapsing polychondritis, schizophrenia, scleroderma, Sjogren’s syndrome, systemic stiffness syndrome, temporal arteritis (also known as giant cell arteritis), vasculitis, vitiligo, Wegener’s granulomatosis, transplant rejection-associated immune reaction(s) (for example, and not limited to, renal transplant rejection, lung transplant rejection, liver transplant rejection), psoriasis, Wiskott-Aldrich syndrome, autoimmune lymphoproliferative syndrome, myasthenia gravis, inflammatory chronic rhinosinusitis, colitis, celiac disease, Barrett’s esophagus, inflammatory gastritis, autoimmune nephritis, autoimmune hepatitis, autoimmune carditis, autoimmune encephalitis, autoimmune mediated hematological disease, asthma, atopic dermatitis, atopy, allergy, allergic rhinitis, scleroderma, bronchitis, pericarditis, 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, systemic inflammatory response syndrome. In some embodiments, the disease or condition is selected from the group consisting of carcinoma, Hodgkin’s lymphoma, and non-Hodgkin’s lymphoma, diffuse large B cell lymphoma, follicular lymphoma, mantle cell lymphoma, blastoma, breast cancer, ER / PR+ breast cancer, Her2+ breast cancer, triple-negative breast cancer, colon cancer, colon cancer with malignant ascites, mucinous tumors, prostate cancer, head and neck cancer, skin cancer, melanoma, genito urinary tract cancer, ovarian cancer, ovarian cancer with malignant ascites, peritoneal carcinomatosis, uterine serous carcinoma, endometrial cancer, cervix cancer, colorectal, uterine cancer, mesothelioma in the peritoneum, kidney cancer, Wilm’s tumor, lung cancer, small-cell lung cancer, non-small cell lung cancer, gastric cancer, stomach cancer, small intestine cancer, liver cancer, hepatocarcinoma, hepatoblastoma, liposarcoma, pancreatic cancer, gall bladder cancer, cancers of the bile duct, esophageal cancer, salivary gland carcinoma, thyroid cancer, epithelial cancer, arrhenoblastoma, adenocarcinoma, sarcoma, and B-cell derived chronic lymphatic leukemia. In some embodiments, the therapeutic agent is an anti-cancer 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 as described herein.

[0031] In some embodiments for treating the subject with the therapeutic agent, the therapeutic agent comprises a masking moiety (MM). In some embodiments, the masking moiety (MM) is capable of being released from the therapeutic agent upon cleavage of the peptide substrate by the mammalian protease. In some embodiments, the masking moiety (MM) interferes with an interaction of the therapeutic agent, in an uncleaved state, to a target tissue or cell. In some embodiments, a bioactivity of the therapeutic agent is capable of being enhanced upon cleavage of the peptide substrate by the mammalian protease. In some embodiments, the masking moiety (MM) is an extended recombinant polypeptide (XTEN). In some embodiments, the XTEN is characterized in that: (i) it comprises at least 100 amino acids; (ii) at least 90% of the amino acid residues of it are selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P); and (iii) it comprises at least 4 different types of amino acids selected from G, A, S, T, E, and P.

[0032] In some embodiments for treating the subject with the therapeutic agent, the subject is determined to have a likelihood of a response to the therapeutic agent by a method as described herein.

[0033] In certain aspects, the present disclosure provides a method for treating a disease or condition in a subject, comprising administering to the subject in need thereof one or more therapeutically effective doses of a therapeutic agent as described herein, or a pharmaceutical composition as described herein.

[0034] In some embodiments for the method for treating the disease or condition in the subject, the subject is selected from the group consisting of mouse, rat, monkey, and human. In some embodiments, the subject is a human. In some embodiments, the subject is determined to have a likelihood of a response to the therapeutic agent or the pharmaceutical composition. In some embodiments, the likelihood of the response is 50% or higher. In some embodiments, the likelihood of the response is determined by a method as described herein.

[0035] In some embodiments for the method for treating the disease or condition in the subject, the disease or condition is a cancer or an inflammatory or autoimmune disease. In some embodiments, the disease or condition is selected from the group consisting of ankylosing spondylitis (AS), arthritis (for example, and 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-Barre syndrome (GBS), Hashimoto’s disease, suppurative scab, Kawasaki disease, IgA nephropathy, idiopathic thrombocytopenic purpura, inflammatory bowel disease (IBD) (for example, and not limited to, Crohn’s disease (CD), clonal disease, ulcerative colitis, collagen colitis, lymphocytic colitis, ischemic colitis, empty colitis, Behcet’s syndrome, infectious colitis, indeterminate colitis, interstitial Cystitis), lupus (for example, and not limited to, systemic lupus erythematosus, discoid lupus, subacute cutaneous lupus erythematosus, cutaneous lupus erythematosus (such as chilblain lupus erythematosus), drug-induced lupus, neonatal lupus, lupus nephritis), mixed connective tissue disease, morphea, multiple sclerosis (MS), severe muscle Force disorder, narcolepsy, neuromuscular angina, pemphigus vulgaris, pernicious anemia, psoriasis, psoriatic arthritis, polymyositis, primary biliary cirrhosis, relapsing polychondritis, schizophrenia, scleroderma, Sjogren’s syndrome, systemic stiffness syndrome, temporal arteritis (also known as giant cell arteritis), vasculitis, vitiligo, Wegener’s granulomatosis, transplant rejection-associated immune reaction(s) (for example, and not limited to, renal transplant rejection, lung transplant rejection, liver transplant rejection), psoriasis, Wiskott-Aldrich syndrome, autoimmune lymphoproliferative syndrome, myasthenia gravis, inflammatory chronic rhinosinusitis, colitis, celiac disease, Barrett’s esophagus, inflammatory gastritis, autoimmune nephritis, autoimmune hepatitis, autoimmune carditis, autoimmune encephalitis, autoimmune mediated hematological disease, asthma, atopic dermatitis, atopy, allergy, allergic rhinitis, scleroderma, bronchitis, pericarditis, 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, systemic inflammatory response syndrome. In some embodiments, the disease or condition is selected from the group consisting of carcinoma, Hodgkin’s lymphoma, and non-Hodgkin’s lymphoma, diffuse large B cell lymphoma, follicular lymphoma, mantle cell lymphoma, blastoma, breast cancer, ER / PR+ breast cancer, Her2+ breast cancer, triple-negative breast cancer, colon cancer, colon cancer with malignant ascites, mucinous tumors, prostate cancer, head and neck cancer, skin cancer, melanoma, genito-urinary tract cancer, ovarian cancer, ovarian cancer with malignant ascites, peritoneal carcinomatosis, uterine serous carcinoma, endometrial cancer, cervix cancer, colorectal, uterine cancer, mesothelioma in the peritoneum, kidney cancer, Wilm’s tumor, lung cancer, small-cell lung cancer, nonsmall cell lung cancer, gastric cancer, stomach cancer, small intestine cancer, liver cancer, hepatocarcinoma, hepatoblastoma, liposarcoma, pancreatic cancer, gall bladder cancer, cancers of the bile duct, esophageal cancer, salivary gland carcinoma, thyroid cancer, epithelial cancer, arrhenoblastoma, adenocarcinoma, sarcoma, and B-cell derived chronic lymphatic leukemia.

[0036] In certain aspects, the present disclosure provides use of a therapeutic agent as described herein in the preparation of a medicament for the treatment of a disease or condition in a subject.

[0037] In certain aspects, the present disclosure provides use of a pharmaceutical composition as described herein in the preparation of a medicament for the treatment of a disease or condition in a subject.

[0038] In some embodiments of the use, the subject is selected from the group consisting of mouse, rat, monkey, and human. In some embodiments, the subject is a human. In some embodiments, the subject is determined to have a likelihood of a response to the therapeutic agent or the pharmaceutical composition. In some embodiments, the likelihood of the response is 50% or higher. In some embodiments, the likelihood of the response is determined by a method as described herein.

[0039] In some embodiments of the use, the disease or condition is a cancer or an inflammatory or autoimmune disease. In some embodiments, the disease or condition is selected from the group consisting of carcinoma, Hodgkin’s lymphoma, and non-Hodgkin’s lymphoma, diffuse large B cell lymphoma, follicular lymphoma, mantle cell lymphoma, blastoma, breast cancer, ER / PR+ breast cancer, Her2+ breast cancer, triple-negative breast cancer, colon cancer, colon cancer with malignant ascites, mucinous tumors, prostate cancer, head and neck cancer, skin cancer, melanoma, genito-urinary tract cancer, ovarian cancer, ovarian cancer with malignant ascites, peritoneal carcinomatosis, uterine serous carcinoma, endometrial cancer, cervix cancer, colorectal, uterine cancer, mesothelioma in the peritoneum, kidney cancer, Wilm’s tumor, lung cancer, small-cell lung cancer, non-small cell lung cancer, gastric cancer, stomach cancer, small intestine cancer, liver cancer, hepatocarcinoma, hepatoblastoma, liposarcoma, pancreatic cancer, gall bladder cancer, cancers of the bile duct, esophageal cancer, salivary gland carcinoma, thyroid cancer, epithelial cancer, arrhenoblastoma, adenocarcinoma, sarcoma, and B-cell derived chronic lymphatic leukemia. In some embodiments, the disease or condition is selected from the group consisting of ankylosing spondylitis (AS), arthritis (for example, and 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-Barre syndrome (GBS), Hashimoto’s disease, suppurative scab, Kawasaki disease, IgA nephropathy, idiopathic thrombocytopenic purpura, inflammatory bowel disease (IBD) (for example, and not limited to, Crohn’s disease (CD), clonal disease, ulcerative colitis, collagen colitis, lymphocytic colitis, ischemic colitis, empty colitis, Behcet’s syndrome, infectious colitis, indeterminate colitis, interstitial Cystitis), lupus (for example, and not limited to, systemic lupus erythematosus, discoid lupus, subacute cutaneous lupus erythematosus, cutaneous lupus erythematosus (such as chilblain lupus erythematosus), drug-induced lupus, neonatal lupus, lupus nephritis), mixed connective tissue disease, morphea, multiple sclerosis (MS), severe muscle Force disorder, narcolepsy, neuromuscular angina, pemphigus vulgaris, pernicious anemia, psoriasis, psoriatic arthritis, polymyositis, primary biliary cirrhosis, relapsing polychondritis, schizophrenia, scleroderma, Sjogren’s syndrome, systemic stiffness syndrome, temporal arteritis (also known as giant cell arteritis), vasculitis, vitiligo, Wegener’s granulomatosis, transplant rejection-associated immune reaction(s) (for example, and not limited to, renal transplant rejection, lung transplant rejection, liver transplant rejection), psoriasis, Wiskott-Aldrich syndrome, autoimmune lymphoproliferative syndrome, myasthenia gravis, inflammatory chronic rhinosinusitis, colitis, celiac disease, Barrett’s esophagus, inflammatory gastritis, autoimmune nephritis, autoimmune hepatitis, autoimmune carditis, autoimmune encephalitis, autoimmune mediated hematological disease, asthma, atopic dermatitis, atopy, allergy, allergic rhinitis, scleroderma, bronchitis, pericarditis, 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, systemic inflammatory response syndrome.

[0040] In some aspects, the present disclosure provides a therapeutic agent (e.g., activatable therapeutic agent, or non-natural, activatable therapeutic agent) comprising a release segment (RS) linked, directly or indirectly, to a biologically active moiety (BM), wherein the RS comprises a peptide substrate having an amino acid sequence susceptible to cleavage by a mammalian protease at a scissile bond, wherein the peptide substrate comprises an amino acid sequence having at most three amino acid substitutions (or at most two amino acid substitutions, or at most one amino acid substitution) with respect to a sequence set forth in Column II or III of Table A (or a subset thereof).

[0041] In some aspects, the present disclosure provides a therapeutic agent (e.g., activatable therapeutic agent, or non-natural, activatable therapeutic agent) comprising a release segment (RS) linked, directly or indirectly, to a biologically active moiety (BM), wherein the RS comprises a peptide substrate having an amino acid sequence susceptible to cleavage by a mammalian protease at a scissile bond, wherein the therapeutic agent is configured for activation at or in proximity to a target tissue or cell in a subject, wherein the target tissue or cell contains therein or thereon, or is associated with in proximity thereto, a reporter sequence capable of being cleaved by the mammalian protease at a cleavage sequence, and wherein the peptide substrate comprises an amino acid sequence having at most three amino acid substitutions (or at most two amino acid substitutions, or at most one amino acid substitution) with respect to the cleavage sequence of the 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, epididymis secretory sperm binding protein, secretogranin-2, angiotensinogen, transgelin-2, pancreatic prohormone, neurosecretory protein VGF, ceruloplasmin, PDZ and LIM domain protein 1, multimerin-1, inter-alpha-trypsin inhibitor heavy chain H2, N-acetylmuramoyl-L-alanine amidase, histone Hl.4, adhesion G-protein coupled receptor G6, mannan-binding lectin serine protease 2, prothrombin, deleted in malignant brain tumors 1 protein, desmoglein-3, calsyntenin-1, alpha-2-macroglobulin, myosin-9, sodium / potassium-transporting ATPase subunit gamma, oncoprotein-induced transcript 3 protein, serglycin, histidine-rich glycoprotein, inter-alpha-trypsin inhibitor heavy chain H5, integrin alpha-IIb, membrane-associated progesterone receptor component 1, histone Hl.2, rho GDP-dissociation inhibitor 2, zinc-alpha-2-glycoprotein, talin-1, secretogranin-1, neutrophil defensin 3, cytochrome P450 2E1, gastric inhibitory polypeptide, transcription initiation factor TFIID subunit 1, integral membrane protein 2B, pigment epithelium-derived factor, voltage-dependent N-type calcium channel subunit alpha-lB, ras GTPase-activating protein nGAP, type I cytoskeletal 17, sulfhydryl oxidase 1, homeobox protein Hox-B2, transcription factor SOX-10, E3 ubiquitin-protein ligase SIAH2, decorin, secreted protein acidic and rich in cysteine (SPARC), laminin gamma 1 chain, vimentin, and nidogen-1 (NIDI).

[0043] In some embodiments of the therapeutic agent, the reporter polypeptide is a polypeptide selected from the group consisting of 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-l 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, epididymis secretory sperm binding protein, zyxin, apolipoprotein C-III, secretogranin-2, angiotensinogen, c-reactive protein, serum albumin, transgelin-2, pancreatic prohormone, neurosecretory protein VGF, ceruloplasmin, PDZ and LIM domain protein 1, tubulin alpha-4 A chain, multimerin-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 Hl.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, probable nonfunctional immunoglobulin kappa variable 2D-24, prothrombin, coagulation factor IX, apolipoprotein LI, deleted in malignant brain tumors 1 protein, desmoglein-3, calsyntenin-1, immunoglobulin lambda constant 3, complement C5, alpha-2-macroglobulin, myosin-9, sodium / potassium-transporting ATPase subunit gamma, immunoglobulin kappa variable 2-28, oncoprotein-induced transcript 3 protein, serglycin, coagulation factor XII, coagulation factor XIII A chain, insulin, histidine-rich glycoprotein, immunoglobulin kappa variable 3-11, immunoglobulin kappa variable 1-39, collagen alpha-l(I) chain, inter-alpha-trypsin inhibitor heavy chain H5, latent-transforming growth factor beta-binding protein 2, integrin alpha-IIb, membrane-associated progesterone receptor component 1, immunoglobulin lambda variable 6-57, immunoglobulin kappa variable 3-15, complement Clr subcomponent-like protein, histone Hl.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 inhibitory polypeptide, immunoglobulin heavy variable 3-15, immunoglobulin lambda variable 2-11, transcription initiation factor TFIID subunit 1, collagen alpha-1 (VII) chain, integral membrane protein 2B, pigment epithelium-derived factor, voltage-dependent N-type calcium channel subunit alpha-lB, immunoglobulin lambda variable 327, ras GTPase-activating protein nGAP, keratin, type I cytoskeletal 17, tubulin beta chain, sulfhydryl oxidase 1, immunoglobulin kappa variable 4-1, complement Clr subcomponent, 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 set forth in Column II or III of Table A (or a subset thereol). In some embodiments, the cleavage sequence does not comprise a methionine residue immediately N-terminal to a scissile bond (contained therein), when the methionine is the first residue at N terminus of the reporter polypeptide. In some embodiments, the target tissue or cell is characterized by an increased amount or activity of the mammalian protease in proximity to the target tissue or cell as compared to a non-target tissue or cell in the subject. In some embodiments, the mammalian proatease is produced at the target tissue or cell. In some embodiments, the peptide substrate comprises an amino acid sequence having at most three amino acid substitutions, or at most two amino acid substitutions, or at most one amino acid substitution with respect to a sequence set forth in Column II or III of Table A (or a subset thereol). In some embodiments, the peptide substrate comprises an amino acid sequence having at most three amino acid substitutions with respect to a sequence set forth in Column II or III of Table A (or a subset thereol). In some embodiments, the scissile bond is not immediately C-terminal to a methionine residue.

[0045] In some embodiments of the therapeutic agent, the peptide substrate contains from six to twenty-five or six to twenty amino acid residues. In some embodiments of the therapeutic agent, the peptide substrate contains from six to twenty-five amino acid residues. In some embodiments of the therapeutic agent, the peptide substrate contains from six to twenty amino acid residues. In some embodiments, the peptide substrate contains from seven to twelve amino acid residues. In some embodiments, the peptide substrate comprises an amino acid sequence having at most two amino acid substitutions with respect to a sequence set forth in Column II or III of Table A (or a subset thereol). In some embodiments, the peptide substrate comprises an amino acid sequence having at most one amino acid substitution with respect to a sequence set forth in Column II or III of Table A (or a subset thereol). In some embodiments, none of the at most three amino acid substitutions, or the at most two amino acid substitutions, or the at most one amino acid substitution is at a position corresponding to an amino acid residue immediately adjacent to a corresponding scissile bond of the corresponding sequence shown in Column II or III of Table A (or a subset thereol). In some embodiments, the peptide substrate comprises an amino acid sequence identical to a sequence set forth in Column II or III of Table A (or a subset thereol). In some embodiments, the peptide substrate does not comprise a methionine residue immediately N-terminal to a scissile bond (contained therein). In some embodiments, the peptide substrate does not comprise 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 thereol) of Column II of Table A. In some embodiments, the peptide substrate comprises two or three sequences set forth in Column II or III of Table A (or a subset thereol). In some embodiments, where the peptide substrate comprises two sequences set forth in Column II or III of Table A (or a subset thereol), the two sequences partially overlap one another. In some embodiments, where the peptide substrate comprises two sequences set forth in Column II or III of Table A (or a subset thereof), the two sequences do not overlap one another. In some embodiments, where the peptide substrate comprises three sequences set forth in Column II or III of Table A (or a subset thereof), two or all of the three sequences do not overlap one another. In some embodiments, where the peptide substrate comprises three sequences set forth in Column II or III of Table A (or a subset thereof), one of the three sequences partially overlaps with another sequence or both other sequences of the three sequences. In some embodiments, where the peptide substrate comprises three sequences set forth in Column II or III of Table A (or a subset thereof), two of the three sequences partially overlap with one another. In some embodiments, where the peptide substrate comprises three sequences set forth in Column II or III of Table A (or a subset thereof), each two of the three sequences partially overlap with one another. In some embodiments, where the peptide substrate comprises three sequences set forth in Column II or III of Table A (or a subset thereof), all of the three sequences partially overlap with one another. In some embodiments, the peptide substrate susceptible to cleavage by the mammalian protease is susceptible to cleavage by a plurality of mammalian proteases comprising the mammalian protease. In some embodiments, the peptide substrate susceptible to cleavage by the plurality of mammalian proteases has at most three amino acid substitutions, or at most two amino acid substitutions, or at most one amino acid substitution with respect to a sequence set forth in Table l(j). In some embodiments, the peptide substrate susceptible to cleavage by the plurality of mammalian proteases has at most three amino acid substitutions with respect to a sequence set forth in Table l(j). In some embodiments, the peptide substrate susceptible to cleavage by the plurality of mammalian proteases has at most two amino acid substitutions with respect to a sequence set forth in Table l(j). In some embodiments, the peptide substrate susceptible to cleavage by the plurality of mammalian proteases has at most one amino acid substitution with respect to a sequence set forth in Table l(j). In some embodiments, none of the at most three amino acid substitutions, or the at most two amino acid substitutions, or the at most one amino acid substitution is at a position corresponding to an amino acid residue immediately adjacent to a corresponding scissile bond of the corresponding sequence set forth in Table l(j). In some embodiments, the peptide substrate susceptible to cleavage by the plurality of mammalian proteases comprises a sequence set forth in Table 10).

[0046] In some embodiments of the therapeutic agent, the release segment (RS) is capable of being cleaved when in proximity to a target tissue or cell, and wherein the target tissue or cell produces the mammalian protease for which the RS is a peptide substrate. In some embodiments, the mammalian protease for cleavage of the release segment (RS) is a serine protease, a cysteine protease, an aspartate protease, a threonine protease, or a metalloproteinase. In some embodiments, the mammalian protease for cleavage of the release segment (RS) is selected from the group consisting of disintegrin and metalloproteinase domaincontaining protein 10 (ADAM 10), 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 motifs 5 (ADAMTS5), Cathepsin B, Cathepsin D, Cathepsin E, Cathepsin K, cathepsin L, cathepsin S, Fibroblast activation protein alpha, Hepsin, kallikrein-2, kallikrein-4, kallikrein-3, Prostate-specific antigen (PSA), kallikrein-13, Legumain, 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 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 type serine protease 1 (MT-SP1), matriptase, 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) linked, directly or indirectly, to the release segment (RS). In some embodiments, the therapeutic agent, in an uncleaved state, has a structural arrangement from N-terminus to C-terminus of BM-RS-MM or MM-RS-BM. 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, the XTEN is characterized in that: (i) it comprises at least 100 amino acids; (ii) at least 90% of the amino acid residues of it are selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P); and (iii) it comprises at least 4 different types of 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 a sequence set forth in Tables 2b-2c. In some embodiments, the masking moiety (MM), when linked to the therapeutic agent, interferes with an interaction of the biologically active moiety (BM) to the target tissue or cell such that a dissociation constant (Kd) of the BM of the therapeutic agent with a target cell marker borne by the target tissue or cell is greater, when the therapeutic agent is in an uncleaved state, compared to a dissociation constant (Kd) of a corresponding biologically active moiety with the target cell marker. In some embodiments, the therapeutic agent effects a broader therapeutic window in delivery of the BM to the target tissue or cell compared to a corresponding biologically active moiety. In some embodiments, the therapeutic agent has a longer terminal half-life compared to that of a corresponding biologically active moiety. In some embodiments, the therapeutic agent is less immunogenic compared to a corresponding biologically active moiety. In some embodiments, the immunogenicity is ascertained by measuring production of IgG antibodies that selectively bind to the biologically active moiety after administration of comparable doses to a subject. In some embodiments, the therapeutic agent has a greater apparent molecular weight factor under a physiological condition compared to a corresponding biologically active moiety.

[0048] In some embodiments of the therapeutic agent, the release segment (RS) is a first release segment (RSI), wherein the scissile bond is a first scissile bond, and wherein the therapeutic agent further comprises a second release segment (RS2) linked, directly or indirectly, to the biologically active moiety (BM), wherein the RS2 comprises a second peptide substrate or cleavage by a mammalian protease at a second scissile bond. In some embodiments, the mammalian protease for cleavage of the RS2 is identical to the mammalian protease for cleavage of the RSI. In some embodiments, the mammalian protease for cleavage of the RS2 is different from the mammalian protease for cleavage of the RSI. In some embodiments, the RS2 has an amino acid sequence identical to that of the RSI. In some embodiments, the RS2 has an amino acid sequence different from that of the RSI. In some embodiments, each of the RSI and the RS2 comprises a peptide substrate for a different mammalian protease selected from the group consisting of disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), disintegrin and metalloproteinase domain-containing protein 12 (ADAM12), disintegrin and metalloproteinase domaincontaining protein 15 (ADAM 15), disintegrin and metalloproteinase domain-containing protein 17 (ADAM17), disintegrin and metalloproteinase domain-containing protein 9 (ADAM9), disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), Cathepsin B, Cathepsin D, Cathepsin E, Cathepsin K, cathepsin L, cathepsin S, Fibroblast activation protein alpha, Hepsin, kallikrein-2, kallikrein-4, kallikrein-3, Prostate-specific antigen (PSA), kallikrein-13, Legumain, 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 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 type serine protease 1 (MT-SP1), matriptase, and u-plasminogen. In some embodiments, each of the RSI and the RS2 comprises 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), legumain, and matriptase. In some embodiments, the second scissile bond is not immediately C-terminal to a methionine residue.

