Protease-cleavable substrates and methods of using same

Abstract

Disclosed are isolated polypeptides comprising cleavable moieties that are substrates for at least two proteases (e.g., MT-SP1 and MMP). Activatable molecules comprising the isolated polypeptides are disclosed. Disclosed are methods of making and using the isolated polypeptides and activatable molecules comprising the isolated polypeptides in various therapeutic, diagnostic, and prophylactic applications. In one aspect, the disclosure provides an isolated polypeptide comprising a substrate, wherein the substrate comprises a first cleavable moiety (CM1) cleavable by a first protease and a second cleavable moiety (CM2) cleavable by a second protease, wherein CM1 comprises the amino acid sequence of HQSRS (SEQ ID NO: 2).

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 370,023, filed August 1, 2022, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format and is incorporated herein by reference in its entirety. The XML copy was created on July 27, 2023, is named CYTX-094-PCT_SL.xml, and is 512,000 bytes in size.

[0003] Technical Field The present disclosure relates generally to polypeptides, including substrates that include one or more cleavable moieties, each of which is cleavable by a protease, and methods of making and using the polypeptides and activatable molecules in a variety of therapeutic, diagnostic, and prophylactic applications. [Background technology]

[0004] background Proteases are enzymes that catalyze the hydrolysis of peptide bonds between amino acid residues. Some proteases are known to cleave specific peptide bonds based on the presence of specific amino acid sequences within a protein. Proteases are naturally present in all living organisms and are involved in a variety of physiological reactions, ranging from simple degradation to highly regulated pathways. Some proteases cleave specific peptide bonds based on the presence of specific amino acid sequences within a protein, while some amino acid sequences are resistant to cleavage by certain proteases.

[0005] Thus, there is a need to identify new substrates for proteases and to use these substrates in a variety of therapeutic, diagnostic and prophylactic applications. Summary of the Invention

[0006] In one aspect, the disclosure provides an isolated polypeptide comprising a substrate, wherein the substrate comprises a first cleavable portion (CM1) cleavable by a first protease and a second cleavable portion (CM2) cleavable by a second protease, wherein CM1 comprises the amino acid sequence of HQSRS (SEQ ID NO:2). In another aspect, the disclosure provides an isolated polypeptide comprising a substrate, wherein the substrate comprises a first cleavable portion (CM1) cleavable by a first protease and a second cleavable portion (CM2) cleavable by a second protease, wherein CM1 comprises the amino acid sequence of HQSR (SEQ ID NO:1), HQSK (SEQ ID NO:3), or HQSRSA (SEQ ID NO:4). In some embodiments, CM1 comprises the amino acid sequence of DHQSRS (SEQ ID NO:5). In some embodiments, CM1 comprises the amino acid sequence of SDHQSRS (SEQ ID NO:6). In some embodiments, CM2 comprises the amino acid sequence of AIALYAD (SEQ ID NO:7), AIALYA (SEQ ID NO:8), or AIAL (SEQ ID NO:9). In some embodiments, CM2 comprises the amino acid sequence of GPWGLL (SEQ ID NO:237) or SGLLSS (SEQ ID NO:238). In some embodiments, CM2 comprises the amino acid sequence of ALAHGLF (SEQ ID NO:74) or ISSGLLSS (SEQ ID NO:75).

[0007] According to some embodiments of the present disclosure, the isolated polypeptide is a molecule in which a portion or component of the molecule is separated from the remainder of the molecule by cleavage of the CM by a protease. In some aspects of the present disclosure, the molecule is activated by cleavage of the CM by a protease. In some aspects, the isolated polypeptide is a molecule in which the CM is cleaved by multiple proteases. In some aspects, the isolated polypeptide is a molecule in which the CM is cleaved by MMP2. In some aspects, the isolated polypeptide is a molecule in which the CM is cleaved by MMP9. In some aspects, the isolated polypeptide is a molecule in which the CM is cleaved by MMP14. In some aspects, the isolated polypeptide is a molecule in which the CM is cleaved by MT-SP1. In some aspects, the isolated polypeptide is a molecule in which the CM is cleaved by two or all of MMP2, MMP9, MMP14, and MT-SP1. In some embodiments, the isolated polypeptide is a molecule that has a % cleavage rate of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, or 100% of the CM, e.g., by any one of MMP2, MMP9, MMP14, or MT-SP1, or any two of MMP2, MMP9, MMP14, and MT-SP1, or by each of MMP2, MMP9, MMP14, and MT-SP1. In some embodiments, the isolated polypeptide is a molecule that has a % cleavability of the CM that is 1.5-fold, 2-fold, 2.5-fold, 3-fold, 5-fold, 7-fold, 8-fold, or 10-fold or more improved over the % cleavability of SEQ ID NO: 78 (see, e.g., Example 2). According to some embodiments of the present disclosure, the isolated polypeptide is a molecule with high in vivo stability such that it is not cleaved in plasma, as demonstrated by less than 50%, less than 40%, or less than 25% in vivo activation after 7 days of in vivo administration (see, e.g., Example 3). According to embodiments of the present disclosure, the isolated polypeptide is administered at a concentration of 1×10 2 M -1 s -1 exceeding kcat / K M (M -1 s -1 According to some embodiments of the present disclosure, the isolated polypeptide is a molecule comprising a CM having 1×10 3 M -1 s -1 exceeding k cat / K M (M -1 s -1 According to an embodiment of the present disclosure, the isolated polypeptide is a molecule comprising a CM having 1×10 4 M -1 s -1 exceeding k cat / K M (M -1 s -1 According to an embodiment of the present disclosure, the isolated polypeptide is a molecule comprising a CM having 1×10 5 M -1 s -1 exceeding k cat / K M (M -1 s -1 ) is a molecule containing CM.

[0008] In some embodiments, CM1 comprises the amino acid sequence of DHQSRS (SEQ ID NO:5). In some embodiments, CM1 comprises the amino acid sequence of SDHQSRS (SEQ ID NO:6). In some embodiments, CM2 comprises the amino acid sequence of AIALYA (SEQ ID NO:8). In some embodiments, CM2 comprises the amino acid sequence of AIALYAD (SEQ ID NO:7). In some embodiments, the substrate comprises the amino acid sequence of AIALYADHQSRS (SEQ ID NO:10), DHQSRSAIALYAD (SEQ ID NO:11), HQSRSAIALYAD (SEQ ID NO:12), DHQSRSAIALYA (SEQ ID NO:13), or HQSRSAIALYA (SEQ ID NO:14). In some embodiments, the substrate comprises the amino acid sequence of DHQSRSAIALYA (SEQ ID NO:13).

[0009] In some embodiments, the substrate comprises the amino acid sequence of HQSRSAIALY (SEQ ID NO: 636). In some embodiments, the substrate comprises the amino acid sequence of HQSRSAIAL (SEQ ID NO: 637). In some embodiments, the substrate comprises the amino acid sequence of AIALHQSRS (SEQ ID NO: 41). In some embodiments, the substrate comprises the amino acid sequence of AIALYAHQSRS (SEQ ID NO: 33) or AIALYHQSRS (SEQ ID NO: 37). In some embodiments, the substrate comprises the amino acid sequence of HQSRSGPRGLL (SEQ ID NO: 638). In some embodiments, the substrate comprises the amino acid sequence of HQSRSGPAGLL (SEQ ID NO: 639). In some embodiments, the substrate comprises the amino acid sequence of HQSRSPWG (SEQ ID NO: 640). In some embodiments, the substrate comprises the amino acid sequence of HQSRAGPWGLL (SEQ ID NO: 645). In some embodiments, the substrate comprises the amino acid sequence of HQSRSAPWGLL (SEQ ID NO: 646). In some embodiments, the substrate comprises the amino acid sequence of HQSRSGAWGLL (SEQ ID NO: 647). In some embodiments, the substrate comprises the amino acid sequence of HQSRSGPWALL (SEQ ID NO:648). In some embodiments, the substrate comprises the amino acid sequence of HQSRSGPWGAL (SEQ ID NO:649). In some embodiments, the substrate comprises the amino acid sequence of HQSRSGPWGLA (SEQ ID NO:650). In some embodiments, the substrate comprises the amino acid sequence of HQSRSAIALYA (SEQ ID NO:14). In some embodiments, the substrate comprises the amino acid sequence of HQSRSPWGL (SEQ ID NO:126). In some embodiments, the substrate comprises the amino acid sequence of PWGLHQSRS (SEQ ID NO:489). In some embodiments, the substrate comprises the amino acid sequence of PWGLLHQSRS (SEQ ID NO:485). In some embodiments, the substrate comprises the amino acid sequence of PWGLHQSR (SEQ ID NO:502). In some embodiments, the substrate comprises the amino acid sequence of PWGLHQSKS (SEQ ID NO:490). In some embodiments, the substrate comprises the amino acid sequence of DHQSKSGPWGLL (SEQ ID NO:178). In some embodiments, the substrate comprises the amino acid sequence of HQSRSGPWGL (SEQ ID NO: 122).In some embodiments, the substrate comprises the amino acid sequence of HQSRSGPYGLL (SEQ ID NO: 138). In some embodiments, the substrate comprises the amino acid sequence of HQSRSGPFGLL (SEQ ID NO: 158). In some embodiments, the substrate comprises the amino acid sequence of HQSRSGPRGLL (SEQ ID NO: 638). In some embodiments, the substrate comprises the amino acid sequence of HQSRSGPAGLL (SEQ ID NO: 639). In some embodiments, the substrate comprises the amino acid sequence of HQSRPWGL (SEQ ID NO: 134).

[0010] In some embodiments, the disclosure includes an isolated polypeptide comprising a substrate, wherein the substrate comprises the amino acid sequence of AQSRSGPWGLL (SEQ ID NO: 641). In some embodiments, the substrate comprises the amino acid sequence of HASRSGPWGLL (SEQ ID NO: 642). In some embodiments, the substrate comprises the amino acid sequence of HQARSGPWGLL (SEQ ID NO: 643). In some embodiments, the substrate comprises the amino acid sequence of HQSASGPWGLL (SEQ ID NO: 644).

[0011] In some embodiments, CM2 comprises the amino acid sequence GPX1GLX2 (sequence number 651), where X1 is W, Y, F, R, K, Q, A, or M, and X2 is any amino acid or is absent.

[0012] In some embodiments, CM2 comprises the amino acid sequence of PXGL, where X is alanine (A), tyrosine (Y), arginine (R), phenylalanine (F), tryptophan (W), lysine (K), glutamine (Q), or methionine (M).

[0013] In certain aspects, the present disclosure can include substitution of any alanine in the disclosed CM sequences with valine.

[0014] In some embodiments, CM2 comprises the amino acid sequence of GPWGLL (SEQ ID NO: 237). In some embodiments, the substrate comprises the amino acid sequence of HQSRSGPWGLL (SEQ ID NO: 15), DHQSRSGPWGLL (SEQ ID NO: 16), or GPWGLLDHQSRS (SEQ ID NO: 17). In some embodiments, the substrate comprises the amino acid sequence of SGLLSS (SEQ ID NO: 238). In some embodiments, CM2 comprises the amino acid sequence of ISSGLLSS (SEQ ID NO: 75). In some embodiments, the substrate comprises the amino acid sequence of ISSGLLSS (SEQ ID NO: 18), DHQSRSISSGLLSS (SEQ ID NO: 19), or DHQSRSSGLLSS (SEQ ID NO: 20). In some embodiments, CM2 comprises the amino acid sequence of ALAHGLF (SEQ ID NO: 74). In some embodiments, the substrate comprises the amino acid sequence of ALAHGLFSDHQSRS (SEQ ID NO: 21) or SDHQSRSALAHGLF (SEQ ID NO: 22).

[0015] In another aspect, the disclosure provides an isolated polypeptide comprising a substrate, wherein the substrate comprises a first cleavable portion (CM1) cleavable by a first protease and a second cleavable portion (CM2) cleavable by a second protease, wherein the substrate comprises an amino acid sequence selected from SEQ ID NOs: 10-73, 83-216, 460-634, and 636-650, optionally wherein the substrate comprises an amino acid sequence selected from SEQ ID NOs: 10-73, 83-216, and 460-634.

[0016] In some embodiments, CM1 and CM2 have a structural arrangement from N-terminus to C-terminus as CM1-CM2. In some embodiments, CM1 and CM2 have a structural arrangement from N-terminus to C-terminus as CM2-CM1. In some embodiments, CM1 and CM2 are indirectly coupled via a connecting peptide. In some embodiments, CM1 and CM2 are directly coupled to each other. In some embodiments, at least a portion of CM1 overlaps with at least a portion of CM2. In some embodiments, the substrate comprises one or more CMs in addition to CM1 and CM2. In some embodiments, the substrate comprises CM1 cleavable by a first protease, CM2 cleavable by a second protease, CM3 cleavable by a third protease, and CM4 cleavable by a fourth protease.

[0017] In some embodiments, the isolated polypeptide is an activatable molecule and further comprises an "active moiety" (AM) that specifically binds to a target. In some embodiments, the AM is a therapeutic macromolecule. In some embodiments, the AM is an antibody or an antigen-binding fragment thereof. In some embodiments, the antibody is a full-length antibody, a single-chain variable fragment (scFv), a diabody (a non-covalent dimer of scFvs), a single-chain antibody (scab), a VHH, a domain antibody (dAb), or a single-domain antibody (nanobody, e.g., a single-domain heavy chain antibody, a single-domain light chain antibody). In some embodiments, the antibody is a monoclonal antibody, a single-chain antibody, a Fab fragment, a F(ab')2 fragment, a single-chain variable fragment (scFv), a diabody (a non-covalent dimer of scFvs), a single-chain antibody (scab), a VHH, a domain antibody (dAb), or a single-domain antibody (nanobody, e.g., a single-domain heavy chain antibody, a single-domain light chain antibody). According to some embodiments of the present disclosure, the isolated polypeptide is an activatable molecule with high in vivo stability such that it is not cleaved in plasma, as demonstrated by less than 50%, less than 40%, or less than 25% in vivo activation after 7 days of in vivo administration (e.g., as shown in Example 3). According to some embodiments of the present disclosure, the isolated polypeptide is an activatable molecule with a masking efficiency of 18-fold, 20-fold, 30-fold, 33-fold, 50-fold, 60-fold, 82-fold, 95-fold, 105-fold, 121-fold, 123-fold, 150-fold, 200-fold, 234-fold, 300-fold, or more (e.g., as shown in Example 4). According to some embodiments of the present disclosure, the activatable molecule is activated by one, two, or all of MMP2, MMP9, MMP14, and MT-SP1.According to some embodiments of the present disclosure, the activatable molecule is activated to a degree that it has a cleavage rate of at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, or 100%, for example, to a degree that it is cleavable by any one of MMP2, MMP9, MMP14, and MT-SP1, or any two of MMP2, MMP9, MMP14, and MT-SP1, or each of MMP2, MMP9, MMP14, and MT-SP1, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, or 100%. In some embodiments, the AM is a cytokine. In some embodiments, the AM is a chimeric antigen receptor. In some aspects, the AM is a drug or agent, such as a therapeutic agent, an imaging agent, or a diagnostic agent.

[0018] In some embodiments, the AM is coupled to CM1. In some embodiments, the AM is directly coupled to CM1. In some embodiments, the AM is indirectly coupled to CM1 via a connecting peptide. In some embodiments, the AM is coupled to CM2. In some embodiments, the AM is directly coupled to CM2. In some embodiments, the AM is indirectly coupled to CM2 via a connecting peptide. In some embodiments, the AM is coupled to CM3. In some embodiments, the AM is directly coupled to CM3. In some embodiments, the AM is indirectly coupled to CM3 via a connecting peptide. In some embodiments, the AM is indirectly coupled to CM1 via one or more components of an activatable protein. In some embodiments, the AM is indirectly coupled to CM2 via one or more components of an activatable protein. In some embodiments, the AM is indirectly coupled to CM3 via one or more components of an activatable protein.

[0019] In some embodiments, the isolated polypeptide further comprises a masking moiety (MM). In some embodiments, the MM has a dissociation constant for binding to the AM that is greater than the dissociation constant of the AM for binding to the target. In some embodiments, the MM does not interfere with or compete with the AM for binding to the target in an activation molecule (i.e., after cleavage of the CM by a protease). In some embodiments, the MM is 2-40 amino acids in length. In some embodiments, the MM does not bind the AM, but interferes with binding of the AM to its binding partner through non-specific interactions. In some embodiments, the MM is a steric mask. In some embodiments, the MM is a protein. In some embodiments, the MM is linked to the substrate such that the isolated polypeptide comprises, from N-terminus to C-terminus, the following structural arrangement: MM-CM1-CM2-AM, MM-CM2-CM1-AM, AM-CM1-CM2-MM, AM-CM2-CM1-MM, MM-CM2-CM1-CM3-AM, MM-CM1-CM2-CM3-AM, MM-CM1-CM3-CM2-AM, MM-CM3-CM1-CM2-AM, or MM-CM3-CM2-CM1-AM. In some embodiments, the MM is directly coupled to CM1. In some embodiments, the MM is directly coupled to CM2. In some embodiments, the MM is directly coupled to CM3.

[0020] In some embodiments, the MM is indirectly coupled to CM1. In some embodiments, the MM is indirectly coupled to CM2. In some embodiments, the MM is indirectly coupled to CM3. In some embodiments, the MM is indirectly coupled to CM1 via a connecting peptide. In some embodiments, the MM is indirectly coupled to CM2 via a connecting peptide. In some embodiments, the MM is indirectly coupled to CM3 via a connecting peptide. In some embodiments, the isolated polypeptide comprises a connecting peptide (LP), and the isolated polypeptide has the following structural arrangement from N-terminus to C-terminus: MM-LP-CM1-CM2-AM, MM-CM1-CM2-LP-AM, MM-LP-CM2-CM1-AM, MM-CM2-CM1-LP-AM, MM-LP-CM2-CM1-CM3-AM, MM-LP-CM1-CM2-CM3-AM, MM-LP-CM1-CM3-CM2-AM, MM-LP-CM3-CM1-CM2-AM, MM-CM2-CM1-CM3-LP-AM, MM-CM1-CM2-CM3-LP-AM, MM-CM1-CM3-CM2-LP-AM, MM-CM3-CM1-CM2-LP-AM, or MM-CM3-CM2-CM1-LP-AM.In some embodiments, the isolated polypeptide comprises a first connecting peptide (LP1) and a second connecting peptide (LP2), and the isolated polypeptide has the following structural arrangement from N-terminus to C-terminus: MM-LP1-CM1-CM2-LP2-AM, MM-LP1-CM2-CM1-LP2-AM, AM-LP2-CM1-CM2-LP1-MM, or AM-LP2-CM2-CM1-LP1-MM, MM-LP1-CM2-CM1-CM3-LP2-AM, MM-LP1-CM1- CM2-CM3-LP2-AM, MM-LP1-CM1-CM3-CM2-LP2-AM, MM-LP1-CM3-CM1-CM2-LP2-AM, MM-LP1-CM3-CM2-CM1-LP2-AM, MM-LP2-CM2-CM1-CM3-LP1-AM, MM-LP2-CM1-CM2-CM3-LP1-AM, MM-LP2-CM1-CM2-CM3-LP1-AM, MM-LP2-CM1-CM3-CM2-LP1-AM, MM-LP2-CM3-CM1-CM2-LP1-AM, or MM-LP2-CM3-CM2-CM1-LP1-AM. In some embodiments, LP1 and LP2 are not identical to each other. In some embodiments, LP1 and LP2 are identical to each other. In some embodiments, each of LP1 and LP2 is a peptide 1 to 20 amino acids in length.

[0021] In some embodiments, the isolated polypeptide comprises an additional connecting peptide (LP3), and the isolated polypeptide has the following structural arrangement from N-terminus to C-terminus: MM-LP-CM1-LP3-CM2-AM, MM-CM1-LP3-CM2-LP-AM, MM-LP-CM2-LP3-CM1-AM, MM-CM2-LP3-CM1-LP-AM, MM-LP-CM2-LP3-CM1-CM3-AM, MM-LP-CM1-LP3-CM2-CM3-AM, MM-LP -CM3-LP3-CM1-CM2-AM, MM-LP-CM3-LP3-CM2-CM1-AM, MM-CM2-LP3-CM1-CM3-LP-AM, MM-CM1-LP3-CM2-CM3-LP-AM, MM-CM1-LP3-CM3-CM2-LP-AM, MM- CM3-LP3-CM1-CM2-LP-AM, MM-CM3-LP3-CM2-CM1-LP-AM, MM-LP-CM2-CM1-LP3-CM3-AM, MM-LP-CM1-CM2-LP3-CM3-AM, MM-LP-CM1-CM3-LP3-CM2-AM, M M-LP-CM3-CM1-LP3-CM2-AM, MM-LP-CM3-CM2-LP3-CM1-AM, MM-CM2-CM1-LP3-CM3-LP-AM, MM-CM1-CM2-LP3-CM3-LP-AM, MM-CM1-CM3-LP3-CM2-LP-A M, MM-CM3-CM1-LP3-CM2-LP-AM, MM-CM3-CM2-LP3-CM1-LP-AM, MM-LP1-CM1-LP3-CM2-LP2-AM, MM-LP1-CM2-LP3-CM1-LP2-AM, AM-LP1-CM1-LP3-CM2- LP2-MM, or AM-LP1-CM2-LP3-CM1-LP2-MM, MM-LP1-CM2-LP3-CM1-CM3-LP2-AM, MM-LP1-CM1-LP3-CM2-CM3-LP2-AM, MM-LP1-CM1-LP3-CM3-CM2-LP2- AM, MM-LP1-CM3-LP3-CM1-CM2-LP2-AM, MM-LP1-CM3-LP3-CM2-CM1-LP2-AM, MM-LP2-CM2-LP3-CM1-CM3-LP1-AM, MM-LP2-CM1-LP3-CM2-CM3-LP1-AM,MM-LP2-CM1-LP3-CM3-CM2-LP1-AM, MM-LP2-CM3-LP3-CM1-CM2-LP1-AM, or MM-LP2-CM3-LP3-CM2-CM1-LP1-AM. In some embodiments, LP1, LP2, and LP3 are not identical to one another. In some embodiments, LP1, LP2, and LP3 are identical to one another. In some embodiments, each of LP1, LP2, and LP3 is a peptide 1 to 20 amino acids in length.

[0022] In some embodiments, the isolated polypeptide comprises an additional connecting peptide (LP4), and the isolated polypeptide has the following structural arrangement from N-terminus to C-terminus: MM-LP-CM2-LP3-CM1-LP4-CM3-AM, MM-LP-CM1-LP3-CM2-LP4-CM3-AM, MM-LP-CM1-LP3-CM3-LP4-CM2-AM, MM-LP-CM3-LP3-CM1-LP4-CM2-AM, MM-LP-CM3-LP3-CM1-LP4-CM2-AM, MM-LP-CM3-LP3-CM2-LP4-CM1-AM, MM-CM2-LP3-CM1- LP4-CM3-LP-AM, MM-CM1-LP3-CM2-LP4-CM3-LP-AM, MM-CM1-LP3-CM3-LP4-CM2-LP-AM, MM-CM3-LP3-CM1-LP4-CM2-LP-AM, MM-CM3-LP3-CM2-LP4-C M1-LP-AM, MM-LP-CM2-LP4-CM1-LP3-CM3-AM, MM-LP-CM1-LP4-CM2-LP3-CM3-AM, MM-LP-CM1-LP4-CM3-LP3-CM2-AM, MM-LP-CM3-LP4-CM1-LP3-CM2 -AM, MM-LP-CM3-LP4-CM2-LP3-CM1-AM, MM-CM2-LP4-CM1-LP3-CM3-LP-AM, MM-CM1-LP4-CM2-LP3-CM3-LP-AM, MM-CM1-LP4-CM3-LP3-CM2-LP-AM, M M-CM3-LP4-CM1-LP3-CM2-LP-AM, MM-CM3-LP4-CM2-LP3-CM1-LP-AM, MM-LP1-CM2-LP3-CM1-LP4-CM3-LP2-AM, MM-LP1-CM1-LP3-CM2-LP4-CM3-LP2 -AM, MM-LP1-CM1-LP3-CM3-LP4-CM2-LP2-AM, MM-LP1-CM3-LP3-CM1-LP4-CM2-LP2-AM, MM-LP1-CM3-LP3-CM2-LP4-CM1-LP2-AM, MM-LP2-CM2-LP3- CM1-LP4-CM3-LP1-AM, MM-LP2-CM1-LP3-CM2-LP4-CM3-LP1-AM, MM-LP2-CM1-LP3-CM3-LP4-CM2-LP1-AM, MM-LP2-CM3-LP3-CM1-LP4-CM2-LP1-AM,or MM-LP2-CM3-LP3-CM2-LP4-CM1-LP1-AM. In some embodiments, LP1, LP2, LP3, and LP4 are not identical to one another. In some embodiments, LP1, LP2, LP3, and LP4 are identical to one another. In some embodiments, each of LP1, LP2, LP3, and LP4 is a peptide 1 to 20 amino acids in length.

[0023] Generally, in each embodiment herein, unless otherwise specified, the polypeptide may include one or more optional linkers between each of the recited elements, and such linkers may be 1 to 30, 6 to 29, 7 to 28, 8 to 27, 9 to 26, 10 to 25, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 amino acids in length.

[0024] In some embodiments, the first protease is a serine protease. In some embodiments, the serine protease is membrane-type serine protease 1 (MT-SP1). In some embodiments, the k of the substrate by MT-SP1 cleavage is cat / K M is at least 1 x 10 3 M -1 s -1 In some embodiments, the k of the substrate by MT-SP1 cleavage is cat / K M is at least 1 x 10 4 M -1 s -1 In some embodiments, the second protease is a matrix metalloproteinase (MMP). In some embodiments, the MMP is MMP2, MMP9, or MMP14. In some embodiments, the kappa cleavage of the substrate by MMP14 cleavage is cat / K M is at least 1 x 10 3 M -1 s -1 In some embodiments, the k of the substrate by MMP14 cleavage is cat / K Mis at least 1 x 10 4 M -1 s -1 In some embodiments, the k of the substrate by MMP9 cleavage is cat / K M is at least 1 x 10 3 M -1 s -1 In some embodiments, the k of the substrate by MMP9 cleavage is cat / K M is at least 1 x 10 4 M -1 s -1 In some embodiments, the k of the substrate by MMP2 cleavage is cat / K M is at least 1 x 10 3 M -1 s -1 In some embodiments, the k of the substrate by MMP2 cleavage is cat / K M is at least 1 x 10 4 M -1 s -1 is.

[0025] In some embodiments, the isolated polypeptide is resistant to in situ cleavage in human bone marrow. In some embodiments, the isolated polypeptide is resistant to in vivo cleavage in human bone marrow.

[0026] In another aspect, the present disclosure provides an isolated polypeptide comprising a cleavable moiety (CM) comprising an amino acid sequence having one or two amino acid mutation(s) in any one of SEQ ID NOs: 10-73, 83-216, 460-634, and 636-650, wherein the CM is a substrate for a protease. In another aspect, the present disclosure provides an isolated polypeptide comprising a cleavable moiety (CM) comprising an amino acid sequence having one or two amino acid mutation(s) in any one of SEQ ID NOs: 10-73, 83-216, and 460-634, wherein the CM is a substrate for a protease. For example, the mutation can comprise a substitution between any one of lysine, arginine, and histidine residues. In certain aspects, the present disclosure can include substitution of any arginine in the disclosed sequences with lysine. In other aspects, the present disclosure also includes substitution of any arginine in the disclosed sequences with an amino acid other than lysine. In certain aspects, the present disclosure may include substitutions of any alanine in the disclosed sequences with valine. For example, the mutation may include a substitution between any one of alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan residues. For example, the mutation may include a substitution between any one of glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine residues. For example, the mutation may include a substitution between any one of arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine, and threonine residues. For example, the mutation may include a substitution between any one of alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine residues. For example, the mutation may include a substitution between any one of serine and threonine residues. For example, the mutation may comprise a substitution between any one of asparagine and glutamine residues. For example, the mutation may comprise a substitution between any one of alanine, valine, leucine, and isoleucine residues. For example, the mutation may comprise a substitution between any one of phenylalanine, tryptophan, and tyrosine residues.

