Activatable double anchoring masking molecules and methods of use thereof

Abstract

Provided herein are activatable target binding proteins comprising a target binding protein (TB) that specifically binds to a target; a masking moiety (MM) coupled to the TB, wherein the MM and the TB are tethered by a non-alpha carbon covalent bond and the MM inhibits binding of the TB to the target when a target binding protein can be activated; and a cleavable moiety (CM) coupled to the TB, wherein the CM is a polypeptide acting as a protease substrate and is located between the TB and the MM.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 323,718, filed on March 25, 2022, which is incorporated herein by reference in its entirety.

[0003] Sequence Listing

[0004] The sequence listing submitted by EFS together with this application is named "4862-121PCT.xml", created on March 22, 2023, with a size of 500,152 bytes, and is incorporated herein by reference in its entirety. Technical Field

[0005] The present disclosure relates to the field of biotechnology, and more particularly, to activatable molecules. Background Art

[0006] Antibody-based therapies have provided effective treatments for a variety of diseases. However, in some cases, toxicity resulting from widespread target expression limits their therapeutic efficacy. In addition, antibody-based therapeutics also exhibit other limitations, such as rapid clearance from the circulation after administration.

[0007] An activatable antibody comprising a masking peptide bound to an antibody is a therapeutic agent with lower toxicity than a conventional antibody. The masking body can inhibit the activity of the antibody by blocking the binding of the antibody to its target molecule. In an activation environment (for example, when the activatable antibody is delivered to a tumor), the masking body is removed so that the antibody can bind to its target molecule to restore its function. However, if a portion of the activatable antibody is in an unmasked state, allowing target binding outside the activation environment, toxicity may occur.

[0008] Therefore, there is a constant need for activatable molecules to minimize target binding outside of the activation context. Summary of the invention

[0009] The present disclosure provides dual-anchored activatable target binding proteins and related compositions and methods.

[0010] In one aspect, the present disclosure provides a dual-anchored activatable target binding protein, comprising: a target binding protein (TB) that specifically binds to a target; a masking moiety (MM) coupled to the TB, wherein the MM inhibits the binding of the TB to the target; and a cleavable moiety (CM) coupled to the TB and located between the TB and the MM, wherein the CM is a polypeptide that acts as a protease substrate and further comprises a non-α carbon covalent bond that tethers the MM and the TB.

[0011] In some embodiments, TB is an antigen binding protein (AB). In some embodiments, the activatable target binding protein has a lower target binding activity than a single anchored activatable target binding protein lacking a non-α carbon covalent bond. In some embodiments, the non-α carbon covalent bond is an isopeptide bond. In some embodiments, the isopeptide bond is between a lysine and a glutamic acid or aspartic acid residue. In some embodiments, the non-α carbon covalent bond is between a functional group substituted to the α carbon in MM and AB. In some embodiments, the isopeptide bond is between the γ-carboxamide group of glutamine and the ε-amino group of the lysine side chain. In some embodiments, the non-α carbon covalent bond is an ester bond between threonine and glutamine. In some embodiments, the non-α carbon covalent bond is a thioester bond between cysteine ​​and glutamine. In some embodiments, the non-α carbon covalent bond is a thioether bond between cysteine ​​and tyrosine. In some embodiments, the non-α carbon covalent bond is formed by a cross-link between histidine and tyrosine (e.g., this type of histidine-tyrosine cross-link is known to exist in cytochrome c oxidase). In some embodiments, the non-alpha carbon covalent bond is a nitrogen-oxygen-sulfur (NOS) bond formed between lysine and cysteine. In some embodiments, the non-alpha carbon covalent bond is a disulfide bond. In some embodiments, the disulfide bond is formed between a first cysteine ​​and a second cysteine, wherein the first cysteine ​​is within the MM and the second cysteine ​​is within the TB, the first cysteine ​​is within a peptide coupled to the MM and the second cysteine ​​is within the TB, or the first cysteine ​​is within the MM and the second cysteine ​​is within a peptide coupled to the TB.

[0012] In some embodiments, the activatable target binding protein further comprises a second CM, wherein the second CM is located between the MM and the non-alpha carbon covalent bond, the second CM is within the MM and is at most 5 amino acids away from the cysteine ​​that forms the non-alpha carbon covalent bond, or the second CM is within the TB and is at most 5 amino acids away from the cysteine ​​that forms the non-alpha carbon covalent bond. In some embodiments, the first CM and the second CM are substrates for different proteases. In some embodiments, the first CM and the second CM are substrates for the same protease.

[0013] In some embodiments, the protease is produced by a tumor of the subject. In some embodiments, AB is an antibody, a Fab fragment, a F(ab') 2Fragment, scFv, scAb, dAb or single domain antibody. In some embodiments, AB is a single domain antibody. In some embodiments, AB is a single domain antibody tagged with Fc. In some embodiments, AB is a bispecific antibody. In some embodiments, the bispecific antibody is a bispecific T cell engager (BiTE) or a dual affinity retargeting antibody (DART). In some embodiments, AB is a multispecific antibody. In some embodiments, a non-α carbon covalent bond is between the MM and the single domain antibody. The present disclosure includes a double-anchored activatable macromolecule comprising a bispecific or multispecific AB, wherein each AB in the bispecific or multispecific AB has a double-anchored MM. The present disclosure also includes a double-anchored activatable macromolecule comprising a bispecific or multispecific AB, wherein at least one AB in the bispecific or multispecific AB has a double-anchored MM and at least one AB in the bispecific or multispecific AB has a single-anchored MM. The present disclosure also includes a dual-anchored activatable macromolecule comprising a bispecific or multispecific AB, wherein at least one of the bispecific or multispecific ABs has a dual-anchored MM and at least one of the bispecific or multispecific ABs does not have a MM.

[0014] In some embodiments, a non-alpha carbon covalent bond is between the MM and the fragment crystallizable region (Fc) region or domain coupled to TB. In some embodiments, the MM may comprise an epitope of TB. In some embodiments, the MM does not comprise a subsequence of four or more consecutive amino acid residues of native TB. In some embodiments, the MM does not comprise a subsequence of four or more consecutive amino acid residues of a target bound by TB. In some embodiments, the MM may comprise a subsequence of less than four consecutive amino acid residues of native TB. In some embodiments, the MM does not comprise an epitope of TB. In some embodiments, the dissociation constant for binding of MM to TB exceeds the dissociation parameter for binding of TB to the target. In some embodiments, the MM is a polypeptide having a length of 2 to 40 amino acids.

[0015] In some embodiments, the activatable target binding protein comprises a linker between MM and CM. In some embodiments, the activatable target binding protein comprises a linker between CM and TB. In some embodiments, the activatable target binding protein comprises a first linker between MM and CM and a second linker between CM and TB.

[0016] In another aspect, the disclosure provides a composition comprising an activatable target binding protein herein. In some embodiments, the composition is a pharmaceutical composition.

[0017] In another aspect, the present disclosure provides a container, a vial, a syringe, an injection pen or a kit comprising at least one dose of a composition herein.

[0018] In another aspect, the disclosure provides a nucleic acid comprising a sequence encoding the activatable target binding protein herein.

[0019] In another aspect, the present disclosure provides a vector comprising a nucleic acid herein.

[0020] In another aspect, the disclosure provides a cell comprising a nucleic acid or a vector herein.

[0021] In another aspect, the disclosure provides a conjugated activatable target binding protein comprising an activatable target binding protein herein conjugated to an agent. In some embodiments, the agent is a therapeutic agent, a targeting moiety, or a detectable moiety.

[0022] In another aspect, the disclosure provides a method of treating a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an activatable target binding protein, composition, or conjugated activatable target binding protein herein. In some embodiments, the subject has been identified or diagnosed as having cancer.

[0023] In another aspect, the present disclosure provides a method of producing an activatable target binding protein, the method comprising: culturing cells in a culture medium under conditions sufficient to produce the activatable target binding protein; and recovering the activatable target binding protein from the cells or the culture medium.

[0024] In some embodiments, the method further comprises isolating the activatable target binding protein recovered from the cells or culture medium. In some embodiments, isolating the activatable target binding protein is performed using a protein purification tag and / or size exclusion chromatography. In some embodiments, the method further comprises formulating the activatable target binding protein into a pharmaceutical composition.

[0025] In another aspect, the present disclosure provides a method for producing a dual-anchored activatable protein, the method comprising: engineering a cysteine ​​residue at a disulfide bonding site in a masking moiety (MM) of the dual-anchored activatable protein; engineering a cysteine ​​residue at a disulfide bonding site in a target binding protein (TB) of the dual-anchored activatable protein, wherein the MM is coupled to the TB and a cleavable moiety (CM) is located between the MM and the TB; expressing the dual-anchored activatable protein; and recovering the dual-anchored activatable protein, wherein in the recovered dual-anchored activatable protein, the MM and the TB are tethered at their disulfide bonding sites. In the present disclosure, the phrases dual-anchored activatable protein and dual-anchored activatable macromolecule are used interchangeably.

[0026] In another aspect, the present disclosure provides a method for producing a dual-anchored activatable macromolecule, the method comprising: engineering an arginine or lysine residue at an isopeptide bonding site in a masking portion (MM) of the dual-anchored activatable macromolecule and / or engineering an aspartic acid or glutamic acid residue at an isopeptide bonding site in a target binding protein (TB) of the dual-anchored activatable macromolecule, wherein the MM is coupled to the TB and the cleavable portion (CM) is located between the MM and the TB; expressing the dual-anchored activatable macromolecule; and recovering the dual-anchored activatable macromolecule, wherein in the recovered dual-anchored activatable macromolecule, the MM and the TB are tethered at their isopeptide bonding sites. In another aspect, the present disclosure provides a method for producing a dual-anchored activatable macromolecule, the method comprising: engineering an isopeptide bonding site so that the γ-carboxamide group of glutamine is available and configured to form an isopeptide bond with the ε-amino group of a lysine side chain.

[0027] In another aspect, the present disclosure provides a method for producing a dual-anchored activatable macromolecule, the method comprising: engineering an aspartic acid or glutamic acid residue at the isopeptide bonding site in a masking portion (MM) of the dual-anchored activatable macromolecule and / or engineering an arginine or lysine residue at the isopeptide bonding site in a target binding protein (TB) of the dual-anchored activatable macromolecule, wherein the MM is coupled to the TB and a cleavable portion (CM) is located between the MM and the TB; expressing the dual-anchored activatable macromolecule; and recovering the dual-anchored activatable macromolecule, wherein in the recovered dual-anchored activatable macromolecule the MM and the TB are tethered at their isopeptide bonding sites.

[0028] In another aspect, the present disclosure provides a method for making a dual-anchored activatable macromolecule, the method comprising providing an MM, the MM comprising an amino acid that forms a non-α-carbon covalent bond, the amino acid that forms a non-α-carbon covalent bond being configured to form a non-α-carbon covalent bond with an amino acid that forms a non-α-carbon covalent bond in a TB, the TB being coupled to the MM.

[0029] In another aspect, the present disclosure provides a method of making a dual-anchored activatable macromolecule, the method comprising providing a MM comprising a cysteine ​​configured to form a non-α carbon covalent bond with an amino acid in a TB that forms a non-α carbon covalent bond, the TB being coupled to the MM. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description which sets forth illustrative embodiments utilizing the principles of the invention and the accompanying drawings, in which:

[0031] Figure 1AThe dynamic equilibrium between the conformational state of complete masker binding (left), the intermediate conformational state of dynamic dissociation of one masker from the binding site of the target binding protein (or antibody) (middle), and the conformational state of dynamic dissociation of two maskers from the binding site of the target binding protein (or antibody) (right) is schematically shown.

[0032] Figure 1B The arrangement of components in an example activatable antibody is shown. The line connecting MM and AB represents a non-alpha carbon covalent bond. Although AB is used as an example, the present disclosure includes the use of any desired protein, is not limited to antibodies, and can include any target binding protein (TB).

[0033] Figures 2A-2D Schematic representation of example activatable molecules is shown.

[0034] Figures 3A-3C Three types of example activatable molecules are shown.

[0035] Figure 4A is a schematic diagram of an illustrative dual-anchored BC2T-Nb, in which the N-terminus of the MM is covalently linked to the Nb via an engineered non-alpha carbon covalent bond (e.g., a disulfide bond), and the C-terminus of the MM is covalently linked to the Nb via the CM. Figure 4B is a schematic diagram of single-anchored BC2T-Nb.

[0036] Figure 4C The cleavage reaction of the doubly anchored BC2T-Nb is depicted, resulting in the MM being single-anchored by an engineered non-alpha carbon covalent bond (e.g., a disulfide bond). The BC2T-Nb molecule is illustrated as a non-limiting proof-of-concept example, and one skilled in the art will appreciate that the structures depicted and described in this disclosure extend to the use of any type of masking moiety and any desired protein without undue experimentation.

[0037] Figure 5A is a schematic diagram of an illustrative dimeric Fc-tagged version of a dual-anchored BC2T-Nb, wherein the N-terminus of the MM is covalently linked to the Nb via an engineered non-alpha carbon covalent bond (eg, a disulfide bond), and the C-terminus of the MM is covalently linked to the Nb via a CM. Figure 5B is a schematic diagram of a dimeric Fc-tagged control molecule (ie, MM and Nb are not tethered via a non-alpha carbon covalent bond, such as a disulfide bond) to show the difference in binding between dual-anchored BC2T-Nb and single-anchored BC2T-Nb. Figure 5C The cleavage reaction of the Fc-tagged dual-anchored BC2T-Nb is depicted to cause the MM to be single-anchored by an engineered non-alpha carbon covalent bond (e.g., a disulfide bond). The BC2T-Nb molecule is illustrated as a non-limiting proof-of-concept example, and those skilled in the art will appreciate that the structures depicted and described in this disclosure extend to the use of any type of masking moiety and any desired protein without undue experimentation.

[0038] Figure 6 is an image of an SDS-PAGE gel run under non-reducing conditions. The gel was loaded as follows: (1) a single domain antibody without MM attached (ProC649; SEQ ID NO: 1); (2) the product of ProC649 and MMP14 (ProC649+MMP14); (3) the product of ProC649 and MMP9 (ProC649+MMP9); (4) a single domain antibody with MM attached to CM 1490DNI (ProC653; SEQ ID NO: 2); (5) the product of ProC653 and MMP14 (ProC653+MMP14); (6) a single domain antibody with MM attached to CM PLGLAG (SEQ ID NO: 17) (ProC654; SEQ ID NO: 3); (7) the product of ProC654 and MMP9 (ProC654+MMP9); (8) MMP14; and (9) MMP9.

[0039] Figure 7 Sensorgram trace binding of intact and activated ProC653 and ProC654 and ProC649 control is shown. ProC649 without MM attached binds to the biotinylated BC2 tag peptide on the biosensor tip. Intact ProC653 and ProC654 do not bind to the biotinylated BC2 tag peptide, but binding is restored after activation with MMP14 or MMP9, respectively.

[0040] Figure 8are images of SDS-PAGE gels run under non-reducing conditions (top) and reducing conditions (bottom). The gels were loaded as follows: (1) single domain antibody without MM attached (ProC649); (2) single domain antibody with MM attached with CM 1490DNI (ProC653); (3) product of ProC653 and uPA (ProC653+uPA); (4) single domain antibody with MM double anchored with engineered cysteines Q3C and Q157C and CM 1490DNI (ProC994; SEQ ID NO: 4); (5) product of ProC994 and uPA (ProC994+uPA); (6) single domain antibody with MM double anchored with engineered cysteines Q3C and W155C and CM1490DNI (ProC995; SEQ ID NO: NO:5); (7) the product of ProC995 and uPA (ProC995+uPA); (8) a single domain antibody having MM double anchored by engineered cysteines D3C and F154C and CM 1490DNI (ProC996; SEQ ID NO:6); (9) the product of ProC996 and uPA (ProC996+uPA).

[0041] Fig. 9 Are images of SDS-PAGE gels run under non-reducing conditions (top) and reducing conditions (bottom). The gel was loaded as follows: (1) Fc-tagged single domain antibody without MM attached (ProC1283; SEQ ID NO:7); (2) the product of ProC1283 and uPA (ProC1283+uPA); (3) Fc-tagged single domain antibody with MM attached with CM 1490DNI (ProC1284; SEQ ID NO:8); (4) the product of ProC1284 and uPA (ProC1284+uPA); (5) Fc-tagged single domain antibody with MM dually anchored with engineered cysteines Q3C and Q157C and CM 1490DNI (ProC1285; SEQ ID NO:9); (6) the product of ProC1285 and uPA (ProC1285+uPA); (7) 1490DNI dual-anchored MM Fc-tagged single domain antibody (ProC1287; SEQ ID NO: 11); (8) the product of ProC1287 and uPA (ProC1287+uPA); (9) uPA.

[0042] Fig.10Results of ELISA binding assays are presented to determine changes in the ability of molecules to bind free masked peptides bound on the plate: Fc-tagged single domain antibody without MM attached (ProC1283), the product of ProC1283 and uPA (ProC1283+uPA), Fc-tagged single domain antibody with MM attached to CM 1490DNI (ProC1284), the product of ProC1284 and uPA (ProC1284+uPA), Fc-tagged single domain antibody with MM dually anchored with engineered cysteines Q3C and Q157C and CM1490DNI (ProC1285); the product of ProC1285 and uPA (ProC1285+uPA), the product of ProC1285 and uPA with engineered cysteines D3C and F154C and CM1490DNI. 1490DNI dual anchored MM Fc-tagged single domain antibody (ProC1287), and the product of ProC1287 and uPA (ProC1287+uPA). The results show that the single domain antibody with dual anchor mask cannot bind to the peptide on the plate. However, treatment of the dual anchor molecule with protease restored molecular binding similar to that of the single anchor molecule (ProC1283).

[0043] Figures 11A-11I An exemplary dual-anchored multispecific activatable antibody is depicted. The ovals indicate components of the AB, which may be the heavy chain variable region (V H ), light chain variable region (V L ), heavy chain constant region (C H ), light chain constant region (C L ), a single variable domain on the heavy chain (VHH) or a single chain variable fragment (scFv). The triangle indicates the MM. The MM is coupled to the AB component via the CM (with an optional linker) and is tethered to the AB via a disulfide bond (or a non-alpha carbon covalent bond) (with an optional linker and / or CM between the MM and the residue that forms a non-alpha carbon covalent bond with the AB).