[0049] In some embodiments of the therapeutic agent, the second peptide substrate contains from six to twenty-five or six to twenty amino acid residues. In some embodiments of the therapeutic agent, the second peptide substrate contains from six to twenty-five amino acid residues. In some embodiments of the therapeutic agent, the second peptide substrate contains from six to twenty amino acid residues. In some embodiments, the second peptide substrate contains from seven to twelve amino acid residues. In some embodiments, the second peptide substrate comprises an amino acid sequence having at most three amino acid substitutions, or at most two amino acid substitutions, or at most one amino acid substitution with respect to a sequence set forth in Column II or III of Table A (or a subset thereof). In some embodiments, the second peptide substrate comprises an amino acid sequence having at most three amino acid substitutions with respect to a sequence set forth in Column II or III of Table A (or a subset thereof), tn some embodiments, the second peptide substrate comprises an amino acid sequence having at most two amino acid substitutions with respect to a sequence set forth in Column II or III of Table A (or a subset thereof), tn some embodiments, the second peptide substrate comprises an amino acid sequence having at most one amino acid substitution with respect to a sequence set forth in Column II or III of Table A (or a subset thereof), tn some embodiments, none of the at most three amino acid substitutions, or the at most two amino acid substitutions, or the at most one amino acid substitution (of the second peptide substrate) is at a position corresponding to an amino acid residue immediately adjacent to a corresponding scissile bond of the corresponding sequence shown in Column II or III of Table A (or a subset thereof), tn some embodiments, the second peptide substrate comprises an amino acid sequence identical to a sequence set forth in Column II or III of Table A (or a subset thereof), tn some embodiments, the second peptide substrate does not comprise a methionine residue immediately N-terminal to a scissile bond (contained therein). In some embodiments, the second peptide substrate does not comprise 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) of Column II of Table A. In some embodiments, the second peptide substrate comprises two or three sequences set forth in Column II or III of Table A (or a subset thereof), tn some embodiments, where the second peptide substrate comprises two sequences set forth in Column II or III of Table A (or a subset thereof), the two sequences (of the second peptide substrate) partially overlap one another. In some embodiments, where the second peptide substrate comprises two sequences set forth in Column II or III of Table A (or a subset thereof), the two sequences (of the second peptide substrate) do not overlap one another. In some embodiments, where the second peptide substrate comprises three sequences set forth in Column II or III of Table A (or a subset thereof), two or all of the three sequences (of the second peptide substrate) do not overlap one another. In some embodiments, where the second peptide substrate comprises three sequences set forth in Column II or III of Table A (or a subset thereof), one of the three sequences (of the second peptide substrate) partially overlaps with another sequence or both other sequences of the three sequences (of the second peptide substrate). In some embodiments, where the second peptide substrate comprises three sequences set forth in Column II or III of Table A (or a subset thereof), two of the three sequences (of the second peptide substrate) partially overlap with one another. In some embodiments, where the second peptide substrate comprises three sequences set forth in Column II or III of Table A (or a subset thereof), each two of the three sequences (of the second peptide substrate) partially overlap with one another. In some embodiments, where the second peptide substrate comprises three sequences set forth in Column II or III of Table A (or a subset thereof), all of the three sequences (of the second peptide substrate) partially overlap with one another. In some embodiments, the second peptide substrate susceptible to cleavage by the mammalian protease is susceptible to cleavage by a plurality of mammalian proteases comprising the mammalian protease. In some embodiments, the second peptide substrate susceptible to cleavage by the plurality of mammalian proteases has at most three amino acid substitutions, or at most two amino acid substitutions, or at most one amino acid substitution with respect to a sequence set forth in Table l(j). In some embodiments, the second peptide substrate susceptible to cleavage by the plurality of mammalian proteases has at most three amino acid substitutions with respect to a sequence set forth in Table 10. In some embodiments, the second peptide substrate susceptible to cleavage by the plurality of mammalian proteases has at most two amino acid substitutions with respect to a sequence set forth in Table l(j). In some embodiments, the second peptide substrate susceptible to cleavage by the plurality of mammalian proteases has at most one amino acid substitution with respect to a sequence set forth in Table l(j). In some embodiments, none of the at most three amino acid substitutions, or the at most two amino acid substitutions, or the at most one amino acid substitution (of the second peptide substrate) is at a position corresponding to an amino acid residue immediately adjacent to a corresponding scissile bond of the corresponding sequence set forth in Table l(j). In some embodiments, the second peptide substrate susceptible to cleavage by the plurality of mammalian proteases comprises a sequence set forth in Table l(j)

[0050] In some embodiments of the therapeutic agent, the second release segment (RS2) is capable of being cleaved when in proximity to the target tissue or cell, and wherein the target tissue or cell produces the mammalian protease for which the RS2 is a peptide substrate. This includes tumor produced proteases and tumor melieu produced proteases. In some embodiments, the mammalian protease for cleavage of the second release segment (RS2) is a serine protease, a cysteine protease, an aspartate protease, a threonine protease or a metalloproteinase. In some embodiments, the mammalian protease for cleavage of the release segment (RS) is selected from the group consisting of disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), disintegrin and metalloproteinase domain-containing protein 12 (ADAM12), disintegrin and metalloproteinase domain-containing protein 15 (ADAM 15), disintegrin and metalloproteinase domain-containing protein 17 (ADAM17), disintegrin and metalloproteinase domaincontaining protein 9 (ADAM9), disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), Cathepsin B, Cathepsin D, Cathepsin E, Cathepsin K, cathepsin L, cathepsin S, Fibroblast activation protein alpha, Hepsin, kallikrein-2, kallikrein-4, kallikrein-3, Prostate-specific antigen (PSA), kallikrein-13, Legumain, 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 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 type serine protease 1 (MT-SP1), matriptase, and u-plasminogen. In some embodiments, the mammalian protease for cleavage of 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), legumain, and matriptase.

[0051] In some embodiments of the therapeutic agent, the masking moiety (MM) is a first masking moiety (MM1), and wherein the therapeutic agent further comprises a second masking moiety (MM2) linked, directly or indirectly, to the second release segment (RS2). In some embodiments, the therapeutic agent, in an uncleaved state, has a structural arrangement from N-terminus to C-terminus of MM1-RS1-BM-RS2-MM2, MM1-RS2-BM-RS1-MM2, MM2-RS1-BM-RS2-MM1, or MM2-RS2-BM-RS1-MM1. In some embodiments of the therapeutic agent, upon cleavage of the second release segment (RS2), the second masking moiety (MM2) is released from the therapeutic agent. In some embodiments, the second masking moiety (MM2) comprises a second extended recombinant polypeptide (XTEN2). In some embodiments, the XTEN2 is characterized in that: (i) it comprises at least 100 amino acids; (ii) at least 90% of the amino acid residues of it are selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P); and (iii) it comprises at least 4 different types of amino acids selected from G, A, S, T, E, and P. In some embodiments, the XTEN2 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from the group of sequences set forth in Tables 2b-2c. In some embodiments, the first masking moiety (MM1) and the second masking moiety (MM2), when both linked in the therapeutic agent, interfere with an interaction of the biologically active moiety (BM) to the target tissue or cell such that a dissociation constant (Ka) of the BM of the therapeutic agent with a target cell marker borne by the target tissue or cell is greater, when the therapeutic agent is in an uncleaved state, compared to a dissociation constant (Kd) of a corresponding biologically active moiety. In some embodiments, the therapeutic agent, in which the biologically active moiety (BM) is linked, directly or indirectly, to one or both of the first masking moiety (MM1) and the second masking moiety (MM2), effects a broader therapeutic window in delivery of the BM to the target tissue or cell compared to a corresponding biologically active moiety. In some embodiments, the therapeutic agent, in which the biologically active moiety (BM) is linked, directly or indirectly, to one or both of the first masking moiety (MM1) and the second masking moiety (MM2), has a longer terminal half-life compared to that of a corresponding biologically active moiety. In some embodiments, the therapeutic agent, in which the biologically active moiety (BM) is linked, directly or indirectly, to one or both of the first masking moiety (MM1) and the second masking moiety (MM2), is less immunogenic compared to a corresponding biologically active moiety. In some embodiments of the therapeutic agent, immunogenicity is ascertained by measuring production of IgG antibodies that selectively bind to the biologically active moiety after administration of comparable doses to a subject. In some embodiments, the therapeutic agent, in which the biologically active moiety (BM) is linked, directly or indirectly, to one or both of the first masking moiety (MM1) and the second masking moiety (MM2), has a greater apparent molecular weight factor under a physiological condition compared to a corresponding biologically active moiety. In some embodiments, the therapeutic agent comprises a fusion polypeptide or conjugate.

[0052] In some embodiments of the therapeutic agent, the biologically active moiety (BM) comprises a biologically active 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 the biologically active peptide (BP) and the release segment (RS). In some embodiments, the recombinant polypeptide comprises the biologically active peptide (BP), the release segment (RS), and the masking moiety (MM). In some embodiments, the recombinant polypeptide, in an uncleaved state, has a structural arrangement from N-terminus to C-terminus of BP-RS-MM or MM-RS-BP. In some embodiments, the recombinant polypeptide comprises the biologically active peptide (BP), the first release segment (RSI), and the second release segment (RS2). In some embodiments, the recombinant polypeptide comprises the biologically active peptide (BP), the first release segment (RSI), the second release segment (RS2), the first masking moiety (MM1), and the second masking moiety (MM2). In some embodiments, the recombinant polypeptide, in an uncleaved state, has a structural arrangement 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 the biologically active peptide (BP), the first release segment (RSI), the second release segment (RS2), the first extended recombinant polypeptide (XTEN1), and the second extended recombinant polypeptide (XTEN2). In some embodiments, the recombinant polypeptide, in an uncleaved state, has a structural arrangement 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 biologically active polypeptide (BP) comprises a binding moiety having a binding affinity for a target cell marker on the target tissue or cell. In some embodiments, the target cell marker is an effector cell antigen expressed on a surface of an effector cell. In some embodiments, the binding moiety is an antibody. In some embodiments, the binding moiety is an antibody selected from the group consisting of Fv, Fab, Fab’, Fab’-SH, nanobody (also known as single domain antibody or Vhh), linear antibody, and single-chain variable fragment (scFv). In some embodiments, the binding moiety is a first binding moiety, wherein the target cell marker is a first target cell marker, and wherein the biologically active polypeptide (BP) further comprises a second binding moiety linked, directly or indirectly to the first binding moiety, wherein the second binding moiety has a binding affinity for a second target cell marker on the target tissue or cell. In some embodiments, the second target cell marker is a marker on a tumor cell or a cancer cell. In some embodiments, the second binding moiety is an antibody. In some embodiments, the second binding moiety is an antibody selected from the group consisting of Fv, Fab, Fab’, Fab’-SH, nanobody (also known as single domain antibody or Vhh), linear antibody, and single-chain variable fragment (scFv).

[0055] Certain aspects of the present disclosure provide an isolated nucleic acid, the isolated nucleic acid comprising: (a) a polynucleotide encoding a recombinant polypeptide as described herein; or (b) a reverse complement of the polynucleotide of (a).

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

[0057] Certain aspects of the present disclosure provide an isolated host cell, the isolated cell comprising the expression vector as described herein. In some embodiments, the host cell is a prokaryote. In some embodiments, the host cell is E. coli or a mammalian cell. In some embodiments, the host cell is E. coli. In some embodiments, the host cell is a mammalian cell.

[0058] Some aspects of the present disclosure provide a pharmaceutical composition, the pharmaceutical composition comprising a therapeutic agent as described herein and one or more pharmaceutically suitable excipients. In some embodiments, the pharmaceutical composition is formulated for oral, intradermal, subcutaneous, intravenous, intra-arterial, intraabdominal, intraperitoneal, intrathecal, or intramuscular administration. In some embodiments, the pharmaceutical composition is in a liquid form or frozen form. In some embodiments, the pharmaceutical composition is in a pre-filled syringe for a single injection. In some embodiments, the pharmaceutical composition is formulated as a lyophilized powder to be reconstituted prior to administration.

[0059] Some aspects of the present disclosure provide a kit, the kit comprising a pharmaceutical composition as described herein, a container, and a label or package insert on or associated with the container.

[0060] In certain aspects, the present disclosure provides a method for preparing a therapeutic agent (e.g., activatable therapeutic agent, or non-natural, activatable therapeutic agent) as provided herein.

[0061] In certain aspects, the present disclosure provides a method for preparing a therapeutic agent (e.g., activatable therapeutic agent, or non-natural, activatable therapeutic agent), the method comprising: (a) culturing a host cell comprising a nucleic acid construct that encodes a recombinant polypeptide under conditions sufficient to express the recombinant polypeptide in the host cell, wherein the recombinant polypeptide comprises a biologically active polypeptide (BP), a release segment (RS), and a masking moiety (MM), wherein: the RS comprises a peptide substrate susceptible for cleavage by a mammalian protease at a scissile bond, wherein the peptide substrate comprises an amino acid sequence having at most three or two amino acid substitutions (or at most one amino acid substitution) with respect to a sequence set forth in Column II or III of Table A (or a subset thereol); and the recombinant polypeptide has a structural arrangement from N-terminus to C-terminus of BP-RS-MM or MM-RS-BP; and (b) recovering the therapeutic agent (e.g., activatable therapeutic agent, or non-natural, activatable therapeutic agent) comprising the recombinant polypeptide.

[0062] In some embodiments of the method for preparing the therapeutic agent, the peptide substrate susceptible to cleavage by the mammalian protease is susceptible to cleavage by a plurality of mammalian proteases comprising the mammalian protease. In some embodiments, the peptide substrate susceptible to cleavage by the plurality of mammalian proteases has at most three amino acid substitutions, or at most two amino acid substitutions, or at most one amino acid substitution with respect to a sequence set forth in Table l(j). In some embodiments, the peptide substrate susceptible to cleavage by the plurality of mammalian proteases comprises a sequence set forth in Table l(j). In some embodiments, the peptide substrate does not comprise SEQ ID NO: 1. In some embodiments, the peptide substrate does not comprise SEQ ID NO: 2. In some embodiments, the peptide substrate does not comprise SEQ ID NO: 3. In some embodiments, the peptide substrate does not comprise SEQ ID NO: 4. In some embodiments, the peptide substrate does not comprise SEQ ID NO: 5. In some embodiments, the peptide substrate does not comprise SEQ ID NO: 6. In some embodiments, the peptide substrate does not comprise SEQ ID NO: 7. In some embodiments, the peptide substrate does not comprise SEQ ID NO: 8. In some embodiments, the masking moiety (MM) comprises an extended recombinant polypeptide (XTEN).

[0063] In some embodiments of the method for preparing the therapeutic agent, the release segment (RS) is a first release segment (RSI), wherein the peptide substrate is a first peptide substrate, wherein the scissile bond is a first scissile bond, wherein the masking moiety (MM) is a first masking moiety (MM1), and wherein the recombinant polypeptide further comprises a second release segment (RS2), and a second masking moiety (MM2), wherein: the RS2 comprises a second peptide substrate susceptible for cleavage by a mammalian protease at a second scissile bond, wherein the second peptide substrate comprises an amino acid sequence having at most three amino acid substitutions, or at most two amino acid substitutions, or at most one amino acid substitution with respect to a sequence set forth in Column II or III of Table A (or a subset thereol); and the recombinant polypeptide has a structural arrangement 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.

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

[0065] In some embodiments of the method for preparing the therapeutic agent, the masking moiety (MM), when linked to the recombinant polypeptide, interferes with an interaction of the BP to a target tissue or cell such that a dissociation constant (Ka) of the BP of the recombinant polypeptide with a target cell marker borne by the target tissue or cell is greater, when the recombinant polypeptide is in an uncleaved state, compared to a dissociation constant (Ka) of a corresponding biologically active peptide, as measured in an in vitro assay under equivalent molar concentrations. In some embodiments, the first masking moiety (MM1) and the second masking moiety (MM2), when both linked in the recombinant polypeptide, interfere with an interaction of the BP to a target tissue or cell such that a dissociation constant (Ka) of the BP of the recombinant polypeptide with a target cell marker borne by the target tissue or cell is greater, when the recombinant polypeptide is in an uncleaved state, compared to a dissociation constant (Ka) of a corresponding biologically active peptide, as measured in an in vitro assay under equivalent molar concentrations. In some embodiments, the in vitro assay is selected from cell membrane integrity assay, mixed cell culture assay, cell-based competitive binding assay, FACS based propidium Iodide assay, trypan Blue influx assay, photometric enzyme release assay, radiometric 51Cr release assay, fluorometric Europium release assay, CalceinAM release assay, photometric MTT assay, XTT assay, WST-1 assay, alamar blue assay, radiometric 3H-Thd incorporation assay, clonogenic assay measuring cell division activity, fluorometric rhodamine 123 assay measuring mitochondrial transmembrane gradient, apoptosis assay monitored by FACS-based phosphatidylserine exposure, ELISA-based TUNEL test assay, sandwich ELISA, caspase activity assay, cell-based LDH release assay, and cell morphology assay, or any combination thereof. In some embodiments, the activatable therapeutic agent is an activatable therapeutic agent or non-natural, activatable therapeutic agent as described herein.

[0066] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive. INCORPORATION BY REFERENCE

[0067] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0069] FIG. 1 illustrates the nomenclature of a peptide biomarker sequence in a reporter polypeptide (e.g., a protein within or adjacent to a target tissue or cell from which a biomarker sequence is generated) (such as any set forth in Table A). The illustrative reporter polypeptide sequence comprises two cleavage sequences, a first cleavage sequence and a second cleavage sequence (such as any set forth in Table A), both capable of being recognized and cleaved by mammalian enzyme(s) (such as mammalian protease(s)). 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 another 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 scissile bond; and the second cleavage sequence, which is C-terminal to the first cleavage sequence, contains a second scissile bond. The first and second scissile bonds (such as indicated with hyphen (-) in Table A) divide the illustrative reporter polypeptide into three portions. By cleaving the illustrative reporter polypeptide with the corresponding enzyme(s) for which both the first and second cleavage sequences are substrates for, an N-terminal fragment (N-terminal to the first scissile bond), a center fragment (between the first and second scissile bonds), and a C-terminal fragment (C-terminal to the second scissile bond) can be obtained. The N-terminal, center, or C-terminal fragment (if present) (such as any set forth in Table A), or a derivative thereof, can function as a peptide biomarker sequence. The first or second cleavage sequence (such as any set forth in Table A) can be incorporated into a release segment of an activatable therapeutic agent (such as any described herein).

[0070] FIG. 2 illustrates the nomenclature of a peptide substrate and a scissile bond thereof for cleavage. The illustrative peptide substrate contains eight consecutive amino acid residues, of which four amino acid residues (with side chain groups, in the order from the N-terminus to the C-terminus, R+, R3, R2, and Ri) are immediately N-terminal to the scissile bond and four amino acid residues (with side chain groups, in the order from the N-terminus to the C-terminus, R’i, R’2, R’3, and R’4) are immediately C-terminal to the scissile bond. For example, mammalian proteases can recognize up to four residues on both sides of the scissile bond. Upon cleavage, the illustrative peptide substrate separates into an N-terminal proteolytic fragment and a C-terminal proteolytic fragment. The four amino acid residues immediately N-terminal to the scissile bond in the illustrative peptide substrate forms the C-terminus of the N-terminal proteolytic fragment; and the four amino acid residues immediately C-terminal to the scissile bond in the illustrative peptide substrate forms the N-terminus of the C-terminal proteolytic fragment.

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

[0072] FIG. 4 illustrates a structural configuration of an exemplary activatable antibody complex (AAC) composition with cross-masking occurring such that target binding by both antibodies or fragments thereof is attenuated in its uncleaved state, and target binding is increased upon cleavage of the release segment (RS) allowing the complex to disassemble. In this figure, the two antibodies or fragments thereof are referred to as the antibody domain 1 (ABDI) and antibody domain 2 (ABD2), respectively.

[0073] FIG. 5 illustrates a structural configuration of an exemplary activatable antibody complex (AAC) composition comprising two antibodies or fragments thereof, a masking moiety (MM), and a release segment (RS).

[0074] FIG. 6 illustrates a structural configuration of an exemplary activatable antibody complex (AAC) composition comprising four antibodies or fragments thereof, two masking moieties (MM) and three release segments (RS).

[0075] FIG. 7 illustrates a structural configuration of an exemplary activatable antibody composition (AA) comprising one antibody or antibody fragment (AB), two masking moieties (MM), and two release segments (RS).

[0076] FIG. 8 illustrates a structural configuration of an XTENylated Protease-Activated T-Cell Engager (XPAT). The illustrative XPAT comprises two binding moieties, each linked to an XTEN via a release segment.

[0077] FIG. 9 illustrates the results of mammalian protease cleavage of release segments having sequence similarities to a sequence found in collagen I. The cleavage site is identified by a star (★) with portions of the sequences identical to the collagen site underlined. A sequence engineered not to be recognized or cleaved by proteases that recognize the collagen-derived cleavage site is set forth as 818-NonClv (RSR-3058) and amino acids that vary from the collagen sequence are shown in black type. DETAILED DESCRIPTION

[0078] In various cancer therapy modalities, agents have been generated that are conditionally activatable in the tumor microenvironment. However, there remains a need for developing more accurate and robust methods for predicting whether administration of these therapies will actually lead to therapeutic responses and outcomes upon administration of prodrugs or other activatable compositions. It is recognized that there is a cascade of events that leads to metastatic growth of cancer cells. A central factor in these events is the interaction between cancer cells and their microenvironment through which the tumor cells proliferate, build new vessels, leave the primary tumor bed and finally enter and persist at secondary sites of metastatic tumor growth. The extracellular matrix (ECM) of the tumor microenvironment consists of a variety of macromolecules, including collagen and glycoproteins. While the basement membranes of the ECM are formed mostly by type IV collagen, type I and type III collagen are the most abundant proteins of the underlying interstitial matrix. In healthy tissue, the ECM undergoes constant remodeling, mediated mainly by matrix-metalloproteinases (MMP), and matrix degradation is balanced by protein formation. This controlled remodeling of the ECM becomes disrupted in cancer development and progression.

[0079] In the process of MMP-mediated ECM degradation, 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 levels of MMP-degraded collagen type I, III and IV (i.e., C1M, C3M and C4M, respectively, Cancer Biomark. 2015;15:783-788) to be 1.5 to 6-fold higher in ovarian and breast cancer patients than in controls. In the present invention, it is demonstrated that cleavage of the ECM by MMPs results in a cleavage product that is highly similar to the MMP cleavage site in protease-cleavable linkers in XPATs. The data presented herein demonstrate that the protease cleavable linker employed in the XPATs of this invention are more efficiently cleaved than the ECM by purified MMPs. As such, it is shown that the presence of ECM peptides in cancer patients can serve as an indicator that the patients’ tumors have a microenvironment that has the appropriate protease (e.g., MMP) activity that can cleave the protease-cleavable linker in an XPAT. In this manner, the presence of the ECM peptides in the sample of a cancer patient thereby predicts whether a given patient or tumor will be able to cleave the XPAT and hence result in treatment of the tumor. This allows for a personalized approach to determine whether an XPAT will be cleaved in a given tumor type by determining whether the subject that has said tumor type has elevated plasma levels of certain cleavage product(s) derived from the extracellular matrix.

[0080] Before the embodiments of the disclosure are described, it is to be understood that such embodiments are provided by way of example only, and that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the invention. Numerous variations, changes, and substitutions will now occur to 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 one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Definitions

[0082] In the context of the present application, the following terms have the meanings ascribed to them unless specified otherwise:

[0083] As used throughout the specification and claims, the terms “a”, “an” and “the” are generally used in the sense that they mean “at least one”, “at least a first”, “one or more” or “a plurality” of the referenced components or steps, except in instances wherein an upper limit is thereafter specifically stated. For example, a “cleavage sequence”, as used herein, means “at least a first cleavage sequence” but includes a plurality of cleavage sequences. The operable limits and parameters of combinations, as with the amounts of any single agent, will be known to those of ordinary skill in the art in light of the present disclosure.

[0084] The term “activatable,” as used herein with respect to a therapeutic agent, generally means that an activity or bioactivity of the therapeutic agent is capable of being enhanced upon activation, for example, via a physical, chemical or physiological process (e.g., enzymatic processes and metabolic processes).

[0085] As used herein, the term “activatable therapeutic agent,” generally refers to a therapeutic agent, of which an activity or bioactivity is capable of being enhanced upon activation, for example, via a physical, chemical or physiological process (e.g., enzymatic processes and metabolic processes). For example, the term “activatable therapeutic agent” may refer to a therapeutic agent in an inactive (or less active) state (at least inactive in one aspect) configured to be activated (i.e., in vitro, in vivo, or ex vivo) into an active (or more active) state (at least in the aspect that is inactive prior to activation). As another example, the term “activatable therapeutic agent” may refer to an active therapeutic agent (at least active in one aspect), of which an activity or bioactivity can be further enhanced (i.e., in vitro, in vivo, or ex vivo). Non-limiting examples of an activatable therapeutic agent include a prodrug, a probody, and a pro-moiety.

[0086] The terms “polypeptide”, “peptide”, and “protein” are used interchangeably herein to generally refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component.

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

[0088] The term “N-terminal end sequence,” as used herein with respect to a polypeptide or polynucleotide sequence of interest, generally means that no other amino acid or nucleotide residues precede the N-terminal end sequence in the polypeptide or polynucleotide sequence of interest at the N-terminal end. The term “C-terminal end sequence,” as used herein with respect to a polypeptide or polynucleotide sequence of interest, generally means that no other amino acid or nucleotide residues follows the C-terminal end sequence in the polypeptide or polynucleotide sequence of interest at the C-terminal end.

[0089] The terms “non-naturally occurring” and “non-natural” are used interchangeably herein. The term “non-naturally occurring” or “non-natural,” as used herein with respect to a therapeutic agent, generally means that the agent is not biologically derived in mammals (including but not limited to human). The term “non-naturally occurring” or “non-natural,” as applied to sequences and as used herein, means polypeptide or polynucleotide sequences that do not have a counterpart to, are not complementary to, or do not have a high degree of homology with a wild-type or naturally-occurring sequence found in a mammal. For example, a non-naturally occurring polypeptide or fragment may share no more than 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50% or even less amino acid sequence identity as compared to a natural sequence when suitably aligned.

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

[0091] A “variant,” when applied to a biologically active protein is a protein with sequence homology to the native biologically active protein that retains at least a portion of the therapeutic and / or biological activity of the biologically active protein. For example, a variant protein may share at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity compared with the reference biologically active protein. As used herein, the term “biologically active protein variant” includes proteins modified deliberately, as for example, by site directed mutagenesis, synthesis of the encoding gene, insertions, or accidentally through mutations and that retain activity.

[0092] The term “sequence variant” means polypeptides that have been modified compared to their native or original sequence by one or more amino acid insertions, deletions, or substitutions. Insertions may be located at either or both termini of the protein, and / or may be positioned within internal regions of the amino acid sequence. A non-limiting example is substitution of an amino acid in an XTEN with a different amino acid. In deletion variants, one or more amino acid residues in a polypeptide as described herein are removed. Deletion variants, therefore, include all fragments of a described polypeptide sequence. In substitution variants, one or more amino acid residues of a polypeptide are removed and replaced with alternative residues. In one aspect, the substitutions are conservative in nature and conservative substitutions of this type are well known in the art. In the context of an antibody or a biologically active polypeptide, a sequence variant would retain at least a portion of the binding affinity or biological activity, respectively, of the unmodified polypeptide.

[0093] The term “moiety” means a component of a larger composition or that is intended to be incorporated into a larger composition, such as a proteinaceous portion joined to a larger polypeptide as a contiguous or non-contiguous sequence. A moiety of a larger composition can confer a desired functionality. For example, an antibody fragment may retain the ability to bind its ligand yet have a smaller molecular size and be in a single-chain format. A masking moiety (including but not limited to an extended recombinant polypeptide (XTEN)) may confer the functionality of increasing molecular weight and / or half-life of a resulting larger composition with which the masking moiety is associated.

