Cleavable polypeptides and methods of use thereof

Abstract

Cleavable polypeptides that include a cleavable moiety that is a substrate for at least one protease (e.g., MT-SP1 and / or MMP) are disclosed. Activatable molecules including the cleavable polypeptides are disclosed. Methods of making and using the cleavable polypeptides and activatable molecules including the cleavable polypeptides in a variety of therapeutic, diagnostic, and prophylactic applications are disclosed.

Description

CYTX-108-WO :: 4862-156.WO1 CLEAVABLE POLYPEPTIDES AND METHODS OF USE THEREOF CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of provisional application U.S.S.N. 63 / 679,348, filed August 5, 2024, pursuant 35 U.S.C. § 119(e), which is incorporated herein by reference in the entirety. REFERENCE TO SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing XML which has been submitted electronically and is hereby incorporated by reference in its entirety. Said Sequence Listing named “4862-156WO1.xml,” created on August 4, 2025, is 212,992 bytes in size. TECHNICAL FIELD

[0003] The present disclosure generally relates to polypeptides that include a cleavable moiety that is a substrate for at least one protease (e.g., a matrix metalloproteinase (MMP) and / or MT-SP1), and to methods of making and using the cleavable polypeptides and activatable molecules in a variety of therapeutic, diagnostic, and prophylactic applications. BACKGROUND

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

[0005] Accordingly, there exists 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 present disclosure provides a cleavable polypeptide comprising a cleavable moiety (CM) comprising an amino acid sequence QXAQX1LX2XBA (SEQ ID NO: 74), wherein XAis N, A, W, or F; X1is G or A; X2is R or K; XBis S, M, V, or L, provided that i) when XA is N, then XB is S, L, or V; ii) when XA is A, then XB is S or M; and iii) when XAis W or F, then XBis M; and wherein the CM is a substrate for a protease. In some aspects, the cleavable polypeptide comprises 2 to 4 CMs. In some aspects, each of theCYTX-108-WO :: 4862-156.WO1 2 to 4 CMs independently comprises an amino acid sequence according to the amino acid sequence QXAQX1LX2XBA (SEQ ID NO: 74). In some aspects, the cleavable polypeptide comprises at least one additional moiety (M) selected from a moiety that is located amino (N) terminally to the CM (Mn), a moiety that is located carboxyl (C) terminally to the CM (Mc), or a combination thereof. In some aspects, the Mn and the Mc are each independently selected from a masking moiety (MM), a target binding moiety (TBM), a therapeutic agent, a detectable moiety, a diagnostic agent, or an affinity tag.

[0007] In one aspect, the present disclosure provides an activatable molecule comprising a) the cleavable polypeptide according to any one or combination of aspects disclosed herein; b) a masking moiety (MM); and c) a target binding moiety (TBM); and d) optionally a half- life extending moiety (EM). In some aspects, the cleavable polypeptide comprises the MM coupled directly or indirectly to the N-terminus of the CM and the TBM coupled directly or indirectly to the C-terminus of the CM, or wherein cleavable polypeptide comprises the TBM coupled directly or indirectly to the N-terminus of the CM and the MM coupled directly or indirectly to the C-terminus of the CM.

[0008] In one aspect, the present disclosure provides a nucleic acid composition comprising one or more nucleic acid molecules encoding a) the cleavable polypeptide according to any one or combination of aspects disclosed herein; b) a masking moiety (MM); and c) a target binding moiety (TBM); and d) optionally a half-life extending moiety (EM). In some aspects, the MM is coupled directly or indirectly to the N-terminus of the CM and the TBM is coupled directly or indirectly to the C-terminus of the CM, or wherein the TBM is coupled directly or indirectly to the N-terminus of the CM and the MM is coupled directly or indirectly to the C-terminus of the CM.

[0009] In each of the foregoing aspects, and unless otherwise stated, the polypeptide may comprise, e.g., one or more optional linkers between each of the elements listed. In some aspects, a linker is a peptide having a length of 5 to 30, 6 to 29, 7 to 28, 8 to 27, 9 to 26, 10 to 25, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 amino acids. In the disclosed structural arrangements in the foregoing paragraphs and throughout this disclosure, one or more linkers may optionally be present between the elements. Further, this disclosure also contemplates and includes activatable molecules in which any one or more of the disclosed elements optionally directly abut each other such that there are no linkers or other amino acid sequences between the elements.CYTX-108-WO :: 4862-156.WO1

[0010] In one aspect, the present disclosure provides a composition comprising the cleavable polypeptide according to any one or combination of aspects disclosed herein or the activatable molecule according to any one or combination of aspects disclosed herein and a carrier, for example a pharmaceutically acceptable carrier or other suitable carrier. In some aspects, the composition comprises an additional agent. In some aspects, the additional agent is a therapeutic, imaging, or diagnostic agent.

[0011] In one aspect, the present disclosure provides a nucleic acid molecule or group of nucleic acid molecules encoding the cleavable polypeptide according to any one or combination of aspects disclosed herein or the activatable molecule according to any one or combination of aspects disclosed herein.

[0012] In one aspect, the present disclosure provides a vector or group of vectors comprising the nucleic acid molecule or group of nucleic acid molecules according to any one or combination of aspects disclosed herein or the nucleic acid composition according to any one or combination of aspects disclosed herein.

[0013] In one aspect, the present disclosure provides a recombinant cell comprising the cleavable polypeptide according to any one or combination of aspects disclosed herein, the activatable molecule according to any one or combination of aspects disclosed herein, the nucleic acid composition according to any one or combination of aspects disclosed herein, the nucleic acid according to any one or combination of aspects disclosed herein, or the vector or group of vectors according to any one or combination of aspects disclosed herein.

[0014] In one aspect, the present disclosure provides an in vitro method of manufacturing the cleavable polypeptide according to any one or combination of aspects disclosed herein or the activatable molecule according to any one or combination of aspects disclosed herein, comprising culturing a recombinant cell under conditions suitable for expressing the cleavable polypeptide or the polypeptide complex and recovering the cleavable polypeptide.

[0015] In one aspect, the present disclosure provides a method of treating, alleviating a symptom of, or delaying the progression of a disease or disorder in a subject, comprising administering a therapeutically effective amount of the cleavable polypeptide according to any one or combination of aspects disclosed herein or the activatable molecule according to any one or combination of aspects disclosed herein, or the composition according to any one or combination of aspects disclosed herein to the subject.

[0016] In one aspect, the present disclosure provides a container, vial, syringe, injector pen, or kit comprising at least one dose of the composition of the cleavable polypeptideCYTX-108-WO :: 4862-156.WO1 according to any one or combination of aspects disclosed herein or the activatable molecule according to any one or combination of aspects disclosed herein, or the composition according to any one or combination of aspects disclosed herein.

[0017] In another aspect, the present disclosure provides a polypeptide complex comprising one or more of the cleavable polypeptides comprising the CMs disclosed herein. In some aspects, the complex comprises one or more of the cleavable polypeptides of the present disclosure bound to a second cleavable polypeptide, e.g., via protein-protein affinity interactions, hydrophobic interactions, disulfide linkage(s), cross-link(s), covalent bond(s), chemical linkage(s), or any other type of binding between two polypeptides.

[0018] In another aspect, the present disclosure provides a conjugated polypeptide comprising the cleavable polypeptide herein conjugated to an agent. In some aspects, the agent is conjugated to the cleavable polypeptide via a conjugating linker. In some aspects, the conjugating linker is cleavable. In some aspects, the conjugating linker is non-cleavable. In some aspects, the conjugating linker comprises an amino acid sequence according to QXAQX1LX2XBA (SEQ ID NO: 74) wherein XAis N, A, W, or F; X1is G or A; X2is R or K; XB is S, M, V, or L, provided that i) when XA is N, then XB is S, L, or V; ii) when XA is A, then XBis S or M; and iii) when XAis W or F, then XBis M. In some aspects, 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.

[0019] In another aspect, the present disclosure provides a method of detecting or diagnosing a disease or health condition in a subject, comprising: contacting the cleavable polypeptide, the activatable molecule, the polypeptide complex, the conjugated polypeptide, or the composition with a sample from the subject; and measuring a level of cleavage of the cleavable polypeptide, thereby detecting or diagnosing the disease or health condition of the subject. In some aspects, the disease is a cancer, an infection, an inflammatory disorder, a cardiovascular disorder, a neurodegenerative disorder, or an autoimmune disorder. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] An understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative aspects, in which the principles of the invention may be utilized, and the accompanying drawings of which:CYTX-108-WO :: 4862-156.WO1

[0021] FIG.1 shows a graph of the in vitro binding affinity and masking efficiency of exemplary cleavable polypeptides with an anti-EGFR TBM that include substrates cleavable by an MMP and / or matriptase (MT-SP1) (activatable antibodies) and an unmasked antibody.

[0022] FIGs.2A-2B show the effects of exemplary cleavable polypeptides with an anti- EGFR TBM that include substrates cleavable by an MMP and / or matriptase (MT-SP1) on tumor regression in mice. The mean tumor volume ± SEM was plotted for each measured time point following administration of the exemplary activatable antibodies or with cetuximab or immunoglobulin (IVIG) control.

[0023] FIG.3 shows graphs illustrating the impact of forced oxidation on substrate cleavage by MT-SP1 for CMs 1002, 5080, 5020, 5030, 5050, and 5070 corresponding to SEQ ID NOs: 3, 5, 10, 15, 25, and 35. DETAILED DESCRIPTION

[0024] While aspects of the subject matter of the present disclosure may be embodied in a variety of forms, the following description is merely intended to disclose some of these forms as specific examples of the subject matter encompassed by the present disclosure. Accordingly, the subject matter of this disclosure is not intended to be limited to the forms or aspects so described.

[0025] Proteases play a critical role in the homeostasis of healthy tissues but are known to be dysregulated within diseases, including 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 provides new opportunities for the development of protease-activatable therapeutic molecules, which are preferentially activated in the local tissue microenvironment. These therapeutics have demonstrated a greater therapeutic window and safety profile with less on- target toxicities occurring in healthy tissues. Hence, there is a need for identification of substrates that act as cleavage recognition sites for proteases that are found to be dysregulated in disease tissues. These substrates or cleavable moieties (CMs) may have multiple cleavage sites for leveraging the activities of multiple disease-associated proteases.

[0026] Understanding the substrate cleavage profile and using these substrates as tools for activation in a specific disease or cancer type will enable the development of new therapeutic protease-activatable molecules. Fine tuning the therapeutic-activatable molecules by using protease substrates with unique cleavage profiles will allow for treatment options for a broader spectrum of patients while offering an improved therapeutic index. For example,CYTX-108-WO :: 4862-156.WO1 “omics” studies have demonstrated the distribution of numerous matrix metalloproteases (MMPs) across numerous cancer types and differences in the expression of the proteases 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 appropriate cleavable moiety selection. Indeed, 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). In addition, membrane type serine protease 1 (MT-SP1) shows great potential for protease-activatable antibody development (Howng, B. et al. “Novel Ex Vivo Zymography Approach for Assessment of Protease Activity in Tissues with Activatable Antibodies,” Pharmaceutics 2021, 13(9), 1390).

[0027] The present disclosure provides cleavable polypeptides comprising a cleavable moiety (CM) that is a substrate for at least one protease, e.g., an MMP and / or MT-SP1. In some aspects, the CMs herein are cleaved in a diseased tissue (e.g., tumor tissue) but less in a healthy tissue. These CMs are useful in a variety of therapeutic, diagnostic and prophylactic applications. In some aspects, the cleavable polypeptides are activatable molecules and further comprise an agent for conjugation or a target binding moiety (TBM) that specifically binds a target. For example, the TBM is a therapeutic macromolecule, a therapeutic agent, an imaging agent, a diagnostic agent, an antibody or antigen-binding fragment, a cytokine, a chemokine, a chimeric antigen receptor, or other molecule used in therapeutic and diagnostic applications.

[0028] Also provided herein are related compositions, kits, nucleic acids, vectors, and recombinant cells, as well as related methods, including methods of using and methods of producing any of the cleavable polypeptides described herein. DEFINITIONS

[0029] 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 are described herein for use in the present disclosure; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and 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, theCYTX-108-WO :: 4862-156.WO1 present specification—including definitions—will control over text incorporated by reference.

[0030] The terms “a” and “an” refer to one or more (i.e., at least one) of the grammatical object of the article. By way of example, “a cell” encompasses one or more cells.

[0031] As used herein, the terms “about” and “approximately,” when used to modify an amount specified in a numeric value or range, indicate that the numeric value as well as reasonable deviations from the value known to the skilled person in the art. For example ± 20%, ± 10%, or ± 5%, are within the intended meaning of the recited value where appropriate.

[0032] Concentrations, amounts, and other numerical data may be expressed or presented herein in a range format. It is to be understood that such a range format is used merely for convenience and brevity and thus should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is 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 values of about 0.01 to about 2.0, but also include individual values and sub-ranges within the indicated range. Thus, included in this numerical range are individual values such as 0.5, 0.7, and 1.5, and sub-ranges such as from 0.5 to 1.7, 0.7 to 1.5, and from 1.0 to 1.5, etc. Furthermore, such an interpretation should apply regardless of the breadth of the range or the characteristics being described. Additionally, it is noted that all percentages are in weight, unless specified otherwise.

[0033] “Including” or “comprising” and their derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The foregoing also applies to words having similar meanings such as “having” and their derivatives.

[0034] “Consisting” and its derivatives, as used herein, are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but exclude the presence of other unstated features, elements, components, groups, integers and / or steps. “Consisting essentially of” is intended to specify the presence of the stated features, elements, components, groups, integers, and / or steps as well as those that do not materially affect the basic and novel characteristic(s) of features, elements,CYTX-108-WO :: 4862-156.WO1 components, groups, integers, and / or steps. Reference to any one of these transition terms (i.e., “comprising,” “consisting,” or “consisting essentially”) provides direct support for replacement to any of the other transition term not specifically used. For example, amending a term from “comprising” to “consisting essentially of” or “consisting of” wouldfind direct support due to this definition for any elements disclosed throughout this disclosure. Based on this definition, any element disclosed herein or incorporated by reference may be included in or excluded from the claimed invention.

[0035] As used herein, a plurality of compounds, elements, or steps may be presented in a common list for convenience. However, these lists should be construed as though each member of the list is individually identified as a separate and unique member. Thus, no individual member of such list should be construed as a de facto equivalent of any other member of the same list solely based on their presentation in a common group without indications to the contrary.

[0036] The term “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 concrete fashion.

[0037] Furthermore, certain molecules, constructs, compositions, elements, moieties, excipients, disorders, conditions, properties, steps, or the like may be discussed in the context of one specific aspect or aspect or in a separate paragraph or section of this disclosure. It is understood that this is merely for convenience and brevity, and any such disclosure is equally applicable to and intended to be combined with any other aspects or aspects found anywhere in the present disclosure and claims, which all form the application and claimed invention at the filing date. For example, a list of constructs, molecules, method steps, kits, or compositions described with respect to a construct, molecule, polypeptide, activatable molecule, composition, or method is intended to and does find direct support for aspects related to constructs, molecules, polypeptides, activatable molecules, compositions, formulations, and methods described in any other part of this disclosure, even if those method steps, active agents, kits, or compositions are not re-listed in the context or section of that aspect or aspect.

[0038] “Activatable” when used in reference to an activatable molecule refers to an activatable molecule that exhibits afirst level of one or more activities, whereupon exposure to a condition that causes cleavage of one or more cleavable moieties results in the generation ofCYTX-108-WO :: 4862-156.WO1 a molecule that exhibits a second level of the one or more activities, where the second level of activity is greater than thefirst level of activity.

[0039] The term “activatable molecule” refers to a molecule that comprises at least one set of a MM, a CM, and an agent for conjugation or a TBM which exhibits attenuated activity compared to the activity of a counterpart “activated” molecule comprising the same agent or TBM. The terms “activated molecule,” and “cleaved activatable molecule,” are used interchangeably herein to refer to the agent-containing or TBM-containing cleavage product that is generated after exposure of the activatable molecule to a CM-specific protease (i.e., after cleavage of the CM by at least one protease). In some aspects, a cleaved activatable molecule may lack a MM due to cleavage of the CM (e.g., by a protease), resulting in release of the MM. For example, when the activatable molecule comprises a MM, a CM, and a TBM, the activatable molecule exhibits attenuated binding to a target as compared to the binding of a counterpart “activated” molecule comprising the same TBM to the same target.

[0040] The term “at least [a certain] % identical to” in the context of two or more nucleic acid or amino acid sequences means that the two or more sequences have nucleotides or amino acid residues in common in the given percent when compared and aligned for maximum correspondence over a comparison window or designated sequences of nucleic acids or amino acids (i.e. the sequences have at least 90 percent (%) identity). Percent identity of nucleic acid or amino acid sequences can be measured using a BLAST sequence comparison algorithm with default parameters, or by manual alignment and visual inspection (see e.g. blast.ncbi.nlm.nih.gov / Blast.cgi). Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, the % sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y where X is the number of amino acid residues scored as identical matches by the sequence in that program’s alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % sequence identity of A to B will not equal the % sequence identity of B to A.CYTX-108-WO :: 4862-156.WO1

[0041] The terms “cleavable moiety” and “CM” are used interchangeably herein to refer to an amino acid sequence that comprises a substrate for a protease.

[0042] The term “cleavable polypeptide” as used herein refers to a polypeptide that is present in a form other than that found in nature. A “cleavable polypeptide” as used herein may be encoded by cDNA, recombinant RNA, recombinant DNA, or a polynucleotide of synthetic origin or some combination thereof. By virtue of its origin, or source of derivation, the “cleavable polypeptide” (1) is not in a naturally occurring organism (e.g., is not an endogenous polypeptide of a naturally occurring organism) and (2) is present in a form not found in nature. In some aspects, the “cleavable polypeptide” is expressed by a cell from a different species. In some aspects, the “cleavable polypeptide” is a therapeutic protein or a diagnostic protein and not a naturally occurring protein. For example, as used herein, the “cleavable polypeptide” is not a plant protein or a protein naturally occurring in bacteria or other natural organisms. The term cleavable polypeptide includes and provides support for activatable molecules including activatable macromolecules, activatable polypeptides, activatable antibodies, activatable cytokines, and the like. The term cleavable polypeptide includes and provides support for activatable molecules in which cleavage of the CM activates the molecule.

[0043] The terms “masking moiety” or “MM” are used interchangeably herein to refer to a polypeptide or protein positioned in an activatable molecule that interferes with binding of the TBM to its target.

[0044] The phrase “masking efficiency” refers to the activity (e.g., EC50) of the activatable molecule divided by the activity of a control molecule, wherein the control molecule may be either cleavage product of the activatable molecule (i.e., the activated molecule) or a recombinant TBM used in the activatable molecule. An activatable molecule having a reduced level of a TBM activity may have a masking efficiency that is greater than 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some aspects, the activatable molecules described herein have a masking efficiency that is greater than 10, greater than 100, greater than 1000, or greater than 5000. In some aspects, the activatable molecule has a masking efficiency that is about 10 to about 1000, or about 10 to about 500, or about 50 to about 250, or about 50 to about 100, as measured by the ratio of the EC50 of the (uncleaved) activatable molecule to the EC50 of control TBM in a cell assay that is responsive to activity of the TBM.

[0045] Unless otherwise specified, a “polynucleotide” as used herein shall mean a polymer of nucleotides, such as, for example, a deoxyribonucleic acid (DNA), cDNA, aCYTX-108-WO :: 4862-156.WO1 ribonucleic acid (RNA), a polynucleotide of synthetic origin, and the like, or some combination thereof. The term “nucleic acid” refers to a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), or a combination thereof, in either a single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses complementary sequences as well as the sequence explicitly indicated. In some aspects, the nucleic acid is DNA. In some aspects, the nucleic acid is RNA.

[0046] Unless otherwise specified, the terms “isolated nucleic acid” and “nucleic acid encoding a cleavable polypeptide” as used herein shall mean a polynucleotide which, by virtue of its origin, (1) is not associated with all or a portion of a polynucleotide in which the “isolated nucleic acid” or “nucleic acid encoding a cleavable polypeptide” is found in nature, (2) is operably linked to a polynucleotide which it is not linked to in nature, and / or (3) does not occur in nature as part of a larger sequence.

[0047] Unless otherwise specified, the phrase “nucleic acid sequence encoding” an amino acid sequence or protein includes all nucleotide sequences that are degenerate versions of each other and thus encode the same amino acid sequence. Persons of ordinary skill in the art can convert amino acid sequences to derive sequences of nucleic acids encoding the amino acid sequences using automated tools (for example, bioinformatics.org / sms2 / rev_trans.html).

[0048] Unless otherwise specified, the terms “polypeptide” and “recombinant polypeptide” are used interchangeably herein to refer to a polymer of amino acids that exists in a form that is not found in nature. As used herein, a “protein” can be a polymer of amino acids, but can also be a complex of two or more polymers of amino acids assembled into a quaternary structure. Polypeptides employed herein may be encoded by cDNA, recombinant RNA, recombinant DNA, or a polynucleotide of synthetic origin or some combination thereof. By virtue of its origin, or source of derivation, the “polypeptide” (1) is not in a naturally occurring organism (e.g., is not an endogenous polypeptide of a naturally occurring organism) and (2) is present in a form not found in nature.

[0049] The terms “N-terminal to” or “N-terminally,” when referring to a position of a first domain or sequence relative to a second domain or sequence in a polypeptide primary amino acid sequence, mean that thefirst domain or sequence is located closer to the N- terminus of the polypeptide primary amino acid sequence than the second domain orCYTX-108-WO :: 4862-156.WO1 sequence. In some aspects, there are additional sequences and / or domains between thefirst domain or sequence and the second domain or sequence.

[0050] The terms “C-terminal to” or “C-terminally,” when referring to a position of afirst domain or sequence relative to a second domain or sequence in a polypeptide primary amino acid sequence, mean that thefirst domain or sequence is located closer to the C-terminus of the polypeptide primary amino acid sequence than the second domain or sequence. In some aspects, there are additional sequences and / or domains between thefirst domain or sequence and the second domain or sequence.

[0051] “Exogenous” refers to any material introduced from or originating from outside a cell, a tissue, or an organism that is not produced by or does not originate from the same cell, tissue, or organism in which it is being introduced.

[0052] As used herein, the term “linker” refers to amino acid sequences that connect or link two polypeptide sequences, e.g., that link two polypeptide domains. Exemplary linkers are described in more detail below.

[0053] The terms “transduced,” “transfected,” or “transformed” refer to a process by which an exogenous nucleic acid is introduced or transferred into a cell. A “transduced,” “transfected,” or “transformed” cell (e.g., mammalian cell) is one that has been transduced, transfected, or transformed with exogenous nucleic acid (e.g., a vector) that includes an exogenous nucleic acid encoding any of the activatable molecules described herein.

[0054] As used herein, the terms “specific binding” and “specifically binds” refer to the non-covalent interactions of the type that occur between a TBM and its target, e.g., an immunoglobulin molecule and an antigen or a cytokine and its receptor, for which the TBM is specific. The strength or affinity of binding interactions can be expressed in terms of the dissociation constant (Kd) of the interaction, wherein a smaller Kd represents a greater affinity. Unless indicated otherwise, as used herein, “affinity” refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a TBM 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 for a TBM and its target are known in the art. Immunological binding properties of selected polypeptides can be quantified using methods well known in the art. One such method entails measuring the rates of antigen-binding site / antigen complex formation and dissociation, wherein those rates depend on the concentrations of the complexCYTX-108-WO :: 4862-156.WO1 partners, the affinity of the interaction, and geometric parameters that equally influence the rate in both directions. Thus, both the “on rate constant” (Kon) and the “off rate constant” (Koff) can be determined by calculation of the concentrations and the actual rates of association and dissociation. (See Nature 361:186-87 (1993)). The ratio of Koff / Kon enables the cancellation of all parameters not related to affinity, and is equal to the dissociation constant Kd. (See, generally, Davies et al. (1990) Annual Rev Biochem 59:439-473). As used herein, a statement that a TBM “specifically binds” to its target refers to a TBM that binds its target with a dissociation constant (Kd) of less than 100 µM (e.g., less than 5 µM or 10 µM). In some examples, the TBM specifically binds its target with a Kdof about 0.01 nM to about 500 nM. In some examples, a TBM is said to specifically bind the target, when the equilibrium binding constant (Kd) is less than or equal to1 micromolar, in some aspects less than or equal to 100 nM, in some aspects less than or equal to 10 nM, and in some aspects less than or equal to 100 pM to about 1 pM, as measured by assays such as radioligand binding assays or similar assays known to those skilled in the art.

[0055] A “half-life extending moiety” or “EM” is a part of an activatable molecule that increases the serum half-life of the molecule attached to the EM.

[0056] An “Fc domain” refers to a contiguous amino acid sequence of a portion of a single heavy chain of an immunoglobulin that excludes its variable domains, e.g., the hinge, the CH2-CH3 domains of IgG, IgA, or IgD, or the CH2-CH3-CH4 domains of IgE or IgM and engineered variants thereof. A pair of Fc domains associate together to form an Fc region of an immunoglobulin.

[0057] 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. Included within the usage of the term “sample,” therefore, is blood and a fraction or component of blood including blood serum, blood plasma, or lymph.

[0058] The term “therapeutic macromolecule” refers to any protein that may be administered to a subject and have a therapeutic effect. In some aspects, the therapeutic macromolecule is a therapeutic polypeptide.

[0059] As generally provided herein, an activatable molecule may comprise MM-CM construct(s), also referred to herein as a prodomain. Accordingly, as used herein, the term “prodomain” refers to a polypeptide domain comprising a masking moiety (MM) and a cleavable moiety (CM). In some aspects, the MM and the CM are separated by a linker, referred to herein as LP1. In some aspects, the prodomain comprises a linker (referred toCYTX-108-WO :: 4862-156.WO1 herein as LP2) that links the CM of the prodomain to the target binding moiety (TBM) in an activatable molecule. In some aspects, the prodomain comprises a linker between the MM and the CM and a linker between the CM and the TBM. In some aspects, the MM and the CM are not separated by a linker. In certain aspects, a prodomain comprises one of the following formulae (where the formulae below represent amino acid sequences in either N- to C-terminal direction or C- to N-terminal direction): MM-LP1-CM, MM-CM-LP2, MM-LP1- CM-LP2, or MM-CM. As used herein and unless otherwise stated, each dash (-) between the components of the activatable molecule represents either a direct linkage or indirect linkage via one or more linking peptides.

[0060] “Treatment” refers to ameliorating at least one symptom of a disorder. CLEAVABLE POLYPEPTIDES

[0061] Proteases are involved in the control of numerous physiological processes, and their dysregulation has been identified in a number of pathologies, such as, for example, oncological, cardiovascular, autoimmune, and neurodegenerative diseases. See, e.g., 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 Discovery, 5 (2006). Protease-activated antibodies have been described in the literature that are activated by native proteases which are more prevalently active in, for example, tumor tissue, and the like, when compared to normal tissue. Id. These prodrugs have incorporated within their structure, a protease substrate that releases active drug following exposure to the appropriate protease and its subsequent cleavage. What appears evident, however, is that the profile of dysregulated protease activity in diseased tissue may differ from one type of disease tissue / disorder to another. Thus, it is desirable to have a collection of substrates that target a variety of different protease activity profiles.

[0062] In one aspect, the present disclosure provides a cleavable polypeptide comprising a cleavable moiety (CM) comprising an amino acid sequence QXAQX1LX2XBA (SEQ ID NO: 74), wherein XA is N, A, W, or F; X1 is G or A; X2 is R or K; XB is S, M, V, or L,CYTX-108-WO :: 4862-156.WO1 provided that i) when XAis N, then XBis S, L, or V; ii) when XAis A, then XBis S or M; and iii) when XA is W or F, then XB is M; and wherein the CM is a substrate for a protease.

[0063] In some aspects, the cleavable polypeptide comprises more than one CM, more than two CMs, more than three CMs, or more than four CMs. In some aspects, the cleavable polypeptide comprises 1 to 8 CMs. In some aspects, the cleavable polypeptide comprises 2 to 4 CMs. In some aspects, each of the CMs, e.g., each of the 2 to 4 CMs, independently comprises an amino acid sequence according to the amino acid sequence QXAQX1LX2XBA (SEQ ID NO: 74). In some aspects, the cleavable polypeptide comprises 3 CMs or 4 CMs.

[0064] In some aspects, the CM comprises a sequence selected from: QNQX1LX2SA (SEQ ID NO: 4), QAQX1LX2MA (SEQ ID NO: 9), QAQX1LX2SA (SEQ ID NO: 14), QNQX1LX2VA (SEQ ID NO: 19), QNQX1LX2LA (SEQ ID NO: 24), QWQX1LX2MA (SEQ ID NO: 29), QFQX1LX2MA (SEQ ID NO: 34),wherein X1 is G or A and X2 is R or K.

[0065] In some aspects, the CM comprises a sequence selected from: QNQALRSA (SEQ ID NO: 5), QNQGLRSA (SEQ ID NO: 6), QNQALKSA (SEQ ID NO: 7), QNQGLKSA (SEQ ID NO: 8), QAQALRMA (SEQ ID NO: 10), QAQGLRMA (SEQ ID NO: 11), QAQALKMA (SEQ ID NO: 12), QAQGLKMA (SEQ ID NO: 13), QAQALRSA (SEQ ID NO: 15), QAQGLRSA (SEQ ID NO: 16), QAQALKSA (SEQ ID NO: 17), QAQGLKSA (SEQ ID NO: 18), QNQALRVA (SEQ ID NO: 20), QNQGLRVA (SEQ ID NO: 21), QNQALKVA (SEQ ID NO: 22), QNQGLKVA (SEQ ID NO: 23), QNQALRLA (SEQ ID NO: 25), QNQGLRLA (SEQ ID NO: 26), QNQALKLA (SEQ ID NO: 27), QNQGLKLA (SEQ ID NO: 28), QWQALRMA (SEQ ID NO: 30), QWQGLRMA (SEQ ID NO: 31), QWQALKMA (SEQ ID NO: 32), QWQGLKMA (SEQ ID NO: 33), QFQALRMA (SEQ ID NO: 35), QFQGLRMA (SEQ ID NO: 36), QFQALKMA (SEQ ID NO: 37), or QFQGLKMA (SEQ ID NO: 38). In cases where the cleavable polypeptide comprises more than one CM, the CMs each independently comprise one of the foregoing sequences.

[0066] In certain aspects, the CM comprises a sequence selected from: QNQALRSA (SEQ ID NO: 5), QNQGLRSA (SEQ ID NO: 6), QNQALKSA (SEQ ID NO: 7), or QNQGLKSA (SEQ ID NO: 8).

[0067] In certain aspects, the CM comprises a sequence selected from: QAQALRMA (SEQ ID NO: 10), QAQGLRMA (SEQ ID NO: 11), QAQALKMA (SEQ ID NO: 12), or QAQGLKMA (SEQ ID NO: 13).CYTX-108-WO :: 4862-156.WO1

[0068] In certain aspects, the CM comprises a sequence selected from: QAQALRSA (SEQ ID NO: 15), QAQGLRSA (SEQ ID NO: 16), QAQALKSA (SEQ ID NO: 17), or QAQGLKSA (SEQ ID NO: 18).

[0069] In certain aspects, the CM comprises a sequence selected from: QNQALRVA (SEQ ID NO: 20), QNQGLRVA (SEQ ID NO: 21), QNQALKVA (SEQ ID NO: 22), or QNQGLKVA (SEQ ID NO: 23).

[0070] In certain aspects, the CM comprises a sequence selected from: QNQALRLA (SEQ ID NO: 25), QNQGLRLA (SEQ ID NO: 26), QNQALKLA (SEQ ID NO: 27), or QNQGLKLA (SEQ ID NO: 28).

[0071] In certain aspects, the CM comprises a sequence selected from: QWQALRMA (SEQ ID NO: 30), QWQGLRMA (SEQ ID NO: 31), QWQALKMA (SEQ ID NO: 32), or QWQGLKMA (SEQ ID NO: 33).

[0072] In certain aspects, the CM comprises a sequence selected from: QFQALRMA (SEQ ID NO: 35), QFQGLRMA (SEQ ID NO: 36), QFQALKMA (SEQ ID NO: 37), or QFQGLKMA (SEQ ID NO: 38).

[0073] In certain aspects, the CM comprises a sequence selected from: QNQALRSA (SEQ ID NO: 5), QAQALRMA (SEQ ID NO: 10), QAQALRSA (SEQ ID NO: 15), QNQALRVA (SEQ ID NO: 20), QNQALRLA (SEQ ID NO: 25), QWQALRMA (SEQ ID NO: 30), or QFQALRMA (SEQ ID NO: 35).

[0074] In some aspects, the CM 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: 4-38 and 74. In some examples, the CM 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: 4-38 and 74. In some examples, the CM 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: 4-38 and 74. In some examples, the CM 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: 4-38 and 74.

[0075] In some aspects, the CM consists of the amino acid sequence of SEQ ID NO: 4. In some aspects, the CM consists of the amino acid sequence of SEQ ID NO: 5. In some aspects, the CM consists of the amino acid sequence of SEQ ID NO: 6. In some aspects, the CM consists of the amino acid sequence of SEQ ID NO: 7. In some aspects, the CM consists of the amino acid sequence of SEQ ID NO: 8. In some aspects, the CM consists of the aminoCYTX-108-WO :: 4862-156.WO1 acid sequence of SEQ ID NO: 9. In some aspects, the CM consists of the amino acid sequence of SEQ ID NO: 10. In some aspects, the CM consists of the amino acid sequence of SEQ ID NO: 11. In some aspects, the CM consists of the amino acid sequence of SEQ ID NO: 12. In some aspects, the CM consists of the amino acid sequence of SEQ ID NO: 13. In some aspects, the CM consists of the amino acid sequence of SEQ ID NO: 14. In some aspects, the CM consists of the amino acid sequence of SEQ ID NO: 15. In some aspects, the CM consists of the amino acid sequence of SEQ ID NO: 16. In some aspects, the CM consists of the amino acid sequence of SEQ ID NO: 17. In some aspects, the CM consists of the amino acid sequence of SEQ ID NO: 18. In some aspects, the CM consists of the amino acid sequence of SEQ ID NO: 19. In some aspects, the CM consists of the amino acid sequence of SEQ ID NO: 20. In some aspects, the CM consists of the amino acid sequence of SEQ ID NO: 21. In some aspects, the CM consists of the amino acid sequence of SEQ ID NO: 22. In some aspects, the CM consists of the amino acid sequence of SEQ ID NO: 23. In some aspects, the CM consists of the amino acid sequence of SEQ ID NO: 24. In some aspects, the CM consists of the amino acid sequence of SEQ ID NO: 25. In some aspects, the CM consists of the amino acid sequence of SEQ ID NO: 26. In some aspects, the CM consists of the amino acid sequence of SEQ ID NO: 27. In some aspects, the CM consists of the amino acid sequence of SEQ ID NO: 28. In some aspects, the CM consists of the amino acid sequence of SEQ ID NO: 29. In some aspects, the CM consists of the amino acid sequence of SEQ ID NO: 30. In some aspects, the CM consists of the amino acid sequence of SEQ ID NO: 31. In some aspects, the CM consists of the amino acid sequence of SEQ ID NO: 32. In some aspects, the CM consists of the amino acid sequence of SEQ ID NO: 33. In some aspects, the CM consists of the amino acid sequence of SEQ ID NO: 34. In some aspects, the CM consists of the amino acid sequence of SEQ ID NO: 35. In some aspects, the CM consists of the amino acid sequence of SEQ ID NO: 36. In some aspects, the CM consists of the amino acid sequence of SEQ ID NO: 37. In some aspects, the CM consists of the amino acid sequence of SEQ ID NO: 38. In some aspects, the CM consists of the amino acid sequence of SEQ ID NO: 74.