[0044] Figures 12A-12B Depicted is the three-dimensional structure of the activatable target binding protein (activatable anti-PDL1 antibody; SEQ ID NOs: 562-563) obtained by BIOVIA Discovery Studios using Dessault Systemes software. Fig. 12A A zoomed-in region of the structure is shown, where solvent accessible residues are within 2-5 angstroms of the head region or N-terminal residues of the masked moiety, and the second view is a 90 degree rotation of the structure. Residues identified by mutagenesis as interacting with the masker are indicated with asterisks. Fig. 12BThe three-dimensional structures of the Fab and propeptide domains (including labeled masking moieties) are shown. The Fab domain is presented in space-filling format, while the propeptide domain is presented in Cα scaffold format.

[0045] Figures 13A-13D Depicted are the J43v2 / anti-mouse PD1 Fab ( Fig.13A ; SEQ ID NO: 568-569), anti-CD166 ( Fig. 13B ; SEQ ID NO: 572-573) and anti-PD1 ( Figures 13C-13D ; SEQ ID NO:570-571) of the antibody structure three-dimensional model based on homology. Figures 13A-13C Modeling using BIOVIA Discovery Studio, Fig.13D Modeled using AlphaFold2 and presented in BIOVIA Discovery Studio. CDRs in each figure are shown in dark grey.

[0046] The figures herein are for illustration purposes only and are not necessarily drawn to scale. DETAILED DESCRIPTION

[0047] Overview

[0048] Provided herein are activatable molecules comprising at least one masker that is directly or indirectly anchored at two points to at least one polypeptide of an otherwise active binding moiety ("activatable dual-anchor masked target binding protein" or "activatable target binding protein"). In some aspects detailed and depicted in the present disclosure, the activatable target binding protein comprises a complex of more than one polypeptide. In some aspects detailed and depicted in the present disclosure, the masker is covalently bonded to one polypeptide of the activatable target binding protein at two anchor positions. In some aspects detailed and depicted in the present disclosure, the masker is covalently bonded to two polypeptides of the activatable target binding protein, for example, anchored to the first polypeptide at one anchor position and anchored to the second polypeptide at the second anchor position. In solution, masked activatable target binding protein molecules lacking the dual anchor structures described herein exist in a dynamic equilibrium state between a conformational state in which one or more masks are actively bound to the binding site of the target binding protein and a conformational state in which one or more masks are not actively bound to the binding site of the target binding protein, such as Figure 1AThis dynamic equilibrium may be referred to herein as "breathing" of the masked activatable target binding protein molecule, wherein one or more binding surfaces of one or more target binding proteins in the masked activatable target binding protein molecule are available for binding to a target or other epitope (including target binding outside the activation environment). Breathing occurs when an antigen binding site is briefly exposed to a portion of the intact molecule due to equilibrium binding of the tethered masker, such as Figure 1A Depicted. The dual-anchor masking structures described herein reduce or inhibit the dynamic dissociation between the masker and the target binding protein, i.e., the "breathing" of the target binding protein molecule can be activated. The dual-anchor masker appears to mask other target binding proteins, improve the masking efficiency and / or reduce the population of molecules in which one or more maskers dynamically dissociate from their corresponding target binding proteins.

[0049] In one aspect, the activatable molecule can be an activatable therapeutic macromolecule ("activatable target binding protein"). In some aspects, the activatable therapeutic macromolecule can be an activatable antibody or any other desired protein, such as a therapeutic protein. The activatable molecule can comprise a target binding protein (TB), a masking moiety (MM), and a cleavable moiety (CM) located between the MM and the TB. In some aspects, the activatable molecule can comprise more than one CM, such as Figure 2C and 2D As shown. For example, the activatable molecule can include a first cleavable portion (CM1) and a second cleavable portion (CM2). In some aspects, the activatable molecule can have a structure including CM1 between MM and TB and including CM2 between MM and a residue that forms a non-α carbon covalent bond with the activatable molecule. Therefore, in some aspects, the present disclosure includes a TB-CM1-MM-CM2 construct, wherein the cleavage of CM1 and CM2 completely cleaves MM from two anchoring sites in the activatable molecule. In some aspects, the cleavage of CM1 and CM2 causes the complete activation of the activatable target binding protein. In some aspects, the cleavage of CM1 and CM2 is necessary for the complete activation of the activatable target binding protein. In some aspects, the cleavage of one of CM1 and CM2 is sufficient to activate the activatable target binding protein.

[0050] In some aspects, the activatable antibody used in the context of the activatable dual-anchored masked antibody disclosed herein may comprise an antigen binding protein (AB), a masking moiety (MM), and one or more cleavable moieties (CM) located between MM and AB. In general, the activatable molecules herein may be dual-anchored, i.e., MM and TB (e.g., AB) are coupled via CM (or CM1 and CM2) and are also tethered by one or more non-α carbon covalent bonds. Such activatable molecules may have lower target binding activity compared to corresponding activatable molecules without non-α carbon covalent bonds (i.e., "single-anchored activatable molecules" or "corresponding activatable target binding proteins"). Compared to single-anchored activatable molecules without non-α carbon covalent bonds (e.g., disulfide bonds) tethering MM and TB, the enhanced masking efficiency of MM in the dual-anchored activatable molecules described herein may lead to improved safety, such as reduced toxicity and reduced target binding outside the activation environment.

[0051] Also provided herein are related compositions, kits, nucleic acids, vectors and recombinant cells, as well as related methods, including methods of using any of the activatable molecules (e.g., activatable macromolecules, such as antibodies and other proteins) described herein and methods of producing any of the activatable molecules (e.g., activatable macromolecules, such as antibodies and other proteins) described herein.

[0052] definition

[0053] 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 the invention belongs. Methods and materials for use in the present disclosure are described herein; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In the event of a conflict, the present specification, including definitions, will prevail.

[0054] The terms "a" or "an" refer to one or more (ie, at least one) grammatical object of the article. By way of example, "a cell" encompasses one or more cells.

[0055] As used herein, the terms "about" and "approximately" when used to modify a numerical value or an amount specified in a range, represent the numerical value and reasonable deviations from the value known to those skilled in the art. For example, ±20%, ±10% or ±5% may be within the intended meaning of the stated value, where appropriate.

[0056] Concentration, amount and other numerical data can be expressed or presented in range format herein.It should be understood that this type of range format is used only for convenience and simplicity, and should therefore be flexibly interpreted as not only including the numerical value of the limit value of the range explicitly stated, but also including all individual numerical values ​​or sub-ranges contained within the range, as if each numerical value and sub-range were explicitly stated.As an illustration, the numerical range of "about 0.01 to 2.0" should be interpreted as not only including the value of about 0.01 to about 2.0 explicitly stated, but also including individual values ​​and sub-ranges within the indicated range.Therefore, individual values, such as 0.5, 0.7 and 1.5, and sub-ranges, such as 0.5 to 1.7, 0.7 to 1.5 and 1.0 to 1.5, etc., are included in this numerical range.In addition, no matter how the width of the range or the features described, this explanation should apply.In addition, it should be noted that, unless otherwise specified, all percentages are calculated by weight.

[0057] In understanding the scope of the present disclosure, the term "comprising" or "including" and their derivatives as used herein are intended to specify the existence of stated features, elements, components, groups, integers and / or steps, but do not exclude the open-ended terms of the existence of other unstated features, elements, components, groups, integers and / or steps. The foregoing also applies to words with similar meanings, such as the terms "comprising", "having" and their derivatives. The term "consisting of" and its derivatives as used herein are intended to specify the existence of stated features, elements, components, groups, integers and / or steps, but exclude the closed terms of the existence of other unstated features, elements, components, groups, integers and / or steps. The term "consisting essentially of" as used herein is intended to specify the existence of stated features, elements, components, groups, integers and / or steps and those features, elements, components, groups, integers and / or steps that do not substantially affect one or more basic and novel features of features, elements, components, groups, integers and / or steps. It should be understood that reference to any of these transitional terms (i.e., "comprising," "consisting of," or "consisting essentially of") provides direct support for substitution with any other transitional term not specifically used. For example, since this definition is directed to any element disclosed throughout this disclosure, modifying the term from "comprising" to "consisting essentially of" or "consisting of" will be directly supported. Based on this definition, any element disclosed or incorporated by reference herein may be included in or excluded from the claimed invention.

[0058] As used herein, for convenience, multiple compounds, elements or steps may be presented in a common list. However, these lists should be interpreted as if each member of the list is individually identified as a separate and unique member. Therefore, individual members of such a list should not be interpreted as de facto equivalents of any other members of the same list based solely on their presentation in a common group without contrary indication.

[0059] In addition, certain molecules, constructs, compositions, elements, parts, excipients, diseases, illnesses, properties, steps, etc. may be discussed in the context of a specific embodiment or aspect or in a separate paragraph or chapter of the disclosure. It should be understood that this is only for convenience and brevity, and any such disclosure is equally applicable to any other embodiment or aspect found anywhere in the disclosure and claims, and is intended to be combined with any other embodiment or aspect described, all of which form the present application and the invention required on the filing date. For example, the list of constructs, molecules, method steps, kits, or compositions described with respect to constructs, compositions, or methods is intended to and does find direct support for the embodiments related to the constructs, compositions, preparations, and methods described in any other part of the disclosure, even if those method steps, activating agents, kits, or compositions are not re-listed in the context or chapter of the embodiment or aspect.

[0060] The term "exemplary" is used herein to mean used as an example, instance or illustration. Any aspect or design described herein as "exemplary" is not necessarily to be construed as being better or superior to other aspects or designs. On the contrary, the word exemplary is used to present concepts in a more specific way.

[0061] Activates target binding protein

[0062] In one aspect, the present disclosure provides an activatable target binding protein (TB), such as an activatable dual-anchored masked antibody ("activatable antibody") or another protein that specifically binds to a target. In some embodiments, the activatable antibody comprises a TB or antigen binding protein (AB) that specifically binds to a target; a cleavable moiety (CM) that is covalently linked (also referred to as "coupled" or "fused") directly or indirectly to the TB (e.g., AB), wherein the CM is a polypeptide that acts as a protease substrate and is located between the TB and the masking moiety (MM), wherein the MM and the TB are tethered by a non-α carbon covalent bond and the MM inhibits the binding of the TB to the target. The term "fusion" and its grammatical variants as used herein refer to the covalent connection of the α carbon backbone of the same polypeptide, such as by recombinant fusion. The term "tethered" and its grammatical variants as used herein refer to the connection of two moieties (e.g., MM and AB in an activatable antibody) by a non-α carbon covalent bond (e.g., a disulfide bond and an amide / isopeptide bond, a bond between functional groups substituted into the α carbon in the MM and AB, or other bonds that do not involve an α carbon backbone bond). As used herein, the term "anchor" and its grammatical variants can include a moiety that is directly or indirectly covalently linked to another moiety (e.g., TB and CM or MM and CM). In some aspects, the isopeptide bond can be between a lysine residue and an aspartic acid or glutamic acid residue, or between the γ-carboxamide group of glutamine and the ε-amino group of a lysine side chain.

[0063] In some embodiments, the activatable antibody provides reduced toxicity and / or adverse side effects, and if the TB is not masked or otherwise inhibited from binding to the target, the binding of the TB (e.g., AB) at a non-treatment site may cause toxicity and / or adverse side effects. In an activatable TB, the MM may interfere with the binding of the TB to its target molecule. In a single-anchored activatable antibody (e.g., a single-anchored activatable antibody to which the MM and AB are not tethered by a non-α carbon covalent bond), the masking effect of the MM on the target binding surface of the AB may be dynamic. Therefore, in a given preparation containing a single-anchored activatable TB molecule, a portion of the activatable TB molecules in the preparation may be exposed by breathing, although the duration is short. In the case where the concentration of the single-anchored activatable TB is high enough, the binding of such exposed activatable TB to the target molecule may be significant and lead to adverse toxicity or effects caused by target binding outside the activation environment.

[0064] As used herein, the term "activatable antibody" or "activatable target binding protein" refers to an activatable antibody or activatable target binding protein in an inactive (uncleaved or native) form, respectively. It is obvious to the skilled person that after the CM of the activatable antibody or activatable target binding protein is modified by at least one protease, a cleaved protein may be produced, wherein the MM does not interfere with the binding between the TB or AB and its target. In some embodiments, the cleavage of the CM by the protease may result in the release of the MM. The term "uncleaved" or "inactive" refers to an activatable TB in the absence of a protease cleavage of the CM, i.e., an activatable TB in its native form. As used throughout the present disclosure, descriptions involving activatable antibodies should be interpreted as also applicable to activatable target binding proteins. Therefore, in one aspect, the dual-anchored activatable target binding protein of the present disclosure may include an MM that inhibits the binding of AB to the target when the activatable target binding protein is in an inactive state.

[0065] As used herein, an activatable TB is "cleaved" or "activated" following modification of the CM by at least one protease. The term "activatable" when used in conjunction with the term "protein" refers to a protein that exhibits reduced binding to a target compared to a corresponding "activated" protein produced by exposing the activatable protein to a cleavage agent (e.g., a protease).

[0066] As used herein, the terms "masking moiety" and "MM" are used interchangeably herein and refer to a peptide or protein that, when positioned in the vicinity of a TB (eg, AB), interferes with the binding of a TB to its target.

[0067] The terms "cleavable moiety" and "CM" are used interchangeably herein and refer to a peptide whose amino acid sequence comprises a substrate for a sequence-specific protease. In the activatable protein, the CM is positioned relative to the MM and TB such that cleavage produces a molecule capable of binding to the biological target of the TB. Thus, the activatable protein has reduced binding to the biological target compared to the activated protein.

[0068] In some embodiments, an activatable TB can be designed by selecting a TB of interest and constructing the remainder of the activatable TB so that when conformationally constrained, the MM provides masking of the TB or reduced binding of the TB to its target. Structural design criteria can be considered to provide this functional feature.

[0069] Activatable antibodies can be provided in a variety of structural configurations. Exemplary formulas of activatable antibodies are provided below. It is contemplated that within an activatable antibody, the N-terminal to C-terminal order of AB, MM, and CM can be reversed. It is also contemplated that CM and MM can overlap in amino acid sequence, for example, such that a CM sequence recognized by a sequence-specific protease is at least partially contained within MM. For example, an activatable antibody can be composed of Figure 1BThe formula in (in order from the amino (N) terminal region to the carboxyl (C) terminal region) is represented. The activatable antibody may further comprise one or more linkers (L) between the MM and the CM and / or between the CM and the AB. The line connecting the MM and the AB represents a non-α carbon covalent bond.

[0070] Exemplary configurations of activatable antibodies are shown in Figures 2A-2D middle. Figure 2A An exemplary activatable antibody 210 is shown, which comprises, in order from the amino (N)-terminal region to the carboxyl (C)-terminal region, MM 211, an optional linker 212, CM 213, an optional linker 214 having the same or different sequence as 212, and AB 215. MM 211 and AB 215 are tethered by a non-alpha carbon covalent bond 216. Figure 2B An exemplary activatable antibody 220 is shown, which comprises, in order from the amino (N)-terminal region to the carboxyl (C)-terminal region, AB 221, an optional linker 222, CM 223, an optional linker 224 having the same or different sequence as 222, and MM 225. AB 221 and MM 225 are tethered by a non-alpha carbon covalent bond 226. Figure 2C An exemplary activatable antibody 230 is shown, which comprises, in order from the amino (N) terminal region to the carboxyl (C) terminal region, an optional linker 231, a CM 232, an optional linker 233, a MM 234, an optional linker 235, a CM 236, an optional linker 237, and an AB 238. MM 234 and AB 238 are tethered by a non-alpha carbon covalent bond 239. Figure 2D An exemplary activatable antibody 241 is shown, which comprises, in order from the amino (N) terminal region to the carboxyl (C) terminal region, AB 241, optional linker 242, CM 243, optional linker 244, MM 245, optional linker 246, CM 247, and optional linker 248. AB 241 and MM 255 are tethered by a non-alpha carbon covalent bond 249.

[0071] In some examples, AB (e.g., Figures 2A-2D In some embodiments, the first polypeptide comprises a MM, a CM, and at least one antibody variable domain selected from the group consisting of a light chain variable domain ("LVD" or "VL") and a heavy chain variable domain ("HVD" or "VH"). In some examples, the AB (e.g. Figures 2A-2DIn some aspects, the AB may comprise multiple polypeptides (e.g., the AB is a complex formed by multiple polypeptides). In some aspects, the AB comprises at least two polypeptides, at least three polypeptides, at least four polypeptides, or more. In this case, the MM may be coupled to the polypeptides of the AB via the CM and tethered to the same polypeptide via a non-alpha carbon covalent bond. Alternatively, the MM may be coupled to the first polypeptide of the AB via the CM and tethered to the second polypeptide of the AB via a non-alpha carbon covalent bond. The activatable antibody may have one or more polypeptides arranged as follows: MM-CM-HVD or MM-CM-LVD or MM-CM-scFv, MM-CM-ScFv-Fab, MM-CM-HVD-scFv, MM-CM-LVD-scFv, MM-CM-scFv-HVD, MM-CM-scFv-LVD, HVD-CM-MM, LVD-CM-MM, scFv-CM-MM, HVD-scFv-CM-MM, LVD-scFv-CM-MM, scFv-HVD-CM-MM, MM-CM-VHH, VHH-CM-MM or scFv-LVD-CM-MM. As used herein and unless otherwise stated, each dash (-) between ACC components represents a direct linkage or a linkage via one or more linkers.

[0072] In some embodiments, the activatable antibody may have two polypeptides. In some examples, the activatable antibody may include a first polypeptide and a second polypeptide, wherein the first polypeptide includes HVD, and the second polypeptide includes any of the following from N-terminal to C-terminal direction: MM-CM-LVD, MM-CM-scFv, MM-CM-LVD-scFv, MM-CM-scFv-LVD, LVD-CM-MM, scFv-CM-MM, LVD-scFv-CM-MM, MM-CM-VHH, VHH-CM-MM or scFv-LVD-CM-MM. In some examples, the activatable antibody may comprise a first polypeptide and a second polypeptide, wherein the first polypeptide comprises LVD, and the second polypeptide comprises any of the following from the N-terminal to the C-terminal direction: MM-CM-HVD, MM-CM-scFv, MM-CM-HVD-scFv, MM-CM-scFv-HVD, HVD-CM-MM, scFv-CM-MM, HVD-scFv-CM-MM, MM-CM-VHH or scFv-HVD-CM-MM. In this case, MM can be tethered to the second polypeptide (i.e., a polypeptide comprising MM) via a non-alpha carbon covalent bond. Alternatively or additionally, MM can be tethered to the first polypeptide (i.e., a polypeptide not comprising MM) via a non-alpha carbon covalent bond.