[0094] The terms “binding domain” and “binding moiety” are used interchangeably herein and each refer to a moiety having specific binding affinity to an antigen (such as an effector cell antigen, or a tumorspecific marker or an antigen of a target cell).

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

[0096] As used herein, a “peptide substrate” generally refers to an amino acid sequence recognized by an enzyme (such as a mammalian protease), leading to cleavage at a peptide bond (or the peptide bond) within the peptide substrate such that two consecutive amino acid residues connected by the peptide bond (or the scissile bond) prior to cleavage are separated upon cleavage. As used herein, a “scissile bond” generally refers to a peptide bond joining consecutive amino acids via an amide linkage that can be cleaved (or is cleaved) by an enzyme (such as a mammalian protease). For example, in the context of a peptide substrate, the scissile bond divides the peptide substrate into a C-terminal proteolytic fragment (or a C-terminal fragment) and an N-terminal proteolytic fragment (or an N-terminal fragment), where the C-terminal proteolytic fragment (or the C-terminal fragment) is N-terminal to the scissile bond in the peptide substrate and the N-terminal proteolytic fragment (or the N-terminal fragment) is C-terminal to the scissile bond in the peptide substrate. For example, the (putative) scissile bond of each cleavage sequence listed in Table A is indicated by a hyphen (-).

[0097] As used herein, the term “scissile bond” generally refers to a peptide bond between two amino acids which is capable of being cleaved by one or more proteases.

[0098] As used herein, the term “mammalian protease” generally means a protease that normally exists in the body fluids, cells, tissues, and may be found in higher levels in certain target tissues or cells, e.g., in diseased tissues (e.g., tumor) of a mammal.

[0099] The term “within”, when referring to a first polypeptide being linked to a second polypeptide, encompasses linking or fusion of an additional component that connects the N-terminus of the first or second polypeptide to the C-terminus of the second or first polypeptide, respectively, as well as insertion of the first polypeptide into the sequence of the second polypeptide. For example, when an RS component is linked “within” an recombinant polypeptide, the RS may be linked to the N-terminus, the C-terminus, or may be inserted between any two amino acids of an XTEN polypeptide.

[00100] The term “linked directly,” as used herein in the context of a therapeutic agent, generally refers to a structure in which a moiety is connected with or attached to another moiety without an intervening tether. The term “linked indirectly,” as used herein in the context of a therapeutic agent, generally refers to a structure in which a moiety of the therapeutic agent is connected with, or attached to, another moiety of the therapeutic agent via an intervening tether. The terms “link,” “linked,” and “linking,” as used herein in the context of a therapeutic agent, generally includes both covalent and non-covalent attachment of a moiety of the therapeutic agent to another moiety of the therapeutic agent.

[00101] “Activity” (such as “bioactivity”) as applied to form(s) of a composition provided herein, generally refers to an action or effect, including but not limited to receptor binding, antagonist activity, agonist activity, a cellular or physiologic response, cell lysis, cell death, or an effect generally known in the art for the effector component of the composition, whether measured by an in vitro, ex vivo or in vivo assay or a clinical effect.

[00102] “Effector cell”, as used herein, includes any eukaryotic cells capable of conferring an effect on a target cell. For example, an effect cell can induce loss of membrane integrity, pyknosis, karyorrhexis, apoptosis, lysis, and / or death of a target cell. In another example, an effector cell can induce division, growth, differentiation of a target cell or otherwise altering signal transduction of a target cell.

[00103] An “effector cell antigen” refers to molecules expressed by an effector cell, including without limitation cell surface molecules such as proteins, glycoproteins or lipoproteins. An effector cell antigen can serve as the binding counterpart of a binding moiety of the subject recombinant polypeptide.

[00104] As used herein, the term “ELISA” refers to an enzyme-linked immunosorbent assay as described herein or as otherwise known in the art.

[00105] A “host cell” generally includes an individual cell or cell culture which can be or has been a recipient for the subject vectors into which exogenous nucleic acid has been introduced, such as those described herein. Host cells include progeny of a single host cell. The progeny may not necessarily be completely identical (in morphology or in genomic of total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells transfected in vivo with a vector of this disclosure.

[00106] The term “isolated”, when used to describe the various polypeptides disclosed herein, generally means polypeptide that has been identified and separated and / or recovered from a component of its natural environment or from a more complex mixture (such as during protein purification). Contaminant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. As is apparent to those of skill in the art, a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, does not require “isolation” to distinguish it from its naturally occurring counterpart. In addition, a “concentrated”, “separated” or “diluted” polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, is distinguishable from its naturally occurring counterpart in that the concentration or number of molecules per volume is generally greater than that of its naturally occurring counterpart. In general, a polypeptide made by recombinant means and expressed in a host cell is considered to be “isolated.”

[00107] An “isolated nucleic acid” is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the polypeptide-encoding nucleic acid. For example, an isolated polypeptide-encoding nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated polypeptide-encoding nucleic acid molecules therefore are distinguished from the specific polypeptide-encoding nucleic acid molecule as it exists in natural cells. However, an isolated polypeptide-encoding nucleic acid molecule includes polypeptide-encoding nucleic acid molecules contained in cells that ordinarily express the polypeptide where, for example, the nucleic acid molecule is in a chromosomal or extra-chromosomal location different from that of natural cells.

[00108] A “chimeric” protein or polypeptide contains at least one fusion polypeptide comprising at least one region in a different position in the sequence than that which occurs in nature. The regions may normally exist in separate proteins and are brought together in the fusion polypeptide; or they may normally exist in the same protein but are placed in a new arrangement in the fusion polypeptide. A chimeric protein may be created, for example, by chemical synthesis, or by recombinantly creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship.

[00109] The terms “fused” and “fusion” are used interchangeably herein, and refers to the joining together of two or more peptide or polypeptide sequences by recombinant means. A "fusion protein" or "chimeric protein” comprises a first amino acid sequence linked to a second amino acid sequence with which it is not naturally linked in nature.

[00110] “Uncleaved” and “uncleaved state” are used interchangeably herein, and refers to a polypeptide that has not been cleaved or digested by a protease such that the polypeptide remains intact.

[00111] “XTENylated” is used to denote a peptide or polypeptide that has been modified by the linking or fusion of one or more XTEN polypeptides (described, below) to the peptide or polypeptide, whether by recombinant or chemical cross-linking means.

[00112] “Crosslinking,” and “conjugating,” are used interchangeably herein, and refer to the covalent joining of two different molecules by a chemical reaction. The crosslinking can occur in one or more chemical reactions, as known in the art.

[00113] In the context of polypeptides, a “linear sequence” or a “sequence” is an order of amino acids in a polypeptide in an amino to carboxyl terminus (N- to C-terminus) direction in which residues that neighbor each other in the sequence are contiguous in the primary structure of the polypeptide. A “partial sequence” is a linear sequence of part of a polypeptide that is known to comprise additional residues in one or both directions.

[00114] “Heterologous” means derived from a genotypically distinct entity from the rest of the entity to which it is being compared. For example, a glycine rich sequence removed from its native coding sequence and operatively linked to a coding sequence other than the native sequence is a heterologous glycine rich sequence. The term “heterologous” as applied to a polynucleotide, a polypeptide, means that the polynucleotide or polypeptide is derived from a genotypically distinct entity from that of the rest of the entity to which it is being compared.

[00115] The terms “polynucleotides”, “nucleic acids”, “nucleotides” and “oligonucleotides” are used interchangeably. They refer to nucleotides of any length, encompassing a singular nucleic acid as well as plural nucleic acids, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined from 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. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.

[00116] As used herein, the term “reporter polypeptide(s)” refers to human poly peptide (s) or protein(s) that, under certain circumstances, can be acted upon to generate a detectable signal (such as being enzymatically digested to produce detectable peptide sequence(s)) that can be identified and characterized from outside of a cell, organ, tissue, or body of a subject. For example, a “reporter polypeptide” can be a human protein capable of being cleaved by protease(s) that are also capable of cleaving activatable therapeutic agent(s) (such as described hereinbelow) comprising peptide substrate. Non-limiting examples of peptide substrates include those described hereinbelow in section “Release Segments (RS).”

[00117] The term “complement of a polynucleotide” denotes a polynucleotide molecule having a complementary base sequence and reverse orientation as compared to a reference sequence, such that it could hybridize with a reference sequence with complete fidelity.

[00118] “Recombinant” as applied to a polynucleotide means that the polynucleotide is the product of various combinations of recombination steps which may include cloning, restriction and / or ligation steps, and other procedures that result in expression of a recombinant protein in a host cell.

[00119] The terms “gene” and “gene fragment” are used interchangeably herein. They refer to a polynucleotide containing at least one open reading frame that is capable of encoding a particular protein after being transcribed and translated. A gene or gene fragment may be genomic or cDNA, as long as the polynucleotide contains at least one open reading frame, which may cover the entire coding region or a segment thereof. A “fusion gene” is a gene composed of at least two heterologous polynucleotides that are linked together.

[00120] The term “homology” or “homologous” or “identity” interchangably refers 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, the default settings may be used, or an appropriate scoring matrix, such as blosum45 or blosum80, may be selected to optimize identity, similarity or homology scores. Preferably, polynucleotides that are homologous are those which hybridize under stringent conditions as defined herein and have 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% sequence identity, when optimally aligned, compared to those sequences. Polypeptides that are homologous preferably have sequence identities that are at least 70%, preferably at least 80%, even more preferably at least 90%, even more preferably at least 95-99% identical when optimally aligned over sequences of comparable length.

[00121] The terms “percent identity,” percentage of sequence identity,” and “% identity,” as applied to polynucleotide sequences, refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences. Percent identity may be measured over the length of an entire defined polynucleotide sequence, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polynucleotide sequence, for instance, 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 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured. The percentage of sequence identity is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of matched positions (at which identical residues occur in both polypeptide sequences), dividing the number of matched positions by the total number of positions in the window of comparison (e.g., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. When sequences of different length are to be compared, the shortest sequence defines the length of the window of comparison. Conservative substitutions are not considered when calculating sequence identity.

[00122] “Percent (%) sequence identity” and “percent (%) identity” with respect to the polypeptide sequences identified herein, is defined as the percentage of amino acid residues in a query sequence that are identical with the amino acid residues of a second, reference polypeptide sequence of comparable length or a portion thereof, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity, thereby resulting in optimal alignment. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve optimal alignment over the full length of the sequences being compared. Percent identity may be measured over the length of an entire defined polypeptide sequence, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, 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 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.

[00123] The term “expression” as used herein refers to a process by which a polynucleotide produces a gene product, for example, an RNA or a polypeptide. It includes without limitation transcription of the 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 an mRNA into a polypeptide. Expression produces a "gene product." As used herein, a gene product can be either a nucleic acid, e.g., a messenger RNA produced by transcription of a gene, or a polypeptide which is translated from a transcript. Gene products described herein further include nucleic acids with post transcriptional modifications, e.g., polyadenylation or splicing, or polypeptides with post translational modifications, e.g., methylation, glycosylation, the addition of lipids, association with other protein subunits, or proteolytic cleavage.

[00124] A “vector” or “expression vector” are used interchangeably and refers to a nucleic acid molecule, preferably self-replicating in an appropriate host, which transfers an inserted nucleic acid molecule into and / or between host cells. The term includes vectors that function primarily for insertion of DNA or RNA into a cell, replication of vectors that function primarily for the replication of DNA or RNA, and expression vectors that function for transcription and / or translation of the DNA or RNA. Also included are vectors that provide more than one of the above functions. An “expression vector” is a polynucleotide which, when introduced into an appropriate host cell, can be transcribed and translated into a polypeptide(s). An “expression system” usually connotes a suitable host cell comprised of an expression vector that can function to yield a desired expression product.

[00125] The terms “ti / 2”, “half-life”, “terminal half-life”, “elimination half-life” and “circulating half-life” are used interchangeably herein and, as used herein, generally means the terminal half-life calculated as ln(2) / Kei. Kei is the terminal elimination rate constant calculated by linear regression of the terminal linear portion of the log concentration vs. time curve. Half-life typically refers to the time required for half the quantity of an administered substance deposited in a living organism to be metabolized or eliminated by normal biological processes. When a clearance curve of a given polypeptide is constructed as a function of time, the curve is usually biphasic with a rapid a-phase and longer beta-phase. The typical beta-phase halflife 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.

[00126] The term “molecular weight” generally refers to the sum of atomic weights of the constituent atoms in a molecule. Molecular weight can be determined theoretically by summing the atomic masses of the constituent atoms in a molecule. When applied in the context of a polypeptide, the molecular weight is calculated by adding, based on amino acid composition, the molecular weight of each type of amino acid in the composition or by estimation from comparison to molecular weight standards in an SDS electrophoresis gel. The calculated molecular weight of a molecule can differ from the apparent molecular weight of a molecule, which generally refers to the molecular weight of a molecule as determined by one or more analytical techniques. “Apparent molecular weight factor” and “apparent molecular weight” are related terms and when used in the context of a polypeptide, the terms refer to a measure of the relative increase or decrease in apparent molecular weight exhibited by a particular amino acid or polypeptide sequence. The apparent molecular weight can be determined, for example, using size exclusion chromatography (SEC) or similar methods by comparing to globular protein standards, as measured in “apparent kD” units. The apparent molecular weight factor is the ratio between the apparent molecular weight and the “molecular weight”; the latter is calculated by adding, based on amino acid composition as described above, or by estimation from comparison to molecular weight standards in an SDS electrophoresis gel. The determination of apparent molecular weight and apparent molecular weight factor is described inter alia in US patent number 8,673,860.

[00127] The terms “hydrodynamic radius” or “Stokes radius” is the effective radius (Rhinnm) of a molecule in a solution measured by assuming that it is a body moving through the solution and resisted by the solution’s viscosity. In the embodiments of the disclosure, the hydrodynamic radius measurements of the XTEN polypeptides correlate with the “apparent molecular weight factor” which is a more intuitive measure. The “hydrodynamic radius” of a protein affects its rate of diffusion in aqueous solution as well as its ability to migrate in gels of macromolecules. The hydrodynamic radius of a protein is determined by its molecular weight as well as by its structure, including shape and compactness. Methods for determining the hydrodynamic radius are well known in the art, such as by the use of size exclusion chromatography (SEC), as described inter alia in U.S. Patent Nos. 6,406,632 and 7,294,513. Most proteins have globular structure, which is the most compact three-dimensional structure a protein can have with the smallest hydrodynamic radius. Some proteins adopt a random and open, unstructured, or ‘linear’ conformation and as a result have a much larger hydrodynamic radius compared to typical globular proteins of similar molecular weight.

[00128] “Physiological conditions” refers to a set of conditions in a living host as well as in vitro conditions, including temperature, salt concentration, pH, that mimic those conditions of a living subject. A host of physiologically relevant conditions for use in in vitro assays have been established. Generally, a physiological buffer contains a physiological concentration of salt and is adjusted to a neutral pH ranging from about 6.5 to about 7.8, and preferably from about 7.0 to about 7.5. A variety of physiological buffers are listed in Sambrook et al. (2001). Physiologically relevant temperature ranges from about 25°C to about 38°C, and preferably from about 35°C to about 37°C.

[00129] The term “binding moiety” is used herein in the broadest sense, and is specifically intended to include the categories of cytokines, cell receptors, antibodies or antibody fragments that have specific affinity for an antigen or ligand such as cell-surface receptors, target cell markers, or antigens or glycoproteins, oligonucleotides, enzymatic substrates, antigenic determinants, or binding sites that may be present in or on the surface of a tissue or cell.

[00130] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical and / or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immuno globulin loci, such methods and other exemplary methods for making monoclonal antibodies being known in the art or described herein.

[00131] An “antibody fragment,” as used herein, generally refers to a molecule other than an intact antibody that comprises a portion of an intact antibody and that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab’, Fab’-SH, F(ab’)2, diabodies, single chain diabodies, linear antibodies, nanobodies (also known as single domain antibodies (including single domain camelid antibodies) or Vhh) single-chain variable fragment (scFv) antibody molecules, and multispecific antibodies formed from antibody fragments.

[00132] “scFv” or “single chain fragment variable” are used interchangeably herein to refer to an antibody fragment format comprising regions of variable heavy (“VH”) and variable light (“VL”) chains or two copies of a VH or VL chain, which are joined together by a short flexible peptide linker. The scFv is not actually a fragment of an antibody, but is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, and can be easily expressed in functional form in E. coli or mammalian cell(s) in either N- to C-termnus orientation; VL-VH or VH-VL.

[00133] The terms “antigen”, “target cell marker” and “ligand” are used interchangeably herein to refer to the structure or binding determinant that a binding moiety, an antibody, antibody fragment or an antibody fragment-based molecule binds to or has binding specificity against.

[00134] The term “epitope” refers to the particular site on an antigen molecule to which an antibody, antibody fragment, or binding moiety binds. An epitope is a ligand of an antibody, antibody fragment, or a binding moiety.

[00135] The term "diagnostic reagent" is used herein to refer to any reagent used in vivo or in vitro for detection of, or screening for a particular disease. This includes but is not limited to assays, antibodies, tests, nucleic acid-based tests including RT-PCR,

[00136] As used herein, “CD3” or “cluster of differentiation 3” means the T cell surface antigen CD3 complex, which includes in individual form or independently combined form all known CD3 subunits, for example CD3 epsilon, CD3 delta, CD3 gamma, CD3 zeta, CD3 alpha and CD3 beta. The extracellular domains of CD3 epsilon, gamma and delta contain an immunoglobulin-like domain, so are therefore considered part of the immunoglobulin superfamily.

[00137] The terms “specific binding” or “specifically bind” or “binding specificity” are used interchangeably herein to refer to the high degree of binding affinity of a binding moiety to its corresponding target. Typically, specific binding as measured by one or more of the assays disclosed herein would have a dissociation constant or Ka of less than about 10'6 M (e.g, of 10'7 M to 10'12 M).

[00138] The term “affinity,” as used herein, generally refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). As used herein “a greater binding affinity” or “increased binding affinity” means a lower Kd value; e.g., 1 x 10'9 M is a greater binding affinity than 1 x 10'8 M, while a “lower binding affinity” means a greater Kd value; e.g., 1 x 10'7 M is a lower binding affinity than 1 x 10'8 M.

[00139] “Inhibition constant”, or “K”, are used interchangeably and mean the dissociation constant of the enzyme-inhibitor complex, or the reciprocal of the binding affinity of the inhibitor to the enzyme.

[00140] “Dissociation constant”, or “Kd”, are used interchangeably and mean the affinity between a ligand “L” and a protein “P”; e.g., how tightly a ligand binds to a particular protein. It can be calculated using the formula Kd = [L][P] / [LP], where [P], [L] and [LP] represent molar concentrations of the protein, ligand and complex, respectively. The term “kon”, as used herein, is intended to refer to the on rate constant for association of an antibody to the antigen to form the antibody / antigen complex as is known in the art. The term “kOff”, as used herein, is intended to refer to the off rate constant for dissociation of an antibody from the antibody / antigen complex as is known in the art. Techniques such as flow cytometry or surface plasmon resonance can be used to detect binding events. The assays may comprise soluble antigens or receptor molecules, or may determine the binding to cell-expressed receptors. Such assays may include cell-based assays, including assays for proliferation, cell death, apoptosis and cell migration. The binding affinity of the subject compositions for the target ligands can be assayed using binding or competitive binding assays, such as Biacore assays with chip-bound receptors or binding proteins or ELISA assays, as described in US Patent 5,534,617, assays described in the Examples herein, radio-receptor assays, reporter gene activity assays, or other assays known in the art. For example, an exemplary reporter gene activity assay can be based on genetically engineered cell(s), generated by stably introducing relevant gene(s) for the receptor(s)- of-interest and the signaling pathway(s)-of-interest, such that binding to the engineered receptor triggers a signaling cascade leading to the activation of the engineered gene pathway with a subsequent production of signature polypeptide(s) (such as an enzyme). The binding affinity constant can then be determined using standard methods, such as Scatchard analysis, as described by van Zoelen, et al., Trends Pharmacol Sciences (1998) 19)12):487, or other methods known in the art.

[00141] A “target cell marker” refers to a molecule expressed by a target cell including but not limited to cell-surface receptors, cytokine receptors, antigens, tumor-associated antigens, glycoproteins, oligonucleotides, enzymatic substrates, antigenic determinants, or binding sites that may be present in the on the surface of a target tissue or cell that may serve as ligands for a binding moiety. Non-limiting examples of target cell markers include the target markers of Table 6.

[00142] The term “target tissue” generally refers to a tissue that is the cause of or is part of a disease condition such as, but not limited to cancer or inflammatory conditions. Sources of diseased target tissue include a body organ, a tumor, a cancerous cell or population of cancerous cells or cells that form a matrix or are found in association with a population of cancerous cells, bone, skin, cells that produce cytokines or factors contributing to a disease condition.

[00143] The term “target cell” generally refers to a cell that has the ligand of a binding moiety, an antibody or antibody fragment of the subject compositions and is associated with or causes a disease or pathologic condition, including cancer cells, tumor cells, and inflammatory cells. The ligand of a target cell is referred to herein as a “target cell marker” or “target cell antigen” and includes, but is not limited to, cell surface receptors or antigens, cytokines, cytokine receptors, MHC proteins, and cytosol proteins or peptides that are exogenously presented. As used herein, “target cell” would not include an effector cell.

[00144] As used herein, an “immunoassay” generally refers to a biochemical test that measures the presence or concentration of a substance in a sample, such as a biological sample, using the reaction of an antibody (or a fragment thereof) to its cognate antigen, for example the specific binding of an antibody to a protein. Both the presence of the antigen or the amount of the antigen present can be measured.

[00145] As used herein, a “mass spectrometer (MS)” generally refers to an apparatus that includes a means for ionizing molecules and detecting charged molecules. A mass spectrum generated by a mass spectrometer can be used to identify molecule(s) of interest based on the molar mass. Non-limiting examples of “mass spectrometer (MS)” include all combinations with liquid chromatography (LC), such as liquid chromatography with mass spectrometry (LC-MS), liquid chromatography with tandom mass spectrometry (LC-MS / MS), etc.

[00146] As used herein, the terms “treatment” or “treating,” or “palliating” or “ameliorating” are used interchangeably herein. These terms generally refer to an approach for obtaining beneficial or desired results including but not limited to a therapeutic benefit and / or a prophylactic benefit. By therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms or improvement in one or more clinical parameters associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder. For prophylactic benefit, the compositions may be administered to a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease may not have been made.

[00147] A “therapeutic effect” or “therapeutic benefit,” as used herein, generally refers to a physiologic effect, including but not limited to the mitigation, amelioration, or prevention of disease or an improvement in one or more clinical parameters associated with the underlying disorder in humans or other animals, or to otherwise enhance physical or mental wellbeing of humans or animals, resulting from administration of a polypeptide of the disclosure other than the ability to induce the production of an antibody against an antigenic epitope possessed by the biologically active protein. For prophylactic benefit, the compositions may be administered to a subject at risk of developing a particular disease, a recurrence of a former disease, condition or symptom of the disease, or to a subject reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease may not have been made.

[00148] The terms “therapeutically effective amount” and “therapeutically effective dose”, as used herein, generally refer to an amount of a drug or a biologically active protein, either alone or as a part of a polypeptide composition, that is capable of having any detectable, beneficial effect on any symptom, aspect, measured parameter or characteristics of a disease state or condition when administered in one or repeated doses to a subject. Such effect need not be absolute to be beneficial. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

[00149] The term “equivalent molar dose” generally means that the amounts of materials administered to a subject have an equivalent amount of moles, based on the molecular weight of the material used in the dose.

[00150] The term “therapeutically effective and non-toxic dose,” as used herein, generally refers to a tolerable dose of the compositions as defined herein that is high enough to cause depletion of tumor or cancer cells, tumor elimination, tumor shrinkage or stabilization of disease without or essentially without major toxic effects in the subject. Such therapeutically effective and non-toxic doses may be determined by dose escalation studies described in the art and should be below the dose inducing severe adverse side effects.

[00151] The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth / proliferation. COMPOSITIONS Therapeutic Agents

[00152] Provided herein, in some embodiments, is a therapeutic agent (or an activatable therapeutic agent, or a non-natural, activatable therapeutic agent) that comprises a release segment (RS) (such as one described hereinbelow in the RELEASE SEGMENTS section or described anywhere else herein) linked, directly or indirectly, to a biologically active moiety (BM) (such as one described hereinbelow in the BIOLOGICALLY ACTIVE Moieties section or described anywhere else herein). The biologically active moiety (BM) can be a biologically active peptide (BP) (such as one described hereinbelow in the BIOLOGICALLY ACTIVE Moieties section or described anywhere else herein). The release segment (RS) can comprise a peptide substrate (such as one described hereinbelow in the Release Segments section or described anywhere else herein) susceptible to cleavage by a mammalian protease (such as one described hereinbelow or described anywhere else herein) at a scissile bond. The therapeutic agent can further comprise a masking moiety (MM) (such as one described hereinbelow in the MASKING MOIETIES section or described anywhere else herein) linked, directly or indirectly, to the release segment (RS). A bioactivity of the therapeutic agent can be enhanced upon cleavage of the peptide substrate by the mammalian protease (thereby releasing the masking moiety). The therapeutic agent, in an uncleaved state, can have a structural arrangement from N-terminus to C-terminus of BM-RS-MM or MM-RS-BM. Upon cleavage of the release segment (RS), the masking moiety (MM) can be released from the therapeutic agent. The masking moiety (MM) can comprise an extended recombinant polypeptide (XTEN). The therapeutic agent, in an uncleaved state, can have a structural arrangement from N-terminus to C-terminus of BM-RS-XTEN or XTEN-RS-BM.