[0076] In some aspects, the CM comprises a sequence selected from: QWQX1LX2SA (SEQ ID NO: 178), QFQX1LX2SA (SEQ ID NO: 183), QWQX1LX2VA (SEQ ID NO: 188), QFQX1LX2VA (SEQ ID NO: 193), QAQX1LX2VA (SEQ ID NO: 198), QWQX1LX2LA (SEQ ID NO: 203), QFQX1LX2LA (SEQ ID NO: 208), QAQX1LX2LA (SEQ ID NO: 213), wherein X1is G or A and X2is R or K.CYTX-108-WO :: 4862-156.WO1

[0077] In some aspects, the CM comprises a sequence selected from: QWQALRSA (SEQ ID NO: 179), QWQGLRSA (SEQ ID NO: 180), QWQALKSA (SEQ ID NO: 181), QWQGLKSA (SEQ ID NO: 182), QFQALRSA (SEQ ID NO: 184), QFQGLRSA (SEQ ID NO: 185), QFQALKSA (SEQ ID NO: 186), QFQGLKSA (SEQ ID NO: 187), QWQALRVA (SEQ ID NO: 189), QWQGLRVA (SEQ ID NO: 190), QWQALKVA (SEQ ID NO: 191), QWQGLKVA (SEQ ID NO: 192), QFQALRVA (SEQ ID NO: 194), QFQGLRVA (SEQ ID NO: 195), QFQALKVA (SEQ ID NO: 196), QFQGLKVA (SEQ ID NO: 197), QAQALRVA (SEQ ID NO: 199), QAQGLRVA (SEQ ID NO: 200), QAQALKVA (SEQ ID NO: 201), QAQGLKVA (SEQ ID NO: 202), QWQALRLA (SEQ ID NO: 204), QWQGLRLA (SEQ ID NO: 205), QWQALKLA (SEQ ID NO: 206), QWQGLKLA (SEQ ID NO: 207), QFQALRLA (SEQ ID NO: 209), QFQGLRLA (SEQ ID NO: 210), QFQALKLA (SEQ ID NO: 211), QFQGLKLA (SEQ ID NO: 212), QAQALRLA (SEQ ID NO:214), QAQGLRLA (SEQ ID NO:215), QAQALKLA (SEQ ID NO:216), QAQGLKLA (SEQ ID NO:217), QNQALRM (SEQ ID NO:218), QNQALR (SEQ ID NO:219), QNQALRS (SEQ ID NO:220), QAQALRM (SEQ ID NO:221), QAQALRS (SEQ ID NO:222), QNQALRV (SEQ ID NO:223), QNQALRL (SEQ ID NO:224), QWQALRM (SEQ ID NO:225), QFQALRM (SEQ ID NO:226), QAQALR (SEQ ID NO:227), QWQALR (SEQ ID NO228), or QFQALR (SEQ ID NO:229)

[0078] In some embodiments, the substrate comprises a combination, a C-terminal truncation variant, a C-terminal extension variant, an N-terminal truncation variant, or an N- terminal extension variant of the amino acid sequences of any one of SEQ ID NOs: 5-8, 10- 13, 15-18, 20-23, 25-28, 30-33, 35-38, 179-182, 184-187, 189-192, 194-197 and 199-202, 204-207, 209-212, 214-229. In some embodiments, the substrate comprises a combination, a C-terminal truncation variant, a C-terminal extension variant, an N-terminal truncation variant, or an N-terminal extension variant of the amino acid sequences of any one of SEQ ID NOs: 5-8, 10-13, 15-18, 20-23, 25-28, 30-33, 35-38, 179-182, 184-187, 189-192, 194-197 and 199-202, 204-207, 209-212, 214-229. Truncation variants of the aforementioned amino acid sequences that are suitable for use in a substrate may be any that retain the recognition site for the corresponding protease. In certain embodiments, the truncation variant comprises a C-terminal deletion and / or an N-terminal deletion of one amino acid residue from an amino acid sequence selected from the group consisting of SEQ ID NOs: 5-8, 10-13, 15-18, 20-23, 25-28, 30-33, 35-38, 179-182, 184-187, 189-192, 194-197 and 199-202, 204-207, 209-212, 214-229. These include C-terminal and / or N-terminal truncation variants comprising at leastCYTX-108-WO :: 4862-156.WO1 1, 2, 3, 4, 5, or more contiguous amino acids of the above-described amino acid sequences that retain a recognition site for a protease. In certain embodiments, the truncation variant comprises a C-terminal deletion and / or an N-terminal deletion of one amino acid residue from an amino acid sequence selected from the group consisting of SEQ ID NOs: 5-8, 10-13, 15-18, 20-23, 25-28, 30-33, 35-38, 179-182, 184-187, 189-192, 194-197 and 199-202, 204- 207, 209-212, 214-229. Extension variants of the aforementioned amino acid sequences that are suitable for use in a substrate may be any that have one or more (e.g., 1, 2, 3, 4, 5 or more) additional amino acids and retain the recognition site for the corresponding protease. In some examples, the additional amino acids are coupled to the C-terminus of the aforementioned amino acid sequences. In some examples, the additional amino acids are coupled to the N-terminus of the aforementioned amino acid sequences. In some examples, the extension variants may comprise additional amino acids coupled to both the C-terminus and the N-terminus of the aforementioned amino acid sequences. In some instances, the C- terminus or N-terminus extension variants can have a C-terminal glycine or an N-terminal serine amino acid.

[0079] 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: 5-8, 10- 13, 15-18, 20-23, 25-28, 30-33, 35-38, 179-182, 184-187, 189-192, 194-197 and 199-202, 204-207, 209-212, 214-229. 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: 5-8, 10-13, 15-18, 20-23, 25-28, 30-33, 35-38, 179-182, 184-187, 189-192, 194-197 and 199-202, 204-207, 209-212, 214-229. 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: 5-8, 10-13, 15-18, 20-23, 25-28, 30-33, 35-38, 179- 182, 184-187, 189-192, 194-197 and 199-202, 204-207, 209-212, 214-229. 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: 5-8, 10-13, 15-18, 20-23, 25-28, 30-33, 35-38, 179-182, 184-187, 189-192, 194-197 and 199-202, 204- 207, 209-212, 214-229. 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: 5-8, 10-13, 15-18, 20-23, 25-28, 30-33, 35-38, 179-182, 184-187, 189-192, 194-197 and 199-202, 204-207, 209-212, 214-229. In some examples, the substrate comprises one, two, three, four, five, six or more additional amino acids at the C-terminus ofCYTX-108-WO :: 4862-156.WO1 the amino acid sequence of any one of SEQ ID NOs: 5-8, 10-13, 15-18, 20-23, 25-28, 30-33, 35-38, 179-182, 184-187, 189-192, 194-197 and 199-202, 204-207, 209-212, 214-229. 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: 5-8, 10-13, 15-18, 20-23, 25-28, 30-33, 35-38, 179-182, 184-187, 189-192, 194-197 and 199-202, 204- 207, 209-212, 214-229. 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: 5-8, 10-13, 15-18, 20-23, 25-28, 30-33, 35-38, 179-182, 184-187, 189-192, 194-197 and 199-202, 204-207, 209-212, 214-229.

[0080] In some embodiments, the substrate comprises a sequence with mutation(s) of one or more amino acid of the amino acid sequence of any one of SEQ ID NOs: 5-8, 10-13, 15- 18, 20-23, 25-28, 30-33, 35-38, 179-182, 184-187, 189-192, 194-197 and 199-202, 204-207, 209-212, 214-229. In some embodiments, the substrate comprises a sequence with mutation(s) of one or more amino acid of the amino acid sequence of any one of SEQ ID NOs: 5-8, 10-13, 15-18, 20-23, 25-28, 30-33, 35-38, 179-182, 184-187, 189-192, 194-197 and 199-202, 204-207, 209-212, 214-229. For example, the substrate comprises a sequence with one-amino acid, two-amino acid, three-amino acid, or four-amino acid mutations of the amino acid sequence of any one of SEQ ID NOs: 5-8, 10-13, 15-18, 20-23, 25-28, 30-33, 35- 38, 179-182, 184-187, 189-192, 194-197 and 199-202, 204-207, 209-212, 214-229. For example, the substrate comprises a sequence with one-amino acid, two-amino acid, three- amino acid, or four-amino acid mutations of the amino acid sequence of any one of SEQ ID NOs: 5-8, 10-13, 15-18, 20-23, 25-28, 30-33, 35-38, 179-182, 184-187, 189-192, 194-197 and 199-202, 204-207, 209-212, 214-229. In some embodiments, the substrate comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5-8, 10-13, 15-18, 20-23, 25-28, 30-33, 35-38, 179-182, 184-187, 189-192, 194-197 and 199-202, 204-207, 209- 212, 214-229 and having one conservative substitution. In some embodiments, the substrate comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5-8, 10-13, 15-18, 20-23, 25-28, 30-33, 35-38, 179-182, 184-187, 189-192, 194-197 and 199-202, 204-207, 209-212, 214-229 and having two conservative substitutions.

[0081] In some embodiments, the substrate consists of a sequence with mutation(s) of one or more amino acid of the amino acid sequence of any one of SEQ ID NOs: 5-8, 10-13,CYTX-108-WO :: 4862-156.WO1 15-18, 20-23, 25-28, 30-33, 35-38, 179-182, 184-187, 189-192, 194-197 and 199-202, 204- 207, 209-212, 214-229. In some embodiments, the substrate consists of a sequence with mutation(s) of one or more amino acid of the amino acid sequence of any one of SEQ ID NOs: 5-8, 10-13, 15-18, 20-23, 25-28, 30-33, 35-38, 179-182, 184-187, 189-192, 194-197 and 199-202, 204-207, 209-212, 214-229. For example, the substrate consists of a sequence with one-amino acid, two-amino acid, three-amino acid, or four-amino acid mutation(s) of the amino acid sequence of any one of SEQ ID NOs: 5-8, 10-13, 15-18, 20-23, 25-28, 30-33, 35-38, 179-182, 184-187, 189-192, 194-197 and 199-202, 204-207, 209-212, 214-229. For example, the substrate consists of a sequence with one-amino acid, two-amino acid, three- amino acid, or four-amino acid mutation(s) of the amino acid sequence of any one of SEQ ID NOs: 5-8, 10-13, 15-18, 20-23, 25-28, 30-33, 35-38, 179-182, 184-187, 189-192, 194-197 and 199-202, 204-207, 209-212, 214-229.

[0082] In some aspects, the cleavability of the CMs are presented as the percentage of the fraction of cleaved CMs (or polypeptides comprising the CMs). In some examples, the cleavability of the CM by a protease (e.g., an MMP) 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 CM by MMP2 is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, or 100% when 500 nM activatable antibody c225 containing a prodomain with the CM being tested was incubated with 10 nM of MMP2 for 4 hours at 37ºC. In some examples, the cleavability of the CM by MMP9 is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, or 100% when 500 nM activatable antibody c225 containing a prodomain with the CM being tested was incubated with 10 nM of MMP9 for 4 hours at 37ºC. In some examples, the cleavability of the CM by MMP14 is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, or 100% when 500 nM activatable antibody c225 containing a prodomain with the CM being tested was incubated with 10 nM of MMP14 for 4 hours at 37ºC. In some examples, the cleavability of the CM 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% when 500 nM activatable antibody c225 containing a prodomain with the CM being tested was incubated with 10 nM of MT-SP1 for 4 hours at 37ºC.

[0083] In some aspects, for specific cleavage by an enzyme, contact between the enzyme and CM is made. When a cleavable polypeptide (e.g., activatable molecule comprising a TBM coupled to a MM and a CM) is in the presence of target and sufficient protease activity,CYTX-108-WO :: 4862-156.WO1 the CM can be cleaved. Sufficient protease activity refers to the ability of the protease to access the CM and effect cleavage.

[0084] In some aspects, a CM according to the present disclosure and a reference polypeptide can be cleaved by the same protease (e.g., an MMP or an MT-SP1), but the CM according to the present disclosure has reduced cleavage or resistance to cleavage (e.g., by a different protease(s) than MT-SP1 or an MMP2, MMP9, and MMP14) in certain tissues in situ compared to a reference polypeptide. For example, the cleavage (e.g., by a different protease than an MT-SP1 or an MMP such as MMP2, MMP9, or MMP14) in situ of the CM is less than 99%, less than 95%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% compared to the cleavage of the reference polypeptide. In some examples, such proteases different than an MT-SP1 or MMP are other proteases in normal tissues, as well as other proteases involved in inflammation and wound healing. In some examples, the cleavage (e.g., by a different protease than an MT-SP1 or an MMP) in situ is measured by increased activity of an activatable molecule comprising the CM or the reference polypeptide in the normal tissue. A CM that is resistant to cleavage by a protease, or a sample or tissue comprising a protease, refers to (i) a CM in which no peptide bond is hydrolyzed by the protease, or no peptide bond is hydrolyzed when incubated in the sample or tissue comprising the protease, or (ii) a CM in which a reduced level of peptide bond is hydrolyzed by the protease, or reduced level of peptide bond is hydrolyzed when incubated in the sample or tissue comprising the protease, compared to a reference CM.

[0085] In some aspects, the CM is cleavable by more than one protease. For example, the CM is cleaved by MT-SP1 and alternatively, or additionally, one or more MMPs (e.g., MMP2, MMP9, and / or MMP14) and by a second or multiple additional proteases. Examples of the additional protease could be any one or more of the following proteases: a disintegrin and metalloprotease (ADAM), an ADAM-like, or a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS, such as, for example, ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAMDEC1, ADAMTS1, ADAMTS4, ADAMTS5); an aspartate protease (such as, for example, BACE, Renin, and the like); an aspartic cathepsin (such as, for example, Cathepsin D, Cathepsin E, and the like); a caspase (such as, for example, Caspase 1, Caspase 2, Caspase 3, Caspase 4, Caspase 5, Caspase 6, Caspase 7, Caspase 8, Caspase 9, Caspase 10, Caspase 14, and the like); a cysteine cathepsin (such as, for example, Cathepsin B, Cathepsin C, Cathepsin K, Cathepsin L, Cathepsin S,CYTX-108-WO :: 4862-156.WO1 Cathepsin V / L2, Cathepsin X / Z / P); a cysteine proteinase (such as, for example, Cruzipain, Legumain, Otubain-2, and the like); a kallikrein-related peptidase (KLK) (such as, for example, KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, KLK14, and the like); a metalloproteinase (such as, for example, Meprin, Neprilysin, prostate-specific membrane antigen (PSMA), bone morphogenetic protein 1 (BMP-1), and the like); a matrix metalloproteinase (MMP, such as, for example, MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP23, MMP24, MMP26, MMP27, and the like); a serine protease (such as, for example, activated protein C, Cathepsin A, Cathepsin G, Chymase, a coagulation factor protease (such as, for example, FVIIa, FIXa, FXa, FXIa, FXIIa, and the like); 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), a Type II transmembrane Serine Protease (TTSP) (such as, for example, DESC1, DPP-4, FAP, Hepsin, Matriptase-2, MT-SP1 / Matriptase, TMPRSS2, TMPRSS3, TMPRSS4, TMPRSS5, TMPRSS6, TMPRSS7, TMPRSS8, TMPRSS9, TMPRSS10, TMPRSS11, and the like), and the like. Specific substrates are described, for example, in WO 2010 / 081173, WO 2015 / 048329, WO 2015 / 116933, WO 2016 / 118629, WO2021207669, WO2021207657, WO2021142029, WO2021061867, WO2020252349, WO2020252358, WO2020236679, WO2020176672, WO2020118109, WO2020092881, WO2020086665, WO2019213444, WO2019183218, WO2019173771, WO2019165143, WO2019075405, WO2019046652, WO2019018828, WO2019014586, WO2018222949, WO2018165619, WO2018085555, WO2017011580, WO2016179335, WO2016179285, WO2016179257, WO2016149201, WO2024 / 030843, WO2024 / 030845, WO2024 / 030847, WO2024 / 030858, WO2024 / 030850, and WO2016014974, each of which is incorporated herein by reference in its entirety.

[0086] In some aspects, the present disclosure provides cleavable moieties that exhibit enhanced cleavability to a matrix metalloproteinase (MMP), e.g., MMP2, MMP9, or MMP14. In certain aspects, the cleavable moieties are cleaved by a second protease, e.g., MT-SP1. In certain aspects, the cleavable moieties are selectively cleavable by certain proteases (e.g., an MMP), but have reduced or no cleavability by another protease. In some aspects, resistance of cleavable moieties to protease cleavage in healthy tissue may reduce systemic toxicities by limiting binding of the activatable molecule to targets that also may beCYTX-108-WO :: 4862-156.WO1 present in healthy tissues. Therefore, cleavable moieties with normal tissue resistance have the potential to demonstrate a greater therapeutic window and safety profile with less on- target toxicities occurring in healthy tissues.

[0087] According to some aspects of the present disclosures, the cleavable polypeptide is a molecule in which cleavage of the CM by a protease results in a part or component of the molecule being separated from the remainder of the molecule. In some aspects of the present disclosure, cleavage of the CM by a protease activates the molecule. In some aspects, the cleavable polypeptide is a molecule in which multiple proteases cleave the CM. In some aspects, the cleavable polypeptide is a molecule in which MMP2 cleaves the CM. In some aspects, the cleavable polypeptide is a molecule in which MMP9 cleaves the CM. In some aspects, the cleavable polypeptide is a molecule in which MMP14 cleaves the CM. In some aspects, the cleavable polypeptide is a molecule in which MT-SP1 cleaves the CM. In some aspects, the cleavable polypeptide is a molecule in which two or all of MMP2, MMP9, MMP14, MT-SP1 cleave the CM. In some aspects, the cleavable polypeptide is a molecule in which the % cleavability of the CM is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, or 100%, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, or 100% cleavable by any one of MMP2, MMP9, MMP14, MT-SP1 or any two of MMP2, MMP9, MMP14, MT-SP1 or each of MMP2, MMP9, MMP14, MT-SP1.

[0088] In some aspects, the cleavable polypeptide is a molecule in which the CM has increased cleavage of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 300%, 400%, or 500% or more by any one of MMP2, MMP9, MMP14, MT-SP1 or any two of MMP2, MMP9, MMP14, MT-SP1 or each of MMP2, MMP9, MMP14, MT-SP1 compared to a cleavable polypeptide comprising a CM comprising a sequence of QNQALRMA (SEQ ID NO: 3). In some aspects, the increased cleavage is an increase of 20% to 500%, 30% to 400%, 40% to 300%, or 50% to 200%, including any other range within a range of 20 to 500%, by any one of MMP2, MMP9, MMP14, MT-SP1 or any two of MMP2, MMP9, MMP14, MT-SP1 or each of MMP2, MMP9, MMP14, MT-SP1 compared to a cleavable polypeptide comprising a CM comprising a sequence of QNQALRMA (SEQ ID NO: 3).

[0089] In some aspects, the cleavable polypeptide is a molecule in which the % cleavability of the CM is improved by 1.5x, 2x, 2.5x, 3x, 5x, 7x, 8x, or 10x or more over theCYTX-108-WO :: 4862-156.WO1 % cleavability of a cleavable polypeptide comprising a CM comprising a sequence of QNQALRMA (SEQ ID NO: 3).

[0090] The CM may be specifically cleaved by a protease (e.g., by an MMP such as MMP2, MMP9, or MMP14) at a desired rate. In some aspects, the cleavable moieties are cleaved by a second protease, e.g., MT-SP1. The rate may be measured as substrate cleavage kinetics (kcat / KM) as disclosed in WO2016118629, which is incorporated by reference in its entirety. In brief, kcatis the turnover number and describes how many substrate molecules are transformed into products per unit time by a protease. The KM value describes the affinity of the substrate to the active site of the protease. The kcat / KMratio provides a measurement of cleavability of the substrate by the protease. In general, the greater the ratio, the higher the rate of cleavability is; conversely, the lower the ratio, the slower the rate of cleavability is. The kcat / KM values may be determined with the following equationt is time (s), and p is protease concentration (M), which assumes that the substrate concentration is below the KM and in excess of the protease concentration.

[0091] According to aspects of the present disclosures, the cleavable polypeptide is a molecule comprising a CM that has a kcat / KM (M-1s-1) of greater than 1 x 102M-1s-1. According to some aspects of the present disclosures, the cleavable polypeptide is a molecule comprising a CM that has a kcat / KM (M-1s-1) of greater than 1 x 103M-1s-1. According to some aspects of the present disclosures, the cleavable polypeptide is a molecule comprising a CM that has a kcat / KM (M-1s-1) of greater than 1 x 104M-1s-1. According to some aspects of the present disclosures, the cleavable polypeptide is a molecule comprising a CM that has a kcat / KM (M-1s-1) of greater than 1 x 105M-1s-1.

[0092] In some aspects, the CM is cleaved by an MMP at a rate that has a kcat / KM value from 1×10 to 1×106M-1s-1, e.g., from 1×10 to 5×10, from 5×10 to 1×102, from 1×102to 5×102, from 5×102to 1×103, from 1×103to 5×103, from 5×103to 1×104, from 1×104to 5×104, from 5×104to 1×105, from 1×105to 5×105, or from 5×105to 1×106M-1s-1. In some aspects, the CM is cleaved by MMP at a rate that has a kcat / KM value of at least 1×10, at least 5×10, at least 1×102, at least 5×102, at least 1×103, at least 5×103, at least 1×104, at least 5×104, at least 1×105, at least 5×105, or at least 1×106. In some aspects, the CM has greaterCYTX-108-WO :: 4862-156.WO1 catalytic efficiency (i.e., a higher kcat / KMvalue) for an MMP compared to a cleavable polypeptide comprising a CM comprising a sequence of QNQALRMA (SEQ ID NO: 3).

[0093] In some aspects, the CM is cleaved by an MMP2 at a rate that has a kcat / KMvalue from 1×10 to 1×106M-1s-1, e.g., from 1×10 to 5×10, from 5×10 to 1×102, from 1×102to 5×102, from 5×102to 1×103, from 1×103to 5×103, from 5×103to 1×104, from 1×104to 5×104, from 5×104to 1×105, from 1×105to 5×105, or from 5×105to 1×106M-1s-1. In some aspects, the CM is cleaved by MMP2 at a rate that has a kcat / KMvalue of at least 1×10, at least 5×10, at least 1×102, at least 5×102, at least 1×103, 5×103, at least 1×104, at least 5×104, at least 1×105, at least 5×105, or at least 1×106. In some aspects, the CM has greater catalytic efficiency (i.e., a higher kcat / KM value) for an MMP2 compared to a cleavable polypeptide comprising a CM comprising a sequence of QNQALRMA (SEQ ID NO: 3).

[0094] In some aspects, the CM is cleaved by an MMP9 at a rate that has a kcat / KM value from 1×10 to 1×106M-1s-1, e.g., from 1×10 to 5×10, from 5×10 to 1×102, from 1×102to 5×102, from 5×102to 1×103, from 1×103to 5×103, from 5×103to 1×104, from 1×104to 5×104, from 5×104to 1×105, from 1×105to 5×105, or from 5×105to 1×106M-1s-1. In some aspects, the CM is cleaved by MMP9 at a rate that has a kcat / KM value of at least 1×10, at least 5×10, at least 1×102, at least 5×102, at least 1×103, at least 5×103, at least 1×104, at least 5×104, at least 1×105, at least 5×105, or at least 1×106. In some aspects, the CM has greater catalytic efficiency (i.e., a higher kcat / KMvalue) for an MMP9 compared to a cleavable polypeptide comprising a CM comprising a sequence of QNQALRMA (SEQ ID NO: 3).

[0095] In some aspects, the CM is cleaved by an MMP14 at a rate that has a kcat / KMvalue from 1×10 to 1×106M-1s-1, e.g., from 1×10 to 5×10, from 5×10 to 1×102, from 1×102to 5×102, from 5×102to 1×103, from 1×103to 5×103, from 5×103to 1×104, from 1×104to 5×104, from 5×104to 1×105, from 1×105to 5×105, or from 5×105to 1×106M-1s-1. In some aspects, the CM is cleaved by MMP14 at a rate that has a kcat / KMvalue of at least 1×10, at least 5×10, at least 1×102, at least 5×102, at least 1×103,at least 1×104, at least 5×104, at least 1×105, at least 5×105, or at least 1×106. In some aspects, the CM has greater catalytic efficiency (i.e., a higher kcat / KM value) for an MMP14 compared to a cleavable polypeptide comprising a CM comprising a sequence of QNQALRMA (SEQ ID NO: 3).

[0096] In some aspects, the CM is cleaved by MT-SP1 at a rate that has a kcat / KM value from 1×10 to 1×106M-1s-1, e.g., from 1×10 to 5×10, from 5×10 to 1×102, from 1×102to 5×102, from 5×102to 1×103, from 1×103to 5×103, from 5×103to 1×104, from 1×104to 5×104, from 5×104to 1×105, from 1×105to 5×105, or from 5×105to 1×106M-1s-1. In someCYTX-108-WO :: 4862-156.WO1 aspects, the CM is cleaved by MT-SP1 at a rate that has a kcat / KMvalue of at least 1×10, at least 5×10, at least 1×102, at least 5×102, at least 1×103, 5×103, at least 1×104, at least 5×104, at least 1×105, at least 5×105, or at least 1×106. In some aspects, the CM has greater catalytic efficiency (i.e., a higher kcat / KM value) for an MT-SP1 compared to a cleavable polypeptide comprising a CM comprising a sequence of QNQALRMA (SEQ ID NO: 3).

[0097] In some aspects, the CM has greater catalytic efficiency for MT-SP1 and MMP2 compared to a cleavable polypeptide comprising a CM comprising a sequence of QNQALRMA (SEQ ID NO: 3). In some aspects, the CM has greater catalytic efficiency for MMP2, MMP9, and MMP14 compared to a cleavable polypeptide comprising a CM comprising a sequence of QNQALRMA (SEQ ID NO: 3).

[0098] According to some aspects of the present disclosure, the cleavable polypeptide is a molecule that has 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 following 7 days of administration in vivo. In some aspects, the CM has an increased in vivo stability of 20%, 30%, 40%, 50% 60%, 70%, 80%, 90%, 100%, 150% or more compared to a cleavable polypeptide comprising a CM comprising a sequence of QNQALRMA (SEQ ID NO: 3), as measured in plasma using a capillary electrophoresis immunoassay. In some aspects, the CM has an increased in vivo stability of 20% to 500%, 30% to 400%, 40% to 300%, or 50% to 200%, including any other range within the range of 20% to 500%, compared to a cleavable polypeptide comprising a CM comprising a sequence of QNQALRMA (SEQ ID NO: 3), as measured in plasma using a capillary electrophoresis immunoassay. ACTIVATABLE MOLECULES

[0099] In some aspects, the cleavable polypeptide comprising a CM further comprises at least one additional moiety (M) selected from a moiety that is located amino (N) terminally to the CM (Mn), a moiety that is located carboxyl (C) terminally to the CM (Mc), or a combination thereof. In some aspects, the Mn and the Mc are each independently selected from a masking moiety (MM), a target binding moiety (TBM), a therapeutic agent, a detectable moiety, a diagnostic agent, or an affinity tag. In some aspects, the cleavable polypeptide comprises the MM coupled directly or indirectly to the N-terminus of the CM and the TBM coupled directly or indirectly to the C-terminus of the CM, or wherein cleavable polypeptide comprises the TBM coupled directly or indirectly to the N-terminus of the CM and the MM coupled directly or indirectly to the C-terminus of the CM.CYTX-108-WO :: 4862-156.WO1

[0100] In some aspects, the MM inhibits binding of the TBM to its target when the cleavable polypeptide is in an uncleaved state. In some aspects, the MM does not interfere or compete with the TBM for binding to a target in a cleaved state. In some aspects, the amino acid sequence of the MM is different from that of the target. In some aspects, the amino acid sequence of the MM is different from that of the target and is no more than 50% identical to the amino acid sequence of a natural binding partner of the TBM. In some aspects, the MM has a dissociation constant for binding to the TBM that is greater than the dissociation constant of the TBM for binding to the target. In some aspects, the MM has a masking efficiency of 175% or more compared to a cleavable polypeptide comprising a CM comprising a sequence of QNQALRMA (SEQ ID NO: 3). In some aspects, the MM has a masking efficiency of 330% or more compared to a cleavable polypeptide comprising a CM comprising a sequence of QNQALRMA (SEQ ID NO: 3).

[0101] In some aspects, the cleavable polypeptide comprises the structural arrangement from N-terminus to C-terminus as follows: MM-CM-TBM or TBM-CM-MM, wherein “-” is a covalent bond. In some aspects, the CM directly couples the MM to the TBM. In some aspects, the CM is coupled to the MM via a linker peptide (LP). In some aspects, the CM is coupled to the TBM via an LP. In some aspects, the cleavable polypeptide comprises a first linking peptide (LP1) and a second linking peptide (LP2), and wherein the cleavable polypeptide has a structural arrangement from N-terminus to C-terminus as follows: MM- LP1-CM-LP2-TBM.

[0102] As used herein, unless otherwise stated, each “-” between elements is a direct or indirect linkage (e.g., via a linker peptide). Thus, inaspects, the elements directly abut each other in amino acid sequence. In alternative aspects, the elements are connected by a linker amino acid sequence.

[0103] In some aspects, the TBM is a biologically active protein. In some aspects, the biologically active protein is a cytokine or a functional fragment thereof. In some aspects, the TBM is a chimeric antigen receptor. In some aspects, the TBM is a therapeutic macromolecule. In some aspects, the TBM is a drug or agent, e.g., a therapeutic, imaging, or diagnostic agent. In some aspects, the TBM is an antibody or antigen binding fragment thereof. In some aspects, the antigen binding fragment thereof is selected from the group consisting of a Fab fragment, a F(ab')2 fragment, a scFv, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody. In some aspects, the antibody is bivalent or multivalent antibody.CYTX-108-WO :: 4862-156.WO1

[0104] In some aspects, the cleavable polypeptide, the activatable molecule, or the polypeptide complex is an activatable molecule comprising a MM coupled directly or indirectly to the TBM via the CM. aspect

[0105] The coupling of any of the components in a cleavable polypeptide, the activatable molecule, or the polypeptide or polypeptide complex (e.g., an activatable molecule) may be direct or indirect. When the two components are coupled directly, the amino acid residue at the C-terminus of a component forms a peptide bond with the amino acid residue at the N- terminus of the other component. When the two components are coupled indirectly, there is a stretch of amino acids between the two components. In some examples, the two components of a polypeptide may be indirectly coupled via one or more other components in the polypeptide, i.e., the one or more other components are between the two coupled components. For indirectly coupling or linking via another component, the one or more other components may be a linker, TBM(s), CM(s), MM(s), or any combination thereof.

[0106] In some aspects, the cleavable polypeptide is an activatable molecule and further comprises a TBM that specifically binds a target. In some aspects, the TBM is a therapeutic macromolecule. In some aspects, the TBM is an antibody or antigen binding fragment thereof. In some aspects, the antibody is a full- length antibody. single-chain variable fragment (scFv), diabody (a noncovalent dimer of scFv), single chain antibody (scAb), a VHH, a domain antibody (dAb) or single domain antibody (nanobody, e.g., single domain heavy chain antibody, single domain light chain antibody). In some aspects, the antibody is a monoclonal antibody, single chain antibody, Fab fragment, F(ab')2fragment, single-chain variable fragment (scFv), diabody (a noncovalent dimer of scFv), single chain antibody (scAb), a VHH, a domain antibody (dAb) or single domain antibody (nanobody, e.g., single domain heavy chain antibody, single domain light chain antibody).

[0107] According to some aspects of the present disclosures, the cleavable polypeptide is an activatable molecule that has 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 following 7 days of administration in vivo (e.g., as exemplified in Example 3). According to some aspects of the present disclosures, the cleavable polypeptide is an activatable molecule that has 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 following 7 days of administration in vivo. In some aspects, the CM has an increased in vivo stability of 20%, 30%, 40%, 50% 60%, 70%, 80%, 90%, 100%, 150% or more compared to a cleavable polypeptide comprisingCYTX-108-WO :: 4862-156.WO1 a CM comprising a sequence of QNQALRMA (SEQ ID NO: 3), as measured in plasma using a capillary electrophoresis immunoassay. In some aspects, the CM has an increased in vivo stability of 20% to 500%, 30% to 400%, 40% to 300%, or 50% to 200% compared to a cleavable polypeptide comprising a CM comprising a sequence of QNQALRMA (SEQ ID NO: 3), as measured in plasma using a capillary electrophoresis immunoassay.

[0108] In some aspects, the cleavable polypeptide is an activatable molecule that has masking efficiency of 20x, 24x, 30x, 50x, 75x, 100x, 150x, 200x, 240x, 246x, 300x, 340x, 346x, or higher. In some aspects, the activatable molecule is activated by one, two, or all of MMP2, MMP9, MMP14 and MT-SP1. In some aspects, the activatable molecule is activated to an extent of having a cleavability percentage 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%, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, or 100% cleavable by any one of MMP2, MMP9, MMP14 and MT-SP1 or any two of MMP2, MMP9, MMP14 and MT- SP1or each of MMP2, MMP9, MMP14 and MT-SP1.

[0109] In some aspects, the cleavable polypeptide is an activatable molecule that has increased cleavage of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 300%, 400%, or 500% or more by any one of MMP2, MMP9, MMP14, MT-SP1 or any two of MMP2, MMP9, MMP14, MT-SP1 or each of MMP2, MMP9, MMP14, MT-SP1 compared to a cleavable polypeptide comprising a CM comprising a sequence of QNQALRMA (SEQ ID NO: 3). In some aspects, the increased cleavage is an increase of 20% to 500%, 30% to 400%, 40% to 300%, or 50% to 200% by any one of MMP2, MMP9, MMP14, MT-SP1 or any two of MMP2, MMP9, MMP14, MT-SP1 or each of MMP2, MMP9, MMP14, MT-SP1 compared to a cleavable polypeptide comprising a CM comprising a sequence of QNQALRMA (SEQ ID NO: 3).

[0110] In some aspects, the cleavable polypeptide is an activatable molecule that has greater catalytic efficiency (i.e., a higher kcat / KMvalue) for an MMP, e.g., MMP2, MMP9, MMP14, or a combination thereof compared to a cleavable polypeptide comprising a CM comprising a sequence of QNQALRMA (SEQ ID NO: 3). In some aspects, the CM has greater catalytic efficiency (i.e., a higher kcat / KM value) for an MT-SP1 compared to a cleavable polypeptide comprising a CM comprising a sequence of QNQALRMA (SEQ ID NO: 3).CYTX-108-WO :: 4862-156.WO1

[0111] In some aspects, the TBM is coupled to the CM. In some aspects, the TBM is coupled directly to the CM. In some aspects, the TBM is coupled to the CM via a linking peptide. In some aspects, the TBM is indirectly coupled to the CM via one or more components of the activatable molecule.

[0112] In some aspects, the MM does not bind the TBM, but still interferes with TBM’s binding to its binding partner through non-specific interactions. In some aspects, the MM is a steric mask. In some aspects, the MM is a protein. In some aspects, the MM is coupled to the CM such that the cleavable polypeptide comprises the structural arrangement from N- terminus to C-terminus as follows: MM-CM-TBM or TBM-CM-MM. In some aspects, the MM is coupled directly to the CM.

[0113] In some aspects, the MM is coupled to the CM via a linking peptide. In some aspects, the cleavable polypeptide comprises a linking peptide (LP) and wherein the cleavable polypeptide has a structural arrangement from N-terminus to C-terminus as follows: MM-LP-CM-TBM or MM-CM-LP-TBM. In some aspects, the cleavable polypeptide comprises a first linking peptide (LP1) and a second linking peptide (LP2), and wherein the cleavable polypeptide has the structural arrangement from N-terminus to C- terminus as follows: MM-LP1-CM-LP2-TBM or TBM-LP2-CM-LP1-MM. In some aspects, the LP1 and LP2 are not identical to each other. In some aspects, the LP1 and LP2 are identical to each other. In some aspects, each of LP1 and LP2 is a peptide of 1 to 20 amino acids in length.

[0114] In general, in each aspect herein, unless otherwise stated, a polypeptide may comprise one or more optional linkers between each of the elements listed, 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.