[0073] In some embodiments, the activatable antibody may have more than two polypeptides. Such an activatable antibody may comprise any combination of the above polypeptides. In some embodiments, the activatable antibody may comprise four polypeptides. In some examples, in two of the polypeptides, each polypeptide may comprise HVD; in the other two polypeptides, each polypeptide may comprise, from N-terminus to C-terminus: MM-CM-LVD, MM-CM-scFv, MM-CM-LVD-scFv, MM-CM-scFv-LVD, LVD-CM-MM, scFv-CM-MM, LVD-scFv-CM-MM, MM-CM-VHH, VHH-CM-MM or scFv-LVD-CM-MM. In some examples, in two of the polypeptides, each polypeptide may comprise LVD; in the other two polypeptides, each polypeptide may comprise, from N-terminal to C-terminal direction: MM-CM-HVD, MM-CM-scFv, MM-CM-HVD-scFv, MM-CM-scFv-HVD, HVD-CM-MM, scFv-CM-MM, HVD-scFv-CM-MM, scFv-HVD-CM-MM. In these cases, the MM may be tethered to the polypeptide comprising the MM via a non-alpha carbon covalent bond. Alternatively or additionally, the MM may be tethered to the polypeptide not comprising the MM via a non-alpha carbon covalent bond.

[0074] In the examples described herein, the HVD and LVD may be contained in an antibody or a fragment thereof (eg, Fab). The activatable antibody may further comprise one or more additional components of an antibody, such as a heavy chain constant region (CH), a light chain constant region (CL), a hinge, an Fc domain, or a combination thereof.

[0075] Figures 3A-3C Three example configurations of activatable antibodies are shown. Figure 3A An exemplary activatable antibody is shown, comprising a Nanobody (ie, a single domain antibody as an exemplary antibody or TB) and a MM coupled via a CM. The Nanobody and the MM are tethered by a non-alpha carbon covalent bond between the masker and the Nanobody.

[0076] Figure 3B An activatable single-chain variable fragment (scFv) comprising a heavy chain variable region (V H ) and light chain variable region (V L ) of scFv, and through CM and V L MM and scFv are also coupled via MM and V H Examples of activatable antibodies also include Figure 3B In one example, the MM is coupled to the V of the scFv via the CM. L MM and scFv are also coupled to VL In another example, the MM is covalently tethered to the non-α carbon of the scFv via the CM. H MM and scFv are also coupled via MM and V H In another example, the MM is covalently tethered to the non-α carbon of the scFv via the CM. H MM and scFv are also coupled via MM and V L The non-α carbons are covalently tethered.

[0077] Figure 3C An activatable full-length antibody is shown, which includes a dimer, each monomer of the dimer includes a heavy chain, a light chain, and an MM coupled to the light chain via a CM. The MM and the activatable full-length antibody are tethered by a non-α carbon covalent bond between the MM and the heavy chain. Examples of activatable antibodies also include Figure 3C In one example, the MM is coupled to the light chain via the CM. The MM and the full-length antibody are tethered by a non-alpha carbon covalent bond between the MM and the light chain. In another example, the MM is coupled to the heavy chain via the CM. The MM and the full-length antibody are tethered by a non-alpha carbon covalent bond between the MM and the light chain. In another example, the MM is coupled to the heavy chain via the CM. The MM and the full-length antibody are tethered by a non-alpha carbon covalent bond between the MM and the light chain.

[0078] Figures 2A-2D The schematic diagrams in Figures 3A-3B are illustrated as non-limiting proof-of-concept examples. Activatable antibodies that are dual-anchored with MM and AB (e.g., via CM and non-α carbon covalent bonds) broadly include any type of activatable antibodies, including activatable full-length antibodies, activatable multispecific antibodies (e.g., bispecific and trispecific antibodies, including multispecific antibodies capable of cross-linking two cells, such as bispecific T cell engagers (BiTEs)) and activatable antibody fragments (e.g., scFv, diabodies, nanobodies, Fabs, etc.). Examples of dual-anchored multispecific activatable antibodies are shown in Figures 11A-11I Although Figures 11A-11I Dual anchors at all masked positions are depicted, but the disclosure also includes constructs comprising one or more dual anchored masks and one or more other masks that are not dual anchored (i.e., anchored to the TB only via the CM (single anchor)). The disclosure also includes constructs comprising one or more dual anchored masks and one or more unmasked TBs. Fig.11A Exemplary bispecific Nanobody tandems are shown. Fig. 11B Shown are exemplary double-chain antibodies comprising two scFvs. In some instances, the two scFvs can be coupled via a peptide linker. In alternative instances, the two scFvs are not coupled via any linker. Fig. 11CAn exemplary bispecific antibody comprising a scFv and a Fab is shown. In some examples, the bispecific antibody can be a bispecific T cell engager (BiTE). Fig. 11C The antibodies in may also comprise one or more Fc domains. Fig.11D An exemplary bispecific F(ab') is shown 2 . Fig.11D An exemplary bispecific antibody is shown. Figures 11E-11I Other examples of dual-anchored trispecific and other multispecific activatable antibodies are shown. In dual-anchored multispecific activatable antibodies, MM can be coupled to AB via CM. MM can be tethered to AB coupled to MM via one or more non-α carbon covalent bonds, or tethered to another component of the activatable antibody. In some examples of multispecific activatable antibodies, all MMs are dual-anchored. In some multispecific activatable examples, only some (but not all) MMs are dual-anchored. Figures 2A-2D The TB in the invention can be any antigen binding protein, including antibodies and proteins described in the target binding protein section below.

[0079] In some embodiments, the activatable target binding protein (e.g., an activatable antibody) may be characterized in that its target binding activity is reduced compared to a control level of target binding activity of the AB not coupled to MM. For example, the activatable TB is characterized in that its target binding activity is reduced by at least 1, 2, 4, 6, 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 5000, or 10000 times compared to a control level of target binding activity of the TB not coupled to MM.

[0080] In some embodiments, the activatable TB (e.g., activatable antibody) may be characterized by a decrease in target binding activity compared to a control level of target binding activity of a TB coupled to a MM but not tethered to the MM by a non-alpha carbon covalent bond (i.e., a single anchored activatable antibody). For example, in some embodiments, the activatable TB is characterized by a decrease in target binding activity by at least 2, 4, 6, 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 5000, 10000, 15000, 20000, 30000, 40000, or 50000 times compared to a control level of target binding activity of a TB coupled to a MM but not tethered to the MM by a non-alpha carbon covalent bond. In some embodiments, activatable TB is characterized by at least a 2-fold decrease in targeted binding activity. In some embodiments, activatable TB is characterized by at least a 4-fold decrease in targeted binding activity. In some embodiments, activatable TB is characterized by at least a 10-fold decrease in targeted binding activity. In some embodiments, activatable TB is characterized by at least a 500-fold decrease in targeted binding activity. In some embodiments, activatable TB is characterized by at least a 100-fold decrease in targeted binding activity. In some embodiments, activatable TB is characterized by at least a 200-fold decrease in targeted binding activity. In some embodiments, activatable TB is characterized by at least a 300-fold decrease in targeted binding activity. In some embodiments, activatable TB is characterized by at least a 500-fold decrease in targeted binding activity. In some embodiments, activatable TB is characterized by at least a 1000-fold decrease in targeted binding activity. In some embodiments, activatable TB is characterized by at least a 5000-fold decrease in targeted binding activity. In some embodiments, activatable TB is characterized by at least a 10000-fold decrease in targeted binding activity. In some embodiments, activatable TB is characterized by at least a 15000-fold decrease in targeted binding activity. In some embodiments, the activatable TB is characterized by at least a 20,000-fold reduction in targeted binding activity. In some embodiments, the activatable TB is characterized by at least a 30,000-fold reduction in targeted binding activity.

[0081] Target binding protein

[0082] The activatable target binding proteins (e.g., activatable antibodies) disclosed herein may include one or more target binding proteins, i.e., proteins that are capable of binding to a target molecule. In some embodiments, the target binding protein (TB) may be a cytokine, a hormone, a growth factor, or an agonist. In some embodiments, the target binding protein (TB) may be an antigen binding protein (AB). In some embodiments, AB may be an antibody or a fragment thereof, such as a monoclonal antibody, a single chain antibody, a Fab fragment, a F(ab')2 In some embodiments, AB can be a full-length antibody. In some embodiments, AB can be an immunologically active fragment. In some embodiments, AB can be an antigen-binding fragment ("Fab"). In some embodiments, AB can be a mouse antibody, other rodent antibodies, chimeric antibodies, humanized antibodies, or fully human monoclonal antibodies. The present disclosure includes structures having one or more polypeptides comprising any of the domains listed above, such as one or more of SDA, Fv, ScFv, Fab, scFab, VHH, and dAb and a combination of one or more of SDA, Fv, scFv, Fab, VHH, scFab, and dAb.

[0083] The term "antibody" is used herein in its broadest sense to include certain types of immunoglobulin molecules that include one or more antigen binding domains that specifically bind to an antigen or epitope. Antibodies specifically include, for example, complete antibodies (e.g., complete immunoglobulins), antibody fragments, bispecific antibodies, and multispecific antibodies. An example of an antigen binding domain is a V domain that is comprised of a V H -V L Dimers form antigen binding domains. Additional examples of antibodies are described herein. Additional examples of antibodies are known in the art.

[0084] In some examples, AB can be a single domain antibody (also called nanobody). A single domain antibody can be an antibody fragment, which is a single monomer variable antibody domain. A single domain antibody can have an antigen affinity similar to that of a corresponding full-length antibody. A single domain antibody can be an Fc-tagged single domain antibody, which comprises an Fc dimer, each Fc monomer being coupled to a single domain antibody.

[0085] In some embodiments, AB can be monospecific, for example, it can only bind to one antigen. In some embodiments, AB can be multispecific (e.g., bispecific or trispecific), for example, it can bind to multiple antigens. In some embodiments, the activatable antibody can be formulated as a part of a former bispecific T cell engager (pro-BITE) molecule or a dual affinity retargeting antibody (DART). In some embodiments, the activatable antibody can be formulated as a part of a T cell or other engineered receptor or other immune effector cell (e.g., NK cell modified by CAR) modified by a former chimeric antigen receptor (pro-CAR). In some embodiments, the activatable antibody can be formulated as a part of a T cell modified by a former chimeric antigen receptor (CAR). In some embodiments, the activatable antibody can be formulated as a part of a NK cell modified by a former chimeric antigen receptor (CAR). In some embodiments, the activatable antibody can be formulated as a part of a T cell bispecific antibody (TCB).

[0086] A "light chain" is composed of a variable domain (VL) and a constant domain (CL). There are two different types or classes of light chains, called kappa or lambda.

[0087] The "heavy chain" consists of a variable domain (VH) and three constant region domains (CH1, CH2, CH3). There are five main classes or isotypes of heavy chains, some of which have several subtypes, and these determine the functional activity of the antibody molecule. The five major classes of immunoglobulins are immunoglobulin M (IgM), immunoglobulin D (IgD), immunoglobulin G (IgG), immunoglobulin A (IgA), and immunoglobulin E (IgE). IgG is by far the most abundant immunoglobulin and has several subclasses (IgG1, IgG2, IgG3, and IgG4 in humans).

[0088] "Fragment antigen binding" (Fab) contains an intact light chain paired with the VH and CH1 domains of the heavy chain.

[0089] When an antibody is cleaved by pepsin below the hinge region, F(ab') is formed. 2 Fragments, in which case the two fragment antigen binding domains (Fab) of an antibody molecule remain linked. F(ab') 2 The fragments contain two complete light chains paired with two VH and CH1 domains of the heavy chain joined together by a hinge region.

[0090] The "crystallizable fragment" (Fc) fragment (also referred to herein as F CThe Fc domain corresponds to the paired CH2 and CH3 domains and is the part of the antibody molecule that interacts with effector molecules and cells. The functional differences between heavy chain isotypes are mainly in the Fc fragment.

[0091] "Single-chain Fv" (scFv) contains only the light chain variable domain (VL) connected to the heavy chain variable domain (VH) by a synthetic peptide. The name single-chain Fv comes from the fragment variable.

[0092] The "hinge region" or "interdomain" is the flexible stretch of amino acids that joins or connects the Fab fragment to the Fc domain.

[0093] The "synthetic hinge region" is the amino acid sequence that joins or connects the Fab fragment to the Fc domain.

[0094] "Prodomain" refers to a polypeptide, a portion of which can inhibit antigen binding, called a masking peptide (MM), and a portion of which contains a protease-cleavable substrate, called a cleavable peptide (CM), which, when linked to a target binding protein (TB), an antibody, an antigen-binding fragment thereof, or an antigen-binding domain (AB), is used to inhibit the binding of TB or AB to an antigen. The prodomain may include a connecting peptide (L1) between the MM and the CM. The prodomain may also include a connecting peptide (L2) located at the carboxyl terminus of the prodomain to facilitate the linkage of the prodomain to the antibody. In certain embodiments, the prodomain comprises one of the following formulae (wherein the following formulae represent amino acid sequences from the N-terminus to the C-terminus): (MM)-(CM), (MM)-L1-(CM), (MM)-(CM)-L2, or (MM)-L1-(CM)-L2.

[0095] TB (e.g., AB) specifically binds to a target. As used herein, the terms "specific binding," "immunological binding," and "immunological binding properties" refer to the type of non-covalent interaction that occurs between an immunoglobulin molecule and an antigen for which the immunoglobulin has specificity. The strength or affinity of the immunological binding interaction can be measured based on the dissociation constant (K) of the interaction. d ) is used to represent the d The smaller the value, the greater the affinity. Methods well known in the art can be used to quantify the immunological binding properties of a selected polypeptide. One such method requires measuring the rates of formation and dissociation of the antigen binding site / antigen complex, where those rates depend on the concentration of the complex partners, the affinity of the interaction, and geometric parameters that affect the rates equally in both directions. Thus, the "association rate constant" (K) can be determined by calculating the concentration and the actual rates of association and dissociation. on ) and the “dissociation rate constant” (K off (See Nature 361: 186-87 (1993)). off / Kon The ratio of 2 can eliminate all parameters not related to affinity and is equal to the dissociation constant K. d (See generally Davies et al. (1990) Annual Rev Biochem 59:439-473). The TB disclosed herein can be used in conjunction with a binding constant (K d )≤1 μM. In some embodiments, the TB disclosed herein can bind specifically to the target at a binding constant (K d )≤100 nM. In some embodiments, the TB disclosed herein can bind specifically to the target at a binding constant (K d )≤10 nM. As measured by, for example, a radioligand binding assay or similar assays known to those skilled in the art, the TB of the present disclosure may be present in a binding constant (K d ) specifically binds to the target at ≤100 pM to about 1 pM.

[0096] The target of TB (e.g., AB) can be a protein or other type of molecule. Example categories of targets of TB include cell surface receptors and secreted binding proteins (e.g., growth factors), soluble enzymes, structural proteins (e.g., collagen, fibronectin), etc. In some instances, the target of TB can be a protein associated with a disease (e.g., cancer) in a subject.

[0097] In some embodiments, the activatable target binding protein or activatable antibody may comprise a serum half-life extending moiety (e.g., a polypeptide that binds a serum protein, such as an immunoglobulin (e.g., IgG) or serum albumin (e.g., human serum albumin (HSA)). The half-life extending moiety may be conjugated to TB.

[0098] In some examples, the half-life extension portion can be a fragment crystallizable region (Fc) of an antibody. Other examples of half-life extension portions include six-cap GST (glutathione S-transferase) glutathione affinity, calmodulin (Calmodulin) binding peptide (CBP), Strep-tag, cellulose binding domain, maltose binding protein, S peptide tag, chitin binding tag, immunoreactive epitope, epitope tag, 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.

[0099] In some embodiments, the serum half-life of the activatable target binding protein or activatable antibody may be longer than the serum half-life of the corresponding protein (e.g., an activatable antibody without a half-life extension portion), for example, the pK of the activatable antibody is longer than the pK of the corresponding antibody. In some embodiments, the serum half-life of the activatable target binding protein or activatable antibody is similar to the serum half-life of the corresponding antibody. In some embodiments, when administered to an organism, the serum half-life of the activatable target binding protein (e.g., activatable antibody) 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.

[0100] Masking part(MM)

[0101] The masking moiety in the activatable macromolecule "masks" or reduces or otherwise inhibits the binding of the activatable macromolecule to its target and / or epitope. In some embodiments, the coupling or modification of the target binding protein (TB) (e.g., AB or other therapeutic or diagnostic protein) to the MM can inhibit the ability of TB to specifically bind to its target and / or epitope by inhibition methods known in the art (e.g., not limited to structural changes and competing antigen binding domains). In some embodiments, the coupling or modification of TB to MM can cause structural changes, thereby reducing or inhibiting the ability of TB to specifically bind to its target and / or epitope. In some embodiments, the coupling or modification of a protein comprising an antigen binding domain to MM sterically blocks, reduces or inhibits the ability of the antigen binding domain to specifically bind to its target and / or epitope.

[0102] The activatable target binding protein (e.g., activatable antibody) herein may comprise one or more masking moieties (MM) capable of interfering with the binding of the target binding protein (e.g., AB) to the target. In general, the MM can be coupled to the target binding protein (e.g., AB) via the CM and optionally one or more linkers described herein. In addition, the MM can also be tethered to the activatable target binding protein (or antibody) as described herein to form an activatable double-anchored masked target binding protein. In some embodiments, the MM prevents the activatable TB from binding to the target; however, when the molecule is activated (when the CM is cleaved by a protease), the MM does not substantially or significantly interfere with the binding of the target binding protein to the target.

[0103] In some embodiments, the MM may interact with the AB (or other desired protein), thereby reducing or inhibiting the interaction between the target binding protein (e.g., AB) and its binding partner. In some embodiments, the MM may comprise at least part or all of the amino acid sequence of a naturally occurring binding partner of the target binding protein (e.g., AB). For example, the MM may be a fragment of a naturally occurring binding partner. The fragment may retain up to 95%, 90%, 80%, 75%, 70%, 60%, 50%, 40%, 30%, 25%, or 20% nucleic acid or amino acid sequence homology with the naturally occurring binding partner. In some embodiments, the MM may be a homologous peptide of the target binding protein (e.g., AB). For example, the MM may comprise a sequence or fragment thereof of an epitope of the target binding protein (e.g., AB). As used herein, the term "naturally occurring" when applied to an object refers to the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence present in an organism (including a virus) that can be isolated from a source in nature and is not intentionally modified by a person in a laboratory or otherwise is naturally occurring.

[0104] In some embodiments, the MM may comprise an amino acid sequence that is not naturally occurring, or an amino acid sequence that does not contain a naturally occurring binding partner or target protein. In certain embodiments, the MM is not a natural binding partner of a target binding protein (e.g., AB). The MM may be a modified binding partner of a target binding protein (e.g., AB) that contains amino acid changes that reduce binding affinity and / or avidity with the target binding protein (e.g., AB). In some embodiments, the MM does not contain or substantially does not contain nucleic acid or amino acid homology with a natural binding partner of AB. In other embodiments, the MM is at most 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% similar to a natural binding partner of a target binding protein (e.g., AB).