[00153] In some embodiments of the therapeutic agent (or the activatable therapeutic agent, or the nonnatural, activatable therapeutic agent), where the release segment (RS) can be a first release segment (RSI), where the peptide substrate (of the RSI) can be a first peptide substrate, and where the scissile bond (of the RSI) can be a first scissile bond, the therapeutic agent can further comprise a second release segment (RS2) (such as one described hereinbelow in the Release Segments section or described anywhere else herein) linked, directly or indirectly, to the biologically active moiety (BM). The second release segment (RS2) can comprise a second peptide substrate (such as one described hereinbelow in the Release Segments section or described anywhere else herein) for cleavage by a mammalian protease (such as one described hereinbelow or described anywhere else herein) at a second scissile bond. A bioactivity of the therapeutic agent can be enhanced upon cleavage of one or both of the first and second peptide substrate by the mammalian protease (thereby releasing one or both of the first and second masking moieties). The mammalian protease for cleavage of the second release segment (RS2) can be identical to the mammalian protease for cleavage of the first release segment (RS 1). The mammalian protease for cleavage of the second release segment (RS2) can be different from the mammalian protease for cleavage of the first release segment (RSI). The second release segment (RS2) can have an amino acid sequence identical to that of the first release segment (RSI). The second release segment (RS2) can have an amino acid sequence different from that of the first release segment (RSI). In some embodiments, the scissile bond (or the first scissile bond, or the scond scissile bond) is not immediately C-terminal to a methionine residue. In some embodiments, the first scissile bond is not immediately C-terminal to a methionine residue. In some embodiments, the second scissile bond is not immediately C-terminal to a methionine residue.

[00154] In some embodiments of the therapeutic agent (or the activatable therapeutic agent, or the nonnatural, activatable therapeutic agent), where the masking moiety (MM) can be a first masking moiety (MM1), the therapeutic agent can further comprise a second masking moiety (MM2) (such as one described hereinbelow in the MASKING MOIETIES section or described anywhere else herein) linked, directly or indirectly, to the second release segment (RS2). The therapeutic agent, in an uncleaved state, can have a structural arrangement from N-terminus to C-terminus of MM1-RS1-BM-RS2-MM2, MM1-RS2-BM-RS1-MM2, MM2-RS1-BM-RS2-MM1, or MM2-RS2-BM-RS1-MM1. Upon cleavage of the second release segment (RS2), the second masking moiety (MM2) can be released from the therapeutic agent. The first masking moiety (MM1) can comprise a first extended recombinant polypeptide (XTEN1). The second masking moiety (MM2) can comprise a second extended recombinant polypeptide (XTEN2). The therapeutic agent, in an uncleaved state, can have a structural arrangement 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.

[00155] In some embodiments of the therapeutic agent (or the activatable therapeutic agent, or the nonnatural, activatable therapeutic agent), the therapeutic agent can comprise a fusion polypeptide (e.g., a recombinant fusion protein) or conjugate (e.g., linked by chemical conjugation). In some embodiments, the therapeutic agent can be configured for activation at or in proximity to a target tissue or cell (such as one described hereinbelow in the TARGET TISSUES OR CELLS section or described anywhere else herein) in a subject. The therapeutic agent can be an anti-cancer agent (such as an activatable anti-cancer agent, or a non-natural, activatable anti-cancer agent). The therapeutic agent can be configured for activation by one or more mammalian proteases (such as one or any combination of those described herein).

[00156] In some embodiments of the therapeutic agent (or the activatable therapeutic agent, or the nonnatural, activatable therapeutic agent), the therapeutic agent can comprise a recombinant polypeptide. The recombinant polypeptide can comprise the biologically active peptide (BP) and the release segment (RS). The recombinant polypeptide can comprise the biologically active peptide (BP), the release segment (RS), and the masking moiety (MM). The recombinant polypeptide, in an uncleaved state, can have a structural arrangement from N-terminus to C-terminus of BP-RS-MM or MM-RS-BP. The recombinant polypeptide can comprise the biologically active peptide (BP), the first release segment (RSI), and the second release segment (RS2). The recombinant polypeptide can comprise the biologically active peptide (BP), the first release segment (RSI), the second release segment (RS2), the first masking moiety (MM1), and the second masking moiety (MM2). The recombinant polypeptide, in an uncleaved state, can have a structural arrangement 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. The recombinant polypeptide can comprise the biologically active peptide (BP), the first release segment (RSI), the second release segment (RS2), the first extended recombinant polypeptide (XTEN1), and the second extended recombinant polypeptide (XTEN2). The recombinant polypeptide, in an uncleaved state, can have a structural arrangement 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. Release Segments (RS)

[00157] In some embodiments of the therapeutic agent (or the activatable therapeutic agent, or the nonnatural, activatable therapeutic agent), the release segment (RS) (or the first release segment (RSI), or the second release segment (RS2), can {each independently) comprise a peptide substrate susceptible to cleavage by a mammalian protease at a scissile bond. The release segment (RS) (or the first release segment (RSI), or the second release segment (RS2)) can (each independently) be cleaved when in proximity to a target tissue or cell (such as one described hereinbelow in the TARGET TISSUES OR CELLS section or described anywhere else herein), where the target tissue or cell can produce a mammalian protease (such as one described hereinbelow in the TARGET TISSUES OR CELLS section or described anywhere else herein) for which the release segment (RS) (or the first release segment (RSI), or the second release segment (RS2)) is a peptide substrate.

[00158] In some embodiments of the therapeutic agent (or the activatable therapeutic agent, or the nonnatural, activatable therapeutic agent), the peptide substrate (or the first peptide substrate, or the second peptide substrate) can have at most four, or at most three, or at most two, or at most one amino acid substitution(s) with respect to a cleavage sequence (such as one set forth in Tables l(a)-l(j) or Table A) of a reporter polypeptide (such as one described hereinbelow in the TARGET TISSUES OR CELLS section or described anywhere else herein). The peptide substrate (or the first peptide substrate, or the second peptide substrate) can have at most four, or at most three, or at most two, or at most one amino acid substitution(s) with respect to a cleavage sequence (such as one set forth in Tables l(a)-l(j) or Table A) of the reporter polypeptide. The peptide substrate (or the first peptide substrate, or the second peptide substrate) can comprise an amino acid sequence identical to a cleavage sequence (such as one set forth in Tables l(a)-l(j) or Table A) of the reporter polypeptide. In some embodiments of the therapeutic agent (or the activatable therapeutic agent, or the non-natural, activatable therapeutic agent), the peptide substrate (or the first peptide substrate, or the second peptide substrate) can comprise an amino acid sequence having at most four, or at most three, or at most two, or at most one amino acid substitution(s) with respect to a sequence set forth in Column II or III of Table A (or a subset thereol) and / or the group set forth in Tables l(a)-l(j) (or any subset thereol). The peptide substrate (or the first peptide substrate, or the second peptide substrate) can comprise an amino acid sequence having at most four, or at most three, or at most two, or at most one amino acid substitution(s) with respect to a sequence set forth in Column II or III of Table A (or a subset thereol) and / or the group set forth in Tables l(a)-l(j) (or any subset thereol). The peptide substrate (or the first peptide substrate, or the second peptide substrate) can comprise an amino acid sequence identical to a sequence set forth in Column II or III of Table A (or a subset thereol) and / or the group set forth in Tables l(a)-l(j) (or any subset thereol). In some embodiments, the peptide substrate (or the first peptide substrate, or the second peptide substrate) comprises two or three sequences set forth in Column II or III of Table A (or a subset thereol). In some embodiments, where the peptide substrate (or the first peptide substrate , or the second peptide substrate) comprises two sequences set forth in Column II or III of Table A (or a subset thereol), the two sequences partially overlap one another. In some embodiments, where the peptide substrate (or the first peptide substrate, or the second peptide substrate) comprises two sequences set forth in Column II or III of Table A (or a subset thereol), the two sequences do not overlap one another. In some embodiments, where the peptide substrate (or the first peptide substrate, or the second peptide substrate) comprises three sequences set forth in Column II or III of Table A (or a subset thereof), two or all of the three sequences do not overlap one another. In some embodiments, where the peptide substrate (or the first peptide substrate, or the second peptide substrate) comprises three sequences set forth in Column II or III of Table A (or a subset thereof), one of the three sequences partially overlaps with another sequence or both other sequences of the three sequences. In some embodiments, where the peptide substrate (or the first peptide substrate, or the second peptide substrate) comprises three sequences set forth in Column II or III of Table A (or a subset thereof), two of the three sequences partially overlap with one another. In some embodiments, where the peptide substrate (or the first peptide substrate, or the second peptide substrate) comprises three sequences set forth in Column II or III of Table A (or a subset thereof), each two of the three sequences partially overlap with one another. In some embodiments, where the peptide substrate (or the first peptide substrate, or the second peptide substrate) comprises three sequences set forth in Column II or III of Table A (or a subset thereof), all of the three sequences partially overlap with one another. In some embodiments, none of the at most four, at most three, at most two, or at most one amino acid substitution(s) is / are at a position corresponding to an amino acid residue immediately adjacent to a scissile bond of a sequence set forth in Column II or III of Table A (or a subset thereof), tn some embodiments, none of the at most four, at most three, at most two, or at most one amino acid substitution(s) is / are at a position corresponding to an amino acid residue immediately adjacent to a scissile bond of a corresponding sequence selected from the group set forth in Tables l(a)-l(i) (or any subset thereof), tn some embodiments, none of the at most four, at most three, at most two, or at most one amino acid substitution(s) is / are at a position corresponding to an amino acid residue immediately adjacent to a scissile bond of a corresponding sequence selected from the group set forth in Table l(j) (or any subset thereof). The peptide substrate (or the first peptide substrate, or the second peptide substrate) can 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 foregoing values. The peptide substrate can contain from six to twenty-five or six to twenty amino acid residues. The peptide substrate can contain from six to twenty-five amino acid residues. The peptide substrate can contain from six to twenty amino acid residues. In some embodiments, the peptide substrate contains from seven to twelve amino acid residues. The peptide substrate can comprise a fragment of an amino acid sequence set forth in Column II or III of Table A (or a subset thereof) and / or the group set forth in Tables l(a)-l(j) (or any subset thereof). The fragment of the peptide substrate can contain at least four amino acid residues and a corresponding scissile bond (such as indicated in Tables l(a)-l(j) or Table A). The fragment of the peptide substrate can 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, a portion of the peptide substrate that is N-terminal of the scissile bond can have at most four, or at most three, or at most two, or at most one amino acid substitution(s) with respect to a C-terminal end sequence containing from four to ten amino acid residues of a sequence set forth in Column IV or V of Table A (or a subset thereof). The portion of the peptide substrate that is N-terminal of the scissile bond can comprise a C-terminal end sequence containing from four to ten amino acid residues of a sequence set forth in Column IV or V of Table A (or a subset thereof). In some cases, a portion of the peptide substrate that is N-terminal of the scissile bond can have at most four, or at most three, or at most two, or at most one amino acid substitution(s) with respect to a C-terminal end sequence containing from four to ten amino acid residues of a sequence set forth in Column IV of Table A (or a subset thereof). The portion of the peptide substrate that is N-terminal of the scissile bond can comprise a C-terminal end sequence containing from four to ten amino acid residues of a sequence set forth in Column IV of Table A (or a subset thereof), tn some cases, a portion of the peptide substrate that is N-terminal of the scissile bond can have at most four, or at most three, or at most two, or at most one amino acid substitution(s) with respect to a C-terminal end sequence containing from four to ten amino acid residues of a sequence set forth in Column V of Table A (or a subset thereof). The portion of the peptide substrate that is N-terminal of the scissile bond can comprise a C-terminal end sequence containing from four to ten amino acid residues of a sequence set forth in Column V of Table A (or a subset thereof), tn some cases, a portion of the peptide substrate that is C-terminal of the scissile bond can have at most four, or at most three, or at most two, or at most one amino acid substitution(s) with respect to an N-terminal end sequence containing from four to ten amino acid residues of a sequence set forth in Column V or VI of Table A (or a subset thereof). The portion of the peptide substrate that is C-terminal of the scissile bond can an N-terminal end sequence containing from four to ten amino acid residues of a sequence set forth in Column V or VI of Table A (or a subset thereof), tn some cases, a portion of the peptide substrate that is C-terminal of the scissile bond can have at most four, or at most three, or at most two, or at most one amino acid substitution(s) with respect to an N-terminal end sequence containing from four to ten amino acid residues of a sequence set forth in Column V of Table A (or a subset thereof). The portion of the peptide substrate that is C-terminal of the scissile bond can an N-terminal end sequence containing from four to ten amino acid residues of a sequence set forth in Column V of Table A (or a subset thereof), tn some cases, a portion of the peptide substrate that is C-terminal of the scissile bond can have at most four, or at most three, or at most two, or at most one amino acid substitution(s) with respect to an N-terminal end sequence containing from four to ten amino acid residues of a sequence set forth in Column VI of Table A (or a subset thereof). The portion of the peptide substrate that is C-terminal of the scissile bond can an N-terminal end sequence containing from four to ten amino acid residues of a sequence set forth in Column VI of Table A (or a subset thereof), tn some embodiments, where the peptide substrate comprises a scissile bond (for cleavage by one or more mammalian proteases), the peptide substrate does not comprise a methionine residue immediately N-terminal to the scissile bond. In some embodiments, where the peptide substrate comprises a plurality of scissile bonds, the peptide substrate does not comprise a methionine residue immediately N-terminal to at least one scissile bond of the plurality of scissile bonds. In some embodiments, where the peptide substrate comprises a plurality of scissile bonds, the peptide substrate does not comprise a methionine residue immediately N-terminal to each scissile bond of the plurality of scissile bonds. In some embodiments, the peptide substrate does not comprise 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) of Column II of Table A

[00159] In some embodiments of the therapeutic agent (or the activatable therapeutic agent, or the nonnatural, activatable therapeutic agent) that comprises (1) a first release segment (RSI) comprising a first peptide substrate and (2) a second release segment (RS2) comprising a second peptide substrate, the second peptide substrate can 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 foregoing values. The second peptide substrate can contain from six to twenty-five or six to twenty amino acid residues. The second peptide substrate can contain from six to twenty-five amino acid residues. The second peptide substrate can contain from six to twenty amino acid residues. The second peptide substrate can contain from seven to twelve amino acid residues. The second peptide substrate can comprise an amino acid sequence having at most four, or at most three, or at most two, or at most one amino acid substitution(s) with respect to a sequence set forth in Column II or III of Table A (or a subset thereof) and / or the group set forth in Tables l(a)-l(j) (or any subset thereof). The second peptide substrate can comprise an amino acid sequence having at most four, or at most three, or at most two, or at most one amino acid substitution(s) with respect to a sequence set forth in Column II or III of Table A (or a subset thereof) and / or the group set forth in Tables l(a)-l(j) (or any subset thereof). The second peptide substrate can comprise an amino acid sequence identical to a sequence set forth in Column II or III of Table A (or a subset thereof) and / or the group set forth in Tables l(a)-l(j) (or any subset thereof), tn some embodiments, the second peptide substrate comprises two or three sequences set forth in Column II or III of Table A (or a subset thereof), tn some embodiments, where the second peptide substrate comprises two sequences set forth in Column II or III of Table A (or a subset thereof), the two sequences (of the second peptide substrate) partially overlap one another. In some embodiments, where the second peptide substrate comprises two sequences set forth in Column II or III of Table A (or a subset thereof), the two sequences (of the second peptide substrate) do not overlap one another. In some embodiments, where the second peptide substrate comprises three sequences set forth in Column II or III of Table A (or a subset thereof), two or all of the three sequences (of the second peptide substrate) do not overlap one another. In some embodiments, where the second peptide substrate comprises three sequences set forth in Column II or III of Table A (or a subset thereof), one of the three sequences (of the second peptide substrate) partially overlaps with another sequence or both other sequences of the three sequences (of the second peptide substrate). In some embodiments, where the second peptide substrate comprises three sequences set forth in Column II or III of Table A (or a subset thereof), two of the three sequences (of the second peptide substrate) partially overlap with one another. In some embodiments, where the second peptide substrate comprises three sequences set forth in Column II or III of Table A (or a subset thereof), each two of the three sequences (of the second peptide substrate) partially overlap with one another. In some embodiments, where the second peptide substrate comprises three sequences set forth in Column II or III of Table A (or a subset thereof), all of the three sequences (of the second peptide substrate) partially overlap with one another. In some embodiments, where the second peptide substrate comprises a scissile bond (for cleavage by one or more mammalian proteases), the second peptide substrate does not comprise a methionine residue immediately N-terminal to the scissile bond. In some embodiments, where the second peptide substrate comprises a plurality of scissile bonds, the second peptide substrate does not comprise a methionine residue immediately N-terminal to at least one scissile bond of the plurality of scissile bonds. In some embodiments, where the second peptide substrate comprises a plurality of scissile bonds, the second peptide substrate does not comprise a methionine residue immediately N-terminal to each scissile bond of the plurality of scissile bonds. In some embodiments, the second peptide substrate does not comprise 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 thereol) of Column II of Table A.

[00160] In some embodiments of the present disclosure, the peptide substrate (or the first peptide substrate, or the second peptide substrate) does not comprise a sequence selected from SEQ ID NOS: 1-8. In some embodiments, the peptide substrate (or the first peptide substrate, or the second peptide substrate) does not comprise a sequence of SEQ ID NO: 1. In some embodiments, the peptide substrate (or the first peptide substrate, or the second peptide substrate) does not comprise a sequence of SEQ ID NO: 2. In some embodiments, the peptide substrate (or the first peptide substrate, or the second peptide substrate) does not comprise a sequence of SEQ ID NO: 3. In some embodiments, the peptide substrate (or the first peptide substrate, or the second peptide substrate) does not comprise a sequence of SEQ ID NO: 4. In some embodiments, the peptide substrate (or the first peptide substrate, or the second peptide substrate) does not comprise a sequence of SEQ ID NO: 5. In some embodiments, the peptide substrate (or the first peptide substrate, or the second peptide substrate) does not comprise a sequence of SEQ ID NO: 6. In some embodiments, the peptide substrate (or the first peptide substrate, or the second peptide substrate) does not comprise a sequence of SEQ ID NO: 7. In some embodiments, the peptide substrate (or the first peptide substrate, or the second peptide substrate) does not comprise a sequence of SEQ ID NO: 8. In some embodiments, the peptide substrate (or the first peptide substrate, or the second peptide substrate) does not comprise a methionine residue immediately N-terminal to a scissile bond (contained therein) (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 comprise a methionine residue immediately N-terminal to one or more scissile bonds (contained therein). In some embodiments, the peptide substrate (or the first peptide substrate, or the second peptide substrate) does not comprise a methionine residue immediately N-terminal to any scissile bond (contained therein). In some embodiments, the peptide substrate (or the first peptide substrate or the second peptide substrate) does not comprise 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 thereol) of Column II of Table A.

[00161] In some embodiments of the therapeutic agent (or the activatable therapeutic agent, or the nonnatural, activatable therapeutic agent), a six to ten consecutive amino acid sequence of a peptide substrate (e.g., a first peptide substrate, a second peptide substrate, etc.) comprises at most four, at most three, at most two, or at most one amino acid substitution(s), with respect to a corresponding six to ten consecutive amino acid sequence of a sequence set forth in Column II or III of Table A (or a subset thereof). In some embodiments, a six to ten consecutive amino acid sequence of a peptide substrate (e.g., a first peptide substrate, a second peptide substrate, etc.) is identical to a corresponding six to ten consecutive amino acid sequence of a sequence set forth in Column II or III of Table A (or a subset thereof), tn some embodiments, an eight to ten consecutive amino acid sequence of a peptide substrate (e.g., a first peptide substrate, a second peptide substrate, etc.) comprises at most three, at most two, or at most one amino acid substitution(s), with respect to a corresponding eight to ten consecutive amino acid sequence of a sequence set forth in Column II or III of Table A (or a subset thereof), tn some embodiments, an eight to ten consecutive amino acid sequence of a peptide substrate (e.g., a first peptide substrate, a second peptide substrate, etc.) is identical to a corresponding eight to ten consecutive amino acid sequence of a sequence set forth in Column II or III of Table A (or a subset thereof), tn some embodiments, an eight consecutive amino acid sequence of a peptide substrate (e.g., a first peptide substrate, a second peptide substrate, etc.) comprises at most three, at most two, or at most one amino acid substitution(s), with respect to a corresponding eight consecutive amino acid sequence of a sequence set forth in Column II or III of Table A (or a subset thereof), tn some embodiments, an eight consecutive amino acid sequence of a peptide substrate (e.g., a first peptide substrate, a second peptide substrate, etc.) is identical to a corresponding eight consecutive amino acid sequence of a sequence set forth in Column II or III of Table A (or a subset thereof), tn some embodiments, a nine consecutive amino acid sequence of a peptide substrate (e.g., a first peptide substrate, a second peptide substrate, etc.) comprises at most three, at most two, or at most one amino acid substitution(s), with respect to a corresponding nine consecutive amino acid sequence of a sequence set forth in Column II or III of Table A (or a subset thereof), tn some embodiments, a nine consecutive amino acid sequence of a peptide substrate (e.g., a first peptide substrate, a second peptide substrate, etc.) is identical to a corresponding nine consecutive amino acid sequence of a sequence set forth in Column II or III of Table A (or a subset thereof), tn some embodiments, a ten consecutive amino acid sequence of a peptide substrate (e.g., a first peptide substrate, a second peptide substrate, etc.) comprises at most three, at most two, or at most one amino acid substitution(s), with respect to a corresponding ten consecutive amino acid sequence of a sequence set forth in Column II or III of Table A (or a subset thereof), tn some embodiments, a ten consecutive amino acid sequence of a peptide substrate (e.g., a first peptide substrate, a second peptide substrate, etc.) is identical to a corresponding ten consecutive amino acid sequence of a sequence set forth in Column II or III of Table A (or a subset thereof).

[00162] tn some embodiments of the therapeutic agent (or the activatable therapeutic agent, or the nonnatural, activatable therapeutic agent), the release segment (RS) (or the first release segment (RSI), or the second release segment (RS2), can {each independently) comprise a peptide substrate (or a first peptide substrate, or a second peptide substrate) for cleavage by a mammalian protease, such as a serine protease, a cysteine protease, an aspartate protease, a threonine protease, or a metalloproteinase. The release segment (RS) (or the first release segment (RSI), or the second release segment (RS2), can (independently) comprise a peptide substrate (or a first peptide substrate, or a second peptide substrate) for cleavage by a mammalian protease selected from the group consisting of 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 (ADAM 17), disintegrin and metalloproteinase domain-containing protein 9 (ADAM9), disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), Cathepsin B, Cathepsin D, Cathepsin E, Cathepsin K, cathepsin L, cathepsin S, Fibroblast activation protein alpha, Hepsin, kallikrein-2, kallikrein-4, kallikrein-3, Prostate-specific antigen (PSA), kallikrein-13, Legumain, 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 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 type serine protease 1 (MT-SP1), matriptase, and u-plasminogen. The release segment (RS) (or the first release segment (RSI), or the second release segment (RS2), can (independently) comprise a peptide substrate (or a first peptide substrate, or a second peptide substrate) for cleavage by a mammalian protease selected from the group consisting of matrix metallopeptidase 1 (MMP1) (for which the sequences listed in Table 1(a), as examples without being limited to, are substrate sequences), matrix metallopeptidase 2 (MMP2) (for which the sequences listed in Table 1(b), as examples without being limited to, are substrate sequences), matrix metallopeptidase 7 (MMP7) (for which the sequences listed in Table 1(c), as examples without being limited to, are substrate sequences), matrix metallopeptidase 9 (MMP9) (for which the sequences listed in Table 1(d), as examples without being limited to, are substrate sequences), matrix metallopeptidase 11 (MMP11) (for which the sequences listed in Table 1(e), as examples without being limited to, are substrate sequences), matrix metallopeptidase 14 (MMP14) (for which the sequences listed in Table 1(f), as examples without being limited to, are substrate sequences), urokinase-type plasminogen activator (uPA) (for which the sequences listed in Table 1(g), as examples without being limited to, are substrate sequences), legumain (for which the sequences listed in Table 1(h), as examples without being limited to, are substrate sequences), and matriptase (for which the sequences listed in Table l(i), as examples without being limited to, are substrate sequences). The release segment (RS) (or the first release segment (RSI), or the second release segment (RS2), can (independently) comprise a peptide substrate (or a first peptide substrate, or a second peptide substrate) for cleavage by a plurality of mammalian proteases. The peptide substrate (or the first peptide substrate, or the second peptide substrate) susceptible to cleavage by the mammalian protease can be susceptible to cleavage by a plurality of mammalian proteases comprising the mammalian protease. The peptide substrate (or the first peptide substrate, or the second peptide substrate) susceptible to cleavage by the plurality of mammalian proteases can have at most four, or at most three, or at most two, or at most one amino acid substitution(s) with respect to a sequence set forth in Table l(j). The peptide substrate (or the first peptide substrate, or the second peptide substrate) susceptible to cleavage by the plurality of mammalian proteases can have at most four, or at most three, or at most two, or at most one amino acid substitution(s) with respect to a sequence set forth in Table l(j). The peptide substrate (or the first peptide substrate, or the second peptide substrate) susceptible to cleavage by the plurality of mammalian proteases can have at most four, or at most three, or at most two, or at most one amino acid substitution(s) with respect to a sequence set forth in Table l(j). The peptide substrate (or the first peptide substrate, or the second peptide substrate) susceptible to cleavage by the plurality of mammalian proteases can comprise a sequence set forth in Table l(j).