[0115] A TBM may be any polypeptide that specifically binds a target. In some examples, the TBM may be a therapeutic macromolecule. In some examples, the TBM is an antibody or an antigen-binding fragment. In some examples, the TBM is an antineoplastic macromolecule. In some examples, the TBM is a cytokine. In some examples, the TBM is a chemokine. In some examples, the TBM is a chimeric antigen receptor. In some aspects, the TBM binds to a tumor-associated moiety. In some aspects, the TBM binds epidermal growth factor receptor (EGFR).

[0116] In some aspects, the cleavable polypeptide comprising a TBM, according to one or more aspects described above, has an in vivo antitumor activity equal to or higher than aCYTX-108-WO :: 4862-156.WO1 cleavable polypeptide comprising a corresponding TBM and comprising a CM comprising a sequence of QNQALRMA (SEQ ID NO: 3). In some aspects, the antitumor activity is a reduction in tumor volume of 25% or more, 30% or more, 40% or more, 50% or more 60% or more, 70% or more, 75% or more, 80% or more, or 90% or more. In some aspects, the cleavable polypeptide has a higher in vivo antitumor activity after at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, or at least 10 days post-administration.

[0117] In some examples, the TBM is a diagnostic macromolecule. For example, the diagnostic macromolecule is a diagnostic polypeptide having 3 to 30, 5 to 25, 7 to 20, or 9 to 15 amino acids in length. Such diagnostic polypeptide may be used, in non-limiting aspects, e.g., for testing cleavage in tissues, and / or assessment of the tissue microenvironment.

[0118] In general, an activatable molecule may be designed by selecting a TBM of interest and constructing the remainder of the activatable molecule so that, when conformationally constrained, the MM provides for masking of the TBM or reduction of binding of the TBM to its target. Structural design criteria can be to be taken into account to provide for this functional feature.

[0119] Activatable molecules may be provided in a variety of structural configurations. Exemplary formulas for activatable molecules are provided below. It is contemplated that the N- to C-terminal order of the TBM, MM and CM may be reversed within an activatable molecule. For example, activatable molecules can be represented by the following formulas (in order from an amino (N) terminal region to carboxyl (C) terminal region): MM-CM-TBM TBM-CM-MM It should be noted that although MM and CM are indicated as distinct components in the formulas above, in all exemplary aspects (including formulae) disclosed herein it is contemplated that the amino acid sequences of the MM and the CM may overlap, e.g., such that the CM is partially contained within the MM. In addition, the formulas above provide for additional amino acid sequences that may be positioned N-terminal or C-terminal to the activatable molecules components. Examples include targeting moieties (e.g., a ligand for a receptor of a cell present in a target tissue) and half-life extending moieties.

[0120] In some aspects, MM, CM, and / or TBM are coupled indirectly via one or more linkers (e.g., a linking peptide (LP)). For example, an activatable molecule may comprise one of the following formulae (in order from an amino (N) terminal region to carboxyl (C) terminal region):CYTX-108-WO :: 4862-156.WO1 MM-LP-CM-TBM MM-CM-LP-TBM MM-LP1-CM-LP2-TBM TBM-LP-CM-MM TBM-CM-LP-MM TBM-LP2-CM-LP1-MM wherein LP1 and LP2 are two linking peptides. In some examples, the LP1 and LP2 are identical to each other. In some examples, the LP1 and LP2 are different from each other.

[0121] In some aspects, the activatable molecule comprise a plurality of CMs, at least one of which comprises the sequence of any of SEQ ID NOs: 4-38 and 74. For example, the CM comprising the sequence of any of SEQ ID NOs: 4-38 and 74 is engineered into a longer cleavage substrate that has a plurality of CMs. Examples of the additional CM(s) in the activatable molecule that are not the CM comprising the sequence of any of SEQ ID NOs: 4- 38 and 74 include those described in WO 2010 / 081173, WO2015 / 066279, WO2015 / 116933, WO2016 / 118629, WO2021207669, WO2021207657, WO2021142029, WO2021061867, WO2020252349, WO2020252358, WO2020236679, WO2020176672, WO2020118109, WO2020092881, WO2020086665, WO2019213444, WO2019183218, WO2019173771, WO2019165143, WO2019075405, WO2019046652, WO2019018828, WO2019014586, WO2018222949, WO2018165619, WO2018085555, WO2017011580, WO2016179335, WO2016179285, WO2016179257, WO2016149201, WO2016014974, WO 2024 / 030843, WO 2024 / 030845, WO 2024 / 030847, WO 2024 / 030858, and WO 2024 / 030850 which are incorporated herein by reference in their entireties for all purposes. In some examples, one or more of the additional CMs are cleavable by legumain.

[0122] In some aspects, the cleavable polypeptide comprises a first CM (CM1) cleavable by a first protease, and a second CM (CM2) cleavable by a second protease. In some aspects, the cleavable polypeptide comprises CM1 cleavable by a first protease, CM2 cleavable by a second protease, and a third CM (CM3) cleavable by a third protease. In some aspects, the cleavable polypeptide comprises CM1 cleavable by a first protease, CM2 cleavable by a second protease, CM3 cleavable by a third protease, and a fourth CM (CM4) cleavable by a fourth protease.

[0123] In some aspects, the activatable molecule comprises a structural arrangement from N-terminus to C-terminus as follows: MM-CM1-CM2-TBM, MM-CM2-CM1-TBM, TBM- CM1-CM2-MM, or TBM-CM2-CM1-MM, MM-CM2-CM1-CM3-TBM, MM-CM1-CM2-CYTX-108-WO :: 4862-156.WO1 CM3-TBM, MM-CM1-CM3-CM2-TBM, MM-CM3-CM1-CM2-TBM, or MM-CM3-CM2- CM1-TBM. Likewise, a CM4 may be inserted any position between the MM and TBM.

[0124] In some aspects, the activatable molecule comprises a linking peptide (LP) and wherein the activatable molecule has a structural arrangement from N-terminus to C-terminus as follows: MM-LP-CM1-CM2-TBM, MM-CM1-CM2-LP-TBM, MM-LP-CM2-CM1-TBM, MM-CM2-CM1-LP-TBM, MM-LP-CM2-CM1-CM3-TBM, MM-LP-CM1-CM2-CM3- TBM, MM-LP-CM1-CM3-CM2-TBM, MM-LP-CM3-CM1-CM2-TBM, MM-LP-CM3- CM2-CM1-TBM, MM-CM2-CM1-CM3-LP-TBM, MM-CM1-CM2-CM3-LP-TBM, MM- CM1-CM3-CM2-LP-TBM, MM-CM3-CM1-CM2-LP-TBM, or MM-CM3-CM2-CM1-LP- TBM. Likewise, a CM4 may be inserted any position between the MM and TBM.

[0125] In some aspects, the activatable molecule comprises a first linking peptide (LP1) and a second linking peptide (LP2), and wherein the activatable molecule has a structural arrangement from N-terminus to C-terminus as follows: MM-LP1-CM1-CM2-LP2-TBM, MM-LP1-CM2-CM1-LP2-TBM, TBM-LP2-CM1-CM2-LP1-MM, or TBM-LP2-CM2-CM1- LP1-MM, MM-LP1-CM2-CM1-CM3-LP2-TBM, MM-LP1-CM1-CM2-CM3-LP2-TBM, MM-LP1-CM1-CM3-CM2-LP2-TBM, MM-LP1-CM3-CM1-CM2-LP2-TBM, MM-LP1- CM3-CM2-CM1-LP2-TBM, MM-LP2-CM2-CM1-CM3-LP1-TBM, MM-LP2-CM1-CM2- CM3-LP1-TBM, MM-LP2-CM1-CM3-CM2-LP1-TBM, MM-LP2-CM3-CM1-CM2-LP1- TBM, or MM-LP2-CM3-CM2-CM1-LP1- TBM. Likewise, a CM4 may be inserted any position between the MM and TBM.

[0126] In some aspects, the activatable molecule comprises an additional linking peptide (LP3) and wherein the activatable molecule has a structural arrangement from N-terminus to C-terminus as follows: MM-LP-CM1-LP3-CM2-TBM, MM-CM1-LP3-CM2-LP-TBM, MM- LP-CM2-LP3-CM1-TBM, MM-CM2-LP3-CM1-LP-TBM, MM-LP-CM2-LP3-CM1-CM3- TBM, MM-LP-CM1-LP3-CM2-CM3-TBM, MM-LP-CM1-LP3-CM3-CM2-TBM, MM-LP- CM3-LP3-CM1-CM2-TBM, MM-LP-CM3-LP3-CM2-CM1-TBM, MM-CM2-LP3-CM1- CM3-LP-TBM, MM-CM1-LP3-CM2-CM3-LP-TBM, MM-CM1-LP3-CM3-CM2-LP-TBM, MM-CM3-LP3-CM1-CM2-LP-TBM, MM-CM3-LP3-CM2-CM1-LP-TBM, MM-LP-CM2- CM1-LP3-CM3-TBM, MM-LP-CM1-CM2-LP3-CM3-TBM, MM-LP-CM1-CM3-LP3- CM2-TBM, MM-LP-CM3-CM1-LP3-CM2-TBM, MM-LP-CM3-CM2-LP3-CM1-TBM, MM-CM2-CM1-LP3-CM3-LP-TBM, MM-CM1-CM2-LP3-CM3-LP-TBM, MM-CM1- CM3-LP3-CM2-LP-TBM, MM-CM3-CM1-LP3-CM2-LP-TBM, MM-CM3-CM2-LP3- CM1-LP-TBM, MM-LP1-CM1-LP3-CM2-LP2-TBM, MM-LP1-CM2-LP3-CM1-LP2-TBM,CYTX-108-WO :: 4862-156.WO1 TBM-LP1-CM1-LP3-CM2-LP2-MM, or TBM-LP1-CM2-LP3-CM1-LP2-MM, MM-LP1- CM2-LP3-CM1-CM3-LP2-TBM, MM-LP1-CM1-LP3-CM2-CM3-LP2-TBM, MM-LP1- CM1-LP3-CM3-CM2-LP2-TBM, MM-LP1-CM3-LP3-CM1-CM2-LP2-TBM, MM-LP1- CM3-LP3-CM2-CM1-LP2-TBM, MM-LP2-CM2-LP3-CM1-CM3-LP1-TBM, MM-LP2- CM1-LP3-CM2-CM3-LP1-TBM, MM-LP2-CM1-LP3-CM3-CM2-LP1-TBM, MM-LP2- CM3-LP3-CM1-CM2-LP1-TBM, or MM-LP2-CM3-LP3-CM2-CM1-LP1- TBM. Likewise, a CM4 may be inserted any position between the MM and TBM.

[0127] In some aspects, the activatable molecule has a structural arrangement from N- terminus to C-terminus as follows: MM-LP-CM2-LP3-CM1-LP4-CM3-TBM, MM-LP-CM1- LP3-CM2-LP4-CM3-TBM, MM-LP-CM1-LP3-CM3-LP4-CM2-TBM, MM-LP-CM3-LP3- CM1-LP4-CM2-TBM, MM-LP-CM3-LP3-CM2-LP4-CM1-TBM, MM-CM2-LP3-CM1- LP4-CM3-LP-TBM, MM-CM1-LP3-CM2-LP4-CM3-LP-TBM, MM-CM1-LP3-CM3-LP4- CM2-LP-TBM, MM-CM3-LP3-CM1-LP4-CM2-LP-TBM, MM-CM3-LP3-CM2-LP4-CM1- LP-TBM, MM-LP-CM2-LP4-CM1-LP3-CM3-TBM, MM-LP-CM1-LP4-CM2-LP3-CM3- TBM, MM-LP-CM1-LP4-CM3-LP3-CM2-TBM, MM-LP-CM3-LP4-CM1-LP3-CM2-TBM, MM-LP-CM3-LP4-CM2-LP3-CM1-TBM, MM-CM2-LP4-CM1-LP3-CM3-LP-TBM, MM- CM1-LP4-CM2-LP3-CM3-LP-TBM, MM-CM1-LP4-CM3-LP3-CM2-LP-TBM, MM-CM3- LP4-CM1-LP3-CM2-LP-TBM, MM-CM3-LP4-CM2-LP3-CM1-LP-TBM, MM-LP1-CM2- LP3-CM1-LP4-CM3-LP2-TBM, MM-LP1-CM1-LP3-CM2-LP4-CM3-LP2-TBM, MM-LP1- CM1-LP3-CM3-LP4-CM2-LP2-TBM, MM-LP1-CM3-LP3-CM1-LP4-CM2-LP2-TBM, MM-LP1-CM3-LP3-CM2-LP4-CM1-LP2-TBM, MM-LP2-CM2-LP3-CM1-LP4-CM3-LP1- TBM, MM-LP2-CM1-LP3-CM2-LP4-CM3-LP1-TBM, MM-LP2-CM1-LP3-CM3-LP4- CM2-LP1-TBM, MM-LP2-CM3-LP3-CM1-LP4-CM2-LP1-TBM, or MM-LP2-CM3-LP3- CM2-LP4-CM1-LP1- TBM. Likewise, a CM4 may be inserted any position between the MM and TBM.

[0128] In some aspects, in the above structural arrangements, the CM1 comprises a sequence of any one of SEQ ID NOs: 4-38 and 74. Alternatively or additionally, in some aspects, in the above structural arrangements, the CM2 comprises a sequence of any one of SEQ ID NOs: 4-38 and 74. Alternatively or additionally, in some aspects, in the above structural arrangements, the CM3 comprises a sequence of any one of SEQ ID NOs: 4-38 and 74. Alternatively or additionally, in some aspects, in the above structural arrangements, the CM4 comprises a sequence of any one of SEQ ID NOs: 4-38 and 74.CYTX-108-WO :: 4862-156.WO1

[0129] In some aspects, where the activatable molecule comprises a plurality of CMs, at least a portion of a first CM overlaps with at least a portion of a second CM in the substrate, such that one or more amino acids in the substrate belongs to both CMs. For example, a substrate with the sequence X1X2X3X4X5X6 (each X is an amino acid), may comprise overlapping CM1 and CM2, in which CM1 is X1X2X3X4and CM2 is X3X4X5X6.

[0130] In some aspects where the activatable molecule comprises a plurality of CMs, two CMs do not overlap in amino acid sequences such that no amino acid in the substrate belongs to both CMs. For example, a substrate with the sequence X1X2X3X4X5X6X7X8 (each X is an amino acid) may comprise non-overlapping CM1 and CM2, in which CM1 is X1X2X3X4and CM2 is X5X6X7X8. In some aspects, the non-overlapping CM1 and CM2 are coupled directly. In some aspects, the non-overlapping CM1 and CM2 are coupled indirectly (e.g., via a linking peptide).

[0131] In some aspects, two CMs, e.g., CM1 and CM2, in a substrate have a structural arrangement from N-terminus to C-terminus as CM1-CM2. In some aspects, two CMs, e.g., CM1 and the CM2 in a substrate have a structural arrangement from N-terminus to C- terminus as CM2-CM1. As used herein, the CM1 and CM2 in the formula CM1-CM2 or CM2-CM1 may be overlapping CM1 and CM2, non-overlapping CM1 and CM2 coupled directly, or non-overlapping CM1 and CM2 coupled indirectly (e.g., via a linking peptide).

[0132] In some aspects, two CMs, e.g., CM2 or CM4 and CM3 or CM4, in a substrate have a structural arrangement from N-terminus to C-terminus as CM2-CM3, CM2-CM4 or CM3-CM4. In some aspects, two CMs, e.g., CM2 and the CM3 in a substrate have a structural arrangement from N-terminus to C-terminus as CM3-CM2 or CM4-CM2 or CM4- CM3. As used herein, the CM2 and CM3 in the formula CM2-CM3 or CM3-CM2 may be overlapping CM2 and CM3, non-overlapping CM2 and CM3 coupled directly, or non- overlapping CM2 and CM3 coupled indirectly (e.g., via a linking peptide). As used herein, the CM2 and CM4 in the formula CM2-CM4 or CM4-CM2 may be overlapping CM2 and CM4, non-overlapping CM2 and CM4 coupled directly, or non-overlapping CM2 and CM4 coupled indirectly (e.g., via a linking peptide). As used herein, the CM4 and CM3 in the formula CM4-CM3 or CM3-CM4 may be overlapping CM4 and CM3, non-overlapping CM4 and CM3 coupled directly, or non-overlapping CM4 and CM3 coupled indirectly (e.g., via a linking peptide).

[0133] As discussed herein, minor variations in the amino acid sequences of polypeptides are contemplated as being encompassed by the present disclosure, providing that theCYTX-108-WO :: 4862-156.WO1 variations in the amino acid sequence maintain at least 75%, in some aspects, at least 80%, at least 90%, at least 95%, and in some aspects, at least 99% identity to the amino acid sequence that is not varied. In particular, conservative amino acid substitutions are contemplated. Conservative substitutions include those that take place within a family of amino acids that are related in their side chains. Genetically encoded amino acids are generally divided into families: (1) acidic amino acids are aspartate, glutamate; (2) basic amino acids are lysine, arginine, histidine; (3) non-polar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. The hydrophilic amino acids include arginine, asparagine, aspartate, glutamine, glutamate, histidine, lysine, serine, and threonine. The hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine and valine. Other families of amino acids include (i) serine and threonine, which are the aliphatic-hydroxy family; (ii) asparagine and glutamine, which are the amide containing family; (iii) alanine, valine, leucine and isoleucine, which are the aliphatic family; and (iv) phenylalanine, tryptophan, and tyrosine, which are the aromatic family. For example, it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the binding or properties of the resulting molecule, especially if the replacement does not involve an amino acid within a framework site. Whether an amino acid change results in a functional peptide can readily be determined by assaying the specific activity of the polypeptide derivative. Assays are described in detail herein. Fragments or analogs of antibodies or immunoglobulin molecules can be readily prepared by those of ordinary skill in the art. Suitable amino- and carboxyl-termini of fragments or analogs occur near boundaries of functional domains. Structural and functional domains can be identified by comparison of the nucleotide and / or amino acid sequence data to public or proprietary sequence databases. In some aspects, computerized comparison methods are used to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and / or function. Methods to identify protein sequences that fold into a known three-dimensional structure are known, e.g., as described in Bowie et al. Science 253:164 (1991). Thus, the foregoing examples demonstrate that those of skill in the art can recognize sequence motifs and structural conformations that may be used to define structural and functional domains in accordance with the disclosure.CYTX-108-WO :: 4862-156.WO1

[0134] Suitable amino acid substitutions include those that: (1) alter susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinities, and (5) confer or modify other physicochemical or functional properties of such analogs. Analogs can include various muteins of a sequence other than the naturally-occurring peptide sequence. For example, single or multiple amino acid substitutions (for example, conservative amino acid substitutions) may be made in the naturally- occurring sequence (for example, in the portion of the polypeptide outside the domain(s) forming intermolecular contacts. A conservative amino acid substitution should not substantially change the structural characteristics of the parent sequence (e.g., a replacement amino acid should not tend to break a helix that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes 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, N.Y. (1991)); and Thornton et at. Nature 354:105 (1991). Antibodies and Antigen-Binding Fragments

[0135] In some aspects, the TBM is an antibody or antigen-binding fragment thereof. The term “antibody” is used herein in its broadest sense and includes certain types of immunoglobulin molecules that include one or more target-binding domains that specifically bind an antigen or epitope. Examples of antibodies include intact antibodies (e.g., intact immunoglobulins), antibody fragments, bispecific, and multi-specific antibodies. One example of a target-binding domain is formed by a VH-VLdimer. Additional examples of an antibody are described herein. Additional examples of an antibody are known in the art.

[0136] A “light chain” includes one variable domain (VL) and one constant domain (CL). There are two different light chains termed kappa or lambda. A “heavy chain” consists of one variable domain (VH) and three constant region domains (CH1, CH2, CH3). There are five main heavy-chain classes or isotypes, some of which have several subtypes, and these determine the functional activity of an antibody molecule. The five major classes of immunoglobulin 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).CYTX-108-WO :: 4862-156.WO1

[0137] In some aspects, the antigen-binding fragment is a Fab fragment, a F(ab')2fragment, a scFv, a scAb, a dAb, a single domain heavy chain antibody, or a single domain light chain antibody. Additional examples of the antigen-binding fragments include a VH domain, a VHH domain, a VNAR domain, and a single chain fragment variable (scFv), BiTE or a component thereof, a (scFv)2, a NANOBODY®, a nanobody-HSA, VHH-scAb, a VHH- Fab, a Dual scFab, a F(ab’)2, a diabody, a CROSSMAB®, a DAF (two-in-one), a DAE (four- in-one), a DUTAMAB®, a DT- IgG, a knobs-in-holes common light chain, a knobs-in-holes assembly, a charge pair, a Fab-arm exchange, a SEEDbody, a LUZ-Y, a FcAb, a kl-body, an orthogonal Fab, a DVD-IgG, a IgG(H)-scFv, a 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, a triple body, a miniantibody, a minibody, a TriBi minibody, scFv-CH3 KIH , Fab-scFv, a F(ab’)2-scFv2, a scFv-KIΉ, a Fab- scFv-Fc, a tetravaient HCAb, a scDiabody-Fc, a Diabody-Fc, a tandem scFv-Fc, a VHH-Fc, a tandem VHH-Fc, a L'ΉH-Fc KiH, a Fab- VHH-Fc, an Intrabody, a dock and lock, an ImmTAC®(immune-mobilizing monoclonal TCRs (T cell receptors) against cancer), an IgG- IgG conjugate, a Cov-X-Body, a scFvl- PEG-scFv2, an Adnectin, a DARPin®, a fibronectin, an IgG, an IgM, an IgA, an IgE, an IgD, a DEP conjugate, TMEAbodyTM, SAFEbody®, TRITAC®, or SHIELD antibody.

[0138] A “fragment antigen binding” (Fab) includes a complete light chain paired with the VH domain and the CH1 domain of a heavy chain. A F(ab′)2 fragment is formed when an antibody is cleaved by pepsin (or otherwise truncated) below the hinge region, in which case the two fragment target-binding domains (Fabs) of the antibody molecule remain linked. A F(ab′)2fragment contains two complete light chains paired with the two VH and CH1 domains of the heavy chains joined together by the hinge region. A “fragment crystallizable” (Fc) fragment (also referred to herein as FCdomain) 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 mainly in the Fc fragment. A “single chain fragment variable” (scFv) contains only the variable domain of a light chain (VL) linked by a stretch of peptide to a variable domain of a heavy chain (VH). The name single-chain Fv is derived from Fragment variable. A “hinge region” or “interdomain” is flexible amino acid stretch that joins or links the Fab fragment to the Fc domain. A “synthetic hinge region” is an amino acid sequence that joins or links a Fab fragment to an Fc domain.CYTX-108-WO :: 4862-156.WO1

[0139] An “Fv” fragment includes a non-covalently-linked dimer of one heavy chain variable domain and one light chain variable domain. A “dual variable domain immunoglobulin G” or “DVD-IgG” refers to multivalent and multispecific target-binding proteins as described, e.g., 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 in, e.g., Garber, Nature Reviews Drug Discovery 13:799-801, 2014.

[0140] A VHH domain is a single monomeric variable antibody domain that can be found in camelids. A VNAR domain is a single monomeric variable antibody domain that can be found in cartilaginous fish. Non-limiting aspects of VHH domains and VNAR domains are described in, e.g., 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 by reference herein in its entirety.

[0141] In some aspects, the TBM may be a mouse, rat, rabbit, goat, camel, donkey, primate, human, or humanized or chimeric polypeptide. In one example, the TBM may be a human polypeptide. In one example, the TBM may be a humanized (e.g., fully humanized) polypeptide.

[0142] The term “humanized” refer to a TBM having an amino acid sequence that includes VH and VL region sequences from a reference protein raised in a non-human species (e.g., a mouse), but also includes modifications in those sequences relative to the reference protein intended to render them more “human-like,” i.e., more similar to human germline variable sequences. In some aspects, a “humanized” TBM is one that immunospecifically binds an antigen of interest and that has a framework (FR) region having substantially the amino acid sequence as that of a human protein, and a complementaryCYTX-108-WO :: 4862-156.WO1 determining region (CDR) having substantially the amino acid sequence as that of a non- human protein contains humanized VH and VL regions.

[0143] The term “human polypeptide” is intended to include TBM s having variable and constant regions generated, assembled, or derived from human immunoglobulin sequences. In some aspects, an TBM may be considered to be “human” even though its amino acid sequence include residues or elements not encoded by human germline immunoglobulin sequences (e.g., include sequence variations, for example that may (originally) have been introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), e.g., in one or more CDRs.

[0144] Examples of antibodies and antigen-binding fragments include those binding to cell surface receptors and secreted binding proteins (e.g., growth factors), soluble enzymes, structural proteins (e.g. collagen, fibronectin) and the like, or an extracellular target (e.g., an extracellular protein target). In some aspects, antibodies and antigen-binding fragments are designed for cellular uptake and are activatable inside a cell.

[0145] Non-limiting examples of antibodies and antigen-binding fragments include those in the Examples, e.g., those comprising a light chain comprising any of the sequences in Table 13, and a heavy chain comprising any of the sequences in Table 13. Multispecific Activatable Antibodies

[0146] In some aspects, the activatable antibodies are multispecific activatable antibodies. In some examples, the multispecific activatable antibodies herein recognize two or more different antigens or epitopes and that include at least one masking moiety (MM) linked to at least one antigen- or epitope-binding domain of the multispecific antibody such that coupling of the MM reduces the ability of the antigen- or epitope-binding domain to bind its target. In some aspects, the MM is coupled to the antigen- or epitope-binding domain of the multispecific antibody via a cleavable moiety (CM) that functions as a substrate for at least one protease, e.g., an MMP. The activatable multispecific antibodies provided herein are stable in circulation, activated at intended sites of therapy and / or diagnosis but not in normal, i.e., healthy tissue, and, when activated, exhibit binding to a target that is at least comparable to the corresponding, unmodified multispecific antibody.

[0147] The multispecific activatable molecules may be used to target a first and a second target tissues. In one aspect, the first and second target tissues are spatially separated, for example, at different sites in the organism. In one aspect, the first and second target tissues are the same tissue temporally separated, for example the same tissue at two different pointsCYTX-108-WO :: 4862-156.WO1 in time, for example the first time point is when the tissue is an early stage tumor, and the second time point is when the tissue is a late stage tumor.

[0148] In some aspects, the multispecific activatable antibody includes a first antibody or antigen-binding fragment thereof (AB1) that binds a first target, where the AB1 is coupled to a masking moiety (MM1) such that coupling of the MM1 reduces the ability of the AB1 to bind the first target, and the multispecific activatable antibody includes a second antibody or antigen-binding fragment thereof (AB2) that binds a second target, where the AB2 is coupled to a masking moiety (MM2) such that coupling of the MM2 reduces the ability of the AB2 to bind the second target. In some aspects, AB1 is coupled to MM1 via CM1, and AB2 is coupled to MM2 via CM2. In some aspects, there are linking peptides between AB1 and CM1, between CM1 and MM1, between AB2 and CM2, and / or between CM2 and MM2. In some aspects, AB1 is directly coupled to CM1, CM1 is directly coupled to MM1, AB2 is directly coupled to CM2, and / or CM2 is directly coupled to MM2.

[0149] For example, the multispecific activatable antibodies can be represented by the following formulas (in order from an amino (N) terminal region to carboxyl (C) terminal region): MM1-CM1-AB1 : MM2-CM2-AB2 AB1-CM1-MM1 : MM2-CM2-AB2 AB1-CM1-MM1 : AB2-CM2-MM2 wherein “:” 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) and each dash (-) between the components of the activatable molecule represents either a direct linkage or indirect linkage via one or more linking peptides.

[0150] In some aspects, the multispecific activatable antibodies are designed to engage immune effector cells, also referred to herein as immune-effector cell engaging multispecific activatable antibodies. In some aspects, the multispecific activatable antibodies are designed to engage leukocytes, also referred to herein as leukocyte engaging multispecific activatable antibodies. In some aspects, the multispecific activatable antibodies are designed to engage T cells, also referred to herein as T-cell engaging multispecific activatable antibodies. In some aspects, the multispecific activatable antibodies engage a surface antigen on a leukocyte, such as on a T cell, on a natural killer (NK) cell, on a myeloid mononuclear cell, on a macrophage, and / or on another immune effector cell. In some aspects, the immune effector cell is a leukocyte. In some aspects, the immune effector cell is a T cell. In some aspects, the immuneCYTX-108-WO :: 4862-156.WO1 effector cell is a NK cell. In some aspects, the immune effector cell is a mononuclear cell, such as a myeloid mononuclear cell. In some aspects, the multispecific activatable antibodies are designed to bind or otherwise interact with more than one target and / or more than one epitope, also referred to herein as multi-antigen targeting activatable antibodies. As used herein, the terms “target” and “antigen” are used interchangeably.

[0151] In some aspects, immune effector cell engaging multispecific activatable antibodies of the disclosure include a targeting antibody or antigen-binding fragment thereof and an immune effector cell engaging antibody or antigen-binding portion thereof, where at least one of the targeting antibody or antigen-binding fragment thereof and / or the immune effector cell engaging antibody or antigen-binding portion thereof is masked.

[0152] In some aspects, the non-immune effector cell engaging antibody is a cancer targeting antibody. In some aspects the non-immune cell effector antibody is an IgG. In some aspects the immune effector cell engaging antibody is a scFv. In some aspects the targeting antibody (e.g., non-immune cell effector antibody) is an IgG and the immune effector cell engaging antibody is a scFv. In some aspects, the immune effector cell is a leukocyte. In some aspects, the immune effector cell is a T cell. In some aspects, the immune effector cell is a NK cell. In some aspects, the immune effector cell is a myeloid mononuclear cell.

[0153] In some aspects of an immune effector cell engaging multispecific activatable antibody, one antigen is typically an antigen present on the surface of a tumor cell or other cell type associated with disease, and another antigen is typically a stimulatory or inhibitory receptor present on the surface of a T-cell, natural killer (NK) cell, myeloid mononuclear cell, macrophage, and / or other immune effector cell.

[0154] One aspect of the disclosure is a multispecific activatable antibody that is activatable in a cancer microenvironment and that includes an antibody, for example an IgG or scFv, directed to a tumor target and an agonist antibody, for example an IgG or scFv, directed to a co-stimulatory receptor expressed on the surface of an activated T cell or NK cell, wherein at least one of the cancer target antibody and / or agonist antibody is masked. In this aspect, the multispecific activatable antibody, once activated by tumor-associated proteases, effectively crosslinks and activates the T cell or NK cell expressed co-stimulatory receptors in a tumor-dependent manner to enhance the activity of T cells that are responding to any tumor antigen via their endogenous T cell antigen or NK-activating receptors. The activation-dependent nature of these T cell or NK cell costimulatory receptors focuses the activity of the activated multispecific activatable antibody to tumor-specific T cells, withoutCYTX-108-WO :: 4862-156.WO1 activating all T cells independent of their antigen specificity. In one aspect, at least the co- stimulatory receptor antibody of the multispecific activatable antibody is masked to prevent activation of auto-reactive T cells that may be present in tissues that also express the antigen recognized by the tumor target-directed antibody in the multispecific activatable antibody, but whose activity is restricted by lack of co-receptor engagement.

[0155] One aspect of the disclosure is a multispecific activatable antibody that is activatable in a disease characterized by T cell overstimulation, such as an autoimmune disease or inflammatory disease microenvironment. Such a multispecific activatable antibody includes an antibody, for example an IgG or scFv, directed to a target comprising a surface antigen expressed in a tissue targeted by a T cell in autoimmune or inflammatory disease and an antibody, for example an IgG or scFv, directed to an inhibitory receptor expressed on the surface of a T cell or NK cell, wherein at least one of the disease tissue target antibody and / or T cell inhibitory receptor antibody is masked. Examples of a tissue antigen targeted by T cells in autoimmune disease include a surface antigen expressed on myelin or nerve cells in multiple sclerosis or a surface antigen expressed on pancreatic islet cells in Type 1 diabetes. In this aspect, the multispecific activatable antibody when localized in the tissue under autoimmune attack or inflammation is activated and co-engages the T cell or NK cell inhibitory receptor to suppress the activity of autoreactive T cells responding to any disease tissue-targeted antigens via their endogenous TCR or activating receptors. In one aspect, at least one or multiple antibodies are masked to prevent suppression of T cell responses in non- disease tissues where the target antigen may also be expressed.

[0156] In some aspects, the multi-antigen targeting antibodies and / or multi-antigen targeting activatable antibodies include at least a first antibody or antigen-binding fragment thereof that binds a first target and / or first epitope and a second antibody or antigen-binding fragment thereof that binds a second target and / or a second epitope. In some aspects, the multi-antigen targeting antibodies and / or multi-antigen targeting activatable antibodies bind two or more different targets. In some aspects, the multi-antigen targeting antibodies and / or multi-antigen targeting activatable antibodies bind two or more different epitopes on the same target. In some aspects, the multi-antigen targeting antibodies and / or multi-antigen targeting activatable antibodies bind a combination of two or more different targets and two or more different epitopes on the same target.CYTX-108-WO :: 4862-156.WO1 Masking Moieties (MMs)

[0157] The activatable molecules herein may comprise one or more masking moieties (MMs) capable of interfering with the binding of the TBMs to the target. A masking moiety in an activatable molecule “masks” or reduces or otherwise inhibits the binding of the activatable molecule to its target. In some aspects, the coupling of a TBM (e.g., an antibody or fragment thereof, or other therapeutic or diagnostic protein) with an MM may inhibit the ability of the TBM to specifically bind its target by means of inhibition known in the art (e.g., structural change, competition for antigen-binding domain, and the like). In some aspects, the coupling of a TBM with an MM may effect a structural change that reduces or inhibits the ability of the TBM to specifically bind its target. In some aspects, the coupling of an activatable molecule comprising a TBM with an MM sterically blocks, reduces or inhibits the ability of the TBM to specifically bind its target and or epitope. In some aspects, when an activatable molecule is not activated, the MM prevents the TBM from target binding; but when the activatable molecule is activated (when the CM is cleaved by a protease), the MM does not substantially or significantly interfere with the TBM’s binding to the target.

[0158] An MM may be coupled to a TBM (e.g., an antibody or fragment thereof, or other therapeutic or diagnostic protein) via the CM described herein, either directly or indirectly (e.g., via one or more linkers described herein). Alternatively, an MM interfering with the target binding of a TBM may be coupled, either directly or indirectly, to a component of the activatable molecule that is not the TBM. For example, the MM is coupled, either directly or indirectly, to a different TBM. In another example, the MM is coupled, either directly or indirectly, with a half-life extending moiety (EM). In either case, in the tertiary or quaternary structure of the activatable structure, the MM may be in a position (e.g., proximal to the TBM to be masked) that allows the MM to mask the TBM.

[0159] In some aspects, an MM interacts with the TBM, thus reducing or inhibiting the interaction between the TBM and its binding partner. In some aspects, the MM comprises at least a partial or complete amino acid sequence of a naturally occurring binding partner of the TBM. The term “naturally occurring” as used herein as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses or bacteria) that can be isolated from a source in nature and that has not been intentionally modified by man in the laboratory or otherwise is naturally occurring.CYTX-108-WO :: 4862-156.WO1

[0160] For example, the MM is a fragment of a naturally occurring binding partner. The fragment may 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 aspects, the MM is a cognate peptide of the TBM. For example, the MM may comprise a sequence of the TBM’s epitope or a fragment thereof.

[0161] In some aspects, the MM comprises an amino acid sequence that is not naturally occurring or does not contain the amino acid sequence of a naturally occurring binding partner or target protein. In certain aspects, the MM is not a natural binding partner of the TBM. In some aspects, the MM does not comprise a subsequence of more than 4, 5, 6, 7, 8, 9 or 10 consecutive amino acid residues of a natural binding partner of the TBM. The MM may be a modified binding partner for the TBM which contains amino acid changes that decrease affinity and / or avidity of binding to the TBM. In some aspects, the MM contains no or substantially no nucleic acid or amino acid homology to the TBM’s natural binding partner. In other aspects the MM has no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% homology to the natural binding partner of the TBM.