[0027] In another aspect, the present disclosure provides a polypeptide complex comprising one or more of the isolated polypeptides herein bound to a second isolated polypeptide. In some aspects, the complex comprises one or more of the isolated polypeptides of the present disclosure bound to a second isolated polypeptide, e.g., via protein-protein affinity interactions, hydrophobic interactions, disulfide bond(s), cross-link(s), covalent bond(s), chemical bond(s), or other types of bonds between the two polypeptides.

[0028] In another aspect, the disclosure provides a conjugated polypeptide comprising an isolated polypeptide or AM herein conjugated to an agent. In some embodiments, the agent is conjugated to the isolated polypeptide or AM via a conjugated linker. In some embodiments, the conjugated linker is cleavable. In some embodiments, the conjugated linker is non-cleavable. In some embodiments, the conjugated linker comprises an amino acid sequence selected from SEQ ID NOs: 10-73, 83-216, 460-634, and 636-650, and optionally comprises an amino acid sequence selected from SEQ ID NOs: 10-73, 83-216, and 460-634. In some embodiments, the agent is a toxin, a microtubule inhibitor, a nucleic acid damaging agent, a dolastatin, an auristatin, a maytansinoid, a duocarmycin, a calicheamicin, or a combination thereof.

[0029] In another aspect, the present disclosure provides a composition comprising an isolated polypeptide, polypeptide complex, or conjugated polypeptide herein and a carrier. In some embodiments, the carrier is a pharmaceutically acceptable carrier. In some embodiments, the composition comprises an additional agent. In some embodiments, the additional agent is a therapeutic agent, an imaging agent, or a diagnostic agent.

[0030] In each of the foregoing embodiments, and unless otherwise specified, the polypeptide may include, for example, one or more optional linkers between each of the recited elements. In some embodiments, the linker is a peptide having a length of 5-30, 6-29, 7-28, 8-27, 9-26, 10-25, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 amino acids. In the structural configurations disclosed in the preceding paragraph and throughout this disclosure, one or more linkers may optionally be present between elements. Furthermore, the present disclosure also contemplates and includes activatable proteins in which any one or more of the disclosed elements optionally directly abut one another, such that there is no linker or other amino acid sequence between the elements.

[0031] In another aspect, the disclosure provides an isolated nucleic acid molecule encoding an isolated polypeptide herein.

[0032] In another aspect, the present disclosure provides a vector comprising the isolated nucleic acid molecule herein.

[0033] In another aspect, the present disclosure provides a cell comprising the isolated nucleic acid molecule or vector herein.

[0034] In another aspect, the disclosure provides a method for producing an activatable molecule comprising a tandem substrate, the method comprising expressing and recovering a polypeptide, including an isolated polypeptide herein.

[0035] In another aspect, the present disclosure provides a method of treating, alleviating the symptoms of, or delaying the progression of a disease or disorder in a subject, comprising administering to the subject a therapeutically effective amount of an isolated polypeptide, polypeptide complex, conjugated polypeptide, or composition herein. In some embodiments, the disease is cancer, an infectious disease, an inflammatory disorder, a cardiovascular disorder, a neurodegenerative disorder, or an autoimmune disorder.

[0036] In another aspect, the present disclosure provides a method for detecting or diagnosing a disease or condition in a subject, the method comprising contacting an isolated polypeptide, polypeptide complex, conjugated polypeptide, or composition herein with a sample from the subject and measuring the level of cleavage of the isolated polypeptide, thereby detecting or diagnosing the disease or condition in the subject. In some embodiments, the disease is cancer, an infectious disease, an inflammatory disorder, a cardiovascular disorder, a neurodegenerative disorder, or an autoimmune disorder.

[0037] 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 may be utilized, and the accompanying drawings of which: [Brief explanation of the drawings]

[0038] [Figure 1A] 1 shows the in vitro masking efficiency of exemplary anti-EGFR activatable antibodies of the present disclosure. These exemplary results demonstrate that substrates affect the masking efficiency of the prodomain of activatable antibodies. [Figure 1B] 1 shows the in vitro masking efficiency of exemplary anti-EGFR activatable antibodies of the present disclosure. These exemplary results demonstrate that substrates affect the masking efficiency of the prodomain of activatable antibodies. [Figure 1C] 1 shows the in vitro masking efficiency of exemplary anti-EGFR activatable antibodies of the present disclosure. These exemplary results demonstrate that substrates affect the masking efficiency of the prodomain of activatable antibodies. [Figure 2A] 1 shows the effect of activatable antibodies with exemplary substrates on H292 xenograft tumors. [Figure 2B] 1 shows the effect of activatable antibodies with exemplary substrates on SUM149 xenograft tumors. [Figure 2C] 1 shows the effect of activatable antibodies with exemplary substrates on H292 xenograft tumors. [Figure 2D]1 shows the effect of activatable antibodies with exemplary substrates on H292 xenograft tumors. [Figure 2E] 1 shows the effect of activatable antibodies with exemplary substrates on H292 xenograft tumors. [Figure 2F] 1 shows the results of an intratumor activation assay using an activatable antibody with an exemplary substrate. [Figure 2G] 1 shows the results of an intratumor activation assay using an activatable antibody with an exemplary substrate. [Figure 2H] 1 shows the results of an intratumor activation assay using an activatable antibody with an exemplary substrate. [Figure 2I] 1 shows the results of an intratumor activation assay using an activatable antibody with an exemplary substrate. [Figure 3] 1 shows the in situ stability in human bone marrow aspirate of an activatable antibody with an exemplary substrate. [Figure 4A] 1 shows the activation of an activatable antibody with an exemplary substrate in colon cancer tumor tissue. [Figure 4B] 1 shows activation of an activatable antibody with an exemplary substrate in head and neck cancer tumor tissue. [Figure 4C] 1 shows activation of an activatable antibody with an exemplary substrate in a triple-negative breast cancer tumor panel. [Figure 5A] 1 shows activation of an activatable antibody with an exemplary substrate in multiple myeloma cells. [Figure 5B] 1 shows activation of activatable antibodies with exemplary substrates in acute myeloid leukemia cells. DETAILED DESCRIPTION OF THE INVENTION

[0039] Detailed Description of the Invention Proteases play a critical role in healthy tissue homeostasis but are known to be dysregulated in diseases such as cancer and autoimmune disorders (Vasiljeva et al. “The multifaceted roles of tumor-associated proteases and harnessing their activity for prodrug activation,” Biol. Chem. 2019 Apr 22). This dysregulation of protease activity offers new opportunities for the development of protease-activatable therapeutic molecules that are preferentially activated in the local tissue microenvironment. These therapeutics demonstrate greater therapeutic scope and safety profiles with less on-target toxicity than occurs in healthy tissue. Therefore, there is a need to identify substrates that function as cleavage recognition sites for proteases found dysregulated in diseased tissues. These substrates, or cleavable moieties (CMs), may possess multiple cleavage sites to exploit the activity of multiple disease-associated proteases.

[0040] Understanding substrate cleavage profiles and using these substrates as activation tools for specific diseases or cancer types will enable the development of novel therapeutic protease-activatable molecules. Fine-tuning therapeutically activatable molecules using protease substrates with unique cleavage profiles will enable treatment options for a wider range of patients while improving their therapeutic index. For example, "omics" studies have demonstrated the distribution of numerous matrix metalloproteases (MMPs) across numerous cancer types and differential expression of MMPs compared to normal tissues (Gobin et al. "A pan-cancer perspective of matrix metalloproteases (MMP) gene expression profile and their diagnostic / prognostic potential," BMC Cancer. 2019 Jun 14;19(1):581), highlighting the need for selecting appropriate cleavable moieties. In fact, the first protease-activatable antibodies were designed using MMP substrates (Bleuez et al., "Exploiting protease activation for therapy," Drug Discovery Today, 2022 Jun;27(6):1743-1754). Furthermore, membrane-type serine protease 1 (MT-SP1) shows great potential for the development of protease-activatable antibodies (Howng, B. et al. “Novel Ex Vivo Zymography Approach for Assessment of Protease Activity in Tissues with Activatable Antibodies,” Pharmaceuticals 2021, 13(9), 1390).

[0041] The present disclosure provides polypeptides comprising substrates that include multiple cleavage moieties (CMs), each cleavable by a protease. For example, a substrate can include a first CM (CM1) that is cleavable by an MMP and a second CM (CM2) that is cleavable by a serine protease. In some aspects, the substrates herein are cleaved in diseased tissue (e.g., tumor tissue) but not in healthy tissue. These substrates are useful for a variety of therapeutic, diagnostic, and preventative applications. In some embodiments, the substrate-containing polypeptide is an activatable molecule and further includes an active moiety (AM) that specifically binds to a target. For example, the AM can be a therapeutic protein, a therapeutic agent, an imaging agent, a diagnostic agent, an antibody or antigen-binding fragment, a cytokine, a chimeric antigen receptor, or other molecule used in therapeutic and diagnostic applications.

[0042] Also provided herein are related compositions, kits, nucleic acids, vectors, and recombinant cells, as well as related methods, including methods of using and producing any of the substrate-containing polypeptides described herein.

[0043] definition 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. Methods and materials for use in the present disclosure are described herein; other suitable methods and materials readily known in the art may also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0044] The terms "a" and "an" refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "a cell" includes one or more cells.

[0045] As used herein, the terms "about" and "approximately," when used to modify a quantity specified by a numerical value or range, refer not only to the numerical value but also to reasonable deviations from that value known to those of ordinary skill in the art. For example, where appropriate, ±20%, ±10%, or ±5% is within the intended meaning of the recited value.

[0046] Concentrations, amounts, and other numerical data may be expressed or presented herein in a range format. It should be understood that such range format is used merely for convenience and brevity and should therefore be interpreted flexibly to include not only the numerical values explicitly recited as range limits, but also all individual numerical values or subranges subsumed within that range, as if each numerical value and subrange were explicitly recited. As an illustration, a numerical range of "about 0.01 to 2.0" should be interpreted to include not only the explicitly recited value of about 0.01 to about 2.0, but also each individual value and subrange within the stated range. Thus, within this numerical range, individual values, such as 0.5, 0.7, and 1.5, as well as subranges, such as 0.5 to 1.7, 0.7 to 1.5, and 1.0 to 1.5, are included. Furthermore, such interpretation should be applied regardless of the breadth or characteristics of the range described. Additionally, it should be noted that all percentages are by weight unless otherwise specified.

[0047] In understanding the scope of the present disclosure, the terms "including" or "comprising," and their derivatives, as used herein, are intended to be open-ended terms specifying the presence of stated features, elements, components, groups, components, and / or steps, but not excluding the presence of other, unstated features, elements, components, groups, components, and / or steps. The above also applies to words of similar meaning, such as "including" and "having," and their derivatives. As used herein, the term "consisting" and its derivatives are intended to be limiting terms specifying the presence of stated features, elements, components, groups, components, and / or steps, but excluding the presence of other, unstated features, elements, components, groups, components, and / or steps. The term "consisting essentially of," as used herein, is intended to specify the presence of recited features, elements, components, groups, ingredients, and / or steps, as well as the presence of features, elements, components, groups, ingredients, and / or steps that do not materially affect the basic and novel characteristic(s) of the features, elements, components, groups, ingredients, and / or steps. Reference to any one of these transitional phrases (i.e., "comprising," "consisting," or "consisting essentially") is understood to provide direct support for the substitution of any other transitional phrase not specifically used. For example, the modification of the term "comprising" to "consisting essentially of" or "consisting of" finds direct support for any element disclosed throughout this disclosure to be so defined. Based on this definition, any element disclosed herein or incorporated by reference may be included in or excluded from the claimed invention.

[0048] As used herein, for convenience, multiple compounds, elements, or steps may be presented in general lists. However, these lists should be construed as though each member of the list were individually identified as a separate and unique member. Accordingly, the individual members of such lists should not be construed as being equally present in de facto terms with any other member of the same list solely based on their presentation in a general grouping, unless otherwise indicated.

[0049] The word "exemplary" is used herein to mean serving as an example, instance, or illustration. Any aspect or design described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other aspects or designs. Rather, use of the word exemplary is intended to present concepts in a more specific manner.

[0050] Additionally, particular molecules, constructs, compositions, elements, moieties, excipients, diseases, conditions, properties, steps, etc. may be discussed in the context of a particular embodiment or aspect of the disclosure or in a separate paragraph or section. This is merely for convenience and brevity, and it is understood that any such disclosure is equally applicable and intended to be combined with any other embodiment or aspect found anywhere in this disclosure and claims, all of which form the application and claimed invention as of the filing date. For example, a listing of a construct, molecule, isolated polypeptide, activatable molecule, composition, or method described with respect to a construct, molecule, method step, kit, or composition is intended to find direct support for the construct, molecule, isolated polypeptide, activatable molecule, composition, formulation, and method-related embodiment described anywhere else in this disclosure, even if those method steps, active agents, kits, or compositions are not re-enumerated in the context or section of that embodiment or aspect.

[0051] As used herein, the term "isolated polynucleotide" refers to a polynucleotide of genomic, cDNA, RNA, mRNA, or synthetic origin, or some combination thereof, and by virtue of its origin, an "isolated polynucleotide" is one that (1) is not associated with all or a portion of a polynucleotide with which it is found in nature; (2) is operably linked to a polynucleotide with which it is not naturally linked; and / or (3) is not naturally occurring as part of a larger sequence. In some embodiments, the polynucleotide comprises a nucleic acid molecule encoding a heavy chain immunoglobulin molecule and a nucleic acid molecule encoding a light chain immunoglobulin molecule. The term isolated polypeptide includes and provides support for activatable molecules, including activatable macromolecules, activatable polypeptides, activatable antibodies, activatable cytokines, and the like. The term isolated polypeptide includes and provides support for activatable molecules, which molecules are activated by cleavage of a CM.

[0052] As used herein, the term "isolated polypeptide" refers to a polypeptide that exists in a form other than that found in nature. As used herein, an "isolated polypeptide" can be encoded by a polynucleotide of cDNA, recombinant RNA, recombinant DNA, messenger RNA, or synthetic origin, or any combination thereof. By virtue of its origin or derivation, an "isolated polypeptide" (1) is not present in a naturally occurring organism (e.g., is not an endogenous polypeptide of a naturally occurring organism), and (2) exists in a form that does not occur in nature. In some embodiments, an "isolated polypeptide" is expressed by cells from a different species. In some embodiments, an "isolated polypeptide" is a therapeutic or diagnostic protein and is not a naturally occurring protein. For example, as used herein, an "isolated polypeptide" is not a plant protein or a protein naturally occurring in a bacterium or other natural organism.

[0053] The term "polypeptide" is used herein as a generic term to refer to naturally occurring proteins, fragments, or analogs of a polypeptide sequence. Thus, proteins, protein fragments, and analogs are species of the polypeptide genus. In some embodiments, polypeptides according to the present disclosure include heavy chain immunoglobulin molecules, light chain immunoglobulin molecules, and antibody molecules formed by combinations including heavy chain immunoglobulin molecules with light chain immunoglobulin molecules, such as kappa light chain immunoglobulin molecules, and vice versa, and fragments and analogs thereof.

[0054] As discussed herein, minor variations in the amino acid sequence of a polypeptide are contemplated as encompassed by the present disclosure, provided that the amino acid sequence variation maintains at least 75%, in some embodiments at least 80%, at least 90%, at least 95%, and in some embodiments at least 99% similarity to the unchanged amino acid sequence. Conservative amino acid substitutions are specifically contemplated. Conservative substitutions include those that occur within a family of amino acids that are related in their side chains. Genetically encoded amino acids are generally divided into the following families: (1) acidic amino acids are aspartic acid and glutamic acid; (2) basic amino acids are lysine, arginine, and histidine; (3) nonpolar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; and (4) uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine. Hydrophilic amino acids include arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine, and threonine. Hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine. Other amino acid families include: (i) the aliphatic hydroxy family, serine and threonine; (ii) the amide-containing family, asparagine and glutamine; (iii) the aliphatic family, alanine, valine, leucine, and isoleucine; and (iv) the aromatic family, phenylalanine, tryptophan, and tyrosine. For example, it is reasonable to expect that isolated substitutions of leucine with isoleucine or valine, aspartic acid with glutamic acid, and threonine with serine, or similar substitutions of amino acids with structurally related amino acids, especially when the substitutions do not involve amino acids in framework regions, will not have a major impact on binding or the properties of the resulting molecule.Whether an amino acid change results in a functional peptide can be readily determined by assaying the specific activity of the polypeptide derivative. Assays are described in detail herein. Fragments or analogs of antibody or immunoglobulin molecules can be readily prepared by one of ordinary skill in the art. Suitable amino and carboxyl termini of fragments or analogs reside near the boundaries of functional domains. Structural and functional domains can be identified by comparing nucleotide and / or amino acid sequence data with suitable or proprietary sequence databases. In some embodiments, computerized comparison methods are used to identify sequence motifs or predicted protein conformational domains that occur in other proteins of known structure and / or function. Methods for identifying protein sequences that fold into known three-dimensional structures are known, for example, as described in Bowie et al. Science 253:164 (1991). Thus, the foregoing examples demonstrate that one of ordinary skill in the art can recognize sequence motifs and structural conformations that can be used to define structural and functional domains in accordance with the present disclosure.

[0055] Suitable amino acid substitutions include (1) those that alter susceptibility to proteolysis, (2) those that reduce susceptibility to oxidation, (3) those that alter binding affinity for forming protein complexes, (4) those that alter binding affinity, and (5) those that confer or modify other physicochemical or functional properties of such analogs. Analogs can include various mutant proteins of a sequence other than the naturally occurring peptide sequence. For example, single or multiple amino acid substitutions (e.g., conservative amino acid substitutions) can be made in the naturally occurring sequence (e.g., in portions of the polypeptide outside the domain(s) that form intramolecular contacts). Conservative amino acid substitutions will not substantially alter the structural characteristics of the parent sequence (e.g., the substituted amino acids will not tend to break helices occurring in the parent sequence or disrupt other types of secondary structure that characterize the parent sequence). Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W.H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, NY (1991)); and Thornton et al. Nature 354:105 (1991).

[0056] The term "sample" is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. Thus, use of the term "sample" includes blood and portions or components of blood, including serum, plasma, or lymph.

[0057] The term "therapeutic macromolecule" refers to any protein or nucleic acid that is administered to a subject and has a therapeutic effect. In some embodiments, a therapeutic macromolecule can be a therapeutic polynucleotide or therapeutic polypeptide, i.e., a polynucleotide or polypeptides that can be used therapeutically.

[0058] As generally provided herein, an activatable molecule can include MM substrate construct(s), also referred to herein as a prodomain. Thus, as used herein, the term "prodomain" refers to a polypeptide domain comprising a masking moiety (MM) and a cleavable substrate. The substrate can comprise one, two, three, or more CMs. In some embodiments, the MM and substrate are separated by a linker, referred to herein as LP1. In some embodiments, the prodomain comprises a linker (referred to herein as LP2) that connects the substrate of the prodomain to the active portion (AM) of the activatable molecule. In some embodiments, the prodomain comprises a linker between the MM and substrate and a linker between the substrate and AM. In some embodiments, the MM and substrate are not separated by a linker. In certain embodiments, the prodomain comprises one of the following formulas (wherein the following formulas represent the amino acid sequences in either the N-terminal to C-terminal direction or the C-terminal to N-terminal direction): MM-LP1-substrate, MM-substrate-LP2, MM-LP1-substrate-LP2, or MM-substrate. As used herein, and unless otherwise specified, each dash (-) between components of an activatable molecule represents either a direct linkage or an indirect linkage via one or more linking peptides.

[0059] Proteases are involved in the control of many physiological processes, and their dysregulation has been identified in many pathological conditions, such as oncological, cardiovascular, autoimmune, and neurodegenerative diseases. For example, O. Vasiljeva, et al., “Monitoring protease activity in biological tissues using antibody prodrugs as sensing probes,” Scientific Reports, 10, 5894 (2020); O. Erster, et al., “Site-specific targeting of antibody activity in vivo mediated by disease-associated proteases,” J. Control Release, 161(3):804-812(2012); L. Desnoyers, et al., “Tumor-specific activation of an EGFR-targeting probody enhances therapeutic index,” Science Translational Medicine, 5(207): 207ra144(2013); and B. Turk “Targeting proteases: successes, failures and future prospects” Nature Reviews Drug See Discovery, 5 (2006). It has been documented that protease-activating antibodies are activated by natural proteases that are more widely active in, for example, tumor tissues compared to normal tissues. See Id. These prodrugs incorporate protease substrates into their structure that release the active drug after exposure to the appropriate protease and subsequent cleavage. However, it is clear that the profile of dysregulated protease activity in diseased tissues may differ depending on the type of diseased tissue / disorder. Therefore, it is desirable to have a collection of substrates that target a variety of different protease activity profiles.

[0060] In some aspects, the present disclosure provides a substrate comprising a first cleavable moiety (CM1) cleavable by a first protease and a second cleavable moiety (CM2) cleavable by a second protease. In some embodiments, the first protease is an MMP (e.g., MMP2, MMP9, or MMP14). In some embodiments, the first protease is a serine protease (e.g., membrane-type serine protease 1 (MT-SP1)). In some embodiments, the first protease is a serine protease (e.g., MT-SP1) and the second protease is an MMP (e.g., MMP2, MMP9, or MMP14). In some embodiments, CM1 is cleavable by a first set of proteases and CM2 is cleavable by a second set of proteases. In some instances, the first and second sets of proteases may overlap, i.e., one or more proteases in the first set are also present in the second set. In some alternative instances, the first and second sets of proteases do not overlap, i.e., the first set does not include a protease in the second set.

[0061] In certain embodiments, a substrate is selectively cleavable by a particular protease (e.g., MT-SP1 and / or MMP) while exhibiting reduced or no cleavability by other proteases. For example, a substrate may be resistant or substantially resistant to cleavage in bone marrow tissue, e.g., bone marrow aspirate. In some embodiments, the resistance of a substrate to protease cleavage in healthy tissue may reduce systemic toxicity by limiting binding of the activatable molecule to targets that may also be present in healthy tissue. Thus, substrates with bone marrow tissue resistance may exhibit less on-target toxicity in healthy tissue, demonstrating a greater therapeutic range and safety profile.

[0062] In certain aspects, the present disclosure provides polypeptides (e.g., isolated polypeptides) that include a substrate that includes multiple cleavable moieties (CMs). A CM is a polypeptide that includes a substrate for a sequence-specific protease. In some aspects, the present disclosure provides polypeptides and polypeptide complexes that include a substrate and an active moiety.

[0063] In some embodiments, the CM1 in the substrate comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the CM1 in the substrate comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the CM1 in the substrate comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the CM1 in the substrate comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the CM1 in the substrate comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, the CM1 in the substrate comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the CM1 in the substrate comprises the amino acid sequence of SEQ ID NO: 77.

[0064] In some embodiments, CM1 in the substrate consists of the amino acid sequence of SEQ ID NO: 1. In some embodiments, CM1 in the substrate consists of the amino acid sequence of SEQ ID NO: 2. In some embodiments, CM1 in the substrate consists of the amino acid sequence of SEQ ID NO: 3. In some embodiments, CM1 in the substrate consists of the amino acid sequence of SEQ ID NO: 4. In some embodiments, CM1 in the substrate consists of the amino acid sequence of SEQ ID NO: 5. In some embodiments, CM1 in the substrate consists of the amino acid sequence of SEQ ID NO: 6. In some embodiments, CM1 in the substrate consists of the amino acid sequence of SEQ ID NO: 77.

[0065] In some embodiments, CM2 in the substrate comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, CM2 in the substrate comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, CM2 in the substrate comprises the amino acid sequence of SEQ ID NO: 9. In some embodiments, CM2 in the substrate comprises the amino acid sequence of SEQ ID NO: 74. In some embodiments, CM2 in the substrate comprises the amino acid sequence of SEQ ID NO: 75. In some embodiments, CM2 in the substrate comprises the amino acid sequence of SEQ ID NO: 76. In some embodiments, CM2 in the substrate comprises the amino acid sequence of SEQ ID NO: 237. In some embodiments, CM2 in the substrate comprises the amino acid sequence of SEQ ID NO: 238. In some embodiments, CM2 in the substrate comprises the amino acid sequence of SEQ ID NO: 635. The substrate may consist of an amino acid sequence selected from SEQ ID NOs: 10-73, 83-216, 460-634, and 636-650.

[0066] In some embodiments, CM2 in the substrate consists of the amino acid sequence of SEQ ID NO: 7. In some embodiments, CM2 in the substrate consists of the amino acid sequence of SEQ ID NO: 8. In some embodiments, CM2 in the substrate consists of the amino acid sequence of SEQ ID NO: 9. In some embodiments, CM2 in the substrate consists of the amino acid sequence of SEQ ID NO: 74. In some embodiments, CM2 in the substrate consists of the amino acid sequence of SEQ ID NO: 75. In some embodiments, CM2 in the substrate consists of the amino acid sequence of SEQ ID NO: 76. In some embodiments, CM2 in the substrate consists of the amino acid sequence of SEQ ID NO: 237. In some embodiments, CM2 in the substrate consists of the amino acid sequence of SEQ ID NO: 238. In some embodiments, CM2 in the substrate consists of the amino acid sequence of SEQ ID NO: 635. The substrate may comprise an amino acid sequence selected from SEQ ID NOs: 10-73, 83-216, 460-634, and 636-650. The substrate may comprise an amino acid sequence selected from SEQ ID NOs: 10-73, 83-216, 460-634, and 636-650. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 11. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 12. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 13. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 15. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 17. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 19. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 20. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 21. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 22. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 23. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 24. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 25.In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 26. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 27. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 28. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 29. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 30. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 31. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 32. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 33. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 34. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 35. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 36. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 37. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 38. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 39. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 40. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 41. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 42. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 43. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 44. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 45. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 46. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 47. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 48. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 49. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 50. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 51. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 52. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 53. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 54. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 55.In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 56. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 57. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 58. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 59. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 60. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 61. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 62. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 63. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 64. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 65. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 66. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 67. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 68. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 69. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 70. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 71. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 72. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 73. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 83. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 84. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 85. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 86. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 87. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 88. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 89. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 90. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 91. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 92. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 93. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 94.In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 95. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 96. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 97. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 98. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 99. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 100. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 101. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 102. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 103. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 104. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 105. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 106. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 107. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 108. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 109. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 110. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 111. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 112. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 113. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 114. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 115. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 116. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 117. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 118. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 119. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 120. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 121. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 122. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 123.In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 124. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 125. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 126. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 127. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 128. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 129. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 130. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 131. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 132. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 133. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 134. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 135. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 136. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 137. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 138. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 139. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 140. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 141. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 142. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 143. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 144. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 145. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 146. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 147. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 148. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 149. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 150. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 151. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 152.In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 153. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 154. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 155. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 156. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 157. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 158. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 158. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 159. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 160. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 161. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 162. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 163. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 164. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 165. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 166. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 167. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 168. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 169. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 170. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 171. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 172. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 173. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 174. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 175. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 176. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 177. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 178. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 179. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 180. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 181. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 182. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 183. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 184. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 185. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 186.In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 187. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 188. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 189. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 190. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 191. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 192. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 193. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 194. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 195. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 196. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 197. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 198. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 199. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 200. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 201. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 202. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 203. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 204. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 205. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 206. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 207. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 208. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 209. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 210. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 211. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 212. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 213. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 214. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 215.In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 216. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 460. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 461. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 462. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 463. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 464. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 465. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 466. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 467. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 468. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 469. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 470. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 471. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 472. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 473. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 474. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 475. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 476. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 477. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 478. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 479. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 480. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 481. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 482. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 483. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 484. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 485. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 486. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 487.In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 488. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 489. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 490. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 491. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 492. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 493. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 494. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 495. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 496. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 497. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 498. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 499. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 500. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 501. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 502. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 503. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 504. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 505. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 506. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 507. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 508. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 509. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 510. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 511. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 512. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 513. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 514. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 515. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 516.In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 517. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 518. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 519. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 520. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 521. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 522. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 523. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 524. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 525. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 526. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 527. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 528. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 529. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 530. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 531. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 532. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 533. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 534. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 535. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 536. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 537. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 538. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 539. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 540. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 541. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 542. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 543. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 544. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 545.In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 546. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 547. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 548. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 549. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 550. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 551. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 552. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 553. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 554. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 555. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 556. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 557. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 558. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 559. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 560. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 561. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 562. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 563. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 564. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 565. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 566. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 567. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 568. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 569. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 570. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 571. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 572. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 573. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 574. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 575. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 576. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 577. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 578. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 579. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 580.In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 581. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 582. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 583. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 584. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 585. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 586. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 587. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 588. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 589. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 590. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 591. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 592. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 593. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 594. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 595. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 596. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 597. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 598. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 599. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 600. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 601. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 602. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 603. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 604. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 605. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 606. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 607. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 608. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 609.In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 610. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 611. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 612. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 613. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 614. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 615. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 616. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 617. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 618. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 619. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 620. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 621. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 622. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 623. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 624. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 625. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 626. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 627. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 628. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 629. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 630. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 631. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 632. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 633. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 634. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 636. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 637. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 638. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 639.In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 640. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 641. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 642. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 643. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 644. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 645. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 646. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 647. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 648. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 649. In some embodiments, the substrate comprises the amino acid sequence of SEQ ID NO: 650.