[00163] In some embodiments of the therapeutic agent (or the activatable therapeutic agent, or the nonnatural, activatable therapeutic agent) that comprises a set of release segments, each release segment in the set can (independently) comprise a peptide substrate for cleavage by a mammalian protease, such as a serine protease, a cysteine protease, an aspartate protease, a threonine protease, or a metalloproteinase. Each release segment in the set can (independently) comprise a peptide substrate for a different mammalian protease (independently) selected from the group consisting of disintegrin and metalloproteinase domaincontaining protein 10 (ADAM 10), 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 domaincontaining protein 9 (ADAM9), disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), Cathepsin B, Cathepsin D, Cathepsin E, Cathepsin K, cathepsin L, cathepsin S, Fibroblast activation protein alpha, Hepsin, kallikrein-2, kallikrein-4, kallikrein-3, Prostate-specific antigen (PSA), kallikrein-13, Legumain, 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 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 type serine protease 1 (MT-SP1), matriptase, and u-plasminogen. Each release segment in the set can (independently) comprise a peptide substrate for a different mammalian protease (independently) selected from the group consisting of matrix metallopeptidase 1 (MMP1) (for which the sequences listed in Table 1(a), as examples without being limited to, are substrate sequences), matrix metallopeptidase 2 (MMP2) (for which the sequences listed in Table 1(b), as examples without being limited to, are substrate sequences), matrix metallopeptidase 7 (MMP7) (for which the sequences listed in Table 1(c), as examples without being limited to, are substrate sequences), matrix metallopeptidase 9 (MMP9) (for which the sequences listed in Table 1(d), as examples without being limited to, are substrate sequences), matrix metallopeptidase 11 (MMP11) (for which the sequences listed in Table 1(e), as examples without being limited to, are substrate sequences), matrix metallopeptidase 14 (MMP14) (for which the sequences listed in Table 1(f), as examples without being limited to, are substrate sequences), urokinase-type plasminogen activator (uPA) (for which the sequences listed in Table 1(g), as examples without being limited to, are substrate sequences), legumain (for which the sequences listed in Table 1(h), as examples without being limited to, are substrate sequences), and matriptase (for which the sequences listed in Table l(i), as examples without being limited to, are substrate sequences). In some cases, at least one release segment (RS) of the set of release segments can (independently) comprise a peptide substrate for cleavage by a plurality of mammalian proteases. The peptide substrate susceptible to cleavage by the plurality of mammalian proteases can have at most four, or at most three, or at most two, or at most one amino acid substitution(s) with respect to a sequence set forth in Table l(j). The peptide substrate susceptible to cleavage by the plurality of mammalian proteases can have at most four, or at most three, or at most two, or at most one amino acid substitution(s) with respect to a sequence set forth in Table l(j). The peptide substrate susceptible to cleavage by the plurality of mammalian proteases can have at most four, or at most three, or at most two, or at most one amino acid substitution(s) with respect to a sequence set forth in Table l(j). The peptide substrate susceptible to cleavage by the plurality of mammalian proteases can comprise a sequence set forth in Table l(j). One of skill in the art will understand that a sequence set forth in Tables l(a)-l(j) may, alternatively or additionally, be cleaved by one or more other proteases with substrate specificity similar to that of a corresponding protease, identified in a corresponding table, as capable of cleaving the sequence. Table 1(a). Exemplary peptide substrates for cleavage by matrix metallopeptidase 1 (MMP1) Name of Reporter Polypeptide SEQ ID IBBB^iBBB Amino Acid Sequence elastin 36 IGPGG-VAAAA alpha-1 -antitrypsin 37 DPQG-DAAQ type I collagen alpha-1 chain 38 DGVRG-LTGPI type V collagen alpha-1 chain 39 RGPSG-HMGRE elastin 40 ISPEA-QAAAA Complement C4-B OR Complement C4-A 41 TPLQ-LFEG type III collagen alpha-1 chain 42 QGPPG-KNGET alpha-2-HS-gly coprotein 43 PPLG-APGL apolipoprotein LI 44 KPLG-DWAA type II collagen alpha-1 chain 45 DGAAG-VKGDR Table 1(b). Exemplary peptide substrates for cleavage by matrix metallopeptidase 2 (MMP2) Name of Reporter Polypeptide SEQ ID Amino Acid Sequence alpha-1 -antichymotrypsin 46 LLSA-LVET pigment epithelium-derived factor 47 QPAH-LTFP SPARC 48 DHPVE-LLARD integrin alpha-IIb 49 QPSR-LQDP Name of Reporter Polypeptide SEQ ID Amino Acid Sequence type I collagen alpha-1 chain 50 DGVRG-LTGPI zyxin 51 QPVS-LANT elastin 52 IGPGG-VAAAA vitronectin 53 LTSD-LQAQ immunoglobulin kappa variable 2-30 54 SPLS-LPVT type IV collagen alpha-1 chain 55 GDPGE-ILGHV Table 1(c). Exemplary peptide substrates for cleavage by matrix metallopeptidase 7 (MMP7) Name of Reporter Polypeptide SEQ ID IBBiB^^IBiBi Amino Acid Sequence elastin 56 IGPGG-VAAAA Complement C4-B OR Complement C4-A 57 TPLQ-LFEG SPARC 58 DHPVE-LLARD type I collagen alpha-1 chain 59 DGVRG-LTGPI immunoglobulin kappa variable 2-30 60 LPVT-LGQP pigment epithelium-derived factor 61 QPAH-LTFP probable non-functional immunoglobulin kappa variable 2D-24 62 SPVT-LGQP immunoglobulin kappa variable 3-20 63 GTLS-LSPG fibrinogen beta chain 64 EEAPS-LRPA type II collagen alpha-1 chain 65 DGAAG-VKGDR Table 1(d). Exemplary peptide substrates for cleavage by matrix metallopeptidase 9 (MMP9) Name of Reporter Polypeptide SEQ ID NO: Amino Acid Sequence type I collagen alpha-1 chain 66 DGVRG-LTGPI elastin 67 IGPGG-VAAAA type III collagen alpha-1 chain 68 QGPPG-KNGET type V collagen alpha-1 chain 69 RGPSG-HMGRE type II collagen alpha-1 chain 70 DGAAG-VKGDR type VI collagen alpha-1 chain 71 KGAKG-YRGPE alpha-2-HS-gly coprotein 72 PPLG-APGL type VI collagen alpha-3 chain 73 IGNRG-PRGET chromogranin-A 74 GPQL-RRGW transcription factor SOX-10 75 SPPG-VDAK Table 1(e). Exemplary peptide substrates for cleavage by matrix metallopeptidase 11 (MMP11) Name of Reporter Polypeptide SEQ ID Amino Acid Sequence alpha-1 -antitrypsin 76 AAGA-MFLE serum amyloid A-l protein 77 AAEA-ISDA fibrinogen alpha chain 78 EAAF-FDTA complement C4-A OR complement C4-B 79 KSHA-LQLN apolipoprotein C-III 80 SARA-SEAE ceruloplasmin 81 PAWA-KEKH serum amyloid A-2 protein 82 AWAA-EVIS Name of Reporter Polypeptide SEQ ID iiiiiii Amino Acid Sequence fibrinogen beta chain 83 EEAPS-LRPA immunoglobulin lambda variable 3-25 84 SEAS-YELT PDZ and LIM domain protein 1 85 PFTA-SPAS Table 1(f). Exemplary peptide substrates for cleavage by matrix metallopeptidase 14 (MMP14) Name of Reporter Polypeptide SEQ ID BBBiiiBB^ Amino Acid Sequence integrin alpha-IIb 86 QPSR-LQDP alpha-1 -antichymotrypsin 87 LLSA-LVET pigment epithelium-derived factor 88 QPAH-LTFP Complement C4-B OR Complement C4-A 89 TPLQ-LFEG zyxin 90 QPVS-LANT type I collagen alpha-1 chain 91 DGVRG-LTGPI SPARC 92 DHPVE-LLARD immunoglobulin kappa variable 2-30 93 SPLS-LPVT immunoglobulin kappa variable 2-30 94 LPVT-LGQP elastin 95 IGPGG-VAAAA Table 1(g). Exemplary peptide substrates for cleavage by urokinase-type plasminogen activator (uPA) Name of Reporter Polypeptide SEQ ID NO: Amino Acid Sequence serum amyloid A-2 protein 96 RSGR-DPNH serum amyloid A-2 protein 97 AAKR-GPGG deleted in malignant brain tumors 1 protein 98 RSKR-DVGS secretogranin-2 99 VSKR-FPVG serum amyloid A-l protein OR serum amyloid A-2 protein 100 VSSR-SFFS haptoglobin 101 PVQR-ILGG fibrinogen alpha chain 102 SSGP-GSTG fibrinogen beta chain 103 FSAR-GHRP complement C4-A OR complement C4-B 104 RQIR-GLEE oncoprotein-induced transcript 3 protein 105 RMRR-GAGG Table 1(h). Exemplary peptide substrates for cleavage by legumain Name of Reporter Polypeptide IBBiiBB bbbBbbb BBii^IBB Amino Acid Sequence neurosecretory protein VGF 106 RKKN-APPE coagulation factor XII 107 GDRN-KPGV Complement C4-B OR Complement C4-A 108 TGRN-GFKS fibrinogen alpha chain 109 GSWN-SGSS tubulin beta chain 110 EPYN-ATLS transthyretin 111 FTAN-DSGP fibrinogen beta chain 112 QGVN-DNEE Name of Reporter Polypeptide llOlii Amino Acid Sequence fibrinogen alpha chain 113 SPRN-PSSA angiotensinogen 114 QQLN-KPEV multimerin-1 115 TSLN-TVGG Table l(i). Exemplary peptide substrates for cleavage by matriptase Name of Reporter Polypeptide liiiiii lliiili Amino Acid Sequence oncoprotein-induced transcript 3 protein 116 RMRR-GAGG deleted in malignant brain tumors 1 protein 117 RSKR-DVGS serum amyloid A-2 protein 118 AAKR-GPGG inter-alpha-trypsin inhibitor heavy chain H5 119 RVPR-QVRL haptoglobin 120 PVQR-ILGG alpha-2-HS-gly coprotein 121 RKTR-TVVQ sulfhydryl oxidase 1 122 PGLR-AAPG gastric inhibitory polypeptide 123 RGPR-YAEG keratin, type I cytoskeletal 17 124 RQVR-TIVE complement C4-A OR complement C4-B 125 RQIR-GLEE Table Hi). Exemplary peptide substrates for cleavage by multiple proteases SEQ ID NO. Amino Acid Sequence Exemplary Proteases That May Cleave the Peptide substrate 1 GPGG-VAAAVSKR-FPVG MMP2, MMP7, uPA 2 GVRG-LTGPVSKR-FPVG MMP2, MMP7, uPA 3 VSKR-FPVGEAGR-SAN-H uPA, matriptase, legumain 4 EAGR-SAN-HGVRG-LTGP matriptase, legumain, MMP1 5 EAGR-SAN-HTPAG-LTGP MMP2, MMP9, matriptase, legumain 6 SPEA-QAAAEAGR-SAN-H MMP1, matriptase, legumain 7 QPAH-LTFPEAGR-SAN-H MMP2, MMP14, legumain, matriptase 8 AGSPGK-DGVRG-LTGP matriptase, MMP2, MMP9 Masking Moieties (MM)

[00164] A masking moiety (MM) of the present disclosure may be capable of specifically or non-specifically interacting with a biologically active moiety (BM) (or any component(s) or fragment(s) thereof) of an activatable therapeutic agent composition (such as described herein), thereby masking the BM (at least in certain cases) by inhibiting or reducing the ability of the BM to bind with designated target(s). In some instances, the masking moiety (MM) may specifically bind to or have specific affinity for the biologically active moiety (e.g., an antibody or antibody fragment), thereby interfering and / or inhibiting binding of the BM to its designed target (e.g., antigen target). In some instances, the masking moiety does not have significant affinity for the biologically active moiety, but exerts it masking effect due to non-specific steric hinderance.

[00165] In some embodiments of the therapeutic agent (or the activatable therapeutic agent, or the nonnatural, activatable therapeutic agent), the masking moiety (MM) (or the first masking moiety (MM1), or the second masking moiety (MM2)), when linked to the corresponding therapeutic agent, can {each independently, individually or collectively) interfere with an interaction of the biologically active moiety (BM) to a target tissue or cell (such as one described hereinbelow in the Target TISSUES OR CELLS section or described anywhere else herein) such that a dissociation constant (Ka) of the BM of the therapeutic agent with a target cell marker (such as one described hereinbelow in the TARGET TISSUES OR CELLS section or described anywhere else herein) borne by the target tissue or cell can be greater, when the therapeutic agent is in an uncleaved state, compared to a dissociation constant (Kd) of a corresponding biologically active moiety (as remaining after the release segment (RS) is cleaved and the MM is released) with the target cell marker. The dissociation constant (Ka) of the biologically active moiety (BM) of the therapeutic agent, when the therapeutic agent is in an uncleaved state, with the target cell marker can be at least (about) 2-fold greater, at least (about) 5-fold greater, at least (about) 10-fold greater, at least (about) 50-fold greater, at least (about) 100-fold greater, at least (about) 200-fold greater, at least (about) 300-fold greater, at least (about) 400-fold greater, at least (about) 500-fold greater, at least (about) 600-fold greater, at least (about) 700-fold greater, at least (about) 800-fold greater, at least (about) 900-fold greater, or at least (about) 1000fold greater, than the dissociation constant (Kd) of the corresponding biologically active moiety with the target cell marker. The dissociation constant (Kd) can be measured in an in vitro assay under equivalent molar concentrations. The in vitro assay can be selected from cell membrane integrity assay, mixed cell culture assay, cell-based competitive binding assay, FACS based propidium Iodide assay, trypan Blue influx assay, photometric enzyme release assay, radiometric 51Cr release assay, fluorometric Europium release assay, CalceinAM release assay, photometric MTT assay, XTT assay, WST-1 assay, alamar blue assay, radiometric 3H-Thd incorporation assay, clonogenic assay measuring cell division activity, fluorometric rhodamine 123 assay measuring mitochondrial transmembrane gradient, apoptosis assay monitored by FACS-based phosphatidylserine exposure, ELISA-based TUNEL test assay, sandwich ELISA, caspase activity assay, cell-based LDH release assay, and cell morphology assay, reporter gene activity assay, or any combination thereof.

[00166] In some embodiments of the therapeutic agent (or the activatable therapeutic agent, or the nonnatural, activatable therapeutic agent), the therapeutic agent can effect an enhancement in a safety profile, for example, improve a maximum tolerable exposure level (MTEL), and / or reduce a side effect (e.g., cytotoxicity), in delivery of the BM to a target tissue or cell (such as one described hereinbelow in the TARGET Tissues or Cells section or described anywhere else herein) compared to a corresponding biologically active moiety (as remaining after the release segment (RS) is cleaved and the MM is released). The therapeutic agent, in which the biologically active moiety (BM) is linked (directly or indirectly) to the masking moiety (MM) (or the first masking moiety (MM1), or the second masking moiety (MM2)) can effect an enhancement in a safety profile, for example, improve a maximum tolerable exposure level (MTEL), and / or reduce a side effect (e.g., cytotoxicity), by at least (about) 2-fold, by at least (about) 5-fold, by at least (about) 10 fold, by at least (about) 50-fold, by at least (about) 100-fold, by at least (about) 200- fold, by at least (about) 300-fold, by at least (about) 400-fold, or by at least (about) 500-fold higher, in delivery of the BM to the target tissue or cell, than the corresponding biologically active moiety.

[00167] In some embodiments of the therapeutic agent (or the activatable therapeutic agent, or the nonnatural, activatable therapeutic agent), the therapeutic agent can have a longer terminal half-life compared to that of a corresponding biologically active moiety. The therapeutic agent, in which the biologically active moiety (BM) is linked (directly or indirectly) to the masking moiety (MM) (or the first masking moiety (MM1), or the second masking moiety (MM2)) can have a terminal half-life of at least (about) 2-fold longer, at least (about) 5-fold longer, at least (about) 10-fold longer, at least (about) 15-fold longer, at least (about) 20-fold longer, at least (about) 50-fold longer, or at least (about) 100-fold longer, than the terminal half-life of the corresponding biologically active moiety.

[00168] In some embodiments, the therapeutic agent can be less immunogenic compared to a corresponding biologically active moiety. The therapeutic agent, in which the biologically active moiety (BM) is linked (directly or indirectly) to the masking moiety (MM) (or the first masking moiety (MM1), or the second masking moiety (MM2)), can be at least (about) 2-fold less immunogenic, at least (about) 5-fold less immunogenic, or at least (about) 10-fold less immunogenic, than the corresponding biologically active moiety. The immunogenicity can be ascertained by measuring production of IgG antibodies that selectively bind to the biologically active moiety after administration of comparable doses to a subject.

[00169] In some embodiments, the therapeutic agent can have a greater apparent molecular weight factor under a physiological condition, compared to a corresponding biologically active moiety. The therapeutic agent, in which the biologically active moiety (BM) is linked (directly or indirectly) to the masking moiety (MM) (or the first masking moiety (MM1), or the second masking moiety (MM2)), can have an apparent molecular weight factor of at least (about) 1.5-fold greater, at least (about) 2-fold greater, at least (about) 5fold greater, at least (about) 8-fold greater, at least (about) 10-fold greater, at least (about) 12-fold greater, at least (about) 15-fold greater, at least (about) 18-fold greater, or at least (about) 20-fold greater, under a physiological condition, than the corresponding biologically active moiety.

[00170] In some embodiments of the therapeutic agent (or the activatable therapeutic agent, or the nonnatural, activatable therapeutic agent) that comprises a first masking moiety (MM1) and a second masking moiety (MM2), the MM1 and the MM2, when both linked in the therapeutic agent, can (each independently, individually or collectively) interfere with an interaction of the biologically active moiety (BM) to a target tissue or cell (such as one described hereinbelow in the Target TISSUES OR CELLS section or described anywhere else herein) such that a dissociation constant (Ka) of the biologically active moiety (BM) of the therapeutic agent with a target cell marker (such as one described hereinbelow in the Target TISSUES OR Cells section or described anywhere else herein) borne by the target tissue or cell can be greater, when the therapeutic agent is in an uncleaved state, compared to a dissociation constant (Kd) of a corresponding biologically active peptide (as remaining after one or both of the first release segment (RS 1) and the second release segment (RS2) is / are cleaved and one or both of the MM1 and the MM2 is / are released). The dissociation constant (Kd) of the biologically active moiety (BM) of the therapeutic agent, when the therapeutic agent is in an uncleaved state, with the target cell marker can be at least (about) 2-fold greater, at least (about) 5-fold greater, at least (about) 10-fold greater, at least (about) 50-fold greater, at least (about) 100-fold greater, at least (about) 200-fold greater, at least (about) 300-fold greater, at least (about) 400-fold greater, at least (about) 500-fold greater, at least (about) 600-fold greater, at least (about) 700-fold greater, at least (about) 800-fold greater, at least (about) 900-fold greater, or at least (about) 1000-fold greater, than the dissociation constant (Kd) of the corresponding biologically active peptide. The dissociation constant (Kd) can be measured in an in vitro assay under equivalent molar concentrations. The in vitro assay can be selected from cell membrane integrity assay, mixed cell culture assay, cell-based competitive binding assay, FACS based propidium Iodide assay, trypan Blue influx assay, photometric enzyme release assay, radiometric 51Cr release assay, fluorometric Europium release assay, CalceinAM release assay, photometric MTT assay, XTT assay, WST-1 assay, alamar blue assay, radiometric 3H-Thd incorporation assay, clonogenic assay measuring cell division activity, fluorometric rhodaminel23 assay measuring mitochondrial transmembrane gradient, apoptosis assay monitored by FACS-based phosphatidylserine exposure, ELISA-based TUNEL test assay, sandwich ELISA, caspase activity assay, cell-based LDH release assay, reporter gene activity assay, and cell morphology assay, or any combination thereof.

[00171] In some embodiments of the therapeutic agent (or the activatable therapeutic agent, or the nonnatural, activatable therapeutic agent) that comprises a first masking moiety (MM1) and a second masking moiety (MM2), the therapeutic agent, in which the biologically active moiety (BM) is linked, directly or indirectly, to one or both of the MM1 and the MM2, can effect an enhancement in a safety profile, for example, improve a maximum tolerable exposure level (MTEL), and / or reduce a side effect (e.g., cytotoxicity), in delivery of the biologically active moiety (BM) to the target tissue or cell compared to a corresponding biologically active moiety (as remaining after one or both of the first release segment (RSI) and the second release segment (RS2) is / are cleaved and one or both of the MM1 and the MM2 is / are released). The therapeutic agent, in which the biologically active moiety (BM) is linked (directly or indirectly) to one or both of the MM1 and the MM2, can effect an enhancement in a safety profile, for example, improve a maximum tolerable exposure level (MTEL), and / or reduce a side effect (e.g., cytotoxicity) by at least (about) 2-fold, by at least (about) 5-fold, by at least (about) 10 fold, by at least (about) 50-fold, by at least (about) 100-fold, by at least (about) 200-fold, by at least (about) 300-fold, by at least (about) 400-fold, or by at least (about) 500-fold higher in delivery of the BM to the target tissue or cell, than the corresponding biologically active moiety.

[00172] In some embodiments of the therapeutic agent (or the activatable therapeutic agent, or the nonnatural, activatable therapeutic agent) that comprises a first masking moiety (MM1) and a second masking moiety (MM2), the therapeutic agent, in which the biologically active moiety (BM) is linked, directly or indirectly, to one or both of the MM1 and the MM2, can have a longer terminal half-life compared to that of a corresponding biologically active moiety (as remaining after one or both of the first release segment (RSI) and the second release segment (RS2) is / are cleaved and one or both of the MM1 and the MM2 is / are released). The therapeutic agent, in which the biologically active moiety (BM) is linked (directly or indirectly) to one or both of the MM1 and the MM2, can have a terminal half-life of at least (about) 2-fold longer, at least (about) 5-fold longer, at least (about) 10-fold longer, at least (about) 15-fold longer, at least (about) 20-fold longer, at least (about) 50-fold longer, at least (about) 100-fold longer, than the terminal half-life of the corresponding biologically active moiety.

[00173] In some embodiments of the therapeutic agent (or the activatable therapeutic agent, or the nonnatural, activatable therapeutic agent) that comprises a first masking moiety (MM1) and a second masking moiety (MM2), the therapeutic agent, in which the biologically active moiety (BM) is linked, directly or indirectly, to one or both of the MM1 and MM2, can be less immunogenic compared to a corresponding biologically active moiety (as remaining after one or both of the first release segment (RSI) and the second release segment (RS2) is / are cleaved and one or both of the MM1 and the MM2 is / are released). The therapeutic agent, in which the biologically active moiety (BM) is linked (directly or indirectly) to one or both of the MM1 and the MM2, can be at least (about) 2-fold less immunogenic, at least (about) 5-fold less immunogenic, or at least (about) 10-fold less immunogenic, than the corresponding biologically active moiety. The immunogenicity can be ascertained by measuring production of IgG antibodies that selectively bind to the biologically active moiety after administration of comparable doses to a subject.

[00174] In some embodiments of the therapeutic agent (or the activatable therapeutic agent, or the nonnatural, activatable therapeutic agent) that comprises a first masking moiety (MM1) and a second masking moiety (MM2), the therapeutic agent, in which the biologically active moiety (BM) is linked, directly or indirectly, to one or both of the MM1 and the MM2, can have a greater apparent molecular weight factor under a physiological condition compared to a corresponding biologically active moiety. The therapeutic agent, in which the biologically active moiety (BM) is linked (directly or indirectly) to one or both of the MM1 and the MM2, can have an apparent molecular weight factor of at least (about) 1.5-fold greater, at least (about) 2-fold greater, at least (about) 5-fold greater, at least (about) 8-fold greater, at least (about) 10fold greater, at least (about) 12-fold greater, at least (about) 15-fold greater, at least (about) 18-fold greater, or at least (about) 20-fold greater, under a physiological condition, than the corresponding biologically active moiety.

[00175] In some embodiments of the therapeutic agent (or the activatable therapeutic agent, or the nonnatural, activatable therapeutic agent), the masking moiety (MM) (or the first masking moiety (MM1), or the second masking moiety (MM2)) can (each independently) comprise an extended recombinant polypeptide (XTEN). The XTEN can be characterized in that: (i) it comprises at least 100 amino acids; (ii) at least 90% of the amino acid residues of it are selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P); and (iii) it comprises at least 4 different types of amino acids selected from G, A, S, T, E, and P. The XTEN can be characterized in that: (i) it comprises at least 150 amino acids; (ii) at least 90% of the amino acid residues of it are selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P); and (iii) it comprises at least 4 different types of amino acids selected from G, A, S, T, E, and P. The extended recombinant polypeptide (XTEN) can (each independently) comprise an amino acid sequence having 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 to a sequence set forth in Tables 2b-2c, or any subset thereof.

[00176] In some embodiments of the therapeutic agent (or the activatable therapeutic agent, or the nonnatural, activatable therapeutic agent) that comprises (1) a first masking moiety (MM1) comprising a first extended recombinant polypeptide (XTEN1) and (2) a second masking moiety (MM2) comprising a second extended recombinant polypeptide (XTEN2), the XTEN2 can be characterized in that: (i) it comprises at least 100 amino acids; (ii) at least 90% of the amino acid residues of it are selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P); and (iii) it comprises at least 4 different types of amino acids selected from G, A, S, T, E, and P. The XTEN2 can be characterized in that: (i) it comprises at least 150 amino acids; (ii) at least 90% of the amino acid residues of it are selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P); and (iii) it comprises at least 4 different types of amino acids selected from G, A, S, T, E, and P. The XTEN2 can comprise an amino acid sequence having 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 to a sequence selected from the group of sequences set forth in Tables 2b-2c, or any subset thereof.