[0162] In some aspects, the MM is a polypeptide that binds to the TBM. In some examples, the MM is an antibody or antibody fragment (e.g., a Fab fragment, a F(ab’)2fragment, a scFv, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody) that binds to the TBM such that interrupts the TBM’s binding to its target. In some examples, the MM is a ligand, a receptor, a fragment thereof (e.g., an extracellular domain of a receptor) of the TBM that binds to the TBM and interrupts the TBM’s binding to its target. In some examples, when the TBM is an antibody or antibody fragment thereof, the MM is an anti-idiotypic antibody or fragment thereof (e.g., scFv) that binds to the idiotype of the TBM. In some examples, the MM is a cytokine or a receptor for a cytokine. In some examples, the MM may have an amino acid sequence that is at least 85% identical to a cytokine or to a receptor for a cytokine.

[0163] In some aspects, the MM does not bind the TBM, but still interferes with TBM’s binding to its binding partner through non-specific interactions such as steric hindrance. For example, the MM is positioned in the activatable molecule such that the tertiary or quaternary structure of the activatable molecule allows the MM to mask the TBM through charge-based interaction, thereby holding the MM in place to interfere with binding partner access to theCYTX-108-WO :: 4862-156.WO1 TBM. Examples of such MMs include an albumin, e.g., human serum albumin (HSA), a fragment crystallizable (Fc) domain, an antibody constant domain (e.g., CH domains), a polymer (e.g., branched or multi-armed polyethylene glycol (PEG)), a latency associated protein (LAP), and any polypeptide or other moieties that sterically interfere TBM-target interactions. In some examples, the MM may recruit a large protein binding partner that sterically interfere TBM-target interactions. For example, the MM is an antibody or a fragment thereof that binds to serum albumin.

[0164] Examples of suitable masking moieties include the full-length or a TBM-binding fragment or mutein of a cognate receptor of the TBM, and TBM-binding antibodies and fragment thereof, e.g., a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody a single chain variable fragment (scFv), single-domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL), a variable domain of camelid-type nanobody (VHH), a dAb and the like. Other exemplary antigen-binding domain that bind the TBM can also be used as an MM include non-immunoglobulin proteins that mimic antibody binding and / or structure such as, anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, kunitz domain peptides, monobodies, and binding domains based on other engineered scaffolds such as SpA, GroEL, fibronectin, lipocallin and CTLA4 scaffolds. As another example, a peptide that is modified by conjugation to a water-soluble polymer, such as PEG, can sterically inhibit or prevent binding of the cytokine to its receptor. Antibodies and antigen-binding domains that bind to, for example, a protein with a long serum half- life such as HSA, immunoglobulin or transferrin, or to a receptor that is recycled to the plasma membrane, such as FcRn or transferrin receptor, can also inhibit the cytokine, particularly when bound to their antigen. In some aspects, the MMs (e.g., those sterically interfere with the TBM-target interaction) can also function as half-life extending elements.

[0165] In some aspects, the MM may have a dissociation constant for binding to the TBM that is no more than the dissociation constant of the TBM to the target. In some aspects, the MM does not interfere or compete with the TBM for binding to the target in in the activated molecule (i.e., following cleavage of the CM by a protease).

[0166] The structural properties of the MMs may be selected according to factors such as the minimum amino acid sequence required for interference with the TBM binding to target, the target protein-protein binding pair of interest, the size of the TBM, the presence or absence of linkers, and the like.CYTX-108-WO :: 4862-156.WO1

[0167] In some aspects, the MM is unique for the coupled TBM. Examples of MMs include MMs that were specifically screened to bind a binding domain of the TBM or fragment thereof (e.g., affinity masks). Methods for screening MMs to obtain MMs unique for the TBM and those that specifically and / or selectively bind a binding domain of a binding partner / target are provided herein and can include protein display methods.

[0168] In some aspects, the MM is a polypeptide of about 2 to 50 amino acids in length. For example, the MM is a polypeptide of from 2 to 40, from 2 to 30, from 2 to 20, from 2 to 10, from 5 to 15, from 10 to 20, from 15 to 25, from 20 to 30, from 25 to 35, from 30 to 40, from 35 to 45, from 40 to 50 amino acids in length. For example, the MM is a polypeptide with 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 length. In some examples, the MM is a polypeptide of more than 50 amino acids in length, e.g., 100, 200, 300, 400, 500, 600, 700, 800, or more amino acids. In some aspects, the MM is a steric mask.

[0169] In some aspects, in an activatable molecule with a TBM and an interfering MM, in the presence of the target of a TBM, there is no binding or substantially no binding of the TBM to the target, or no more than 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 50% binding of the TBM to its target, as compared to the binding of an counterpart molecule without the interfering MM, 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 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 when measured in vitro immunoabsorbant assay, e.g., as described in US20200308243A1.

[0170] The binding affinity of the TBM towards the target or binding partner with an interfering MM may be 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 times lower than the binding affinity of the TBM towards its binding partner without an interfering MM, or between 5-10, 10-100, 10-1,000, 10-10,000, 10-100,000, 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, 1,000-1,000,000, 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 than the binding affinity of the TBM towards its binding partner when there is no interfering MM.

[0171] The dissociation constant (Kd) of the MM towards the TBM it masks, may be greater than the dissociation constant of the TBM towards the target. The dissociationCYTX-108-WO :: 4862-156.WO1 constant of the MM towards the masked TBM 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 greater than the dissociation constant of the TBM towards the target. Conversely, the binding affinity of the MM towards the masked TBM may be lower than the binding affinity of the TBM towards the target. The binding affinity of MM towards the TBM 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 TBM towards the target.

[0172] In some aspects, the Kd of the activatable molecule comprising an MM and a CM towards the TBM’s 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 greater, or between 5-10, 10-100, 10-1,000, 10-10,000, 10-100,000, 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, 1,000-1,000,000, 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 greater than the Kd of a counterpart molecule that is substantially the same as the activatable molecule but does not comprise the MM or CM towards the TBM’s target. Conversely, the binding affinity of the activatable molecule comprising an MM and a CM towards the TBM’s 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 greater, or between 5-10, 10-100, 10-1,000, 10-10,000, 10-100,000, 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, 1,000-1,000,000, 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 than the binding affinity of a counterpart molecule that is substantially the same as the activatable molecule but does not comprise the MM or CM towards the TBM’s target.

[0173] In some aspects, when the TBM is coupled with an MM and is in the presence of the target, the specific binding of the TBM to its target is reduced or inhibited, as compared to the specific binding of the TBM not coupled with the MM. When compared to the binding of the TBM not coupled with the MM to the target, the target-binding ability of the TBM coupled with the 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 when measured in vivo or in an in vitro assay.CYTX-108-WO :: 4862-156.WO1

[0174] In some aspects, the MM comprises a non-binding steric moiety (NB) that does not bind the TBM but is able to interfere the binding between the TBM and its target via steric hindrance. In some aspects, the MM comprises a binding partner (BP) for a NB, where the BP recruits or otherwise attracts the NB to the activatable molecule.

[0175] In some aspects, the MM contains genetically encoded or genetically non-encoded amino acid(s). Examples of genetically non-encoded amino acids include D-amino acids, β- amino acids, and γ-amino acids. In specific aspects, the MMs contain no more than 50%, 40%, 30%, 20%, 15%, 10%, 5% or 1% of genetically non-encoded amino acids.

[0176] In some aspects, once released from the activatable molecule and in a free state, the MM may have a biological activity or a therapeutic effect, such as binding capability. For example, the free peptide may bind with the same or a different binding partner. In certain aspects, the free MM may exert a therapeutic effect, providing a secondary function to the compositions disclosed herein. In some aspects, once uncoupled from the activatable molecule and in a free state, the MM may advantageously not exhibit biological activity. For example, in some aspects the MM after cleavage from the activatable molecule does not elicit an immune response in the subject.

[0177] Suitable MMs may be identified and / or further optimized through a screening procedure from a library of candidate activatable molecule having variable MMs. For example, a TBM and a CM are selected to provide for a desired enzyme / target combination, and the amino acid sequence of the MM can be identified by the screening procedure described below to identify an MM that provides for an activatable phenotype. For example, a random peptide library (e.g., of peptides comprising 2 to 40 amino acids or more) is used in the screening methods disclosed herein to identify a suitable MM.

[0178] In some aspects, MMs with specific binding affinity for a TBM are identified through a screening procedure that includes providing a library of peptide scaffolds comprising candidate MMs wherein each scaffold is made up of a transmembrane protein and the candidate MM. The library may then be contacted with an entire or portion of a protein such as a full length protein, a naturally occurring protein fragment, or a non-naturally occurring fragment containing a protein (also capable of binding the binding partner of interest), and identifying one or more candidate MMs having detectably bound protein. The screening may be performed by one more rounds of magnetic-activated sorting (MACS) or fluorescence-activated sorting (FACS), as well as determination of the binding affinity of MM towards the TBM and subsequent determination of the masking efficiency, e.g., asCYTX-108-WO :: 4862-156.WO1 described in WO2009025846 and US20200308243A1, which are incorporated herein by reference in their entireties.

[0179] Examples of suitable MMs are disclosed in WO2021207657, WO2021142029, WO2021061867, WO2020252349, WO2020252358, WO2020236679, WO2020176672, WO2020118109, WO2020092881, WO2020086665, WO2019213444, WO2019183218, WO2019173771, WO2019165143, WO2019075405, WO2019046652, WO2019018828, WO2019014586, WO2018222949, WO2018165619, WO2018085555, WO2017011580, WO2016179335, WO2016179285, WO2016179257, WO2016149201, and WO2016014974, which are incorporated herein by reference in their entireties.

[0180] In some aspects, the TBM in an activatable molecule is an antibody or antigen- binding fragment that specifically binds a tumor-associated moiety, for example, EGFR. In some examples, such an activatable molecule comprises an MM that comprises the amino acid sequence of SEQ ID NO: 39. In some examples, such an activatable molecule comprises an MM that comprises a sequence according to SEQ ID NO: 39. In some examples, such an activatable molecule comprises an MM that consists of the amino acid sequence of SEQ ID NO: 39.

[0181] In some aspects, the present disclosure includes an activatable antibody comprising an anti-EGFR antibody coupled directly or indirectly to a CM, wherein the CM is directly or indirectly coupled to an MM that comprises or consists of a sequence according to SEQ ID NO: 39.

[0182] In some aspects, the activatable molecule has an in vivo antitumor activity that is equal to or higher than an activatable molecule comprising a CM comprising a sequence of QNQALRMA (SEQ ID NO: 3). In some aspects, the antitumor activity is a reduction in tumor volume of 25% or more, 30% or more, 40% or more, 50% or more 60% or more, 70% or more, 75% or more, 80% or more, or 90% or more. In some aspects, the activatable molecule has a higher in vivo antitumor activity after at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, or at least 10 days post-administration. Linkers

[0183] The activatable molecules may comprise one or more linkers. The linkers may be linking peptides that comprise a stretch of amino acid sequence that link two components in the activatable molecule. The linkers may be non-cleavable by any protease. In some aspects, one or more linkers may be introduced into the activatable molecule to provide flexibility at one or more of the junctions between domains, between moieties, between moieties andCYTX-108-WO :: 4862-156.WO1 domains, or at any other junctions where a linker would be beneficial. In some aspects, where the activatable molecule is provided as a conformationally constrained construct, a flexible linker is inserted to facilitate formation and maintenance of a structure in the activatable molecule. Any of the linkers described herein may provide the desired flexibility to facilitate the inhibition of the binding of a target, or to facilitate cleavage of a CM by a protease. In some aspects, linkers included in the activatable molecule are all or partially flexible, such that the linker can include a flexible linker as well as one or more portions that confer less flexible structure to provide for a desired activatable molecule. Some linkers may include cysteine residues, which may form disulfide bonds and reduce flexibility of the construct.

[0184] In some aspects, a linker coupled to an MM may have a length that allows the MM to be in a position in the tertiary or quaternary to effectively mask a TBM, (e.g., proximal to the TBM to be masked) that allows the MM to mask the TBM.

[0185] In most instances, the linker’s length may be determined by counting, in a N- to C- direction, the number of amino acids from the N-terminus of the linker adjacent to the C- terminal amino acid of the preceding component, to the C-terminus of the linker adjacent to the N-terminal amino acid of the following component (i.e., where the linker length does not include either the C-terminal amino acid of the preceding component or the N-terminal amino acid of the following component).

[0186] In some aspects, a linker may include a total of 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 5, 2 to 4, 2 to 3, 4 to 25, 4 to 24, 4 to 22, 4 to 20, 4 to 18, 4 to 16, 4 to 15, 4 to 14, 4 to 12, 4 to 10, 4 to 8, 4 to 6, 4 to 5, 5 to 25, 5 to 24, 5 to 22, 5 to 20, 5 to 18, 5 to 16, 5 to 15, 5 to 14, 5 to 12, 5 to 10, 5 to 8, 5 to 6, 6 to 25, 6 to 24, 6 to 22, 6 to 20, 6 to 18, 6 to 16, 6 to 15, 6 to 14, 6 to 12, 6 to 10, 6 to 8, 8 to 25, 8 to 24, 8 to 22, 8 to 20, 8 to 18, 8 to 16, 8 to 15, 8 to 14, 8 to 12, 8 to 10, 10 to 25, 10 to 24, 10 to 22, 10 to 20, 10 to 18, 10 to 16, 10 to 15, 10 to 14, 10 to 12, 12 to 25, 12 to 24, 12 to 22, 12 to 20, 12 to 18, 12 to 16, 12 to 15, 12 to 14, 14 to 25, 14 to 24, 14 to 22, 14 to 20, 14 to 18, 14 to 16, 14 to 15, 15 to 25, 15 to 24, 15 to 22, 15 to 20, 15 to 18, 15 to 16, 16 to 25, 16 to 24, 16 to 22, 16 to 20, 16 to 18, 18 to 25, 18 to 24, 18 to 22, 18 to 20, 20 to 25, 20 to 24, 20 to 22, 22 to 25, 22 to 24, or 24 to 25 amino acids). In some aspects, the linker may include 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.CYTX-108-WO :: 4862-156.WO1

[0187] In some aspects, a linker is rich in glycine (Gly or G) residues. In some aspects, the linker is rich in serine (Ser or S) residues. In some aspects, the linker is rich in glycine and serine residues. In some aspects, the linker has 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).

[0188] In some aspects, the linker has one or more Gly-Gly-Gly-Ser (GGGS) (SEQ ID NO: 120) sequences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGGS sequences (SEQ ID NO: 120)). In some aspects, the linker has one or more Gly-Gly-Gly-Gly-Ser (GGGGS) (SEQ ID NO: 126) sequences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGGGS sequences (SEQ ID NO: 126)). In some aspects, the linker has one or more Gly-Gly-Ser-Gly (GGSG) (SEQ ID NO: 113) sequences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGSG sequences (SEQ ID NO: 113)). Examples of the linkers include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, (GGS)n, (GSGGS)n (SEQ ID NO: 177) and (GGGS)n (SEQ ID NO: 120), where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine- serine polymers may be relatively unstructured, and therefore may be able to serve as a neutral link between components. Glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem.11173-142 (1992)). Exemplary flexible linkers include one of or a combination of one or more of: GGSG (SEQ ID NO: 113), GGSGG (SEQ ID NO: 114), GSGSG (SEQ ID NO: 115), GSGGG (SEQ ID NO: 116), GGGSG (SEQ ID NO: 117), GSSSG (SEQ ID NO: 118), GSSGGSGGSGG (SEQ ID NO: 119), GGGS (SEQ ID NO: 120), GGGSGGGS (SEQ ID NO: 121), GGGSGGGSGGGS (SEQ ID NO: 122), GGGGSGGGGSGGGGS (SEQ ID NO: 123), GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 124), GGGGSGGGGS (SEQ ID NO: 125), GGGGS (SEQ ID NO: 126), GS, GGGGSGS (SEQ ID NO: 127), GGGGSGGGGSGGGGSGS (SEQ ID NO: 128), GGSLDPKGGGGS (SEQ ID NO: 129), PKSCDKTHTCPPCPAPELLG (SEQ ID NO: 130), SKYGPPCPPCPAPEFLG (SEQ ID NO: 131), GKSSGSGSESKS (SEQ ID NO: 132), GSTSGSGKSSEGKG (SEQ ID NO: 133), GSTSGSGKSSEGSGSTKG (SEQ ID NO: 134), GSTSGSGKPGSGEGSTKG (SEQ ID NO: 135), GSTSGSGKPGSSEGST (SEQ ID NO: 136), GGGSSGGS (SEQ ID NO: 137), GGGGSGGGGSS (SEQ ID NO: 138), GGGSSGGSGGSSGGS (SEQ ID NO: 139), and GSTSGSGKPGSSEGST (SEQ ID NO: 140).CYTX-108-WO :: 4862-156.WO1

[0189] Examples of linkers may further include a sequence that is 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 example linkers described herein. An ordinarily skilled artisan will recognize that design of an activatable molecules can include linkers that are all or partially flexible, such that the linker can include a flexible linker as well as one or more portions that confer less flexible structure to provide for a desired activatable molecules structure.

[0190] In some aspects, an activatable molecule may include one, two, three, four, five, six, seven, eight, nine, or ten linker sequence(s) (e.g., the same or different linker sequences of any of the exemplary linker sequences described herein or known in the art). In some aspects, a linker may comprise sulfo-SIAB, SMPB, and sulfo-SMPB, wherein the linkers react with primary amines sulfhydryls. Half-life extending moieties (EMs)

[0191] The activatable molecule may further comprise one or more half-life extending moieties (EMs). In some examples, the half-life extending moiety is a serum half-life extending moiety, i.e., capable of extending the serum half-life of the molecule attached to the EM.

[0192] In some examples, the EM comprises a fragment crystallizable region (Fc domain) of an antibody. For example, the EM is the Fc domain of an IgG (e.g., IgG1, IgG2, IgG3, or IgG4). In some examples, the EM comprises a dimer formed by two Fc domains. The Fc domain may be a wild type peptide or a mutant. For example, the EM comprises a dimer formed by two Fc domain mutants. In such cases, the two Fc domain mutants are a Fc domain hole mutant and a Fc domain knob mutant. The knob and hole mutants may interact with each other to facilitate the dimerization of the two Fc domains. In some aspects, the knob and hole mutants may comprise one or more amino acid modifications within the interface between two Fc domains (e.g., in the CH3 domain). In one example, the modifications comprise 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 in the other IgG Fc domain (numbering according to EU numbering system). Example of Fc mutants also include SEQ ID NOs: 141 and 142.

[0193] Examples of the Fc domain mutants also include those described in U.S. Pat. Nos. 7,695,936, which is incorporated herein by reference in its entirety. In one example, theCYTX-108-WO :: 4862-156.WO1 modifications comprise amino acid substitution T366Y in one IgG Fc domain, and the amino acid substitutions Y407T in the other IgG Fc domain. In one example, the modifications comprise amino acid substitution T366W in one IgG Fc domain, and the amino acid substitutions Y407A in the other IgG Fc domain. In one example, the modifications comprise amino acid substitution F405A in one IgG Fc domain, and the amino acid substitutions T394W in the other IgG Fc domain. In one example, the modifications comprise amino acid substitution 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 modifications comprise amino acid substitution T366W and F405W in one IgG Fc domain, and the amino acid substitutions T394S and Y407A in the other IgG Fc domain. In one example, the modifications comprise amino acid substitution F405W and Y407A in one IgG Fc domain, and the amino acid substitutions T366W and T394S in the other IgG Fc domain. In one example, the modifications comprise amino acid substitution F405W in one IgG Fc domain, and the amino acid substitutions T394S in the other IgG Fc domain. The mutation positions in the Fc domains are numbered according to EU numbering system. The IgG Fc domain may comprise a sequence of SEQ ID NOs: 143-146 (IgG1, IgG2, IgG3 or IgG4). In these sequences, amino acids 1-107 correspond to EU numbering 341-447.

[0194] In some examples, the Fc domains mutants may have reduced effector function. Examples of such Fc domains include those disclosed in in US20190135943, which incorporated herein by reference in its entirety.

[0195] Further examples of EMs include immunoglobulin (e.g., IgG), serum albumin (e.g., human serum albumin (HSA), an antigen binding polypeptide that binds human serum albumin, a polyethylene glycol molecule, transferrin, 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, Immuno- reactive Epitopes, Epitope Tags, E2Tag, HA Epitope Tag, Myc Epitope, FLAG Epitope, AU1 and AU5 Epitopes, Glu-Glu Epitope, KT3 Epitope, IRS Epitope, Btag Epitope, Protein Kinase-C Epitope, and VSV Epitope.

[0196] In some aspects, the serum half-life of an activatable molecule comprising an EM is longer than that of a counterpart molecule that is substantially the same as the activatable molecule but does not comprise the EM, e.g., the pK of the activatable molecule is longer than that of the reference molecule. In some examples, the activatable molecule with an EM may have a serum half-life that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,CYTX-108-WO :: 4862-156.WO1 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, 100-fold longer than the serum half-life of the reference counterpart molecule. In some aspects, the serum half-life of the activatable molecule with an EM is 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 when administered to an organism. Conjugation to Agents

[0197] In some aspects, the present disclosure provides conjugated polypeptides. In some aspects, the cleavable polypeptide further comprises an agent conjugated to the cleavable polypeptide. In some aspects, the agent is conjugated to the cleavable polypeptide via a conjugating linker. In some aspects, the conjugating linker is cleavable. In some aspects, the conjugating linker is non-cleavable. In some aspects, the agent is selected from the group consisting of: a toxin, a microtubule inhibitor, a nucleic acid damaging agent, a dolastatin, an auristatin, a maytansinoid, a duocarmycin, and a calicheamicin.

[0198] In some aspects, a conjugated polypeptide comprises a cleavable polypeptide herein conjugated to one or more agent, e.g., a targeting moiety to facilitate delivery to a cell or tissue of interest, a therapeutic agent (e.g., an antineoplastic agent such as chemotherapeutic or anti-neoplastic agent), a toxin, or a fragment thereof. The agents may be conjugated to a component of the activatable molecules. In some aspects, the conjugated polypeptide is an antibody-drug conjugate (ADC), which comprises an antibody or antigen- binding fragment thereof conjugated with a drug. In some examples, the antibody or antigen- binding fragment thereof are conjugated with the drug via a CM disclosed herein. In some examples, the antibody or antigen-binding fragment thereof is an activatable antibody or antigen-binding fragment thereof (e.g., coupled with a MM via a CM), which is further conjugated with a drug (e.g., via a cleavable or non-cleavable conjugating linker).

[0199] The term “agent” is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials. Examples of the agent include toxin, a microtubule inhibitor, a nucleic acid damaging agent, a dolastatin, an auristatin, a maytansinoid, a duocarmycin, a calicheamicin, or a combination thereof.In some aspects, the activatable molecule is conjugated to a cytotoxic agent, e.g., a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof) or a radioactive isotope.CYTX-108-WO :: 4862-156.WO1

[0200] Examples of cytotoxic agents include that can be conjugated to the activatable molecules dolastatins and derivatives thereof (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 tyramine, auristatin quinolone), maytansinoids (e.g., DM-1, DM-4), maytansinoid derivatives, duocarmycin, alpha-amanitin, turbostatin, phenstatin, hydroxyphenstatin, spongistatin 5, spongistatin 7, halistatin 1, halistatin 2, halistatin 3, halocomstatin, pyrrolobenzimidazoles (PBI), cibrostatin6, doxaliform, cemadotin analogue (CemCH2-SH), Pseudomonas toxin A (PES8) variant, Pseudomonase toxin A (ZZ-PE38) variant, ZJ-101, anthracycline, doxorubicin, daunorubicin, bryostatin, camptothecin, 7-substituted campothecin, 11- difluoromethylenedioxycamptothecin, combretastatins, debromoaplysiatoxin, KahaMide-F, discodermolide, and Ecteinascidins. In some aspects, the agent is DM1 or DM4. In some aspects, the agent is a duocarmycin or derivative thereof. In some aspects, the agent is a calicheamicin or derivative thereof. In some aspects, the agent is a pyrrolobenzodiazepine.

[0201] Examples of enzymatically active toxins that can be conjugated to the activatable molecules include diphtheria toxin, exotoxin A chain from Pseudomonas aeruginosa, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleuriies fordii proteins, dianfhin proteins, Phytolaca Americana proteins (e.g., PAPI, PAPII, and PAP-8), momordica charantia inhibitor, curcin, crotirs, sapaonaria officinalis inhibitor, geionin, mitogeliin, restrictocin, phenomycin, neomycin, and tricothecenes. A variety of radionuclides are available for the production of radioconjugated molecules. Examples of radionuclides include212Bi,131I,131In,90Y, and186Re.

[0202] Examples of anti-neoplastics that can be conjugated to the activatable molecules include: adriamycin, cerubidine, bleomycin, alkeran, velban, oncovin, fluorouracil, methotrexate, thiotepa, bisantrene, novantrone, thioguanine, procarabizine, and cytarabine.

[0203] Examples of antivirals that can be conjugated to the activatable molecules include acyclovir, vira A, and symmetrel. Examples of antifungals that can be conjugated to the activatable molecules include: nystatin. Examples of detection reagents that can be conjugated to the activatable molecules include: fluorescein and derivatives thereof, fluorescein isothiocyanate (FITC). Examples of antibacterials that can be conjugated to the activatable molecules include: aminoglycosides, streptomycin, neomycin, kanamycin, amikacin, gentamicin, and tobramycin. Examples of 3beta,16beta,17alpha-trihydroxycholest-CYTX-108-WO :: 4862-156.WO1 5-en-22-one 16-O-(2-O-4-methoxybenzoyl-beta-D-xylopyranosyl)-(1-->3)-(2-O-acetyl- alpha-L-arabinopyranoside) (OSW-1) that can be conjugated to the activatable molecules include: s-nitrobenzyloxycarbonyl derivatives of O6-benzylguanine, toposisomerase inhibitors, hemiasterlin, cephalotaxine, homoharringionine, pyrrol obenzodiazepine dimers (PBDs), functionalized pyrrolobenzodiazepenes, calcicheamicins, podophyiitoxins, taxanes, and vinca alkoids. Examples of radiopharmaceuticals that can be conjugated to the activatable molecules include:123I,89Zr,125I,131I,201T1,62Cu,18F,68Ga,13N,15O,38K,82Rb,111In,133Xe,11C, and99mTc (Technetium). Examples of heavy metals that can be conjugated to the activatable molecules include: barium, gold, and platinum. Examples of anti- mycoplasmals that can be conjugated to the activatable molecules include: tylosin, spectinomycin, streptomycin B, ampicillin, sulfanilamide, polymyxin, and chloramphenicol.

[0204] In some aspects, the agent is a nucleic acid damaging agent, such as a DNA alkylator or DNA intercalator, or other DNA damaging agent.

[0205] Additional examples of the agents that can be conjugated include those in Table 1 below. Table 1: Exemplary Pharmaceutical Agents for Conjugation CYTOTOXIC AGENTS Auristatins Turbostatin Auristatin E Phenstatins Monomethyl auristatin D (MMAD) Hydroxyphenstatin Monomethyl auristatin E (MMAE) Spongistatin 5 Desmethyl auristatin E (DMAE) Spongistatin 7 Auristatin F Halistatin 1 Monomethyl auristatin F (MMAF) Halistatin 2 Desmethyl auristatin F (DMAF) Halistatin 3 Auristatin derivatives, e.g., amides thereof Modified Bryostatins Auristatin tyramine Halocomstatins Auristatin quinoline Pyrrolobenzimidazoles (PBI) Dolastatins Cibrostatin6 Dolastatin derivatives Doxaliform Dolastatin 16 DmJ Anthracyclins analogues Dolastatin 16 Dpv Maytansinoids, e.g. DM-1; DM-4 Maytansinoid derivatives Cemadotin analogue (CemCH2-SH) Duocarmycin Pseudomonas toxin A (PE38) variant Duocarmycin derivatives Pseudomonas toxin A (ZZ-PE38) variant Alpha-amanitin ZJ-101 Anthracyclines OSW-1 Doxorubicin 4-Nitrobenzyloxycarbonyl Derivatives of O6-BenzylguanineCYTX-108-WO :: 4862-156.WO1 Daunorubicin Topoisomerase inhibitors Bryostatins Hemiasterlin Camptothecin Cephalotaxine Camptothecin derivatives Homoharringtonine 7-substituted Camptothecin Pyrrolobenzodiazepine dimers (PBDs) 10, 11- Functionalized pyrrolobenzodiazepenes Difluoromethylenedioxycamptothecin Combretastatins Calicheamicins Debromoaplysiatoxin Podophyllotoxins Kahalalide-F Taxanes Discodermolide Vinca alkaloids Ecteinascidins CONJUGATABLE DETECTION REAGENTS ANTIVIRALS Fluorescein and derivatives thereof Acyclovir Fluorescein isothiocyanate (FITC) Vira A Symmetrel RADIOPHARMACEUTICALS 125I ANTIFUNGALS131I Nystatin89Zr 111In ADDITIONAL ANTI-NEOPLASTICS123I Adriamycin131I Cerubidine99mTc (Technetium) Bleomycin201Tl Alkeran133Xe Velban11C Oncovin62Cu Fluorouracil18F Methotrexate68Ga Thiotepa13N Bisantrene15O Novantrone38K Thioguanine82Rb Procarabizine Cytarabine HEAVY METALS ANTI-BACTERIALS Barium Aminoglycosides Gold Streptomycin Platinum Neomycin Kanamycin ANTI-MYCOPLASMALS Amikacin Tylosin Gentamicin Spectinomycin Tobramycin Streptomycin B NanoparticlesCYTX-108-WO :: 4862-156.WO1 Spectinomycin Ampicillin Sulfanilamide Polymyxin Chloramphenicol

[0206] In some aspects, the activatable molecule comprises a signal peptide. If comprising multiple polypeptides, the activatable molecule may comprise multiple signal peptides, e.g., one signal peptide for each of the multiple polypeptides. A signal peptide may be a peptide (e.g., 10-30 amino acids long) present at a terminus (e.g., the N-terminus or C- terminus) of a newly synthesized proteins that are destined toward the secretory pathway. In some aspects, the signal peptide is conjugated to the activatable molecule via a spacer. In some aspects, the spacer is conjugated to the activatable molecule in the absence of a signal peptide.

[0207] Those of ordinary skill in the art will recognize that a large variety of possible agents may be conjugated to any of the activatable molecules described herein. The agents may be conjugated to another component of the activatable molecule by a conjugating linker. Conjugation may include any chemical reaction that binds the two molecules so long as the activatable molecule and the other moiety retain their respective activities. Conjugation may include many chemical mechanisms, e.g., covalent binding, affinity binding, intercalation, coordinate binding, and complexation. In some aspects, the binding may be covalent binding. Covalent binding may be achieved either by direct condensation of existing side chains or by the incorporation of external bridging molecules. Many bivalent or polyvalent linking agents may be useful in conjugating any of the activatable molecules described herein. For example, conjugation may include organic compounds, such as thioesters, carbodiimides, succinimide esters, glutaraldehyde, diazobenzenes, and hexamethylene diamines. In some aspects, the activatable molecules may include, or otherwise introduce, one or more non-natural amino acid residues to provide suitable sites for conjugation.

[0208] In some aspects, an agent is attached by disulfide bonds (e.g., disulfide bonds on a cysteine molecule) to the activatable molecule. Since many cancers naturally release high levels of glutathione, a reducing agent, glutathione present in the cancerous tissue microenvironment can reduce the disulfide bonds, and subsequently release the agent at the site of delivery.CYTX-108-WO :: 4862-156.WO1

[0209] In some aspects, when the agent binds its target in the presence of complement within the target site (e.g., diseased tissue (e.g., cancerous tissue)), the amide or ester bond attaching the agent to the linker is cleaved, resulting in the release of the agent in its activated form. These agents when administered to a subject, may accomplish delivery and release of the agent at the target site (e.g., diseased tissue (e.g., cancerous tissue)). These agents may be effective for the in vivo delivery of any of the agents described herein.

[0210] In some aspects, the one or more agents are conjugated to a component of the activatable molecule (e.g., to a TBM, or a CM) via a conjugating linker. The conjugating linker may be a peptide or chemical moiety linking the agent and the activatable molecule. In some examples, the conjugating linker is cleavable (e.g., by an enzyme such as a protease). In some examples, the conjugating linker is non-cleavable (e.g., cannot be cleaved by an enzyme such as a protease). In some aspects, the conjugating linker is non-cleavable by enzymes of the complement system. In some aspects, two or more conjugating linkers are present. The two or more conjugating linkers may be the same, i.e., cleavable or non- cleavable. The two or more conjugating linkers may be different, i.e., at least one cleavable and at least one non-cleavable. For example, the agent is released without complement activation since complement activation ultimately lyses the target cell. In such aspects, the conjugate and / or agent is to be delivered to the target cell (e.g., hormones, enzymes, corticosteroids, neurotransmitters, or genes). Furthermore, the conjugating linker may be mildly susceptible to cleavage by serum proteases, and the conjugate and / or agent is released slowly at the target site.

[0211] In some aspects, the agent is conjugated to a component of the activatable molecule via a maleimide caproyl-valine-citrulline linker or a maleimide PEG-valine- citrulline linker. In some aspects, the agent is conjugated to a component of the activatable molecule via a maleimide caproyl-valine-citrulline linker. In some aspects, the agent is conjugated to a component of the activatable molecule via a maleimide PEG-valine-citrulline linker. In some aspects, the agent is monomethyl auristatin D (MMAD) conjugated to a component of the activatable molecule via a maleimide PEG-valine-citrulline-para- aminobenzyloxycarbonyl linker, and this linker payload construct is vc-MMAD. In some aspects, the agent is monomethyl auristatin E (MMAE) conjugated to a component of the activatable molecule via a maleimide PEG-valine-citrulline-para-aminobenzyloxycarbonyl linker, and this linker payload construct is vc-MMAE.CYTX-108-WO :: 4862-156.WO1

[0212] In some aspects, the agent is designed such that the agent is delivered to the target site (e.g., diseased tissue (e.g., cancerous tissue)) but the conjugate and / or agent is not released.

[0213] In some aspects, the agent is attached to a TBM either directly or via amino acids (e.g., D-amino acids), peptides, thiol-containing moieties, or other organic compounds that may be modified to include functional groups that can subsequently be utilized in attachment to TBM by methods described herein.

[0214] In some aspects, an activatable molecule includes at least one point of conjugation for an agent. In some aspects, all possible points of conjugation are available for conjugation to an agent. In some aspects, the one or more points of conjugation may include sulfur atoms involved in disulfide bonds, sulfur atoms involved in interchain disulfide bonds, sulfur atoms involved in interchain sulfide bonds but not sulfur atoms involved in intrachain disulfide bonds, and / or sulfur atoms of cysteine or other amino acid residues containing a sulfur atom. In such cases, residues may occur naturally in the activatable molecule structure or may be incorporated into the activatable molecule using methods including site-directed mutagenesis, chemical conversion, or mis-incorporation of non-natural amino acids.

[0215] The present disclosure also provides methods and materials for preparing an activatable molecule with one or more conjugated agents. In some aspects, an activatable molecule is modified to include one or more interchain disulfide bonds. For example, disulfide bonds may undergo reduction following exposure to a reducing agent such as, without limitation, TCEP, DTT, or β-mercaptoethanol. In some cases, the reduction of the disulfide bonds is only partial. As used herein, the term partial reduction refers to situations where an activatable molecule is contacted with a reducing agent and a fraction of all possible sites of conjugation undergo reduction (e.g., not all disulfide bonds are reduced). In some aspects, an activatable molecule is partially reduced following contact with a reducing agent if less than 99%, (e.g., less than 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5%) of all possible sites of conjugation are reduced. In some aspects, the activatable molecule having a reduction in one or more interchain disulfide bonds may be conjugated to a drug reactive with free thiols.