[0067] The substrate may consist of an amino acid sequence selected from SEQ ID NOs: 10-73, 83-216, 460-634, and 636-650. The substrate may consist of an amino acid sequence selected from SEQ ID NOs: 10-73, 83-216, 460-634, and 636-650. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 10. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 11. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 12. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 13. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 14. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 15. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 16. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 17. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 18. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 19. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 20. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO:21. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO:22. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO:23. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO:24. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO:25. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO:26. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO:27. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO:28. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO:29. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO:30. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO:31. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO:32. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO:33. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO:34. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO:35.In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 36. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 37. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 38. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 39. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 40. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 41. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 42. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 43. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 44. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 45. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 46. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 47. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 48. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 49. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 50. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 51. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 52. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 53. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 54. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 55. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 56. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 57. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 58. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 59. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 60. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 61. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 62. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 63. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 64.In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 65. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 66. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 67. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 68. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 69. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 70. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 71. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 72. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 73. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 83. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 85. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 86. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 87. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 88. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 89. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 90. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO:91. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO:92. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO:93. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO:94. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO:95. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO:96. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO:97. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO:98. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO:99. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO:100. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO:101. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO:102.In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 103. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 104. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 105. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 106. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 107. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 108. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 109. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 110. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 111. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 112. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 113. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 114. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 115. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 116. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 117. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 118. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 119. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 120. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 121. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 122. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 123. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 124. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 125. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 126. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 127. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 128. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 129. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 130.In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 131. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 132. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 133. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 134. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 135. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 136. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 137. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 138. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 139. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 140. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 141. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 142. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 143. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 144. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 145. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 146. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 147. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 148. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 149. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 150. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 151. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 152. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 153. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 154. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 155. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 156. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 157. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 158.In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 159. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 160. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 161. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 162. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 163. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 164. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 164. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 165. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 166. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 167. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 168. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 169. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 170. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 171. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 172. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 173. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 174. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 175. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 176. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 177. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 178. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 179. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 180. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 181. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 182. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 183. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 184. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 185. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 186. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 187. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 188. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 189. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 190. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 191.In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 192. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 193. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 194. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 195. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 196. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 197. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 198. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 199. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 200. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 201. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 202. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 203. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 204. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 205. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 206. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 207. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 208. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 209. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 210. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 211. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 212. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 213. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 214. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 215. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 216. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 460. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 461. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 462.In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 463. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 464. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 465. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 466. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 467. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 468. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 469. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 470. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 471. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 472. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 473. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 474. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 475. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 476. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 477. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 478. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 479. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 480. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 481. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 482. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 483. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 484. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 485. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 486. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 487. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 488. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 489. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 490.In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 491. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 492. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 493. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 494. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 495. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 496. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 497. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 498. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 499. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 500. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 501. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 502. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 503. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 504. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 505. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 506. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 507. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 508. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 509. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 510. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 511. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 512. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 513. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 514. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 515. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 516. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 517. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 518.In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 519. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 520. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 521. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 522. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 523. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 524. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 525. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 526. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 527. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 528. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 529. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 530. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 531. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 532. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 533. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 534. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 535. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 536. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 537. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 538. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 539. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 540. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 541. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 542. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 543. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 544. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 545. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 546.In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 547. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 548. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 549. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 550. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 551. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 552. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 5. 53. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 554. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 555. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 556. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 557. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 558. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 559. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 560. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 561. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 562. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 563. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 564. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 565. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 566. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 567. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 568. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 569. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 570. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 571. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 572. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 573. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 574. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 575. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 576. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 577. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 578. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 579. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 580. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 581.In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 582. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 583. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 584. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 585. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 586. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 587. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 588. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 589. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 590. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 591. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 592. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 593. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 594. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 595. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 596. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 597. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 598. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 599. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 600. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 601. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 602. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 603. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 604. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 605. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 606. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 607. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 608. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 609.In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 610. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 611. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 612. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 613. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 614. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 615. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 616. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 617. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 618. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 619. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 620. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 621. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 622. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 623. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 624. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 625. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 626. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 627. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 628. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 629. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 630. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 631. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 632. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 633. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 634. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 636. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 637. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 638.In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 639. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 640. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 641. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 642. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 643. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 644. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 645. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 646. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 647. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 648. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 649. In some embodiments, the substrate consists of the amino acid sequence of SEQ ID NO: 650.

[0068] In some embodiments, the substrate comprises a combination of the amino acid sequences of any one of SEQ ID NOS: 10-73, 83-216, 460-634, and 636-650, a C-terminal truncation mutant, a C-terminal extension mutant, an N-terminal truncation mutant, or an N-terminal extension mutant. In some embodiments, the substrate comprises a combination of the amino acid sequences of any one of SEQ ID NOS: 10-73, 83-216, and 460-634, a C-terminal truncation mutant, a C-terminal extension mutant, an N-terminal truncation mutant, or an N-terminal extension mutant. Truncation mutants of the aforementioned amino acid sequences suitable for use in substrates can be any that retain the recognition site of the corresponding protease. These include C- and / or N-terminal truncation mutants containing at least one, two, three, four, five, or more consecutive amino acids of the aforementioned amino acid sequences that retain the recognition site of the protease. In certain embodiments, the truncation mutant comprises a C-terminal and / or N-terminal deletion of one amino acid residue from an amino acid sequence selected from the group consisting of SEQ ID NOs: 10-73, 83-216, 460-634, and 636-650. In certain embodiments, the truncation mutant comprises a C-terminal and / or N-terminal deletion of one amino acid residue from an amino acid sequence selected from the group consisting of SEQ ID NOs: 10-73, 83-216, and 460-634. Extension mutants of the foregoing amino acid sequences suitable for use in substrates can be any that have one or more (e.g., 1, 2, 3, 4, 5, or more) additional amino acids and retain a recognition site for the corresponding protease. In some examples, the additional amino acid is coupled to the C-terminus of the foregoing amino acid sequence. In some examples, the additional amino acid is coupled to the N-terminus of the foregoing amino acid sequence. In some examples, the extension mutant can include additional amino acids coupled to both the C-terminus and the N-terminus of the foregoing amino acid sequence. Optionally, the C-terminal or N-terminal extension mutants can have a C-terminal glycine or N-terminal serine amino acid.

[0069] In some embodiments, the substrate comprises one, two, three, four, five, six, or more amino acids in addition to the amino acid sequence of any one of SEQ ID NOs: 10-73, 83-216, 460-634, and 636-650. In some embodiments, the substrate comprises one, two, three, four, five, six, or more amino acids in addition to the amino acid sequence of any one of SEQ ID NOs: 10-73, 83-216, and 460-634. In some examples, the substrate comprises one, two, three, four, five, six, or more additional amino acids at the N-terminus of the amino acid sequence of any one of SEQ ID NOs: 10-73, 83-216, 460-634, and 636-650. In some examples, the substrate comprises one, two, three, four, five, six, or more additional amino acids at the N-terminus of the amino acid sequence of any one of SEQ ID NOs: 10-73, 83-216, and 460-634. In some examples, the substrate comprises one, two, three, four, five, six or more additional amino acids at the C-terminus of the amino acid sequence of any one of SEQ ID NOs: 10-73, 83-216, 460-634, and 636-650. In some examples, the substrate comprises one, two, three, four, five, six or more additional amino acids at the C-terminus of the amino acid sequence of any one of SEQ ID NOs: 10-73, 83-216, and 460-634. In some examples, the substrate comprises one, two, three, four, five, six or more additional amino acids at the N-terminus and one, two, three, four, five, six or more additional amino acids at the C-terminus of the amino acid sequence of any one of SEQ ID NOs: 10-73, 83-216, 460-634, and 636-650. In some examples, the substrate comprises 1, 2, 3, 4, 5, 6 or more additional amino acids at the N-terminus and 1, 2, 3, 4, 5, 6 or more additional amino acids at the C-terminus of the amino acid sequence of any one of SEQ ID NOs: 10-73, 83-216, and 460-634.

[0070] In some embodiments, the substrate comprises a sequence having one or more amino acid mutation(s) of the amino acid sequence of any one of SEQ ID NOs: 10-73, 83-216, 460-634, and 636-650. In some embodiments, the substrate comprises a sequence having one or more amino acid mutation(s) of the amino acid sequence of any one of SEQ ID NOs: 10-73, 83-216, and 460-634. For example, the substrate comprises a sequence having one, two, three, four, or five amino acid mutation(s) of the amino acid sequence of any one of SEQ ID NOs: 10-73, 83-216, 460-634, and 636-650. For example, the substrate comprises a sequence having one, two, three, four, or five amino acid mutation(s) of the amino acid sequence of any one of SEQ ID NOs: 10-73, 83-216, and 460-634. In some embodiments, the substrate comprises an amino acid sequence with one conservative substitution selected from the group consisting of SEQ ID NOs: 10-73, 83-216, 460-634, and 636-650. In some embodiments, the substrate comprises an amino acid sequence with one conservative substitution selected from the group consisting of SEQ ID NOs: 10-73, 83-216, and 460-634.

[0071] In some embodiments, the substrate consists of a sequence having one or more amino acid mutation(s) of the amino acid sequence of any one of SEQ ID NOs: 10-73, 83-216, 460-634, and 636-650. In some embodiments, the substrate consists of a sequence having one or more amino acid mutation(s) of the amino acid sequence of any one of SEQ ID NOs: 10-73, 83-216, and 460-634. For example, the substrate consists of a sequence having one, two, three, four, or five amino acid mutation(s) of the amino acid sequence of any one of SEQ ID NOs: 10-73, 83-216, 460-634, and 636-650. For example, the substrate consists of a sequence having one, two, three, four, or five amino acid mutation(s) of the amino acid sequence of any one of SEQ ID NOs: 10-73, 83-216, and 460-634.

[0072] In some embodiments, the substrate contains a total of 3 to 25 amino acids. For example, the substrate may contain a total of 3 to 25, 3 to 20, 3 to 15, 3 to 10, 3 to 5, 5 to 25, 5 to 20, 5 to 15, 5 to 10, 10 to 25, 10 to 20, 10 to 15, 15 to 25, 15 to 20, or 20 to 25 amino acids. In some embodiments, the substrate consists of a total of 3 to 25 amino acids. For example, the substrate may consist of a total of 3 to 25, 3 to 20, 3 to 15, 3 to 10, 3 to 5, 5 to 25, 5 to 20, 5 to 15, 5 to 10, 10 to 25, 10 to 20, 10 to 15, 15 to 25, 15 to 20, or 20 to 25 amino acids.

[0073] The substrate can be specifically cleaved by one or more proteases (e.g., MT-SP1 and / or MMP) at a desired rate, which can be determined by the substrate cleavage kinetics (k), as disclosed in WO2016118629. cat / KM), which is incorporated by reference in its entirety. cat is the turnover number, which represents how many substrate molecules are converted to products by a protease per unit time. M The k value represents the affinity of the substrate for the active site of the protease. cat / K M The ratio provides a measure of the cleavability of the substrate by the protease. Generally, the higher the ratio, the higher the cleavage rate. Conversely, the lower the ratio, the slower the cleavage rate. cat / K M The value can be determined by the following formula:

number

[0074] In some embodiments, the substrate is between 1×10 and 1×10 6 M -1 s -1, e.g., 1×10 to 5×10, 5×10 to 1×10 2 , 1×10 2 ~5×10 2 , 5×10 2 ~1×10 3 , 1×10 3 ~5×10 3 , 5×10 3 ~1×10 4 , 1×10 4 ~5×10 4 , 5×10 4 ~1×10 5 , 1×10 5 ~5×10 5 , or 5 × 10 5 ~1×10 6 M -1 s -1 k cat / K M In some embodiments, the substrate is cleaved by MT-SP1 at a rate having a value of at least 1 x 10, at least 5 x 10, at least 1 x 10 2 , at least 5 × 10 2 , at least 1 x 10 3 , 5×10 3 , at least 1 x 10 4 , at least 5 × 10 4 , at least 1 x 10 5 , at least 5 × 10 5 , or at least 1 × 10 6 M -1 s -1 k cat / K M It is cleaved by MT-SP1 at a rate of 100 kJ / s.

[0075] In some embodiments, the substrate is between 1×10 and 1×10 6 M -1 s -1 , e.g., 1×10 to 5×10, 5×10 to 1×10 2 , 1×10 2 ~5×10 2 , 5×10 2 ~1×10 3 , 1×10 3 ~5×10 3 , 5×10 3~1×10 4 , 1×10 4 ~5×10 4 , 5×10 4 ~1×10 5 , 1×10 5 ~5×10 5 , or 5 × 10 5 ~1×10 6 M -1 s -1 k cat / K M In some embodiments, the substrate is cleaved by the MMP at a rate having a value of at least 1 x 10, at least 5 x 10, at least 1 x 10 2 , at least 5 × 10 2 , at least 1 x 10 3 , 5×10 3 , at least 1 x 10 4 , at least 5 × 10 4 , at least 1 x 10 5 , at least 5 × 10 5 , or at least 1 × 10 6 M -1 s -1 k cat / K M It is cleaved by MMPs at a rate having a value.

[0076] In some embodiments, the substrate is between 1×10 and 1×10 6 M -1 s -1 , e.g., 1×10 to 5×10, 5×10 to 1×10 2 , 1×10 2 ~5×10 2 , 5×10 2 ~1×10 3 , 1×10 3 ~5×10 3 , 5×10 3 ~1×10 4 , 1×10 4 ~5×10 4 , 5×10 4 ~1×10 5 , 1×10 5 ~5×10 5 , or 5 × 10 5 ~1×10 6 M-1 s -1 k cat / K M In some embodiments, the substrate is cleaved by MMP2 at a rate having a value of at least 1 x 10, at least 5 x 10, at least 1 x 10 2 , at least 5 × 10 2 , at least 1 x 10 3 , 5×10 3 , at least 1 x 10 4 , at least 5 × 10 4 , at least 1 x 10 5 , at least 5 × 10 5 , or at least 1 × 10 6 M -1 s -1 k cat / K M It is cleaved by MMP2 at a rate of

[0077] In some embodiments, the substrate is between 1×10 and 1×10 6 M -1 s -1 , e.g., 1×10 to 5×10, 5×10 to 1×10 2 , 1×10 2 ~5×10 2 , 5×10 2 ~1×10 3 , 1×10 3 ~5×10 3 , 5×10 3 ~1×10 4 , 1×10 4 ~5×10 4 , 5×10 4 ~1×10 5 , 1×10 5 ~5×10 5 , or 5 × 10 5 ~1×10 6 M -1 s -1 k cat / K M In some embodiments, the substrate is cleaved by MMP9 at a rate having a value of at least 1 x 10, at least 5 x 10, at least 1 x 10 2 , at least 5 × 10 2 , at least 1 x 103 , 5×10 3 , at least 1 x 10 4 , at least 5 × 10 4 , at least 1 x 10 5 , at least 5 × 10 5 , or at least 1 × 10 6 M -1 s -1 k cat / K M It is cleaved by MMP9 at a rate of

[0078] In some embodiments, the substrate is between 1×10 and 1×10 6 M -1 s -1 , e.g., 1×10 to 5×10, 5×10 to 1×10 2 , 1×10 2 ~5×10 2 , 5×10 2 ~1×10 3 , 1×10 3 ~5×10 3 , 5×10 3 ~1×10 4 , 1×10 4 ~5×10 4 , 5×10 4 ~1×10 5 , 1×10 5 ~5×10 5 , or 5 × 10 5 ~1×10 6 M -1 s -1 k cat / K M In some embodiments, the substrate is cleaved by MMP14 at a rate having a value of at least 1 x 10, at least 5 x 10, at least 1 x 10 2 , at least 5 × 10 2 , at least 1 x 10 3 , 5×10 3 , at least 1 x 10 4 , at least 5 × 10 4 , at least 1 x 10 5 , at least 5 × 10 5 , or at least 1 × 10 6 M -1 s-1 k cat / K M It is cleaved by MMP14 at a rate of

[0079] In some embodiments, the substrate is between 1×10 and 1×10 6 M -1 s -1 , e.g., 1×10 to 5×10, 5×10 to 1×10 2 , 1×10 2 ~5×10 2 , 5×10 2 ~1×10 3 , 1×10 3 ~5×10 3 , 5×10 3 ~1×10 4 , 1×10 4 ~5×10 4 , 5×10 4 ~1×10 5 , 1×10 5 ~5×10 5 , or 5 × 10 5 ~1×10 6 M -1 s -1 k cat / K M In some embodiments, the substrate is cleaved by proteases in bone marrow tissue (e.g., bone marrow aspirate) at a rate having a value of at least 1 x 10, at least 5 x 10, at least 1 x 10 2 , at least 5 × 10 2 , at least 1 x 10 3 , 5×10 3 , at least 1 x 10 4 , at least 5 × 10 4 , at least 1 x 10 5 , at least 5 × 10 5 , or at least 1 × 10 6 M -1 s -1 k cat / K M It is cleaved by proteases in bone marrow tissue (eg, bone marrow aspirate) at a rate having a value.

[0080] In some embodiments, substrate cleavability is expressed as a percentage of the proportion of the substrate (or polypeptide comprising the substrate) that is cleaved, e.g., as measured by capillary electrophoresis as described in Example 2. In some examples, the cleavability of the substrate by the protease is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, or 100%. In some examples, when 500 nM of activatable antibody c225 comprising the prodomain of the substrate being tested is incubated with 10 nM of MT-SP1 at 37°C for 1.5 hours, the cleavability of the substrate by MT-SP1 is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, or 100%. In some examples, when 500 nM of activatable antibody c225 containing the prodomain of the substrate to be tested is incubated with 10 nM of MMP for 1.5 hours at 37° C., the cleavability of the substrate by MMP is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%. In some examples, when 500 nM of activatable antibody c225 containing the prodomain of the substrate to be tested is incubated with 10 nM of MMP2 for 1.5 hours at 37° C., the cleavability of the substrate by MMP2 is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, or 100%. In some examples, when 500 nM of activatable antibody c225 containing the prodomain of the substrate to be tested is incubated with 10 nM of MMP9 for 1.5 hours at 37° C., the cleavability of the substrate by MMP9 is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%. In some examples, when 500 nM of activatable antibody c225 containing the prodomain of the substrate to be tested is incubated with 10 nM of MMP14 for 1.5 hours at 37° C., the cleavability of the substrate by MMP14 is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, or 100%.In some examples, the cleavability of the substrate by the protease in bone marrow tissue (e.g., in bone marrow aspirate) is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, or 100% when incubated with bone marrow tissue (e.g., bone marrow aspirate) at 37°C for 1.5 hours or 4 hours.

[0081] In some embodiments, contact between the enzyme and the substrate is effected for specific cleavage by the enzyme. When a substrate-containing polypeptide (e.g., an activatable molecule comprising an AM coupled to an MM and a substrate) is in the presence of a target and sufficient protease activity, the substrate can be cleaved. Sufficient protease activity refers to the ability of the protease to access and cleave the substrate.

[0082] In some embodiments, a substrate according to the present disclosure and a reference polypeptide may be cleaved by the same protease, but the substrate according to the present disclosure has reduced cleavage or resistance to cleavage (e.g., by a different protease) in a particular tissue in situ compared to the reference polypeptide. In some examples, a substrate according to the present disclosure and a reference polypeptide may be cleaved by MT-SP1 and / or an MMP, but the substrate according to the present disclosure has reduced resistance to cleavage or cleavage (e.g., by a protease(s) different from MT-SP1 and / or MMP2, MMP9, and MMP14) in bone marrow in situ compared to the reference polypeptide. For example, the substrate may be cleaved in bone marrow in situ (e.g., by a protease different from MT-SP1 and / or an MMP) by less than 99%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or 1% of the cleavage of the reference polypeptide. In some examples, such proteases distinct from MT-SP1 and / or MMPs may be other proteases in bone marrow or other normal tissues, as well as other proteases involved in inflammation and wound healing. In some examples, cleavage in bone marrow in situ (e.g., by proteases distinct from MT-SP1 and / or MMPs) can be measured by an increase in the activity of an activatable molecule comprising a substrate or reference polypeptide in bone marrow, e.g., by the method described in Example 6. A substrate that is resistant to cleavage by a protease, or a sample or tissue containing a protease, refers to (i) a substrate whose peptide bond is not hydrolyzed by the protease or whose peptide bond is not hydrolyzed when incubated in a sample or tissue containing the protease, or (ii) a substrate whose peptide bond is hydrolyzed at a reduced level by the protease or whose peptide bond is hydrolyzed at a reduced level when incubated in a sample or tissue containing the protease, compared to a reference substrate.

[0083] In some embodiments, the substrate is cleavable by more than two proteases. For example, the substrate can be cleaved by a serine protease (e.g., MT-SP1) and an MMP (e.g., MMP2, MMP9, and / or MMP14), as well as one or more additional proteases. The additional proteases can include any one or more of the following proteases: a disintegrin and metalloproteinase (ADAM), ADAM-like, or a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS, e.g., ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAMDECl, ADAMTS1, ADAMTS4, ADAMTS5); asparagine Acid proteases (e.g., BACE, renin, etc.); aspartic cathepsins (e.g., cathepsin D, cathepsin E, etc.); caspases (e.g., caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 14, etc.); cysteine cathepsins (e.g., cathepsin B, cathepsin C, cathepsin K, cathepsin L, cathepsin S, cathepsin V / L2, cathepsin psin X / Z / P, etc.); cysteine proteinases (e.g., cruzipain, legumain, otubein-2, etc.); kallikrein-related peptidases (KLK) (e.g., KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, KLK14, etc.); metalloproteinases (e.g., meprin, neprilysin, prostate-specific membrane antigen (PSMA), bone morphogenetic protein 1 (BMP-1), etc.); matrix metalloproteinases (M MPs, such as MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP23, MMP24, MMP26, MMP27, etc.; serine proteases (such as activated protein C, cathepsin A, cathepsin G, chymase, coagulation factor proteases (such as FVIIa, FIXa, FXa, FXIa, FXIIa, etc.));Elastase, granzyme B, guanidinobenzoatase, HtrA1, proteinase 3, neutrophil elastase, neutrophil serine protease 4 (NSP4), lactoferrin, marapsin, NS3 / 4A, PACE4, plasmin, prostate-specific antigen (PSA), tissue plasminogen activator (tPA), thrombin, tryptase, urokinase-type plasminogen activator (uPA), type II transmembrane serine proteases (TTSPs) (e.g., DESC1, DPP-4, FAP, hepsin, matriptase-2, MT-SP1 / matriptase, TMPRSS2, TMPRSS3, TMPRSS4, TMPRSS5, TMPRSS6, TMPRSS7, TMPRSS8, TMPRSS9, TMPRSS10, TMPRSS11, etc.), etc. Particular substrates are described, for example, in WO2010 / 081173, WO2015 / 048329, WO2015 / 116933, and WO2016 / 118629, each of which is incorporated herein by reference in its entirety.

[0084] In some embodiments, at least a portion of CM1 in a substrate overlaps with at least a portion of CM2 in the substrate, such that one or more amino acids in the substrate belong to both CM1 and CM2. For example, a substrate with the sequence X1X2X3X4X5X6 (where each X is an amino acid) may contain overlapping CM1 and CM2, where CM1 is X1X2X3X4 and CM2 is X3X4X5X6.

[0085] In some embodiments, CM1 and CM2 in a substrate do not overlap in amino acid sequence, such that no amino acid in the substrate belongs to both CM1 and CM2. For example, a substrate with the sequence X1X2X3X4X5X6X7X8 (where each X is an amino acid) may contain non-overlapping CM1 and CM2, where CM1 is X1X2X3X4 and CM2 is X5X6X7X8. In some embodiments, non-overlapping CM1 and CM2 are directly coupled. In some embodiments, non-overlapping CM1 and CM2 are indirectly coupled (e.g., via a linking peptide).

[0086] In some embodiments, CM1 and CM2 in a substrate have a structural arrangement from N-terminus to C-terminus as CM1-CM2. In some embodiments, CM1 and CM2 in a substrate have a structural arrangement from N-terminus to C-terminus as CM2-CM1. As used herein, CM1 and CM2 in the formula CM1-CM2 or CM2-CM1 can be overlapping CM1 and CM2, directly coupled non-overlapping CM1 and CM2, or indirectly coupled non-overlapping CM1 and CM2 (e.g., via a linking peptide).

[0087] activatable molecules In some embodiments, the polypeptide or polypeptide complex comprising the substrate is an activatable molecule. The activatable molecule may comprise an active moiety (AM) that specifically binds to a target. The AM may be coupled to a substrate. In some embodiments, the activatable molecule comprises a masking moiety (MM) coupled to the AM via the substrate.

[0088] The coupling of two components in a polypeptide or polypeptide complex (e.g., an activatable molecule) can be direct or indirect. When two components are directly coupled, the C-terminal amino acid residue of one component forms a peptide bond with the N-terminal amino acid residue of the other component. When two components are indirectly coupled, there is a stretch of amino acids between the two components. In some examples, two components of a polypeptide can be indirectly coupled via one or more other components in the polypeptide. That is, one or more other components are between the two coupled components. When indirectly coupled or linked via another component, the one or more other components can be a linker, AM(s), CM(s), MM(s), or any combination thereof.

[0089] As used herein, the term "activatable molecule" refers to a molecule that comprises at least one set of a MM, a substrate, and an AM, and that binds to a target attenuated compared to when a corresponding "activated" molecule comprising the same AM binds to the same target. The terms "activated molecule" and "cleaved activatable molecule" are used interchangeably herein and refer to an AM-containing cleavage product that is generated after exposure of an activatable molecule to a substrate-specific protease (i.e., after cleavage of the substrate by at least one protease). In some embodiments, the cleaved activatable molecule lacks the MM due to cleavage of the substrate (e.g., by the protease), which may result in release of the MM.

[0090] An AM can be any polypeptide that specifically binds to a target. In some examples, an AM can be a therapeutic macromolecule. In some examples, an AM can be an antibody or an antigen-binding fragment. In some examples, an AM can be an anti-tumor macromolecule. In some examples, an AM can be a cytokine. In some examples, an AM can be a chimeric antigen receptor.

[0091] In some examples, the AM can be a diagnostic macromolecule. For example, the diagnostic macromolecule can be a diagnostic polypeptide having a length of 3 to 30, 5 to 25, 7 to 20, or 9 to 15 amino acids. Such diagnostic polypeptides can be used, in non-limiting embodiments, for example, to test for cleavage in tissues and / or to evaluate the tissue microenvironment.