[00177] In some embodiments, the XTEN (or the XTEN1, or the XTEN2) can (each independently) comprise, or can (each independently) be formed from, a plurality of non-overlapping sequence motifs. At least one of the non-overlapping sequence motifs can be recurring (or repeated at least two times in the corresponding XTEN). At least one of the non-overlapping sequence motifs can be non-recurring (or found only once within the corresponding XTEN). The plurality of non-overlapping sequence motifs can comprise (i) a set of (recurring) non-overlapping sequence motifs, where each motif of the set is repeated at least two times in the corresponding XTEN and (ii) a non-overlapping (non-recurring) sequence motif that occurs (or is found) only once within the corresponding XTEN. Each non-overlapping sequence motif can be from 9 to 14 (or 10 to 14, or 11 to 13) amino acids in length. Each non-overlapping sequence motif can be 12 amino acids in length. The plurality of non-overlapping sequence motifs can comprise a set of non-overlapping (recurring) sequence motifs, where each motif of the set can be (1) repeated at least two times in the corresponding XTEN and (2) between 9 and 14 amino acids in length. The set of (recurring) nonoverlapping sequence motifs can comprise 12-mer sequence motifs selected from the group set forth in Table 2a. The set of (recurring) non-overlapping sequence motifs can comprise 12-mer sequence motifs selected from the group set forth in Table 2a. The set of (recurring) non-overlapping sequence motifs can comprise at least two, at least three, or all four of 12-mer sequence motifs of the group set forth in Table 2a. Table 2a. Exemplary 12-mer sequence motifs for construction of the XTENs Motif Family* iiiiiii Amino Acid Sequence AD 126 GESPGGSSGSES AD 127 GSEGSSGPGESS AD 128 GSSESGSSEGGP AD 129 GSGGEPSESGSS AE, AM 130 GSPAGSPTSTEE AE, AM, AQ 131 GSEPATSGSETP AE, AM, AQ 132 GTSESATPESGP AE, AM, AQ 133 GTSTEPSEGSAP AF, AM 134 GSTSESPSGTAP AF, AM 135 GTSTPESGSASP AF, AM 136 GTSPSGESSTAP AF, AM 137 GSTSSTAESPGP AG, AM 138 GTPGSGTASSSP AG, AM 139 GSSTPSGATGSP AG, AM 140 GSSPSASTGTGP AG, AM 141 GASPGTSSTGSP AQ 142 GEPAGSPTSTSE AQ 143 GTGEPSSTPASE AQ 144 GSGPSTESAPTE AQ 145 GSETPSGPSETA AQ 146 GPSETSTSEPGA AQ 147 GSPSEPTEGTSA BC 148 GSGASEPTSTEP BC 149 GSEPATSGTEPS BC 150 GTSEPSTSEPGA BC 151 GTSTEPSEPGSA BD 152 GSTAGSETSTEA BD 153 GSETATSGSETA BD 154 GTSESATSESGA BD 155 GTSTEASEGSAS *Denotes individual motif sequences that, when used together in various permutations, results in a “family sequence” Table 2b. Exemplary XTEN polypeptides XTEN Name SEQ ID iiiiiii Amino Acid Sequence AE144 156 GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTST EPSEGSAPGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSE GSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAP AE144_ 1A 157 SPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTE PSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGS ETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPG AE144_ 2A 158 TSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSES ATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEG SAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPG XTEN Name SEQ ID lliilli Amino Acid Sequence AE144_ 2B 159 TSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSES ATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEG SAPGTSTEPSEGSAPGTS ESATPESGPGTSESATPESGPG AE144_ 3A 160 SPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTE PSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEG SAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPG AE144_ 3B 161 SPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTE PSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEG SAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPG AE144_ 4A 162 TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSES ATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTS TEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPG AE144_ 4B 163 TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSES ATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTS TEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPG AE144_ 5A 164 TSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSES ATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGS ETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEG AE144_ 6B 165 TSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPA TSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEG SAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG AE288_ 1 166 GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSE SATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSG SETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAP GTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEP ATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSG SETPGTSESATPESGPGTSTEPSEGSAP AE288_ 2 167 GSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTST EPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE GSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGP GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTST EPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPT STEEGTSESATPESGPGTSTEPSEGSAP AE576 168 GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTST EPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSG SETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAP GSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTST EPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSE GSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGP GSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTST EPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPT STEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGP GSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPA GSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSE GSAP AE624 169 MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGS PAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEP SEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSE TPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGS PAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEP SEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGS APGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGS EPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEP XTEN Name SEQ ID lliilli Amino Acid Sequence SEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTST EEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGS EPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGS PTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGS AP AE864 170 GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTST EPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSG SETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAP GSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTST EPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSE GSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGP GSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTST EPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPT STEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGP GSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPA GSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSE GSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETP GTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEP ATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSE GSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETP GSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEP ATSGSETPGTSESATPESGPGTSTEPSEGSAP AE865 171 GGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTS TEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATS GSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSA PGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTS TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPS EGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESG PGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTS TEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSP TSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESG PGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSP AGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPS EGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSET PGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSE PATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPS EGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSET PGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSE PATSGSETPGTSESATPESGPGTSTEPSEGSAP AE866 172 PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTS TEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATS GSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSA PGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTS TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPS EGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESG PGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTS TEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSP TSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESG PGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSP AGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPS EGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSET PGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSE PATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPS XTEN Name SEQ ID lliilli Amino Acid Sequence EGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSET PGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSE PATSGSETPGTSESATPESGPGTSTEPSEGSAPG AE1152 173 GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTST EPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSG SETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAP GSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTST EPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSE GSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGP GSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTST EPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPT STEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGP GSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPA GSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSE GSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETP GTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEP ATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSE GSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETP GSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEP ATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSG SETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP GSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPA GSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATP ESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEE GTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTST EPSEGSAP AE144 A 174 STEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPAT SGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGS APGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGS AE144 B 175 SEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTE PSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPE SGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPG AE180 A 176 TSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTE EGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTS ESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPS EGSAPGTSTEPSEGSAPGSEPATS AE216 A 177 PESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSET PGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTS ESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATS GSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSET PGTSESAT AE252 A 178 ESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEE GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPA GSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATP ESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAP GTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSE AE288 A 179 TPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPES GPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEG TSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAG SPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPE SGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPG TSTEPSEGSAPGSEPATSGSETPGTSESA XTEN Name SEQ ID lliilli Amino Acid Sequence AE324 A 180 PESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESG PGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSE PATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESAT PESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTE EGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSE PATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPS EGSAPGSEPATS AE360 A 181 PESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTE EGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSE PATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPS EGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESG PGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTS ESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSP TSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAT AE396 A 182 PESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESG PGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTS TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATS GSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESG PGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTS TEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATS GSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPS AE432 A 183 EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSET PGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTS ESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESAT PESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESG PGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTS ESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSP TSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESG PGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTS TEPSEGSAPGSEPATS AE468 A 184 EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSA PGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTS ESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSP TSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESG PGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS TEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESAT PESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESG PGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSP AGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAT AE504 A 185 EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA PGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTS ESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESAT PESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESG PGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSE PATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESAT PESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTE EGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSE PATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPS EGSAPGSEPATSGSETPGTSESATPESGPGTSTEPS AE540 A 186 TPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPES GPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPG TSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSES ATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPE XTEN Name SEQ ID lliilli Amino Acid Sequence SGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEG SPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPA TSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEG SAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPG SPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSES ATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTS TEEGTSTEPSEGSAPGTSTEP AE576 A 187 TPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSE TPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGT STEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEP SEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSE TPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGS PAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESA TPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPES GPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPG TSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSES ATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGS ETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPG TSESA AE612 A 188 GSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESG PGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS TEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPS EGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESG PGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS TEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSP TSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSET PGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSP AGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSP TSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESG PGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTS TEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAT AE648 A 189 PESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSA PGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTS ESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPS EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA PGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTS ESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESAT PESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESG PGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSE PATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESAT PESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTE EGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSE PATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPS EGSAPGSEPATSGSETPGTSESAT AE684 A 190 EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESG PGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTS TEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESAT PESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSA PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSP AGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESAT PESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESG PGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTS TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATS XTEN Name SEQ ID lliilli Amino Acid Sequence GSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESG PGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTS TEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATS GSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSA PGSEPATS AE720 A 191 TSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEG SAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPG TSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTE PSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTS TEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTE PSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGS ETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG TSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSES ATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPE SGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEG TSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAG SPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPE SGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTE AE756 A 192 TSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEG SAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPG TSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTE PSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTS TEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTE PSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGS ETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG TSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSES ATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPE SGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEG TSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAG SPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPE SGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPG TSTEPSEGSAPGSEPATSGSETPGTSES AE792 A 193 EGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTE EGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTS TEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESAT PESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESG PGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTS ESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPS EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSA PGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTS ESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSP TSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESG PGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS TEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESAT PESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESG PGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSP AGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAT PESGPGTSTEPS AE828 A 194 PESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSA PGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTS ESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPS XTEN Name SEQ ID lliilli Amino Acid Sequence EGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESG PGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTS ESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESAT PESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA PGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTS ESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESAT PESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTE EGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSE PATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPS EGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESG PGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTS ESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSP TSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESAT AE869 195 GSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEG TSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPA TSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEG SAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPG TSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTE PSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPE SGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPG TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAG SPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPE SGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPG SPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTE PSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGS ETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPG SEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTE PSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGS ETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPG SEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGR AE144_ RI 196 SAGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTE EGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSE PATSGSETPGSPAGSPTSTEEGTSESATPESGPGTESASR AE288_ RI 197 SAGSPTGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATP ESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETP GTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSE SATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSG SETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETP GTSESATPESGPGTSTEPSEGSAPSASR AE432_ RI 198 SAGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTE EGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSE PATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPS EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESG PGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTS TEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESAT PESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSA PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSP AGSPTSTEEGTESASR AE576_ RI 199 SAGSPTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSE GSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAP GTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEP ATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSE GSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEE XTEN Name SEQ ID lliilli Amino Acid Sequence GTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSE SATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPT STEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEE GSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSE SATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSE GSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP SASR AE864_ RI 200 SAGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTE EGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSE PATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPS EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESG PGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTS TEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESAT PESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSA PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSP AGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESAT PESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESG PGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTS TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATS GSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESG PGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTS TEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATS GSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSA PGSEPATSGSETPGTSESATPESGPGTESASR AE712 201 PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTS TEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATS GSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSA PGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTS TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPS EGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESG PGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTS TEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSP TSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESG PGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSP AGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPS EGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSET PGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSE PATSGSETPGTSESATPESGPGSPAGSPTSTEAHHH AE864_ R2 202 GSPGAGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTE EGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSE PATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPS EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESG PGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTS TEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESAT PESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSA PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSP AGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESAT PESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESG PGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTS TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATS GSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESG PGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTS TEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATS XTEN Name SEQ ID lliilli Amino Acid Sequence GSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSA PGSEPATSGSETPGTSESATPESGPGTESASR AE288_ 3 203 SPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPA TSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEG SAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPG SPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSES ATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTS TEEGTSTEPSEGSAPGTSTEPSEGSAPG AE284 204 GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSE SATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSG SETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAP GTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEP ATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSG SETPGTSESATPESGPGTSTEPSE AE292 205 SPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPA TSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEG SAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPG SPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSES ATPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTS TEEGTSTEPSEGSAPGTSTEPSEGSAPGGSAP AE293 206 PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTS TEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATS GSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSA PGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTS TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPS EGSAPGTSESATPESGPGTSESATPEGAAEPEA AE300 207 PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTS TEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATS GSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSA PGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTS TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPS EGSAPGTSESATPESGPGTSESATPESGPGSPAGAAEPEA AE864_ 2 208 AGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPS EGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSET PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSP AGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPS EGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSA PGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSE PATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPS EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTE EGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSE PATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSP TSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSA PGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTS ESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATS GSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSA PGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSE PATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPATS GSETPGTSESATPESGPGTSTEPSEGAAEPEA AE867 209 GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTST EPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSG SETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAP GSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTST XTEN Name SEQ ID lliilli Amino Acid Sequence EPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSE GSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGP GSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTST EPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPT STEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGP GSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPA GSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSE GSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETP GTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEP ATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSE GSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETP GSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEP ATSGSETPGTSESATPESGPGTSTEPSEGAAEPEA AE867_ 2 210 SPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGT STEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPAT SGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGS APGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGT STEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEP SEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPES GPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPG TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAG SPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPE SGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPG SPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTE PSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGS ETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPG SEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTE PSEGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGS ETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPG SEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG AE868 211 PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTS TEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATS GSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSA PGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTS TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPS EGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESG PGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTS TEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSP TSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESG PGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSP AGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPS EGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSET PGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSE PATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPS EGSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSET PGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSE PATSGSETPGTSESATPESGPGTSTEPSEGAAEPEA AE584 212 PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTS TEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATS GSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSA PGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTS TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPS EGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESG XTEN Name SEQ ID lliilli Amino Acid Sequence PGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTS TEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSP TSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESG PGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSP AGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPS EGSAPGAAEPEA Table 2c. Exemplary XTEN polypeptides Exemplary Use ' SEQ ID NO. Amino Acid Sequence C-terminal XTEN 213 PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEE GTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPG SEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGT STEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTS ESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTST EPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAG SPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEP SEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPS EGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATP ESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPE SGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTST EEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESG PGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGP GTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPG TSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGT SESATPESGPGTSESATPESGPGftabTSESATPESGPGSEPATSGPTES GSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTESTPSEGSAPG SEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGEPEA C-terminal XTEN 214 PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEE GTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPG SEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGT STEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTS ESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTST EPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAG SPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEP SEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPS EGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATP ESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPE SGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTST EEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESG PGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGP GTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPG TSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGT SESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGPTESGSE PATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEP ATSGSETPGTSESATPESGPGTSTEPSEGSAPGEPEA C-terminal XTEN 215 PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEE GTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPG SEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGT STEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTS ESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTST EPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAG SPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEP Exemplary Use ’ SEQID NO. Amino Acid Sequence SEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPS EGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATP ESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPE SGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTST EEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESG PGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGP GTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPG TSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGT SESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSE PATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTESTPSEGSAPGSEP ATSGSETPGTSESATPESGPGTSTEPSEGSAPGEPEA N-terminal XTEN 216 ASSPAGSPTSTESGTSESATPESGPGTETEPSEGSAPGTSESATPESGP GSEPATSGSETPGTSESATPESGPGSTPAESGSETPGTSESATPESGPG TSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGESPATSGSTPEGT SESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTS ESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEP ATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSA P N-terminal XTEN 217 ASSPAGSPTSTESGTSESATPESGPGTSTEPSEGSAPGTSESATPESGP GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPG TSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGESPATSGSTPEGT SESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTS ESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEP ATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSA P N-terminal XTEN 218 ASSPAGSPTSTESGTSESATPESGPGTSTEPSEGSAPGTSESATPESGP GSEPATSGSETPGTSESATPESGPGSTPAESGSETPGTSESATPESGPG TSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGEEPATSGSTPEGT SESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTS ESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEP ATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSA P N-terminal XTEN 219 ASSPAGSPTSTESGTSESATPESGPGTSTEPSEGSAPGTSESATPESGP GSEPATSGSETPGTSESATPESGPGSTPAESGSETPGTSESATPESGPG TSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGT SESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTS ESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEP ATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSA P C-terminal XTEN 220 PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEE GTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPG SEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGT STEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTS ESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTST EPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAG SPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEP SEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPS EGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATP ESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPE SGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTST EEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESG PGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGP GTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPG Exemplary Use ’ SEQID NO. Amino Acid Sequence TSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGT SESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSE PATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTESTPSEGSAPGSEP ATSGSETPGTSESATPESGPGTSTEPSEGSAPG C-terminal XTEN 221 PGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGP GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPG TSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGT SESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTS ESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPA GSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSES ATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESA TPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATS GSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSE GSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGS ETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTESTPSEGS APGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG N-terminal XTEN 222 SAGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPT STEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSTPAESGS ETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGS APGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSA PGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSTET PGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGP GSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPG TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGT STEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTS ESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSE SATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAG SPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESA TPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESAT PESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSG SETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEG SAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSE TPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSA PGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEE GTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPG TSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGS PAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSP AGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTST EPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTSTE PSEGSAPGTSESATPESGPGTESAS C-terminal XTEN 223 SAGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPT STEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGS ETPGSEPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGS APGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSA PGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETP GTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPG SPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGT STEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTS TEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSE SATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSES ATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGS PTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESAT PESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATP Exemplary Use ’ SEQID NO. Amino Acid Sequence ESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGS ETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGS APGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSET PGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAP GSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEG TSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGT STEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSTETPGS PAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSP AGSPTSTEEGTSTEPSEGSAPGTATESPEGSAPGTSESATPESGPGTS TEPSEGSAPGTSAESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTS TEPSEGSAPGTSESATPESGPGTESAS N-terminal XTEN 224 GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEG TSTEPSEGSAPGTSTEPSEGSAPATSESATPESGPGSEPATSGSETPGS EPATSGSETPGSPAGSPTSTEEGTSESASPESGPGTSTEPSEGSAPGTS TEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSE SATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTE PSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGS PTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPS EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE GSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPE SGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPES GPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTE EGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGP GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPG TSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGT SESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTS ESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEP ATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPA TSGSETPGTSESATPESGPGTSTEPSEGSAP N-terminal XTEN 225 GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEG TSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSESATSGSETPGS EPATSGSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTS TEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSE SATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTE PSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGS PTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPS EGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE GSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPE SGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPES GPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTE EGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGP GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPG TSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGT SESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTS ESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEP ATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGSEPA TSGSETPGTSESATPESGPGTSTEPSEGSAP N-terminal XTEN (with His-tag) 226 SPAGSPTSTESGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGS EPATSGSETPGTSESATPESGPGSTPAESGSETPGTSESATPESGPGTS TEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSE SATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSES ATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEPAT SGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSAP Exemplary Use ’ SEQID NO. Amino Acid Sequence C-terminal XTEN 227 PGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGP GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPG TSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGT SESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTS ESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPA GSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSES ATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESA TPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATS GSETPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSE GSAPGTSESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGS ETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTESTPSEGS APGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGEPEA C-terminal XTEN 228 TPESGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATS GSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSE GSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTS TEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPES GPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESG PGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGP GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPG SEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGT SESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSP AGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSE SATPESGPGSEPATSGSETPGSESATSGSETPGSPAGSPTSTEEGTSTE PSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESA C-terminal XTEN 229 GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPG SPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGS PAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTS TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGSAPGTST EPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEEGTSES ATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEP SEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPS EGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATS GSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSE GSAPGTSESASPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPAGSPTS TEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSE TPGTSESATPESGPGSEPATSGSETPGTSESATPESGP C-terminal XTEN 230 GSETPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSE GSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPE SGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSTEPSEGS APGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTE EGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP GTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPG TSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGS EPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS TEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGSPA GSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPA TSGSTETGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEP SEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATS C-terminal XTEN 231 EGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATP ESGPGSEPATSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPE SGPGTSESATPESGPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSE TPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA PGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEE Exemplary Use ’ SEQID NO. Amino Acid Sequence GTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPG SEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGS PAGSPTSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTS TEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEP ATSGSETPGTSESASPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSES ATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSPAGS PTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESAT N-terminal 232 ASSPAGSPTSTESGTSESATPESGPGTSTEPSEGSAPGTSESATPESGP GSEPATSGSETPGTSESATPESGPGSTPAESGSETPGTSESATPESGPG TSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGT SESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTS ESATPESGPGTSESATPESGPGTSESATPESGPGSEPATSGSETPGSEP ATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGGSA P

[00178] Additional examples of XTEN sequences that can be used according to the present disclosure are disclosed in U.S. Patent Publication Nos. 2010 / 0239554 Al, 2010 / 0323956 Al, 2011 / 0046060 Al, 2011 / 0046061 Al, 2011 / 0077199 Al, 2011 / 0172146 Al, 2018 / 0244736 Al, 2018 / 0346952 Al, and 2019 / 0153115 Al; U.S. Patent Nos. 8,673,860, 9,371,369, 9,926,351, 9,249,211, and 9,976,166; and International Patent Publication Nos. WO 2010 / 091122 Al, WO 2010 / 144502 A2, WO 2010 / 144508 Al, WO 2011 / 028228 Al, WO 2011 / 028229 Al, WO 2011 / 028344 A2, WO 2014 / 011819 A2, WO 2015 / 023891, WO 2016 / 077505 A2, WO 2017 / 040344 A2, and WO 2019 / 126576 Al.

[00179] In general, XTEN are polypeptides with non-naturally occurring, substantially non-repetitive sequences having a low degree or no secondary or tertiary structure under physiologic conditions, as well as additional properties described in the paragraphs that follow. XTEN can 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 foregoing. As used herein, XTEN specifically excludes whole antibodies or antibody fragments (e.g. single-chain antibodies and Fc fragments). XTEN polypeptides have utility as fusion partners in that they serve in various roles, conferring certain desirable properties when linked to a composition comprising, for example, one or more biologically active moieties (such as one described herein). The resulting compositions have enhanced properties, such as enhanced pharmacokinetic, physicochemical, pharmacologic, and improved toxicological and pharmaceutical properties compared to the corresponding one or more biologically active moieties not linked to XTEN, making them useful in the treatment of certain conditions for which the one or more biologically active moieties are known in the art to be used.

[00180] The unstructured characteristic and physicochemical properties of the XTEN result, in part, from the overall amino acid composition that is disproportionately limited to 4-6 types of hydrophilic amino acids, the sequence of the amino acids in a quantifiable, substantially non-repetitive design, and from the resulting length of the XTEN polypeptide. In an advantageous feature common to XTEN but uncommon to native polypeptides, the properties of XTEN disclosed herein may not be tied to an absolute primary amino acid sequence, as evidenced by the diversity of the exemplary sequences of Tables 2b-2c that, within varying ranges of length, possess similar properties and confer enhanced properties on the compositions to which they are linked, many of which are documented in the Examples. Indeed, it is specifically contemplated that the compositions of the disclosure not be limited to those XTEN specifically enumerated in Tables 8 or 10, but, rather, the embodiments at least include sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, when optimally aligned, to the sequences of Tables 2b-2c as they exhibit the properties of XTEN described herein. It has been established that such XTEN have properties more like non-proteinaceous, hydrophilic polymers (such as polyethylene glycol, or “PEG”) than they do proteins. The XTEN of the present disclosure exhibit one or more of the following advantageous properties: defined and uniform length (for a given sequence), conformational flexibility, reduced or lack of secondary structure, high degree of random coil formation, high degree of aqueous solubility, high degree of protease resistance, low immunogenicity, low binding to mammalian receptors, a defined degree of charge, and increased hydrodynamic (or Stokes) radii; properties that are similar to certain hydrophilic polymers (e.g., polyethylene glycol) that make them particularly useful as fusion partners.

[00181] XTEN, as described herein, are designed to behave like denatured peptide sequences under physiological conditions, despite the extended length of the polymer. “Denatured” describes the state of a peptide in solution that is characterized by a large conformational freedom of the peptide backbone. Most peptides and proteins adopt a denatured conformation in the presence of high concentrations of denaturants or at elevated temperature. Peptides in denatured conformation have, for example, characteristic circular dichroism (CD) spectra and are characterized by a lack of long-range interactions as determined by NMR. “Denatured conformation” and “unstructured conformation” are used synonymously herein. In some embodiments, the disclosure provides compositions that comprise XTEN sequences that, under physiologic conditions, resemble denatured sequences that are substantially devoid of secondary structure under physiologic conditions. “Substantially devoid,” as used in this context, means that 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 secondary structure, as measured or determined by the methods described herein, including algorithms or spectrophotometric assays.

[00182] A variety of well-established methods and assays are known in the art for determining and confirming the physicochemical properties of the subject XTEN and the subject polypeptide compositions into 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 uniformity, melting properties, contamination and water content. The methods to measure 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, refractometry, and UV / Visible spectroscopy. In particular, secondary structure can be measured spectrophotometrically, e.g., by circular dichroism spectroscopy in the “far-UV” spectral region (190-250 nm). Secondary structure elements, such as alpha-helix and beta-sheet, each give rise to a characteristic shape and magnitude of CD spectra, as does the lack of these structure elements. Secondary structure can also be predicted for a polypeptide sequence via certain computer programs or algorithms, such as the well-known Chou-Fasman algorithm (Chou, P. Y., et al. (1974) Biochemistry, 13: 222-45) and the Gamier-Osguthorpe-Robson algorithm (“GOR IV algorithm”) (Gamier J, Gibrat JF, Robson B. (1996), GOR method for predicting protein secondary structure from amino acid sequence. Methods Enzymol 266:540553), as described in US Patent Application Publication No. 20030228309A1. For a given sequence, the algorithms can predict whether there exists some or no secondary structure at all, expressed as the total and / or percentage of residues of the sequence that form, for example, alpha-helices or beta-sheets or the percentage of residues of the sequence predicted to result in random coil formation (which lacks secondary structure). Polypeptide sequences can be analyzed using the Chou-Fasman algorithm using sites on the world wide web at, for example, fasta.bioch.virginia.edu / fasta_www2 / fasta_www.cgi?rm=miscl 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). Random coil can be determined by a variety of methods, including by using intrinsic viscosity measurements, which scale with chain length in a conformation-dependent way (Tanford, C., Kawahara, K. & Lapanje, S. (1966) J. Biol. Chem. 241 , 1921-1923), as well as by size-exclusion chromatography (Squire, P. G., 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). Additional methods are disclosed in Arnau, et al., Prot Expr and Purif (2006) 48, 1-13.

[00183] In some embodiments of the present disclosure, the activatable therapeutic agent is an activatable antibody (AA) composition, where the masking moiety (MM) refers to an amino acid sequence coupled to an antibody or antibody fragment (AB) and positioned such that it reduces the ability of the AB to bind its designated binding target by specifically binding to the antigen-binding domain of the AB (such as the complementarity-determining region(s) (CDR(s)). Such binding can be non-covalent. In some embodiments, the activatable antibody composition can be prevented from binding to the designated binding target by binding the MM to an N- or C-terminus of the activatable antibody composition.

[00184] Alternatively, the MM may not specifically bind the AB, but rather interfere with AB-target binding through non-specific interactions such as steric hindrance. For example, the MM may be positioned in the uncleaved activatable antibody composition such that the tertiary or quaternary structure of the activatable antibody allows the MM to mask the AB through charge-based interaction, thereby holding the MM in place to interfere with target access to the AB. The masking moiety (MM) can interfere or / and inhibit binding of the antibody or antibody fragment (AB) to the target allosterically or sterically.

[00185] When the antibody or antibody fragment (AB) is modified with a MM and is in the presence of the target, specific binding of the AB to its target can be reduced or inhibited, as compared to the specific binding of the AB, not modified with an MM, to the target. A dissociation constant (Ka) of the AB modified with a MM towards the AB’s target can be generally greater than a corresponding Ka of the AB, not modified with a MM, towards the target. Conversely, a binding affinity of the AB modified with a MM towards the target can be generally lower than a binding affinity of the AB, not modified with a MM, towards the target. In some embodiments, the masking moiety (MM) of the activatable antibody composition can have an equilibrium dissociation constant (Ka) for binding to the antibody or a fragment thereof which is greater than the equilibrium dissociation of the antibody or the fragment thereof for binding to its designated binding target (near or at a diseased site in a subject).