[0216] The present disclosure also provides methods and materials for conjugating a therapeutic agent to a particular location on an activatable molecule. In some aspects, an activatable molecule is modified so that the therapeutic agents can be conjugated to theCYTX-108-WO :: 4862-156.WO1 activatable molecule at particular locations on the activatable molecule. For example, an activatable molecule is partially reduced in a manner that facilitates conjugation to the activatable molecule. In such cases, partial reduction of the activatable molecule may occur in a manner that conjugation sites in the activatable molecule are not reduced. In some aspects, the conjugation site(s) on the activatable molecule may be selected to facilitate conjugation of an agent at a particular location on the activatable molecule. Various factors can influence the “level of reduction” of the activatable molecule upon treatment with a reducing agent. For example, without limitation, the ratio of reducing agent to activatable molecule, length of incubation, incubation temperature, and / or pH of the reducing reaction solution can require optimization in order to achieve partial reduction of the activatable molecule with the methods and materials described herein. Any appropriate combination of factors (e.g., ratio of reducing agent to activatable molecule, the length and temperature of incubation with reducing agent, and / or pH of reducing agent) may be used to achieve partial reduction of the activatable molecule (e.g., general reduction of possible conjugation sites or reduction at specific conjugation sites).

[0217] An effective ratio of reducing agent to activatable molecule can be any ratio that at least partially (i.e., partially or fully) reduces the activatable molecule in a manner that allows conjugation to an agent (e.g., general reduction of possible conjugation sites or reduction at specific conjugation sites). In some aspects, the ratio of reducing agent to activatable molecule is in a range from about 20:1 to 1:1, from 10:1 to 1:1, from 9:1 to 1:1, from 8:1 to 1:1, from 7:1 to 1:1, from 6:1 to 1:1, from 5:1 to 1:1, from 4:1 to 1:1, from 3:1 to 1:1, from 2:1 to 1:1, from 20:1 to 1:1.5, from 10:1 to 1:1.5, from 9:1 to 1:1.5, from 8:1 to 1:1.5, from 7:1 to 1:1.5, from 6:1 to 1:1.5, from 5:1 to 1:1.5, from 4:1 to 1:1.5, from 3:1 to 1:1.5, from 2:1 to 1:1.5, from 1.5:1 to 1:1.5, or from 1:1 to 1:1.5.

[0218] An effective incubation time and temperature for treating an activatable molecule with a reducing agent may be any time and temperature that at least partially reduces the activatable molecule in a manner that allows conjugation of an agent to an activatable molecule (e.g., general reduction of possible conjugation sites or reduction at specific conjugation sites). In some aspects, the incubation time and temperature for treating an activatable molecule is in a range from about 1 hour at 37 °C to about 12 hours at 37 °C (or any subranges therein).

[0219] An effective pH for a reduction reaction for treating an activatable molecule with a reducing agent can be any pH that at least partially reduces the activatable molecule in aCYTX-108-WO :: 4862-156.WO1 manner that allows conjugation of the activatable molecule to an agent (e.g., general reduction of possible conjugation sites or reduction at specific conjugation sites).

[0220] When a partially-reduced activatable molecule is contacted with an agent containing thiols, the agent may conjugate to the interchain thiols in the activatable molecule. An agent can be modified in a manner to include thiols using a thiol-containing reagent (e.g., cysteine or N-acetyl cysteine). For example, the activatable molecule can be partially reduced following incubation with reducing agent (e.g., TEPC) for about 1 hour at about 37 °C at a desired ratio of reducing agent to activatable molecule. An effective ratio of reducing agent to activatable molecule may be any ratio that partially reduces at least two interchain disulfide bonds located in the activatable molecule in a manner that allows conjugation of a thiol- containing agent (e.g., general reduction of possible conjugation sites or reduction at specific conjugation sites).

[0221] In some aspects, an activatable molecule is reduced by a reducing agent in a manner that avoids reducing any intrachain disulfide bonds. In some aspects of, an activatable molecule is reduced by a reducing agent in a manner that avoids reducing any intrachain disulfide bonds and reduces at least one interchain disulfide bond.

[0222] In some aspects, the agent is a detectable moiety such as, for example, a label or other marker. For example, the agent is or includes a radiolabeled amino acid, one or more biotinyl moieties that can be detected 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 aspects, detectable moieties are attached by spacer molecules. In some aspects, the detectable label may include an imaging agent, a contrasting agent, an enzyme, a fluorescent label, a chromophore, a dye, one or more metal ions, or a ligand-based label. In some aspects, the imaging agent may comprise a radioisotope. In some aspects, the radioisotope is indium or technetium. In some aspects, the contrasting agent may comprise iodine, gadolinium or iron oxide. In some aspects, the enzyme may comprise horseradish peroxidase, alkaline phosphatase, or β-galactosidase. In some aspects, 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 aspects, the luminescent label may comprise an N- methylacrydium derivative. In some aspects, the label may comprise an Alexa Fluor®CYTX-108-WO :: 4862-156.WO1 label, such as Alex Fluor® 680 or Alexa Fluor® 750. In some aspects, the ligand-based label may comprise biotin, avidin, streptavidin or one or more haptens.

[0223] 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 material include 125I, 131I, 35S or 3H.

[0224] In some aspects, the agent is conjugated to the activatable molecule using a carbohydrate moiety, sulfhydryl group, amino group, or carboxylate group. In some aspects, the agent is conjugated to the activatable molecule via a linker and / or a CM described herein. In some aspects, the agent is conjugated to a cysteine or a lysine in the activatable molecule. In some aspects, the agent is conjugated to another residue of the activatable molecule, such as those residues disclosed herein.

[0225] In some aspects, a variety of bifunctional protein-coupling agents are used to conjugate the agent to the activatable molecule 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., glutareldehyde), bis-azido compounds (e.g., bis (p-azidobenzoyl) hexanediamine), bis- diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., tolyene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4- dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987). In some aspects, a carbon-14-labeled 1-isothiocyanatobenzyl- 3-methyldiethylene triaminepentaacetic acid (MX-DTPA) chelating agent can be used to conjugate a radionucleotide to the activatable molecule. (See, e.g., WO94 / 11026).

[0226] Suitable conjugating linkers also include those described in the literature. (See, for example, Ramakrishnan, S. et al., Cancer Res.44:201-208 (1984) describing use of MBS (M- maleimidobenzoyl-N-hydroxysuccinimide ester). See also, U.S. Patent No.5,030,719, describing use of halogenated acetyl hydrazide derivative coupled to an activatable moleculeCYTX-108-WO :: 4862-156.WO1 by way of an oligopeptide. In some aspects, suitable conjugating linkers include: (i) EDC (1- ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride; (ii) SMPT (4- succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pridyl-dithio)-toluene (Pierce Chem. Co., Cat. (21558G); (iii) SPDP (succinimidyl-6 [3-(2-pyridyldithio) propionamido] hexanoate (Pierce Chem. Co., Cat #21651G); (iv) Sulfo-LC-SPDP (sulfosuccinimidyl 6 [3-(2- pyridyldithio)-propianamide] hexanoate (Pierce Chem. Co. Cat. #2165-G); and (v) sulfo- NHS (N-hydroxysulfo-succinimide: Pierce Chem. Co., Cat. #24510) conjugated to EDC. Additional example agents include SMCC, sulfo-SMCC, SPDB, and sulfo-SPDB.

[0227] Exemplary conjugating linkers for attachment to reduced activatable molecules include those having certain reactive groups capable of reaction with a sulfhydryl group of a reduced antibody or fragment. 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 by Mitra and Lawton, 1979, J. Amer. Chem. Soc.101: 3097-3110).

[0228] Exemplary conjugating linkers for attachment to neither oxidized nor reduced activatable molecules include those having certain functional groups capable of reaction with the primary amino groups present in unmodified lysine residues in the activatable molecules. Such reactive groups include NHS carboxylic or carbonic esters, sulfo-NHS carboxylic or carbonic esters, 4-nitrophenyl carboxylic or carbonic esters, pentafluorophenyl carboxylic or carbonic esters, acyl imidazoles, isocyanates, and isothiocyanates, and other dehydrating agents utilized for carboxamide formation. In these instances, the functional groups present in the suitable conjugating linkers include primary and secondary amines, hydrazines, hydroxylamines, and hydrazides.

[0229] The agent may be attached to the conjugating linker before or after the conjugating linker is attached to the activatable molecule. In certain applications, an activatable molecule-conjugating linker intermediate is first produced in which the conjugating linker is free of an associated agent. Depending upon the particular application, a specific agent is then covalently attached to the conjugating linker. In some aspects, the TBM is first attached to the MM, CM and associated linking peptides and then attached to the conjugating linker for conjugation purposes.

[0230] In specific aspects, branched conjugating linkers that have multiple sites for attachment of agents are utilized. For multiple site conjugating linkers, a single covalent attachment to an activatable molecule may result in an activatable molecule-linkerCYTX-108-WO :: 4862-156.WO1 intermediate capable of binding an agent at a number of sites. The sites may be aldehyde or sulfhydryl groups or any chemical site to which agents can be attached.

[0231] In some aspects, higher specific activity (or higher ratio of agents to activatable molecule) can be achieved by attachment of a single site conjugating linker at a plurality of sites on the activatable molecule. This plurality of sites may be introduced into the activatable molecule by either of two methods. First, one may generate multiple aldehyde groups and / or sulfhydryl groups in the same activatable molecule. Second, one may attach to an aldehyde or sulfhydryl of the activatable molecule a branched conjugating linker having multiple functional sites for subsequent attachment to conjugating linkers. The functional sites of the branched conjugating linker or multiple site conjugating linker may be aldehyde or sulfhydryl groups, or may be any chemical site to which conjugating linkers may be attached. Still higher specific activities may be obtained by combining these two approaches, that is, attaching multiple site conjugating linkers at several sites on the activatable molecule.

[0232] Peptide conjugating linkers that are susceptible to cleavage by enzymes of the complement system, such as but not limited to u-plasminogen activator, tissue plasminogen activator, trypsin, plasmin, or another enzyme having proteolytic activity may be used in one aspect of the present disclosure. According to one method of the present disclosure, an agent is attached via a conjugating linker susceptible to cleavage by complement. The antibody is selected from a class that can activate complement. The antibody-agent conjugate, thus, activates the complement cascade and releases the agent at the target site. According to another method of the present disclosure, an agent is attached via a conjugating linker susceptible to cleavage by enzymes having a proteolytic activity such as a u-plasminogen activator, a tissue plasminogen activator, plasmin, or trypsin. These cleavable conjugating linkers are useful in conjugated activatable molecules that include an extracellular toxin, e.g., by way of non-limiting example, any of the extracellular toxins shown in Table 1.

[0233] Non-limiting examples of cleavable linker sequences include any cleavable sequence disclosed herein or incorporated herein by reference as well as the exemplary sequences provided in Table 2. Table 2: Exemplary Conjugating Linker Sequences for Conjugation Types of Cleavable Sequences Amino Acid Sequence Plasmin cleavable sequences Pro-urokinase PRFKIIGG (SEQ ID NO: 147)CYTX-108-WO :: 4862-156.WO1 PRFRIIGG (SEQ ID NO: 148) TGFβ SSRHRRALD (SEQ ID NO: 149) Plasminogen RKSSIIIRMRDVVL (SEQ ID NO: 150) Staphylokinase SSSFDKGKYKKGDDA (SEQ ID NO: 151) SSSFDKGKYKRGDDA (SEQ ID NO: 152) Factor Xa cleavable sequences IEGR (SEQ ID NO: 153) IDGR (SEQ ID NO: 154) GGSIDGR (SEQ ID NO: 155) MMP cleavable sequences Gelatinase A PLGLWA (SEQ ID NO: 156) Collagenase cleavable sequences Calf skin collagen (α1(I) chain) GPQGIAGQ (SEQ ID NO: 157) Calf skin collagen (α2(I) chain) GPQGLLGA (SEQ ID NO: 158) Bovine cartilage collagen (α1(II) chain) GIAGQ (SEQ ID NO: 159) Human liver collagen (α1(III) chain) GPLGIAGI (SEQ ID NO: 160) Human α2M GPEGLRVG (SEQ ID NO: 161) Human PZP YGAGLGVV (SEQ ID NO:162) AGLGVVER (SEQ ID NO: 163) AGLGISST (SEQ ID NO: 164) Rat α1M EPQALAMS (SEQ ID NO: 165) QALAMSAI (SEQ ID NO: 166) Rat α2M AAYHLVSQ (SEQ ID NO: 167) MDAFLESS (SEQ ID NO: 168) Rat α1I3(2J) ESLPVVAV (SEQ ID NO: 169) Rat α1I3(27J) SAPAVESE (SEQ ID NO: 170) Human fibroblast collagenase DVAQFVLT (SEQ ID NO: 171) (autolytic cleavages) VAQFVLTE (SEQ ID NO: 172) AQFVLTEG (SEQ ID NO: 173) PVQPIGPQ (SEQ ID NO:174)

[0234] In addition, the agents may be attached via disulfide bonds (for example, the disulfide bonds on a cysteine molecule) to the activatable molecule. Since many tumors naturally release high levels of glutathione (a reducing agent) this can reduce the disulfide bonds with subsequent release of the agent at the site of delivery. In some aspects, the reducing agent that would modify a CM would also modify the conjugating linker of the conjugated activatable molecule.

[0235] In some aspects, the conjugating linker is constructed in such a way as to optimize the spacing between the agent and the activatable molecule. This may be accomplished by use of a conjugating linker of the general structure: W – (CH2)n – Q wherein W is either -NH-CH2- or -CH2-;CYTX-108-WO :: 4862-156.WO1 Q is an amino acid, a polypeptide having between 2 to 20 amino acids; and n is an integer from 0 to 20.

[0236] In some aspects, the conjugating linker may comprise a spacer element and a cleavable element. The spacer element serves to position the cleavable element away from the core of the activatable molecule such that the cleavable element is more accessible to the enzyme responsible for cleavage. Certain of the branched linkers described above may serve as spacer elements.

[0237] Throughout this discussion, it should be understood that attachment of the conjugating linker to the agent (or of spacer element to cleavable element, or cleavable element to agent) need not be by a particular mode of attachment or reaction. Any reaction providing a product of suitable stability and biological compatibility is acceptable.

[0238] In some aspects, when release of an agent is desired, an activatable molecule that is an antibody of a class that can activate complement is used. The resulting conjugate retains both the ability to bind antigen and activate the complement cascade. Thus, according to this aspect of the present disclosure, an agent is joined to one end of the cleavable conjugating linker or cleavable element and the other end of the conjugating linker group is attached to a specific site on the activatable molecule. For example, if the agent has a hydroxyl group or an amino group, it is attached to the carboxyl terminus of a peptide, amino acid or other suitably chosen conjugating linker via an ester or amide bond, respectively. For example, such agents are attached to the linker peptide via a carbodiimide reaction. If the agent contains functional groups that would interfere with attachment to the conjugating linker, these interfering functional groups can be blocked before attachment and deblocked once the product conjugate or intermediate is made. The opposite or amino terminus of the linker is then used either directly or after further modification for binding to an activatable molecule that is capable of activating complement.

[0239] Conjugating linkers (or spacer elements of conjugating linkers) may be of any desired length, one end of which can be covalently attached to specific sites on the activatable molecule. The other end of the conjugating linker or spacer element may be attached to an amino acid or peptide conjugating linker.

[0240] Thus when these conjugates bind antigen in the presence of complement the amide or ester bond that attaches the agent to the linker will be cleaved, resulting in release of the agent in its active form. These conjugates, when administered to a subject, will accomplish delivery and release of the agent at the target site, and are particularly effectiveCYTX-108-WO :: 4862-156.WO1 for the in in vivo delivery of pharmaceutical agents, antibiotics, antimetabolites, antiproliferative agents and the like.

[0241] In some aspects, release of the agent without complement activation is desired since activation of the complement cascade will ultimately lyse the target cell. Hence, this approach is useful when delivery and release of the agent should be accomplished without killing the target cell. Such is the goal when delivery of cell mediators such as hormones, enzymes, corticosteroids, neurotransmitters, genes or enzymes to target cells is desired. These conjugates may be prepared by attaching the agent to an activatable molecule that is not capable of activating complement via a linker that is mildly susceptible to cleavage by serum proteases. When this conjugate is administered to an individual, antigen-antibody complexes will form quickly whereas cleavage of the agent will occur slowly, thus resulting in release of the compound at the target site.

[0242] In some aspects, the activatable molecule is conjugated to one or more therapeutic agents using certain biochemical cross-linkers. Cross-linking reagents form molecular bridges that tie together functional groups of two different molecules. To link two different proteins in a step-wise manner, hetero-bifunctional cross-linkers can be used that eliminate unwanted homopolymer formation.

[0243] Peptidyl conjugating linkers cleavable by lysosomal proteases are also useful, for example, Val-Cit, Val-Ala or other dipeptides. In addition, acid-labile conjugating linkers cleavable in the low-pH environment of the lysosome may be used, for example: bis-sialyl ether. Other suitable conjugating linkers include cathepsin-labile substrates, particularly those that show optimal function at an acidic pH.

[0244] Exemplary hetero-bifunctional cross-linkers are referenced in Table 3. Table 3: Exemplary Hetero-Bifunctional Cross-Linkers HETERO-BIFUNCTIONAL CROSS-LINKERS Spacer Arm Length after Advantages and cross-linking Linker Reactive Toward Applications (Angstroms) SMPT Primary amines Greater stability 11.2 Å Sulfhydryls SPDP Primary amines Thiolation 6.8 Å Sulfhydryls Cleavable cross-linking LC-SPDP Primary amines Extended spacer arm 15.6 ÅCYTX-108-WO :: 4862-156.WO1 Sulfhydryls Sulfo-LC-SPDP Primary amines Extender spacer arm 15.6 Å Sulfhydryls Water-soluble SMCC Primary amines Stable maleimide reactive 11.6 Å group Sulfhydryls Enzyme-antibody conjugation Hapten-carrier protein conjugation Sulfo-SMCC Primary amines Stable maleimide reactive 11.6 Å group Sulfhydryls Water-soluble Enzyme-antibody conjugation MBS Primary amines Enzyme-antibody 9.9 Å conjugation Sulfhydryls Hapten-carrier protein conjugation Sulfo-MBS Primary amines Water-soluble 9.9 Å Sulfhydryls SIAB Primary amines Enzyme-antibody 10.6 Å conjugation Sulfhydryls Sulfo-SIAB Primary amines Water-soluble 10.6 Å Sulfhydryls SMPB Primary amines Extended spacer arm 14.5 Å Sulfhydryls Enzyme-antibody conjugation Sulfo-SMPB Primary amines Extended spacer arm 14.5 Å Sulfhydryls Water-soluble EDE / Sulfo- Primary amines Hapten-Carrier conjugation 0 NHS Carboxyl groups ABH Carbohydrates Reacts with sugar groups 11.9 Å Nonselective

[0245] In some aspects, the agent is designed so that the agent is delivered to the target but not released. This may be accomplished by attaching an agent to an activatable molecule either directly or via a non-cleavable conjugating linker.

[0246] These non-cleavable conjugating linkers may include amino acids, peptides, D- amino acids or other organic compounds that may be modified to include functional groups that can subsequently be utilized in attachment to activatable molecules by the methods described herein.

[0247] In some aspects, a compound is attached to activatable molecules that do not activate complement. When using activatable molecules that are incapable of complementCYTX-108-WO :: 4862-156.WO1 activation, this attachment may be accomplished using conjugating linkers that are susceptible to cleavage by activated complement or using linkers that are not susceptible to cleavage by activated complement.

[0248] The cleavable polypeptides disclosed herein can also be formulated as immunoliposomes. Liposomes containing the antibody are prepared by methods known in the art, such as 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 U.S. Pat. Nos.4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556. Particularly useful liposomes can be generated by the reverse-phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG- derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. A component of an activatable molecule can be conjugated to the liposomes as described in Martin et al., J. Biol. Chem., 257: 286-288 (1982) via a disulfide-interchange reaction.

[0249] The agents described above may contain components that have different attributes, thus leading to conjugates with differing physio-chemical 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. Further, the SMPT contains a sterically-hindered disulfide bond, and can form conjugates with increased stability. Disulfide linkages, are in general, less stable than other linkages because the disulfide linkage is cleaved in vitro, resulting in less conjugate available. Sulfo-NHS, in particular, can enhance the stability of carbodiimide couplings. Carbodiimide couplings (such as EDC) when used in conjunction with sulfo-NHS, forms esters that are more resistant to hydrolysis than the carbodiimide coupling reaction alone.

[0250] Those of ordinary skill in the art will recognize that a large variety of possible agents can be conjugated to the activatable molecule of the disclosure. (See, for example, “Conjugate Vaccines”, Contributions to Microbiology and Immunology, J. M. Cruse and R. E. Lewis, Jr (eds), Carger Press, New York, (1989), the entire contents of which are incorporated herein by reference). In general, an effective conjugation of an agent (e.g., cytotoxic agent) to an activatable molecule can be accomplished by any chemical reaction that will bind the agent to the activatable molecule while also allowing the agent and the activatable molecule to retain functionality.CYTX-108-WO :: 4862-156.WO1 OPTIONAL COMPONENTS

[0251] In some aspects, a spacer (or “header”) is employed in an activatable molecule of the present disclosure. As used herein, the term “spacer” refers to an amino acid residue or amino acid sequence incorporated at a free terminus of the activatable molecule. In some aspects, a spacer comprises one or more glutamine (Q) residues. In some aspects, residues in the spacer minimize aminopeptidase and / or exopeptidase action to prevent cleavage of N- terminal amino acids. Illustrative and non-limiting spacer amino acid sequences may comprise or consist of any of the following exemplary amino acid sequences: QGQSGS (SEQ ID NO: 87); GQSGS (SEQ ID NO: 88); QSGS (SEQ ID NO: 89); SGS; GS; S; QGQSGQG (SEQ ID NO: 90); GQSGQG (SEQ ID NO: 91); QSGQG (SEQ ID NO: 92); SGQG (SEQ ID NO: 93); GQG; QG; G; QGQSGQ (SEQ ID NO: 94); GQSGQ (SEQ ID NO: 95); QSGQ (SEQ ID NO: 96); QGQSG (SEQ ID NO: 97); QGQS (SEQ ID NO: 98); SGQ; GQ; and Q. In some aspects, spacer sequences are omitted. NUCLEIC ACIDS AND VECTORS

[0252] In some aspects, the present disclosure further provides nucleic acids comprising sequences that encode the cleavable polypeptides and polypeptide complexes (e.g., activatable molecules) herein, or components or fragment thereof. In some aspects, the present disclosures provides a nucleic acid composition comprising one or more nucleic acid molecules encoding: 1 the cleavable polypeptide of any one of the aspects described herein; 2 a masking moiety (MM); and 3 a target binding moiety (TBM); and 4 optionally a half-life extending moiety (EM); wherein the MM is coupled directly or indirectly to the N-terminus of the CM and the TBM is coupled directly or indirectly to the C-terminus of the CM, or wherein the TBM is coupled directly or indirectly to the N-terminus of the CM and the MM is coupled directly or indirectly to the C-terminus of the CM. In some aspects, the present disclosure provides a nucleic acid molecule or group of nucleic acid molecules encoding the cleavable polypeptide or the activatable molecule of any one of the aspects disclosed herein. In some aspects, the present disclosure provides a vector or group of vectors comprising the nucleic acid molecule or group of nucleic acid molecules or the nucleic acid composition of one of the aspects disclosed herein.CYTX-108-WO :: 4862-156.WO1

[0253] In some aspects, the nucleic acid comprises DNA, RNA, or a combination of DNA and RNA bases in single stranded or double stranded form. In some aspects, the nucleic acid comprises or consists of a deoxyribonucleic acid (DNA). In some aspects, the nucleic acid comprises or consists of a ribonucleic acid (RNA). The polynucleotide sequence of the nucleic acid may be codon optimized for optimal expression from the desired host cell species. In some aspects, the nucleic acid composition encodes a protein comprising a heavy chain amino acid sequence according to SEQ ID NO: 41 and a light chain amino acid sequence selected from the group consisting of SEQ ID NOs: 45-51. In some aspects, the CM is encoded by a sequence selected from SEQ ID NOs: 67-73. In some aspects, the one or more nucleic acid sequences encoding the protein comprise a sequence according to SEQ ID NO: 53 and a sequence selected from the group consisting of SEQ ID NOs: 57-63.

[0254] In some aspects, the present disclosure further provides a nucleic acid molecule or group of nucleic acid molecules encoding the cleavable or the activatable molecule of any of the aspects described herein. In some aspects, the present disclosure further provides a vector or group of vectors comprising the nucleic acid molecule or group of the nucleic acid molecules or the nucleic acid composition of any of the aspects described herein.

[0255] The nucleic acids may comprise coding sequences for the CMs. The nucleic acids may further comprise coding sequences for other components in an activatable molecule, e.g., the TBMs, the MMs, the EM and / or the linker(s). In cases where the activatable molecule comprises multiple polypeptides, the nucleic acids may comprise coding sequences for the multiple polypeptides. In some examples, the coding sequence for one of the polypeptides is comprised in a nucleic acid molecule, and the coding sequence for another one of the polypeptides is comprised in another nucleic acid molecule. In some examples, the coding sequences for two or more of the multiple polypeptides are comprised in the same nucleic acid molecule.

[0256] Modifications may be introduced into a nucleotide sequence by standard techniques known in the art, such as site-directed mutagenesis and polymerase chain reaction (PCR)-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having 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), non-polar amino acids (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan),CYTX-108-WO :: 4862-156.WO1 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), hydrophobic amino acids (e.g., alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine). Other families of amino acids include: aliphatic-hydroxy amino acids (e.g., serine and threonine), amide family (e.g., asparagine and glutamine), aliphatic family (e.g., alanine, valine, leucine and isoleucine), and aromatic family (e.g., phenylalanine, tryptophan, and tyrosine).

[0257] The present disclosure further provides vectors and sets of vectors comprising any of the nucleic acids described herein. One skilled in the art will be capable of selecting suitable vectors or sets of vectors (e.g., expression vectors) for making any of the activatable molecules described herein, and using the vectors or sets of vectors to express any of the activatable molecules described herein. For example, in selecting a vector or a set of vectors, the type of cell may be selected such that the vector(s) may need to be able to integrate into a chromosome of the cell and / or replicate in it. Example vectors that can be used to produce an activatable molecule are also described herein. As used herein, the term “vector” refers to a polynucleotide capable of inducing the 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 able to deliver nucleic acids and fragments thereof into a host cell, and includes regulatory sequences (e.g., promoter, enhancer, poly(A) signal). Exogenous polynucleotides may be inserted into the expression vector in order to be expressed. The term “vector” also includes artificial chromosomes, plasmids, retroviruses, and baculovirus vectors.

[0258] Methods for constructing suitable vectors that comprise 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.

[0259] Examples of vectors include plasmids, transposons, cosmids, and viral vectors (e.g., any adenoviral vectors (e.g., pSV or pCMV vectors), adeno-associated virus (AAV) vectors, lentivirus vectors, and retroviral vectors), and any Gateway® vectors. A vector may, for example, include sufficient cis-acting elements for expression; other elements for expression may be supplied by the host mammalian cell or in an in vitro expression system.CYTX-108-WO :: 4862-156.WO1 Skilled practitioners will be capable of selecting suitable vectors and mammalian cells for making any activatable molecule described herein.

[0260] In some aspects, the cleavable polypeptides may be made biosynthetically using recombinant DNA technology and expression in eukaryotic or prokaryotic species. CELLS

[0261] In some aspects, the present disclosure provides recombinant host cells comprising any of the vectors or nucleic acids described herein. As used herein, the term “recombinant host cell” refers to a cell into which an exogenous polynucleotide has been introduced. For example, in some aspects the exogenous polynucleotide is an expression vector. For example, a prokaryotic host cell is a genetically modified prokaryotic host cell (e.g., a bacterium), by virtue of introduction into a suitable prokaryotic host cell of a heterologous nucleic acid, e.g., an exogenous nucleic acid that is foreign to (not found in nature in) the prokaryotic host cell, or a recombinant nucleic acid that is not normally found in the prokaryotic host cell; and a subject eukaryotic host cell is a genetically modified eukaryotic host cell, by virtue of introduction into a suitable eukaryotic host cell of a heterologous nucleic acid, e.g., an exogenous nucleic acid that is foreign to the eukaryotic host cell, or a recombinant nucleic acid that is not naturally found in the eukaryotic host cell. In some aspects, the recombinant host cell comprises the cleavable polypeptide, the activatable molecule, the nucleic acid composition, the nucleic acid, or the vector or group of vectors of any one of the foregoing aspects or aspects.

[0262] The cells may be used to produce the cleavable polypeptides (e.g., activatable molecules) described herein. In some aspects, the cell may be an animal cell, a mammalian cell (e.g., a human cell), a rodent cell (e.g., a mouse cell, a rat cell, a hamster cell, or a guinea pig cell), a non-human primate cell, an insect cell, a bacterial cell, a fungal cell, or a plant cell. In some aspects, the cell is a eukaryotic cell. As used herein, the term “eukaryotic cell” refers to a cell having a distinct, membrane-bound nucleus. Such cells may include, for example, mammalian (e.g., rodent, non-human primate, or human), insect, fungal, or plant cells. In some aspects, the eukaryotic cell is a yeast cell, such as Saccharomyces cerevisiae. In some aspects, the eukaryotic cell is a higher eukaryote, such as mammalian, avian, plant, or insect cells. Non-limiting examples of mammalian cells include Chinese hamster ovary (CHO) cells and human embryonic kidney cells (e.g., HEK293 cells). In some aspects, the cell is a prokaryotic cell, e.g., an E. coli cell.CYTX-108-WO :: 4862-156.WO1

[0263] Suitable methods of introducing any of the nucleic acids and / or vectors (e.g., any of the vectors or any of the sets of vectors described herein) described herein into a cell include any method that is known in the art. Non-limiting examples of methods that can be used to introducing a nucleic acid into a cell include: lipofection, calcium phosphate transfection, cationic polymer transfection, viral transduction (e.g., adenoviral transduction, lentiviral transduction), nanoparticle transfection, and electroporation.

[0264] In some aspects, the introducing step includes introducing into a cell a vector (e.g., any of the vectors or sets of vectors described herein) including a nucleic acid encoding the monomers that make up any activatable molecule described herein. COMPOSITIONS AND KITS

[0265] The present disclosure provides a composition comprising the cleavable polypeptide or the activatable molecule of any of the aspects described herein and a pharmaceutically acceptable carrier. In some aspects, the composition further comprising at least one additional agent.

[0266] The present disclosure also provides compositions comprising the nucleic acid molecule or group of nucleic acid molecules or the vector or group of vectors of any of the aspects described herein. Also provided herein is a nucleic acid composition comprising one or more nucleic acid molecules encoding: a) the cleavable polypeptide of any one of the above aspects; b) a masking moiety (MM); and c) a target binding moiety (TBM); and d) optionally a half-life extending moiety (EM); wherein the MM is coupled directly or indirectly to the N-terminus of the CM and the TBM is coupled directly or indirectly to the C-terminus of the CM, or wherein the TBM is coupled directly or indirectly to the N-terminus of the CM and the MM is coupled directly or indirectly to the C-terminus of the CM. Nucleic acid compositions and compositions comprising the nucleic acid molecule or group of nucleic acid molecules or the vector or group of vectors of any of the aspects described herein are useful for producing the cleavable polypeptides and the activatable molecules by methods that include, for example, transfection of a mammalian cell, transformation of a bacterial cell, as well as, in vitro transcription and / or translation processes, and the like. In some aspects, nucleic acid compositions and compositions comprising the nucleic acid molecule or group of nucleic acid molecules or theCYTX-108-WO :: 4862-156.WO1 vector or group of vectors of any of the aspects described herein comprise a carrier (e.g., water, and the like) and, optionally, an additional agent (e.g., a buffer, and the like).

[0267] Also provided herein are pharmaceutical compositions that comprise any of the cleavable polypeptides or the activatable molecules of any of the aspects described herein. In some aspects, the pharmaceutical compositions comprise at least one additional therapeutic agent.

[0268] The present disclosure also provides compositions and kits comprising the cleavable polypeptides (e.g., activatable molecules or conjugated polypeptides) described herein. The compositions and kits may further comprise one or more excipients, carriers, reagents, instructions needed for the use of the activatable molecules.

[0269] In some aspects, the compositions comprise the cleavable polypeptides, derivatives, fragments, analogs and homologs thereof. The compositions may comprise the cleavable polypeptide (for example, the activatable molecule) and a carrier. In some aspects, the carrier is a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” includes 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 solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0270] A composition may be formulated to be compatible with its intended route of administration or other applicable use. 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 may include one or more of the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid orCYTX-108-WO :: 4862-156.WO1 sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH may be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. In some, any of the activatable molecules described herein are prepared with carriers that protect against rapid elimination from the body, e.g., sustained and controlled release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic-co-glycolic acid, and polylactic acid. Methods for preparation of such compositions and formulations are apparent to those skilled in the art. For example, the activatable molecules may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules) or in macroemulsions.

[0271] Sustained-release preparations may be prepared. Suitable examples of sustained- release preparations include semipermeable matrices of solid hydrophobic polymers containing the cleavable polypeptide (e.g., the activatable molecule), which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides, copolymers of L-glutamic acid and y 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 time periods.

[0272] In some aspects, 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 dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor®EL (CAS No. 61791-12-6) (BASF, Parsippany, N.J.), which is a mixture of polyoxyethylated triglycerides,CYTX-108-WO :: 4862-156.WO1 by reacting castor oil with ethylene oxide in a molar ratio of 1 : 35, that acts as a nonionic surfactant, or phosphate buffered saline (PBS). The composition may be sterile and should be fluid and of a viscosity that facilitates easy syringeability. It may 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 (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. For dispersed particulate compositions, the proper fluidity can be maintained, for example, by the use of a coating on the particles such as lecithin, and by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In some aspects, the compositions may further comprise one or more antibacterial and / or antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In some aspects, isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and the like, as well as salts, such as, for example, sodium chloride and the like may be included in the composition. Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

[0273] In some aspects, the composition may comprise a sterile injectable solution. Sterile injectable solutions may be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions may be prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0274] In some aspects, the composition may comprise an oral composition. Oral compositions may include an inert diluent or an edible carrier. They may be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions may also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents,CYTX-108-WO :: 4862-156.WO1 and / or adjuvant materials may be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primojel®(sodium starch glycolate), or corn starch; a lubricant such as magnesium stearate; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0275] In some aspects, the composition are formulated for administration by inhalation. For example, the compounds may be delivered in the form of an aerosol spray from pressured container or dispenser that contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0276] In some aspects, the composition are formulated for systemic administration. For example, systemic administration may be by intravenous, as well by transmucosal or transdermal means. 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, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration may be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds may be formulated into ointments, salves, gels, or creams as generally known in the art.

[0277] In some aspects, the composition is 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.

[0278] In one aspect, the composition is prepared with carriers that protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers may be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic-co-glycolic acid and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.

[0279] It may be advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated toCYTX-108-WO :: 4862-156.WO1 produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the disclosure may be dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

[0280] In some aspects, the compositions are included in a container, vial, syringe, injector pen, pack, or dispenser, optionally together with instructions for administration.

[0281] Also provided herein are kits that include any of the cleavable polypeptides (e.g., activatable molecules or conjugated polypeptides) described herein, any of the compositions that include any of the polypeptides described herein, or any of the compositions that include any of the polypeptides described herein. Also provided are kits that include one or more second therapeutic agent(s) in addition to a polypeptide described herein. The second therapeutic agent(s) may be provided in a dosage administration form that is separate from the polypeptides herein. Alternatively, the second therapeutic agent(s) may be formulated together with the polypeptides herein.

[0282] Any of the kits described herein can include instructions for using any of the compositions (e.g., pharmaceutical compositions) and / or any of the cleavable polypeptides (e.g., activatable molecules or conjugated polypeptides) described herein. In some aspects, the kits can include instructions for performing any of the methods described herein. In some aspects, the kits can include at least one dose of any of the compositions (e.g., pharmaceutical compositions) described herein. In some aspects, the kits can provide a syringe for administering any of the compositions described herein.