[0092] As used herein, the terms "specific binding" and "specifically binds" refer to the type of non-covalent interaction that occurs between an AM and a target for which the AM is specific, such as an immunoglobulin molecule and an antigen or a cytokine and its receptor. The strength or affinity of a binding interaction is determined by the dissociation constant (K d ) and can be expressed in terms of smaller K drepresents a greater affinity. Unless otherwise indicated, as used herein, "affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between a member of an AM and its target. Affinity can be measured by common methods known in the art, including those described herein. Affinity can be determined, for example, using surface plasmon resonance (SPR) technology (e.g., BIACORE®) or biolayer interferometry (e.g., FORTEBIO®). Additional methods for determining the affinity of an AM for its target are known in the art. The immunological binding properties of a selected polypeptide can be quantified by methods well known in the art. One such method involves measuring the rates of antigen-binding site / antigen complex formation and dissociation, which depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that affect the rate equally in both directions. Thus, the "on-rate constant" (k on ) and "off rate constant" (k off Both k and k can be determined by calculating the concentration and the actual rates of association and dissociation. (See Nature 361:186-87 (1993)). off / k on The ratio of α to β allows for the release of all parameters not related to affinity and gives the dissociation constant K d (See generally Davies et al. (1990) Annual Rev Biochem 59:439-473.) As used herein, the statement that an AM "specifically binds" to its target means that the AM binds to its target with a dissociation constant (K) of less than 100 μM (e.g., less than 5 μM or 10 μM). d ) that binds to its target. In some examples, the AM has a K of about 0.01 nM to about 500 nM. d In some instances, the AM specifically binds to its target at an equilibrium binding constant (K), as measured by an assay such as a radioligand binding assay or similar assay known to one of skill in the art. d) is said to specifically bind to a target when the ion concentration is ≦1 μM, in some embodiments ≦100 nM, in some embodiments ≦10 nM, and in some embodiments ≦100 pM to about 1 pM.

[0093] In general, an activatable molecule can be designed by selecting an AM of interest and constructing the remainder of the activatable molecule such that, when conformationally constrained, the MM provides masking of the AM or reduced binding of the AM to its target. Structural design criteria can be taken into account to provide this functional feature.

[0094] The activatable molecule can be provided in various structural configurations. Exemplary formulas of activatable molecules are provided below. It is contemplated that the order of the AM, MM, and substrate from N-terminus to C-terminus may be reversed within the activatable molecule. For example, the activatable molecule can be represented by the following formula (from the amino (N)-terminal region to the carboxyl (C)-terminal region): MM-substrate-AM, AM-substrate-MM MM-CM1-CM2-AM, MM-CM2-CM1-AM, AM-CM1-CM2-MM, or AM-CM2-CM1-MM As used herein, unless otherwise specified, each dash (-) between components of an activatable molecule represents a direct linkage, an indirect linkage via one or more linkers, or an overlap between the components. It should be noted that although the MM and substrate are shown as separate components in the formulas above, in all exemplary embodiments (including formulas) disclosed herein, it is contemplated that the amino acid sequences of the MM and substrate may overlap, e.g., such that the substrate is fully or partially contained within the MM. Additionally, the formulas above provide additional amino acid sequences that may be located at the N- or C-terminus of an activatable molecule component. Examples include targeting moieties (e.g., a target for a cellular receptor present in a target tissue) and half-life extending moieties.

[0095] In some embodiments, the MM, substrate, and / or AM are indirectly coupled via one or more linkers (e.g., connecting peptides (LPs)). For example, the activatable molecule can comprise any of the following formulas (in order from the amino (N)-terminal region to the carboxyl (C)-terminal region): MM-LP-Substrate-AM MM-substrate-LP-AM MM-LP1-substrate-LP2-AM MM-LP2-substrate-LP1-AM AM-LP-substrate-MM AM-Substrate-LP-MM AM-LP2-substrate-LP1-MM AM-LP1-substrate-LP2-MM MM-LP-CM1-CM2-AM MM-LP-CM2-CM1-AM MM-CM1-CM2-LP-AM MM-CM2-CM1-LP-AM MM-LP1-CM1-CM2-LP2-AM MM-LP1-CM2-CM1-LP2-AM MM-LP2-CM1-CM2-LP1-AM MM-LP2-CM2-CM1-LP1-AM AM-LP-CM1-CM2-MM AM-LP-CM2-CM1-MM AM-CM1-CM2-LP-MM AM-CM2-CM1-LP-MM AM-LP2-CM1-CM2-LP1-MM AM-LP2-CM2-CM1-LP1-MM AM-LP1-CM1-CM2-LP2-MM AM-LP1-CM2-CM1-LP2-MM wherein LP1 and LP2 are two connecting peptides. In some embodiments, LP1 and LP2 are identical to each other. In some embodiments, LP1 and LP2 are not identical to each other. As used herein, and unless otherwise specified, each dash (-) between components of an activatable molecule represents a direct linkage or an indirect linkage via one or more linkers, or an overlap between the components.

[0096] In some embodiments, a substrate comprises more than two CMs, for example, a substrate can comprise CM1 cleavable by a first protease, CM2 cleavable by a second protease, and CM3 cleavable by a third protease. Examples of additional CM(s) in the substrate that are not CMs comprising any of the sequences of SEQ ID NOs: 10-73, 83-216, 460-634, and 636-650, such as CM3, include those described in WO2010 / 081173, WO2021207669, WO2021207657, WO2021142029, WO2021061867, WO2020252349, WO2020252358, WO2020236679, WO2020176672, WO2020118109, WO2020092881, WO2020086665, WO2019213444, WO2019 183218, WO2019173771, WO2019165143, WO2019075405, WO2019046652, WO2019018828, WO2019014586, WO2018222949, WO2018165619, WO2018085555, WO2017011580, WO2016179335, WO2016179285, WO2016179257, WO2016149201, and WO2016014974, which are incorporated by reference herein in their entireties for all purposes. In some examples, one or more additional CMs may be cleavable by legumain. In some examples, a CM cleavable by legumain may comprise any one of the sequences of SEQ ID NOs: 10 to 73, 83 to 216, 460 to 634, and 636 to 650, and an N- or C-terminal asparagine (Asn) residue. In some examples, a CM cleavable by legumain may comprise any one of the sequences of SEQ ID NOs: 10 to 73, 83 to 216, and 460 to 634, and an N- or C-terminal asparagine (Asn) residue.

[0097] In some embodiments, the activatable molecule comprises the following structural arrangement from N- to C-terminus: MM-CM1-CM2-AM, MM-CM2-CM1-AM, AM-CM1-CM2-MM, or AM-CM2-CM1-MM, MM-CM2-CM1-CM3-AM, MM-CM1-CM2-CM3-AM, MM-CM1-CM3-CM2-AM, MM-CM3-CM1-CM2-AM, or MM-CM3-CM2-CM1-AM. Similarly, CM4 can be inserted anywhere between MM and AM.

[0098] In some embodiments, the activatable molecule comprises a connecting peptide (LP), and the activatable molecule has the following structural arrangement from N-terminus to C-terminus: MM-LP-CM1-CM2-AM, MM-CM1-CM2-LP-AM, MM-LP-CM2-CM1-AM, MM-CM2-CM1-LP-AM, MM-LP-CM2-CM1-CM3-AM, MM-LP-CM1-CM2-CM3-AM, MM-LP-CM1-CM3-CM2-AM, MM-LP-CM3-CM1-CM2-AM, MM-CM2-CM1-CM3-LP-AM, MM-CM1-CM2-CM3-LP-AM, MM-CM1-CM3-CM2-LP-AM, MM-CM3-CM1-CM2-LP-AM, or MM-CM3-CM2-CM1-LP-AM. Similarly, CM4 can be inserted at any position between MM and AM.

[0099] In some embodiments, the activatable molecule comprises a first connecting peptide (LP1) and a second connecting peptide (LP2), and the activatable molecule has the following structural arrangement from N-terminus to C-terminus: MM-LP1-CM1-CM2-LP2-AM, MM-LP1-CM2-CM1-LP2-AM, AM-LP2-CM1-CM2-LP1-MM, or AM-LP2-CM2-CM1-LP1-MM, MM-LP1-CM2-CM1-CM3-LP2-AM, MM-LP1-CM1-CM2 -CM3-LP2-AM, MM-LP1-CM1-CM3-CM2-LP2-AM, MM-LP1-CM3-CM1-CM2-LP2-AM, MM-LP1-CM3-CM2-CM1-LP2-AM, MM-LP2-CM2-CM1-CM3-LP1-AM, MM-LP2-CM1-CM2-CM3-LP1-AM, MM-LP2-CM1-CM2-CM3-LP1-AM, MM-LP2-CM1-CM3-CM2-LP1-AM, MM-LP2-CM1-CM3-CM2-LP1-AM, MM-LP2-CM3-CM1-CM2-LP1-AM, or MM-LP2-CM3-CM2-CM1-LP1-AM. Similarly, CM4 can be inserted anywhere between MM and AM.

[0100] In some embodiments, the activatable molecule comprises an additional connecting peptide (LP3), and the activatable molecule has the following structural arrangement from N-terminus to C-terminus: MM-LP-CM1-LP3-CM2-AM, MM-CM1-LP3-CM2-LP-AM, MM-LP-CM2-LP3-CM1-AM, MM-CM2-LP3-CM1-LP-AM, MM-LP-CM2-LP3-CM1-CM3-AM, MM-LP-CM1-LP3-CM2-CM3-AM, MM-LP-CM1-LP3-CM3-CM2-AM, MM-LP-CM3- LP3-CM1-CM2-AM, MM-LP-CM3-LP3-CM2-CM1-AM, MM-CM2-LP3-CM1-CM3-LP-AM, MM-CM1-LP3-CM2-CM3-LP-AM, MM-CM1-LP3-CM3-CM2-LP-AM, MM-CM3- LP3-CM1-CM2-LP-AM, MM-CM3-LP3-CM2-CM1-LP-AM, MM-LP-CM2-CM1-LP3-CM3-AM, MM-LP-CM1-CM2-LP3-CM3-AM, MM-LP-CM1-CM3-LP3-CM2-AM, MM-L P-CM3-CM1-LP3-CM2-AM, MM-LP-CM3-CM2-LP3-CM1-AM, MM-CM2-CM1-LP3-CM3-LP-AM, MM-CM1-CM2-LP3-CM3-LP-AM, MM-CM1-CM3-LP3-CM2-LP-AM, M M-CM3-CM1-LP3-CM2-LP-AM, MM-CM3-CM2-LP3-CM1-LP-AM, MM-LP1-CM1-LP3-CM2-LP2-AM, MM-LP1-CM2-LP3-CM1-LP2-AM, AM-LP1-CM1-LP3-CM2-LP 2-MM, or AM-LP1-CM2-LP3-CM1-LP2-MM, MM-LP1-CM2-LP3-CM1-CM3-LP2-AM, MM-LP1-CM1-LP3-CM2-CM3-LP2-AM, MM-LP1-CM1-LP3-CM3-CM2-LP2-A M, MM-LP1-CM3-LP3-CM1-CM2-LP2-AM, MM-LP1-CM3-LP3-CM2-CM1-LP2-AM, MM-LP2-CM2-LP3-CM1-CM3-LP1-AM, MM-LP2-CM1-LP3-CM2-CM3-LP1-AM,MM-LP2-CM1-LP3-CM3-CM2-LP1-AM, MM-LP2-CM3-LP3-CM1-CM2-LP1-AM, or MM-LP2-CM3-LP3-CM2-CM1-LP1-AM. Similarly, CM4 can be inserted anywhere between MM and AM.

[0101] In some embodiments, the activatable molecule has the following structural arrangement from N-terminus to C-terminus: MM-LP-CM2-LP3-CM1-LP4-CM3-AM, MM-LP-CM1-LP3-CM2-LP4-CM3-AM, MM-LP-CM1-LP3-CM3-LP4-CM2-AM, MM-LP-CM3-LP3-CM1-LP4-CM2-AM, MM-LP-CM3-LP3-CM2-LP4-CM1-AM, MM-CM2-LP3-CM1-LP4-CM3-LP-AM, MM-CM1-LP3-CM2-LP4-CM 3-LP-AM, MM-CM1-LP3-CM3-LP4-CM2-LP-AM, MM-CM3-LP3-CM1-LP4-CM2-LP-AM, MM-CM3-LP3-CM2-LP4-CM1-LP-AM, MM-LP-CM2-LP4-CM1-LP3-CM3- AM, MM-LP-CM1-LP4-CM2-LP3-CM3-AM, MM-LP-CM1-LP4-CM3-LP3-CM2-AM, MM-LP-CM3-LP4-CM1-LP3-CM2-AM, MM-LP-CM3-LP4-CM2-LP3-CM1-AM, MM- CM2-LP4-CM1-LP3-CM3-LP-AM, MM-CM1-LP4-CM2-LP3-CM3-LP-AM, MM-CM1-LP4-CM3-LP3-CM2-LP-AM, MM-CM3-LP4-CM1-LP3-CM2-LP-AM, MM-CM3-L P4-CM2-LP3-CM1-LP-AM, MM-LP1-CM2-LP3-CM1-LP4-CM3-LP2-AM, MM-LP1-CM1-LP3-CM2-LP4-CM3-LP2-AM, MM-LP1-CM1-LP3-CM3-LP4-CM2-LP2-A M, MM-LP1-CM3-LP3-CM1-LP4-CM2-LP2-AM, MM-LP1-CM3-LP3-CM2-LP4-CM1-LP2-AM, MM-LP2-CM2-LP3-CM1-LP4-CM3-LP1-AM, MM-LP2-CM1-LP3-CM2-LP4-CM3-LP1-AM, MM-LP2-CM1-LP3-CM3-LP4-CM2-LP1-AM, MM-LP2-CM1-LP3-CM3-LP4-CM2-LP1-AM, MM-LP2-CM3-LP3-CM1-LP4-CM2-LP1-AM, or MM-LP2-CM3-LP3-CM2-LP4-CM1-LP1-AM. Similarly, CM4 can be inserted anywhere between MM and AM.

[0102] In some embodiments, two CMs in a substrate, e.g., CM1 and CM2, have a structural arrangement from N-terminus to C-terminus as CM1-CM2. In some embodiments, two CMs in a substrate, e.g., CM1 and CM2, have a structural arrangement from N-terminus to C-terminus as CM2-CM1. As used herein, CM1 and CM2 in the formula CM1-CM2 or CM2-CM1 can be overlapping CM1 and CM2, directly coupled non-overlapping CM1 and CM2, or indirectly coupled non-overlapping CM1 and CM2 (e.g., via a linking peptide).

[0103] In some embodiments, two CMs in a substrate, e.g., CM2 or CM4 and CM3 or CM4, have a structural arrangement from N-terminus to C-terminus as CM2-CM3, CM2-CM4, or CM3-CM4. In some embodiments, two CMs in a substrate, e.g., CM2 and CM3, have a structural arrangement from N-terminus to C-terminus as CM3-CM2, CM4-CM2, or CM4-CM3. As used herein, CM2 and CM3 in the formula CM2-CM3 or CM3-CM2 can be overlapping CM2 and CM3, directly coupled non-overlapping CM2 and CM3, or indirectly coupled non-overlapping CM2 and CM3 (e.g., via a connecting peptide). As used herein, CM2 and CM4 in the formula CM2-CM4 or CM4-CM2 can be overlapping CM2 and CM4, directly coupled non-overlapping CM2 and CM4, or indirectly coupled non-overlapping CM2 and CM4 (e.g., via a connecting peptide). As used herein, the CM4 and CM3 in the formula CM4-CM3 or CM3-CM4 may be overlapping CM4 and CM3, directly coupled non-overlapping CM4 and CM3, or indirectly coupled (e.g., via a linking peptide) non-overlapping CM4 and CM3.

[0104] Antibodies and antigen-binding fragments In some embodiments, the AM is an antibody or an antigen-binding fragment thereof. The term "antibody" is used herein in its broadest sense and includes certain types of immunoglobulin molecules that contain one or more target-binding domains that specifically bind to an antigen or epitope. Examples of antibodies include intact antibodies (e.g., intact immunoglobulins), antibody fragments, bispecific, and multispecific antibodies. An example of a target-binding domain is a V H -V L It is formed by a dimer. Additional examples of antibodies are described herein. Additional examples of antibodies are known in the art.

[0105] The "light chain" contains one variable domain (VL) and one constant domain (CL). Two different light chains exist, called kappa or lambda. The "heavy chain" consists of one variable domain (VH) and three constant region domains (CH1, CH2, and CH3). There are five main classes or isotypes of heavy chains, some of which have multiple subtypes, which determine the functional activity of antibody molecules. The five major classes of immunoglobulins are immunoglobulin M (IgM), immunoglobulin D (IgD), immunoglobulin G (IgG), immunoglobulin A (IgA), and immunoglobulin E (IgE). IgG is by far the most abundant immunoglobulin and has several subclasses (IgG1, IgG2, IgG3, and IgG4 in humans).

[0106] In some embodiments, the antigen-binding fragment is a Fab fragment, a F(ab')2 fragment, a scFv, a scAb, a dAb, a single-domain heavy chain antibody, or a single-domain light chain antibody. Additional examples of antigen-binding fragments include VH domains, VHH domains, VNAR domains, and single-chain variable fragments (scFv), BiTEs or components thereof, (scFv)2, NANOBODY®, nanobody-HSA, VHH-scAb, VHH-Fab, double-scFab, F(ab')2, diabodies, CROSSMAB®, DAF(2-in-1), DAE(4-in-1), DUTAMAB (registered trademark), DT-IgG, knob-in-hole common light chain, knob-in-hole assembly, charge pair, Fab-arm exchange, SEED body, LUZ-Y, FcAb, kl-body, orthogonal Fab, DVD-IgG, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, ZYBODY™, DVI-IgG, diabody-CH3, triplebody, miniantibody, minibody, TriBi minibody, scFv-CH3 KIH, Fab-scFv, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, sc diabody-Fc, diabody-Fc, tandem scFv-Fc, VHH-Fc, tandem VHH-Fc, L'HH-Fc Includes KiH, Fab-VHH-Fc, intrabody, Dock and Lock, ImmTAC® (immune mobilizing monoclonal TCR (T cell receptor) against cancer), IgG-IgG conjugate, Cov-X-body, scFvl-PEG-scFv2, Adnectin, DARPin®, fibronectin, IgG, IgM, IgA, IgE, IgD, DEP conjugate, TMEAbody™, SAFEbody®, TRITAC®, or SHIELD antibody.

[0107] A "fragment antigen binding" (Fab) contains an intact light chain paired with the VH and CH1 domains of a heavy chain. F(ab')2 fragments are formed when an antibody is cleaved with pepsin (or otherwise cleaved) below the hinge region, in which case the two fragment target binding domains (Fab) of the antibody molecule remain linked. F(ab')2 fragments contain two intact light chains paired with the two VH and CH1 domains of the heavy chain joined together by the hinge region. A "fragment crystallizable" (Fc) fragment (referred to herein as Fc) is a fragment of an antibody molecule that is capable of binding to a light chain. C The Fc domain (also called the Fc domain) corresponds to the paired CH2 and CH3 domains and is the part of the antibody molecule that interacts with effector molecules and cells. The functional differences between heavy chain isotypes lie primarily in the Fc fragment. A "single-chain fragment variable" (scFv) contains only the variable domain of the light chain (VL) connected to the variable domain of the heavy chain (VH) by a peptide stretch. The name single-chain Fv comes from the variable part of the fragment. The "hinge region" or "interdomain" is a flexible stretch of amino acids that joins or links the Fab fragment to the Fc domain. The "synthetic hinge region" is the amino acid sequence that joins or links the Fab fragment to the Fc domain.

[0108] An "Fv" fragment comprises a non-covalent dimer of one heavy-chain variable domain and one light-chain variable domain. "Dual variable domain immunoglobulin G" or "DVD-IgG" refers to a multivalent and multispecific target-binding protein described, for example, in DiGiammarino et al., Methods Mol. Biol. 899:145-156, 2012; Jakob et al., MABs 5:358-363, 2013; and U.S. Patent Nos. 7,612,181, 8,258,268, 8,586,714, 8,716,450, 8,722,855, 8,735,546, and 8,822,645 (each of which is incorporated by reference in its entirety). Examples of DARTs are described, for example, in Garber, Nature Reviews Drug Discovery 13:799-801, 2014.

[0109] VHH domains are single monomeric variable antibody domains that can be found in Camelidae. VNAR domains are single monomeric variable antibody domains that can be found in cartilaginous fish. Non-limiting embodiments of VHH domains and VNAR domains are described, for example, in Cromie et al., Curr. Top. Med. Chem. 15:2543-2557, 2016; De Genst et al., Dev. Comp. Immunol. 30:187-198, 2006; De Meyer et al., Trends Biotechnol 32:263-270, 2014; Kijanka et al., Nanomedicine 10:161-174, 2015; Kovaleva et al., Expert. Opin. Biol. Ther. 14:1527-1539, 2014; Krah et al., Immunopharmacol. Immunotoxicol. 38:21-28, 2016; Mujic-Delic et al., Trends Pharmacol.Sci.35:247-255,2014, Muyldermans,J.Biotechnol.74:277-302,2001,Muyldermans et al.,Trends Biocheni.Sci.26:230-235,2001, Muyldermans,Ann.Rev.Biochem.82:775-797,2013, Rahbarizadeh et al.,Immunol,invest.40:299-338,2011, Van Audenhove et al.,EBioMedicine 8:40-48,2016, Van Bockstaele et al.,Curr.Opin.Investig.Drugs 10:1212-1224,2009, Vincke et al.Methods Mol. Biol. 911:15-26, 2012, and Wesolowski et al. Med. Microbiol. Immunol. 198:157-174, 2009, each of which is incorporated herein by reference in its entirety.

[0110] In some embodiments, the AM may be a mouse, rat, rabbit, goat, camel, donkey, primate, human, or humanized or chimeric polypeptide. In one example, the AM may be a human polypeptide. In one example, the AM may be a humanized (e.g., fully humanized) polypeptide.

[0111] The term "humanized" refers to an AM having an amino acid sequence that includes VH and VL region sequences derived from a reference protein produced in a non-human species (e.g., a mouse), but also includes modifications in those sequences intended to make them more "human-like," i.e., more similar to human germline variable sequences, as compared to the reference protein. In some embodiments, a "humanized" AM is a protein that immunospecifically binds to an antigen of interest, has framework (FR) regions having amino acid sequences substantially that of a human protein, and complementarity-determining regions (CDRs) having amino acid sequences substantially that of a non-human protein, and includes humanized VH and VL regions.

[0112] The term "human polypeptide" is intended to include AMs having variable and constant regions generated, assembled, or derived from human immunoglobulin sequences. In some embodiments, an AM may be considered "human" even if its amino acid sequence includes residues or elements, e.g., in one or more CDRs, that are not encoded by human germline immunoglobulin sequences (including sequence diversity that may be (naturally) introduced by, e.g., random or site-specific mutagenesis in vitro or by somatic mutation in vivo).

[0113] Examples of antibodies and antigen-binding fragments include those that bind to cell surface receptors and secreted binding proteins (e.g., growth factors), soluble enzymes, structural proteins (e.g., collagen, fibronectin), etc., or extracellular targets (e.g., extracellular protein targets). In some embodiments, the antibodies and antigen-binding fragments are designed to be taken up by cells and are activatable within the cells.

[0114] Exemplary antibodies and antigen-binding fragments include those in Example 1, e.g., those comprising a light chain comprising a sequence selected from SEQ ID NOs: 217, 219, 221, 222, and 224-235, and a heavy chain comprising a sequence selected from SEQ ID NOs: 218, 220, and 223. In some examples, a polypeptide (e.g., an isolated polypeptide) herein comprises a sequence selected from SEQ ID NOs: 221, 222, and 224-235.

[0115] Multispecific activatable antibodies In some embodiments, the activatable antibody is a multispecific activatable antibody. In some examples, the multispecific activatable antibodies herein recognize two or more different antigens or epitopes and comprise at least one masking moiety (MM) linked to at least one antigen- or epitope-binding domain of the multispecific antibody, where coupling of the MM reduces the ability of the antigen- or epitope-binding domain to bind to its target. In some embodiments, the MM is coupled to the antigen- or epitope-binding domain of the multispecific antibody via a substrate that is cleavable by one or more proteases, e.g., MT-SP1 and / or MMP. The activatable multispecific antibodies provided herein are stable in circulation, are activated at the intended site of therapy and / or diagnosis but not in normal, i.e., healthy tissue, and, upon activation, exhibit target binding at least comparable to that of the corresponding unmodified multispecific antibody.

[0116] The multispecific activatable molecule can be used to target first and second target tissues. In one embodiment, the first and second target tissues are spatially separated, e.g., at different sites in an organism. In one embodiment, the first and second target tissues are the same tissue separated in time, e.g., the same tissue at two different time points, e.g., the first time point is when the tissue is an early-stage tumor and the second time point is when the tissue is a later-stage tumor.

[0117] In some embodiments, the multispecific activatable antibody comprises a first antibody or antigen-binding fragment thereof (AB1) that binds to a first target, where AB1 is coupled to a masking moiety (MM1) such that the coupling of the masking moiety reduces the ability of AB1 to bind to the first target, and the multispecific activatable antibody comprises a second antibody or antigen-binding fragment thereof (AB2) that binds to a second target, where AB2 is coupled to MM2 such that the coupling of the masking moiety (MM2) reduces the ability of AB2 to bind to the second target. In some embodiments, AB1 is coupled to MM1 via Substrate 1, and AB2 is coupled to MM2 via Substrate 2. In some embodiments, a linking peptide is present between AB1 and Substrate 1, between Substrate 1 and MM1, between AB2 and Substrate 2, and / or between Substrate 2 and MM2. In some embodiments, AB1 is directly coupled to Substrate 1, Substrate 1 is directly coupled to MM1, AB2 is directly coupled to Substrate 2, and / or Substrate 2 is directly coupled to MM2.

[0118] For example, a multispecific activatable antibody can be represented by the following formula (from the amino (N) terminal region to the carboxyl (C) terminal region): MM1-substrate 1-AB1:MM2-substrate 2-AB2 AB1-Substrate 1-MM1:MM2-Substrate 2-AB2 AB1-substrate 1-MM1:AB2-substrate 2-MM2 MM1-Substrate 1-AB1:AB2-Substrate 2-MM2 where ":" separates two polypeptides, which may be two independent polypeptides on two different molecules, or two polypeptides on the same molecule (e.g., two polypeptide chains of the same protein). As used herein, and unless otherwise specified, each dash (-) between components of an activatable molecule represents a direct linkage or an indirect linkage via one or more linking peptides, or an overlap between the components.

[0119] In some embodiments, the multispecific activatable antibody is designed to engage an immune effector cell, also referred to herein as an immune effector cell-engaging multispecific activatable antibody. In some embodiments, the multispecific activatable antibody is designed to engage a leukocyte, also referred to herein as a leukocyte-engaging multispecific activatable antibody. In some embodiments, the multispecific activatable antibody is designed to engage a T cell, also referred to herein as a T cell-engaging multispecific activatable antibody. In some embodiments, the multispecific activatable antibody engages a surface antigen on a leukocyte, such as a T cell, a natural killer (NK) cell, a myelomononuclear cell, a macrophage, and / or another immune effector cell. In some embodiments, the immune effector cell is a leukocyte. In some embodiments, the immune effector cell is a T cell. In some embodiments, the immune effector cell is an NK cell. In some embodiments, the immune effector cell is a mononuclear cell, such as a myelomononuclear cell. In some embodiments, multispecific activatable antibodies are designed to bind to or interact with more than one target and / or more than one epitope, and are also referred to herein as multi-antigen targeted activatable antibodies. As used herein, the terms "target" and "antigen" are used interchangeably.

[0120] In some embodiments, immune effector cell-engaging multispecific activatable antibodies of the present disclosure include a targeting antibody or antigen-binding fragment thereof and an immune effector cell-engaging antibody or antigen-binding portion thereof, wherein at least one of the targeting antibody or antigen-binding fragment and / or the immune effector cell-engaging antibody or antigen-binding portion thereof is masked.

[0121] In some embodiments, the non-immune effector cell-engaging antibody is a cancer-targeting antibody. In some embodiments, the non-immune cell effector antibody is an IgG. In some embodiments, the immune effector cell-engaging antibody is an scFv. In some embodiments, the targeting antibody (e.g., non-immune cell effector antibody) is an IgG and the immune effector cell-engaging antibody is an scFv. In some embodiments, the immune effector cell is a leukocyte. In some embodiments, the immune effector cell is a T cell. In some embodiments, the immune effector cell is a NK cell. In some embodiments, the immune effector cell is a myelomononuclear cell.