[0105] In some embodiments, the MM may not specifically bind to the AB (or other activatable protein), but rather interfere with the binding of the target binding protein (e.g., AB) to its binding partner through non-specific interactions such as steric hindrance. For example, the MM may be located in the activatable target binding protein such that the tertiary or quaternary structure of the activatable target binding protein allows the MM to mask the target binding protein through charge-based interactions, thereby holding the MM in place to interfere with the access of the binding partner to the target binding protein.

[0106] In some embodiments, the dissociation constant of the MM binding to the target binding protein (eg, AB) does not exceed the dissociation constant of the target binding protein to the target. In some embodiments, the MM does not interfere with or compete for the binding of the target binding protein to the target after cleavage of the CM.

[0107] The structural properties of the MM can be selected based on factors such as the minimal amino acid sequence required to interfere with protein binding to the target, the target protein-protein binding pair of interest, the size of the target binding protein, the presence or absence of a linker, and the like.

[0108] In some embodiments, the MM is unique to the coupled target binding protein. Examples of MMs include MMs that are specifically screened to bind to a binding domain of a target binding protein or fragment thereof (e.g., affinity masking). Methods for screening MMs to obtain MMs that are unique to a target binding protein and those that specifically and / or selectively bind to a binding domain of a binding partner / target are provided herein and may include protein display methods.

[0109] As used herein, the term "masking efficiency" or "ME" refers to the activity (e.g., EC50) of an activatable target binding protein (e.g., activatable AB) divided by the activity of a control target binding protein (e.g., antibody), wherein the control target binding protein (e.g., control antibody) can be a cleavage product of the activatable target binding protein (e.g., activatable antibody), or a target binding protein (e.g., antibody) or a fragment thereof used as a target binding protein for the activatable target binding protein. An activatable target binding protein with a reduced level of target binding (e.g., antibody) activity can have a masking efficiency greater than 10. In some embodiments, the activatable target binding protein (e.g., activatable antibody) described herein can have a masking efficiency greater than 10, 100, 1000, 5000, 10,000, or 15,000.

[0110] In some embodiments, the MM can be a polypeptide of about 2 to 50 amino acids in length. For example, the MM can be a polypeptide of 2 to 40, 2 to 30, 2 to 20, 2 to 10, 5 to 15, 10 to 20, 15 to 25, 20 to 30, 25 to 35, 30 to 40, 35 to 45, 40 to 50 amino acids in length. For example, the MM can be a polypeptide of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length. In some examples, the MM can be a polypeptide that is greater than 50 amino acids in length, such as 100, 200, 300, 400, 500, 600, 700, 800 or more amino acids.

[0111] In some embodiments, when the MM is tethered to its coupled target binding protein (e.g., AB), in the presence of the target of the target binding protein, the target binding protein has no binding or substantially no binding to the target, or the binding of the target binding protein to the target is at most 0.001%, 0.01%, 0.02%, 0.03%, 0.04%, 0.06%, 0.07%, 0.08%, 0.10%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.20%, 0.21%, 0.22%, 0.23%, 0.24%, 0.25%, 0.26%, 0.27%, 0.28%, 0.29%, 0.30%, 0.31%, 0.32%, 0.33%, 0.34%, 0.35%, 0.36%, 0.37%, 0.38%, 0.39%, 0.40%, 0.41%, 0.42%, 0.43%, 0.44%, 0.45%, 0.46%, 0.47%, 0.48%, 0.49%, 0.50%, 0.51%, 0.52%, 0.53%, 0.54%, 0.56%, 0.57%, 0.58%, 0.59%, 0.60%, 0.61%, 0.62%, 0.63%, 0.64%, 0.65%, 0.66%, 0.67%, 0.68%, 0.69%, 0.70%, 0.71%, 0.72%, 0.73%, 0.74%, 0.75%, 0. 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 longer.

[0112] The binding affinity of TB (e.g. AB) to a target or binding partner when TB (e.g. AB) is tethered to a MM coupled to TB (e.g. AB) 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, or 50,000,000 times lower than the binding affinity of TB (e.g. AB) to its binding partner when it is not coupled to MM, or 5-10, 10-100, 10-1,000 times lower than the binding affinity of TB (e.g. AB) to its binding partner when it is not coupled to MM. 0, 10-10,000, 10-100,000, 10-1,000,000, 10-10,000,000, 100-1,000, 100-10,000, 100-10,000,000, 100-1,000,000, 100-10,000,000, 1,000-10,000, 1,000-10,000, 1,000 0-100,000, 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.

[0113] The binding affinity of TB (e.g. AB) for a target or binding partner when TB (e.g. AB) is tethered to a MM to which it is coupled 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 or 50,000,000 times lower than the binding affinity of TB (e.g. AB) for its binding partner when TB (e.g. AB) is cleaved from the MM by a protease.

[0114] The binding affinity of TB (e.g. AB) to a target or binding partner when TB (e.g. AB) is tethered to a MM coupled to TB (e.g. AB) 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, or 50,000,000 times lower than the binding affinity of TB (e.g. AB) to its binding partner when TB (e.g. AB) is not coupled to a MM and is not tethered, or 5-10, 10-100, 10-20 times lower than the binding affinity of TB (e.g. AB) to its binding partner when it is not coupled to a MM and is not tethered. -1,000, 10-10,000, 10-100,000, 10-1,000,000, 10-10,000,000, 100-1,000, 100-10,000, 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.

[0115] The dissociation constant (K) of the MM to its coupled and tethered (double anchored) TB (e.g., AB) d ) may be greater than the K of TB (e.g., AB) for the target d MM vs. TB (e.g. AB) K d Comparable TB (e.g. AB) K for target dAB) to the target.

[0116] In some embodiments, the MM may contain genetically encoded amino acids or genetically non-encoded amino acids. Examples of genetically non-encoded amino acids are, but are not limited to, D-amino acids, β-amino acids, and γ-amino acids. In specific embodiments, the MM contains up to 50%, 40%, 30%, 20%, 15%, 10%, 5%, or 1% of genetically non-encoded amino acids.

[0117] In some embodiments, once uncoupled from TB and in a free state, MM may have biological activity or therapeutic effects, such as binding ability. For example, free peptides may bind to the same or different binding partners. In certain embodiments, free MM (e.g., MM not coupled or tethered to TB) may exert therapeutic effects, thereby providing a secondary function for the compositions disclosed herein. In some embodiments, once uncoupled from TB and in a free state, MM may advantageously not exhibit biological activity. For example, in some embodiments, MM in a free state does not induce an immune response in a subject.

[0118] TB and MM may contain one or more cysteine ​​residues capable of forming a non-alpha carbon covalent bond between TB and MM. In some embodiments, one or more non-alpha carbon covalent bonds may be formed between sulfur atoms of cysteine ​​or other amino acid residues containing sulfur atoms. Such residues may be naturally present in activatable TB (e.g., in TB and MM), or may be incorporated into activatable TB by site-directed mutagenesis, chemical conversion, or misincorporation of non-natural amino acids.

[0119] In some embodiments, the cysteine ​​residues may be in a position that provides a conformationally restricted activatable TB, but after cleavage of the CM, the MM does not substantially or significantly interfere with target binding of the activated TB.

[0120] The position of cysteine ​​residues in the activatable target binding protein can be determined based on the structure of the activatable target binding protein or its components (e.g., crystal structure, or other structural models based on other techniques, such as NMR, spectroscopy or computational methods). Any of a variety of homology-based computational protein models can be used to generate a three-dimensional structure of a target binding protein, with or without MM, including, for example, Rosetta modeling software (rosettacommons.org), DiscoveryStudio (Dassault Systemes BIOVIA), BioLuminate, PIPER, Prime (Schrodinger), AlphaFold Colab (Google), SWISSMODELER, etc. An example of a three-dimensional structure obtained using Discovery Studio software is Figures 12A-12B The three-dimensional structure of the activatable target binding protein (activatable anti-PDL1 antibody) depicted in FIG. For example, the region where MM interacts with TB can be identified, and cysteine ​​residues (naturally occurring or introduced) in this region can be used to form non-α carbon covalent bonds tethering TB and MM. In general, the Cα atom of the disulfide-bonded cysteine ​​residue is in the Therefore, as a first approximation, MM and TB residues with Cα distances within this range would be good candidates for cysteine ​​mutation. Any of a variety of disulfide bond prediction programs, including, for example, MODIP (Dani, Ramakrishnan, Varadarajan 2003), Disulfide by Design (Craig and Dombkowski, 2013), and SSbondPre (Gao, Dong, Li, Liu, and Liu, 2020), can be used to further identify MM and TB residues that are highly likely to form disulfide bonds.

[0121] In some embodiments, a non-alpha carbon covalent bond, for example, a disulfide bond tethering MM and TB (e.g., AB), can be formed by a first cysteine ​​and a second cysteine. In one example, the first cysteine ​​is within the MM and the second cysteine ​​is within the TB. In another example, the first cysteine ​​is within the peptide coupled to the MM and the second cysteine ​​is within the TB. In another example, the first cysteine ​​is within the MM and the second cysteine ​​is within the peptide coupled to the TB. In such an example, the peptide coupled to the MM or TB can be a linker or a peptide. In one example, the peptide coupled to the MM or TB can be a leader peptide, which is a peptide position located near the end (e.g., N-terminus or C-terminus) of the MM or TB. The leader peptide may be located between the signal peptide and the MM or TB.

[0122] In some embodiments, the one or more cysteines that form a disulfide bond with the MM may be cysteines that occur naturally in the activatable target binding protein. In some embodiments, the one or more cysteines that form a disulfide bond may be engineered into the activatable TB (eg, AB).

[0123] Suitable MMs can be identified and / or further optimized from a library of candidate activatable TBs with variable MMs by a screening procedure. For example, TBs and CMs can be selected to provide a desired enzyme / target combination, and the amino acid sequence of the MM can be identified by a screening procedure described below to identify MMs that provide an activatable phenotype. For example, a random peptide library (e.g., a random peptide library comprising peptides of 2 to 40 amino acids or more) can be used in the screening methods disclosed herein to identify suitable MMs.

[0124] In some embodiments, MMs with specific binding affinity for TB (e.g., AB) can be identified by a screening procedure comprising providing a peptide scaffold library consisting of candidate MMs, wherein each scaffold consists of a transmembrane protein and a candidate MM. The library can then be contacted with the entire protein or a portion (e.g., a full-length protein, a naturally occurring protein fragment, or a non-naturally occurring fragment containing the protein (also capable of binding the binding partner of interest)), and one or more candidate MMs with detectably bound protein are identified. Screening can be performed by one or more rounds of magnetic activated sorting (MACS) or fluorescence activated sorting (FACS), and determining the binding affinity of the MMs for TB and subsequently determining the masking efficiency, such as described in WO2009025846 and US20200308243A1, which are incorporated herein by reference in their entirety.

[0125] In some embodiments, MM can be selected for use with specific antibodies or antibody fragments. Additional suitable MMs are disclosed in: WO2021207657, WO2021142029, WO2021061867, WO2020252349, WO2020252358, WO2020236679, WO2020176672, WO2020118109, WO2020092881, WO2020086665, WO2019213444, WO2019183218, WO2019173771, WO2019 165143, WO2019075405, WO2019046652, WO2019018828, WO2019014586, WO2018222949, WO2018165619, WO2018085555, WO2017011580, WO2016179335, WO2016179285, WO2016179257, WO2016149201, WO2016014974 and WO2016118629.

[0126] Cleavable fraction (CM)

[0127] The activatable target binding protein may comprise one or more cleavable moieties (CM) as defined above.

[0128] In some embodiments, the activatable TB may comprise a CM between the TB (e.g., AB) and the MM. The CM and TB of the activatable target binding protein (e.g., AB) may be selected such that the TB comprises a binding portion for a given target, while the CM comprises a substrate for one or more proteases, wherein the one or more proteases are co-localized with the target in a tissue (e.g., at a treatment site or diagnostic site in a subject). In some embodiments, the activatable TB may have special uses, where, for example, one or more proteases capable of cleaving a site in the CM are present at relatively higher levels in target-containing tissues at a treatment site or diagnostic site than in tissues at non-treatment sites (e.g., in healthy tissues).

[0129] In some embodiments, the CM herein may include substrates for proteases that have been reported in one or more cancers. See, for example, La Roca et al., British J. Cancer 90(7):1414-1421, 2004. Substrates suitable for use in the CM components employed herein include those more commonly found in cancerous cells and tissues. Thus, in certain embodiments, the CM may include substrates for proteases that are more commonly found in diseased tissues associated with cancer. Examples of cancer include gastric cancer, breast cancer, osteosarcoma, esophageal cancer, breast cancer, HER2-positive cancer, Kaposi sarcoma, hairy cell leukemia, chronic myeloid leukemia (CML), follicular lymphoma, renal cell carcinoma (RCC), melanoma, neuroblastoma, basal cell carcinoma, cutaneous T-cell lymphoma, nasopharyngeal adenocarcinoma, ovarian cancer, bladder cancer, BCG-resistant non-muscle invasive bladder cancer (NMIBC), endometrial cancer, pancreatic cancer, non-small cell lung cancer (NSCLC), colorectal cancer, esophageal cancer, gallbladder cancer, glioma, head and neck cancer, uterine cancer, cervical cancer or testicular cancer, etc. In some embodiments, the CM component comprises a substrate for a protease that is more prevalent in tumor tissue. For example, the protease can be produced by a tumor of a subject.

[0130] In some embodiments, the activatable TB may include a first CM (CM1) and a second CM (CM2) between the MM and the TB (e.g., AB) so that the MM can be completely dissociated from the TB by CM cleavage. In some examples, the first CM and the second CM may include substrates of the same protease. In some examples, the first CM and the second CM may include substrates of different proteases. In some examples, the first CM and the second CM may have the same sequence. In some examples, the first CM and the second CM may have different sequences.

[0131] The second CM (CM2) may be located at a position in the activatable TB where its cleavage facilitates the dissociation of the MM from the TB. In some embodiments, the CM2 may be located between the MM and the non-α carbon covalent bond. In some embodiments, the second CM may be within the MM and at most 10 amino acids away from the non-α carbon covalent bond. In some embodiments, the second CM may be within the TB and at most 10 amino acids away from the non-α carbon covalent bond. The second CM may be located near a cysteine ​​that forms a non-α carbon covalent bond (e.g., there are 0 amino acids between the second CM and the cysteine). Alternatively, the second CM may be located at a maximum of 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids away from a cysteine ​​that forms a non-α carbon covalent bond (e.g., a disulfide bond). The activatable target binding protein may have an enhanced masking efficiency due to the tethering between the TB and the MM, thereby minimizing the toxicity and effects caused by target binding outside the activation environment, and may be fully activated by a protease to provide the desired activity (e.g., therapeutic effect, target detection, etc.).

[0132] CMs suitable for use herein that can activate TB include any protease substrate known in the art. In some examples, the CM may include substrates for serine proteases (e.g., u-type plasminogen activator (uPA, also known as urokinase)), matriptase (also referred to herein as MT-SP1 or MTSP1). In some examples, the CM may include substrates for matrix metalloproteinases (MMPs). In some examples, the CM may include substrates for cysteine ​​proteases (CPs) (e.g., legumin).

[0133] In some embodiments, the CM may comprise substrates for a disintegrin and metalloproteinase (ADAM) or a disintegrin and metalloproteinase with a thrombospondin motif (ADAMTS) (e.g., ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADEMDEC1, ADAMTS1, ADAMTS4, ADAMTS5), an aspartic protease (e.g., BACE, renin), an aspartic cathepsin (e.g., cathepsin D, cathepsin E), an apoptotic protease (e.g., apoptotic cathepsin A, cathepsin B, cathepsin C, cathepsin G, cathepsin K, cathepsin L, cathepsin S, cathepsin V / L2, cathepsin X / Z / P), cysteine ​​proteases (e.g., Cruzipain, legumin, Otubain-2), Protease, DESC1, DPP-4, FAP, Elastase, FVIIa, FiXA, FXa, FXIa, FXIIa, Granzyme B, Guanidinobenzoic acid enzyme, Hepsin, HtrA1, Human neutrophil elastase, KLK (e.g., KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, KLK14), Metalloproteinase (e.g., Meprin, Enkephalinase, PSMA, BMP-1), Lactoferrin, Marapsin , interstitial proteinase-2, MT-SP1 / interstitial proteinase, NS3 / 4A, PACE4, plasmin, PSA, MMPs (e.g., MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP23, MMP24, MMP26, MMP27), TMPRSS2, TMPRSS3, TMPRSS4, tPA, thrombin, tryptase, and uPA.

[0134] In some embodiments, the protease substrate in the CM may comprise a polypeptide sequence that is substantially non-identical (eg, no more than 90%, 80%, 70%, 60%, or 50% identical) to any polypeptide sequence naturally cleaved by the same protease.

[0135] In some embodiments, the CM may be or include the sequence of LSGRSDNH (SEQ ID NO: 214) or PLGLAG (SEQ ID NO: 17). In some embodiments, the CM may be or include the consensus sequence of any one of SEQ ID NOs: 317-327, 329-335, 340-347, 352-363, 371-378, 394-401, 410-419, 425-433, 436-449, 453-456, 458-469, 473, 475-482, 485-495 disclosed in WO2015116933 (which is incorporated herein by reference in its entirety) and SEQ ID NOs: 1-162, 268-306 disclosed in WO2015116933 (which is incorporated herein by reference in its entirety).

[0136] In some embodiments, the CM may be or include SEQ ID NOs: 14-52, 126-154, 159, 315-316, 328, 336-339, 348-351, 364-370, 379-393, 402-409, 420-424, 434-435, 450-452, 457, 470-472, 474, 483, 484 disclosed in WO2015116933, SEQ ID NOs: 163-267, 307-384, 402-445, 665-683 disclosed in WO2016118629 (which are incorporated herein by reference in their entirety), ID NO: 20-21, 411, 480-482, 351-369, 18, 71, 370-380, 412-415, 468, 547-554, 319-346 and WO2020118109 (which is incorporated herein by reference in its entirety) disclosed in SEQ ID NO: 1-16, 50-56, 60-63, 20, 70-76, 78-115, 120-128, 130-132, 135-140, 141, 152, 21-23, 17-19, 25-43 in any one of the sequences. In some examples, the CM of the cysteine ​​protease may be or include the sequence of AAN, SAN or GPTN (SEQ ID NO: 301). Examples of CM also include WO 2010 / 081173, WO2021207669, WO2021207657, WO2021142029, WO2021061867, WO2020252349, WO2020252358, WO2020236 679. WO2020176672, WO2020118109, WO2020092881, WO2020086665, WO2019213444, WO2019183218, WO2019173771, WO201 9165143, WO2019075405, WO2019046652, WO2019018828, WO2019014586, WO2018222949, WO2018165619, WO2018085555, WO2017011580, WO2016179335, WO2016179285, WO2016179257, WO2016149201, WO2016014974 (which are incorporated herein by reference in their entirety).