[0283] Also provided herein are cleavable polypeptides (e.g., activatable molecules or conjugated polypeptides) produced by any of the methods described herein. Also provided are compositions that comprise any of the polypeptides produced by any of the methods described herein. Also provided herein are kits that include at least one dose of any of the compositions described herein. METHODS OF PRODUCING CLEAVABLE POLYPEPTIDES

[0284] Provided herein are methods of producing the cleavable polypeptides (e.g., activatable molecules or conjugated polypeptides) described herein that include: (a) culturing any of the recombinant host cells described herein in a liquid culture medium under conditions sufficient to produce the cleavable polypeptides; and (b) recovering the cleavable polypeptides from the host cell and / or the liquid culture medium.CYTX-108-WO :: 4862-156.WO1

[0285] Methods of culturing cells are well known in the art. In some aspects, cells may be maintained in vitro under conditions that favor cell proliferation, cell differentiation and cell growth. For example, the recombinant cells may be cultured by contacting a cell (e.g., any of the cells described herein) with a cell culture medium that includes the necessary growth factors and supplements sufficient to support cell viability and growth.

[0286] In some aspects, the method may further include isolating the recovered cleavable polypeptides (e.g., activatable molecules or conjugated polypeptides). The isolation of the cleavable polypeptides may be performed using any separation or purification technique for separating protein species, e.g., affinity tag-based protein purification (e.g., polyhistidine (His) tag, glutathione-S-transferase tag, and the like), 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, and the like.

[0287] Compositions and methods described herein may involve use of non-reducing or partially-reducing conditions that allow disulfide bonds to form between the MM and the TBM of the activatable molecules.

[0288] In some aspects, the method further includes formulating the cleavable polypeptides into a composition. Various formulations are known in the art and are described herein. Any cleavable polypeptides described herein can be formulated for any route of administration (e.g., intravenous, intratumoral, subcutaneous, intradermal, oral (e.g., inhalation), transdermal (e.g., topical), transmucosal, or intramuscular). METHODS OF USING CLEAVABLE POLYPEPTIDES

[0289] In some aspects, the present disclosure further provides methods of using the cleavable polypeptides described herein. In some aspects the present disclosure provides a method of treating, alleviating a symptom of, or delaying the progression of a disease or disorder in a subject, comprising administering a therapeutically effective amount of the cleavable polypeptide or the activatable molecule according to any of the foregoing aspects or aspects.

[0290] In some aspects, the present disclosure provides the cleavable polypeptide or the activatable molecule according to any of the foregoing aspects or aspects for use as a medicament or for use in therapy, optionally for treating a cancer, an infection, an inflammatory disorder, a cardiovascular disorder, a neurodegenerative disorder, or anCYTX-108-WO :: 4862-156.WO1 autoimmune disorder, optionally with an additional agent which is optionally a therapeutic agent.

[0291] In some aspects, the present disclosure provides methods of the treating a disease (e.g., a cancer (e.g., any of the cancers described herein)) in a subject including administering a therapeutically effective amount of any of the polypeptides (e.g., activatable molecules or conjugated polypeptides) described herein to the subject. In some aspects, the disclosure provides methods of preventing, delaying the progression of, treating, alleviating a symptom of, or otherwise ameliorating disease in a subject by administering a therapeutically effective amount of an polypeptides (e.g., activatable molecules or conjugated polypeptides) described herein to a subject in need thereof. In some aspects, the disorder being treated may be a cancer or autoimmune disease or to ameliorate at least one symptom of a cancer or autoimmune disease. As used herein, the term “subject” refers to any mammal. In some aspects, 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, a rat, a hamster or a 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 aspects, the subject is a human. The terms subject and patient are used interchangeably herein. In some aspects, the subject has been previously identified or diagnosed as having the disease (e.g., cancer (e.g., any of the cancers described herein)).

[0292] A therapeutically effective amount of a cleavable polypeptide (e.g., activatable molecule or conjugated polypeptide) of the disclosure relates generally to the amount needed to achieve a therapeutic objective. As noted above, this may be a binding interaction between the TBM and its target that, in certain cases, interferes with the functioning of the targets. The amount required to be administered will furthermore depend on the binding affinity of the polypeptides for its specific target, and will also depend on the rate at which an administered polypeptide is depleted from the free volume other subject to which it is administered. Common ranges for therapeutically effective dosing of a polypeptides of the disclosure may be, by way of non-limiting example, from 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 higher. The structure of the polypeptides of the present disclosure makes it possible to reduce the dosage of the polypeptide that is administered to a subject compared to conventional activatable molecules and compared to conventional antibodies. For example, the administered dose on a unit dosage basis or total dosage over a dosage regimen period may be reduced by 10, 20, 30,CYTX-108-WO :: 4862-156.WO1 40, or 50% compared to the corresponding dose of a corresponding conventional therapeutic molecules.

[0293] Common dosing frequencies may range, for example, from once or twice daily, weekly, biweekly, or monthly.

[0294] Efficaciousness of treatment is determined in association with any known method for diagnosing or treating the particular disorder. Methods for the screening of polypeptides that possess the desired specificity include, but are not limited to, enzyme linked immunosorbent assay (ELISA) and other immunologically mediated techniques known within the art.

[0295] The cleavable polypeptides used in any of the aspects of these methods and uses may be administered at any stage of the disease. For example, such a polypeptide may be administered to a patient suffering cancer of any stage, from early to metastatic. In some aspects, the cleavable polypeptides and formulations thereof may be administered to a subject suffering from or susceptible to a disease or disorder associated with aberrant target expression and / or activity.

[0296] A subject suffering from or susceptible to a disease or disorder associated with aberrant target expression and / or activity may be identified using any of a variety of methods known in the art. For example, subjects suffering from cancer or other neoplastic condition may be identified using any of a variety of clinical and / or laboratory tests such as, physical examination and blood, urine and / or stool analysis to evaluate health status. For example, subjects suffering from inflammation and / or an inflammatory disorder may be identified using any of a variety of clinical and / or laboratory tests such as physical examination and / or bodily fluid analysis, e.g., blood, urine and / or stool analysis, to evaluate health status.

[0297] In some aspects, administration of a polypeptide to a patient suffering from a disease or disorder associated with aberrant target expression and / or activity may be considered successful if any of a variety of laboratory or clinical objectives is achieved. For example, administration of a polypeptide to a patient suffering from a disease or disorder associated with aberrant target expression and / or activity may be considered successful if one or more of the symptoms associated with the disease or disorder is alleviated, reduced, inhibited or does 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 may be considered successful if the disease or disorder enters remission or does not progress to a further, i.e., worse, state.CYTX-108-WO :: 4862-156.WO1

[0298] As used herein, the term “treat” includes reducing the severity, frequency or the number of one or more (e.g., 1, 2, 3, 4, or 5) symptoms or signs of a disease (e.g., a cancer (e.g., any of the cancers described herein)) in the subject (e.g., any of the subjects described herein). In some aspects where the disease is cancer, treating results in reducing cancer growth, inhibiting cancer progression, inhibiting cancer metastasis, or reducing the risk of cancer recurrence in a subject having cancer.

[0299] In some aspects, the CM comprises a substrate for a protease that is active, e.g., upregulated or otherwise unregulated, in a disease condition or diseased tissue. Exemplary disease conditions include, for example, a cancer (e.g., where the diseased tissue is a tumor tissue) and an inflammatory or autoimmune condition (e.g., where the diseased tissue is inflamed tissue). In some aspects, the CM comprises a substrate for an extracellular protease. In some aspects, the CM comprises a substrate for an intracellular protease. In some aspects, the CM is a substrate for an intracellular protease and an extracellular protease. In some aspects, the disease may be a cancer. In some aspects, the subject may have been identified or diagnosed as having a cancer. Examples of cancer include: solid tumor, hematological tumor, sarcoma, a leukemia (e.g., hairy cell leukemia, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), stomach cancer, urothelial carcinoma, 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 carcinoma (NSCLC), squamous cell head and neck carcinoma, endometrial cancer, bladder cancer, cervical cancer, and liver cancer. Metastases of the aforementioned cancers may also be treated or prevented in accordance with the methods described herein.

[0300] In some aspects, the disease may be an autoimmune disease or condition. In some aspects, the subject may have been identified or diagnosed as having an autoimmune disease or condition or is at heightened 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 transplantation), autoimmune diseases, infectious disease, chronic inflammation, or transplant rejection. In some aspects, the disease is a cardiovascular disorder. In some aspects, the disease is a neurodegenerative disorder.CYTX-108-WO :: 4862-156.WO1

[0301] In some aspects, the methods herein may result in a reduction in the number, severity, or frequency of one or more symptoms of the cancer in the subject (e.g., as compared to the number, severity, or frequency of the one or more symptoms of the cancer in the subject prior to treatment).

[0302] The methods may further comprise administering to a subject one or more additional agents. In some aspects, the cleavable polypeptides (e.g., activatable molecules or conjugated polypeptides) may be administered during and / or after treatment in combination with one or more additional agents. In some aspects, the polypeptide may be formulated into 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 is formulated into a separate therapeutic composition, and the polypeptide and the additional agent are administered simultaneously, or the polypeptide and the additional agent are administered at different times during a treatment regimen. For example, the polypeptide may be administered prior to the administration of the additional agent, subsequent to the administration of the additional agent, or in an alternating fashion. The polypeptide and additional agent(s) may be administered in single doses or in multiple doses.

[0303] One of more of the polypeptides herein may be co-formulated with, and / or co- administered with, one or more anti-inflammatory drugs, immunosuppressants, or metabolic or enzymatic inhibitors. In some aspects, one or more polypeptides herein may be combined with one or more polypeptides of other types.

[0304] The present disclosure also provides methods of detecting presence or absence of a cleaving agent and / or the target in a subject or a sample. Such methods may comprise (i) contacting a subject or biological sample with an activatable molecule, wherein the activatable molecule includes a detectable label that is positioned on a portion of the activatable molecule that is released following cleavage of the CM and (ii) measuring a level of activated molecule in the subject or biological sample, wherein a detectable level of activated molecule in the subject or biological sample indicates that the cleaving agent, the target or both the cleaving agent and the target are absent and / or not sufficiently present in the subject or biological sample, such that the target binding and / or protease cleavage of the activatable molecule cannot be detected in the subject or biological sample, and wherein a reduced detectable level of activated molecule in the subject or biological sample indicates that the cleaving agent and the target are present in the subject or biological sample.CYTX-108-WO :: 4862-156.WO1

[0305] Such detection methods may be adapted to also provide for detection of the presence or absence of a target that is capable of binding the TBM of the activatable molecules when cleaved. Thus, the assays can be adapted to assess the presence or absence of a cleaving agent and the presence or absence of a target of interest. The presence or absence of the cleaving agent can be detected by the presence of and / or an increase in detectable label of the activatable molecules as described above, and the presence or absence of the target can be detected by detection of a target- TBM complex e.g., by use of a detectably labeled anti- target antibody.

[0306] In some aspects, activatable molecules are also useful in in situ imaging for the validation of activatable molecule activation, e.g., by protease cleavage, and binding to a particular target. In situ imaging is a technique that enables localization of proteolytic activity and target in biological samples such as cell cultures or tissue sections. Using this technique, it is possible to confirm both binding to a given target and proteolytic activity based on the presence of a detectable label (e.g., a fluorescent label).

[0307] These techniques are useful with any frozen cells or tissue derived from a disease site (e.g. tumor tissue) or healthy tissues. These techniques are also useful with fresh cell or tissue samples.

[0308] In these techniques, an activatable molecule may be labeled with a detectable label. The detectable label may be a fluorescent dye, (e.g. a fluorophore, Fluorescein Isothiocyanate (FITC), Rhodamine Isothiocyanate (TRITC), an Alexa Fluor® label), 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 (e.g. horseradish peroxidase or alkaline phosphatase).

[0309] Detection of the label in a sample that has been incubated with the labeled, activatable molecule indicates that the sample contains the target and contains a protease that is specific for the CM of the activatable molecule. In some aspects, the presence of the protease can be confirmed using broad spectrum protease inhibitors such as those described herein, and / or by using an agent that is specific for the protease, for example, an antibody such as A11, which is specific for the protease matriptase and inhibits the proteolytic activity of matriptase; see e.g., International Publication Number WO 2010 / 129609, published 11 November 2010. The same approach of using broad spectrum protease inhibitors such as those described herein, and / or by using a more selective inhibitory agent can be used to identify a protease that is specific for the CM of the activatable molecule. In some aspects,CYTX-108-WO :: 4862-156.WO1 the presence of the target can be confirmed using an agent that is specific for the target, e.g., another antibody, or the detectable label can be competed with unlabeled target. In some aspects, unlabeled activatable molecule may be used, with detection by a labeled secondary antibody or more complex detection system.

[0310] Similar techniques are also useful for in vivo imaging where detection of the fluorescent signal in a subject, e.g., a mammal, including a human, indicates that the disease site contains the target and contains a protease that is specific for the CM of the activatable molecule.

[0311] These techniques are also useful in kits and / or as reagents for the detection, identification or characterization of protease activity in a variety of cells, tissues, and organisms based on the protease-specific CM in the activatable molecule.

[0312] A reduced level of detectable label may be, for example, a reduction of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or a reduction of substantially 100%. In some aspects, the detectable label may be conjugated to a component of the polypeptide, e.g., the TBM. In some aspects, measuring the level of polypeptide in the subject or sample may be accomplished using a secondary reagent that specifically binds the activated molecule, wherein the reagent comprises a detectable label. The secondary reagent may be an antibody comprising a detectable label.

[0313] In some aspects, the cleavable polypeptides may also be useful in the detection of the target in patient samples and accordingly are useful as diagnostics. For example, the polypeptides may be used in in vitro assays, e.g., ELISA, to detect target levels in a patient sample. For example, a polypeptide may be immobilized on a solid support (e.g., the well(s) of a microtiter plate). The immobilized polypeptide may serve as a capture protein for any target that may be present in a test sample. Prior to contacting the immobilized polypeptide with a patient sample, the solid support may be rinsed and treated with a blocking agent such as milk protein or albumin to prevent nonspecific adsorption of the analyte.

[0314] In some aspects, based on the results obtained using the polypeptides in an in vitro diagnostic assay, the stage of a disease in a subject may be determined based on expression levels of the target protein (e.g., antigen). For a given disease, samples of blood may be taken from subjects diagnosed as being at various stages in the progression of the disease, and / or at various points in the therapeutic treatment of the disease. Using a population of samples that provides statistically significant results for each stage of progression or therapy, a range ofCYTX-108-WO :: 4862-156.WO1 concentrations of the target protein (e.g., antigen) that may be considered characteristic of each stage is designated.

[0315] Polypeptides herein may also be used in diagnostic and / or imaging methods. In some aspects, such methods may be in vitro methods. In some aspects, such methods may be in vivo methods. In some aspects, such methods may be in situ methods. In some aspects, such methods may be ex vivo methods. For example, polypeptides having a CM may be used to detect the presence or absence of an enzyme capable of cleaving the CM. Such polypeptides may be used in diagnostics, which can include in vivo detection (e.g., qualitative or quantitative) of enzyme activity (or, in some aspects, an environment of increased reduction potential such as that which can provide for reduction of a disulfide bond) through measured accumulation of activated antibodies (i.e., antibodies resulting from cleavage of a polypeptide) in a given cell or tissue of a given host organism. Such accumulation of activated proteins indicates not only that the tissue expresses enzymatic activity (or an increased reduction potential depending on the nature of the CM) but also that the tissue expresses target to which the activated protein binds. In some examples, the polypeptides may be used for detecting protease activity with an assay that does not rely on target binding, e.g., a quantitative ex vivo zymography (QZ) assay as described in Howng et al., “Novel Ex Vivo Zymography Approach for Assessment of Protease Activity in Tissues with Activatable Antibodies,” Pharmaceutics.2021 Sep 2; 13(9):1390, which is incorporated by reference herein in its entirety.

[0316] For example, the CM may 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 at a biologically confined site (e.g., such as in an abscess, in an organ, and the like), and the like. The TBM may be one that binds a target protein (e.g., antigen). Using methods familiar to one skilled in the art, a detectable label (e.g., a fluorescent label or radioactive label or radiotracer) may be conjugated to a TBM or other region of a polypeptide. Suitable detectable labels may be discussed in the context of the above screening methods and additional specific examples are provided below. Using a TBM specific to a protein or peptide of the disease state, along with a protease whose activity is elevated in the disease tissue of interest, polypeptides may exhibit an increased rate of binding to disease tissue relative to tissues where the CM specific enzyme is not present at a detectable level or is present at a lower level than in disease tissue or is inactive (e.g., in zymogen form or in complex with an inhibitor). Since small proteins and peptides are rapidly cleared from the blood by the renal filtration system, and because theCYTX-108-WO :: 4862-156.WO1 enzyme specific for the CM is not present at a detectable level (or is present at lower levels in non-disease tissues or is present in inactive conformation), accumulation of activated protein in the disease tissue may be enhanced relative to non-disease tissues.

[0317] In some aspects, the cleavable polypeptides may be useful for in vivo imaging where detection of the fluorescent signal in a subject, e.g., a mammal, including a human, indicates that the disease site contains the target and contains a protease that is specific for the CM of the polypeptide. The in vivo imaging may be used to identify or otherwise refine a patient population suitable for treatment with a polypeptide of the disclosure. For example, patients that test positive for both the target and a protease that cleaves the substrate in the CM of the polypeptide being tested (e.g., accumulate activated proteins at the disease site) are identified as suitable candidates for treatment with such a polypeptide comprising such a CM. Likewise, patients that test negative may be identified as suitable candidates for another form of therapy (i.e., not suitable for treatment with the polypeptide being tested). In some aspects, such patients that test negative with respect to a first polypeptide can be tested with other polypeptides comprising different CMs until a suitable polypeptide for treatment is identified (e.g., a polypeptide comprising a CM that is cleaved by the patient at the site of disease).

[0318] In some aspects, in situ imaging may be useful in methods to identify which patients to treat. For example, in in situ imaging, the polypeptides may be used to screen patient samples to identify those patients having the appropriate protease(s) and target(s) at the appropriate location, e.g., at a tumor site. In some aspects, in situ imaging is used to identify or otherwise refine a patient population suitable for treatment with a polypeptide of the disclosure. For example, patients that test positive for both the target and a protease that cleaves the substrate in the CM of the polypeptide being tested (e.g., accumulate activated antibodies at the disease site) are identified as suitable candidates for treatment with such a polypeptide comprising such a CM. Likewise, patients that test negative for either or both of the target and the protease that cleaves the CM used in the polypeptide being tested using these methods are identified as suitable candidates for another form of therapy (i.e., not suitable for treatment with the polypeptide being tested). In some aspects, such patients that test negative with respect to a first polypeptide can be tested with other polypeptides comprising different CMs until a suitable polypeptide for treatment is identified (e.g., a polypeptide comprising a CM that is cleaved by the patient at the site of disease).

[0319] The present application also provides aspects as set forth in the following numbered Statements:CYTX-108-WO :: 4862-156.WO1

[0320] Statement 1. A cleavable polypeptide comprising a cleavable moiety (CM) comprising an amino acid sequence QXAQX1LX2XBA (SEQ ID NO: 74), wherein XA is N, A, W, or F; X1is G or A; X2is R or K; XBis S, M, V, or L, provided that: i) when XA is N, then XB is S, L, or V; ii) when XAis A, then XBis S or M; and iii) when XA is W or F, then XB is M; and wherein the CM is a substrate for a protease.

[0321] Statement 2. The cleavable polypeptide of Statement 1, wherein the cleavable polypeptide comprises 2 to 4 CMs.

[0322] Statement 3. The cleavable polypeptide of Statement 2, wherein each of the 2 to 4 CMs independently comprises an amino acid sequence according to the amino acid sequence QXAQX1LX2XBA (SEQ ID NO: 74).

[0323] Statement 4. The cleavable polypeptide of any one of Statements 1-3, wherein the cleavable polypeptide comprises 3 CMs.

[0324] Statement 5. The cleavable polypeptide of any one of Statements 1-4, wherein the cleavable polypeptide comprises 4 CMs.

[0325] Statement 6. The cleavable polypeptide of any one of Statements 1-5, wherein the CM comprises a sequence selected from: QNQX1LX2SA (SEQ ID NO: 4), QAQX1LX2MA (SEQ ID NO: 9), QAQX1LX2SA (SEQ ID NO: 14), QNQX1LX2VA (SEQ ID NO: 19), QNQX1LX2LA (SEQ ID NO: 24), QWQX1LX2MA (SEQ ID NO: 29), QFQX1LX2MA (SEQ ID NO: 34),wherein X1is G or A and X2is R or K.

[0326] Statement 7. The cleavable polypeptide of any one of Statements 1-6, wherein the CM comprises a sequence selected from: QNQALRSA (SEQ ID NO: 5), QNQGLRSA (SEQ ID NO: 6), QNQALKSA (SEQ ID NO: 7), QNQGLKSA (SEQ ID NO: 8), QAQALRMA (SEQ ID NO: 10), QAQGLRMA (SEQ ID NO: 11), QAQALKMA (SEQ ID NO: 12), QAQGLKMA (SEQ ID NO: 13), QAQALRSA (SEQ ID NO: 15), QAQGLRSA (SEQ ID NO: 16), QAQALKSA (SEQ ID NO: 17), QAQGLKSA (SEQ ID NO: 18), QNQALRVA (SEQ ID NO: 20), QNQGLRVA (SEQ ID NO: 21), QNQALKVA (SEQ ID NO: 22), QNQGLKVA (SEQ ID NO: 23), QNQALRLA (SEQ ID NO: 25), QNQGLRLA (SEQ ID NO: 26), QNQALKLA (SEQ ID NO: 27), QNQGLKLA (SEQ ID NO: 28), QWQALRMA (SEQ ID NO: 30), QWQGLRMA (SEQ ID NO: 31), QWQALKMA (SEQ ID NO: 32), QWQGLKMA (SEQ ID NO: 33), QFQALRMA (SEQ ID NO: 35), QFQGLRMA (SEQ ID NO: 36), QFQALKMA (SEQ ID NO: 37), or QFQGLKMA (SEQ ID NO: 38), optionallyCYTX-108-WO :: 4862-156.WO1 wherein the substrate comprises an amino acid sequence with one-amino acid or two-amino acid mutation(s) of any one of SEQ ID NOs: 5-8, 10-13, 15-18, 20-23, 25-28, 30-33, 35-38.

[0327] Statement 8. The cleavable polypeptide of any one of Statements 1-7, wherein the CM comprises a sequence selected from: QNQALRSA (SEQ ID NO: 5), QNQGLRSA (SEQ ID NO: 6), QNQALKSA (SEQ ID NO: 7), or QNQGLKSA (SEQ ID NO: 8).

[0328] Statement 9. The cleavable polypeptide of any one of Statements 1-7, wherein the CM comprises a sequence selected from: QAQALRMA (SEQ ID NO: 10), QAQGLRMA (SEQ ID NO: 11), QAQALKMA (SEQ ID NO: 12), or QAQGLKMA (SEQ ID NO: 13).

[0329] Statement 10. The cleavable polypeptide of any one of Statements 1-7, wherein the CM comprises a sequence selected from: QAQALRSA (SEQ ID NO: 15), QAQGLRSA (SEQ ID NO: 16), QAQALKSA (SEQ ID NO: 17), or QAQGLKSA (SEQ ID NO: 18).

[0330] Statement 11. The cleavable polypeptide of any one of Statements 1-7, wherein the CM comprises a sequence selected from: QNQALRVA (SEQ ID NO: 20), QNQGLRVA (SEQ ID NO: 21), QNQALKVA (SEQ ID NO: 22), or QNQGLKVA (SEQ ID NO: 23).

[0331] Statement 12. The cleavable polypeptide of any one of Statements 1-7, wherein the CM comprises a sequence selected from: QNQALRLA (SEQ ID NO: 25), QNQGLRLA (SEQ ID NO: 26), QNQALKLA (SEQ ID NO: 27), or QNQGLKLA (SEQ ID NO: 28).

[0332] Statement 13. The cleavable polypeptide of any one of Statements 1-7, wherein the CM comprises a sequence selected from: QWQALRMA (SEQ ID NO: 30), QWQGLRMA (SEQ ID NO: 31), QWQALKMA (SEQ ID NO: 32), or QWQGLKMA (SEQ ID NO: 33).

[0333] Statement 14. The cleavable polypeptide of any one of Statements 1-7, wherein the CM comprises a sequence selected from: QFQALRMA (SEQ ID NO: 35), QFQGLRMA (SEQ ID NO: 36), QFQALKMA (SEQ ID NO: 37), or QFQGLKMA (SEQ ID NO: 38).

[0334] Statement 15. The cleavable polypeptide of any one of Statements 1-7, wherein the CM comprises a sequence selected from: QNQALRSA (SEQ ID NO: 5), QAQALRMA (SEQ ID NO: 10), QAQALRSA (SEQ ID NO: 15), QNQALRVA (SEQ ID NO: 20), QNQALRLA (SEQ ID NO: 25), QWQALRMA (SEQ ID NO: 30), or QFQALRMA (SEQ ID NO: 35).

[0335] Statement 16. The cleavable polypeptide of any preceding Statement, wherein the protease is a membrane type serine protease 1 (MT-SP1).

[0336] Statement 17. The cleavable polypeptide of any one of Statements 1-15, wherein the protease is a matrix metalloprotease (MMP).CYTX-108-WO :: 4862-156.WO1

[0337] Statement 18. The cleavable polypeptide of any one of Statements 1-15 and 17, wherein the protease is a matrix metalloprotease 2 (MMP2).

[0338] Statement 19. The cleavable polypeptide of any one of Statements 1-15, and 17, wherein the protease is a matrix metalloprotease 9 (MMP9).

[0339] Statement 20. The cleavable polypeptide of any one of Statements 1-15 and 17, wherein the protease is a matrix metalloprotease 14 (MMP14).

[0340] Statement 21. The cleavable polypeptide of any one of Statements 1-16, wherein the kcat / KM of the substrate by MT-SP1 cleavage is at least 1 × 103M-1s-1.

[0341] Statement 22. The cleavable polypeptide of any one of Statements 1-15 and 17, wherein the kcat / KM of the substrate by MMP2 cleavage is at least 1 × 103M-1s-1.

[0342] Statement 23. The cleavable polypeptide of any one of Statements 1-15 and 17, wherein the kcat / KM of the substrate by MMP9 cleavage is at least 1 × 103M-1s-1.

[0343] Statement 24. The cleavable polypeptide of any one of Statements 1-15 and 17, wherein the kcat / KM of the substrate by MMP14 cleavage is at least 1 × 103M-1s-1.

[0344] Statement 25. The cleavable polypeptide of any one of Statements 1-8 and 10, wherein the kcat / KM of the substrate by MMP2 cleavage is at least 1 × 104M-1s-1.

[0345] Statement 26. The cleavable polypeptide of any one of Statements 1-7, 9, and 10, wherein the kcat / KM of the substrate by MMP14 cleavage is at least 1 × 104M-1s-1.

[0346] Statement 27. The cleavable polypeptide of any one of Statements 1-6, wherein the kcat / KM of the substrate by MMP9 cleavage is at least 1 × 104M-1s-1.

[0347] Statement 28. The cleavable polypeptide of any one of Statements 1-6, wherein the kcat / KM of the substrate by MT-SP1 cleavage is at least 1 × 104M-1s-1.

[0348] Statement 29. The cleavable polypeptide of any one of Statements 1-8 and 15, wherein the CM has a higher catalytic efficiency (kcat / KM) for MT-SP1, MMP2, and MMP9 compared to a cleavable polypeptide comprising a CM comprising a sequence of QNQALRMA (SEQ ID NO: 3).

[0349] Statement 30. The cleavable polypeptide of any one of Statements 1-7, 9, and 15, wherein the CM has a higher catalytic efficiency (kcat / KM) for MMP2, MMP9, and MMP14 compared to a cleavable polypeptide comprising a CM comprising a sequence of QNQALRMA (SEQ ID NO: 3).

[0350] Statement 31. The cleavable polypeptide of any one of Statements 1-7, 10, and 13- 15, wherein the CM has a higher catalytic efficiency (kcat / KM) for MMP2, MMP9, andCYTX-108-WO :: 4862-156.WO1 MMP14 compared to a cleavable polypeptide comprising a CM comprising a sequence of QNQALRMA (SEQ ID NO: 3).

[0351] Statement 32. The cleavable polypeptide of any one of Statements 1-7, 11, and 15, wherein the CM has a higher catalytic efficiency (kcat / KM) for MMP2 and MMP9 compared to a cleavable polypeptide comprising a CM comprising a sequence of QNQALRMA (SEQ ID NO: 3).

[0352] Statement 33. The cleavable polypeptide of any one of Statements 1-8, 10-12, and 15, wherein the CM retains about 100%, about 99%, about 95%, or about 90% cleavage by MT-SP1 under oxidizing conditions compared to non-oxidizing conditions, wherein the oxidizing conditions comprise incubation with 0.05% tert-butyl hydroperoxide (tBHP) at 40 ºC for 3h.

[0353] Statement 34. The cleavable polypeptide of any one of Statements 1-7, 9, and 14- 15, wherein the CM has 5% to 50%, 10% to 40%, or 20 to 30% lower methionine oxidation under oxidizing conditions compared to a cleavable polypeptide comprising a CM comprising a sequence of QNQALRMA (SEQ ID NO: 3), wherein the oxidizing conditions comprise incubation with 0.05% tert-butyl hydroperoxide (tBHP) at 40 ºC for 3h.

[0354] Statement 35. The cleavable polypeptide of any one of Statements 1-7, 12, and 15, wherein the CM has 5% to 50%, 10% to 40%, or 20 to 30% lower asparagine deamidation under deamidating conditions compared to a cleavable polypeptide comprising a CM comprising a sequence of QNQALRMA (SEQ ID NO: 3), wherein the deamidation conditions comprise incubation at pH 8.5 and 40°C for 7 days.

[0355] Statement 36. The cleavable polypeptide of any preceding Statements, wherein the cleavable polypeptide comprises at least one additional moiety (M) selected from a moiety that is located amino (N) terminally to the CM (Mn), a moiety that is located carboxyl (C) terminally to the CM (Mc), or a combination thereof.

[0356] Statement 37. The cleavable polypeptide of any preceding Statements, wherein the Mn and the Mc are each independently selected from a masking moiety (MM), a target binding moiety (TBM), a therapeutic agent, a detectable moiety, a diagnostic agent, or an affinity tag.

[0357] Statement 38. The cleavable polypeptide of Statement 37, wherein the cleavable polypeptide comprises the MM coupled directly or indirectly to the N-terminus of the CM and the TBM coupled directly or indirectly to the C-terminus of the CM, or whereinCYTX-108-WO :: 4862-156.WO1 cleavable polypeptide comprises the TBM coupled directly or indirectly to the N-terminus of the CM and the MM coupled directly or indirectly to the C-terminus of the CM.

[0358] Statement 39. The cleavable polypeptide of Statement 38, wherein the MM inhibits binding of the TBM to its target.

[0359] Statement 40. The cleavable polypeptide of Statement 38, wherein the amino acid sequence of the MM is different from that of the target.

[0360] Statement 41. The cleavable polypeptide of Statement 38, wherein the amino acid sequence of the MM is different from that of the target and is no more than 50% identical to the amino acid sequence of a natural binding partner of the TBM.

[0361] Statement 42. The cleavable polypeptide of any one of Statements 37-41, wherein the MM has a dissociation constant for binding to the TBM that is greater than the dissociation constant of the TBM for binding to the target.

[0362] Statement 43. The cleavable polypeptide of any one of Statements 37-42, wherein the MM is 2 to 40 amino acids in length.

[0363] Statement 44. The cleavable polypeptide of any one of Statements 37-43, wherein the MM is 20 to 200 amino acids in length.

[0364] Statement 45. The cleavable polypeptide of any one of Statements 37-44, wherein the cleavable polypeptide comprises the structural arrangement from N-terminus to C- terminus as follows: MM-CM-TBM or TBM-CM-MM.

[0365] Statement 46. The cleavable polypeptide of any one of Statements 37-45, wherein the CM directly couples the MM to the TBM.

[0366] Statement 47. The cleavable polypeptide of any one of Statements 37-45, wherein the CM is coupled to the MM via a linker peptide (LP).

[0367] Statement 48. The cleavable polypeptide of any one of Statements 35-45, wherein the CM is coupled to the TBM via an LP.

[0368] Statement 49. The cleavable polypeptide of any one of Statements 47-48, wherein the cleavable polypeptide comprises a first linking peptide (LP1) and a second linking peptide (LP2), and wherein the cleavable polypeptide has a structural arrangement from N- terminus to C-terminus as follows: MM-LP1-CM-LP2-TBM.

[0369] Statement 50. The cleavable polypeptide of any one of Statements 37-49, wherein the TBM is a biologically active protein.

[0370] Statement 51. The cleavable polypeptide of Statement 50, wherein the biologically active protein is a cytokine or a functional fragment thereofCYTX-108-WO :: 4862-156.WO1

[0371] Statement 52. The cleavable polypeptide of any one of Statements 37-49, wherein the TBM is a chimeric antigen receptor.

[0372] Statement 53. The cleavable polypeptide of any one of Statements 37-49, wherein the TBM is a therapeutic macromolecule.

[0373] Statement 54. The cleavable polypeptide of any one of Statements 37-49, wherein the TBM is an antibody or antigen binding fragment thereof.

[0374] Statement 55. The cleavable polypeptide of Statement 54, wherein the antigen binding fragment thereof is selected from the group consisting of a Fab fragment, a F(ab')2 fragment, a scFv, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody.

[0375] Statement 56. The cleavable polypeptide of Statement 54, wherein the antibody is bivalent antibody.

[0376] Statement 57. The cleavable polypeptide of Statement 54, wherein the antibody is a multivalent antibody.

[0377] Statement 58. The cleavable polypeptide of Statement 49, wherein the LP1 and the LP2 are not identical to each other.

[0378] Statement 59. The cleavable polypeptide of Statement 49, wherein the LP1 and the LP2 are identical to each other.

[0379] Statement 60. The cleavable polypeptide of any one of Statements 49 and 58-59, wherein the LP1 and the LP2 are each independently 1 to 20 amino acids in length.

[0380] Statement 61. The cleavable polypeptide of any preceding Statement, wherein the cleavable polypeptide further comprises a half-life extending moiety (EM).

[0381] Statement 62. The cleavable polypeptide of Statement 61, wherein the EM is an Fc region of an IgG, a serum albumin, a polyethylene glycol molecule, an antigen binding polypeptide that binds human serum albumin, or transferrin.

[0382] Statement 63. The cleavable polypeptide of any preceding Statement, further comprising an agent conjugated to the cleavable polypeptide.

[0383] Statement 64. The cleavable polypeptide of Statement 63, wherein the agent is conjugated to the cleavable polypeptide via a conjugating linker.

[0384] Statement 65. The cleavable polypeptide of Statement 64, wherein the conjugating linker is cleavable.

[0385] Statement 66. The cleavable polypeptide of Statement 64, wherein the conjugating linker is non-cleavable.CYTX-108-WO :: 4862-156.WO1

[0386] Statement 67. The cleavable polypeptide of any one of Statements 63-66, wherein the agent is selected from the group consisting of: a toxin, a microtubule inhibitor, a nucleic acid damaging agent, a dolastatin, an auristatin, a maytansinoid, a duocarmycin, and a calicheamicin.

[0387] Statement 68. The cleavable polypeptide of Statement 67, wherein the agent is a toxin.

[0388] Statement 69. The cleavable polypeptide of Statement 67, wherein the agent is a microtubule inhibitor.

[0389] Statement 70. The cleavable polypeptide of Statement 67, wherein the agent is a nucleic acid damaging agent.

[0390] Statement 71. The cleavable polypeptide of Statement 67, wherein the agent is a dolastatin.

[0391] Statement 72. The cleavable polypeptide of Statement 67, wherein the agent is an auristatin.

[0392] Statement 73. The cleavable polypeptide of Statement 67, wherein the agent is a maytansinoid.

[0393] Statement 74. The cleavable polypeptide of Statement 67, wherein the agent is a duocarmycin.

[0394] Statement 75. The cleavable polypeptide of Statement 67, wherein the agent is a calicheamicin.

[0395] Statement 76. The cleavable polypeptide of any one of Statements 38-75, wherein the TBM binds to a tumor-associated moiety.