[00186] When the antibody or antibody fragment (AB) is modified with a release segment (RS) and a masking moiety (MM) and is in the presence of the target but not sufficient protease or protease activity to cleave the RS, specific binding of the modified AB to the target can be generally reduced or inhibited, as compared to the specific binding of the AB modified with a RS and a MM in the presence of the target and sufficient protease or protease activity to cleave the RS. For example, when the modified antibody is an activatable antibody composition and comprises a release segment (RS), the AB can be unmasked upon cleavage of the RS, in the presence of protease, preferably a disease-specific protease. Thus, the MM is one that when the activatable antibody composition is uncleaved provides for masking of the AB from target binding, but does not substantially or significantly interfere or compete for binding of the target to the AB when the activatable antibody composition is in the cleaved conformation. A schematic of an exemplary activatable antibody (AA) composition is provided in FIG. 3. As illustrated, the release segment (RS) is positioned such that in a cleaved (or relatively active state) and in the presence of a target, the antibody or antibody fragment (AB) binds a target, while in an uncleaved (or relatively inactive state) in the presence of the target, specific binding of the AB to its target is reduced or inhibited. The specific binding of the antibody or antibody fragment (AB) to its target can be reduced due to the due to the inhibition or masking of the AB’s ability to specifically bind its target by the masking moiety (MM).

[00187] In some embodiments of the activatable antibody compositions, where an antibody or antibody fragment (AB) is capable of specifically binding its designated binding target, a coupling of the masking moiety (MM) to the antibody or antibody fragment (AB) can reduce the ability of the AB to bind its designated binding target as compared to the ability of the AB not coupled to the MM to bind the designated binding target (for example, when assayed in vitro using a target displacement assay). Such coupling of the MM to the AB can reduce the ability of the AB to bind its designated binding target for a duration.

[00188] The masking moiety (MM) can be provided in a variety of different forms. In certain embodiments, the MM can be selected to be a known binding partner of the antibody or antibody fragment (AB), provided that the MM binds the AB with less affinity and / or avidity than the target protein to which the AB is designed to bind following cleavage of the release segment (RS) so as to reduce interference of MM in target-AB binding. Stated differently, as discussed above, the MM is one that masks the AB from target binding when the activatable antibody composition is uncleaved, but does not substantially or significantly interfere or compete for binding for target when the activatable antibody composition is in the cleaved conformation. In a specific embodiment, the AB and MM do not contain the amino acid sequences of a naturally-occurring binding partner pair, such that at least one of the AB and MM does not have the amino acid sequence of a member of a naturally occurring binding partner. The masking moiety (MM) may not comprise more than 50% amino acid sequence identity to a natural binding partner of the antibody or antibody fragment (AB). The masking moiety (MM) can comprise a consensus sequence specific for binding to a class of antibodies against a designated binding target (e.g., diseased target). The MM can be a polypeptide of no more than 40 (e.g., from 2 to 40) amino acids in length. The MM can be coupled to the activatable antibody composition by covalent binding.

[00189] In some embodiments, the present disclosure provides for an activatable antibody complex (AAC) composition (as illustrated in FIG. 4) comprising: (1) two antibodies or antibody fragments (ABI and AB2), each capable of specifically binding its designated binding target, (2) at least one masking moiety (MM) coupled to either ABI or AB2, capable of inhibiting the specific binding of ABI and AB2 to their designated binding target(s), and (3) at least one release segment (RS) coupled to either ABI or AB2, capable of being specifically cleaved by a protease whereby activating the AAC composition. In some embodiments, when the AAC is in an uncleaved state, the MM can inhibit the specific binding of ABI and AB2 to their designated binding target(s) and when the AAC is in a cleaved state, the MM does not inhibit the specific binding of ABI and AB2 to their designated binding targets. The two ABs can bind different targets, or different epitopes on the same target.

[00190] In some embodiments, the MM does not inhibit cellular entry of the activatable antibody composition.

[00191] In some embodiments, the masking moiety (MM) can comprise an anti-albumin domain, such as a single domain antibody (sdAb) anti-albumin domain. In some embodiments, the anti-albumin domain can comprise non-CDR loops, CDR loops, or any combination thereof. In some embodiments, the anti-albumin domain can comprise both non-CDR loops and CDR loops. The non-CDR loops can be capable of binding to one or more antibody or antibody fragment (AB) (for example, and not limited to, the CDRs of the AB) of an activatable antibody (AA) composition, thereby masking the AB (at least in some cases) by inhibiting or reducing the ability of the AB to bind to its designated target(s). The CDR loops can be capable of binding albumin (e.g., human serum albumin), thereby (at least in some cases) masking the AB in the activatable antibody (AA) composition from binding to its designated target(s) via steric or allosteric hindrance and / or conferring half-life extension for the AA composition. In some embodiments, the non-CDR loops can be engineered into different position of the anti-albumin sdAb domain. In some embodiments, the MM can (1) inhibit or reduce the ability of the AB to bind to its designated target(s) via (la) specific binding to the target recognition region of the AB and / or (lb) steric masking of target recognition region of the AB, and / or the MM can (2) confer half-life extension for the AA containing the AB via binding to albumin. The MM can be coupled (directly or indirectly) to the activatable antibody composition by covalent binding.

[00192] As illustrated in the schematic shown in FIG. 5, an exemplary activatable antibody complex (AAC) composition can comprise: (1) at least two antibodies or antibody fragments (ABI and AB2), each capable of specifically binding their designated binding target(s), (2) at least one masking moiety (MM) coupled to ABI or AB2, capable of inhibiting the specific binding of ABI or AB2 to their designated binding target(s), and (3) at least one release segment (RS) coupled to ABI or AB2, capable of being specifically cleaved by a protease whereby activating the activatable antibody complex (AAC) composition. In some embodiments, when the AA is in an uncleaved state, the MM can inhibit the specific binding of ABI or AB2 to their designated binding target(s), and when the activatable antibody complex (AAC) composition is in a cleaved state, the MM does not inhibit the specific binding of ABI or AB2 to their designated binding target(s). In some embodiments, the masking moiety (MM) can be coupled to both ABI and AB2 via two separate release segments (RS). In other words, the MM can be placed between ABI and AB2, coupled either to the C end of ABI and the N end of AB2, or coupled to the N end of ABI and the C end of AB2.

[00193] In some embodiments of the present disclosure, the activatable therapeutic agent is an activatable antibody (AA) composition, where the masking moiety (MM) refers to an amino acid sequence coupled to an antibody or antibody fragment (AB) (for example, but not limited to, an scFv, an sdAb, or a fragment thereol) and positioned such that it reduces the ability of the AB to dimerize with another antibody or antibody fragment, preventing the formation of an antibody or an antibody fragment capable of binding to target. Such binding can be non-covalent. In some embodiments, the activatable antibody composition can be prevented from binding to the designated binding target by binding the MM to an N- or C-terminus of the activatable antibody composition.

[00194] When the antibody or antibody fragment (AB) is modified with a MM and is in the presence of the target, specific binding of the AB to its dimerization partner can be reduced or inhibited, as compared to the specific binding of the AB, not modified with an MM, to its dimerization partner. A dissociation constant (Kd)of the AB modified with a MM towards its dimerization partner can be generally greater than a corresponding Ka of the AB, not modified with a MM, towards its dimerization partner. Conversely, a binding affinity of the AB modified with a MM towards its dimerization partner can be generally lower than a binding affinity of the AB, not modified with a MM, towards its dimerization partner. In some embodiments, the masking moiety (MM) of the activatable antibody composition can have an equilibrium dissociation constant (Ka) for binding to the antibody or a fragment thereof which is greater than the equilibrium dissociation of the antibody or the fragment thereof for binding to its designated dimerization partner.

[00195] When the antibody or antibody fragment (AB) is modified with a release segment (RS) and a masking moiety (MM) and is in the presence of the target but not sufficient protease or protease activity to cleave the RS, 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(s) can be generally reduced or inhibited, as compared to the specific dimerization ability of the AB modified with a RS and a MM and the subsequent ability of the dimer to bind to its designated binding target(s) in the presence of the target and sufficient protease or protease activity to cleave the RS. For example, when the modified antibody is an activatable antibody composition and comprises a release segment (RS), the AB can be unmasked upon cleavage of the RS, in the presence of protease, preferably a disease-specific protease. Thus, the MM is one that when the activatable antibody composition is uncleaved provides for masking of the AB from dimerization with another AB and for reduction or inhibition of binding of the resulting dimer to its designated binding target(s), but does not substantially or significantly interfere or compete for dimerization to another AB and for reduction or inhibition of binding of the resulting dimer to its designated binding target(s) when the activatable antibody composition is in the cleaved conformation.

[00196] The masking moiety can be provided in different forms. In some embodiments, the masking domain can be an inhibitory antibody or antibody fragment (IAB; for example, but not limited to, a VL or VH domain), provided that the MM binds the AB with less affinity and / or avidity than the dimerization partner with which AB is designed to dimerize following cleavage of the release segment (RS) so as to reduce interference of MM in AB - AB dimerization. Stated differently, as discussed above, the MM is one that masks the AB from dimerization to another AB when the activatable antibody composition is uncleaved, but does not substantially or significantly interfere or compete for dimerization with another AB when the activatable antibody composition is in the cleaved conformation. The MM can be coupled to the activatable antibody composition by covalent binding.

[00197] In some embodiments, the present disclosure provides for an activatable antibody complex (AAC) composition (as illustrated in FIG. 6) comprising: (1) two antibodies or antibody fragments (ABI and AB2) (2) two masking moieties (MM) coupled to one each to ABI and AB2, capable of reducing or inhibiting the specific dimerization of ABI and AB2 and subsequent binding of AB1-AB2 complex to their designated binding target(s), (3) at least three release segments (RS) coupled to ABI, AB2 and MMs capable of being specifically cleaved by a protease whereby activating the AAC composition, (4) at least one additional antibody or antibody fragment (AB3 and / or AB4; for example, but not limited to, an scFv or an sdAb), coupled to ABI and / or AB2. In some embodiments, when the AAC is in an uncleaved state, the MM can inhibit or reduce the specific dimerization of ABI and AB2 and subsequently inhibit or reduce the binding of the resulting AB1-AB2 dimer to its designated binding target(s) and when the AAC is in a cleaved state, the MM does not reduce or inhibit the specific dimerization of ABI and AB2 and does not reduce or inhibit the subsequent binding of the AB1-AB2 dimer to its designated binding target(s). When more than one additional AB is coupled to ABI and / or AB2, the additional ABs can bind the same target or different targets.

[00198] In some embodiments, the MM can comprise a coiled-coil domain, for example, but not limited to to, (1) high affinity parallel heterodimeric leucine zipper coiled-coil domain, containing or devoid of cysteines, (2) low affinity parallel heterodimeric coiled-coil leucine zipper domain, containing or devoid of cysteines, (3) disulfide-linked covalent coiled-coil domain, (4) antiparallel heterodimeric leucine zipper coiled-coil domain, (5) helix-turn-helix homodimeric leucine zipper coiled coil domain. The MM can be coupled (directly or indirectly) to the activatable antibody composition by covalent binding. In some embodiments, the MM can reduce or inhibit the binding of AB to its intended target(s) via steric or allosteric hindrance.

[00199] In some embodiments, the present disclosure provides for an activatable antibody complex (AAC) composition (as illustrated in FIG. 7) comprising: (1) at least one antibody or antibody fragment (AB), (2) at least one masking moiety (MM) coupled to AB, capable of inhibiting the specific binding of AB to its designated binding target, and (3) at least one release segment (RS) coupled to AB, capable of being specifically cleaved by a protease whereby 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(s) and when the AAC is in a cleaved state, the MM does not reduce or inhibit the specific binding of AB to its designated binding target(s).

[00200] In some embodiments, the activatable therapeutic agent may incorporate a cleavage sequence as described herein, and / or be administered to a patient who is identified as being a likely responder to the therapeutic agent based on the identification of a peptide biomarker in a biological sample from the subject (as described further herein). Biologically Active Moieties (BM)

[00201] In some embodiments of the therapeutic agent (or the activatable therapeutic agent, or the nonnatural, activatable therapeutic agent), the biologically active moiety (BM) can comprise a biologically active peptide (BP). The biologically active peptide (BP) can comprise an antibody, a cytokine, a cell receptor, or a fragment thereof. The biologically active polypeptide (BP) can comprise a binding moiety having a binding affinity for a target cell marker on a target tissue or cell. The target cell marker can be an effector cell antigen expressed on a surface of an effector cell. The binding moiety can be an antibody. The antibody can be selected from the group consisting of Fv, Fab, Fab’, Fab’-SH, nanobody (also known as single domain antibody or Vhh), linear antibody, and single-chain variable fragment (scFv).

[00202] In some embodiments of the therapeutic agent (or the activatable therapeutic agent, or the nonnatural, activatable therapeutic agent), where the binding moiety can be a first binding moiety, and wherein the target cell marker can be a first target cell marker, the biologically active polypeptide (BP) can further comprise a second binding moiety linked, directly or indirectly to the first binding moiety. The second binding moiety can have a binding affinity for a second target cell marker on the target tissue or cell. The second target cell marker can be a marker on a tumor cell or a cancer cell. The second binding moiety can be an antibody. The second binding moiety can be an antibody selected from the group consisting of Fv, Fab, Fab’, Fab’-SH, nanobody (also known as single domain antibody or Vhh), linear antibody, and singlechain variable fragment (scFv).

[00203] In some embodiments as disclosed herein, a biologically active moiety (BM) or a biologically active peptide (BP) can exhibit a binding specificity to a given target (or a given number of targets) or / and another desired biological characteristic, when used in vivo or when utilized in an in vitro assay. For example, the BM or BP can be an agonist, a receptor, a ligand, an antagonist, an enzyme, an antibody (e.g., mono- or bispecific), or a hormone. Of particular interest are BM or BP used, or known to be useful, for a disease or disorder where the native BM or BP have a relatively short terminal half-life and for which an enhancement of a pharmacokinetic parameter (which optionally could be released from a conjugate or a fusion polypeptide by cleavage of a spacer sequence) would permit less frequent dosing or an enhanced pharmacologic effect. Also of interest are BM or BP that have a relatively narrow therapeutic window between the minimum effective dose or blood concentration (Cmm) and the maximum tolerated dose or blood concentration (Cmax). In such cases, the linking of the BM or BP within a conjugate or a fusion polypeptide comprising a select masking moiety, such as XTEN, can result in an improvement in these properties, making them more useful as therapeutic or preventive agents compared to the BM or BP not linked to a masking moiety, such as XTEN. The BM or BP encompassed by the inventive compositions described herein can have utility in the treatment in various therapeutic or disease categories, including but not limited to glucose and insulin disorders, metabolic disorders, cardiovascular diseases, coagulation and bleeding disorders, growth disorders or conditions, endocrine disorders, eye diseases, kidney diseases, liver diseases, tumorigenic conditions, inflammatory conditions, autoimmune conditions, etc.

[00204] In some embodiments of the compositions disclosed herein, where the biologically active moiety is a biologically active peptide (BP), the BP can comprise a peptide sequence that exhibits at least (about) 80% sequence identity (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 to an amino acid sequence of a glucose regulating peptide or a glucagon- like peptide (native or synthetic analog) set forth in Tables 3a-3c (such as one described more fully hereinbelow in the GLUCOSE REGULATING PEPTIDES section), or to an amino acid sequence of a protein relating to metabolic disorders and cardiology set forth in Table 3d (such as one described more fully hereinbelow in the METABOLIC DISEASE AND CARDIOVASCULAR PROTEINS section), or to an amino acid sequence of a growth hormone set forth in Table 3f (such as one described more fully hereinbelow in the GROWTH Hormone Proteins section), or to an amino acid sequence of a cytokine set forth in Table 3g (such as one described more fully hereinbelow in the CYTOKINES section), or to an amino acid sequence of a transduction domain in Table 3h (such as one described more fully hereinbelow). In some embodiments of the compositions of this disclosure, the sequence of the BP can comprise one or more substitutions shown in Table 4 (such as one described more fully hereinbelow).

[00205] In some embodiments of the compositions disclosed herein, where the biologically active moiety is a biologically active peptide (BP), the BP can comprise an antibody (e.g., a monospecific, bispecific, trispecific, or multispecific antibody) (as defined hereinabove, the term “antibody” includes, among other things, an antibody fragment) (such as one described more fully hereinbelow in the ANTIBODIES section). The antibody can comprise a binding domain (or binding moiety) having binding affinity for an effector cell antigen. The effector cell antigen can be expressed on the surface of an effector cell selected from a plasma cell, a T cell, a B cell, a cytokine induced killer cell (CIK cell), a mast cell, a dendritic cell, a regulatory T cell (RegT cell), a helper T cell, a myeloid cell, and a NK cell. The effector cell antigen can be expressed on or within an effector cell. The effector cell antigen can be expressed on a T cell, such as a CD4+, CD8+, or natural killer (NK) cell. The effector cell antigen can be expressed on the surface of a T cell. The effector cell antigen can be expressed on a B cell, master cell, dendritic cell, or myeloid cell. The binding domain (or binding moiety) can comprise VH and VL regions derived from a monoclonal antibody capable of binding human CD3. In some embodiments, where the binding domain (or binding moiety) having binding affinity for CD3, the binding domain (or binding moiety) can have binding affinity for a member of the CD3 complex, which includes in individual form or independently combined form all known CD3 subunits of the CD3 complex; for example, CD3 epsilon, CD3 delta, CD3 gamma, CD3 zeta, CD3 alpha and CD3 beta. The binding domain (or binding moiety) having binding affinity for CD3 can have binding affinity for 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) binding human CD3 can be derived from an anti-CD3 antibody selected from the group of antibodies set forth in Tables 5a-5e. The binding domain (or binding moiety) binding human CD3 can comprise VH a...

Claims

1. A method for assessing a likelihood of a subject being responsive to a therapeutic agent that is activatable by a mammalian protease expressed in said subject having a disease or disorder, the method comprising:a. determining, in a biological sample from said subject affected by the disease or disorder, a presence or an amount of a proteolytic peptide product produced by action of said mammalian protease, wherein said peptidei. comprises at least five or six consecutive amino acid residues shown in a sequence set forth in Column V of Table A; orii. comprises at least five or six consecutive amino acids shown in a sequence set forth in Column IV of Table A; oriii. comprises at least five or six consecutive amino acids shown in a sequence set forth in Column VI of Table A; andb. designating said subject as being likely to respond to said therapeutic agent when said peptide of (i), (ii) or (iii) is present and / or if its amount exceeds a threshold value.

2. The method of claim 1, wherein said therapeutic agent comprises a peptide substrate having an amino acid sequence that is susceptible to cleavage by said mammalian protease at a scissile bond.

3. The method of claim 2, wherein said 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 said sequence of the peptide substrate that is either N-terminal or C-terminal side of said scissile bond.

4. The method of claim 1 or 2, wherein said sequence of the peptide substrate is susceptible to cleavage by said mammalian protease at a scissile bond, and wherein said polypeptide of (i), (ii), or (iii) is a cleavage product of a reporter polypeptide comprising a substrate sequence that is susceptible to cleavage by the same mammalian protease at a scissile bond and where said reporter polypeptide comprises a sequence set forth in Column II or III of Table A.

5. The method of claim 1 or 2, wherein said sequence of the peptide substrate is susceptible to cleavage by said mammalian protease at a scissile bond, and wherein said polypeptide of (i), (ii), or (iii) is a cleavage product of a human protein that comprises a portion containing at least five or six consecutive amino acid residues of said peptide substrate sequence that includes the scissile bond.

6. The method of any one of claims 1-5, wherein said polypeptide of (i) comprises at least seven, at least eight, at least nine, or at least ten consecutive amino acid residues shown in a sequence set forth in Column V of Table A.

7. The method of any one of claims 1-6, wherein said polypeptide of (ii) comprises at least seven, at least eight, at least nine, or at least ten consecutive amino acids shown in a sequence set forth in Column IV of Table A.

8. The method of any one of claims 1-7, wherein said polypeptide of (iii) comprises at least seven, at least eight, at least nine, or at least ten consecutive amino acids shown in a sequence set forth in Column VI of Table A.

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

10. The method of any one of claims 1-9, wherein said threshold is zero or nominal.

11. The method of any one of claims 1-10, wherein said biological sample comprises a serum or plasmasample.

12. The method of any one of claims 1-11, wherein said mammalian protease is a serine protease, a cysteine protease, an aspartate protease, a threonine protease, or a metalloproteinase.

13. The method of claim 12, wherein said mammalian protease is selected from the group consisting of disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), disintegrin and metalloproteinase domain-containing protein 12 (ADAM12), disintegrin and metalloproteinase domaincontaining protein 15 (ADAM 15), disintegrin and metalloproteinase domain-containing protein 17 (ADAM17), disintegrin and metalloproteinase domain-containing protein 9 (ADAM9), disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), Cathepsin B, Cathepsin D, Cathepsin E, Cathepsin K, cathepsin L, cathepsin S, Fibroblast activation protein alpha, Hepsin, kallikrein-2, kallikrein-4, kallikrein-3, Prostate-specific antigen (PSA), kallikrein-13, Legumain, 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 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 type serine protease 1 (MT-SP1), matriptase, and u-plasminogen.

14. The method of claim 12, wherein said 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), legumain, and matriptase.

15. The method of any one of claims 1-14, wherein said mammalian protease is preferentially expressedor activated in a target tissue or cell.

16. The method of claim 15, wherein said target tissue or cell is a tumor.

17. The method of claim 15 or 16, wherein said target tissue or cell produces or is co-localized withsaid mammalian protease.

18. The method of any one of claims 15-17, wherein said target tissue or cell contains therein or thereon, or is associated with in proximity thereto, a reporter polypeptide.

19. The method of claim 4 or 18, wherein said reporter polypeptide is a polypeptide selected from the group consisting of 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, epididymis secretory sperm binding protein, secretogranin-2, angiotensinogen, transgelin-2, pancreatic prohormone, neurosecretory protein VGF, ceruloplasmin, PDZ and LIM domain protein 1, multimerin-1, inter-alpha-trypsin inhibitor heavy chain H2, N-acetylmuramoyl-L-alanine amidase, histone Hl.4, adhesion G-protein coupled receptor G6, mannan-binding lectin serine protease 2, prothrombin, deleted in malignant brain tumors 1 protein, desmoglein-3, calsyntenin-1, alpha-2-macroglobulin, myosin-9, sodium / potassium-transporting ATPase subunit gamma, oncoprotein-induced transcript 3 protein, serglycin, histidine-rich glycoprotein, inter-alpha-trypsin inhibitor heavy chain H5, integrin alpha-IIb, membrane-associated progesterone receptor component 1, histone Hl.2, rho GDP-dissociation inhibitor 2, zinc-alpha-2-glycoprotein, talin-1, secretogranin-1, neutrophil defensin 3, cytochrome P450 2E1, gastric inhibitory polypeptide, transcription initiation factor TFIID subunit 1, integral membrane protein 2B, pigment epithelium-derived factor, voltage-dependent N-type calcium channel subunit alpha-IB, ras GTPase-activating protein nGAP, type I cytoskeletal 17, sulfhydryl oxidase 1, homeobox protein Hox-B2, transcription factor SOX-10, E3 ubiquitin-protein ligase SIAH2, decorin, secreted protein acidic and rich in cysteine (SPARC), laminin gamma 1 chain, vimentin, and nidogen-1 (NIDI).

20. The method of claim 4 or 18, wherein said reporter polypeptide is a polypeptide selected from the group consisting of 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-l 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, epididymis secretory sperm binding protein, zyxin, apolipoprotein C-III, secretogranin-2, angiotensinogen, c-reactive protein, serum albumin, transgelin-2, pancreatic prohormone, neurosecretory protein VGF, ceruloplasmin, PDZ and LIM domain protein 1, tubulin alpha-4 A chain, multimerin-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 230, insulin-like growth factor II, apolipoprotein A-II, probable non-functional immunoglobulin kappa variable 2D-24, prothrombin, coagulation factor IX, apolipoprotein LI, deleted in malignant brain tumors1 protein, desmoglein-3, calsyntenin-1, immunoglobulin lambda constant 3, complement C5, alpha-2-macroglobulin, myosin-9, sodium / potassium-transporting ATPase subunit gamma, immunoglobulin kappa variable 2-28, oncoprotein-induced transcript 3 protein, serglycin, coagulation factor XII, coagulation factor XIII A chain, insulin, histidine-rich glycoprotein, immunoglobulin kappa variable 3-11, immunoglobulin kappa variable 1-39, collagen alpha-l(I) chain, inter-alpha-trypsin inhibitor heavy chain H5, latent-transforming growth factor beta-binding protein 2, integrin alpha-IIb, membrane-associated progesterone receptor component 1, immunoglobulin lambda variable 6-57, immunoglobulin kappa variable 3-15, complement Clr subcomponent-like protein, histone Hl.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 inhibitory polypeptide, immunoglobulin heavy variable 3-15, immunoglobulin lambda variable 2-11, transcription initiation factor TFIID subunit 1, collagen alpha-1 (VII) chain, integral membrane protein 2B, pigment epithelium-derived factor, voltage-dependent N-type calcium channel subunit alpha-IB, immunoglobulin lambda variable 3-27, ras GTPase-activating protein nGAP, keratin, type I cytoskeletal 17, tubulin beta chain, sulfhydryl oxidase 1, immunoglobulin kappa variable 4-1, complement Clr subcomponent, 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 of any one of claims 18-20, wherein said reporter polypeptide comprises a sequence set forth in Columns II-VI of Table A.

22. The method of any one of claims 15-18, wherein said target tissue or cell is characterized by an increased amount or activity of said mammalian protease in proximity to said target tissue or cell as compared to a non-target tissue or cell in said subject.

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

24. The method of claim 23, wherein said disease or condition is a cancer or an inflammatory or autoimmune disease.

25. The method of claim 24, wherein said disease or condition is selected from the group consisting of carcinoma, Hodgkin’s lymphoma, and non-Hodgkin’s lymphoma, diffuse large B cell lymphoma, follicular lymphoma, mantle cell lymphoma, blastoma, breast cancer, ER / PR+ breast cancer, Her2+ breast cancer, triple-negative breast cancer, colon cancer, colon cancer with malignant ascites, mucinous tumors, prostate cancer, head and neck cancer, skin cancer, melanoma, genito-urinary tract cancer, ovarian cancer, ovarian cancer with malignant ascites, peritoneal carcinomatosis, uterine serous carcinoma, endometrial cancer, cervix cancer, colorectal, uterine cancer, mesothelioma in the peritoneum, kidney cancer, Wilm’s tumor,lung cancer, small-cell lung cancer, non-small cell lung cancer, gastric cancer, stomach cancer, small intestine cancer, liver cancer, hepatocarcinoma, hepatoblastoma, liposarcoma, pancreatic cancer, gall bladder cancer, cancers of the bile duct, esophageal cancer, salivary gland carcinoma, thyroid cancer, epithelial cancer, arrhenoblastoma, adenocarcinoma, sarcoma, and B-cell derived chronic lymphatic leukemia.