[0137] In some embodiments, the CM may be or include a sequence of any one of the sequences in the following table or be encompassed by a consensus sequence of the sequences.

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145] In some embodiments, the CM may be or include a combination of the above-mentioned exemplary sequences, a C-terminal truncated variant, or an N-terminal truncated variant. The truncated variants of the above-mentioned amino acid sequences suitable for use in the CM may be any truncated variants that retain the recognition site of the corresponding protease. These include C-terminal and / or N-terminal truncated variants that retain the recognition site of the protease and contain at least 3 consecutive amino acids of the above-mentioned amino acid sequence or at least 4, 5, 6, 7, 8, 9 or 10 amino acids of the aforementioned amino acid sequence. In certain embodiments, the truncated variants of the above-mentioned amino acid sequence may correspond to any of the above-mentioned amino acid sequences, but the C-terminus and / or N-terminus are truncated by 1 to 10 amino acids, 1 to 9 amino acids, 1 to 8 amino acids, 1 to 7 amino acids, 1 to 6 amino acids, 1 to 5 amino acids, 1 to 4 amino acids or 1 to 3 amino acids, and: (1) have at least three amino acid residues; and (2) retain the recognition site of the protease. In some of the aforementioned embodiments, the truncated CM is an N-terminal truncated CM. In some embodiments, the truncated CM is a C-terminal truncated CM. In some embodiments, the truncated C is a C-terminally and N-terminally truncated CM.

[0146] In some embodiments, the CM may comprise a total of 3 amino acids to 25 amino acids. In some embodiments, the CM may comprise a total of 3 to 25, 3 to 20, 3 to 15, 3 to 10, 3 to 5, 5 to 25, 5 to 20, 5 to 15, 5 to 10, 10 to 25, 10 to 20, 10 to 15, 15 to 25, 15 to 20, or 20 to 25 amino acids.

[0147] In some embodiments, CM is treated with at least one protease at a concentration of about 0.001-1500×10 4 M -1 S -1or at least 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, 7.5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 200, 250, 500, 750, 1000, 1250, or 1500×10 4 M -1 S -1 The rate of specific cleavage can be determined by the substrate cleavage kinetics (k cat / K m ) is measured as disclosed in WO2016118629.

[0148] Connector (L)

[0149] Activatable TB (e.g., activatable antibodies or other therapeutic proteins) may include one or more linkers (L). The linker may include an amino acid sequence connecting two components in the activatable TB. The linker may not be cleaved by any protease. In some embodiments, one or more linkers (e.g., flexible linkers) may be introduced into the activatable TB to provide flexibility at one or more junctions between domains, at junctions between parts, at junctions between parts and domains, or at any other junctions where the linker will be beneficial. In some embodiments, when the activatable TB is provided in the form of a conformationally restricted construct, a flexible linker may be inserted to facilitate the formation and maintenance of the structure in the uncleaved activatable TB. Any linker described herein may provide the required flexibility to facilitate the inhibition of target binding, or facilitate the cleavage of CM by a protease. In some embodiments, the linker included in the activatable TB may be flexible in whole or in part, so that the linker may include a flexible linker and one or more parts that impart a less flexible structure to provide the desired activatable TB. Some linkers may include cysteine ​​residues that may form non-α carbon covalent bonds and reduce the flexibility of the construct.

[0150] In most cases, the linker length is determined by counting 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 in the N to C direction (i.e., wherein the linker length does not include the C-terminal amino acid of the preceding component or the N-terminal amino acid of the following component).

[0151] In some embodiments, the linkers may include 1 to 50, 1 to 40, 1 to 30, 1 to 25 total (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, 20 to 24, 20 to 22, 22 to 25, 22 to 24, or 24 to 25 amino acids). In some embodiments, the linker may include 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 total.

[0152] In some embodiments, the linker may be rich in glycine (Gly or G) residues. In some embodiments, the linker may be rich in serine (Ser or S) residues. In some embodiments, the linker may be rich in glycine and serine residues. In some embodiments, the linker may have one or more pairs of glycine-serine residues (GS) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more pairs of GS).

[0153] In some embodiments, the linker may have one or more Gly-Gly-Gly-Ser (GGGS) (SEQ ID NO: 18) sequences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more GGGS sequences). In some embodiments, the linker may have one or more Gly-Gly-Gly-Gly-Ser (GGGGS) (SEQ ID NO: 535) sequences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more GGGGS sequences). In some embodiments, the linker may have one or more Gly-Gly-Ser-Gly (GGSG) (SEQ ID NO: 537) sequences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more GGSG (SEQ ID NO: 537) sequences). Examples of linkers may include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, (GGS)n, (GSGGS)n (SEQ ID NO: 536), and (GGGS)n (SEQ ID NO: 18), 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 are relatively unstructured and therefore may be able to act as neutral links between components. Glycine is even closer to alanine than is significantly larger. -ψ space and are much less constrained than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Exemplary flexible linkers include one or a combination of one or more of the following: GGSG (SEQ ID NO: 537), GGSGG (SEQ ID NO: 538), GSGSG (SEQ ID NO: 539), GSGGG (SEQ ID NO: 540), GGGSG (SEQ ID NO: 541), GSSSG (SEQ ID NO: 542), GSSGGSGGSGG (SEQ ID NO: 543), GGGS (SEQ ID NO: 18), GGGSGGGS (SEQ ID NO: 544), GGGSGGGSGGGS (SEQ ID NO: 545), GGGGSGGGGSGGGGS (SEQ ID NO: 546), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 547), GGGGSGGGGS (SEQ ID NO: 548), GGGGS (SEQ ID NO: 535), GS, GGGGSGS (SEQ ID NO: 536). NO:549), GGGSGGGGSGGGGSGS (SEQ ID NO:550), GGSLDPK GGGGS (SEQ ID NO:551), PKSCDKTHTCPPCPAPELLG (SEQ ID NO:552), SKYGPPCPPCPAPEFLG (SEQ ID NO:553), GKSSGS GSESKS (SEQ ID NO:554),GSTGSSGKSSEGKG(SEQ ID NO:555),GSTGSSGKSSEGSGSTKG(SEQ ID NO:556),GSTGSSGKPGS GEGSTKG(SEQ ID NO:557),GSTGSSGKPGSSEGST(SEQ ID NO:558),GSTGSSGKPGSSEGST(SEQ ID NO:559), GGGSSGSGS (SEQ ID NO:15), GGGGSGGGGSS (SEQ ID NO:560) and GGGSSGGSGGSSGGS (SEQ ID NO:561).Examples of linkers may also include sequences that are at least 70% identical (e.g., at least 72%, at least 74%, at least 75%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the exemplary linkers described herein. One of ordinary skill will recognize that the design of an activatable TB may include a fully or partially flexible linker such that a linker may include a flexible linker and one or more portions that impart a less flexible structure to provide the desired activatable TB structure.

[0154] In some embodiments, the activatable TB may include one, two, three, four, five, six, seven, eight, nine, or ten linker sequences (e.g., the same or different linker sequences of any exemplary linker sequences described herein or known in the art). In some embodiments, the linker may include Sulfo-SIAB ( Sulfosuccinimidyl (4-iodoethyl acyl)aminobenzoate ), SMPB (succinimidyl 4-(N-maleimidophenyl) butyrate), and sulfo-SMPB (sulfosuccinimidyl 4-(N-maleimidophenyl) butyrate), where the linker reacts with a primary amine thiol.

[0155] Conjugating agent

[0156] In some aspects, the activatable TB (e.g., activatable AB) may also include one or more additional agents, e.g., a targeting moiety that facilitates delivery to cells or tissues of interest, a therapeutic agent (e.g., an anti-tumor agent such as a chemotherapeutic agent or an anti-tumor agent), a toxin, or a fragment thereof. The additional agent may be conjugated to the activatable TB. The term "agent" is used herein to refer to a chemical compound, a mixture of chemical compounds, a biomacromolecule, or an extract made from a biological material.

[0157] In some embodiments, the activatable TB may be conjugated to a cytotoxic agent, such as a toxin (eg, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof) or a radioactive isotope.

[0158] Examples of cytotoxic agents that can be conjugated to activatable TB include dolastatin and its derivatives (e.g., auristatin E, AFP, monomethyl auristatin D (MMAD), monomethyl auristatin F (MMAF), monomethyl auristatin E (MMAE), demethyl auristatin E (DMAE), auristatin F, demethyl auristatin F (DMAF), dolastatin 16 (DmJ), dolastatin 16 (Dpv), auristatin derivatives (e.g., auristatin tyramine, auristatin quinolone (auristatin quinolone), maytansinoids (e.g., DM-1, DM-4), maytansinoid derivatives, duocarmycin, alpha-amanitin, turbostatin, phenstatin, hydroxyphenstatin, spongistatin 5, spongistatin 7, halistatin 1 1), halocomstatin 2, halocomstatin 3, halocomstatin, pyrrolobenzimidazole (PBI), cibrostatin 6, doxaliform, cemadotin analog (CemCH2-SH), Pseudomonas toxin A (PES8) variant, Pseudomonas toxin A (ZZ-PE38) variant, ZJ-101, anthracycline, doxorubicin, daunorubicin, bryostatin, camptothecin, 7-substituted camptothecin, 10,11-difluoromethylenedioxycamptothecin, combretastatin, debromoaplysiatoxin, KahaMide-F, discodermolide and ecteinascidin.

[0159] Examples of enzymatically active toxins that can be conjugated to activatable TB include: diphtheria toxin, exotoxin A chain from Pseudomonas aeruginosa, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleuriies fordii protein, dianthin protein, Phytoiaca Americana proteins (e.g., PAPI, PAPII, and PAP-8), momordica charantia inhibitors, curcin, crotirs, sapaonaria officinalis inhibitors, gelonin, mitogeliin, restrictocin, phenomycin, neomycin, and tricothecene.

[0160] Examples of anti-tumor agents that can be conjugated to activatable TB include: adriamycin, cerubidine, bleomycin, alkeran, velban, oncovin, fluorouracil, methotrexate, thiotepa, bisantrene, novantrone, thioguanine, procarabizine, and cytarabine.

[0161] Examples of antiviral agents that can be conjugated to activatable TB include: acyclovir, vira A, and symmetrel. Examples of antifungal agents that can be conjugated to activatable TB include: nystatin. Examples of detection agents that can be conjugated to activatable TB include: fluorescein and its derivatives, fluorescein isothiocyanate (FITC). Examples of antibacterial agents that can be conjugated to activatable TB include: aminoglycosides, streptomycin, neomycin, kanamycin, amikacin, gentamicin, and tobramycin. 3β,16β,17α-trihydroxycholest-5-en-22-one 16-O-(2-O-4-methoxybenzoyl-β-D-xylopyranosyl)-(1-->3)-(2-O-acetyl-α-L-arabinopyranoside) (OSW-1) that can be conjugated to activatable TB include: s-nitrobenzyloxycarbonyl derivatives of O6-benzylguanine, topoisomerase inhibitors, hemiasterlin, cephalotaxine, homoharringionine, pyrrolobenzodiazepines Dimer (PBD), functionalized pyrrolobenzodiazepine calcicheamicin, podophyllotoxin, taxanes, and vinca alkaloids. Examples of radiopharmaceuticals that can be conjugated to activatable TB include: 123 I. 89 Zr, 125 I. 131 I. 99 mTc, 201 T1, 62 Cu, 18 F. 68 Ga, 13 N. 15 O. 38 K. 82 Rb, 111 In, 133 Xe, 11 C and 99mTc (technetium). Examples of heavy metals that can be conjugated to activatable TB include: barium, gold, and platinum. Examples of anti-mycoplasma agents that can be conjugated to activatable TB include: tylosine, spectinomycin, streptomycin B, ampicillin, sulfanilamide, polymyxin, and chloramphenicol.

[0162] In some embodiments, the activatable TB may include a signal peptide. The signal peptide may be a peptide (e.g., 10-30 amino acids long) located at the end (e.g., N-terminus or C-terminus) of a newly synthesized protein that is destined for the secretory pathway. In some embodiments, the signal peptide may be conjugated to the activatable TB via a spacer. In some embodiments, the spacer is conjugated to the activatable TB in the absence of a signal peptide.

[0163] Those of ordinary skill in the art will recognize that a large number of possible agents can be conjugated to any activatable TB described herein. An agent can be conjugated to another component of the activatable TB through a conjugated portion. Conjugation can include any chemical reaction that will bind the two molecules, as long as the activatable TB and the other parts retain their corresponding activities. Conjugation can include many chemical mechanisms, such as covalent binding, affinity binding, embedding, coordination binding and complexing. In some embodiments, the binding can be covalent binding. Covalent binding can be achieved by direct condensation of existing side chains or by incorporation of external bridging molecules. Many divalent or multivalent linkers can be used to conjugate any activatable TB described herein. For example, conjugation can include organic compounds, such as thioesters, carbodiimides, succinimide esters, glutaraldehyde, diazobenzene and hexamethylenediamine. In some embodiments, the activatable TB may include or otherwise introduce one or more non-natural amino acid residues to provide a suitable conjugation site.

[0164] In some embodiments, the agent and / or conjugate can be attached to the antigen binding domain via a non-alpha carbon covalent bond (e.g., a disulfide bond on a cysteine ​​molecule). Because many cancers naturally release high levels of the reducing agent glutathione, the glutathione present in the cancerous tissue microenvironment can reduce the non-alpha carbon covalent bonds, subsequently releasing the agent and / or conjugate at the delivery site.

[0165] In some embodiments, when the conjugate binds to its target in the presence of complement at the target site (e.g., diseased tissue (e.g., cancerous tissue)), the amide bond or ester bond attaching the conjugate and / or agent to the linker is cleaved, thereby releasing the conjugate and / or agent in its active form. When administered to a subject, these conjugates and / or agents can achieve delivery and release of the conjugate and / or agent at the target site (e.g., diseased tissue (e.g., cancerous tissue)). These conjugates and / or agents can effectively deliver any conjugate and / or agent described herein in vivo.

[0166] In some embodiments, the conjugated moiety cannot be cleaved by enzymes of the complement system. For example, the conjugate and / or agent is released without complement activation, because complement activation will eventually dissolve the target cell. In such embodiments, the conjugate and / or agent will be delivered to the target cell (e.g., hormone, enzyme, corticosteroid, neurotransmitter or gene). In addition, the conjugated moiety is slightly susceptible to cleavage by serum proteases, and the conjugate and / or agent is slowly released at the target site.

[0167] In some embodiments, the conjugate and / or agent can be designed such that the conjugate and / or agent is delivered to a target site (eg, diseased tissue (eg, cancerous tissue)), but the conjugate and / or agent is not released.

[0168] In some embodiments, the conjugate and / or agent can be attached directly to the antigen binding domain, or via amino acids (e.g., D-amino acids), peptides, thiol-containing moieties, or other organic compounds that can be modified to include functional groups that can then be used to attach to the antigen binding domain by the methods described herein.

[0169] In some embodiments, the activatable TB may include at least one conjugation point of the agent. In some embodiments, all possible conjugation points can be used for conjugation with the agent. In some embodiments, one or more conjugation points may include a sulfur atom involved in a non-α carbon covalent bond, a sulfur atom involved in an interchain non-α carbon covalent bond, a sulfur atom involved in an interchain sulfur bond, and a sulfur atom involved in a non-intrachain non-α carbon covalent bond, and / or a sulfur atom of cysteine ​​or other amino acid residues containing sulfur atoms. In such cases, the residue may be naturally present in the protein construct structure, or may be incorporated into the protein construct using methods including site-directed mutagenesis, chemical conversion, or misincorporation of non-natural amino acids.

[0170] The present disclosure also provides methods and materials for preparing activatable TB with one or more conjugating agents. In some embodiments, activatable TB can be modified to include one or more interchain disulfide bonds. For example, disulfide bonds can be reduced after exposure to a reducing agent, such as but not limited to TCEP, DTT or β-mercaptoethanol. In some cases, the reduction of disulfide bonds is only partial. As used herein, the term partial reduction refers to a situation in which an activatable TB is contacted with a reducing agent and a portion of all possible conjugation sites are reduced (e.g., not all disulfide bonds are reduced). In some embodiments, 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 conjugation sites are reduced after contact with a reducing agent, then the activatable TB is partially reduced. In some embodiments, an activatable TB having one or more interchain disulfide bonds reduced can be conjugated to a drug reactive with a free thiol.

[0171] The present disclosure also provides methods and materials for conjugating therapeutic agents to specific positions on activatable TB. In some embodiments, activatable TB can be modified so that therapeutic agents can be conjugated to activatable TB at specific positions of activatable TB. For example, activatable TB can be partially reduced in a manner that is conducive to binding to activatable TB. In such cases, partial reduction of activatable TB can occur in a manner where the conjugation site in activatable TB is not reduced. In some embodiments, one or more conjugation sites on activatable TB are selected to facilitate conjugation of the agent at a specific position on the protein construct. Various factors can affect the "reduction level" of activatable TB after treatment with a reducing agent. For example, without limitation, the ratio of reducing agent to activatable TB, the duration of incubation, the incubation temperature, and / or the pH value of the reduction reaction solution may need to be optimized to achieve partial reduction of activatable TB using the methods and materials described herein. Any appropriate combination of factors (e.g., the ratio of reducing agent to activatable TB, the duration and temperature of incubation with reducing agent, and / or the pH value of reducing agent) can be used to achieve partial reduction of activatable TB (e.g., general reduction of possible conjugation sites or reduction at specific conjugation sites).

[0172] The effective ratio of reducing agent to activatable TB can be any ratio that at least partially reduces the activatable TB in a manner that allows conjugation with the agent (eg, general reduction of possible conjugation sites or reduction at a specific conjugation site). In some embodiments, the ratio of reducing agent to activatable TB can be in the range of about 20:1 to 1:1, 10:1 to 1:1, 9:1 to 1:1, 8:1 to 1:1, 7:1 to 1:1, 6:1 to 1:1, 5:1 to 1:1, 4:1 to 1:1, 3:1 to 1:1, 2:1 to 1:1, 20:1 to 1:1.5, 10:1 to 1:1.5, 9:1 to 1:1.5, 8:1 to 1:1.5, 7:1 to 1:1.5, 6:1 to 1:1.5, 5:1 to 1:1.5, 4:1 to 1:1.5, 3:1 to 1:1.5, 2:1 to 1:1.5, 1.5:1 to 1:1.5, or 1:1 to 1:1.5.