[0396] Statement 77. The cleavable polypeptide of Statement 38 or Statement 76, wherein the cleavable polypeptide has an in vivo antitumor activity that is equal to or higher than a cleavable polypeptide comprising a CM comprising a sequence of QNQALRMA (SEQ ID NO: 3).

[0397] Statement 78. The cleavable polypeptide of Statement 77, wherein the antitumor activity is a reduction in tumor volume of 25% or more, 30% or more, 40% or more, 50% or more 60% or more, 70% or more, 75% or more, 80% or more, or 90% or more.

[0398] Statement 79. The cleavable polypeptide of any one of Statements 76-78, wherein the TBM binds epidermal growth factor receptor (EGFR).

[0399] Statement 80. An activatable molecule comprising: a) the cleavable polypeptide of any one of Statements 1-35;CYTX-108-WO :: 4862-156.WO1 b) a masking moiety (MM); and c) a target binding moiety (TBM); and d) an optional half-life extending moiety (EM); wherein the cleavable polypeptide comprises the MM coupled directly or indirectly to the N-terminus of the CM and the TBM coupled directly or indirectly to the C-terminus of the CM, or wherein cleavable polypeptide comprises the TBM coupled directly or indirectly to the N-terminus of the CM and the MM coupled directly or indirectly to the C-terminus of the CM.

[0400] Statement 81. The activatable molecule of Statement 80, further comprising an agent conjugated to the activatable molecule.

[0401] Statement 82. The activatable molecule of Statement 81, wherein the agent is conjugated to the cleavable polypeptide via a conjugating linker.

[0402] Statement 83. The activatable molecule of Statement 82, wherein the conjugating linker is cleavable.

[0403] Statement 84. The activatable molecule of Statement 82, wherein the conjugating linker is non-cleavable.

[0404] Statement 85. The activatable molecule of any one of Statements 81-84, wherein the agent is selected from the group consisting of: a toxin, a microtubule inhibitor, a nucleic acid damaging agent, a dolastatin, an auristatin, a maytansinoid, a duocarmycin, and a calicheamicin.

[0405] Statement 86. The activatable molecule of any one of Statements 80-85, wherein the TBM binds to a tumor-associated moiety.

[0406] Statement 87. The activatable molecule of Statement 86, wherein the activatable molecule has an in vivo antitumor activity that is equal to or higher than a cleavable polypeptide comprising a CM comprising a sequence of QNQALRMA (SEQ ID NO: 3).

[0407] Statement 88. The activatable molecule of Statement 87, wherein the antitumor activity is a reduction in tumor volume of 25% or more, 30% or more, 40% or more, 50% or more 60% or more, 70% or more, 75% or more, 80% or more, or 90% or more.

[0408] Statement 89. The activatable molecule of any one of Statements 80-88, wherein the TBM binds epidermal growth factor receptor (EGFR).

[0409] Statement 90. A nucleic acid composition comprising one or more nucleic acid molecules encoding: a) the cleavable polypeptide of any one of Statements 1-35;CYTX-108-WO :: 4862-156.WO1 b) a masking moiety (MM); and c) a target binding moiety (TBM); and d) optionally a half-life extending moiety (EM); wherein the MM is coupled directly or indirectly to the N-terminus of the CM and the TBM is coupled directly or indirectly to the C-terminus of the CM, or wherein the TBM is coupled directly or indirectly to the N-terminus of the CM and the MM is coupled directly or indirectly to the C-terminus of the CM; and a carrier.

[0410] Statement 91. The nucleic acid composition of Statement 90, wherein the EM is present and is coupled directly to the TBM.

[0411] Statement 92. The nucleic acid composition of Statement 90, wherein the EM is present and is coupled indirectly to the TBM.

[0412] Statement 93. The nucleic acid composition of Statement 90, wherein the EM is present and is encoded by an additional nucleic acid molecule.

[0413] Statement 94. The activatable molecule of any one of Statements 80-89 or the nucleic acid composition of any one of Statements 90-93, having a structural arrangement from N-terminus to C-terminus as follows: MM-CM-TBM or TBM-CM-MM, wherein “-” is a direct linkage or indirect linkage via one or more linking peptides.

[0414] Statement 95. The activatable molecule of any one of Statements 80-89 or the nucleic acid composition of any one of Statements 90-93, wherein the CM directly couples the MM to the TBM.

[0415] Statement 96. The activatable molecule of any one of Statements 80-89 or the nucleic acid composition of any one of Statements 90-93, wherein the CM is coupled to the MM via a linker peptide (LP).

[0416] Statement 97. The activatable molecule of any one of Statements 80-89 or the nucleic acid composition of any one of Statements 90-93, wherein the CM is coupled to the TBM via an LP.

[0417] Statement 98. The activatable molecule of any one of Statements 80-89 or the nucleic acid composition of any one of Statements 90-93, comprising a first linking peptide (LP1) and a second linking peptide (LP2), and a structural arrangement from N-terminus to C-terminus as follows: MM-LP1-CM-LP2-TBM, and wherein (i) LP1 and LP2 are the same or different; (ii) the LP1 and the LP2 are each independently 1 to 20 amino acids in length, or (iii) a combination of (i) and (ii).CYTX-108-WO :: 4862-156.WO1

[0418] Statement 99. The activatable molecule of any one of Statements 80-89 or the nucleic acid composition of any one of Statements 90-93, having an EM, wherein the EM is selected from the Fc region of an IgG, serum albumin, an antigen binding polypeptide that binds human serum albumin, or transferrin.

[0419] Statement 100. The activatable molecule of any one of Statements 80-89 or the nucleic acid composition of any one of Statements 90-93, wherein the TBM is an antibody or antigen binding fragment thereof.

[0420] Statement 101. The activatable molecule of any one of Statements 80-89 or the nucleic acid composition of any one of Statements 90-93, wherein the TBM is an antigen binding fragment selected from the group consisting of a Fab fragment, a F(ab')2 fragment, a scFv, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody.

[0421] Statement 102. The activatable molecule of any one of Statements 80-89 or the nucleic acid composition of any one of Statements 90-93, wherein the activatable molecule comprises a heavy chain amino acid sequence according to SEQ ID NO: 41 and a light chain amino acid sequence selected from the group consisting of SEQ ID NOs: 45-51.

[0422] Statement 103. The nucleic acid composition of any one of Statements 90-93, wherein the CM is encoded by a sequence selected from SEQ ID NOs: 67-73.

[0423] Statement 104. The nucleic acid composition of any one of Statements 90-93, wherein the one or more nucleic acid sequences comprise a sequence according to SEQ ID NO: 53 and a sequence selected from the group consisting of SEQ ID NOs: 57-63.

[0424] Statement 105. A composition comprising the cleavable polypeptide of any one of Statements 1-79 or the activatable molecule of any one of Statements 80-89 and a pharmaceutically acceptable carrier.

[0425] Statement 106. The composition of Statement 105, further comprising at least one additional agent.

[0426] Statement 107. The composition of Statement 106, wherein the at least one additional agent is a therapeutic agent.

[0427] Statement 108. A nucleic acid molecule or group of nucleic acid molecules encoding the cleavable polypeptide of any one of Statements 1-79 or the activatable molecule of any one of Statements 80-89.CYTX-108-WO :: 4862-156.WO1

[0428] Statement 109. A vector or group of vectors comprising the nucleic acid molecule or group of nucleic acid molecules of Statement 108 or the nucleic acid composition of any one of Statements 90-93.

[0429] Statement 110. A recombinant cell comprising the cleavable polypeptide of any one of Statements 1-79, the activatable molecule of any one of Statements 80-89, the nucleic acid composition of any one of Statements 90-93, the nucleic acid or group of nucleic acid molecules of Statement 108, or the vector or group of vectors comprising of Statement 109.

[0430] Statement 111. An in vitro method of manufacturing the cleavable polypeptide of any one of Statements 1-79 or the activatable molecule of any one of Statements 80-89, comprising culturing the recombinant cell of Statement 110 under conditions suitable for expressing the cleavable polypeptide or the polypeptide complex and recovering the cleavable polypeptide.

[0431] Statement 112. A method of treating, alleviating a symptom of, or delaying the progression of a disease or disorder in a subject, comprising administering a therapeutically effective amount of the cleavable polypeptide of any one of Statements 1-79, the activatable molecule of any one of Statements 80-89, or the composition of any one of Statements 105- 107 to the subject.

[0432] Statement 113. The method of Statement 112, wherein the disease or disorder is a cancer, an infection, an inflammatory disorder, a cardiovascular disorder, a neurodegenerative disorder, or an autoimmune disorder.

[0433] Statement 114. The method of Statement 112 or Statement 113, wherein the disease is a cancer.

[0434] Statement 115. The cleavable polypeptide of any one of Statements 1-79, the activatable molecule of any one of Statements 80-89, or the composition of any one of Statements 105-107 for use as a medicament or for use in therapy, optionally for treating a cancer, an infection, an inflammatory disorder, a cardiovascular disorder, a neurodegenerative disorder, or an autoimmune disorder, optionally with an additional agent which is optionally a therapeutic agent.

[0435] Statement 116. A container, vial, syringe, injector pen, or kit comprising at least one dose of the composition of the cleavable polypeptide of any one of Statements 1-79 or the activatable molecule of any one of Statements 80-89, or the composition of any one of Statements 105-107.CYTX-108-WO :: 4862-156.WO1 EXAMPLES Example 1: Exemplary Protease-Cleavable Substrates and Activatable Antibodies

[0436] The studies set forth below describe exemplary cleavable polypeptides comprising a CM of the present disclosure that are cleavable by at least one matrix metalloprotease (MMP) and by matriptase (MT-SP1). The protease-cleavable substrates may be cleavable by one or more proteases in addition to at least one MMP and MT-SP1.

[0437] Exemplary cleavable polypeptides forming activatable antibodies were constructed such that each cleavable polypeptide included one of the CMs listed in Table 4 and a carboxy terminal moiety (Mc) comprising an antibody or antigen binding fragment thereof as a target binding moiety (TBM), which is based on a mouse / human chimeric monoclonal antibody that specifically binds to epidermal growth factor receptor (EGFR). The exemplary cleavable polypeptides also include an amino terminal moiety (Mn) comprising a masking moiety (MM). The exemplary cleavable polypeptides are listed in Table 5. Table 4: MMP / MT-SP1 Substrates (CMs) Name Sequence SEQ ID NO: 2001 ISSGLLSGRSDNH 1 2011 ISSGLLSGRSDNP 2 1002 QNQALRMA 3 5080 QNQALRSA 5 5020 QAQALRMA 10 5030 QAQALRSA 15 5040 QNQALRVA 20 5050 QNQALRLA 25 5060 QWQALRMA 30 5070 QFQALRMA 35 Table 5: Activatable Antibody Sequences Light chain Light chain TBM: Light chain / EGFR MM substrate (CM) Heavy chain CISPRGCPDGPYVMY ISSGLLSGRSDNH C225v5-3954-2001 LC / (SEQ ID NO: 39) (2001) C225VH1-HC (SEQ ID NO: 1) SEQ ID NO: 42 / SEQ ID NO: 40 CISPRGCPDGPYVMY ISSGLLSGRSDNP C225v5-3954-12011 / (SEQ ID NO: 39) (2011) C225VH1-HC (SEQ ID NO: 2) SEQ ID NO: 43 / SEQ ID NO: 40 CISPRGCPDGPYVMY QNQALRMA (1002) C2253954100242 / (SEQ ID NO: 39) (SEQ ID NO: 3) C225VH1-HCCYTX-108-WO :: 4862-156.WO1 SEQ ID NO: 44 / SEQ ID NO: 40 CISPRGCPDGPYVMY QNQALRSA (5080) LC_C225-3954-5080 / (SEQ ID NO: 39) (SEQ ID NO: 5) HC_C225VH2 SEQ ID NO: 45 / SEQ ID NO: 41 CISPRGCPDGPYVMY QAQALRMA (5020) LC_C225-3954-5020 / (SEQ ID NO: 39) (SEQ ID NO: 10) HC_C225VH2 SEQ ID NO: 46 / SEQ ID NO: 41 CISPRGCPDGPYVMY QAQALRSA (5030) LC_C225-3954-5030 / (SEQ ID NO: 39) (SEQ ID NO: 15) HC_C225VH2 SEQ ID NO: 47 / SEQ ID NO: 41 CISPRGCPDGPYVMY QNQALRVA (5040) LC_C225-3954-5040 / (SEQ ID NO: 39) (SEQ ID NO: 20) HC_C225VH2 SEQ ID NO: 48 / SEQ ID NO: 41 CISPRGCPDGPYVMY QNQALRLA (5050) LC_C225-3954-5050 / (SEQ ID NO: 39) (SEQ ID NO: 25) HC_C225VH2 SEQ ID NO: 49 / SEQ ID NO: 41 CISPRGCPDGPYVMY QWQALRMA LC_C225-3954-5060 / (SEQ ID NO: 39) (5060) HC_C225VH2 (SEQ ID NO: 30) SEQ ID NO: 50 / SEQ ID NO: 41 CISPRGCPDGPYVMY QFQALRMA (5070) LC_C225-3954-5070 / (SEQ ID NO: 39) (SEQ ID NO: 35) HC_C225VH2 SEQ ID NO: 51 / SEQ ID NO: 41 Example 2: In Vitro Cleavage of Exemplary Activatable Antibodies with Protease Cleavable Substrates

[0438] This study evaluated the in vitro cleavage of activatable antibodies, as described above in Example 1, containing exemplary protease-cleavable substrates of the disclosure that are cleavable by at least one matrix metalloprotease (MMP) and by matriptase (MT- SP1).

[0439] The cleavage of the activatable antibodies having the substrates of the present disclosure, along with control substrates 2001 (WO 2016 / 118629) and 1002 (WO 2015 / 048329), was measured against MT-SP1, MMP2, MMP9, and MMP14. Each activatable antibody (500 nM) was incubated with 10 nM of a single protease for 4 hours at 37ºC as indicated in the tables below. Human recombinant proteases were purchased from R&D Systems: MMP2 (catalog No: 902-MP), MMP9 (catalog No: 911-MP), MMP14CYTX-108-WO :: 4862-156.WO1 (catalog No: 918-MP), and MT-SP1 (catalog No: 3946-SEB). MMPs were activated according to the manufacturer’s instructions. Protease concentrations were determined by active site titration. Activity assays for MMP2 and MMP9 were performed in the buffer: 50 mM Tris-HCl (pH 7.5), 10 mM CaCl2, 150 mM NaCl, 0.05% (w / v) Brij-35. Activity assays were performed for MMP14 using 50 mM HEPES (pH 6.8), 10 mM CaCl2, and 0.5 mM MgCl2. Activity assays were performed for MT-SP1 using buffer 50 mM TRIS-HCl (pH 7.4), 150 mM NaCl, 0.05% Tween 20. Following incubation, the presence of cleavage product was determined by capillary electrophoresis using a LabChip GXII Touch system (Revvity) with the HT Protein Express 100 protocol (Revvity). LabChip HT Protein Express Chips (Revvity #760499) were set up using the protocol of the Protein Express Assay Reagent Kit (Revvity #CLS960008). The fraction of cleaved activatable antibody was determined by quantifying the fraction of the higher mobility polypeptide corresponding to the cleaved activatable antibody using the LabChip GX Reviewer software (Revvity). The fraction of activatable antibody, and hence, substrate that is cleaved by each particular protease is presented as a “cleavage percentage” in Table 6 and Table 7. Table 6: In Vitro Activation of Activatable Antibodies with Exemplary Protease- Cleavable Substrates Cleavage (%) Substrate (CM) CM Sequence MT-SP1 MMP2 MMP9 MMP1 4 2001ISSGLLSGRSDNH(SEQ ID NO: 1) 27 44 - 80 1002 QNQALRMA (SEQ ID NO: 3) 32 24 - 46 5080 QNQALRSA (SEQ ID NO: 5)38 81 - 345020 QAQALRMA (SEQ ID NO: 10) 21 58 5 100 5030QAQALRSA(SEQ ID NO: 15) 34 100 29 100 “ – “ Denotes no cleavage detected. Table 7: In Vitro Activation of Activatable Antibodies with Exemplary Protease- Cleavable Substrates Substrate Cleavage (%) (CM) CM Sequence MT-SP1 MMP2 MMP9 MMP1 4CYTX-108-WO :: 4862-156.WO1 2001ISSGLLSGRSDNH(SEQ ID NO: 1) 27 36 - 85 1002 QNQALRMA (SEQ ID NO: 3)36 18 - 505040 QNQALRVA (SEQ ID NO: 20) 7 27 - 26 5050QNQALRLA(SEQ ID NO: 25) 10 - - 32 5060 QWQALRMA (SEQ ID NO: 30) 32 28 - 86 5070 QFQALRMA (SEQ ID NO: 35) 37 20 - 67 “ – “ Denotes no cleavage detected.

[0440] The data of Table 6 and Table 7 indicate that each of substrates 5080, 5020, 5030, 5040, 5050, 5060, and 5070 have a unique protease cleavage profile for the proteases employed in the study.

[0441] Relative to control substrate 1002, substrate 5080 was similarly cleavable by MT- SP1 and MMP14. Cleavage of substrate 5080 by MMP2, however, was 238% greater than control substrate 1002.

[0442] Relative to control substrate 1002, substrate 5020 demonstrated a 34% decrease in cleavage by MT-SP1 while exhibiting 142% and 117% increases in cleavage by MMP2 and MMP14, respectively. Substrate 5020 was also slightly cleaved by MMP9, whereas no cleavage of substrate 1002 was observed.

[0443] Relative to control substrate 1002, substrate 5030 was similarly cleavable by MT- SP1. Cleavage of substrate 5030 by MMP2 and MMP14 was drastically increased relative to substrate 1002, with increases in cleavage of 317% and 117%, respectively. Substrate 5030 also demonstrated considerable cleavage by MMP9, demonstrating substrate 5030 to be broadly cleavable by MMPs.

[0444] Relative to control substrate 1002, substrate 5040 demonstrated an 81% decrease in cleavage by MT-SP1, a 48% decrease in cleavage by MMP14, and a 50% increase in cleavage by MMP2.

[0445] Relative to control substrate 1002, substrate 5050 demonstrated a 72% decrease in cleavage by MT-SP1 and a 36% decrease in cleavage by MMP14. Substrate 5050 was resistant to cleavage by MMP2, with no detectable cleavage.CYTX-108-WO :: 4862-156.WO1

[0446] Relative to control substrate 1002, substrate 5060 was similarly cleavable by MT- SP1. Cleavage of substrate 5060 by MMP2 and MMP14 increased 56% and 72%, respectively, relative to substrate 1002.

[0447] Relative to control substrate 1002, substrate 5070 was similarly cleaved by MT- SP1 and MMP2, with a 34% increase in cleavage by MMP14.

[0448] In addition, an exemplary study was performed to determine the cleavability kinetics (i.e., kcat / KM(M-1s-1)) of the indicated CMs with the indicated protease enzymes. The percent cleavage assay (Tables 6 and 7) is a relative measurement performed at the specified protease concentration and timepoint. The kcat / KMparameter is determined over multiple protease concentrations and timepoints, which can result in improved sensitivity of cleavage detection. The results of this in vitro study are summarized in Table 8.

[0449] Relative to control substrate 1002, substrate 5080 was similarly cleaved by MT- SP1, MMP9, and MMP14 with a kcat / KMof at least 1x103M-1s-1for MT-SP1 and MMP14 1 to control substrate 1002, substrate 5080demonstrated increased cleavability by MMP2 with a kcat / KMof at least 1x104M-1s-1.

[0450] Relative to control substrate 1002, substrate 5020 was similarly cleaved by MT- SP1 with a kcat / KMof at least 1x103M-1s-1. Relative to control substrate 1002, substrate 5020 demonstrated increased cleavability by MMPs with a kcat / KM of at least 5x103M-1s-1, 1x103M-1s-1, and 1x104M-1s-1for MMP2, MMP9, and MMP14, respectively.

[0451] Relative to control substrate 1002, substrate 5030 was similarly cleaved by MT- SP1 with a kcat / KMof at least 1x103M-1s-1. Relative to control substrate 1002, substrate 5030 cleavability by MMPs with a kcat / KM of at least 1x104M-1s-1, 5x103M-1s-1, and 1x104M-1s-1for MMP2, MMP9, and MMP14, respectively.

[0452] Substrate 5040 demonstrated reduced cleavability by MT-SP1 compared to the control substrate with a kcat / KMof at least 1x103M-1s-1for. Relative to control substrate 1002, substrate 5040 was similarly cleaved by MMPs with a kcat / KM of at least 1x103M-1s-1, 1x102M-1s-1, and 1x103M-1s-1for MMP2, MMP9, and MMP14, respectively.

[0453] Relative to control substrate 1002, substrate 5050 was similarly cleaved by MMP9 and MMP14 with a kcat / KMof at least 1x102M-1s-1for MMP9 and at least 1x103M-1s-1for MMP14. Substrate 5050 had reduced cleavability by MT-SP1 and MMP2 compared to control substrate 1002 with a kcat / KMof at least at least 1x103M-1s-1.

[0454] Relative to control substrate 1002, substrate 5060 was similarly cleaved by MT- SP1, MMP2, and MMP9 with a kcat / KMof at least 1x103M-1s-1for MT-SP1 and MMP2 and atCYTX-108-WO :: 4862-156.WO1 least 1x102M-1s-1for MMP9. Relative to control substrate 1002, substrate 5060 demonstrated increased cleavability by MMP14 with a kcat / KM of at least 5x103M-1s-1.

[0455] Relative to control substrate 1002, substrate 5070 was similarly cleaved by MT- SP1, MMP2, and MMP9 with a kcat / KM of at least 1x103M-1s-1for MT-SP1 and MMP2 and at least 1x102M-1s-1for MMP9. Relative to control substrate 1002, substrate 5070 demonstrated increased cleavability by MMP14 with a kcat / KM of at least 5x103M-1s-1.

[0456] Substrates 5040 and 5050 demonstrated reduced cleavability by MT-SP1 compared to the control substrate 1002 with a kcat / KM of at least 1x103M-1s-1.

[0457] Relative to the control substrate, substrates 5020, 5030, 5060, and 5070 demonstrated increased cleavability by MMP14 with a kcat / KM of at least 5x103M-1s-1.

[0458] Relative to the control substrate, substrates 5080 and 5030 demonstrated increased cleavability by MMP2 with a kcat / KM of at least 1x104M-1s-1.

[0459] Relative to the control substrate, substrates 5020 and 5030 demonstrated increased cleavability by MMP9 and MMP14 with a kcat / KM of at least 1x103M-1s-1for MMP9 and at least 1x104M-1s-1for MMP14. Table 8: In Vitro Activation of Cleavable Polypeptides with Exemplary Protease- Cleavable Substrates kcat / KM(M-1s-1) Substrate (CM) CM Sequence MT-SP1 MMP2 MMP9 MMP14 1002 QNQALRMA 2.38 3.39 ) E+03 E+03 1.75E 3.19 (SEQ ID NO: 3 +02 E+03 5080 QNQALRSA 2.68 1.76 2.80 2.77 (SEQ ID NO: 5) E+03 E+04 E+02 E+03 5020 QAQALRMA 1.53 8.68 1.76 1.07 (SEQ ID NO: 10) E+03 E+03 E+03 E+04 5030 QAQALRSA 2.33 4.16 6.16 1.01 (SEQ ID NO: 15) E+03 E+04 E+03 E+04 5040 QNQALRVA 2.00 4.54 2.04 2.81 (SEQ ID NO: 20) E+02 E+03 E+02 E+03 5050QNQALRLA2.53 5.98 1.35 2.21 (SEQ ID NO: 25) E+02 E+02 E+02 E+03 5060 QWQALRMA 1.62 5.18 2.68 9.96 (SEQ ID NO: 30) E+03 E+03 E+02 E+03 5070 QFQALRMA 1.66 3.75 3.27 6.76 (SEQ ID NO: 35) E+03 E+03 E+02 E+03CYTX-108-WO :: 4862-156.WO1 Example 3: In Vivo Stability of Activatable Antibodies with Exemplary Protease- Cleavable Substrates

[0460] This study evaluated the in vivo stability of activatable antibodies, described in Example 1, above.

[0461] The stability of activatable antibodies containing the exemplary substrates was measured by administering a dose of the activatable antibodies to mice, and then measuring the cleaved activatable antibody in the plasma by a capillary electrophoresis immunoassay. The stability was compared to an activatable antibody, as described in Example 1, but with control substrates 2001 (WO 2016 / 118629) and 1002 (WO 2015 / 048329).

[0462] In this study, nu / nu mice of about 7-8 weeks of age were administered intraperitoneally with the indicated test article at a dosage of 10 mg / kg. After 7 days following the administration, terminal blood was collected by cardiac puncture and processed to plasma within 1 hour of collection. The collected sample was diluted 1:50 (v:v) in phosphate-buffered saline solution and denatured and analyzed using the WesTMWestern Blot protocol (Protein Simple) with the A110UK goat anti-human IgG antibody (American Qualex) and an anti-goat secondary antibody (Jackson ImmunoResearch). The fraction of cleaved activatable antibody was determined by quantifying the fraction of the higher mobility polypeptide corresponding to the cleaved activatable antibody using the Compass software (Protein Simple). The results of these exemplary assays are summarized in Table 9 and Table 10. Table 9: In Vivo Stability of Activatable Antibodies with Exemplary Substrates Substrate (CM) CM Sequence In Vivo % Activation (nu / nu, day 7) 2001ISSGLLSGRSDNH(SEQ ID NO: 1) 37 1002 QNQALRMA (SEQ ID NO: 3) 38 5080 QNQALRSA (SEQ ID NO: 5) 38 5020QAQALRMA(SEQ ID NO: 10) 64 5030 QAQALRSA (SEQ ID NO: 15)59Table 10: In Vivo Stability of Activatable Antibodies with Exemplary Substrates Substrate (CM) CM Sequence In Vivo % Activation (nu / nu, day 7)CYTX-108-WO :: 4862-156.WO1 2001ISSGLLSGRSDNH(SEQ ID NO: 1) 34 1002 QNQALRMA (SEQ ID NO: 3)285050 QNQALRLA (SEQ ID NO: 25) 26 5060QWQALRMA(SEQ ID NO: 30) 47 5070 QFQALRMA (SEQ ID NO: 35) 48

[0463] Substrate 5080 demonstrated similar in vivo stability to substrate 1002, with both substrates showing 38% activation (Table 9). Substrates 5060 and 5070 demonstrated moderately decreased stability relative to substrate 1002. Substrate 5050 was slightly more stable in vivo relative to substrate 1002 (Table 10). Substrates 5020 and 5030 demonstrated decreased in vivo stability relative to substrate 1002 (Table 9). Example 4: Masking Efficiency of Activatable Antibodies with Exemplary Substrates

[0464] This study evaluated the in vitro masking efficiency of activatable antibodies as described in Example 1.

[0465] A solid-phase binding assay (ELISA) was used to demonstrate the EGFR binding activity of anti-EGFR activatable antibodies that include substrates cleavable by an MMP and / or matriptase (MT-SP1) using recombinant EGFR. The binding to EGFR of the activatable antibodies with the indicated substrates was measured and compared to the unmasked control antibody. A summary of these exemplary results is shown in FIG.1 and Table 11. Table 11: In Vitro Binding Activity and Masking Efficiency of Activatable Antibodies Substrate EC50 Masking Efficiency N / A 0.136 1 1002 32.47 239 5080 16.46 121 5020 9.39 69 5030 21.56 159 5040 20.59 151 5050 39.07 287 5060 17.66 130 5070 21.87 161CYTX-108-WO :: 4862-156.WO1

[0466] These results demonstrate that the substrates had a slight effect on the apparent masking efficiency of the masking moiety in the activatable antibody. Substrates 5080, 5030, 5040, 5050, 5060, and 5070 demonstrated a similar masking efficiency relative to the control substrate 1002. Substrate 5020 reduced apparent masking efficiency by 71% relative to substrate 1002. Example 5: In Vivo Efficacy of Cleavable Polypeptides with Exemplary Substrates

[0467] This study evaluated the in vivo efficacy of the cleavable polypeptides, described in Example 1, using the H292 (human lung cancer cell line) xenograft model.

[0468] In these studies, H292 subcutaneous xenograft tumors in female nu / nu mice of 6-8 weeks of age were grown to a volume of 96-257 mm3. The H292 cell line is responsive to the anti-EGFR antibody cetuximab. The mice were then randomized into groups of 8 mice each and, each group was dosed intraperitoneally on day 1 with 7.5 mg / kg of the indicated test article as noted in FIGs.2A and 2B. The mean tumor volume ± SEM was plotted for each time point following administration of the test article, as shown in FIGs.2A and 2B. Each mouse was treated with activatable antibodies with the indicated substrates, or with cetuximab or immunoglobulin (IVIG) control. The efficacy was determined with activatable antibodies having a control substrate, e.g., 2001 (WO 2016 / 118629), 2011 (WO 2016 / 118629), and 1002 (WO 2015 / 048329)

[0469] As depicted in FIG.2A, the cleavable polypeptides with an anti-EGFR TBM that include the CM 5080 demonstrated improved efficacy in reducing tumor volume relative to an activatable antibody including the CM 1002. Efficacy with a 5080 CM-containing cleavable polypeptide with an anti-EGFR TBM was comparable to cetuximab, which lacks both a substrate and mask.

[0470] As depicted in FIG.2B, the cleavable polypeptides with an anti-EGFR TBM including either CM 5050 or 5060 demonstrated improved efficacy in reducing tumor volume relative to the cleavable polypeptide with an anti-EGFR TBM including CM 1002. Example 6: Forced Stability and In Vitro Protease Cleavage of Activatable Antibodies with Exemplary Substrates

[0471] This study evaluated the forced stability and in vitro protease cleavage of activatable antibodies, as described in Example 1.

[0472] For forced oxidation and deamidation, the activatable antibodies were formulated at 1 mg / mL in phosphate buffered saline (pH 7.2) containing 6% sucrose. Forced deamidation was performed through incubation of the activatable antibodies at pH 8.5 andCYTX-108-WO :: 4862-156.WO1 40°C for 7 days, whereas forced oxidation was performed through incubation of the activatable antibodies with 0.05% tert-butyl hydroperoxide (tBHP) at 40°C for 3h. Peptide mapping with liquid chromatography tandem mass spectrometry (LC-MS / MS) was performed to characterize the percent of deamidation or oxidation in the exemplary substrates following forced deamidation, forced oxidation, or in the initial samples prior to treatment (T0) as shown in Table 12.

[0473] In control substrate 1002, a significant percentage of methionine oxidation (44.54%) was identified in the forced oxidation condition relative to the T0condition (1.96%). In control substrate 1002, a lower percentage of asparagine deamidation (8.42%) was identified in the forced deamidation condition relative to the T0condition (0.76%) compared to the percentage of methionine oxidation that was identified in the forced oxidation condition relative to the T0condition. In the exemplary substrates 5080 and 5050, the site of oxidation was removed through replacement of methionine in the control substrate (1002) with serine or leucine in substrates 5080 and 5050, respectively. The percentage of forced asparagine deamidation in these exemplary substrates was not significantly changed by the methionine replacement. In the exemplary substrates 5020 and 5070, the site of deamidation was removed through replacement of the asparagine in the control substrate (1002) with alanine or phenylalanine in substrates 5020 and 5070, respectively. The percentage of forced methionine oxidation in the exemplary substrate 5020 was not significantly altered by the alanine substitution, whereas the percentage of forced methionine oxidation in the exemplary substrate 5070 was reduced by approximately 15% compared to the control substrate 1002. Methionine oxidation is eliminated in 5080 and 5050 by removal of the methionine relative to control substrate 1002. Asparagine deamidation is eliminated in 5020 and 5070 by removal of the asparagine relative to control substrate 1002. These forced stability studies demonstrate that 5020, which has a Met at the same position as in 1002, exhibits lower Met oxidation (40.46% vs.44.54%), 5050, which has an Asn at the same position as 1002, exhibits slightly lower Asn deamidation (7.91% vs 8.42%), and 5070, which has a Met at the same position as in 1002, exhibits lower Met oxidation (29.21% vs 44.54%). Table 12: Peptide mapping of substrate deamidation and methionine oxidation in stress- tested activatable antibodies with exemplary substrates Substra pH 8.5 0.05% tBHP Substrate te Initial Sequence Modification (T0) 40°C 7d 40°C 3hCYTX-108-WO :: 4862-156.WO1 QNQALRM Asn 1002 A (SEQ ID Deamidation 0.76% 8.42% 0.21% NO: 3) Met Oxidation 1.96% 6.20% 44.54% QNQALRSA Asn D 0.35% 8.52% 0.41% 5080 (SEQ ID eamidation NO: 5) Met Oxidation Met not present QAQALRM Asn Asn not present 5020 A (SEQ ID Deamidation NO: 10) Met Oxidation 1.34% 4.53% 40.46% QNQALRL Asn Deami 0.41% 7.91% 0.39% 5050 A (SEQ ID dation NO: 25) Met Oxidation Met not present QFQALRM Asn Asn not present 5070 A (SEQ ID Deamidation NO: 35) Met Oxidation 3.55% 7.36% 29.21%