[0122] In some embodiments of immune effector cell-engaging multispecific activatable antibodies, one antigen is typically present on the surface of a tumor cell or other cell type associated with the disease, and the other antigen is typically a stimulatory or inhibitory receptor present on the surface of T cells, natural killer (NK) cells, myelomononuclear cells, macrophages, and / or other immune effector cells.

[0123] One embodiment of the present disclosure is a multispecific activatable antibody that is activatable in the cancer microenvironment and includes an antibody (e.g., IgG or scFv) directed against a tumor target and an agonistic antibody (e.g., IgG or scFv) directed against a costimulatory receptor expressed on the surface of activated T cells or NK cells, wherein at least one of the cancer-targeting antibody and / or agonistic antibody is masked. In this embodiment, when activated by tumor-associated proteases, the multispecific activatable antibody effectively crosslinks and activates T cell- or NK cell-expressed costimulatory receptors in a tumor-dependent manner, enhancing the activity of T cells that respond to any tumor antigen via endogenous T cell antigens or NK activating receptors. Due to the activation-dependent nature of these T cell or NK cell costimulatory receptors, the activity of the activated multispecific activatable antibody is focused on tumor-specific T cells without activating all T cells regardless of antigen specificity. In one embodiment, at least the costimulatory receptor antibody of the multispecific activatable antibody is masked to prevent activation of autoreactive T cells that may be present in tissues expressing the antigen recognized by the tumor-targeting antibody of the multispecific activatable antibody, but whose activity is limited by the lack of co-receptor engagement.

[0124] One embodiment of the present disclosure is a multispecific activatable antibody that can be activated in diseases characterized by T cell overstimulation, such as autoimmune or inflammatory disease microenvironments. Such a multispecific activatable antibody includes an antibody (e.g., IgG or scFv) directed to a target comprising a surface antigen expressed in tissues targeted by T cells in autoimmune or inflammatory diseases, and an antibody (e.g., IgG or scFv) directed to an inhibitory receptor expressed on the surface of T cells or NK cells, where at least one of the disease tissue-targeting antibody and / or the T cell inhibitory receptor antibody is masked. Examples of tissue antigens targeted by T cells in autoimmune diseases include surface antigens expressed on myelin or nerve cells in multiple sclerosis, or surface antigens expressed on pancreatic islet cells in type 1 diabetes. In this embodiment, the multispecific activatable antibody is activated upon localization to tissues under autoimmune attack or inflammation, co-engaging with T cell or NK cell inhibitory receptors to suppress the activity of autoreactive T cells that respond to antigens targeted to the diseased tissue via endogenous TCRs or activating receptors. In one embodiment, at least one or more antibodies are masked to prevent suppression of T cell responses in non-diseased tissue where the target antigen may also be expressed.

[0125] In some embodiments, the multi-antigen targeting antibody and / or multi-antigen targeting activatable antibody comprises at least a first antibody or antigen-binding fragment that binds to a first target and / or a first epitope and a second antibody or antigen-binding fragment that binds to a second target and / or a second epitope. In some embodiments, the multi-antigen targeting antibody and / or multi-antigen targeting activatable antibody binds to two or more different targets. In some embodiments, the multi-antigen targeting antibody and / or multi-antigen targeting activatable antibody binds to two or more different epitopes on the same target. In some embodiments, the multi-antigen targeting antibody and / or multi-antigen targeting activatable antibody binds to a combination of two or more different targets and two or more different epitopes on the same target.

[0126] Masking part (MM) The activatable molecules herein may contain one or more masking moieties (MMs) that can interfere with binding of the AM to its target. The masking moiety in the activatable molecule "masks" or reduces or otherwise inhibits binding of the activatable molecule to its target. In some embodiments, coupling of an AM (e.g., an antibody or fragment thereof, or other therapeutic or diagnostic protein) with a MM can inhibit the ability of the AM to specifically bind to its target by inhibitions known in the art (e.g., conformational changes, competition for antigen-binding domains, etc.). In some embodiments, coupling of an AM with a MM can result in a conformational change that reduces or inhibits the ability of the AM to specifically bind to its target. In some embodiments, coupling of a protein that includes an AM with a MM sterically blocks, reduces, or inhibits the ability of the AM to specifically bind to its target and / or epitope. In some embodiments, the MM prevents target binding of the AM when the activatable molecule is not activated, but does not substantially or significantly interfere with target binding of the AM when the activatable molecule is activated (when the substrate is cleaved by one or more proteases).

[0127] The MM can be coupled directly or indirectly (e.g., via one or more linkers described herein) to an AM (e.g., an antibody or fragment thereof, or other therapeutic or diagnostic protein) via a substrate described herein. Alternatively, the MM that interferes with target binding of the AM can be coupled directly or indirectly to a component of the activatable molecule that is not an AM. For example, the MM can be coupled directly or indirectly to a different AM. In another example, the MM can be coupled directly or indirectly to a half-life extending moiety (EM). In either case, in the tertiary or quaternary structure of the activatable structure, the MM can be in a position that allows the MM to mask the AM (e.g., proximal to the AM to be masked).

[0128] In some embodiments, the MM interacts with the AM, thereby reducing or inhibiting the interaction between the AM and its binding partner. In some embodiments, the MM comprises at least a partial or complete amino acid sequence of the naturally occurring binding partner of the AM. As used herein, the term "naturally occurring" when applied to an object refers to the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence that can be isolated from a natural source and exists in an organism (including a virus or bacterium) that has not been intentionally modified by humans in a laboratory is naturally occurring.

[0129] For example, the MM can be a fragment of a naturally occurring binding partner. The fragment can retain at least 95%, at least 90%, at least 80%, at least 75%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 25%, or at least 20% nucleic acid or amino acid sequence homology to the naturally occurring binding partner. In some embodiments, the MM is a cognate peptide of the AM. For example, the MM can include the sequence of an epitope of the AM or a fragment thereof.

[0130] In some embodiments, the MM comprises an amino acid sequence that is not naturally occurring or that does not contain the amino acid sequence of a naturally occurring binding partner or target protein. In certain embodiments, the MM is not a natural binding partner of the AM. In some embodiments, the MM does not contain a subsequence of more than 4, 5, 6, 7, 8, 9, or 10 consecutive amino acid residues of the natural binding partner of the AM. The MM can be a modified binding partner of the AM that contains amino acid changes that reduce binding affinity and / or avidity to the AM. In some embodiments, the MM contains no, or substantially no, nucleic acid or amino acid homology with the natural binding partner of the AM. In other embodiments, the MM has no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% homology with the natural binding partner of the AM.

[0131] In some embodiments, the MM is a polypeptide that binds to an AM. In some examples, the MM can be an antibody or antibody fragment (e.g., a Fab fragment, F(ab')2 fragment, scFv, scAb, dAb, single-domain heavy chain antibody, and single-domain light chain antibody) that binds to the AM and inhibits binding of the AM to its target. In some examples, the MM can be a ligand of an AM, a receptor, or a fragment thereof (e.g., the extracellular domain of a receptor) that binds to the AM and inhibits binding of the AM to its target. In some examples, when the AM is an antibody or antibody fragment thereof, the MM can be an anti-idiotype antibody or a fragment thereof (e.g., an scFv) that binds to the idiotype of the AM. In some examples, the MM can be a cytokine or a cytokine receptor. In some examples, the MM can have an amino acid sequence that is at least 85% identical to a cytokine or cytokine receptor.

[0132] In some embodiments, the MM does not bind the AM but interferes with the binding of the AM to its binding partner through nonspecific interactions, such as steric hindrance. For example, the MM can be positioned in the activatable molecule such that the tertiary or quaternary structure of the activatable molecule allows the MM to mask the AM through charge-based interactions, thereby holding the MM in place and preventing binding partners from accessing the AM. Examples of such MMs include albumin, e.g., human serum albumin (HSA), crystallizable fragment (Fc) domains, antibody constant domains (e.g., CH domains), polymers (e.g., branched or multi-arm polyethylene glycol (PEG)), latency-associated protein (LAP), and any polypeptide or other moiety that sterically interferes with the AM-target interaction. In some examples, the MM may recruit a large protein binding partner that sterically interferes with the AM-target interaction. For example, the MM can be an antibody or fragment thereof that binds to serum albumin.

[0133] Examples of suitable masking moieties include full-length or AM-binding fragments or muteins of the AM's cognate receptor, as well as AM-binding antibodies and fragments thereof, e.g., polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, single-chain variable fragments (scFv), heavy chain variable domains (VH), light chain variable domains (VL), single-domain antibodies such as variable domains of camelid nanobodies (VHH), dAbs, etc. Other exemplary antigen-binding domains that bind to AMs can also be used as MMs, including non-immunoglobulin proteins that mimic the binding and / or structure of antibodies, e.g., anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, finomers, Kunitz domain peptides, monobodies, and binding domains based on other engineered scaffolds such as SpA, GroEL, fibronectin, lipocalin, and CTLA4 scaffolds. As another example, peptides modified by conjugation to water-soluble polymers such as PEG can sterically inhibit or prevent cytokines from binding to their receptors. For example, antibodies and antigen-binding domains that bind to proteins with long serum half-lives, such as HSA, immunoglobulins, and transferrin, or receptors that recycle to the cell membrane, such as FcRn and the transferrin receptor, can also inhibit cytokines, particularly when bound to antigens. In some embodiments, MMs (e.g., those that sterically interfere with AM-target interactions) can also function as half-life extenders.

[0134] In some embodiments, the MM can have a dissociation constant for binding to the AM that is equal to or less than the dissociation constant of the AM for its target. In some embodiments, the MM does not interfere with or compete with the AM for binding to its target in an activation molecule (i.e., after cleavage of the substrate by a protease).

[0135] The structural characteristics of the MM can be selected according to factors such as the minimum amino acid sequence required to interfere with binding of the target to the AM, the target protein-protein binding pair of interest, the size of the AM, and the presence or absence of a linker.

[0136] In some embodiments, the MM can be unique to the bound AM. Examples of MMs include MMs (e.g., affinity masked) that have been specifically screened for binding to the binding domain of an AM or a fragment thereof. Methods for screening MMs to obtain MMs that are unique to an AM and that specifically and / or selectively bind to the binding domain of a binding partner / target are provided herein and can include protein display methods.

[0137] As used herein, the term "masking efficiency" refers to the activity of an activatable molecule (e.g., EC 50 ) divided by the activity of a control molecule, where the control molecule can be either a cleavage product of the activatable molecule (i.e., the activated molecule) or the AM used in the activatable molecule. Activatable molecules with reduced levels of AM activity can have a masking efficiency of greater than 10. In some embodiments, the activatable molecules described herein have a masking efficiency of greater than 10, 100, 1000, or 5000.

[0138] In some embodiments, MM is a polypeptide between about 2 and 50 amino acids in length. For example, MM can be a polypeptide between 2 and 40, 2 and 30, 2 and 20, 2 and 10, 5 and 15, 10 and 20, 15 and 25, 20 and 30, 25 and 35, 30 and 40, 35 and 45, or 40 and 50 amino acids in length. For example, MM can be a polypeptide having a length of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids. In some examples, the MM can be a polypeptide that is more than 50 amino acids in length, for example, 100, 200, 300, 400, 500, 600, 700, 800, or more amino acids in length. In some embodiments, the MM is a steric mask.

[0139] In some embodiments, an activatable molecule having an AM and an interfering MM exhibits a sustained release of the AM in the presence of a target of the AM for at least 0.1, 0.5, 1, 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours, or for 5, 10, 15, 30, 45, 60, 90, 120, 150, or 160 hours, as measured by an in vitro immunosorbent assay, e.g., as described in US20200308243A1. has no or substantially no binding of the AM to its target for 180 days or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or has 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 50% or less binding of the AM to its target compared to the binding of a corresponding molecule without the interfering MM.

[0140] The binding affinity of an AM with an interfering MM for a target or binding partner is at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, or 50,000,000 times lower than the binding affinity of an AM without an interfering MM for its binding partner, or at least 5-10, 10-100, 10-1,000, 10-10,000 times lower than the binding affinity of an AM without an interfering MM for its binding partner. 00, 10~100,000, 10~1,000,000, 10~10,000,000, 10~10,000,000, 100~1,000,000, 100~10,000,000, 1,000~10,000,000, 1,000~10,000, 1,000~100,000, It may be 1,000-1,000,000, 1000-10,000,000, 10,000-100,000, 10,000-1,000,000, 10,000-10,000,000, 100,000-1,000,000, or 100,000-10,000,000 times lower.

[0141] The dissociation constant (K d ) may be greater than the dissociation constant of the AM for the target. The dissociation constant of the MM for the masked AM may be 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 100,000, 1,000,000, or even 10,000,000 times greater than the dissociation constant of the AM for the target. Conversely, the binding affinity of the MM for the masked AM may be lower than the binding affinity of the AM for the target. The binding affinity of the MM for the AM may be at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 100,000, 1,000,000, or even 10,000,000 times lower than the binding affinity of the AM for the target.

[0142] In some embodiments, the K for the target of the AM of an activatable molecule comprising a MM and a substrate. d is the K for the target of the AM of a counterpart molecule that is essentially the same as the activatable molecule but does not contain the MM or substrate. dis at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000, or more than 5-10, 10-100, 10-1,000, 10-10,000, 10-100,000, 10-1,000,000, 10-10,000,000, 10-10,000,000, 00, 100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000, 1,000-10,000, 1,000-100,000, 1,000-1,000,000, 1000-10,000,000, 10,000-100,000, 10,000-10,000,000, 100,000-1,000,000, or 100,000-10,000,000 times larger. Conversely, the binding affinity of an activatable molecule that includes a MM and a substrate for an AM target is at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000, or more less, or 5-10, 10-100, 10-1,000, 10-10 ... 0~100,000, 10~1,000,000, 10~10,000,000, 100~1,000, 100~10,000, 100~100,000, 100~1,000,000, 100~10,000,000, 1,000~10,000, 1,000~100,000, 1, It may be 1,000-1,000,000, 1000-10,000,000, 10,000-100,000, 10,000-1,000,000, 10,000-10,000,000, 100,000-1,000,000, or 100,000-10,000,000 times lower.

[0143] In some embodiments, when an AM is coupled to a MM and in the presence of a target, specific binding of the AM to its target is reduced or inhibited compared to specific binding of an AM not coupled to a MM. When compared to the binding of an AM not coupled to a MM to its target, the target binding ability of an AM coupled to a MM may be reduced by at least 50%, 60%, 70%, 80%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours, or 5, 10, 15, 30, 45, 60, 90, 120, 150, or 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or more, as measured in an in vivo or in vitro assay.

[0144] In some embodiments, the MM comprises a non-binding steric moiety (NB) that does not bind to the AM but can interfere with binding between the AM and its target through steric hindrance, hi some embodiments, the MM comprises a binding partner (BP) for the NB, which recruits or otherwise attracts the NB to an activatable molecule.

[0145] In some embodiments, the MM comprises genetically encoded amino acid(s) or non-genetically encoded amino acids. Examples of non-genetically encoded amino acids include D-amino acids, β-amino acids, and γ-amino acids. In certain embodiments, the MM comprises no more than 50%, 40%, 30%, 20%, 15%, 10%, 5%, or 1% non-genetically encoded amino acids.

[0146] In some embodiments, the MM may have biological activity or therapeutic effects, e.g., binding ability, once released from the activatable molecule and in a free state. For example, the free peptide may bind to the same or a different binding partner. In certain embodiments, the free MM may exert a therapeutic effect and provide a secondary function to the compositions disclosed herein. In some embodiments, the MM may advantageously not exhibit biological activity once released from the activatable molecule. For example, in some embodiments, the MM does not induce an immune response in a subject after being cleaved from the activatable molecule.

[0147] Suitable MMs can be identified and / or optimized through screening procedures from libraries of candidate activatable molecules with tunable MMs. For example, AMs and substrates can be selected to provide a desired enzyme / target combination, and the amino acid sequences of the MMs can be identified by screening procedures described below to identify MMs that provide an activatable phenotype. For example, random peptide libraries (e.g., of peptides containing 2-40 or more amino acids) can be used in the screening methods disclosed herein to identify suitable MMs.

[0148] In some embodiments, MMs with specific binding affinity to an AM can be identified by a screening procedure that includes providing a library of peptide scaffolds containing candidate MMs, each scaffold consisting of a transmembrane protein and a candidate MM. The library can then be contacted with all or part of a protein, such as a full-length protein, a naturally occurring protein fragment, or a non-naturally occurring fragment containing the protein (which can also bind to a binding partner of interest), to identify one or more candidate MMs with detectable binding proteins. Screening can be performed by one or more rounds of magnetically activated sorting (MACS) or fluorescence-activated sorting (FACS), and determining the binding affinity of the MMs to the AM and subsequent masking efficiency, as described, for example, in WO2009025846 and US20200308243A1 (incorporated herein by reference in their entireties).

[0149] Examples of suitable MMs are WO2021207657, WO2021142029, WO2021061867, WO2020252349, WO2020252358, WO2020236679, WO2020176672, WO2020118109, WO2020092881, WO2020086665, WO2019213444, WO2019183218, WO2019173771, WO2019165143, WO20190 75405, WO2019046652, WO2019018828, WO2019014586, WO2018222949, WO2018165619, WO2018085555, WO2017011580, WO2016179335, WO2016179285, WO2016179257, WO2016149201, and WO2016014974, which are incorporated herein by reference in their entireties.

[0150] In some embodiments, the AM in the activatable molecule is an antibody or antigen-binding fragment that specifically binds to EGFR. In some examples, such an activatable molecule comprises an MM comprising the amino acid sequence of SEQ ID NO: 81. In some examples, such an activatable molecule comprises an MM comprising the amino acid sequence of SEQ ID NO: 82. In some examples, such an activatable molecule comprises an MM consisting of the amino acid sequence of SEQ ID NO: 81. In some examples, such an activatable molecule comprises an MM consisting of the amino acid sequence of SEQ ID NO: 82.

[0151] In some embodiments, the present disclosure includes an activatable antibody comprising an anti-EGFR antibody directly or indirectly coupled to a substrate, wherein the substrate is directly or indirectly coupled to an MM comprising or consisting of the amino acid sequence of SEQ ID NO:82. Linker

[0152] An activatable molecule may contain one or more linkers. A linker may be a connecting peptide comprising a stretch of amino acid sequence that connects two components in an activatable molecule. The linker cannot be cleaved by any protease. In some embodiments, one or more linkers can be introduced into an activatable molecule to provide flexibility at one or more junctions between domains, between moieties, between moieties and domains, or at any other junction where a linker would be beneficial. In some embodiments, when an activatable molecule is provided as a conformationally constrained construct, a flexible linker can be inserted to facilitate the formation and maintenance of structure in the activatable molecule. Any of the linkers described herein can provide the desired flexibility to facilitate inhibition of target binding or facilitate cleavage of a substrate by a protease. In some embodiments, the linker included in an activatable molecule is fully or partially flexible, and as a result, the linker can include, in addition to a flexible linker, one or more moieties that impart a less flexible structure to provide a desirable activatable molecule. Some linkers may contain cysteine residues, which may form disulfide bonds and reduce the flexibility of the construct.

[0153] In some embodiments, the linker coupled to the MM may be positioned in a tertiary or quaternary position where the MM can effectively mask the AM (e.g., proximal to the AM to be masked) and may have a length that allows the MM to mask the AM.

[0154] In most cases, the length of a linker can be determined by counting the number of amino acids in the N to C direction from the N-terminus of the linker adjacent to the C-terminal amino acid of the previous building block to the C-terminus of the linker adjacent to the N-terminal amino acid of the next building block (i.e., the length of the linker does not include either the C-terminal amino acid of the previous building block or the N-terminal amino acid of the next building block).

[0155] In some embodiments, the linkers are 1 to 50, 1 to 40, 1 to 30, 1 to 25 (e.g., 1 to 24, 1 to 22, 1 to 20, 1 to 18, 1 to 16, 1 to 15, 1 to 14, 1 to 12, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 25, 2 to 24, 2 to 22, 2 to 20, 2 to 18, 2 to 16, 2 to 15, 2 to 14, 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 2 ~5, 2~4, 2~3, 4~25, 4~24, 4~22, 4~20, 4~18, 4~16, 4~15, 4~14, 4~12, 4~10, 4~8, 4~6, 4~5, 5~25, 5~24, 5~22, 5~20, 5~18, 5~16, 5~15, 5~14, 5~12, 5~10, 5~8, 5~6, 6~25, 6~24, 6~22, 6~20, 6~18, 6~16, 6~15, 6~14, 6~ 12, 6-10, 6-8, 8-25, 8-24, 8-22, 8-20, 8-18, 8-16, 8-15, 8-14, 8-12, 8-10, 10-25, 10-24, 10-22, 10-20, 10-18, 10-16, 10-15, 10-14, 10-12, 12-25, 12-24, 12-22, 12-20, 12-18, 12-16, 12-15, 12-14, 14-25, 14- In some embodiments, the linker may comprise a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids.

[0156] In some embodiments, the linker can be rich in glycine (Gly or G) residues. In some embodiments, the linker can be rich in serine (Ser or S) residues. In some embodiments, the linker can be rich in glycine and serine residues. In some embodiments, the linker can have one or more glycine-serine residue pairs (GS) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GS pairs).

[0157] In some embodiments, a linker can have one or more Gly-Gly-Gly-Ser (GGGS) (SEQ ID NO: 402) sequences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGGS sequences (SEQ ID NO: 402)). In some embodiments, a linker can have one or more Gly-Gly-Gly-Gly-Ser (GGGGS) (SEQ ID NO: 408) sequences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGGGS sequences (SEQ ID NO: 408)). In some embodiments, a linker can have one or more Gly-Gly-Ser-Gly (GGSG) (SEQ ID NO: 395) sequences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGSG sequences (SEQ ID NO: 402)). Exemplary linkers may include glycine polymers (G)n, glycine-serine polymers (e.g., (GS)n, (GGS)n, (GSGGS)n (SEQ ID NO: 652), and (GGGS)n (SEQ ID NO: 402), where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured and can function as neutral links between components. Glycine has access to significantly more phi-psi space than alanine and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)).Exemplary flexible linkers include GGSG (SEQ ID NO:395), GGSGG (SEQ ID NO:396), GSGSG (SEQ ID NO:397), GSGGG (SEQ ID NO:398), GGGSG (SEQ ID NO:399), GSSSG (SEQ ID NO:400), GSSGGSGGSGG (SEQ ID NO:401), GGGS (SEQ ID NO:402), GGGSGGGS (SEQ ID NO:403), GGGSGGGSGGGS (SEQ ID NO:404), GGGGSGGGGSGGGGGS (SEQ ID NO:405), GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:406), GGGGSGGGGS (SEQ ID NO:407), GGGGS (SEQ ID NO:408), GS, GGGGSGS (SEQ ID NO:409), GGGGSGGGGSGGGGSGS (SEQ ID NO:410), G and combinations of one or more of: GSLDPKGGGGS (SEQ ID NO: 411), PKSCDKTHTCPPCPAPELLG (SEQ ID NO: 412), SKYGPPCPPCPAPEFLG (SEQ ID NO: 413), GKSSGSGSESKS (SEQ ID NO: 414), GSTSGSGKSSEGKG (SEQ ID NO: 415), GSTSGSGKSSEGSGSTKG (SEQ ID NO: 416), GSTSGSGKPGSGEGSTKG (SEQ ID NO: 417), GSTSGSGKPGSSEGST (SEQ ID NO: 418), GGGSSGGS (SEQ ID NO: 419), GGGGSGGGGSS (SEQ ID NO: 420), GGGSSGGSGGSSGGS (SEQ ID NO: 421), and GSTSGSGKPGSSEGST (SEQ ID NO: 422).

[0158] Exemplary linkers may further include sequences that are at least 70% identical (e.g., at least 72%, at least 74%, at least 75%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the exemplary linkers described herein. Those skilled in the art will recognize that the design of activatable molecules can include linkers that are all or partially flexible, such that the linker can include one or more moieties that confer a less flexible structure, as well as flexible linkers, to provide the desired activatable molecule structure.

[0159] In some embodiments, the activatable molecule can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 linker sequence(s) (e.g., linker sequences the same as or different from any of the exemplary linker sequences described herein or known in the art). In some embodiments, the linkers can include sulfo-SIAB, SMPB, and sulfo-SMPB, which linkers react with primary amine sulfhydryls.

[0160] Half-life extender (EM) The activatable molecule may further comprise a half-life extending moiety (EM). In some instances, the half-life extending moiety may be a serum half-life extending moiety, i.e., capable of extending the serum half-life of a molecule conjugated with the EM.

[0161] In some examples, the EM may comprise a fragment crystallizable region (Fc domain) of an antibody. For example, the EM may be an Fc domain of an IgG (e.g., IgG1, IgG2, IgG3, or IgG4). In some examples, the EM may comprise a dimer formed by two Fc domains. The Fc domain may be a wild-type peptide or a mutant. For example, the EM may comprise a dimer formed by two Fc domain mutants. In such cases, the two Fc domain mutants may be an Fc domain hole mutant and an Fc domain knob mutant. The knob and hole mutants may interact with each other to promote dimerization of the two Fc domains. In some embodiments, the knob and hole mutants may comprise one or more amino acid modifications within the interface between the two Fc domains (e.g., in the CH3 domain). In one example, the modifications include the amino acid substitution T366W, and optionally the amino acid substitution S354C, in one IgG Fc domain, and the amino acid substitutions T366S, L368A, Y407V, and optionally Y349C (numbering according to the EU numbering system) in the other IgG Fc domain. Examples of Fc variants also include SEQ ID NOs: 423-424.

[0162] Examples of Fc domain variants also include those described in U.S. Patent No. 7,695,936, which is incorporated herein by reference in its entirety. In one example, the modification comprises the amino acid substitution T366Y in one IgG Fc domain and the amino acid substitution Y407T in the other IgG Fc domain. In one example, the modification comprises the amino acid substitution T366W in one IgG Fc domain and the amino acid substitution Y407A in the other IgG Fc domain. In one example, the modification comprises the amino acid substitution F405A in one IgG Fc domain and the amino acid substitution T394W in the other IgG Fc domain. In one example, the modification comprises the amino acid substitutions T366Y and F405A in one IgG Fc domain and the amino acid substitutions T394W and Y407T in the other IgG Fc domain. In one example, the modification comprises amino acid substitutions T366W and F405W in one IgG Fc domain and amino acid substitutions T394S and Y407A in the other IgG Fc domain. In one example, the modification comprises amino acid substitutions F405W and Y407A in one IgG Fc domain and amino acid substitutions T366W and T394S in the other IgG Fc domain. In one example, the modification comprises amino acid substitution F405W in one IgG Fc domain and amino acid substitution T394S in the other IgG Fc domain. The mutation positions in the Fc domains are numbered according to the EU numbering system. The IgG Fc domain may comprise the sequence of SEQ ID NOs: 425-428 (IgG1, IgG2, IgG3, or IgG4). In these sequences, amino acids 1-107 correspond to EU numbers 341-447.

[0163] In some examples, the Fc domain variants may have reduced effector function. Examples of such Fc domains include those disclosed in US20190135943 (incorporated herein by reference in its entirety).

[0164] Further examples of EMs include immunoglobulins (e.g., IgG), serum albumins (e.g., human serum albumin (HSA)), hexa-HAT GST (glutathione S-transferase) glutathione affinity, calmodulin-binding peptide (CBP), Strep-tag, cellulose-binding domain, maltose-binding protein, S-peptide tag, chitin-binding tag, immunoreactive epitope, epitope tag, E2 tag, HA epitope tag, Myc epitope, FLAG epitope, AU1 and AU5 epitope, Glu-Glu epitope, KT3 epitope, IRS epitope, B tag epitope, protein kinase-C epitope, and VSV epitope.

[0165] In some embodiments, the serum half-life of an activatable molecule that includes an EM is longer than the serum half-life of a counterpart molecule that is substantially the same as the activatable molecule but does not include an EM, e.g., the pK of the activatable molecule is longer than the pK of the reference molecule. In some examples, an activatable molecule with an EM may have a serum half-life that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 4-fold, 6-fold, 8-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold longer than the serum half-life of the reference counterpart molecule. In some embodiments, the serum half-life of an activatable molecule having an EM when administered to an organism can be at least 15 days, 12 days, 11 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, 20 hours, 18 hours, 16 hours, 14 hours, 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, 3 hours, 2 hours, or 1 hour.