26. The method of claim 24, wherein said disease or condition is selected from the group consisting of ankylosing spondylitis (AS), arthritis (for example, and 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-Barre syndrome (GBS), Hashimoto’s disease, suppurative scab, Kawasaki disease, IgA nephropathy, idiopathic thrombocytopenic purpura, inflammatory bowel disease (IBD) (for example, and not limited to, Crohn’s disease (CD), clonal disease, ulcerative colitis, collagen colitis, lymphocytic colitis, ischemic colitis, empty colitis, Behcet’s syndrome, infectious colitis, indeterminate colitis, interstitial Cystitis), lupus (for example, and not limited to, systemic lupus erythematosus, discoid lupus, subacute cutaneous lupus erythematosus, cutaneous lupus erythematosus (such as chilblain lupus erythematosus), drug-induced lupus, neonatal lupus, lupus nephritis), mixed connective tissue disease, morphea, multiple sclerosis (MS), severe muscle Force disorder, narcolepsy, neuromuscular angina, pemphigus vulgaris, pernicious anemia, psoriasis, psoriatic arthritis, polymyositis, primary biliary cirrhosis, relapsing polychondritis, schizophrenia, scleroderma, Sjogren’s syndrome, systemic stiffness syndrome, temporal arteritis (also known as giant cell arteritis), vasculitis, vitiligo, Wegener’s granulomatosis, transplant rejection-associated immune reaction(s) (for example, and not limited to, renal transplant rejection, lung transplant rejection, liver transplant rejection), psoriasis, Wiskott-Aldrich syndrome, autoimmune lymphoproliferative syndrome, myasthenia gravis, inflammatory chronic rhinosinusitis, colitis, celiac disease, Barrett’s esophagus, inflammatory gastritis, autoimmune nephritis, autoimmune hepatitis, autoimmune carditis, autoimmune encephalitis, autoimmune mediated hematological disease, asthma, atopic dermatitis, atopy, allergy, allergic rhinitis, scleroderma, bronchitis, pericarditis, 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, systemic inflammatory response syndrome.

27. The method of any one of claims 1-26, wherein said therapeutic agent is an anti-cancer agent.

28. The method of any one of claims 1-27, wherein said therapeutic agent is an activatable therapeuticagent.

29. The method of any one of claims 1-28, wherein said therapeutic agent further comprises a masking moiety (MM).

30. The method of claim 29, wherein said masking moiety (MM) is capable of being released from said therapeutic agent upon cleavage of said peptide substrate by said mammalian protease.

31. The method of claim 29 or 30, wherein said masking moiety (MM) interferes with an interaction of said therapeutic agent, in an uncleaved state, to a target tissue or cell.

32. The method of any one of claims 29-31, wherein a bioactivity of said therapeutic agent is capable of being enhanced upon cleavage of said peptide substrate by said mammalian protease.

33. The method of any one of claims 29-32, wherein said masking moiety (MM) is an extended recombinant polypeptide (XTEN).

34. The method of claim 33, wherein said XTEN is characterized in that:(i) it comprises at least 100 amino acids;(ii) at least 90% of the amino acid residues of it are selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P); and(iii) it comprises at least 4 different types of amino acids selected from G, A, S, T, E, and P.

35. The method of any one of claims 1-34, further comprises, assessing if a subject will be responsive to a therapeutic subsequent to (b), by contacting said therapeutic agent with said mammalian protease.

36. The method of any one of claims 1-34, wherein (a) comprises detecting said polypeptide of (i), (ii) or (iii) in an immuno-assay.

37. The method of claim 36, wherein said immuno-assay utilizes an antibody that specifically binds to said polypeptide of (i), (ii) or (iii), or an epitope thereof.

38. The method of any one of claims 1-37, wherein (a) comprises detecting said polypeptide of (i), (ii) or (iii) by using a mass spectrometer (MS).

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

40. A method for treating a subject with an activatable therapeutic agent, the method comprising:(a) identifying said subject as having a likelihood of a response to said activatable therapeutic agent based on identification of a peptide biomarker in a biological sample from said subject, which activatable therapeutic agent comprises a peptide substrate sequence susceptible to cleavage by a mammalian protease at a scissile bond; and(b) administering said activatable therapeutic agent to said subject based on said identification of said subject in (a);wherein said peptide biomarker comprises a portion identical to at least four consecutive amino acid residues of said peptide substrate sequence that is either N-terminal or C-terminal of said scissile bond.

41. The method of claim 40, wherein said peptide biomarker is derived from a reporter polypeptide, which reporter polypeptide comprises a sequence set forth in Columns II-VI of Table A.

42. The method of claim 40 or 41, wherein said peptide biomarker has an amino acid sequence that is identical to a sequence of a reporter polypeptide, which reporter polypeptide comprises a sequence set forth in Columns II-VI of Table A.

43. The method of any one of claims 40-42, wherein said peptide substrate sequence contains from six to twenty-five or six to twenty amino acid residues.

44. The method of claim 43, wherein said peptide substrate sequence contains from seven to twelve amino acid residues.

45. The method of any one of claims 40-44, wherein said peptide substrate sequence comprises an amino acid sequence having at most three amino acid substitutions, at most two amino acid substitutions, or at most one amino acid substitution with respect to a sequence set forth in Column II or III of Table A, wherein none of said amino acid substitution is at a position corresponding to an amino acid residue immediately adjacent to a corresponding scissile bond as indicated in Table A.

46. The method of claim 45, wherein said peptide substrate sequence comprises an amino acid sequence set forth in Column II or III of Table A.

47. The method of any one of claims 40-46, wherein said peptide substrate sequence susceptible to cleavage by said mammalian protease is susceptible to cleavage by a plurality of mammalian proteases comprising said mammalian protease.

48. The method of claim 47, wherein said peptide substrate sequence susceptible to cleavage by said plurality of mammalian proteases has at most three amino acid substitutions, at most two amino acid substitutions, or at most one amino acid substitution with respect to a sequence set forth in Table l(j), wherein none of said amino acid substitution is at a position corresponding to an amino acid residue immediately adjacent to a corresponding scissile bond.

49. The method of claim 47 or 48, wherein said peptide substrate sequence susceptible to cleavage by said plurality of mammalian proteases comprises a sequence set forth in Table 1 (j).

50. The method of claim 45, wherein said peptide substrate sequence has an amino acid sequence identical to a fragment of a sequence set forth in Column II or III of Table A, wherein said fragment comprises at least four consecutive amino acid residues immediately adjacent to a corresponding scissile bond as indicated in Table A.

51. The method of claim 50, wherein said 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 of any one of claims 40-51, wherein a portion of said peptide substrate sequence that is N-terminal of said scissile bond has at most three amino acid substitutions, at most two amino acid substitutions, or at most one amino acid substitution with respect to a C-terminal end sequence containing from four to ten amino acid residues of a sequence set forth in Column IV or V of Table A, wherein none of said amino acid substitution is at a position corresponding to an amino acid residue immediately adjacent to a corresponding scissile bond.

53. The method of claim 52, wherein said portion of said peptide substrate sequence that is N-terminal of said scissile bond comprises a C-terminal end sequence containing from four to ten amino acid residues of a sequence set forth in Column TV or V of Table A.

54. The method of any one of claims 40-52, wherein a portion of said peptide substrate sequence that is C-terminal of said scissile bond has at most three amino acid substitutions, at most two amino acid substitutions, or at most one amino acid substitution with respect to an N-terminal end sequence containing from four to ten amino acid residues of a sequence set forth in Column V or VI of Table A, wherein none of said amino acid substitution is at a position corresponding to an amino acid residue immediately adjacent to a corresponding scissile bond.

55. The method of claim 54, wherein said portion of said peptide substrate sequence that is C-terminal of said scissile bond comprises an N-terminal end sequence containing from four to ten amino acid residues of a sequence set forth in Column V or VI of Table A.

56. The method of any one of claim 40-55, wherein said likelihood of said response is determined by a method according to any one of claims 1-39.

57. A method for treating a subject in need of a therapeutic agent that is activatable by a mammalian protease expressed in said subject, the method comprising:administering an effective amount of said therapeutic agent to said subject, wherein said subject has been shown to express in a biological sample from said subject:(i) a polypeptide comprising at least five or six consecutive amino acid residues shown in a sequence set forth in Column V of Table A; or(ii) a polypeptide comprising at least five or six consecutive amino acids shown in a sequence set forth in Column IV of Table A; or(iii) a polypeptide comprising at least five or six consecutive amino acids shown in a sequence set forth in Column VI of Table A; or(iv) expression level of polypeptide (i), (ii) or (iii) exceeds a threshold.

58. The method of claim 57, wherein said polypeptide sequence of (i) comprises at least seven, at least eight, at least nine, or at least ten consecutive amino acid residues shown in a sequence set forth in Column V of Table A.

59. The method of claim 57 or 58, wherein said polypeptide of (ii) comprises at least seven, at least eight, at least nine, or at least ten consecutive amino acids shown in a sequence set forth in Column IV of Table A.

60. The method of any one of claims 57-59, wherein said polypeptide of (iii) comprises at least seven, at least eight, at least nine, or at least ten consecutive amino acids shown in a sequence set forth in Column VI of Table A.

61. The method of any one of claims 57-60, wherein said subject has been shown to express in said biological sample any two of (i)-(iii).

62. The method of any one of claims 57-61, wherein said therapeutic agent comprises a peptide substrate sequence susceptible to cleavage by said mammalian protease.

63. The method of claim 62, wherein said peptide substrate sequence is susceptible to cleavage by said mammalian protease at a scissile bond, and wherein said polypeptide of (i), (ii), or (iii) comprises a portioncontaining at least four consecutive amino acid residues of said peptide substrate sequence that is either N-terminal or C-terminal of said scissile bond.

64. The method of claim 63, wherein a portion of said peptide substrate sequence that is N-terminal of said scissile bond has at most three amino acid substitutions, at most two amino acid substitutions, or at most one amino acid substitution with respect to a C-terminal end sequence containing from four to ten amino acid residues of a sequence set forth in Column IV or V of Table A, wherein none of said amino acid substitution is at a position corresponding to an amino acid residue immediately adjacent to a corresponding scissile bond.

65. The method of claim 63 or 64, wherein said portion of said peptide substrate sequence that is N-terminal of said scissile bond comprises a C-terminal end sequence containing from four to ten amino acid residues of a sequence set forth in Column IV or V of Table A.

66. The method of any one of claims 63-65, wherein a portion of said peptide substrate sequence that is C-terminal of said scissile bond has at most three amino acid substitutions, at most two amino acid substitutions, or at most one amino acid substitution with respect to an N-terminal end sequence containing from four to ten amino acid residues of a sequence set forth in Column V or VI of Table A, wherein none of said amino acid substitution is at a position corresponding to an amino acid residue immediately adjacent to a corresponding scissile bond.

67. The method of any one of claims 63-66, wherein said portion of said peptide substrate sequence that is C-terminal of said scissile bond comprises an N-terminal end sequence containing from four to ten amino acid residues of a sequence set forth in Column V or VI of Table A.

68. The method of any one of claims 57-67, wherein said threshold is zero or nominal.

69. The method of any one of claims 40-68, wherein said biological sample comprises a serum or plasma sample.

70. The method of any one of claims 40-69, wherein said mammalian protease is a serine protease, a cysteine protease, an aspartate protease, a threonine protease, or a metalloproteinase.

71. The method of claim 70, wherein said mammalian protease is selected from the group consisting of disintegrin and metalloproteinase domain-containing protein 10 (ADAM 10), disintegrin and metalloproteinase domain-containing protein 12 (ADAM12), disintegrin and metalloproteinase domaincontaining protein 15 (ADAM 15), disintegrin and metalloproteinase domain-containing protein 17 (ADAM17), disintegrin and metalloproteinase domain-containing protein 9 (ADAM9), disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), Cathepsin B, Cathepsin D, Cathepsin E, Cathepsin K, cathepsin L, cathepsin S, Fibroblast activation protein alpha, Hepsin, kallikrein-2, kallikrein-4, kallikrein-3, Prostate-specific antigen (PSA), kallikrein-13, Legumain, 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 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 type serine protease 1 (MT-SP1), matriptase, and u-plasminogen.

72. The method of claim 70, wherein said mammalian protease is selected from the group consisting ofmatrix 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.

73. The method of any one of claims 40-71, wherein said mammalian protease is preferentially expressed or activated in a target tissue or cell.

74. The method of claim 73, wherein said target tissue or cell is a tumor.

75. The method of claim 73 or 74, wherein said target tissue or cell produces or is co-localized withsaid mammalian protease.

76. The method of any one of claims 73-75, wherein said target tissue or cell contains therein or thereon, or is associated with in proximity thereto, a reporter polypeptide.

77. The method of claim 76, wherein said reporter polypeptide is a polypeptide selected from the groupconsisting of 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, epididymis secretory sperm binding protein, secretogranin-2, angiotensinogen, transgelin-2, pancreatic prohormone, neurosecretory protein VGF, ceruloplasmin, PDZ and LIM domain protein 1, multimerin-1, inter-alpha-trypsin inhibitor heavy chain H2, N-acetylmuramoyl-L-alanine amidase, histone Hl.4, adhesion G-protein coupled receptor G6, mannan-binding lectin serine protease 2, prothrombin, deleted in malignant brain tumors 1 protein, desmoglein-3, calsyntenin-1, alpha-2-macroglobulin, myosin-9, sodium / potassium-transporting ATPase subunit gamma, oncoprotein-induced transcript 3 protein, serglycin, histidine-rich glycoprotein, inter-alpha-trypsin inhibitor heavy chain H5, integrin alpha-IIb, membrane-associated progesterone receptor component 1, histone Hl.2, rho GDP-dissociation inhibitor 2, zinc-alpha-2-glycoprotein, talin-1, secretogranin-1, neutrophil defensin 3, cytochrome P450 2E1, gastric inhibitory polypeptide, transcription initiation factor TFIID subunit 1, integral membrane protein 2B, pigment epithelium-derived factor, voltage-dependent N-type calcium channel subunit alpha-IB, ras GTPase-activating protein nGAP, type I cytoskeletal 17, sulfhydryl oxidase 1, homeobox protein Hox-B2, transcription factor SOX-10, E3 ubiquitin-protein ligase SIAH2, decorin, secreted protein acidic and rich in cysteine (SPARC), laminin gamma 1 chain, vimentin, and nidogen-1 (NIDI).

78. The method of claim 77, wherein said reporter polypeptide is a polypeptide selected from the group consisting of 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-l 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, epididymis secretory sperm binding protein, zyxin, apolipoprotein C-III, secretogranin-2, angiotensinogen, c-reactive protein, serum albumin, transgelin-2, pancreatic prohormone, neurosecretory protein VGF, ceruloplasmin, PDZ and LIM domain protein 1, tubulin alpha-4 A chain, multimerin-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 Hl.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 230, insulin-like growth factor II, apolipoprotein A-II, probable non-functional immunoglobulin kappa variable 2D-24, prothrombin, coagulation factor IX, apolipoprotein LI, deleted in malignant brain tumors 1 protein, desmoglein-3, calsyntenin-1, immunoglobulin lambda constant 3, complement C5, alpha-2-macroglobulin, myosin-9, sodium / potassium-transporting ATPase subunit gamma, immunoglobulin kappa variable 2-28, oncoprotein-induced transcript 3 protein, serglycin, coagulation factor XII, coagulation factor XIII A chain, insulin, histidine-rich glycoprotein, immunoglobulin kappa variable 3-11, immunoglobulin kappa variable 1-39, collagen alpha-l(I) chain, inter-alpha-trypsin inhibitor heavy chain H5, latent-transforming growth factor beta-binding protein 2, integrin alpha-IIb, membrane-associated progesterone receptor component 1, immunoglobulin lambda variable 6-57, immunoglobulin kappa variable 3-15, complement Clr subcomponent-like protein, histone Hl.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 inhibitory polypeptide, immunoglobulin heavy variable 3-15, immunoglobulin lambda variable 2-11, transcription initiation factor TFIID subunit 1, collagen alpha-1 (VII) chain, integral membrane protein 2B, pigment epithelium-derived factor, voltage-dependent N-type calcium channel subunit alpha-IB, immunoglobulin lambda variable 3-27, ras GTPase-activating protein nGAP, keratin, type I cytoskeletal 17, tubulin beta chain, sulfhydryl oxidase 1, immunoglobulin kappa variable 4-1, complement Clr subcomponent, 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 of any one of claims 76-78, wherein said reporter polypeptide comprises a sequence set forth in Columns II-VI of Table A.

80. The method of any one of claims 73-79, wherein said target tissue or cell is characterized by an increased amount or activity of said mammalian protease in proximity to said target tissue or cell as compared to a non-target tissue or cell in said subject.

81. The method of any one of claims 40-80, wherein said subject is suffering from, or is suspected of suffering from, a disease or condition characterized by an increased expression or activity of said mammalian protease in proximity to a target tissue or cell as compared to a corresponding non-target tissue or cell in said subject.

82. The method of claim 81, wherein said disease or condition is a cancer or an inflammatory or autoimmune disease.

83. The method of claim 82, wherein said disease or condition is selected from the group consisting of ankylosing spondylitis (AS), arthritis (for example, and 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-Barre syndrome (GBS), Hashimoto’s disease, suppurative scab, Kawasaki disease, IgA nephropathy, idiopathic thrombocytopenic purpura, inflammatory bowel disease (IBD) (for example, and not limited to, Crohn’s disease (CD), clonal disease, ulcerative colitis, collagen colitis, lymphocytic colitis, ischemic colitis, empty colitis, Behcet’s syndrome, infectious colitis, indeterminate colitis, interstitial Cystitis), lupus (for example, and not limited to, systemic lupus erythematosus, discoid lupus, subacute cutaneous lupus erythematosus, cutaneous lupus erythematosus (such as chilblain lupus erythematosus), drug-induced lupus, neonatal lupus, lupus nephritis), mixed connective tissue disease, morphea, multiple sclerosis (MS), severe muscle Force disorder, narcolepsy, neuromuscular angina, pemphigus vulgaris, pernicious anemia, psoriasis, psoriatic arthritis, polymyositis, primary biliary cirrhosis, relapsing polychondritis, schizophrenia, scleroderma, Sjogren’s syndrome, systemic stiffness syndrome, temporal arteritis (also known as giant cell arteritis), vasculitis, vitiligo, Wegener’s granulomatosis, transplant rejection-associated immune reaction(s) (for example, and not limited to, renal transplant rejection, lung transplant rejection, liver transplant rejection), psoriasis, Wiskott-Aldrich syndrome, autoimmune lymphoproliferative syndrome, myasthenia gravis, inflammatory chronic rhinosinusitis, colitis, celiac disease, Barrett’s esophagus, inflammatory gastritis, autoimmune nephritis, autoimmune hepatitis, autoimmune carditis, autoimmune encephalitis, autoimmune mediated hematological disease, asthma, atopic dermatitis, atopy, allergy, allergic rhinitis, scleroderma, bronchitis, pericarditis, 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, systemic inflammatory response syndrome.

84. The method of claim 83, wherein said disease or condition is selected from the group consisting of carcinoma, Hodgkin’s lymphoma, and non-Hodgkin’s lymphoma, diffuse large B cell lymphoma, follicular lymphoma, mantle cell lymphoma, blastoma, breast cancer, ER / PR+ breast cancer, Her2+ breast cancer,triple-negative breast cancer, colon cancer, colon cancer with malignant ascites, mucinous tumors, prostate cancer, head and neck cancer, skin cancer, melanoma, genito-urinary tract cancer, ovarian cancer, ovarian cancer with malignant ascites, peritoneal carcinomatosis, uterine serous carcinoma, endometrial cancer, cervix cancer, colorectal, uterine cancer, mesothelioma in the peritoneum, kidney cancer, Wilm’s tumor, lung cancer, small-cell lung cancer, non-small cell lung cancer, gastric cancer, stomach cancer, small intestine cancer, liver cancer, hepatocarcinoma, hepatoblastoma, liposarcoma, pancreatic cancer, gall bladder cancer, cancers of the bile duct, esophageal cancer, salivary gland carcinoma, thyroid cancer, epithelial cancer, arrhenoblastoma, adenocarcinoma, sarcoma, and B-cell derived chronic lymphatic leukemia.

85. The method of any one of claims 40-84, wherein said therapeutic agent is an anti-cancer agent.

86. The method of any one of claims 40-85, wherein said therapeutic agent is an activatable therapeuticagent.

87. The method of claim 86, wherein said therapeutic agent is a non-natural, activatable therapeutic agent.

88. The method of any one of claims 40-86, wherein said therapeutic agent comprises a masking moiety (MM).

89. The method of claim 88, wherein said masking moiety (MM) is capable of being released from said therapeutic agent upon cleavage of said peptide substrate sequence by said mammalian protease.

90. The method of claim 88 or 89, wherein said masking moiety (MM) interferes with an interaction of said therapeutic agent, in an uncleaved state, to a target tissue or cell.

91. The method of any one of claims 88-90, wherein a bioactivity of said therapeutic agent is capable of being enhanced upon cleavage of said peptide substrate sequence by said mammalian protease.

92. The method of any one of claims 88-90, wherein said masking moiety (MM) is an extended recombinant polypeptide (XTEN).

93. The method of claim 92, wherein said XTEN is characterized in that:(i) it comprises at least 100 amino acids;(ii) at least 90% of the amino acid residues of it are selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P); and(iii) it comprises at least 4 different types of amino acids selected from G, A, S, T, E, and P.

94. The method of any one of claims 57-93, wherein said subject is determined to have a likelihood of a response to a therapeutic agent.

95. The method of any one of claims 57-94 for treating a disease or condition in a subject, comprising administering to said subject in need thereof one or more therapeutically effective doses of the activatable therapeutic or a pharmaceutical composition comprised of the activatable therapeutic.

96. The method of claim 95, wherein said subject is selected from the group consisting of mouse, rat, monkey, and human.

97. The method of claim 95, wherein said subject is a human.

98. The method of any one of claims 95-97, wherein said subject is determined to have a likelihood of a response to said therapeutic agent or said pharmaceutical composition.

99. The method of claim 98 wherein said likelihood of said response is 50% or higher.

100. The method of claim 99 or 100, wherein said likelihood of said response is determined by a methodaccording to any one of claims 1-39.

101. The method of any one of claims 95-100, wherein said disease or condition is a cancer or an inflammatory or autoimmune disease.

102. The method of claim 101, wherein said disease or condition is selected from the group consisting of ankylosing spondylitis (AS), arthritis (for example, and 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-Barre syndrome (GBS), Hashimoto’s disease, suppurative scab, Kawasaki disease, IgA nephropathy, idiopathic thrombocytopenic purpura, inflammatory bowel disease (IBD) (for example, and not limited to, Crohn’s disease (CD), clonal disease, ulcerative colitis, collagen colitis, lymphocytic colitis, ischemic colitis, empty colitis, Behcet’s syndrome, infectious colitis, indeterminate colitis, interstitial Cystitis), lupus (for example, and not limited to, systemic lupus erythematosus, discoid lupus, subacute cutaneous lupus erythematosus, cutaneous lupus erythematosus (such as chilblain lupus erythematosus), drug-induced lupus, neonatal lupus, lupus nephritis), mixed connective tissue disease, morphea, multiple sclerosis (MS), severe muscle Force disorder, narcolepsy, neuromuscular angina, pemphigus vulgaris, pernicious anemia, psoriasis, psoriatic arthritis, polymyositis, primary biliary cirrhosis, relapsing polychondritis, schizophrenia, scleroderma, Sjogren’s syndrome, systemic stiffness syndrome, temporal arteritis (also known as giant cell arteritis), vasculitis, vitiligo, Wegener’s granulomatosis, transplant rejection-associated immune reaction(s) (for example, and not limited to, renal transplant rejection, lung transplant rejection, liver transplant rejection), psoriasis, Wiskott-Aldrich syndrome, autoimmune lymphoproliferative syndrome, myasthenia gravis, inflammatory chronic rhinosinusitis, colitis, celiac disease, Barrett’s esophagus, inflammatory gastritis, autoimmune nephritis, autoimmune hepatitis, autoimmune carditis, autoimmune encephalitis, autoimmune mediated hematological disease, asthma, atopic dermatitis, atopy, allergy, allergic rhinitis, scleroderma, bronchitis, pericarditis, 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, systemic inflammatory response syndrome.

103. The method of claim 101, wherein said disease or condition is selected from the group consisting of carcinoma, Hodgkin’s lymphoma, and non-Hodgkin’s lymphoma, diffuse large B cell lymphoma, follicular lymphoma, mantle cell lymphoma, blastoma, breast cancer, ER / PR+ breast cancer, Her2+ breast cancer, triple-negative breast cancer, colon cancer, colon cancer with malignant ascites, mucinous tumors, prostate cancer, head and neck cancer, skin cancer, melanoma, genito-urinary tract cancer, ovarian cancer,ovarian cancer with malignant ascites, peritoneal carcinomatosis, uterine serous carcinoma, endometrial cancer, cervix cancer, colorectal, uterine cancer, mesothelioma in the peritoneum, kidney cancer, Wilm’s tumor, lung cancer, small-cell lung cancer, non-small cell lung cancer, gastric cancer, stomach cancer, small intestine cancer, liver cancer, hepatocarcinoma, hepatoblastoma, liposarcoma, pancreatic cancer, gall bladder cancer, cancers of the bile duct, esophageal cancer, salivary gland carcinoma, thyroid cancer, epithelial cancer, arrhenoblastoma, adenocarcinoma, sarcoma, and B-cell derived chronic lymphatic leukemia.

104. Use of a diagnostic reagent in the practice of a method of any one of claims 1-39 for assessing a likelihood of a subject being responsive to a therapeutic agent that is activatable by a mammalian protease expressed in said subject having a disease or disorder.

105. Use of a diagnostic reagent in the practice of a method of any one of claims 40-103 for assessing a likelihood of a subject being responsive to a therapeutic agent that is activatable by a mammalian protease expressed in said subject having a disease or disorder.

106. A kit for the practice of a method of claims 1-103 for assessing a likelihood of a subject being responsive to a therapeutic agent that is activatable by a mammalian protease expressed in said subject having a disease or disorder comprising a reagent for detecting the presence or amount of a proteolytic peptide product produced by action of said mammalian protease.

Images