[0474] Protease cleavage assessment with MT-SP1 was performed with the activatable antibodies, as described in Example 1, following forced oxidation or for the T0 condition using the experimental approach described in Example 2. Briefly, each activatable antibody (500 nM) was incubated with 10 nM of MT-SP1 at 37ºC, and percent cleavage product was determined by capillary electrophoresis following 0.5h, 1.5h, 4h, and 24h incubation. As shown in FIG.3, the percent cleavage of the control substrate 1002 by MT-SP1 was reduced following forced oxidation compared to the T0 condition. A reduction in percent cleavage by MT-SP1 also was observed for exemplary substrates 5020 and 5070 following forced oxidation compared to the T0 condition. Substrates 1002, 5020, and 5070 demonstrate 44.54%, 40.46%, and 29.21% methionine oxidation, respectively, in the forced oxidation condition as described in Table 12. In contrast, the exemplary substrates 5080, 5030, and 5050 did not demonstrate a significant change in percent cleavage by MT-SP1 following forced oxidation compared to the T0 condition. Therefore, replacement of the methionine site of oxidation in the control substrate 1002 with an amino acid less susceptible to oxidation in the exemplary substrates 5080, 5030, and 5050 maintains the same level of cleavability by MT-SP1. Table 13: Exemplary Sequences SEQ ID DESCRIPTION / SEQUENCE NO NOTES 1 CM 2001 ISSGLLSGRSDNH 2 CM 2011 ISSGLLSGRSDNP 3 CM 1002 QNQALRMA 4 CM 5080 Consensus QNQX1LX2SA X1 = A or GCYTX-108-WO :: 4862-156.WO1 X2 = R or K CM 5080 QNQALRSA CM 5081 QNQGLRSA CM 5082 QNQALKSA CM 5083 QNQGLKSA CM 5020 Consensus QAQX1LX2MA X1= A or G X2= R or K CM 5020 QAQALRMA CM 5021 QAQGLRMA CM 5022 QAQALKMA CM 5023 QAQGLKMA CM 5030 Consensus QAQX1LX2SA X1= A or G X2= R or K CM 5030 QAQALRSA CM 5031 QAQGLRSA CM 5032 QAQALKSA CM 5033 QAQGLKSA QNQX1LX2VA CM 5040 Consensus X1 = A or G X2 = R or K CM 5040 QNQALRVA CM 5041 QNQGLRVA CM 5042 QNQALKVA CM 5043 QNQGLKVA QNQX1LX2LA CM 5050 Consensus X1 = A or G X2 = R or K CM 5050 QNQALRLA CM 5051 QNQGLRLA CM 5052 QNQALKLA CM 5053 QNQGLKLA QWQX1LX2MA CM 5060 Consensus X1= A or G X2= R or K CM 5060 QWQALRMA CM 5061 QWQGLRMA Substrate 5062 QWQALKMA Substrate 5063 QWQGLKMA QFQX1LX2MA CM 5070 Consensus X1 = A or G X2 = R or K CM 5070 QFQALRMA CM 5071 QFQGLRMA CM 5072 QFQALKMA CM 5073 QFQGLKMA EGFR Mask CISPRGCPDGPYVMYCYTX-108-WO :: 4862-156.WO1 C225VH1-HC QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYG VHWVRQSPGKGLEWLGVIWSGGNTDYNTPFT SRLSINKDNSKSQVFFKMNSLQSQDTAIYYCA RALTYYDYEFAYWGQGTLVTVSAASTKGPSV FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK HC_C225VH2 QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYG VHWVRQSPGKGLEWLGVIWSGGNTDYNTPFT SRLSINKDNSKSQVFFKMNSLQSQDTAIYYCA RALTYYDYEFAYWGQGTLVTVSSASTKGPSV FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK C225v5-3954-2001 LC QGQSGQCISPRGCPDGPYVMYGSSGGSGGSG GSGISSGLLSGRSDNHGSSGTQILLTQSPVILSV SPGERVSFSCRASQSIGTNIHWYQQRTNGSPRL LIKYASESISGIPSRFSGSGSGTDFTLSINSVESE DIADYYCQQNNNWPTTFGAGTKLELKRTVAA PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLS STLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC C225v5-3954-2011 QGQSGQCISPRGCPDGPYVMYGSSGGSGGSG GSGISSGLLSGRSDNPGSSGTQILLTQSPVILSV SPGERVSFSCRASQSIGTNIHWYQQRTNGSPRL LIKYASESISGIPSRFSGSGSGDFTLSINSVESEDI ADYYCQQNNNWPTTFGAGTKLELKRTVAAPS VFIFPPSDEQLKSGTASWCLLNNFYPREAKVQ WKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGECCYTX-108-WO :: 4862-156.WO1 C22539541002 LC QGQSGQCISPRGCPDGPYVMYGSSGGSGGSG GSGQNQALRMAGSSGTQILLTQSPVILSVSPGE RVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKY ASESISGIPSRFSGSGSGTDFTLSINSVESEDIAD YYCQQNNNWPTTFGAGTKLELKRTVAAPSVF IFPPSDEQLKSGTASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC LC_C225-3954-5080 QGQSGQGCISPRGCPDGPYVMYGGGSSGGSQ NQALRSAGGGSQILLTQSPVILSVSPGERVSFS CRASQSIGTNIHWYQQRTNGSPRLLIKYASESI SGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQ QNNNWPTTFGAGTKLELKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDN ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC LC_C225-3954-5020 QGQSGQGCISPRGCPDGPYVMYGGGSSGGSQ AQALRMAGGGSQILLTQSPVILSVSPGERVSFS CRASQSIGTNIHWYQQRTNGSPRLLIKYASESI SGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQ QNNNWPTTFGAGTKLELKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDN ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC LC_C225-3954-5030 QGQSGQGCISPRGCPDGPYVMYGGGSSGGSQ AQALRSAGGGSQILLTQSPVILSVSPGERVSFS CRASQSIGTNIHWYQQRTNGSPRLLIKYASESI SGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQ QNNNWPTTFGAGTKLELKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDN ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC LC_C225-3954-5040 QGQSGQGCISPRGCPDGPYVMYGGGSSGGSQ NQALRVAGGGSQILLTQSPVILSVSPGERVSFS CRASQSIGTNIHWYQQRTNGSPRLLIKYASESI SGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQ QNNNWPTTFGAGTKLELKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDN ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC LC_C225-3954-5050 QGQSGQGCISPRGCPDGPYVMYGGGSSGGSQ NQALRLAGGGSQILLTQSPVILSVSPGERVSFS CRASQSIGTNIHWYQQRTNGSPRLLIKYASESI SGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQ QNNNWPTTFGAGTKLELKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDN ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGECCYTX-108-WO :: 4862-156.WO1 LC_C225-3954-5060 QGQSGQGCISPRGCPDGPYVMYGGGSSGGSQ WQALRMAGGGSQILLTQSPVILSVSPGERVSFS CRASQSIGTNIHWYQQRTNGSPRLLIKYASESI SGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQ QNNNWPTTFGAGTKLELKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDN ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC LC_C225-3954-5070 QGQSGQGCISPRGCPDGPYVMYGGGSSGGSQ FQALRMAGGGSQILLTQSPVILSVSPGERVSFS CRASQSIGTNIHWYQQRTNGSPRLLIKYASESI SGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQ QNNNWPTTFGAGTKLELKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDN ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC C225VH1-HC CAAGTACAACTGAAACAGTCAGGCCCGGGG CTGGTGCAACCCTCCCAATCTCTGAGCATCA CTTGTACCGTCAGTGGATTTAGCCTCACAAA CTATGGTGTCCATTGGGTACGCCAGAGCCCG GGAAAGGGTCTTGAATGGCTCGGCGTCATCT GGAGCGGCGGCAACACTGACTACAACACTC CATTTACTTCTCGGCTGTCCATTAACAAGGA TAATAGTAAGTCCCAAGTGTTCTTCAAAATG AATAGCCTGCAATCCCAAGACACTGCCATTT ATTACTGCGCCCGCGCTCTCACATACTACGA CTATGAGTTTGCATACTGGGGTCAAGGAAC GCTGGTTACAGTTTCAGCAGCTAGCACCAAG GGCCCATCGGTCTTCCCCCTGGCACCCTCCT CCAAGAGCACCTCTGGGGGCACAGCGGCCC TGGGCTGCCTGGTCAAGGACTACTTCCCCGA ACCGGTGACGGTGTCGTGGAACTCAGGCGC CCTGACCAGCGGCGTGCACACCTTCCCGGCT GTCCTACAGTCCTCAGGACTCTACTCCCTCA GCAGCGTGGTGACCGTGCCCTCCAGCAGCTT GGGCACCCAGACCTACATCTGCAACGTGAA TCACAAGCCCAGCAACACCAAGGTGGACAA GAAAGTTGAGCCCAAATCTTGTGACAAAAC TCACACATGCCCACCGTGCCCAGCACCTGAA CTCCTGGGGGGACCGTCAGTCTTCCTCTTCC CCCCAAAACCCAAGGACACCCTCATGATCTC CCGGACCCCTGAGGTCACATGCGTGGTGGT GGACGTGAGCCACGAAGACCCTGAGGTCAA GTTCAACTGGTACGTGGACGGCGTGGAGGT GCATAATGCCAAGACAAAGCCGCGGGAGGA GCAGTACAACAGCACGTACCGTGTGGTCAG CGTCCTCACCGTCCTGCACCAGGACTGGCTG AATGGCAAGGAGTACAAGTGCAAGGTCTCC AACAAAGCCCTCCCAGCCCCCATCGAGAAACYTX-108-WO :: 4862-156.WO1 ACCATCTCCAAAGCCAAAGGGCAGCCCCGA GAACCACAGGTGTACACCCTGCCCCCATCCC GGGAGGAGATGACCAAGAACCAGGTCAGCC TGACCTGCCTGGTCAAAGGCTTCTATCCCAG CGACATCGCCGTGGAGTGGGAGAGCAATGG GCAGCCGGAGAACAACTACAAGACCACGCC TCCCGTGCTGGACTCCGACGGCTCCTTCTTC CTCTACAGCAAGCTCACCGTGGACAAGAGC AGGTGGCAGCAGGGGAACGTCTTCTCATGC TCCGTGATGCATGAGGCTCTGCACAACCACT ACACGCAGAAGAGCCTCTCCCTGTCTCCGGG TAAAT HC_C225VH2 CAGGTGCAGCTGAAACAATCTGGACCTGGC CTGGTGCAGCCTAGCCAGAGCCTGTCTATCA CCTGTACCGTGTCCGGCTTCAGCCTGACCAA TTACGGCGTGCACTGGGTCCGACAGTCTCCA GGCAAAGGACTGGAATGGCTGGGAGTGATT TGGAGCGGCGGCAACACCGACTACAACACC CCATTCACCAGCAGACTGAGCATCAACAAG GACAACAGCAAGAGCCAGGTGTTCTTCAAG ATGAACAGCCTGCAGAGCCAGGACACCGCC ATCTACTACTGTGCTAGAGCCCTGACCTACT ACGACTACGAGTTCGCCTATTGGGGCCAGG GCACACTGGTCACAGTTAGCAGCGCTAGCA CAAAGGGCCCCAGCGTTTTCCCACTGGCTCC TAGCAGCAAGTCTACCTCTGGCGGAACAGC CGCTCTGGGCTGTCTGGTCAAGGACTACTTT CCCGAGCCAGTGACCGTGTCCTGGAATAGC GGAGCACTGACATCTGGCGTGCACACATTTC CAGCCGTGCTGCAGTCTAGCGGCCTGTACTC TCTGAGCAGCGTCGTGACAGTGCCAAGCAG CTCTCTGGGCACCCAGACCTACATCTGCAAT GTGAACCACAAGCCTAGCAACACCAAGGTG GACAAGAAGGTGGAACCCAAGAGCTGCGAC AAGACCCACACCTGTCCTCCATGTCCTGCTC CAGAACTGCTCGGCGGACCTTCCGTGTTCCT GTTTCCTCCAAAGCCTAAGGACACCCTGATG ATCAGCAGAACCCCTGAAGTGACCTGCGTG GTGGTGGATGTGTCTCACGAGGACCCCGAA GTGAAGTTCAATTGGTACGTGGACGGCGTG GAAGTGCACAACGCCAAGACCAAGCCTAGA GAGGAACAGTACAACAGCACCTACAGAGTG GTGTCCGTGCTGACAGTGCTGCATCAGGACT GGCTGAACGGCAAAGAGTACAAGTGCAAGG TGTCCAACAAGGCCCTGCCTGCTCCTATCGA GAAAACCATCAGCAAGGCCAAGGGCCAGCC TAGGGAACCCCAGGTTTACACACTGCCTCCA AGCCGGGAAGAGATGACCAAGAATCAGGTGCYTX-108-WO :: 4862-156.WO1 TCCCTGACCTGCCTCGTGAAGGGCTTCTACC CTTCCGATATCGCCGTGGAATGGGAGAGCA ATGGCCAGCCTGAGAACAACTACAAGACAA CCCCTCCTGTGCTGGACAGCGACGGCTCATT CTTCCTGTACAGCAAGCTGACCGTGGACAA GTCCAGATGGCAGCAGGGCAACGTGTTCAG CTGCTCCGTGATGCACGAGGCCCTGCACAAC CACTACACCCAGAAGTCCCTGTCTCTGAGCC CCGGCTGA C225v5-3954-2001 LC CAGGTGCAGCTGAAACAATCTGGACCTGGC CTGGTGCAGCCTAGCCAGAGCCTGTCTATCA CCTGTACCGTGTCCGGCTTCAGCCTGACCAA TTACGGCGTGCACTGGGTCCGACAGTCTCCA GGCAAAGGACTGGAATGGCTGGGAGTGATT TGGAGCGGCGGCAACACCGACTACAACACC CCATTCACCAGCAGACTGAGCATCAACAAG GACAACAGCAAGAGCCAGGTGTTCTTCAAG ATGAACAGCCTGCAGAGCCAGGACACCGCC ATCTACTACTGTGCTAGAGCCCTGACCTACT ACGACTACGAGTTCGCCTATTGGGGCCAGG GCACACTGGTCACAGTTAGCAGCGCTAGCA CAAAGGGCCCCAGCGTTTTCCCACTGGCTCC TAGCAGCAAGTCTACCTCTGGCGGAACAGC CGCTCTGGGCTGTCTGGTCAAGGACTACTTT CCCGAGCCAGTGACCGTGTCCTGGAATAGC GGAGCACTGACATCTGGCGTGCACACATTTC CAGCCGTGCTGCAGTCTAGCGGCCTGTACTC TCTGAGCAGCGTCGTGACAGTGCCAAGCAG CTCTCTGGGCACCCAGACCTACATCTGCAAT GTGAACCACAAGCCTAGCAACACCAAGGTG GACAAGAAGGTGGAACCCAAGAGCTGCGAC AAGACCCACACCTGTCCTCCATGTCCTGCTC CAGAACTGCTCGGCGGACCTTCCGTGTTC...

Claims

CYTX-108-WO :: 4862-156.WO1 WHAT IS CLAIMED IS:

1. A cleavable polypeptide comprising a cleavable moiety (CM) comprising an amino acid sequence QXAQX1LX2XBA (SEQ ID NO: 74), wherein XA is N, A, W, or F; X1 is G or A; X2is R or K; XBis S, M, V, or L, provided that: i) when XA is N, then XB is S, L, or V; ii) when XA is A, then XB is S or M; and iii) when XA is W or F, then XB is M; and wherein the CM is a substrate for a protease.

2. The cleavable polypeptide of claim 1, wherein the cleavable polypeptide comprises 2 to 4 CMs.

3. The cleavable polypeptide of claim 2, wherein each of the 2 to 4 CMs independently comprises an amino acid sequence according to the amino acid sequence QXAQX1LX2XBA (SEQ ID NO: 74).

4. The cleavable polypeptide of any one of claims 1-3, wherein the cleavable polypeptide comprises 3 CMs.

5. The cleavable polypeptide of any one of claims 1-4, wherein the cleavable polypeptide comprises 4 CMs.

6. The cleavable polypeptide of any one of claims 1-5, wherein the CM comprises a sequence selected from: QNQX1LX2SA (SEQ ID NO: 4), QAQX1LX2MA (SEQ ID NO: 9), QAQX1LX2SA (SEQ ID NO: 14), QNQX1LX2VA (SEQ ID NO: 19), QNQX1LX2LA (SEQ ID NO: 24), QWQX1LX2MA (SEQ ID NO: 29), QFQX1LX2MA (SEQ ID NO: 34),wherein X1is G or A and X2is R or K.

7. The cleavable polypeptide of any one of claims 1-6, wherein the CM comprises a sequence selected from: QNQALRSA (SEQ ID NO: 5), QNQGLRSA (SEQ ID NO: 6), QNQALKSA (SEQ ID NO: 7), QNQGLKSA (SEQ ID NO: 8), QAQALRMA (SEQ ID NO: 10), QAQGLRMA (SEQ ID NO: 11), QAQALKMA (SEQ ID NO: 12),CYTX-108-WO :: 4862-156.WO1 QAQGLKMA (SEQ ID NO: 13), QAQALRSA (SEQ ID NO: 15), QAQGLRSA (SEQ ID NO: 16), QAQALKSA (SEQ ID NO: 17), QAQGLKSA (SEQ ID NO: 18), QNQALRVA (SEQ ID NO: 20), QNQGLRVA (SEQ ID NO: 21), QNQALKVA (SEQ ID NO: 22), QNQGLKVA (SEQ ID NO: 23), QNQALRLA (SEQ ID NO: 25), QNQGLRLA (SEQ ID NO: 26), QNQALKLA (SEQ ID NO: 27), QNQGLKLA (SEQ ID NO: 28), QWQALRMA (SEQ ID NO: 30), QWQGLRMA (SEQ ID NO: 31), QWQALKMA (SEQ ID NO: 32), QWQGLKMA (SEQ ID NO: 33), QFQALRMA (SEQ ID NO: 35), QFQGLRMA (SEQ ID NO: 36), QFQALKMA (SEQ ID NO: 37), or QFQGLKMA (SEQ ID NO: 38), optionally wherein the substrate comprises an amino acid sequence with one- amino acid or two-amino acid mutation(s) of any one of SEQ ID NOs: 5-8, 10-13, 15-18, 20-23, 25-28, 30-33, 35-38.

8. The cleavable polypeptide of any one of claims 1-7, wherein the CM comprises a sequence selected from: QNQALRSA (SEQ ID NO: 5), QNQGLRSA (SEQ ID NO: 6), QNQALKSA (SEQ ID NO: 7), or QNQGLKSA (SEQ ID NO: 8).

9. The cleavable polypeptide of any one of claims 1-7, wherein the CM comprises a sequence selected from: QAQALRMA (SEQ ID NO: 10), QAQGLRMA (SEQ ID NO: 11), QAQALKMA (SEQ ID NO: 12), or QAQGLKMA (SEQ ID NO: 13).

10. The cleavable polypeptide of any one of claims 1-7, wherein the CM comprises a sequence selected from: QAQALRSA (SEQ ID NO: 15), QAQGLRSA (SEQ ID NO: 16), QAQALKSA (SEQ ID NO: 17), or QAQGLKSA (SEQ ID NO: 18).

11. The cleavable polypeptide of any one of claims 1-7, wherein the CM comprises a sequence selected from: QNQALRVA (SEQ ID NO: 20), QNQGLRVA (SEQ ID NO: 21), QNQALKVA (SEQ ID NO: 22), or QNQGLKVA (SEQ ID NO: 23).

12. The cleavable polypeptide of any one of claims 1-7, wherein the CM comprises a sequence selected from: QNQALRLA (SEQ ID NO: 25), QNQGLRLA (SEQ ID NO: 26), QNQALKLA (SEQ ID NO: 27), or QNQGLKLA (SEQ ID NO: 28).CYTX-108-WO :: 4862-156.WO1 13. The cleavable polypeptide of any one of claims 1-7, wherein the CM comprises a sequence selected from: QWQALRMA (SEQ ID NO: 30), QWQGLRMA (SEQ ID NO: 31), QWQALKMA (SEQ ID NO: 32), or QWQGLKMA (SEQ ID NO: 33).

14. The cleavable polypeptide of any one of claims 1-7, wherein the CM comprises a sequence selected from: QFQALRMA (SEQ ID NO: 35), QFQGLRMA (SEQ ID NO: 36), QFQALKMA (SEQ ID NO: 37), or QFQGLKMA (SEQ ID NO: 38).

15. The cleavable polypeptide of any one of claims 1-7, wherein the CM comprises a sequence selected from: QNQALRSA (SEQ ID NO: 5), QAQALRMA (SEQ ID NO: 10), QAQALRSA (SEQ ID NO: 15), QNQALRVA (SEQ ID NO: 20), QNQALRLA (SEQ ID NO: 25), QWQALRMA (SEQ ID NO: 30), or QFQALRMA (SEQ ID NO: 35).

16. The cleavable polypeptide of any preceding claim, wherein the protease is a membrane type serine protease 1 (MT-SP1).

17. The cleavable polypeptide of any one of claims 1-15, wherein the protease is a matrix metalloprotease (MMP).

18. The cleavable polypeptide of any one of claims 1-15 and 17, wherein the protease is a matrix metalloprotease 2 (MMP2).

19. The cleavable polypeptide of any one of claims 1-15, and 17, wherein the protease is a matrix metalloprotease 9 (MMP9).

20. The cleavable polypeptide of any one of claims 1-15 and 17, wherein the protease is a matrix metalloprotease 14 (MMP14).

21. The cleavable polypeptide of any one of claims 1-16, wherein the kcat / KMof the substrate by MT-SP1 cleavage is at least 1 × 103M-1s-1.CYTX-108-WO :: 4862-156.WO1 22. The cleavable polypeptide of any one of claims 1-15 and 17, wherein the kcat / KMof the substrate by MMP2 cleavage is at least 1 × 103M-1s-1.

23. The cleavable polypeptide of any one of claims 1-15 and 17, wherein the kcat / KM of the substrate by MMP9 cleavage is at least 1 × 103M-1s-1.

24. The cleavable polypeptide of any one of claims 1-15 and 17, wherein the kcat / KMof the substrate by MMP14 cleavage is at least 1 × 103M-1s-1.

25. The cleavable polypeptide of any one of claims 1-8 and 10, wherein the kcat / KMof the substrate by MMP2 cleavage is at least 1 × 104M-1s-1.

26. The cleavable polypeptide of any one of claims 1-7, 9, and 10, wherein the kcat / KM of the substrate by MMP14 cleavage is at least 1 × 104M-1s-1.

27. The cleavable polypeptide of any one of claims 1-6, wherein the kcat / KMof the substrate by MMP9 cleavage is at least 1 × 104M-1s-1.

28. The cleavable polypeptide of any one of claims 1-6, wherein the kcat / KM of the substrate by MT-SP1 cleavage is at least 1 × 104M-1s-1.

29. The cleavable polypeptide of any one of claims 1-8 and 15, wherein the CM has a higher catalytic efficiency (kcat / KM) for MT-SP1, MMP2, and MMP9 compared to a cleavable polypeptide comprising a CM comprising a sequence of QNQALRMA (SEQ ID NO: 3).

30. The cleavable polypeptide of any one of claims 1-7, 9, and 15, wherein the CM has a higher catalytic efficiency (kcat / KM) for MMP2, MMP9, and MMP14 compared to a cleavable polypeptide comprising a CM comprising a sequence of QNQALRMA (SEQ ID NO: 3).

31. The cleavable polypeptide of any one of claims 1-7, 10, and 13-15, wherein the CM has a higher catalytic efficiency (kcat / KM) for MMP2, MMP9, and MMP14 compared to aCYTX-108-WO :: 4862-156.WO1 cleavable polypeptide comprising a CM comprising a sequence of QNQALRMA (SEQ ID NO: 3).

32. The cleavable polypeptide of any one of claims 1-7, 11, and 15, wherein the CM has a higher catalytic efficiency (kcat / KM) for MMP2 and MMP9 compared to a cleavable polypeptide comprising a CM comprising a sequence of QNQALRMA (SEQ ID NO: 3).

33. The cleavable polypeptide of any one of claims 1-8, 10-12, and 15, wherein the CM retains about 100%, about 99%, about 95%, or about 90% cleavage by MT-SP1 under oxidizing conditions compared to non-oxidizing conditions, wherein the oxidizing conditions comprise incubation with 0.05% tert-butyl hydroperoxide (tBHP) at 40 ºC for 3h.

34. The cleavable polypeptide of any one of claims 1-7, 9, and 14-15, wherein the CM has 5% to 50%, 10% to 40%, or 20 to 30% lower methionine oxidation under oxidizing conditions compared to a cleavable polypeptide comprising a CM comprising a sequence of QNQALRMA (SEQ ID NO: 3), wherein the oxidizing conditions comprise incubation with 0.05% tert-butyl hydroperoxide (tBHP) at 40 ºC for 3h.

35. The cleavable polypeptide of any one of claims 1-7, 12, and 15, wherein the CM has 5% to 50%, 10% to 40%, or 20 to 30% lower asparagine deamidation under deamidating conditions compared to a cleavable polypeptide comprising a CM comprising a sequence of QNQALRMA (SEQ ID NO: 3), wherein the deamidation conditions comprise incubation at pH 8.5 and 40°C for 7 days.

36. The cleavable polypeptide of any preceding claims, wherein the cleavable polypeptide comprises at least one additional moiety (M) selected from a moiety that is located amino (N) terminally to the CM (Mn), a moiety that is located carboxyl (C) terminally to the CM (Mc), or a combination thereof.CYTX-108-WO :: 4862-156.WO1 37. The cleavable polypeptide of any preceding claims, wherein the Mn and the Mc are each independently selected from a masking moiety (MM), a target binding moiety (TBM), a therapeutic agent, a detectable moiety, a diagnostic agent, or an affinity tag.

38. The cleavable polypeptide of claim 37, wherein the cleavable polypeptide comprises the MM coupled directly or indirectly to the N-terminus of the CM and the TBM coupled directly or indirectly to the C-terminus of the CM, or wherein cleavable polypeptide comprises the TBM coupled directly or indirectly to the N-terminus of the CM and the MM coupled directly or indirectly to the C-terminus of the CM.

39. The cleavable polypeptide of claim 38, wherein the MM inhibits binding of the TBM to its target.

40. The cleavable polypeptide of claim 38, wherein the amino acid sequence of the MM is different from that of the target.

41. The cleavable polypeptide of claim 38, wherein the amino acid sequence of the MM is different from that of the target and is no more than 50% identical to the amino acid sequence of a natural binding partner of the TBM.

42. The cleavable polypeptide of any one of claims 37-41, wherein the MM has a dissociation constant for binding to the TBM that is greater than the dissociation constant of the TBM for binding to the target.

43. The cleavable polypeptide of any one of claims 37-42, wherein the MM is 2 to 40 amino acids in length.

44. The cleavable polypeptide of any one of claims 37-43, wherein the MM is 20 to 200 amino acids in length.CYTX-108-WO :: 4862-156.WO1 45. The cleavable polypeptide of any one of claims 37-44, wherein the cleavable polypeptide comprises the structural arrangement from N-terminus to C-terminus as follows: MM-CM- TBM or TBM-CM-MM.

46. The cleavable polypeptide of any one of claims 37-45, wherein the CM directly couples the MM to the TBM.

47. The cleavable polypeptide of any one of claims 37-45, wherein the CM is coupled to the MM via a linker peptide (LP).

48. The cleavable polypeptide of any one of claims 35-45, wherein the CM is coupled to the TBM via an LP.

49. The cleavable polypeptide of any one of claims 47-48, wherein the cleavable polypeptide comprises a first linking peptide (LP1) and a second linking peptide (LP2), and wherein the cleavable polypeptide has a structural arrangement from N-terminus to C-terminus as follows: MM-LP1-CM-LP2-TBM.

50. The cleavable polypeptide of any one of claims 37-49, wherein the TBM is a biologically active protein.

51. The cleavable polypeptide of claim 50, wherein the biologically active protein is a cytokine or a functional fragment thereof 52. The cleavable polypeptide of any one of claims 37-49, wherein the TBM is a chimeric antigen receptor.

53. The cleavable polypeptide of any one of claims 37-49, wherein the TBM is a therapeutic macromolecule.CYTX-108-WO :: 4862-156.WO1 54. The cleavable polypeptide of any one of claims 37-49, wherein the TBM is an antibody or antigen binding fragment thereof.

55. The cleavable polypeptide of claim 54, wherein the antigen binding fragment thereof is selected from the group consisting of a Fab fragment, a F(ab')2 fragment, a scFv, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody.

56. The cleavable polypeptide of claim 54, wherein the antibody is bivalent antibody.

57. The cleavable polypeptide of claim 54, wherein the antibody is a multivalent antibody.

58. The cleavable polypeptide of claim 49, wherein the LP1 and the LP2 are not identical to each other.

59. The cleavable polypeptide of claim 49, wherein the LP1 and the LP2 are identical to each other.

60. The cleavable polypeptide of any one of claims 49 and 58-59, wherein the LP1 and the LP2 are each independently 1 to 20 amino acids in length.

61. The cleavable polypeptide of any preceding claim, wherein the cleavable polypeptide further comprises a half-life extending moiety (EM).

62. The cleavable polypeptide of 61, wherein the EM is an Fc region of an IgG, a serum albumin, a polyethylene glycol molecule, an antigen binding polypeptide that binds human serum albumin, or transferrin.

63. The cleavable polypeptide of any preceding claim, further comprising an agent conjugated to the cleavable polypeptide.CYTX-108-WO :: 4862-156.WO1 64. The cleavable polypeptide of claim 63, wherein the agent is conjugated to the cleavable polypeptide via a conjugating linker.

65. The cleavable polypeptide of claim 64, wherein the conjugating linker is cleavable.

66. The cleavable polypeptide of claim 64, wherein the conjugating linker is non-cleavable.

67. The cleavable polypeptide of any one of claims 63-66, wherein the agent is selected from the group consisting of: a toxin, a microtubule inhibitor, a nucleic acid damaging agent, a dolastatin, an auristatin, a maytansinoid, a duocarmycin, and a calicheamicin.

68. The cleavable polypeptide of claim 67, wherein the agent is a toxin.

69. The cleavable polypeptide of claim 67, wherein the agent is a microtubule inhibitor.

70. The cleavable polypeptide of claim 67, wherein the agent is a nucleic acid damaging agent.

71. The cleavable polypeptide of claim 67, wherein the agent is a dolastatin.

72. The cleavable polypeptide of claim 67, wherein the agent is an auristatin.

73. The cleavable polypeptide of claim 67, wherein the agent is a maytansinoid.

74. The cleavable polypeptide of claim 67, wherein the agent is a duocarmycin.

75. The cleavable polypeptide of claim 67, wherein the agent is a calicheamicin.

76. The cleavable polypeptide of any one of claims 38-75, wherein the TBM binds to a tumor-associated moiety.CYTX-108-WO :: 4862-156.WO1 77. The cleavable polypeptide of claim 38, wherein the cleavable polypeptide has an in vivo antitumor activity that is equal to or higher than a cleavable polypeptide comprising a CM comprising a sequence of QNQALRMA (SEQ ID NO: 3).

78. The cleavable polypeptide of claim 77, wherein the antitumor activity is a reduction in tumor volume of 25% or more, 30% or more, 40% or more, 50% or more 60% or more, 70% or more, 75% or more, 80% or more, or 90% or more.

79. The cleavable polypeptide of any one of claims 76-78, wherein the TBM binds epidermal growth factor receptor (EGFR).

80. An activatable molecule comprising: a) the cleavable polypeptide of any one of claims 1-35; b) a masking moiety (MM); and c) a target binding moiety (TBM); and d) an optional half-life extending moiety (EM); wherein the cleavable polypeptide comprises the MM coupled directly or indirectly to the N-terminus of the CM and the TBM coupled directly or indirectly to the C-terminus of the CM, or wherein cleavable polypeptide comprises the TBM coupled directly or indirectly to the N-terminus of the CM and the MM coupled directly or indirectly to the C-terminus of the CM.

81. The activatable molecule of claim 80, further comprising an agent conjugated to the activatable molecule 82. The activatable molecule of claim 81, wherein the agent is conjugated to the cleavable polypeptide via a conjugating linker.

83. The activatable molecule of claim 82, wherein the conjugating linker is cleavable.

84. The activatable molecule of claim 82, wherein the conjugating linker is non-cleavable.CYTX-108-WO :: 4862-156.WO1 85. The activatable molecule of any one of claims 81-84, wherein the agent is selected from the group consisting of: a toxin, a microtubule inhibitor, a nucleic acid damaging agent, a dolastatin, an auristatin, a maytansinoid, a duocarmycin, and a calicheamicin.

86. The activatable molecule of any one of claims 80-85, wherein the TBM binds to a tumor- associated moiety.

87. The activatable molecule of claim 86, wherein the activatable molecule has an in vivo antitumor activity that is equal to or higher than a cleavable polypeptide comprising a CM comprising a sequence of QNQALRMA (SEQ ID NO: 3).

88. The activatable molecule of claim 87, wherein the antitumor activity is a reduction in tumor volume of 25% or more, 30% or more, 40% or more, 50% or more 60% or more, 70% or more, 75% or more, 80% or more, or 90% or more.

89. The activatable molecule of any one of claims 80-88, wherein the TBM binds epidermal growth factor receptor (EGFR).

90. A nucleic acid composition comprising one or more nucleic acid molecules encoding: a) the cleavable polypeptide of any one of claims 1-35; b) a masking moiety (MM); and c) a target binding moiety (TBM); and d) optionally a half-life extending moiety (EM); wherein the MM is coupled directly or indirectly to the N-terminus of the CM and the TBM is coupled directly or indirectly to the C-terminus of the CM, or wherein the TBM is coupled directly or indirectly to the N-terminus of the CM and the MM is coupled directly or indirectly to the C-terminus of the CM; and a carrier.

91. The nucleic acid composition of claim 90, wherein the EM is present and is coupled directly to the TBM.CYTX-108-WO :: 4862-156.WO1 92. The nucleic acid composition of claim 90, wherein the EM is present and is coupled indirectly to the TBM.

93. The nucleic acid composition of claim 90, wherein the EM is present and is encoded by an additional nucleic acid molecule.

94. The activatable molecule of any one of claims 80-89 or the nucleic acid composition of any one of claims 90-93, having a structural arrangement from N-terminus to C-terminus as follows: MM-CM-TBM or TBM-CM-MM, wherein “-” is a direct linkage or indirect linkage via one or more linking peptides.

95. The activatable molecule of any one of claims 80-89 or the nucleic acid composition of any one of claims 90-93, wherein the CM directly couples the MM to the TBM.

96. The activatable molecule of any one of claims 80-89 or the nucleic acid composition of any one of claims 90-93, wherein the CM is coupled to the MM via a linker peptide (LP).

97. The activatable molecule of any one of claims 80-89 or the nucleic acid composition of any one of claims 90-93, wherein the CM is coupled to the TBM via an LP.

98. The activatable molecule of any one of claims 80-89 or the nucleic acid composition of any one of claims 90-93, comprising a first linking peptide (LP1) and a second linking peptide (LP2), and a structural arrangement from N-terminus to C-terminus as follows: MM-LP1-CM-LP2-TBM, and wherein (i) LP1 and LP2 are the same or different; (ii) the LP1 and the LP2 are each independently 1 to 20 amino acids in length, or (iii) a combination of (i) and (ii).

99. The activatable molecule of any one of claims 80-89 or the nucleic acid composition of any one of claims 90-93, having an EM, wherein the EM is selected from the Fc region of an IgG, serum albumin, an antigen binding polypeptide that binds human serum albumin, or transferrin.CYTX-108-WO :: 4862-156.WO1 100. The activatable molecule of any one of claims 80-89 or the nucleic acid composition of any one of claims 90-93, wherein the TBM is an antibody or antigen binding fragment thereof.

101. The activatable molecule of any one of claims 80-89 or the nucleic acid composition of any one of claims 90-93, wherein the TBM is an antigen binding fragment selected from the group consisting of a Fab fragment, a F(ab')2 fragment, a scFv, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody.

102. The activatable molecule of any one of claims 80-89 or the nucleic acid composition of any one of claims 90-93, wherein the activatable molecule comprises a heavy chain amino acid sequence according to SEQ ID NO: 41 and a light chain amino acid sequence selected from the group consisting of SEQ ID NOs: 45-51.

103. The nucleic acid composition of any one of claims 90-93, wherein the CM is encoded by a sequence selected from SEQ ID NOs: 67-73.

104. The nucleic acid composition of any one of claims 90-93, wherein the one or more nucleic acid sequences comprise a sequence according to SEQ ID NO: 53 and a sequence selected from the group consisting of SEQ ID NOs: 57-63.

105. A composition comprising the cleavable polypeptide of any one of claims 1-79 or the activatable molecule of any one of claims 80-89 and a pharmaceutically acceptable carrier.

106. The composition of claim 105, further comprising at least one additional agent.

107. The composition of claim 106, wherein the at least one additional agent is a therapeutic agent.CYTX-108-WO :: 4862-156.WO1 108. A nucleic acid molecule or group of nucleic acid molecules encoding the cleavable polypeptide of any one of claims 1-79 or the activatable molecule of any one of claims 80-89.

109. A vector or group of vectors comprising the nucleic acid molecule or group of nucleic acid molecules of claim 108 or the nucleic acid composition of any one of claims 90-93.

110. A recombinant cell comprising the cleavable polypeptide of any one of claims 1-79, the activatable molecule of any one of claims 80-89, the nucleic acid composition of any one of claims 90-93, the nucleic acid or group of nucleic acid molecules of claim 108, or the vector or group of vectors comprising of claim 109.

111. An in vitro method of manufacturing the cleavable polypeptide of any one of claims 1-79 or the activatable molecule of any one of claims 80-89, comprising culturing the recombinant cell of claim 110 under conditions suitable for expressing the cleavable polypeptide or the polypeptide complex and recovering the cleavable polypeptide.

112. A method of treating, alleviating a symptom of, or delaying the progression of a disease or disorder in a subject, comprising administering a therapeutically effective amount of the cleavable polypeptide of any one of claims 1-79, the activatable molecule of any one of claims 80-89, or the composition of any one of claims 105-107 to the subject.

113. The method of claim 112, wherein the disease or disorder is a cancer, an infection, an inflammatory disorder, a cardiovascular disorder, a neurodegenerative disorder, or an autoimmune disorder.

114. The method of claim 112 or claim 113, wherein the disease is a cancer.CYTX-108-WO :: 4862-156.WO1 115. The cleavable polypeptide of any one of claims 1-79, the activatable molecule of any one of claims 80-89, or the composition of any one of claims 105-107 for use as a medicament or for use in therapy, optionally for treating a cancer, an infection, an inflammatory disorder, a cardiovascular disorder, a neurodegenerative disorder, or an autoimmune disorder, optionally with an additional agent which is optionally a therapeutic agent.

116. A container, vial, syringe, injector pen, or kit comprising at least one dose of the composition of the cleavable polypeptide of any one of claims 1-79 or the activatable molecule of any one of claims 80-89, or the composition of any one of claims 105-107.

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