[0166] Conjugates In some aspects, the present disclosure provides conjugated polypeptides. In some embodiments, a conjugated polypeptide, as used herein, comprises a substrate-containing polypeptide conjugated to one or more agents, such as a targeting moiety that facilitates delivery to a cell or tissue of interest, a therapeutic agent (e.g., an anti-tumor agent such as a chemotherapeutic agent or an anti-neoplastic agent), a toxin, or a fragment thereof. The agent may be conjugated to an activatable molecular component. In some embodiments, the conjugated polypeptide is an antibody-drug conjugate (ADC) comprising an antibody or antigen-binding fragment thereof conjugated to a drug. In some examples, the antibody or antigen-binding fragment thereof can be conjugated to a drug via a substrate disclosed herein. In some examples, the antibody or antigen-binding fragment thereof can be an activatable antibody or antigen-binding fragment thereof (e.g., coupled to an MM via a substrate), which is further conjugated to a drug (e.g., via a cleavable or non-cleavable conjugate linker).

[0167] The term "agent" is used herein to refer to a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological material. Agents include toxins, microtubule inhibitors, nucleic acid damaging agents, dolastatins, auristatins, maytansinoids, duocarmycins, calicheamicins, or combinations thereof.

[0168] In some embodiments, the activatable molecule is conjugated to a cytotoxic agent, such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof) or a radioisotope.

[0169] Examples of cytotoxic agents that can be conjugated to an activatable molecule include dolastatin and its derivatives (e.g., auristatin E, AFP, monomethyl auristatin D (MMAD), monomethyl auristatin F (MMAF), monomethyl auristatin E (MMAE), desmethyl auristatin E (DMAE), auristatin F, desmethyl auristatin F (DMAF), dolastatin 16 (DmJ), dolastatin 16 (Dpv), auristatin derivatives (e.g., auristatin quinolones, auristatin quinolones), maytansinoids (e.g., DM-1, DM-4), maytansinoid derivatives, duocarmycins, alpha-amanitin, turbostatin, phenstatin, hydroxyphenstatin, spongistatin 5, spongistatin 7, halistatin 1, halistatin 2, halistatin 3, halocomestatin mstatin, pyrrolobenzimidazole (PBI), cibrostatin, doxaliform, cemadotin analog (CemCH2-SH), Pseudomonas toxin A (PES8) variant, Pseudomonas toxin A (ZZ-PE38) variant, ZJ-101, anthracyclines, doxorubicin, daunorubicin, bryostatin, camptothecin, 7-substituted campothecin, 11-difluoromethylenedioxycamptothecin In some embodiments, the drug is DM1 or DM4. In some embodiments, the drug is a duocarmycin or a derivative thereof. In some embodiments, the drug is a calicheamicin or a derivative thereof. In some embodiments, the drug is a pyrrolobenzodiazepine.

[0170] Examples of enzymatically active toxins that can be conjugated to activatable molecules include diphtheria toxin, exotoxin A chain from Pseudomonas aeruginosa, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleuries fordii protein, dianfhin protein, Phytolacca Americana protein (e.g., PAPI, PAPII, and PAP-8), momordica charantia inhibitor, curcin, crotirs, sapaonaria officinalis inhibitor, geoionin, mitogeliin, restrictocin, phenomycin, neomycin, and trichothecin. A wide variety of radionuclides are available for the production of radioconjugated molecules. Examples of radionuclides include: 212 Bi, 131 I, 131 In, 90 Y, and 186 Re is an example.

[0171] Examples of anti-tumor drugs that can be conjugated to an activatable molecule include adriamycin, cerbidine, bleomycin, alkeran, velban, oncovin, fluorouracil, methotrexate, thiotepa, bisantrene, novantrone, thioguanine, procarabidine, and cytarabine.

[0172] Examples of antiviral drugs that can be conjugated to activatable molecules include acyclovir, Vira A, and Symmetrel. An example of an antifungal agent that can be conjugated to an activatable molecule includes nystatin. An example of a detection reagent that can be conjugated to an activatable molecule includes fluorescein and its derivative, fluorescein isothiocyanate (FITC). Examples of antibacterial drugs that can be conjugated to an activatable molecule include aminoglycosides, streptomycin, neomycin, kanamycin, amikacin, gentamicin, and tobramycin. Examples of 3β,16β,17α-trihydroxycholest-5-en-22-one 16-O-(2-O-4-methoxybenzoyl-β-D-xylopyranosyl)-(1-->3)-(2-O-acetyl-α-L-arabinopyranoside) (OSW-1) that can be conjugated to an activatable molecule include s-nitrobenzyloxycarbonyl derivatives of O6-benzylguanine, topoisomerase inhibitors, hemiasterlin, cephalotaxine, homoharringionine, pyrrolobenzodiazepine dimers (PBDs), functionalized pyrrolobenzodiazepines, calicheamicins, podophyllotoxins, taxanes, and vinca alkaloids. Examples of radiopharmaceuticals that can be conjugated to an activatable molecule include 123 I, 89 Zr, 125 I, 131 I, 201 T1, 62 Cu, 18 F, 68 Ga, 13 N, 15 O. 38 K. 82 Rb, 111 In, 133 Xe, 11 C, and 99Examples of heavy metals that can be conjugated to activatable molecules include mTc (technetium). Examples of heavy metals that can be conjugated to activatable molecules include barium, gold, and platinum. Examples of anti-mycoplasma agents that can be conjugated to activatable molecules include tylosin, spectinomycin, streptomycin B, ampicillin, sulfanilamide, polymyxin, and chloramphenicol.

[0173] In some embodiments, the agent is a nucleic acid damaging agent, such as a DNA alkylating agent or a DNA intercalator, or other DNA damaging agent.

[0174] Further examples of drugs that can be conjugated include those in Table 1 below. [Table 1-1] [Table 1-2]

[0175] In some embodiments, the activatable molecule comprises a signal peptide. When comprising multiple polypeptides, the activatable molecule may comprise multiple signal peptides, e.g., one signal peptide for each of the multiple polypeptides. The signal peptide may be a peptide (e.g., 10-30 amino acids in length) present at the terminus (e.g., N-terminus or C-terminus) of a newly synthesized protein destined for the secretory pathway. In some embodiments, the signal peptide may be conjugated to the activatable molecule via a spacer. In some embodiments, the spacer may be conjugated to the activatable molecule in the absence of a signal peptide.

[0176] Those skilled in the art will recognize that a wide variety of potential drugs can be conjugated to any of the activatable molecules described herein. Drugs may be conjugated to another component of the activatable molecule via a conjugate linker. Conjugation can include any chemical reaction that links two molecules, so long as the activatable molecule and the other moiety retain their respective activities. Conjugation can include many chemical mechanisms, such as covalent bonding, affinity bonding, intercalation, coordinate bonding, and complexation. In some embodiments, the bond can be covalent. Covalent bonding can be achieved by direct condensation of existing side chains or by incorporation of an external crosslinking molecule. Many bivalent or multivalent linking agents can be useful for conjugating any of the activatable molecules described herein. For example, conjugation can include organic compounds such as thioesters, carbodiimides, succinimide esters, glutaraldehyde, diazobenzene, hexamethylenediamine, and the like. In some embodiments, the activatable molecule can include or otherwise incorporate one or more non-natural amino acid residues to provide suitable sites for conjugation.

[0177] In some embodiments, the drug may be attached to an activatable molecule by a disulfide bond (e.g., a disulfide bond on a cysteine molecule). Because many cancers naturally release high levels of glutathione, a reducing agent, glutathione present in the cancer tissue microenvironment can reduce the disulfide bond, followed by release of the drug at the delivery site.

[0178] In some embodiments, when the drug binds to its target in the presence of complement within the target site (e.g., diseased tissue (e.g., cancer tissue)), the amide or ester bond connecting the drug to the linker is cleaved, resulting in release of the co-drug in its activated form. When administered to a subject, these drugs can achieve delivery and release of the drug at the target site (e.g., diseased tissue (e.g., cancer tissue)). These drugs can be effective for in vivo delivery of any of the drugs described herein.

[0179] In some embodiments, one or more agents are conjugated to a component of an activatable molecule (e.g., an AM) via a conjugate linker. The conjugate linker may be a peptide or chemical moiety that connects the agent and the activatable molecule. In some examples, the conjugate linker may be cleavable (e.g., by an enzyme such as a protease). In some examples, the conjugate linker may be non-cleavable (e.g., not cleavable by an enzyme such as a protease). In some embodiments, the conjugate linker may not be cleavable by an enzyme of the complement system. In some embodiments, there are two or more conjugate linkers. The two or more conjugate linkers may be the same, i.e., cleavable or non-cleavable. The two or more conjugate linkers may be different, i.e., at least one is cleavable and at least one is non-cleavable. For example, the agent may be released without complement activation, as complement activation ultimately lyses the target cell. In such embodiments, the conjugate and / or drug will be delivered to the target cell (e.g., hormones, enzymes, corticosteroids, neurotransmitters, or genes). Additionally, the conjugate linker may be mildly susceptible to cleavage by serum proteases, allowing the conjugate and / or drug to be slowly released at the target site.

[0180] In some embodiments, the drug is conjugated to the activatable molecular component via a maleimidocaproyl-valine-citrulline linker or a maleimidoPEG-valine-citrulline linker. In some embodiments, the drug is conjugated to the activatable molecular component via a maleimidocaproyl-valine-citrulline linker. In some embodiments, the drug is conjugated to the activatable molecular component via a maleimidoPEG-valine-citrulline linker. In some embodiments, the drug is conjugated to the activatable molecular component via a maleimidoPEG-valine-citrulline-para-aminobenzyloxycarbonyl linker, where the linker-payload construct is vc-MMAD. In some embodiments, the drug is monomethylauristatin D (MMAD) conjugated to the activatable molecular component via a maleimidoPEG-valine-citrulline-para-aminobenzyloxycarbonyl linker, where the linker-payload construct is vc-MMAE.

[0181] In some embodiments, the agent can be designed so that the agent is delivered to a target site (e.g., diseased tissue (e.g., cancerous tissue)) but the conjugate and / or agent is not released.

[0182] In some embodiments, the agent may be attached directly to the AM or may be attached via an amino acid (e.g., a D-amino acid), a peptide, a thiol-containing moiety, or other organic compound that can be modified by the methods described herein to contain a functional group that is then available for attachment to the AM.

[0183] In some embodiments, the activatable molecule comprises at least one point of conjugation of a drug. In some embodiments, all available points of conjugation are available for conjugation to a drug. In some embodiments, one or more points of conjugation can include sulfur atoms involved in disulfide bonds, sulfur atoms involved in interchain disulfide bonds, sulfur atoms involved in interchain but not intrachain disulfide bonds, and / or sulfur atoms of cysteine or other amino acid residues containing sulfur atoms. In such cases, the residues can be naturally occurring in the protein construct structure or can be incorporated into the protein construct using methods including site-directed mutagenesis, chemical conversion, or misincorporation of unnatural amino acids.

[0184] The present disclosure also provides methods and materials for preparing activatable molecules with one or more conjugated agents. In some embodiments, the activatable molecules can be modified to contain one or more interchain disulfide bonds. For example, the disulfide bonds can be reduced after exposure to a reducing agent, such as, but not limited to, TCEP, DTT, or β-mercaptoethanol. In some cases, the reduction of the disulfide bonds can be only partial. As used herein, the term partial reduction refers to a situation in which an activatable molecule is contacted with a reducing agent and a portion of all possible conjugation sites undergo reduction (e.g., not all disulfide bonds are reduced). In some embodiments, an activatable molecule can be partially reduced if, after contact with a reducing agent, all available sites on the conjugate are reduced by less than 99% (e.g., less than 98%, less than 97%, less than 96%, less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, or less than 5%). In some embodiments, an activatable molecule having a reduction in one or more interchain disulfide bonds can be conjugated to an agent that is reactive with free thiols.

[0185] The present disclosure also provides methods and materials for conjugating a therapeutic agent to a specific position on an activatable molecule. In some embodiments, an activatable molecule may be modified to allow a therapeutic agent to be conjugated to the activatable molecule at a specific position on the activatable molecule. For example, the activatable molecule may be partially reduced in a manner that facilitates conjugation to the activatable molecule. In such cases, partial reduction of the activatable molecule may occur without reducing the conjugation site within the activatable molecule. In some embodiments, the conjugation site(s) on the activatable molecule may be selected to facilitate conjugation of the agent at a specific position on the protein construct. Upon treatment with a reducing agent, various factors may affect the "level of reduction" of the activatable molecule. For example, achieving partial reduction of an activatable molecule using the methods and materials described herein may require optimization of, but is not limited to, the ratio of reducing agent to activatable molecule, the length of incubation, the incubation temperature, and / or the pH of the reduction reaction solution. Any suitable combination of factors (e.g., the ratio of reducing agent to activatable molecule, the length and temperature of incubation with the reducing agent, and / or the pH of the reducing agent) can be used to achieve partial reduction of the activatable molecule (e.g., overall reduction of available conjugation sites or reduction of specific conjugation sites).

[0186] An effective ratio of reducing agent to activatable molecule can be any ratio that at least partially (i.e., partially or completely) reduces the activatable molecule in a manner that allows for conjugation to a drug (e.g., overall reduction of available conjugation sites or reduction of specific conjugation sites). In some embodiments, the ratio of reducing agent to activatable molecule can be in the range of about 20:1 to 1:1, 10:1 to 1:1, 9:1 to 1:1, 8:1 to 1:1, 7:1 to 1:1, 6:1 to 1:1, 5:1 to 1:1, 4:1 to 1:1, 3:1 to 1:1, 2:1 to 1:1, 20:1 to 1:1.5, 10:1 to 1:1.5, 9:1 to 1:1.5, 8:1 to 1:1.5, 7:1 to 1:1.5, 6:1 to 1:1.5, 5:1 to 1:1.5, 4:1 to 1:1.5, 3:1 to 1:1.5, 2:1 to 1:1.5, 1.5:1 to 1:1.5, or 1:1 to 1:1.5.

[0187] Effective incubation times and temperatures for treating an activatable molecule with a reducing agent can be any time and temperature that at least partially reduces the activatable molecule (e.g., overall reduction of available conjugation sites or reduction of specific conjugation sites) in a manner that allows for conjugation of an agent to the activatable molecule. In some embodiments, incubation times and temperatures for treating an activatable molecule can range from about 1 hour at 37°C to about 12 hours at 37°C (or any subrange therein).

[0188] The effective pH of the reduction reaction for treating an activatable molecule with a reducing agent can be any pH that at least partially reduces the activatable molecule (e.g., overall reduction of available conjugation sites or reduction of specific conjugation sites) in a manner that allows for conjugation of the activatable molecule to a drug.

[0189] When a partially reduced activatable molecule contacts a thiol-containing drug, the drug can be conjugated to the interchain thiol within the activatable molecule. The drug can be modified to contain a thiol using a thiol-containing reagent (e.g., cysteine or N-acetylcysteine). For example, the activatable molecule can be partially reduced after incubation with a reducing agent (e.g., TEPC) at a desired ratio of reducing agent to activatable molecule for about 1 hour at about 37°C. An effective ratio of reducing agent to activatable molecule can be any ratio that partially reduces at least two interchain disulfide bonds located within the activatable molecule in a manner that allows conjugation of the thiol-containing drug (e.g., reducing all available conjugation sites or reducing specific conjugation sites).

[0190] In some embodiments, the activatable molecule can be reduced by a reducing agent in a manner that avoids reduction of any intrachain disulfide bonds, hi some embodiments, the activatable molecule can be reduced by a reducing agent in a manner that avoids reduction of any intrachain disulfide bonds and reduces at least one interchain disulfide bond.

[0191] In some embodiments, the agent may be a detectable moiety, such as, for example, a label or other marker. For example, the agent may be or may include a radiolabeled amino acid, one or more biotinyl moieties detectable by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or calorimetric methods), one or more radioisotopes or radionuclides, one or more fluorescent labels, one or more enzymatic labels, and / or one or more chemiluminescent agents. In some embodiments, the detectable moiety may be attached by a spacer molecule. In some embodiments, the detectable label may include an imaging agent, a contrast agent, an enzyme, a fluorescent label, a chromophore, a dye, one or more metal ions, or a ligand-based label. In some embodiments, the contrast agent may include a radioisotope. In some embodiments, the radioisotope may be indium or technetium. In some embodiments, the contrast agent may include iodine, gadolinium, or iron oxide. In some embodiments, the enzyme may include horseradish peroxidase, alkaline phosphatase, or β-galactosidase. In some embodiments, the fluorescent label may comprise yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), green fluorescent protein (GFP), modified red fluorescent protein (mRFP), red fluorescent protein tdimer2 (RFP tdimer2), HCRED, or a europium derivative. In some embodiments, the luminescent label may comprise an N-methylacridium derivative. In some embodiments, the label may comprise an Alexa Fluor® label, such as Alex Fluor® 680 or Alexa Fluor® 750. In some embodiments, the ligand-based label may comprise biotin, avidin, streptavidin, or one or more haptens.

[0192] Further examples of detectable labels also include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive materials include 125 I, 131 I, 35 S, or 3 Contains H.

[0193] In some embodiments, an agent may be conjugated to an activatable molecule using a carbohydrate moiety, a sulfhydryl group, an amino group, or a carboxylate group. In some embodiments, an agent may be conjugated to an activatable molecule via a linker and / or a substrate described herein. In some embodiments, an agent may be conjugated to a cysteine or lysine within the activatable molecule. In some embodiments, an agent may be conjugated to another residue of the activatable molecule, such as a residue disclosed herein.

[0194] In some embodiments, a variety of bifunctional protein coupling agents can be used to conjugate agents to activatable molecules, including N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., triene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). In some embodiments, radionucleotides can be conjugated to activatable molecules using a carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) chelator. (See, e.g., WO 94 / 11026).

[0195] Suitable conjugate linkers include those described in the literature (see, e.g., Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984) describing the use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester). See also U.S. Pat. No. 5,030,719, describing the use of halogenated acetylhydrazide derivatives attached to activatable molecules via oligopeptides. In some embodiments, suitable conjugate linkers include (i) EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride); (ii) SMPT (4-succinimidyloxycarbonyl-alpha-methyl-α-(2-pridyl-dithio)-toluene (Pierce Chem. Co., Catalog No. (21558G); (iii) SPDP (succinimidyl-6[3-(2-pyridyldithio)propionamido]hexanoate (Pierce Chem. Co., Catalog No. 21651G); (iv) sulfo-LC-SPDP (sulfosuccinimidyl-6[3-(2-pyridyldithio)-propionamido]hexanoate (Pierce Chem. Co., Catalog No. 2165-G); and (v) sulfo-NHS (N-hydroxysulfosuccinimide) conjugated to EDC (Pierce Chem. Co., Catalog No. 2165-G). Chem. Co., catalog number 24510. Additional exemplary agents include SMCC, sulfo-SMCC, SPDB, and sulfo-SPDB.

[0196] Exemplary conjugate linkers for attachment to reduced activatable molecules include those having specific reactive groups capable of reacting with sulfhydryl groups on reduced antibodies or fragments. Such reactive groups include reactive haloalkyl groups (including, for example, haloacetyl groups), p-mercuribenzoate groups, and groups capable of Michael-type addition reactions (including, for example, maleimides and groups of the type described in Mitra and Lawton, 1979, J. Amer. Chem. Soc. 101:3097-3110).

[0197] Exemplary conjugate linkers that do not bind to either oxidized or reduced activatable molecules include those with specific functional groups that can react with primary amino groups present on unmodified lysine residues of activatable molecules. Such reactive groups include NHS carboxylic acid or carbonate esters, sulfo-NHS carboxylic acid or carbonate esters, 4-nitrophenyl carboxylic acid or carbonate esters, pentafluorophenyl carboxylic acid or carbonate esters, acylimidazoles, isocyanates, and isothiocyanates, as well as other dehydrating agents that are utilized to form carboxamides. In these cases, functional groups present on suitable conjugate linkers include primary and secondary amines, hydrazines, hydroxylamines, and hydrazides.

[0198] The drug can be attached to the conjugate linker before or after the conjugate linker is attached to the activatable molecule. In certain applications, it may be desirable to first generate an activatable molecule-conjugate linker intermediate in which the conjugate linker does not contain an association agent. Depending on the particular application, a specific drug can be covalently attached to the conjugate linker. In some embodiments, the AM is first attached to the MM, substrate, and associated linking peptide, and then attached to the conjugate linker for conjugation purposes.

[0199] In certain embodiments, a branched conjugate linker is utilized that has multiple sites for drug attachment. In the case of a multi-site conjugate linker, a single covalent bond to an activatable molecule can result in an activatable molecule-linker intermediate that can attach drugs at many sites. The sites can be aldehyde groups, sulfhydryl groups, or any chemical site to which a drug can be attached.

[0200] In some embodiments, higher specific activity (or higher drug to activatable molecule ratio) can be achieved by attaching a single-site conjugate linker to multiple sites on the activatable molecule. These multiple sites can be introduced into the activatable molecule by one of two methods. First, multiple aldehyde and / or sulfhydryl groups can be generated within the same activatable molecule. Second, a branched conjugate linker with multiple functional sites for subsequent attachment to a conjugate linker can be attached to the aldehydes or sulfhydryls of the activatable molecule. The functional sites of the branched or multi-site conjugate linker can be aldehyde or sulfhydryl groups, or any chemical site to which a conjugate linker can be attached. Combining these two approaches, i.e., attaching a multi-site conjugate linker to several sites on the activatable molecule, can potentially achieve even higher specific activity.

[0201] In one embodiment of the present disclosure, a peptide conjugate linker susceptible to cleavage by an enzyme of the complement system, such as, but not limited to, u-plasminogen activator, tissue plasminogen activator, trypsin, plasmin, or another enzyme with proteolytic activity, can be used. According to one method of the present disclosure, a drug is attached via a conjugate linker susceptible to cleavage by complement. The antibody is selected from a class capable of activating complement. Thus, the antibody-drug conjugate activates the complement cascade and releases the drug at the target site. According to another method of the present disclosure, a drug is attached via a conjugate linker susceptible to cleavage by an enzyme with proteolytic activity, such as u-plasminogen activator, tissue plasminogen activator, plasmin, or trypsin. These cleavable conjugate linkers are useful in conjugated activatable molecules containing exotoxins, for example, any of the exotoxins listed in Table 1, as non-limiting examples.

[0202] Non-limiting examples of cleavable linker sequences include any of the cleavable sequences disclosed herein or incorporated by reference herein, as well as the exemplary sequences provided in Table 2. [Table 2]

[0203] Additionally, drugs can be attached to activatable molecules via disulfide bonds (e.g., disulfide bonds on cysteine molecules). Many tumors naturally release high levels of glutathione (a reducing agent), which can reduce the disulfide bond, followed by release of the drug at the delivery site. In some embodiments, the reducing agent that modifies the substrate will also modify the conjugate linker of the conjugated activatable molecule.

[0204] In some embodiments, it may be necessary to construct the conjugate linker in such a way as to optimize the spacing between the drug and the activatable molecule, which can be achieved by using conjugate linkers of the general structure: W-(CH2)nQ During the ceremony, W is either -NH-CH2- or -CH2-; Q is an amino acid, a polypeptide having 2 to 20 amino acids, and n is an integer of 0 to 20.

[0205] In some embodiments, the conjugate linker may comprise a spacer element and a cleavable element. The spacer element serves to space the cleavable element away from the core of the activatable molecule, making it more accessible to the enzyme responsible for cleavage. Some of the branched linkers described above may function as spacer elements.

[0206] It should be understood throughout this discussion that the attachment of a conjugate linker to a drug (or the attachment of a spacer element to a cleavable element, or the cleavable element to a drug) does not have to be by any particular bond or reaction mode: any reaction that provides a product with suitable stability and biological compatibility is acceptable.

[0207] In some embodiments, when drug release is desired, an activatable molecule is used that is a class of antibody capable of activating complement. The resulting conjugate retains both the ability to bind to an antigen and the ability to activate the complement cascade. Thus, according to this embodiment of the present disclosure, a drug is attached to one end of a cleavable conjugate linker or cleavable element, and the other end of the conjugate linker group is bound to a specific site on the activatable molecule. For example, if the drug has a hydroxyl or amino group, it can be attached to the carboxyl terminus of a peptide, amino acid, or other appropriately selected conjugate linker via an ester or amide bond, respectively. For example, such a drug can be attached to a linker peptide via a carbodiimide reaction. If the drug contains functional groups that interfere with binding to the conjugate linker, these interfering functional groups can be blocked prior to conjugation and then unblocked once the product conjugate or intermediate is produced. The opposite amino terminus of the linker is used, either directly or after further modification, to attach to an activatable molecule capable of activating complement.

[0208] The conjugate linker (or spacer element of the conjugate linker) can be of any length, and one end can be covalently attached to a specific site on the activatable molecule. The other end of the conjugate linker or spacer element can be attached to an amino acid or peptide conjugate linker.

[0209] Thus, when these conjugates bind to an antigen in the presence of complement, the amide or ester bond connecting the drug to the linker is cleaved, resulting in the release of the drug in an active form. When administered to a subject, these conjugates achieve delivery and release of the drug at the target site and are particularly useful for the in vivo delivery of pharmaceuticals, antibiotics, antimetabolites, antiproliferative agents, and the like.

[0210] In some embodiments, it is desirable to release a drug without complement activation, because activation of the complement cascade ultimately lyses target cells. This approach is therefore useful when drug delivery and release must be achieved without killing the target cells. This is the goal when it is desired to deliver cellular mediators, such as hormones, enzymes, corticosteroids, neurotransmitters, genes, or enzymes, to target cells. These conjugates can be prepared by attaching a drug to an activatable molecule that cannot activate complement via a linker that is only mildly susceptible to cleavage by serum proteases. When this conjugate is administered to an individual, the antigen-antibody complex forms rapidly, while the drug is slowly cleaved, resulting in the release of the compound at the target site.

[0211] In some embodiments, activatable molecules can be conjugated to one or more therapeutic agents using specific biochemical cross-linking reagents. Cross-linking reagents form molecular bridges that link functional groups of two different molecules. To conjugate two different proteins in a stepwise fashion, heterobifunctional cross-linkers can be used, which eliminate the formation of undesired homopolymers.

[0212] Peptidyl conjugate linkers that can be cleaved by lysosomal proteases, such as Val-Cit, Val-Ala, or other dipeptides, are also useful.In addition, acid-labile conjugate linkers that can be cleaved in the low pH environment of lysosomes, such as bis-sialyl ether, can also be used.Other suitable conjugate linkers include cathepsin-labile substrates, particularly those that function optimally at acidic pH.

[0213] Exemplary heterobifunctional crosslinkers are shown in Table 3. [Table 3-1] [Table 3-2]

[0214] In some embodiments, agents can be designed to be delivered to a target but not released, which can be achieved by attaching the agent to an activatable molecule directly or via a non-cleavable conjugate linker.

[0215] These non-cleavable conjugate linkers may include amino acids, peptides, D-amino acids, or other organic compounds that may be modified by the methods described herein to contain functional groups that are subsequently available for attachment to activatable molecules.

[0216] In some embodiments, the compound may be conjugated to an activatable molecule that does not activate complement. When an activatable molecule that cannot activate complement is used, this conjugation may be achieved using a conjugate linker that is susceptible to cleavage by activated complement, or using a linker that is not susceptible to cleavage by activated complement.

[0217] The substrate-containing polypeptides disclosed herein can also be formulated as immunoliposomes. Antibody-containing liposomes can be prepared by methods known in the art, such as those described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82:3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77:4030 (1980); and US Pat. Nos. 4,485,045 and 4,544,545. Liposomes with improved circulation time are described in U.S. Pat. No. 5,013,556. Particularly useful liposomes can be generated by the reverse-phase evaporation method using a lipid composition containing phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Components of the activatable molecule can be conjugated to liposomes via a disulfide exchange reaction as described in Martin et al., J. Biol. Chem., 257:286-288 (1982).