[0173] The effective incubation time and temperature for treating the activatable TB with a reducing agent can be any time and temperature that at least partially reduces the activatable TB in a manner that allows the agent to conjugate with the activatable TB (e.g., general reduction of possible conjugation sites or reduction at specific conjugation sites). In some embodiments, the incubation time and temperature for treating the activatable TB can be in the range of about 1 hour at 37° C. to about 12 hours at 37° C. (or any sub-range therein).

[0174] The effective pH value for the reduction reaction of treating the activatable TB with the reducing agent can be any pH value that at least partially reduces the activatable TB in a manner that allows conjugation of the activatable TB with the agent (eg, general reduction of possible conjugation sites or reduction at specific conjugation sites).

[0175] When the partially reduced activatable TB is contacted with a thiol-containing agent, the agent can be conjugated to the interchain thiol in the activatable TB. The agent can be modified using a thiol-containing agent (e.g., cysteine ​​or N-acetylcysteine) in a manner that includes a thiol. For example, the activatable TB can be partially reduced after incubation with a reducing agent (e.g., TEPC) at a desired ratio of reducing agent to activatable TB for about 1 hour at about 37° C. The effective ratio of reducing agent to activatable TB can be any ratio that partially reduces at least two interchain disulfide bonds located in the activatable TB in a manner that allows conjugation of the thiol-containing agent (e.g., general reduction of possible conjugation sites or reduction at specific conjugation sites).

[0176] In some embodiments, the activatable TB can be reduced by a reducing agent in a manner that avoids reduction of any intrachain disulfide bonds. In some embodiments, the activatable TB can be reduced by a reducing agent in a manner that avoids reduction of any intrachain disulfide bonds and reduces at least one interchain disulfide bond.

[0177] In some embodiments, the agent (e.g., an agent conjugated to an activatable TB) is a detectable moiety, such as an image marker or other marker. For example, the agent can be or include a radiolabeled amino acid, one or more biotinyl moieties detectable by labeled avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity detectable by optical or calorimetric methods), one or more radioisotopes or radionuclides, one or more fluorescent labels, one or more enzyme labels, and / or one or more chemiluminescent agents. In some embodiments, the detectable moiety can be attached by a spacer molecule. In some embodiments, the detectable label can include an imaging agent, a contrast agent, an enzyme, a fluorescent label, a chromophore, a dye, one or more metal ions, or a ligand-based label. In some embodiments, the imaging agent can include a radioisotope. In some embodiments, the radioisotope can be indium or technetium. In some embodiments, the contrast agent can include iodine, gadolinium, or iron oxide. In some embodiments, the enzyme can include horseradish peroxidase, alkaline phosphatase, or beta-galactosidase. In some embodiments, the fluorescent label may include 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 europium derivatives. In some embodiments, the luminescent label may include N-methylacridine derivatives. In some embodiments, the label may include Alexa Fluor. Tags, such as Alex 680 or Alexa 750. In some embodiments, the ligand-based label may comprise biotin, avidin, streptavidin, or one or more haptens.

[0178] In some embodiments, the agent can be conjugated to the activatable TB using a carbohydrate moiety, a thiol group, an amino group, or a carboxylate group. In some embodiments, the agent can be conjugated to the activatable TB via a linker and / or a CM as described herein. In some embodiments, the agent can be conjugated to a cysteine ​​or lysine in the activatable TB. In some embodiments, the agent can be conjugated to another residue of the activatable TB (e.g., those disclosed herein).

[0179] In some embodiments, a variety of bifunctional protein coupling agents can be used to conjugate the agent to the activatable TB, including but not limited to N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl diimidoadipate hydrochloride), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). In some embodiments, carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) chelator can be used to conjugate radionucleotides to activatable TB (see, e.g., WO94 / 11026).

[0180] Suitable conjugated moieties include those described in the literature. (See, e.g., Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984), which describes the use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester). See also U.S. Pat. No. 5,030,719, which describes the use of halogenated acetyl hydrazide derivatives coupled to activatable TB via oligopeptides. In some embodiments, suitable conjugated moieties include: (i) EDC (1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride; (ii) SMPT (4-succinimidyloxycarbonyl-α-methyl-α-(2-pyridyl-disulfide)-toluene (Pierce Chem., Catalog No. (21558G)); (iii) SPDP (succinimidyl-6-[3-(2-pyridyldisulfide) propionamido] hexanoate (Pierce Chem., Catalog No. 21651G); (iv) sulfo-LC-SPDP (6-[3-(2-pyridyldisulfide)-propionamido] hexanoic acid sulfosuccinimidyl ester (Pierce Chem., Catalog No. 2165-G); and (v) sulfo-NHS (N-hydroxysulfo-succinimide: Pierce Chem.) conjugated to EDC. Chem., Cat. No. 24510). Additional exemplary conjugated moieties include SMCC, Sulfo-SMCC (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate), SPDB, and Sulfo-SPDB.

[0181] The conjugated moieties described above may contain components with different properties, thus making the conjugates have different physicochemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates. The solubility of the linker containing NHS esters is less than that of sulfo-NHS esters. In addition, SMPT contains a disulfide bond with steric hindrance and can form a conjugate with increased stability. In general, the stability of disulfide bonds is less than that of other bonds because disulfide bonds are cleaved in vitro, resulting in less available conjugates. In particular, sulfo-NHS can enhance the stability of carbodiimide coupling. Compared to a single carbodiimide coupling reaction, carbodiimide coupling (e.g., EDC) forms an ester that is more resistant to hydrolysis when used in conjunction with sulfo-NHS.

[0182] One of ordinary skill in the art will recognize a variety of possible moieties that can be coupled to the activatable TB of the present disclosure. (See, e.g., "Conjugate Vaccines", Contributions to Microbiology and Immunology, JM Cruse and RE Lewis, Jr (eds.), Carger Press, New York, (1989), the entire contents of which are incorporated herein by reference). In general, effective conjugation of an agent (e.g., a cytotoxic agent) to an activatable TB can be achieved by any chemical reaction that binds the agent to the activatable TB while also allowing the agent and activatable TB to retain functionality.

[0183] Nucleic acids and vectors

[0184] In some aspects, the present disclosure also provides a nucleic acid comprising a sequence encoding an activatable target binding protein (e.g., an activatable antibody) or a component or fragment thereof. The nucleic acid may comprise a coding sequence of TB, CM, MM, and a linker in an activatable TB. In the case where the activatable TB comprises multiple peptides, for example, the activatable TB comprises multiple peptides, the nucleic acid may comprise a coding sequence of multiple peptides. In some instances, the coding sequence of one of the peptides is contained in one nucleic acid, and the coding sequence of another peptide is contained in another nucleic acid. In some instances, the coding sequences of two or more peptides in the multiple peptides are contained in the same nucleic acid.

[0185] Unless otherwise specified, "protein encoding nucleic acid sequence" includes all nucleotide sequences that are degenerate forms of each other and therefore encode the same amino acid sequence. The term "nucleic acid" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in single-stranded or double-stranded form, or a combination thereof. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides with similar binding properties to the reference nucleotides. Unless otherwise indicated, a specific nucleic acid sequence also implicitly encompasses complementary sequences as well as sequences explicitly indicated. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is RNA.

[0186] When referring to the position of a first domain or sequence relative to a second domain or sequence in a polypeptide primary amino acid sequence, the term "N-terminal positioning" means that the first domain is positioned closer to the N-terminus of the polypeptide primary amino acid sequence. In some embodiments, additional sequences and / or domains may be present between the first domain or sequence and the second domain or sequence.

[0187] When referring to the position of a first domain or sequence relative to a second domain or sequence in a polypeptide primary amino acid sequence, the term "C-terminal positioning" means that the first domain is located closer to the C-terminus of the polypeptide primary amino acid sequence. In some embodiments, additional sequences and / or domains may be present between the first domain or sequence and the second domain or sequence.

[0188] Modifications can be introduced into the 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 amino acid substitutions in which the amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include: amino acids with acidic side chains (e.g., aspartic acid and glutamic acid), amino acids with basic side chains (e.g., lysine, arginine, and histidine), nonpolar amino acids (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), uncharged polar amino acids (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine), hydrophilic amino acids (e.g., arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine, and threonine), hydrophobic amino acids (e.g., alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine). Other amino acid families 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), aromatic family (e.g., phenylalanine, tryptophan, and tyrosine).

[0189] The present disclosure also provides vectors and vector groups comprising any nucleic acid described herein. Those skilled in the art will be able to select a vector or vector group (e.g., an expression vector) suitable for preparing any activatable TB described herein, and use the vector or vector group to express any activatable TB described herein. For example, when selecting a vector or vector group, a cell type can be selected so that one or more vectors may need to be able to be integrated into the chromosome of the cell and / or replicate in the cell. Exemplary vectors that can be used to produce activatable TB are also described herein. As used herein, the term "vector" refers to a polynucleotide capable of inducing expression of a recombinant protein (e.g., a first or second monomer) in a cell (e.g., any cell described herein). "Vector" is capable of delivering nucleic acids and fragments thereof into a host cell, and includes regulatory sequences (e.g., promoters, enhancers, polyadenylic acid (poly (A)) signals). Exogenous polynucleotides can be inserted into an expression vector for expression. The term "vector" also includes artificial chromosomes, plasmids, retroviruses, and baculovirus vectors.

[0190] Methods for constructing suitable vectors comprising 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., ed., "Molecular Cloning: A Laboratory Manual," 2nd ed., Cold Spring Harbor Press, 1989 and Ausubel et al., ed., "Current Protocols in Molecular Biology," Current Protocols, 1993.

[0191] Examples of vectors include plasmids, transposons, cosmids, and viral vectors (e.g., any adenoviral vector (e.g., pSV or pCMV vector), adeno-associated virus (AAV) vector, lentiviral vector, and retroviral vector), as well as any Vector. The vector may, for example, include cis-acting elements sufficient to achieve expression; other elements for expression may be provided by the host mammalian cell or in an in vitro expression system. A skilled practitioner will be able to select vectors and mammalian cells suitable for preparing any activatable TB described herein.

[0192] In some embodiments, activatable TB can be produced biosynthetically using recombinant DNA technology and expression in eukaryotic or prokaryotic species.

[0193] cell

[0194] In some aspects, the disclosure provides a recombinant host cell comprising any vector or nucleic acid described herein. The cell can be used to produce an activatable TB (e.g., an activatable antibody) as described herein. In some embodiments, the cell can 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 embodiments, the cell can be a eukaryotic cell. As used herein, the term "eukaryotic cell" refers to a cell having a distinct, membrane-surrounded nucleus. Such cells may include, for example, mammals (e.g., rodents, non-human primates, or humans), insects, fungi, or plant cells. In some embodiments, the eukaryotic cell is a yeast cell, such as Saccharomyces cerevisiae. In some embodiments, the eukaryotic cell is a higher eukaryotic cell, such as a mammal, a bird, a plant, or an insect cell. Non-limiting examples of mammalian cells include Chinese hamster ovary (CHO) cells and human embryonic kidney cells (e.g., HEK293 cells). In some embodiments, the cell is a prokaryotic cell.

[0195] Methods for introducing nucleic acids and vectors (e.g., any vector or any vector group described herein) into cells are known in the art. Examples of methods that can be used to introduce nucleic acids into cells include: liposome transfection, transfection, calcium phosphate transfection, cationic polymer transfection, viral transduction (e.g., adenoviral transduction, lentiviral transduction), nanoparticle transfection, and electroporation.

[0196] In some embodiments, the introducing step comprises introducing into the cell a vector (eg, any vector or set of vectors described herein) comprising a nucleic acid encoding a monomer comprising any of the TB-activatable monomers described herein.

[0197] Compositions and kits

[0198] The present disclosure also provides compositions and kits comprising the activatable TB described herein. The compositions and kits may also include one or more excipients, carriers, reagents, instructions required for use of the activatable TB.

[0199] In some embodiments, the composition can be a pharmaceutical composition comprising an activatable TB, an antibody, a derivative, a fragment, an analog, and a homologue thereof. The pharmaceutical composition can comprise an activatable TB (e.g., an antibody) and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents that are compatible with drug administration. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, which is a standard reference text in the field and is incorporated herein by reference. Suitable examples of such carriers or diluents include water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles, such as fixed oils, can also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agents are incompatible with the active compound, it is expected to be used in the composition. Supplementary active compounds can also be incorporated into the composition.

[0200] The pharmaceutical composition can be formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (e.g., topical), transmucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous applications may include one or more of the following components: sterile diluents, such as water for injection, saline solutions, fixed oils, polyethylene glycols, glycerol, propylene glycol, or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetates, citrates, or phosphates, and agents for adjusting tension, such as sodium chloride or dextrose. pH values ​​may be adjusted with acids or bases (e.g., hydrochloric acid or sodium hydroxide). Parenteral preparations may be packaged in ampoules, disposable syringes, or multidose vials made of glass or plastic. In some cases, any of the activatable TBs described herein are prepared with carriers that prevent rapid elimination from the body, such as sustained and controlled release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid, and polylactic-co-glycolic acid. Methods for preparing such pharmaceutical compositions and formulations will be apparent to those skilled in the art.

[0201] In some embodiments, pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where soluble in water) or dispersions and sterile powders for preparing sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ), phosphate buffered saline (PBS), etc. The composition can be sterile and should be fluid, and have a viscosity that is convenient for injection. It must be stable under the conditions of manufacture and storage and must be preserved to prevent the contamination of microorganisms such as bacteria and fungi. The carrier can be a solvent or a dispersion medium, which contains, for example, water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol, etc.) and their suitable mixtures. For dispersed particulate compositions, for example, by using a coating such as lecithin on the particles, and by maintaining the required particle size in the case of dispersions, and by using a surfactant, appropriate fluidity can be maintained. In some embodiments, the pharmaceutical composition may also include one or more antibacterial agents and / or antibacterial agents and antifungal agents (such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.). In some embodiments, isotonic agents, such as sugars, polyols (such as mannitol, sorbitol), etc., and salts, such as sodium chloride, etc., may be included in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0202] In some embodiments, the pharmaceutical composition may include a sterile injection solution. A sterile injection solution may be prepared by incorporating the required amount of active compound and a composition listed above or a combination thereof into an appropriate solvent as required, followed by filtration sterilization. Typically, dispersions may be prepared by incorporating the active compound into a sterile vehicle containing other components required in an alkaline dispersion medium and those components listed above. In the case of a sterile powder for the preparation of a sterile injection solution, the preparation method is vacuum drying and freeze drying to obtain a powder of the active ingredient plus any other required components from a previously sterile filtered solution.

[0203] In some embodiments, the pharmaceutical composition may include an oral composition. The oral composition may include an inert diluent or an edible carrier. They may be encapsulated in a gelatin capsule or compressed into a tablet. For the purpose of oral therapeutic administration, the active compound may be blended with an excipient and used in the form of a tablet, lozenge or capsule. Oral compositions may also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is taken orally and gargled, coughed out or swallowed. Pharmaceutically compatible binders and / or adjuvant materials may be included as part of the composition. Tablets, pills, capsules, lozenges, etc. may contain any of the following ingredients or compounds of similar nature: a binder, such as microcrystalline cellulose, tragacanth or gelatin; an excipient, such as starch or lactose; a disintegrant such as alginic acid, Primogel or corn starch; a lubricant, such as magnesium stearate or Sterotes; a glidant, such as colloidal silicon dioxide; a sweetener, such as sucrose or saccharin; or a flavoring agent, such as mint, methyl salicylate or orange flavoring.

[0204] In one embodiment, the pharmaceutical compositions may be formulated for administration by inhalation. For example, the compounds may be delivered in the form of an aerosol spray from a pressured container or dispenser that contains a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer.

[0205] In one embodiment, the pharmaceutical composition can be formulated for systemic administration. For example, systemic administration can be intravenous, or it can be transmucosal or transdermal. For transmucosal or transdermal administration, a penetrant suitable for the barrier to be penetrated can be used in the formulation. Such penetrants are generally known in the art, and include, for example, detergents, bile salts, and fusidic acid derivatives for transmucosal administration. Transmucosal administration can be achieved by using nasal sprays or suppositories. For transdermal administration, the active compound can be formulated into an ointment, salves, gels, or creams, as generally known in the art.

[0206] In some embodiments, the pharmaceutical compositions may be prepared in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0207] In one embodiment, the pharmaceutical composition can be prepared with a carrier that prevents rapid elimination from the body, such as sustained and controlled release formulations, including implants and microencapsulated delivery systems. Biodegradable biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid, and polylactic acid-co-glycolic acid. Methods for preparing such preparations will be apparent to those skilled in the art.

[0208] Oral or parenteral compositions are preferably formulated in unit dosage form for ease of administration and uniformity of dosage. Unit dosage form as used herein refers to physically discrete units suitable as unitary dosages for subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the unit dosage forms of the present disclosure are dictated by and directly dependent on the unique characteristics of the active compound and the specific therapeutic effect to be achieved, and the inherent limitations in the art of compounding such active compounds for treating individuals.

[0209] In some embodiments, a composition (eg, a pharmaceutical composition) may be included in a container, vial, syringe, pen, pack, or dispenser, optionally together with instructions for administration.

[0210] Also provided herein are kits comprising any activatable TB described herein, any compositions comprising any activatable TB described herein, or any pharmaceutical compositions comprising any activatable TB described herein. Also provided are kits comprising one or more second therapeutic agents in addition to the activatable TB described herein. The one or more second therapeutic agents may be provided in a dosage administration form separate from the activatable TB. Alternatively, the one or more second therapeutic agents may be formulated together with the activatable TB.

[0211] Any kit described herein may include instructions for using any composition (e.g., pharmaceutical composition) and / or any activatable TB described herein. In some embodiments, the kit may include instructions for performing any method described herein. In some embodiments, the kit may include at least one dose of any composition (e.g., pharmaceutical composition) described herein. In some embodiments, the kit may provide a syringe for administering any pharmaceutical composition described herein.

[0212] Also provided herein are activatable TB produced by any of the methods described herein. Also provided are compositions (e.g., pharmaceutical compositions) comprising any activatable TB produced by any of the methods described herein. Also provided herein are kits comprising at least one dose of any of the compositions (e.g., pharmaceutical compositions) described herein.

[0213] Methods for producing activatable target binding proteins

[0214] Provided herein are methods for producing any activatable TB described herein, the methods comprising: (a) culturing any recombinant host cell described herein in a liquid culture medium under conditions sufficient to produce the activatable TB; and (b) recovering the activatable TB from the host cell and / or the liquid culture medium.