[0218] The above-mentioned drugs may contain components with different properties, thus resulting in conjugates with different physicochemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates. NHS ester-containing linkers are less soluble than sulfo-NHS esters. Furthermore, SMPT can contain sterically hindered disulfide bonds to form conjugates with increased stability. Disulfide bonds are generally less stable than other bonds because they are cleaved in vitro, resulting in fewer usable conjugates. Sulfo-NHS can particularly enhance the stability of carbodiimide coupling. When carbodiimide coupling (e.g., EDC) is used in conjunction with sulfo-NHS, it forms esters that are more resistant to hydrolysis than carbodiimide coupling alone.

[0219] Those skilled in the art will recognize that a wide variety of possible drugs can be conjugated to the activatable molecules herein. (See, e.g., "Conjugate Vaccines," Contributions to Microbiology and Immunology, JM Cruse and RE Lewis, Jr. (eds), Carger Press, New York, (1989), the entire contents of which are incorporated herein by reference.) In general, effective conjugation of a drug (e.g., a cytotoxic agent) to an activatable molecule can be achieved by any chemical reaction that conjugates the drug to the activatable molecule while also allowing the drug and activatable molecule to retain functionality.

[0220] Nucleic acids and vectors In some aspects, the present disclosure further provides nucleic acids comprising sequences encoding the substrate-containing polypeptides and polypeptide complexes (e.g., activatable molecules) herein, or components or fragments thereof. The nucleic acid may comprise a coding sequence for the substrate. The nucleic acid may further comprise coding sequences for other components within the activatable molecule, such as AM, MM, EM, and / or linker(s). When the activatable molecule comprises multiple polypeptides, the nucleic acid may comprise coding sequences for the multiple polypeptides. In some examples, the coding sequence for one of the polypeptides is contained in a nucleic acid molecule and the coding sequence for another of the polypeptides is contained in a separate nucleic acid molecule. In some examples, the coding sequences for two or more of the multiple polypeptides are contained in the same nucleic acid molecule.

[0221] Unless otherwise specified, a "protein-encoding nucleic acid sequence" includes all nucleotide sequences that are degenerate versions of each other and therefore encode the same amino acid sequence. The term "nucleic acid" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), or a combination thereof, in single- or double-stranded form. Unless otherwise limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the referenced nucleotides. Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses complementary sequences in addition to the sequence explicitly indicated. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is RNA.

[0222] Modifications to nucleotide sequences can be introduced by standard techniques known in the art, such as site-directed mutagenesis and polymerase chain reaction (PCR)-mediated mutagenesis. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with acidic side chains (e.g., aspartate and glutamate), amino acids with basic side chains (e.g., lysine, arginine, and histidine), nonpolar amino acids (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), uncharged polar amino acids (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine), hydrophilic amino acids (e.g., arginine, asparagine, aspartate, glutamine, glutamate, histidine, lysine, serine, and threonine), and hydrophobic amino acids (e.g., alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine). Other families of amino acids include the aliphatic hydroxyamino acids (e.g., serine and threonine), the amide family (e.g., asparagine and glutamine), the aliphatic family (e.g., alanine, valine, leucine, and isoleucine), and the aromatic family (e.g., phenylalanine, tryptophan, and tyrosine).

[0223] The present disclosure further provides vectors and vector sets comprising any of the nucleic acids described herein. Those skilled in the art will be able to select an appropriate vector or vector set (e.g., expression vectors) to generate any of the activatable molecules described herein and use the vector or vector set to express any of the activatable molecules described herein. For example, when selecting a vector or vector set, the cell type can be selected so that the vector(s) can be integrated into the chromosome of the cell and / or replicate therein. Examples of vectors that can be used to generate activatable molecules are also described herein. As used herein, the term "vector" refers to a polynucleotide that can induce expression of a recombinant protein (e.g., a first or second monomer) in a cell (e.g., any of the cells described herein). A "vector" is capable of delivering nucleic acids and their fragments into a host cell and contains control sequences (e.g., promoters, enhancers, poly(A) signals). An exogenous polynucleotide can be inserted into an expression vector for expression. The term "vector" also encompasses artificial chromosomes, plasmids, retroviruses, and baculovirus vectors.

[0224] Methods for constructing vectors containing any of the nucleic acids described herein and suitable for transforming cells (e.g., mammalian cells) are well known in the art. See, e.g., Sambrook et al., Eds. "Molecular Cloning: A Laboratory Manual," 2nd Ed., Cold Spring Harbor Press, 1989 and Ausubel et al., Eds. "Current Protocols in Molecular Biology," Current Protocols, 1993.

[0225] Examples of vectors include plasmids, transposons, cosmids, and viral vectors (e.g., any adenoviral vector (e.g., pSV or pCMV vector), adeno-associated viral (AAV) vector, lentiviral vector, and retroviral vector), as well as any Gateway® vector. A vector can, for example, contain sufficient cis-acting elements for expression; other elements for expression can be supplied by the host mammalian cell or in an in vitro expression system. One of skill in the art would be able to select suitable vectors and mammalian cells for producing any of the activatable molecules described herein.

[0226] In some embodiments, the substrate-containing polypeptide can be made biosynthetically using recombinant DNA technology and expression in eukaryotic or prokaryotic species.

[0227] cell In some aspects, the disclosure provides recombinant host cells comprising any of the vectors or nucleic acids described herein. The cells can be used to produce substrate-containing polypeptides (e.g., activatable molecules) described herein. In some embodiments, the cells can be animal cells, mammalian cells (e.g., human cells), rodent cells (e.g., mouse cells, rat cells, hamster cells, or guinea pig cells), non-human primate cells, insect cells, bacterial cells, fungal cells, or plant cells. In some embodiments, the cells can be eukaryotic cells. As used herein, the term "eukaryotic cell" refers to a cell having a distinct, membrane-bound nucleus. Such cells can include, for example, mammalian (e.g., rodent, non-human primate, or human), insect, fungal, or plant cells. In some embodiments, the eukaryotic cell is a yeast cell, such as Saccharomyces cerevisiae. In some embodiments, the eukaryotic cell is a higher eukaryotic cell, such as a mammalian, avian, plant, or insect cell. Non-limiting examples of mammalian cells include Chinese hamster ovary (CHO) cells and human embryonic kidney cells (e.g., HEK293 cells). In some embodiments, the cell may be a prokaryotic cell, such as an E. coli cell.

[0228] Methods for introducing nucleic acids and vectors (e.g., any of the vectors or sets of vectors described herein) into cells are known in the art. Examples of methods that can be used to introduce nucleic acids into cells include lipofection, transfection, calcium phosphate transfection, cationic polymer transfection, viral transduction (e.g., adenoviral transduction, lentiviral transduction), nanoparticle transfection, and electroporation.

[0229] In some embodiments, the introducing step comprises introducing into the cell a vector (e.g., any of the vectors or sets of vectors described herein) containing nucleic acids encoding the monomers that make up any of the activatable molecules described herein.

[0230] Compositions and Kits The present disclosure also provides compositions and kits that include the substrate-containing polypeptides (e.g., activatable molecules or conjugated polypeptides) described herein. The compositions and kits may further include one or more excipients, carriers, reagents, or instructions required for use of the activatable molecules.

[0231] In some embodiments, the composition may be a pharmaceutical composition comprising a substrate-containing polypeptide, its derivatives, fragments, analogs, and homologs. The pharmaceutical composition may comprise a substrate-containing polypeptide and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Suitable examples of such carriers or diluents include water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles, such as liposomes and fixed oils, may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Any conventional media or agent is contemplated in the compositions, except insofar as it is incompatible with the active compound. Supplementary active compounds may also be incorporated into the compositions.

[0232] Pharmaceutical compositions can be formulated to be compatible with the intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (e.g., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can contain one or more of the following components: a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; an antibacterial agent such as benzyl alcohol or methylparaben; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating agent such as ethylenediaminetetraacetic acid (EDTA); a buffer such as acetic acid, citric acid, or phosphate; and an agent for adjusting osmotic pressure such as sodium chloride or dextrose. pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be enclosed in glass or plastic ampoules, disposable syringes, or multiple-dose vials. In some cases, any of the activatable molecules described herein are prepared with carriers that protect them from rapid elimination from the body, such as sustained-release and controlled-release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic-co-glycolic acid, and polylactic acid can be used. Methods for preparing such pharmaceutical compositions and formulations are readily apparent to those skilled in the art. For example, the activatable molecules may be incorporated into microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or macroemulsions.

[0233] Sustained-release preparations may also be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the substrate-containing polypeptide, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactides, copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers (e.g., injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter periods.

[0234] In some embodiments, pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor® EL (CAS No. 61791-12-6) (BASF, Parsippany, NJ), which is a mixture of polyoxyethylated triglycerides prepared by reacting castor oil with ethylene oxide in a molar ratio of 1:35, acting as a non-ionic surfactant, or phosphate-buffered saline (PBS). The composition may be sterile, liquid, and of a viscosity that is easy to inject. It must be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. In the case of dispersed particle compositions, proper fluidity can be maintained, for example, by the use of a coating on the particles, such as lecithin, and by maintaining the required particle size in the case of dispersions, and by the use of surfactants. In some embodiments, the pharmaceutical composition may further contain one or more antibacterial and / or antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In some embodiments, isotonic agents, such as sugars, polyalcohols such as mannitol, sorbitol, and salts such as sodium chloride, may be included in the composition. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

[0235] In some embodiments, pharmaceutical compositions can comprise sterile injectable solutions.Sterile injectable solutions can be prepared by incorporating the required amount of active compound into a suitable solvent, along with one or a combination of the above-listed ingredients as needed, followed by filtration sterilization.Generally, dispersion can be prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the other necessary ingredients listed above.For the preparation of sterile injectable powder, the preparation method is vacuum drying and freeze-drying, which allows the powder of active ingredient plus any additional desired ingredients to be obtained from the solution that has been previously sterile-filtered.

[0236] In some embodiments, the pharmaceutical composition may include an oral composition. The oral composition may include an inert diluent or an edible carrier. They may be enclosed in gelatin capsules or compressed into tablets. For oral therapeutic administration, the active compound may be incorporated with an excipient and used in the form of tablets, lozenges, or capsules. Oral compositions may also be prepared using a fluid carrier for use as a mouthwash; the fluid carrier containing the compound is poured into the mouth, swished, and expectorated or swallowed. Pharmaceutically compatible binding agents and / or adjuvants may be included as part of the composition. Tablets, pills, capsules, troches and the like may contain any of the following ingredients: a binder such as microcrystalline cellulose, gum tragacanth, or gelatin; an excipient such as starch or lactose; a disintegrating agent such as alginic acid, Primogel® (sodium starch glycolate), or corn starch; a lubricant such as magnesium stearate; a glidant such as colloidal silicon dioxide; a sweetener such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavor, or compounds of a similar nature.

[0237] In some embodiments, the pharmaceutical compositions can be formulated for administration by inhalation. For example, the compounds can be delivered in the form of an aerosol spray from a pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0238] In some embodiments, the pharmaceutical composition may be formulated for systemic administration. For example, systemic administration may be intravenous administration, transmucosal administration, or transdermal administration. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated may be used in the formulation. Such penetrants are generally known in the art, and include, for example, detergents, bile salts, and fusidic acid derivatives for transmucosal administration. Transmucosal administration can be achieved by using nasal sprays or suppositories. For transdermal administration, the active compound may generally be formulated into ointments, salves, gels, or creams, as is known in the art.

[0239] In some embodiments, pharmaceutical compositions can be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0240] In one embodiment, the pharmaceutical composition can be prepared with a carrier that protects the composition against rapid elimination from the body, such as a controlled-release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic-co-glycolic acid, and polylactic acid. Methods for preparing such formulations will be apparent to those skilled in the art.

[0241] For ease of administration and uniformity of dosage, it may be advantageous to formulate oral or parenteral compositions in dosage unit form.Dosage unit form as used herein refers to a physically separate unit suitable as a unit dosage for the subject to be treated, and each unit contains a predetermined amount of active compound calculated to produce desired therapeutic effect in association with required pharmaceutical carrier.The specification of dosage unit form of the present disclosure is determined by and can directly depend on the unique characteristics of active compound and the specific therapeutic effect to be achieved, and the inherent limitations in the art of compounding such active compound for individual treatment.

[0242] In some embodiments, the compositions (eg, pharmaceutical compositions) may be included in a container, vial, syringe, injector pen, pack, or dispenser, optionally together with instructions for administration.

[0243] Also provided herein are kits containing any of the substrate-containing polypeptides (e.g., activatable molecules or conjugated polypeptides) described herein, any of the compositions containing any of the polypeptides described herein, or any of the pharmaceutical compositions containing any of the polypeptides described herein. Also provided are kits that include, in addition to the polypeptides described herein, one or more second therapeutic agent(s). The second therapeutic agent(s) may be provided in a dosage form separate from the polypeptides described herein. Alternatively, the second therapeutic agent(s) may be prepared together with the polypeptides described herein.

[0244] Any of the kits described herein can include instructions for using any of the compositions (e.g., pharmaceutical compositions) and / or any of the substrate-containing polypeptides (e.g., activatable molecules or conjugated polypeptides) described herein. In some embodiments, the kits can include instructions for practicing any of the methods described herein. In some embodiments, the kits can include at least one dose of any of the compositions (e.g., pharmaceutical compositions) described herein. In some embodiments, the kits can provide a syringe for administering any of the pharmaceutical compositions described herein.

[0245] Also provided herein are substrate-containing polypeptides (e.g., activatable molecules or conjugated polypeptides) produced by any of the methods described herein. Also provided are compositions (e.g., pharmaceutical compositions) containing any of the polypeptides produced by any of the methods described herein. Also provided herein are kits including at least one dose of any of the compositions (e.g., pharmaceutical compositions) described herein.

[0246] Method for producing substrate-containing polypeptide Provided herein are methods for producing a substrate-containing polypeptide (e.g., an activatable molecule or conjugated polypeptide) described herein, the methods comprising: (a) culturing any of the recombinant host cells described herein in a liquid medium under conditions sufficient to produce the substrate-containing polypeptide; and (b) recovering the substrate-containing polypeptide from the host cell and / or the liquid medium.

[0247] Methods for culturing cells are well known in the art. In some embodiments, cells can be maintained in vitro under conditions that favor cell proliferation, cell differentiation, and cell growth. For example, recombinant cells can be cultured by contacting cells (e.g., any of the cells described herein) with a cell culture medium containing necessary growth factors and sufficient supplements to support cell viability and growth.

[0248] In some embodiments, the method may further include isolating the recovered substrate-containing polypeptide (e.g., the activatable molecule or conjugated polypeptide). Isolation of the substrate-containing polypeptide can be carried out using any separation or purification technique for separating protein species, such as affinity tag-based protein purification (e.g., polyhistidine (His) tag, glutathione-S-transferase tag, etc.), ammonium sulfate precipitation, polyethylene glycol precipitation, size exclusion chromatography, ligand affinity chromatography (e.g., Protein A chromatography), ion exchange chromatography (e.g., anion or cation), hydrophobic interaction chromatography, etc.

[0249] The compositions and methods described herein may involve the use of non-reducing or partially reducing conditions that allow for the formation of disulfide bonds between the MM and AM of the activatable molecule.

[0250] In some embodiments, the method further comprises formulating the isolated polypeptide into a pharmaceutical composition. Various formulations are known in the art and are described herein. Any of the isolated polypeptides described herein can be prepared for any route of administration (e.g., intravenous, intratumoral, subcutaneous, intradermal, oral (e.g., inhalation), transdermal (e.g., topical), transmucosal, or intramuscular).

[0251] Methods of Using Substrate-Containing Polypeptides In some aspects, the present disclosure further provides methods of using the substrate-containing polypeptides herein. In some embodiments, the present disclosure provides methods of treating a disease in a subject (e.g., cancer (e.g., any of the cancers described herein)) comprising administering to the subject a therapeutically effective amount of any of the polypeptides described herein (e.g., activatable molecules or conjugated polypeptides). In some embodiments, the present disclosure provides methods of preventing, slowing the progression of, treating, alleviating symptoms, or otherwise ameliorating a disease in a subject by administering to a subject in need thereof a therapeutically effective amount of a polypeptide (e.g., an activatable molecule or conjugated polypeptide) described herein. The term "treatment" means improving at least one symptom of a disorder. In some embodiments, the disease being treated may be cancer or an autoimmune disease, and may be for the purpose of alleviating at least one symptom of cancer or an autoimmune disease. As used herein, the term "subject" means any mammal. In some embodiments, the subject is a feline (e.g., a cat), a canine (e.g., a dog), an equine (e.g., a horse), a rabbit, a pig, a rodent (e.g., a mouse, rat, hamster, or guinea pig), a non-human primate (e.g., a simian (e.g., a monkey (e.g., a baboon, a marmoset), or an ape (e.g., a chimpanzee, a gorilla, an orangutan, or a gibbon)), or a human. In some embodiments, the subject is a human. The terms subject and patient are used interchangeably herein. In some embodiments, the subject has been previously identified or diagnosed as having a disease (e.g., a cancer (e.g., any of the cancers described herein)).

[0252] A therapeutically effective amount of a substrate-containing polypeptide (e.g., an activatable molecule or conjugated polypeptide) of the present disclosure generally relates to the amount necessary to achieve a therapeutic goal. As described above, this may be a binding interaction between the AM and its target, which, in certain cases, interferes with the function of the target. The amount required to be administered further depends on the binding affinity of the polypeptide for its specific target and the rate at which the administered polypeptide is depleted from the free pool of other subjects to which it is administered. A typical range for a therapeutically effective dosage of a polypeptide of the present disclosure may be, by way of non-limiting example, about 0.001, 0.01, 0.1, 0.3, 0.5, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 mg / kg body weight or more. The structure of the polypeptide of the present disclosure allows for a reduced dosage of the polypeptide to be administered to a subject compared to conventional activatable molecules and conventional antibodies. For example, the dose administered on a unit dosage basis or the total dose over a dosing regimen can be reduced by 10, 20, 30, 40, or 50% compared to the corresponding dose of the corresponding conventional therapeutic molecule.

[0253] Typical dosing frequencies may range, for example, once or twice daily, once or twice weekly, once or twice every two weeks, or once or twice monthly.

[0254] The effectiveness of treatment is determined in association with any known method for diagnosing or treating the particular disorder. Methods for screening for polypeptides possessing the desired specificity include, but are not limited to, enzyme-linked immunosorbent assays (ELISAs) and other immunologically mediated techniques known in the art.

[0255] In another embodiment, polypeptides directed to two or more targets are used in methods known in the art related to target localization and / or quantification (e.g., for use in measuring the levels of one or more targets in an appropriate physiological sample, for use in diagnostic methods, for use in protein imaging, etc.) In a given embodiment, polypeptides directed to two or more targets, or derivatives, fragments, analogs, or homologs thereof, comprising an antigen-binding domain derived from an antibody, are utilized as pharmacologically active compounds (hereinafter referred to as "therapeutic agents").

[0256] The substrate-containing polypeptides used in any of these method and use embodiments can be administered at any stage of disease. For example, such polypeptides can be administered to patients suffering from any stage of cancer, from early to metastatic. In some embodiments, substrate-containing polypeptides and formulations thereof can be administered to subjects suffering from or susceptible to a disease or disorder associated with aberrant target expression and / or activity.

[0257] A subject suffering from or susceptible to a disease or disorder associated with aberrant target expression and / or activity can be identified using any of a variety of methods known in the art. For example, a subject suffering from cancer or other neoplastic conditions can be identified using any of a variety of clinical and / or laboratory tests, such as a physical examination and blood, urine, and / or stool analysis to assess health status. For example, a subject suffering from inflammation and / or an inflammatory disorder can be identified using any of a variety of clinical and / or laboratory tests, such as a physical examination and / or body fluid analysis, e.g., blood, urine, and / or stool analysis, to assess health status.

[0258] In some embodiments, administration of a polypeptide to a patient suffering from a disease or disorder associated with aberrant target expression and / or activity can be considered successful if any of a variety of laboratory or clinical objectives are achieved. For example, administration of a polypeptide to a patient suffering from a disease or disorder associated with aberrant target expression and / or activity can be considered successful if one or more of the symptoms associated with the disease or disorder are alleviated, reduced, suppressed, or do not progress to a further, i.e., worse, state. Administration of a polypeptide to a patient suffering from a disease or disorder associated with aberrant target expression and / or activity can be considered successful if the disease or disorder goes into remission or does not progress to a further, i.e., worse, state.

[0259] As used herein, the term "treatment" includes reducing the severity, frequency, or number of one or more (e.g., 1, 2, 3, 4, or 5) symptoms or signs of a disease (e.g., cancer (e.g., any of the cancers described herein)) in a subject (e.g., any of the subjects described herein). In some embodiments where the disease is cancer, treatment results in a reduction in cancer growth, inhibition of cancer progression, inhibition of cancer metastasis, or a reduced risk of cancer recurrence in a subject with cancer.

[0260] In some embodiments, the substrate comprises a substrate of a protease that is active, e.g., upregulated or otherwise unregulated, in a disease state or diseased tissue. Exemplary disease states include, for example, cancer (e.g., where the diseased tissue is tumor tissue) and inflammatory or autoimmune conditions (e.g., where the diseased tissue is inflamed tissue). In some embodiments, the substrate comprises a substrate of an extracellular protease. In some embodiments, the substrate comprises a substrate of an intracellular protease. In some embodiments, the substrate is an intracellular protease and an extracellular protease. In some embodiments, the disease may be cancer. In some embodiments, the subject may have been identified or diagnosed with cancer. Examples of cancer include solid tumors, hematological tumors, sarcomas, leukemias (e.g., hairy cell leukemia, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL)), gastric cancer, urothelial cancer, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, brain cancer, colon cancer, bone cancer, lung cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung cancer (NSCLC), squamous cell head and neck cancer, endometrial cancer, bladder cancer, cervical cancer, and liver cancer. Metastasis of the aforementioned cancers can also be treated or prevented according to the methods described herein.

[0261] In some embodiments, the disease may be an autoimmune disease or condition. In some embodiments, the subject may have been identified or diagnosed with an autoimmune disease or condition, or may be at high risk of developing an autoimmune disease or condition. Examples of autoimmune diseases include type 1 diabetes, rheumatoid arthritis (RA), psoriasis / psoriatic arthritis, multiple sclerosis, systemic lupus erythematosus, inflammatory bowel disease (e.g., Crohn's disease, ulcerative colitis), chronic inflammation, or transplant rejection (e.g., in kidney, liver, or heart transplants), autoimmune disease, infectious disease, chronic inflammation, or transplant rejection. In some embodiments, the disease is a cardiovascular disease. In some embodiments, the disease is a neurodegenerative disorder.

[0262] In some embodiments, the methods herein can result in a reduction in the number, severity, or frequency of one or more symptoms of cancer in a subject (e.g., compared to the number, severity, or frequency of one or more symptoms of cancer in the subject before treatment).

[0263] The method may further include administering one or more additional agents to the subject. In some embodiments, the substrate-containing polypeptide (e.g., an activatable molecule or a conjugated polypeptide) may be administered in combination with one or more additional agents during and / or after treatment. In some embodiments, the polypeptide may be formulated in a single therapeutic composition, and the polypeptide and additional agent(s) may be administered simultaneously. Alternatively, the polypeptide and additional agent(s) may be separate from each other, e.g., each formulated in a separate therapeutic composition, and the polypeptide and additional agent(s) are administered simultaneously, or at different times during the treatment regimen. For example, the polypeptide may be administered before the administration of the additional agent, after the administration of the additional agent, or alternately. The polypeptide and additional agent(s) may be administered in a single dose or multiple doses.

[0264] One or more of the polypeptides herein may be co-formulated with and / or co-administered with one or more anti-inflammatory agents, immunosuppressants, or metabolic or enzyme inhibitors. In some embodiments, one or more polypeptides herein may be combined with one or more polypeptides of other types.

[0265] The present disclosure also provides methods for detecting the presence or absence of a cleavage agent and / or target in a subject or sample. Such methods may include (i) contacting a subject or biological sample with an activatable molecule, where the activatable molecule comprises a detectable label disposed on a portion of the activatable molecule that is released after cleavage of the substrate, and (ii) measuring the level of the activated molecule in the subject or biological sample, where a detectable level of the activated molecule in the subject or biological sample indicates that the cleavage agent, the target, or both the cleavage agent and the target are absent and / or sufficiently absent in the subject or biological sample; thus, target binding and / or protease cleavage of the activatable molecule cannot be detected in the subject or biological sample, and a decrease in the detectable level of the activated molecule in the subject or biological sample indicates that the cleavage agent and the target are present in the subject or biological sample.

[0266] Such detection methods can also be adapted to provide for the detection of the presence or absence of a target that, when cleaved, can bind to the AM of the activatable molecule. Thus, the assay can be adapted to assess the presence or absence of the cleaving agent and the presence or absence of the target of interest. The presence or absence of the cleaving agent can be detected by the presence and / or increase of the detectable label of the activatable molecule, as described above, and the presence or absence of the target can be detected by detecting the target-AM complex, for example, using a detectably labeled anti-target antibody.

[0267] In some embodiments, activatable molecules are also useful for in situ imaging, e.g., to verify activation of the activatable molecule by protease cleavage and binding to a specific target. In situ imaging is a technique that allows for the localization of proteolytic activity and targets in biological samples, such as cell cultures or tissue sections. This technique can be used to confirm both binding to a specific target and proteolytic activity based on the presence of a detectable label (e.g., a fluorescent label).

[0268] These techniques are useful for any frozen cells or tissues, whether from a diseased site (such as tumor tissue) or healthy tissue. These techniques are also useful for fresh cell or tissue samples.

[0269] In these techniques, the activatable molecule can be labeled with a detectable label, which can be a fluorescent dye (e.g., a fluorophore, fluorescein isothiocyanate (FITC), rhodamine isothiocyanate (TRITC), Alexa Fluor® labels), a near-infrared (NIR) dye (e.g., Qdot® nanocrystals), a colloidal metal, a hapten, a radioactive marker, biotin, and an amplification reagent such as streptavidin, or an enzyme (such as horseradish peroxidase or alkaline phosphatase).

[0270] Detection of label in a sample incubated with a labeled activatable molecule indicates that the sample contains a target, a protease specific for the activatable molecule's substrate. In some embodiments, the presence of the protease can be confirmed using a broad-spectrum protease inhibitor as described herein and / or a protease-specific agent, e.g., an antibody such as A11, which is specific for the protease matriptase and inhibits the proteolytic activity of matriptase. See, e.g., International Publication No. WO 2010 / 129609, published November 11, 2010. The same approach of using a broad-spectrum protease inhibitor as described herein and / or using a more selective inhibitor can be used to identify proteases specific for the activatable molecule's substrate. In some embodiments, the presence of the target can be confirmed using a target-specific agent, e.g., another antibody, or the detectable label can be competed with unlabeled target. In some embodiments, an unlabeled activatable molecule can be used with detection by a labeled secondary antibody or a more complex detection system.

[0271] Similar techniques may be useful for in vivo imaging, where detection of a fluorescent signal in a subject, e.g., a mammal, including a human, indicates that the disease site contains a target, a protease specific for the substrate of the activatable molecule.

[0272] These techniques are also useful as kits and / or reagents for detecting, identifying, or characterizing protease activity in various cells, tissues, and organisms based on protease-specific substrates of activatable molecules.

[0273] The reduction in the level of the detectable label can be, for example, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or substantially 100% reduction. In some embodiments, the detectable label can be conjugated to a component of the polypeptide, such as an AM. In some embodiments, measuring the level of the polypeptide in a subject or sample can be achieved using a secondary reagent that specifically binds to the activated protein, wherein the reagent comprises a detectable label. The secondary reagent can be an antibody comprising a detectable label.

[0274] In some embodiments, the substrate-containing polypeptide is also useful for detecting targets in patient samples, and thus is useful as a diagnostic agent. For example, the polypeptide can be used in an in vitro assay, such as an ELISA, to detect target levels in patient samples. For example, the polypeptide can be immobilized on a solid support (e.g., the well(s) of a microtiter plate). The immobilized polypeptide can serve as a capture protein for any target that may be present in a test sample. Before contacting the immobilized polypeptide with a patient sample, the solid support can be rinsed and treated with a blocking agent, such as milk protein or albumin, to prevent nonspecific adsorption of the analyte.

[0275] In some embodiments, based on the results obtained using the polypeptide in an in vitro diagnostic assay, the stage of disease in a subject can be determined based on the expression level of a target protein (e.g., an antigen). For a particular disease, blood samples can be taken from subjects diagnosed at various stages of disease progression and / or various time points during therapeutic treatment of the disease. A population of samples that provides statistically significant results for each stage of progression or treatment is used to designate a concentration range of the target protein (e.g., antigen) that can be considered characteristic of each stage.

[0276] The polypeptides herein can also be used in diagnostic and / or imaging methods. In some embodiments, such methods can be in vitro methods. In some embodiments, such methods can be in vivo methods. In some embodiments, such methods can be in situ methods. In some embodiments, such methods can be ex vivo methods. For example, a polypeptide bearing a substrate can be used to detect the presence or absence of an enzyme capable of cleaving the substrate. Such polypeptides can be used for diagnostics, which can include in vivo detection (e.g., qualitative or quantitative) of enzyme activity (or, in some embodiments, an environment of elevated reduction potential, such as can provide for reduction of disulfide bonds) by the measured accumulation of activated antibodies (i.e., antibodies resulting from cleavage of the polypeptide) in a given cell or tissue of a given host organism. Such accumulation of activated protein indicates not only that the tissue expresses enzyme activity (or an increase in reduction potential, depending on the nature of the substrate), but also that the tissue expresses the target to which the activated protein binds. In some examples, the polypeptides can be used to detect protease activity in assays that do not rely on target binding, such as the quantitative ex vivo zymography (QZ) assay described in Howng et al., “Novel Ex Vivo Zymography Approach for Assessment of Protease Activity in Tissues with Activatable Antibodies,” Pharmaceuticals. 2021 Sep 2;13(9):1390, which is incorporated herein by reference in its entirety.

[0277] For example, the substrate can be selected to be a protease substrate for a protease found at the site of a tumor, at the site of a viral or bacterial infection in a biologically restricted site (e.g., in an abscess, in an organ, etc.), etc. The AM can be one that binds to a target protein (e.g., an antigen). A detectable label (e.g., a fluorescent label, a radioactive label, or a radioactive tracer) can be conjugated to the AM or other region of the polypeptide using methods well known to those skilled in the art. Suitable detectable labels can be discussed in the context of the screening methods described above, and additional specific examples are provided below. By using an AM specific for a disease-state protein or peptide together with a protease whose activity is elevated in the diseased tissue of interest, the polypeptide can exhibit an increased rate of binding to the diseased tissue compared to tissue in which the substrate-specific enzyme is absent at detectable levels, present at lower levels than in the diseased tissue, or inactive (e.g., in the proenzyme form or complexed with an inhibitor). Because small proteins and peptides are rapidly cleared from the blood by the renal filtration system and substrate-specific enzymes are not present at detectable levels (or are present at lower levels or in an inactive conformation in non-diseased tissue), the accumulation of activated proteins in diseased tissues may be increased compared with non-diseased tissues.

[0278] In some embodiments, substrate-containing polypeptides may be useful for in vivo imaging, where detection of a fluorescent signal in a subject, e.g., a mammal, including a human, indicates that a disease site contains the target and a protease specific for the polypeptide's substrate. In vivo imaging can be used to identify or narrow down patient populations suitable for treatment with the polypeptides of the present disclosure. For example, patients who test positive for both the target being tested and the protease that cleaves the substrate within the polypeptide's substrate (e.g., accumulate activated protein at the disease site) are identified as suitable candidates for treatment with such a polypeptide containing such a substrate. Similarly, patients who test negative may be identified as suitable candidates for a different treatment (i.e., not suitable for treatment with the polypeptide being tested). In some embodiments, patients who test negative for a first polypeptide may be tested with other polypeptides containing different substrates until a suitable polypeptide for treatment (e.g., polypeptide B containing a substrate that is cleaved at the patient's disease site) is identified.

[0279] In some embodiments, in situ imaging can be useful in methods for identifying which patients to treat. For example, in situ imaging can use polypeptides to screen patient samples to identify patients with the appropriate protease(s) and target(s) in the appropriate location, e.g., tumor site. In some embodiments, in situ imaging is used to identify or narrow the patient population suitable for treatment with the polypeptides of the present disclosure. For example, patients who test positive for both the target being tested and the protease that cleaves the substrate within the polypeptide's substrate (e.g., accumulate activating antibodies at the disease site) are identified as suitable candidates for treatment with such a polypeptide containing such a substrate. Similarly, patients who test negative for either or both the protease and target that cleaves the substrate used for the polypeptide tested using these methods are identified as suitable candidates for an alternative treatment (i.e., not suitable for treatment with the polypeptide being tested). In some embodiments, patients who test negative for a first polypeptide can be tested with other polypeptides containing different substrates until a suitable polypeptide for treatment (e.g., polypeptide B containing a substrate that is cleaved at the patient's disease site) is identified.

[0280] The present application also provides aspects and embodiments described in the following numbered statements:

[0281] Statement 1. An isolated polypeptide comprising a substrate, the substrate comprising a first cleavable portion (CM1) cleavable by a first protease and a second cleavable portion (CM2) cleavable by a second protease, wherein the CM1 comprises the amino acid sequence of HQSR (SEQ ID NO: 1) or HQSK (SEQ ID NO: 3). In certain aspects, the present disclosure may include substitution of any alanine in the disclosed CM sequence with valine. In some aspects, the isolated polypeptide comprises a first cleavable portion (CM1) cleavable by a first protease and a second cleavable portion (CM2) cleavable by a second protease, wherein the CM1 comprises the amino acid sequence of AQSRSGPWGLL (SEQ ID NO: 641), HASRSGPWGLL (SEQ ID NO: 642), HQARSGPWGLL (SEQ ID NO: 643), or HQSASGPWGLL (SEQ ID NO: 644). According to some embodiments of the present disclosure, the isolated polypeptide is a molecule in which a portion or component of the molecule is separated from the remainder of the molecule by cleavage of the CM by a protease. In some aspects of the present disclosure, the molecule is activated by cleavage of the CM by a protease. In some aspects, the isolated polypeptide is a molecule in which the CM is cleaved by multiple proteases. In some aspects, the isolated polypeptide is a molecule in which the CM is cleaved by MMP2. In some aspects, the isolated polypeptide is a molecule in which the CM is cleaved by MMP9. In some aspects, the isolated polypeptide is a molecule in which the CM is cleaved by MMP14. In some aspects, the isolated polypeptide is a molecule in which the CM is cleaved by MT-SP1. In some aspects, the isolated polypeptide is a molecule in which the CM is cleaved by two or all of MMP2, MMP9, MMP14, and MT-SP1.In some embodiments, the isolated polypeptide is a molecule that has a % cleavage rate of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, or 100% of the CM, e.g., by any one of MMP2, MMP9, MMP14, or MT-SP1, or any two of MMP2, MMP9, MMP14, and MT-SP1, or by each of MMP2, MMP9, MMP14, and MT-SP1. In some embodiments, the isolated polypeptide is a molecule that has a % cleavability of the CM that is 1.5-fold, 2-fold, 2.5-fold, 3-fold, 5-fold, 7-fold, 8-fold, or 10-fold or more improved over the % cleavability of SEQ ID NO: 78 (see, e.g., Example 2). According to some embodiments of the present disclosure, the isolated polypeptide is a molecule with high in vivo stability such that it is not cleaved in plasma, as demonstrated by less than 50%, less than 40%, or less than 25% in vivo activation after 7 days of in vivo administration (see, e.g., Example 3). According to embodiments of the present disclosure, the isolated polypeptide is 1 x 10 2 M -1 s -1 exceeding k cat / K M (M -1 s -1 According to some embodiments of the present disclosure, the isolated polypeptide is a molecule comprising a CM having 1×10 3 M -1 s -1 exceeding k cat / K M (M -1 s -1 According to an embodiment of the present disclosure, the isolated polypeptide is a molecule comprising a CM having 1×10 4 M -1 s -1 exceeding k cat / K M (M -1 s -1 According to an embodiment of the present disclosure, the isolated polypeptide is a molecule comprising a CM having 1×105 M -1 s -1 exceeding k cat / K M (M -1 s -1 ) is a molecule containing CM.

[0282] Statement 2. The isolated polypeptide of statement 1, wherein said CM1 comprises the amino acid sequence of HQSRS (SEQ ID NO: 2).

[0283] Statement 3. The isolated polypeptide of statement 1, wherein said CM1 comprises the amino acid sequence of HQSRSA (SEQ ID NO: 4).

[0284] Statement 4. The isolated polypeptide of statement 1, wherein said CM1 comprises the amino acid sequence of DHQSRS (SEQ ID NO: 5).

[0285] Statement 5. The isolated polypeptide of statement 2, wherein said CM1 comprises the amino acid sequence of SDHQSRS (SEQ ID NO: 6).

[0286] Statement 6. The isolated polypeptide of any one of statements 1 to 5, wherein said CM2 comprises the amino acid sequence of AIAL (SEQ ID NO: 9).

[0287] Statement 7. The isolated polypeptide of statements 1-5, wherein said CM2 comprises the amino acid sequence of AIALY (SEQ ID NO: 635).

[0288] Statement 8. The isolated polypeptide of any one of statements 1-5, wherein said CM2 comprises the amino acid sequence of AIALYA (SEQ ID NO: 8).

[0289] Statement 9. The isolated polypeptide of statement 8, wherein said CM2 comprises the amino acid sequence of AIALYAD (SEQ ID NO: 7).

[0290] Statement 10. The substrate is selected from the group consisting of AIALYADHQSRS (SEQ ID NO: 10), DHQSRSAIALYAD (SEQ ID NO: 11), HQSRSAIALYAD (SEQ ID NO: 12), DHQSRSAIALYA (SEQ ID NO: 13), HQSRSAIALY (SEQ ID NO: 636), HQSRSAIAL (SEQ ID NO: 637), AIALYAHQSRS (SEQ ID NO: 33), AIALYHQSRS (SEQ ID NO: 37), HQSRSAIALY (SEQ ID NO: 636), HQSRSAIAL (SEQ ID NO: 637), AIALHQSRS (SEQ ID NO: 41), HQSRSGPRGLL (SEQ ID NO: 638), HQSRSGPAGLL (SEQ ID NO: 639), HQSRSPWG (SEQ ID NO: 640), HQSRAGPWGLL (SEQ ID NO: 641). 45), HQSRSAPWGLL (SEQ ID NO: 646), HQSRSGAWGLL (SEQ ID NO: 647), HQSRSGPWALL (SEQ ID NO: 648), HQSRSGPWGAL (SEQ ID NO: 649), HQSRSGPWGLA (SEQ ID NO: 650), HQSRPWGL (SEQ ID NO: 134), or HQSRSAIALYA (SEQ ID NO: 14); and optionally, the substrate comprises the amino acid sequence of AIALYADHQSRS (SEQ ID NO: 10), DHQSRSAIALYAD (SEQ ID NO: 11), HQSRSAIALYAD (SEQ ID NO: 12), DHQSRSAIALYA (SEQ ID NO: 13), or HQSRSAIALYA (SEQ ID NO: 14).

[0291] Statement 11. The isolated polypeptide of statement 1, wherein the substrate comprises the amino acid sequence of DHQSRSAIALYA (SEQ ID NO: 13).

[0292] Statement 12. The isolated polypeptide of any one of statements 1-5, where...

Claims

1. An isolated polypeptide comprising a substrate, wherein the substrate comprises a first cleavable portion (CM1) cleavable by a first protease and a second cleavable portion (CM2) cleavable by a second protease, and the CM1 comprises the amino acid sequence of HQSR (SEQ ID NO: 1) or HQSK (SEQ ID NO: 3).

2. The isolated polypeptide of claim 1, wherein the CM1 comprises the amino acid sequence of HQSRS (SEQ ID NO: 2).

3. The isolated polypeptide of claim 1, wherein the CM1 comprises the amino acid sequence of HQSRSA (SEQ ID NO: 4).

4. The isolated polypeptide of claim 1 , wherein the CM1 comprises the amino acid sequence of DHQSRS (SEQ ID NO: 5).

5. The isolated polypeptide of claim 4, wherein the CM1 comprises the amino acid sequence of SDHQSRS (SEQ ID NO: 6).

6. The isolated polypeptide of any one of claims 1 to 5, wherein the CM2 comprises the amino acid sequence of AIAL (SEQ ID NO: 9).

7. 7. The isolated polypeptide of any one of claims 1 to 6, wherein the CM2 comprises the amino acid sequence of AIALY (SEQ ID NO: 635).

8. The isolated polypeptide of any one of claims 1 to 6, wherein the CM2 comprises the amino acid sequence of AIALYA (SEQ ID NO: 8).

9. The isolated polypeptide of any one of claims 1 to 6, wherein the CM2 comprises the amino acid sequence of AIALYAD (SEQ ID NO: 7).

10. The substrates are selected from the group consisting of AIALYADHQSRS (SEQ ID NO: 10), DHQSRSAIALYAD (SEQ ID NO: 11), HQSRSAIALYAD (SEQ ID NO: 12), DHQSRSAIALYA (SEQ ID NO: 13), HQSRSAIALY (SEQ ID NO: 636), HQSRSAIAL (SEQ ID NO: 637), AIALHQSRS (SEQ ID NO: 41), AIALYAHQSRS (SEQ ID NO: 33), AIALYHQSRS (SEQ ID NO: 37), HQSRSGPRGLL (SEQ ID NO: 638), HQSRSGPAGLL (SEQ ID NO: 639), HQSRPWGL (SEQ ID NO: 134), HQSRSPWG (SEQ ID NO: 640), HQSRAGPWGLL (SEQ ID NO: 645), H 2. The isolated polypeptide of claim 1, comprising the amino acid sequence of QSRSAPWGLL (SEQ ID NO: 646), HQSRSGAWGLL (SEQ ID NO: 647), HQSRSGPWALL (SEQ ID NO: 648), HQSRSGPWGAL (SEQ ID NO: 649), HQSRSGPWGLA (SEQ ID NO: 650), or HQSRSAIALYA (SEQ ID NO: 14), and optionally the substrate comprises the amino acid sequence of AIALYADHQSRS (SEQ ID NO: 10), DHQSRSAIALYAD (SEQ ID NO: 11), HQSRSAIALYAD (SEQ ID NO: 12), DHQSRSAIALYA (SEQ ID NO: 13), or HQSRSAIALYA (SEQ ID NO: 14).

11. 2. The isolated polypeptide of claim 1, wherein the substrate comprises the amino acid sequence DHQSRSAIALYA (SEQ ID NO: 13).

12. 6. The isolated polypeptide of any one of claims 1 to 5, wherein the CM2 comprises the amino acid sequence of GPWGLL (SEQ ID NO: 237).

13. 13. The isolated polypeptide of claim 12, wherein the substrate comprises the amino acid sequence HQSRSGPWGLL (SEQ ID NO: 15), DHQSRSGPWGLL (SEQ ID NO: 16), or GPWGLLDHQSRS (SEQ ID NO: 17).

14. The CM2 comprises the amino acid sequence PXGL, where X is alanine (A), tyrosine (Y), arginine (R), phenylalanine (F), tryptophan (W), lysine (K), glutamine (Q), or methionine (M), or the CM2 comprises the amino acid sequence GPX １ GLX ２ (SEQ ID NO: 651), wherein X １ is W, Y, F, R, K, Q, A, or M, and X ２ The isolated polypeptide of any one of claims 1 to 5, wherein is any amino acid or is absent.

15. 15. The isolated polypeptide of claim 14, wherein the substrate comprises the amino acid sequence of HQSRSPWGL (SEQ ID NO: 126), PWGLHQSRS (SEQ ID NO: 489), PWGLLHQSRS (SEQ ID NO: 485), PWGLHQSR (SEQ ID NO: 502), PWGLHQSKS (SEQ ID NO: 490), HQSRPWGL (SEQ ID NO: 134), DHQSKSGPWGLL (SEQ ID NO: 178), HQSRSGPWGL (SEQ ID NO: 122), HQSRSGPYGLL (SEQ ID NO: 138), HQSRSGPFGLL (SEQ ID NO: 158), HQSRSGPRGLL (SEQ ID NO: 638), or HQSRSGPAGLL (SEQ ID NO: 639).

16. 6. The isolated polypeptide of any one of claims 1 to 5, wherein the CM2 comprises the amino acid sequence SGLLSS (SEQ ID NO: 238).

17. 17. The isolated polypeptide of claim 16, wherein the CM2 comprises the amino acid sequence of ISSGLLSS (SEQ ID NO: 75).

18. 17. The isolated polypeptide of claim 16, wherein the substrate comprises the amino acid sequence of ISSGLLSSDHQSRS (SEQ ID NO: 18), DHQSRSISSGLLSS (SEQ ID NO: 19), or DHQSRSSGLLSS (SEQ ID NO: 20).

19. 6. The isolated polypeptide of any one of claims 1 to 5, wherein the CM2 comprises the amino acid sequence of ALAHGLF (SEQ ID NO: 74).

20. 20. The isolated polypeptide of claim 19, wherein the substrate comprises the amino acid sequence ALAHGLFSDHQSRS (SEQ ID NO: 21) or SDHQSRSALAHGLF (SEQ ID NO: 22).

21. 1. An isolated polypeptide comprising a substrate, said substrate comprising a first cleavable moiety (CM1) cleavable by a first protease or set of proteases and a second cleavable moiety (CM2) cleavable by a second protease or set of proteases, said substrate comprising an amino acid sequence selected from SEQ ID NOs: 10-73, 83-216, 460-634, 636-650, and optionally comprising an amino acid sequence selected from SEQ ID NOs: 10-73, 83-216, and 460-634.

22. 20. The isolated polypeptide of any one of claims 1 to 9, 12, 14, 16, 17, and 19, wherein the CM1 and CM2 have a structural arrangement from N-terminus to C-terminus as CM1-CM2.

23. 20. The isolated polypeptide of any one of claims 1 to 9, 12, 14, 16, 17, and 19, wherein the CM1 and CM2 have a structural arrangement from N-terminus to C-terminus as CM2-CM1.

24. 24. The isolated polypeptide of claim 22 or 23, wherein the CM1 and the CM2 are indirectly coupled via a connecting peptide.

25. 24. The isolated polypeptide of claim 22 or 23, wherein the CM1 and the CM2 are directly coupled to each other.

26. 20. The isolated polypeptide of any one of claims 1 to 9, 12, 14, 16, 17, and 19, wherein at least a portion of CM1 overlaps with at least a portion of CM2.

27. 1. An isolated polypeptide comprising a substrate, said substrate comprising a first cleavable portion (CM1) cleavable by a first protease and a second cleavable portion (CM2) cleavable by a second protease, said substrate comprising an amino acid sequence having one or two amino acid mutation(s) of any one of SEQ ID NOs: 10-73, 83-216, 460-634, 636-650, optionally SEQ ID NOs: 10-73, 83-216, and 460-634.

28. 28. The isolated polypeptide of any one of claims 1 to 27, wherein the isolated polypeptide is an activatable molecule and further comprises an active moiety (AM) that specifically binds to a target.

29. 29. The isolated polypeptide of claim 28, wherein the AM is an antibody or an antigen-binding fragment thereof.

30. The antigen-binding fragment may be a Fab fragment, F(ab') ２ 30. The isolated polypeptide of claim 29, wherein the polypeptide is selected from the group consisting of a fragment, an scFv, an scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody.

31. 29. The isolated polypeptide of claim 28, wherein the AM is a therapeutic macromolecule.

32. 29. The isolated polypeptide of claim 28, wherein the AM is a cytokine or a chimeric antigen receptor.

33. 33. The isolated polypeptide of any one of claims 28 to 32, wherein the AM is coupled to the CM1.

34. 34. The isolated polypeptide of claim 33, wherein the AM is directly coupled to the CM1.

35. 34. The isolated polypeptide of claim 33, wherein the AM is indirectly coupled to the CM1 via a connecting peptide.

36. 36. The isolated polypeptide of any one of claims 28 to 35, wherein the AM is coupled to the CM2.

37. 37. The isolated polypeptide of claim 36, wherein the AM is directly coupled to the CM2.

38. 37. The isolated polypeptide of claim 36, wherein the AM is indirectly coupled to the CM2 via a connecting peptide.

39. 39. The isolated polypeptide of any one of claims 27 to 38, further comprising a masking moiety (MM).

40. 40. The isolated polypeptide of claim 39, wherein the MM has a dissociation constant for binding to the AM that is greater than the dissociation constant of the AM for binding to the target.

41. 41. The isolated polypeptide of claim 39 or 40, wherein the MM is 2 to 40 amino acids in length.

42. 42. The isolated polypeptide of any one of claims 39-41, wherein the MM is linked to the substrate such that the isolated polypeptide comprises, from N-terminus to C-terminus, the following structural arrangement: MM-CM1-CM2-AM, MM-CM2-CM1-AM, AM-CM1-CM2-MM, or AM-CM2-CM1-MM.

43. 43. The isolated polypeptide of claim 42, wherein the MM is directly coupled to the CM1.

44. 43. The isolated polypeptide of claim 42, wherein the MM is indirectly coupled to the CM1 via a connecting peptide.

45. 43. The isolated polypeptide of claim 42, wherein the MM is directly coupled to the CM2.

46. 43. The isolated polypeptide of claim 42, wherein the MM is indirectly coupled to the CM2 via a connecting peptide.

47. 47. The isolated polypeptide of any one of claims 39-46, wherein the isolated polypeptide comprises a first connecting peptide (LP1) and a second connecting peptide (LP2), and wherein the isolated polypeptide has the following structural arrangement from N-terminus to C-terminus: MM-LP1-CM1-CM2-LP2-AM, MM-LP1-CM2-CM1-LP2-AM, AM-LP2-CM1-CM2-LP1-MM, or AM-LP2-CM2-CM1-LP1-MM.

48. 48. The isolated polypeptide of claim 47, wherein LP1 and LP2 are not identical to each other.

49. 48. The isolated polypeptide of claim 47, wherein LP1 and LP2 are identical to each other.

50. 50. The isolated polypeptide of any one of claims 47 to 49, wherein each of LP1 and LP2 is a peptide of 1 to 20 amino acids in length.

51. 51. The isolated polypeptide of any one of claims 1 to 50, wherein the first protease is a serine protease.

52. 52. The isolated polypeptide of claim 51, wherein the serine protease is membrane-type serine protease 1 (MT-SP1).

53. k of the substrate by MT-SP1 cleavage ｃａｔ / K Ｍ is at least 1 × 10 ３ M －１ s －１ 53. The isolated polypeptide of claim 52, wherein

54. k of the substrate by MT-SP1 cleavage ｃａｔ / K Ｍ is at least 1 × 10 ４ M －１ s －１ 53. The isolated polypeptide of claim 52, wherein

55. 55. The isolated polypeptide of any one of claims 1 to 54, wherein the second protease is a matrix metalloproteinase (MMP).

56. 56. The isolated polypeptide of claim 55, wherein the MMP is MMP2, MMP9, or MMP14.

57. k of the substrate by MMP2 cleavage ｃａｔ / K Ｍ is at least 1 × 10 ３ M －１ s －１ 57. The isolated polypeptide of claim 56,

58. The k of the substrate due to the MMP2 cleavage ｃａｔ / K Ｍ is at least 1 × 10 ４ M －１ s －１ 57. The isolated polypeptide of claim 56,

59. k of the substrate by MMP9 cleavage ｃａｔ / K Ｍ is at least 1 × 10 ３ M －１ s －１ 57. The isolated polypeptide of claim 56,

60. The k of the substrate due to MMP9 cleavage ｃａｔ / K Ｍ is at least 1 × 10 ４ M －１ s －１ 57. The isolated polypeptide of claim 56,

61. k of the substrate by MMP14 cleavage ｃａｔ / K Ｍ is at least 1 × 10 ３ M －１ s －１ 55. The isolated polypeptide of claim 54, wherein:

62. The k of the substrate by MMP14 cleavage ｃａｔ / K Ｍ is at least 1 × 10 ４ M －１ s －１ 57. The isolated polypeptide of claim 56,

63. 63. The isolated polypeptide of any one of claims 1 to 62, wherein the substrate is resistant to in situ cleavage in human bone marrow.

64. 63. The isolated polypeptide of any one of claims 1 to 62, wherein the substrate is resistant to in vivo cleavage in human bone marrow.

65. 65. The isolated polypeptide of any one of claims 1-64, further comprising one or more additional cleavable moieties, optionally wherein at least a portion of the substrate overlaps with at least a portion of a second cleavable moiety.

66. 66. A polypeptide complex comprising one or more isolated polypeptides according to any one of claims 1 to 65 bound to a second isolated polypeptide.

67. A conjugated polypeptide comprising the isolated polypeptide of any one of claims 1 to 65 conjugated to an agent.

68. 68. The conjugated polypeptide of claim 67, wherein the drug is conjugated to the AM via a conjugate linker.

69. 69. The conjugated polypeptide of claim 68, wherein the conjugated linker is cleavable.

70. 69. The conjugated polypeptide of claim 68, wherein the conjugated linker is non-cleavable.

71. 70. The conjugated polypeptide of claim 69, wherein the conjugate linker comprises an amino acid sequence selected from SEQ ID NOs: 10-73, 83-216, 460-634, 636-650.

72. 72. The conjugated polypeptide of any one of claims 67-71, wherein the agent is a toxin, a microtubule inhibitor, a nucleic acid damaging agent, a dolastatin, an auristatin, a maytansinoid, a duocarmycin, a calicheamicin, or a combination thereof.

73. 72. A composition comprising an isolated polypeptide according to any one of claims 1 to 65, a polypeptide complex according to claim 66, a conjugated polypeptide according to any one of claims 67 to 72, and a carrier.

74. 74. The composition of claim 73, wherein the carrier is a pharmaceutically acceptable carrier.

75. 75. The composition of claim 74, comprising an additional agent.

76. 76. The composition of claim 75, wherein the additional agent is a therapeutic agent.

77. 66. An isolated nucleic acid molecule encoding the isolated polypeptide of any one of claims 1 to 65.

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

79. 79. A cell comprising the isolated nucleic acid molecule of claim 77 or the vector of claim 78.

80. 66. A method for producing an activatable molecule comprising a tandem substrate, comprising expressing and recovering an activatable molecule comprising the isolated polypeptide of any one of claims 1 to 65.

81. 72. A method of treating, alleviating a symptom of, or delaying progression of a disease or disorder in a subject, comprising administering to the subject a therapeutically effective amount of an isolated polypeptide of any one of claims 1-65, a polypeptide complex of claim 66, a conjugated polypeptide of any one of claims 67-72, or a composition of any one of claims 73-76.

82. 82. The method of claim 81, wherein the disease is cancer, an infectious disease, an inflammatory disease, a cardiovascular disease, a neurodegenerative disease, or an autoimmune disease.

83. 80. The isolated polypeptide of any one of claims 1 to 65, the polypeptide complex of claim 66, the conjugated polypeptide of any one of claims 67 to 72, the composition of any one of claims 73 to 76, the nucleic acid molecule of claim 77, the vector of claim 78 or the cell of claim 79 for use as a pharmaceutical or in therapy, optionally in combination with an additional agent which may be a therapeutic agent, for treating cancer, an infectious disease, an inflammatory disease, a cardiovascular disease, a neurodegenerative disease or an autoimmune disease, optionally in combination with an additional agent which may be a therapeutic agent.

84. 1. A method for detecting or diagnosing a disease or condition in a subject, comprising: contacting a sample from said subject with an isolated polypeptide of any one of claims 1 to 65, a polypeptide complex of claim 66, a conjugated polypeptide of any one of claims 67 to 72, or a composition of any one of claims 73 to 76; detecting or diagnosing said disease or condition in said subject by measuring the level of cleavage of said isolated polypeptide.

85. 85. The method of claim 84, wherein the disease is cancer, an infectious disease, an inflammatory disease, a cardiovascular disease, a neurodegenerative disease, or an autoimmune disease.

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