[0215] Methods for culturing cells are well known in the art. In some embodiments, cells can be maintained in vitro under conditions that are conducive to cell proliferation, cell differentiation, and cell growth. For example, recombinant cells can be cultured by contacting cells (e.g., any cells described herein) with a cell culture medium that includes necessary growth factors and supplements sufficient to support cell viability and growth.

[0216] In some embodiments, the method may further include isolating the recovered activatable TB. Isolation of the activatable TB may be performed using any separation or purification technique for isolating protein species, such as affinity tag-based protein separation (e.g., polyhistidine (His) tag, glutathione-S-transferase tag, etc.), ammonium sulfate precipitation, polyethylene glycol precipitation, size exclusion chromatography, ligand affinity chromatography (e.g., protein A chromatography), ion exchange chromatography (e.g., anion or cation), hydrophobic interaction chromatography, etc.

[0217] The compositions and methods described herein may involve the use of non-reducing or partially reducing conditions that allow for the formation of a non-alpha carbon covalent bond, such as a disulfide bond, between the MM and the AB that can activate the TB.

[0218] In some embodiments, the dual-anchored activatable macromolecule of the present disclosure (e.g., dual-anchored activatable TB or dual-anchored activatable antibody) is prepared by a method comprising: engineering a cysteine ​​residue at a disulfide bonding site in the MM of the dual-anchored activatable macromolecule; engineering a cysteine ​​residue at a disulfide bonding site in the TB of the dual-anchored activatable macromolecule, wherein the MM is coupled to the TB and the CM is located between the MM and the TB. In some aspects, the present disclosure includes expressing an activatable macromolecule having an engineered cysteine ​​residue at a disulfide bonding site in the TB of the dual-anchored activatable macromolecule and recovering the dual-anchored activatable macromolecule, wherein in the recovered dual-anchored activatable macromolecule, the MM is tethered to the TB at its disulfide bonding site.

[0219] In some embodiments, the dual-anchored activatable macromolecule of the present disclosure (e.g., a dual-anchored activatable TB or a dual-anchored activatable antibody) is prepared by a method comprising coupling a MM comprising cysteine ​​to a TB, wherein the cysteine ​​forms a non-α-carbon covalent bond with an amino acid in the TB that forms a non-α-carbon covalent bond.

[0220] In some embodiments, the dual-anchored activatable macromolecule of the present disclosure (e.g., a dual-anchored activatable TB or a dual-anchored activatable antibody) is prepared by a method comprising coupling TB to an MM comprising an amino acid that forms a non-α-carbon covalent bond, wherein the MM forms a non-α-carbon covalent bond with the amino acid that forms a non-α-carbon covalent bond in the TB.

[0221] In some aspects, the present disclosure includes a method of making a dual-anchored activatable macromolecule, the method including providing a MM, the MM including an amino acid that forms a non-α carbon covalent bond, the amino acid that forms a non-α carbon covalent bond is configured to form a non-α carbon covalent bond with an amino acid that forms a non-α carbon covalent bond in a TB, the TB being coupled to the MM. In some aspects, the present disclosure includes a method of making a dual-anchored activatable macromolecule, the method including providing a MM including a cysteine, the cysteine ​​being configured to form a non-α carbon covalent bond with an amino acid that forms a non-α carbon covalent bond in a TB, the TB being coupled to the MM.

[0222] In some aspects, the present disclosure includes a method for identifying the position of an amino acid inserted into a double-anchored activatable macromolecule to form a non-α carbon covalent bond. In some aspects, the method includes analyzing the structural arrangement of TB and MM in the double-anchored activatable macromolecule and identifying the position of an amino acid inserted into TB to form a non-α carbon covalent bond. In some aspects, the method includes analyzing the structural arrangement of TB and MM in the double-anchored activatable macromolecule and identifying the position of an amino acid inserted into MM to form a non-α carbon covalent bond. In some aspects, the method includes analyzing the structural arrangement of TB and MM in the double-anchored activatable macromolecule and identifying the position of an amino acid inserted into a peptide coupled to TB to form a non-α carbon covalent bond. In some aspects, the method includes analyzing the structural arrangement of TB and MM in the double-anchored activatable macromolecule and identifying the position of an amino acid inserted into a peptide coupled to MM to form a non-α carbon covalent bond. In some aspects, one or more amino acids of non-α carbon covalent bonds formed are cysteine.

[0223] In some aspects, the present disclosure includes a method for identifying the position of an amino acid that is inserted into a double-anchored activatable macromolecule to form a non-α carbon covalent bond, the method comprising drawing a three-dimensional structure of a TB coupled to an MM, identifying a first amino acid of a TB close to a second amino acid of the MM, wherein the first amino acid is a position for inserting an amino acid that forms a non-α carbon covalent bond in the TB. In some aspects, the second amino acid is a position for inserting an amino acid that forms a non-α carbon covalent bond in the MM. In some aspects, in the three-dimensional structure of the TB coupled to the MM, the α carbon atom of the first amino acid is within 2 to 15 angstroms of the α carbon atom of the second amino acid. In some aspects, in the three-dimensional structure of the TB coupled to the MM, the α carbon atom of the first amino acid is within 15, 10, 9, 8, 7, 6, 5, 4, 3 or 2 angstroms of the α carbon atom of the second amino acid. In the case where one or both amino acids are in a flexible loop, the above-mentioned distance between Cα applies to the case where the two amino acids are closest to each other. In some aspects, non-α carbon covalent bonds include, but are not limited to:

[0224] 1. Disulfide bond between two Cys residues.

[0225] 2.Isopeptide bonds between Lys and glutamic acid or glutamine residues.

[0226] 3. Ester bond between Thr and glutamine.

[0227] 4. Thioester bond between Cys and Gln.

[0228] 5.Thioether bond between Cys and Tyr.

[0229] 6. His-Tyr cross-links between His and Tyr (eg, known to exist in cytochrome c oxidase-type proteins).

[0230] 7. Lys-Cys NOS (nitrogen-oxygen-sulfur) crosslinks.

[0231] In some aspects, the disclosure includes a method of making a dual-anchored activatable macromolecule, the method comprising coupling a MM comprising a cysteine ​​to a TB, wherein the cysteine ​​forms a non-α carbon covalent bond with a non-α carbon covalent bond-forming amino acid in the TB.

[0232] In some aspects, the present disclosure includes a method of making a dual-anchored activatable macromolecule, the method comprising coupling a TB to an MM comprising an amino acid that forms a non-α carbon covalent bond, wherein the MM forms a non-α carbon covalent bond with the amino acid that forms a non-α carbon covalent bond in the TB.

[0233] In some embodiments, the method further comprises formulating the isolated activatable TB into a pharmaceutical composition. Various formulations are known in the art and are described herein. Any isolated activatable TB described herein can be formulated for any route of administration (e.g., intravenous, intratumoral, subcutaneous, intradermal, oral, inhaled, intranasal, intrapulmonary, intrathecal, infusion, transdermal, topical, transmucosal, or intramuscular).

[0234] Treatment

[0235] In some aspects, the present disclosure also provides a method of treating a disease (e.g., cancer (e.g., any cancer described herein), an inflammatory disorder, condition or disease, or an autoimmune disorder, condition or disease) in a subject, the method comprising administering a therapeutically effective amount of any activatable TB described herein to the subject. In some embodiments, the present disclosure provides a method of preventing a disease in a subject, delaying the progression of a disease, treating a disease, alleviating a symptom of a disease, or otherwise ameliorating a disease by administering a therapeutically effective amount of an activatable TB described herein to a subject in need thereof. The term "treating" refers to ameliorating at least one symptom of a disorder. In some embodiments, the disorder being treated is cancer and is to ameliorate at least one symptom of cancer. As used herein, the term "subject" refers to any mammal. In some embodiments, the subject is a feline (e.g., a cat), a canine (e.g., a dog), an equine (e.g., a horse), a rabbit, a pig, a rodent (e.g., a mouse, a rat, a hamster, or a guinea pig), a non-human primate (e.g., a monkey (e.g., a baboon, a marmoset) or an ape (e.g., a chimpanzee, a gorilla, an orangutan, or a gibbon)), or a human. In some embodiments, the subject is a human. The terms subject and patient are used interchangeably herein. In some embodiments, the subject has been previously identified or diagnosed as having a disease (e.g., a cancer (e.g., any of the cancers described herein)).

[0236] The activatable TB used in any of the embodiments of these methods and uses can be administered at any stage of the disease. For example, such activatable TB can be administered to cancer patients at any stage, from early stage to metastatic. In some embodiments, the activatable TB and its formulations can be administered to subjects suffering from or susceptible to a disease or condition associated with aberrant target expression and / or activity.

[0237] Any of a variety of methods known in the art can be used to identify subjects suffering from or susceptible to diseases or conditions associated with abnormal target expression and / or activity. For example, any of a variety of clinical and / or laboratory tests (e.g., physical examination and blood, urine and / or stool analysis) can be used to assess health status to identify subjects suffering from inflammatory disorders, disorders or diseases or autoimmune disorders, disorders or diseases, cancer or other tumor disorders. For example, any of a variety of clinical and / or laboratory tests (e.g., physical examination and / or body fluid analysis, such as blood, urine and / or stool analysis) can be used to assess health status to identify subjects suffering from inflammation and / or inflammatory disorders.

[0238] In some embodiments, administration of an activatable TB to a patient with a disease or condition associated with aberrant target expression and / or activity is considered successful if any of a variety of laboratory or clinical goals are achieved. For example, administration of an activatable TB to a patient with a disease or condition associated with aberrant target expression and / or activity is considered successful if one or more symptoms associated with the disease or condition are alleviated, mitigated, suppressed, or do not progress to a further (i.e., worse) state. Administration of an activatable TB to a patient with a disease or condition associated with aberrant target expression and / or activity is considered successful if the disease or condition enters remission or does not progress to a further (i.e., worse) state.

[0239] As used herein, the term "treating" includes reducing the severity, frequency, or number of one or more (e.g., 1, 2, 3, 4, or 5) symptoms or signs of a disease (e.g., cancer (e.g., any cancer described herein)) in a subject (e.g., any subject described herein). In some embodiments where the disease is cancer, treatment reduces cancer growth, inhibits cancer progression, inhibits cancer metastasis, or reduces the risk of cancer recurrence in a subject with cancer.

[0240] In some embodiments, the disease can be cancer. In some embodiments, the subject can have been identified or diagnosed as having cancer. Examples of cancer include: solid tumors, hematological tumors, sarcomas, osteosarcomas, glioblastomas, neuroblastomas, melanomas, rhabdomyosarcomas, Ewing sarcomas, sarcoma), osteosarcoma, B cell tumors, multiple myeloma, lymphoma (e.g., B cell lymphoma, B cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, cutaneous T cell lymphoma), leukemia (e.g., hairy cell leukemia, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL)), myelodysplastic syndrome (MDS), Kaposi's sarcoma, retinoblastoma, gastric cancer, urothelial cancer, lung cancer, renal cell carcinoma, gastric cancer and esophageal cancer, pancreatic cancer, prostate cancer, brain cancer, colon cancer, bone cancer, lung cancer, breast cancer, colorectal cancer, ovarian cancer, nasopharyngeal adenocarcinoma, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma, endometrial cancer, bladder cancer, cervical cancer, liver cancer, and hepatocellular carcinoma. In some embodiments, the cancer is a lymphoma. In some embodiments, the lymphoma is Burkitt's lymphoma. In some aspects, the subject has been identified or diagnosed as having a familial cancer syndrome, such as Li-Fraumeni Syndrome, familial breast cancer-ovarian cancer (BRCA1 or BRAC2 mutation) syndrome, etc. The disclosed method can also be used to treat non-solid cancers. Exemplary solid tumors include malignancies (e.g., sarcomas, adenocarcinomas, and carcinomas) of various organ systems, such as lung, breast, lymph, gastrointestinal (e.g., colon) and urogenital (e.g., kidney, urothelial or testicular tumors) tracts, pharynx, prostate, and ovarian malignancies. Exemplary adenocarcinomas include colorectal cancer, renal cell carcinoma, liver cancer, non-small cell lung cancer, and small intestinal cancer. Other examples of cancers treatable by the compositions and methods herein include: acute lymphoblastic leukemia in adults; acute lymphoblastic leukemia in children; acute myeloid leukemia in adults; adrenocortical carcinoma; adrenocortical carcinoma in children; AIDS-related lymphoma; AIDS-related malignancies; anal cancer; cerebellar astrocytoma in children; cerebral astrocytoma in children; extrahepatic bile duct cancer; bladder cancer; bladder cancer in children; osteosarcoma / malignant fibrous histiocytoma bone cancer; brain stem glioma in children; brain tumors in adults; brain stem glioma brain tumors in children; cerebellar astrocytoma brain tumors in children; cerebral astrocytoma / malignant glioma brain tumors in children; ependymoma brain tumors in children; medulloblastoma brain tumors in children; supratentorial primitive neuroectodermal tumor brain tumors in children; optic pathway and hypothalamic glioma brain tumors in children; brain tumors in children (other); breast cancer; breast cancer and pregnancy; breast cancer in children; breast cancer in men; bronchial adenoma / carcinoid in children; carcinoid tumors in children; gastrointestinal carcinoid tumors;Adrenocortical carcinoma; Islet cell carcinoma; Carcinoma of unknown primary; Primary central nervous system lymphoma; Cerebellar astrocytoma, childhood; Cerebral astrocytoma / glioblastoma, childhood; Cervical cancer; Childhood cancer; Chronic lymphocytic leukemia; Chronic myeloid leukemia; Chronic myeloproliferative disorder; Clear cell sarcoma of the tendon sheath; Colon cancer; Childhood colorectal cancer; Cutaneous T-cell lymphoma; Endometrial carcinoma; Childhood ependymoma; Ovarian epithelial carcinoma; Esophageal cancer; Childhood esophageal cancer; Ewing family tumors; Childhood extracranial germ cell tumors; Extragonadal germ cell tumors tumors; extrahepatic bile duct cancer; intraocular melanoma eye cancer; retinoblastoma eye cancer; gallbladder cancer; stomach (gastric) cancer; childhood stomach (gastric) cancer; gastrointestinal carcinoid tumors; childhood extracranial germ cell tumors; extragonadal germ cell tumors; ovarian germ cell tumors; gestational trophoblastic tumors; childhood brain stem gliomas; childhood optic pathway and hypothalamic gliomas; hairy cell leukemia; head and neck cancer; adult (primary) hepatocellular (liver) cancer; childhood (primary) hepatocellular (liver) cancer; adult Hodgkin's lymphoma (Hodgkin's Lymphoma; Hodgkin's lymphoma in children; Hodgkin's lymphoma in pregnancy; Hypopharyngeal cancer; Hypothalamic and optic pathway gliomas in children; Intraocular melanoma; Islet cell carcinoma (endocrine pancreas); Kaposi's sarcoma; Kidney cancer; Laryngeal cancer; Laryngeal cancer in children; Acute lymphoblastic leukemia in adults; Acute lymphoblastic leukemia in children; Acute myeloid leukemia in adults; Acute myeloid leukemia in children; Chronic lymphocytic leukemia; Chronic myeloid leukemia; Hairy cell leukemia; Lip and oral cancer; Liver cancer (primary) in adults; Liver cancer (primary) in children; Non-small cell lung cancer; Small cell lung cancer; Acute lymphoblastic leukemia in adults; Acute lymphoblastic leukemia in children ; Chronic lymphocytic leukemia; AIDS-related lymphoma; Central nervous system (primary) lymphoma; Cutaneous T-cell lymphoma; Hodgkin's lymphoma, adult; Hodgkin's lymphoma, childhood; Hodgkin's lymphoma, pregnancy; Non-Hodgkin's lymphoma, adult; Non-Hodgkin's lymphoma, childhood; Non-Hodgkin's lymphoma, pregnancy; Primary central nervous system lymphoma; Macroglobulinemia, Waldenstrom's; Male breast cancer; Malignant mesothelioma, adult; Malignant mesothelioma, childhood; Malignant thymoma; Medulloblastoma, childhood; Melanoma; Intraocular melanoma; Merkel cell carcinoma CellCarcinoma); Malignant Mesothelioma; Metastatic Squamous Cell Carcinoma of the Neck with Occult Primary; Multiple Endocrine Neoplasia Syndrome in Children; Multiple Myeloma / Plasma Cell Neoplasm; Mycosis Fungoides; Myelodysplastic Syndrome; Chronic Myeloid Leukemia; Acute Myeloid Leukemia in Children; Multiple Myeloma; Chronic Myeloproliferative Disorders; Nasal and Paranasal Sinus Cancer; Nasopharyngeal Carcinoma; Nasopharyngeal Carcinoma in Children; Neuroblastoma; Non-Hodgkin's Lymphoma in Adults; Non-Hodgkin's Lymphoma in Children; Non-Hodgkin's Lymphoma in Pregnancy; Non-Small Cell Lung Cancer;Oral cancer, childhood; oral cavity and lip cancer; oropharyngeal cancer; osteosarcoma / malignant fibrous histiocytoma of bone; ovarian cancer, childhood; ovarian epithelial cancer; ovarian germ cell tumors; ovarian low-grade malignant potential tumors; pancreatic cancer; pancreatic cancer, childhood; islet cell pancreatic cancer; paranasal sinus and nasal cavity cancer; parathyroid cancer; penile cancer; pheochromocytoma; pineal and supratentorial primitive neuroectodermal tumors, childhood; pituitary tumors; plasma cell neoplasms / multiple myeloma; pleuropulmonary blastoma; pregnancy and breast cancer; pregnancy and Hodgkin lymphoma; pregnancy and non Hodgkin lymphoma; primary central nervous system lymphoma; primary liver cancer, adults; primary liver cancer, children; prostate cancer; colorectal cancer; renal cell (kidney) cancer; renal cell cancer, children; transitional cell carcinoma of the renal pelvis and ureter; retinoblastoma; rhabdomyosarcoma, children; salivary gland cancer; salivary gland cancer, children; Ewing family tumor sarcoma; Kaposi sarcoma; sarcoma (osteosarcoma) / malignant fibrous histiocytoma of bone; rhabdomyosarcoma, children; soft tissue sarcoma, adults; soft tissue sarcoma, children; Sezary syndrome Syndrome); skin cancer; childhood skin cancer; skin cancer (melanoma); Merkel cell skin cancer; small cell lung cancer; small intestinal cancer; adult soft tissue sarcoma; childhood soft tissue sarcoma; metastatic squamous cell carcinoma of the neck with occult primary site; stomach (gastric) cancer; childhood stomach (gastric) cancer; childhood supratentorial primitive neuroectodermal tumor; cutaneous T-cell lymphoma; testicular cancer; childhood thymoma; malignant thymoma; thyroid cancer; childhood thyroid cancer; transitional cell carcinoma of the renal pelvis and ureter; gestational trophoblastic tumor; childhood cancer of unknown primary site; unusual childhood cancer; transitional cell carcinoma of the ureter and renal pelvis; urethral cancer; uterine sarcoma; vaginal cancer; childhood optic pathway and hypothalamic glioma; vulvar cancer; Waldenstrom's macroglobulinemia; Wilms' Tumor; diffuse large B-cell lymphoma (DLBCL); and mantle cell lymphoma (MCL). Metastasis of the above-mentioned cancers can also be treated or prevented according to the methods described herein. ;

[0241] In some embodiments, the methods herein may result in a decrease in the number, severity, or frequency of one or more symptoms of cancer in a subject (eg, compared to the number, severity, or frequency of one or more symptoms of cancer in the subject prior to treatment).

[0242] The method may also include administering one or more additional agents to the subject. In some embodiments, activatable TB may be administered in combination with one or more additional agents during and / or after treatment. In some embodiments, activatable TB may be formulated into a single therapeutic composition, and activatable TB and one or more additional agents may be administered simultaneously. Alternatively, activatable TB and one or more additional agents may be separated from each other, for example, each formulated into a separate therapeutic composition, and activatable TB and additional agents are administered simultaneously, or activatable TB and additional agents are administered at different times during the treatment regimen. For example, activatable TB may be administered before administering additional agents, after administering additional agents, or in an alternating manner. Activatable TB and one or more additional agents may be administered in a single dose or multiple doses.

[0243] The present disclosure also provides methods for detecting the presence or absence of a cleavage agent and a target in a subject or sample. Such methods may include: (i) contacting a subject or biological sample with an activatable TB, wherein the activatable TB includes a detectable marker located on a portion of the activatable TB that is released after CM cleavage; and (ii) measuring the level of activated (cleaved) TB in the subject or biological sample, wherein a detectable level of activated TB in the subject or biological sample indicates that a cleavage agent, a target, or both a cleavage agent and a target are not present and / or insufficiently present in the subject or biological sample, such that target binding and / or protease cleavage of the activatable TB cannot be detected in the subject or biological sample, and wherein a decrease in the detectable level of activated (cleaved) TB in the subject or biological sample indicates the presence of a cleavage agent and a target in the subject or biological sample.

[0244] The reduction in the level of the detectable marker can 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 substantial reduction of 100%. In some embodiments, the detectable marker can be conjugated to a component (e.g., AB) of the activatable TB. In some embodiments, a secondary reagent that specifically binds to the activated TB can be used to measure the level of the activatable TB in a subject or sample, wherein the reagent comprises a detectable marker. The secondary reagent can be a TB comprising a detectable marker.

[0245] In some embodiments, the activatable TB can also be used to detect targets in patient samples and can therefore be used as diagnostic agents. For example, the activatable TB can be used in in vitro assays, such as ELISA, to detect target levels in patient samples. For example, the activatable TB can be fixed on a solid support (e.g., a well in a microtiter plate). The fixed activatable TB can be used as a capture TB for any target that may be present in a test sample. Before contacting the fixed TB with the patient sample, the solid support can be rinsed and treated with a blocking agent (e.g., milk protein or albumin) to prevent nonspecific adsorption of the analyte.

[0246] In some embodiments, based on the results obtained in an in vitro diagnostic assay using an activatable TB, a subject's disease can be staged based on the expression level of the target antigen. For a given disease, blood samples are drawn from subjects diagnosed as being at various stages of disease progression and / or at various time points during therapeutic treatment of the disease. Using a population of samples that provide statistically significant results for each stage of progression or treatment, a range of antigen concentrations that can be considered characteristic of each stage is specified.

[0247] The activatable TB herein can also be used for diagnosis and / or imaging methods. In some embodiments, such methods can be in vitro methods. In some embodiments, such methods can be in vivo methods. In some embodiments, such methods can be in situ methods. In some embodiments, such methods can be ex vivo methods. For example, an activatable TB with CM can be used to detect the presence or absence of an enzyme capable of cleaving CM. Such activatable TB can be used in diagnosis, which may include detecting (e.g., qualitatively or quantitatively) enzyme activity in vivo (or in some embodiments, an environment with an increased reduction potential, such as an environment that can provide disulfide bond reduction) by measuring the accumulation of activated TB (i.e., TB produced by the cleavage of activatable TB) in a given cell or tissue of a given host organism. This accumulation of activated TB not only indicates that the tissue expresses enzyme activity (or an increased reduction potential according to the nature of the CM substrate), but also indicates that the tissue expresses the target to which the activated TB binds.

[0248] For example, CM can be selected as a protease substrate for a protease found at a tumor site, a site of viral or bacterial infection at a biorestricted site (e.g., such as an abscess, an organ, etc.), etc. The AB can be an AB that binds a target antigen. Using methods familiar to those skilled in the art, a detectable label (e.g., a fluorescent label or a radioactive label or a radioactive tracer) can be conjugated to the AB or other regions of the activatable TB. Suitable detectable labels can be discussed in the context of the above screening methods, and additional specific examples are provided below. Using a TB (e.g., AB) specific for a protein or peptide of a disease state and a protease with increased activity in the disease tissue of interest, the activatable TB will exhibit an increased binding rate to the disease tissue relative to tissues in which the CM-specific enzyme is not present at a detectable level or is present at a level lower than that in the disease tissue or is inactive (e.g., in a zymogen form or in a complex with an inhibitor). Because the kidney filtration system rapidly clears small proteins and polypeptides from the blood, and because the enzyme specific for CM is not present at a detectable level (or is present at a lower level in non-disease tissue or in an inactive conformation), the accumulation of activated TB in the disease tissue relative to non-disease tissue is increased.

[0249] In some embodiments, the activatable TB can be used for in vivo imaging, wherein the detection of a fluorescent signal in a subject (e.g., a mammal, including a human) indicates that the disease site contains the target and contains a protease specific for the CM of the activatable TB. In vivo imaging can be used to identify or otherwise refine a patient population suitable for treatment with the activatable TB of the present disclosure. For example, a patient who tests positive for both the target and the protease that cleaves the substrate in the CM of the activatable TB being tested (e.g., activated TB accumulates at the disease site) is identified as a candidate suitable for treatment with such an activatable TB comprising such CM. Similarly, a patient who tests negative can be identified as a suitable candidate for another therapy (i.e., not suitable for treatment with the activatable TB tested). In some embodiments, such patients who test negative for the first activatable TB can be tested with other activatable TBs containing different CMs until an activatable TB suitable for treatment (e.g., an activatable TB comprising CM that is cleaved by the patient at the disease site) is identified.

[0250] In some embodiments, in situ imaging can be used for methods of identifying patients to be treated. For example, in in situ imaging, activatable TB can be used to screen patient samples to identify those patients with appropriate proteases and targets in appropriate locations, such as tumor sites. In some embodiments, in situ imaging is used to identify or otherwise refine the patient population suitable for treatment with activatable TB of the present disclosure. For example, patients whose proteases for the target and the substrates in the CM of the activatable TB being tested for cleavage are all tested positive (e.g., activated TB accumulates at the disease site) are identified as candidates suitable for treatment with such activatable TBs comprising such CMs. Similarly, patients whose proteases for the target and the substrates in the CM used in the activatable TB being tested for cleavage using these methods are tested negative for one or both are identified as suitable candidates for another therapy (i.e., not suitable for treatment with the activatable TB tested). In some embodiments, such patients whose first activatable TB test is negative can be tested with other activatable TBs comprising different CMs until an activatable TB suitable for treatment (e.g., an activatable TB comprising a CM cleaved by a patient at the disease site) is identified.

[0251] In some embodiments, the present disclosure includes any one or combination of the following non-limiting numbered items:

[0252] 1. A dual-anchored activatable target binding protein, the dual-anchored activatable target binding protein comprising:

[0253] a target binding protein (TB) that specifically binds to the target;

[0254] a masking moiety (MM) coupled to the TB, wherein the MM inhibits binding of the AB to the target; and

[0255] a cleavable moiety (CM) coupled to the TB and positioned between the TB and the MM, wherein the CM is a polypeptide that serves as a protease substrate, and

[0256] Also included are non-alpha carbon covalent bonds tethering the MM and the TB.

[0257] 2. The activatable target binding protein of item 1, wherein the TB is an antigen binding protein (AB).

[0258] 3. The activatable target binding protein of item 1 or item 2, wherein the activatable target binding protein has lower target binding activity compared to a single-anchored activatable target binding protein lacking the non-α-carbon covalent bond.

[0259] 4. The activatable target binding protein of any one or combination of items 1-3, wherein the non-α carbon covalent bond is an ester bond or a thioester bond.

[0260] 5. The activatable target binding protein of claim 4, wherein the ester bond is between threonine and glutamine.

[0261] 6. The activatable target binding protein of item 4, wherein the thioester bond is between cysteine ​​and glutamine or tyrosine.

[0262] 7. The activatable target binding protein of any one or combination of items 1-3, wherein the non-α carbon covalent bond is a cross-link between histidine and tyrosine or a cross-link between lysine and cysteine.

[0263] 8. The activatable target binding protein of any one or combination of items 1-3, wherein the non-α carbon covalent bond is an isopeptide bond.

[0264] 9. The activatable target binding protein of claim 8, wherein the isopeptide bond is between a lysine and a glutamic acid or aspartic acid residue.

[0265] 10. The activatable target binding protein of claim 8, wherein the isopeptide bond is between the γ-carboxamide group of glutamine and the ε-amino group of the lysine side chain.

[0266] 11. The activatable target binding protein of any one or combination of items 1-3, wherein the non-α carbon covalent bond is a disulfide bond.

[0267] 12. The activatable target binding protein of claim 11, wherein the disulfide bond is formed between the first cysteine ​​and the second cysteine, wherein

[0268] The first cysteine ​​is within the MM and the second cysteine ​​is within the TB;

[0269] The first cysteine ​​is within a peptide coupled to the MM and the second cysteine ​​is within the TB, or

[0270] The first cysteine ​​is within the MM and the second cysteine ​​is within a peptide coupled to the TB.

[0271] 13. The activatable target binding protein of any one or combination of items 1-12, further comprising a second CM, wherein

[0272] The second CM is located between the MM and the non-α carbon covalent bond;

[0273] The second CM is within the MM and is at most 5 amino acids away from the cysteine ​​forming the non-alpha carbon covalent bond, or

[0274] The second CM is within the TB and is at most 5 amino acids away from the cysteine ​​forming the non-alpha carbon covalent bond.

[0275] 14. The activatable target binding protein of any one or combination of items 1-13, wherein the first CM and the second CM are substrates of different proteases.

[0276] 15. The activatable target binding protein of any one or combination of items 1-13, wherein the first CM and the second CM are substrates of the same protease.

[0277] 16. The activatable target binding protein of any one or combination of items 1-12, wherein the protease is produced by a tumor of the subject.

[0278] 17. The activatable target binding protein of any one or combination of items 2-16, wherein the AB is an antibody, a Fab fragment, a F(ab') 2 fragment, scFv, scAb, dAb, VHH or single domain antibody.

[0279] 18. The activatable target binding protein of any one or combination of items 2-17, wherein the AB is a single domain antibody.

[0280] 19. The activatable target binding protein of any one or combination of items 2-18, wherein the AB is an Fc-tagged single domain antibody.

[0281] 20. The activatable target binding protein of any one or combination of items 2-17, wherein the AB is a bispecific antibody.

[0282] 21. The activatable target binding protein of claim 20, wherein the bispecific antibody is a bispecific T cell engager (BiTE) or a dual affinity retargeting antibody (DART).

[0283] 22. The activatable target binding protein of any one or combination of items 2-17, wherein the AB is a multispecific antibody.

[0284] 23. The activatable target binding protein of any one or combination of items 18-19, wherein the non-alpha carbon covalent bond is between the MM and the single domain antibody.

[0285] 24. An activatable target binding protein according to any one or a combination of items 1-22, wherein the non-α carbon covalent bond is between the MM and the Fc domain.

[0286] 25. The activatable target binding protein of any one or combination of items 1-24, wherein the MM comprises an epitope of the TB.

[0287] 26. The activatable target binding protein of any one or combination of items 1-24, wherein the MM does not comprise four or more consecutive amino acids of an epitope of the TB.

[0288] 27. The activatable target binding protein of any one or combination of items 1-26, wherein the dissociation constant of the binding of the MM to the TB exceeds the dissociation parameter of the binding of the TB to the target.

[0289] 28. The activatable target binding protein of any one or combination of items 1-27, wherein the MM is a polypeptide with a length of 2 to 40 amino acids.

[0290] 29. The activatable target binding protein of any one or combination of items 1-28, wherein the activatable target binding protein comprises a linker between the MM and the CM.

[0291] 30. The activatable target binding protein of any one or combination of items 1-29, wherein the activatable target binding protein comprises a linker between the CM and the TB.

[0292] 31. The activatable target binding protein of any one or combination of items 1-29, wherein the activatable target binding protein comprises a first linker between the MM and the CM and a second linker between the CM and the TB.

[0293] 32. A composition comprising the activatable target binding protein of any one or a combination of items 1 to 31 and a carrier.

[0294] 33. The composition of claim 32, wherein the composition is a pharmaceutical composition.

[0295] 34. A container, vial, syringe, injection pen or kit comprising at least one dose of the composition of item 32 or 33.

[0296] 35. A nucleic acid comprising a sequence encoding the activatable target binding protein of any one of items 1 to 31.

[0297] 36. A vector comprising the nucleic acid of item 35.

[0298] 37. A cell comprising the nucleic acid of item 35 or the vector of item 36.

[0299] 38. A conjugated activatable target binding protein, comprising the activatable target binding protein of any one or a combination of items 1 to 31 conjugated to an agent.

[0300] 39. The conjugated, activatable target binding protein of claim 38, wherein the agent is a therapeutic agent, a targeting moiety, or a detectable moiety.

[0301] 40. A method for treating a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an activatable target binding protein of any one or a combination of items 1 to 31, a composition of item 32 or 33, or a conjugated activatable target binding protein of item 38 or 39.

[0302] 41. The method of claim 40, wherein the subject has been identified or diagnosed as having cancer, an inflammatory disorder, condition or disease, or an autoimmune disorder, condition or disease.

[0303] 42. A method for producing an activatable target binding protein, the method comprising:

[0304] Cultivating the cell of item 37 in a culture medium under conditions sufficient to produce the activatable target binding protein; and

[0305] The activatable target binding protein is recovered from the cells or the culture medium.

[0306] 43. The method of item 42, further comprising isolating the activatable target binding protein recovered from the cells or the culture medium.

[0307] 44. The method of claim 43, wherein the separation is performed using a protein purification tag and / or size exclusion chromatography.

[0308] 45. The method of item 43 or item 44, further comprising formulating the activatable target binding protein into a pharmaceutical composition.

[0309] 46. ​​A method for producing a dual-anchored activatable macromolecule, the method comprising: engineering a cysteine ​​residue at a disulfide bonding site in a masking portion (MM) of the dual-anchored activatable macromolecule; engineering a cysteine ​​residue at a disulfide bonding site in a target binding protein (TB) of the dual-anchored activatable macromolecule, wherein the MM is coupled to the TB and a cleavable portion (CM) is located between the MM and the TB; expressing the dual-anchored activatable macromolecule; and recovering the dual-anchored activatable macromolecule, wherein in the recovered dual-anchored activatable macromolecule the MM and the TB are tethered at their disulfide bonding sites.

[0310] 47. A method for producing a doubly anchored activatable macromolecule, the method comprising: engineering an arginine or lysine residue at an isopeptide bonding site in a masking portion (MM) of the doubly anchored activatable macromolecule and / or engineering an aspartic acid or glutamic acid residue at an isopeptide bonding site in a target binding protein (TB) of the doubly anchored activatable macromolecule, wherein the MM is coupled to the TB and a cleavable portion (CM) is located between the MM and the TB; expressing the doubly anchored activatable macromolecule; and recovering the doubly anchored activatable macromolecule, wherein in the recovered doubly anchored activatable macromolecule the MM and the TB are tethered at their isopeptide bonding sites.

[0311] 48. A method for producing a doubly anchored activatable macromolecule, the method comprising: engineering an aspartic acid or glutamic acid residue at an isopeptide bonding site in a masking moiety (MM) of the doubly anchored activatable macromolecule and / or engineering an arginine or lysine residue at an isopeptide bonding site in a target binding protein (TB) of the doubly anchored activatable macromolecule, wherein the MM is coupled to the TB and a cleavable moiety (CM) is located between the MM and the TB; expressing the doubly anchored activatable macromolecule; and recovering the doubly anchored activatable macromolecule, wherein in the recovered doubly anchored activatable macromolecule the MM and the TB are tethered at their isopeptide bonding sites.

[0312] 49. A method of making a dual-anchored activatable macromolecule, the method comprising providing a MM comprising a non-α carbon covalent bond forming amino acid, wherein the non-α carbon covalent bond forming amino acid is configured to form a non-α carbon covalent bond with a non-α carbon covalent bond forming amino acid in a TB, wherein the TB is coupled to the MM.

[0313] 50. The method of item 49, wherein the amino acid forming an α-carbon covalent bond of the MM is threonine and the amino acid forming an α-carbon covalent bond of the TB is glutamine.

[0314] 51. The method of item 49, wherein the amino acid forming an α-carbon covalent bond of the MM is glutamine and the amino acid forming an α-carbon covalent bond of the TB is threonine.

[0315] 52. The method of item 49, wherein the amino acid forming an α-carbon covalent bond of the MM is cysteine ​​and the amino acid forming an α-carbon covalent bond of the TB is glutamine or tyrosine.

[0316] 53. The method of item 49, wherein the amino acid forming an α-carbon covalent bond of the MM is glutamine or tyrosine and the amino acid forming an α-carbon covalent bond of the TB is cysteine.

[0317] 54. The method of item 49, wherein the amino acid forming an alpha-carbon covalent bond of the MM is histidine and the amino acid forming an alpha-carbon covalent bond of the TB is tyrosine.

[0318] 55. The method of item 49, wherein the amino acid forming an alpha-carbon covalent bond of the MM is tyrosine and the amino acid forming an alpha-carbon covalent bond of the TB is histidine.

[0319] 56. The method of item 49, wherein the amino acid forming an α-carbon covalent bond of the MM is lysine and the amino acid forming an α-carbon covalent bond of the TB is cysteine, glutamic acid or aspartic acid.

[0320] 57. The method of item 49, wherein the amino acid forming an α-carbon covalent bond of the MM is cysteine, glutamic acid or aspartic acid and the amino acid forming an α-carbon covalent bond of the TB is lysine.

[0321] 58. The method of item 49, wherein the amino acid forming the α-carbon covalent bond of the MM is glutamine and the amino acid forming the α-carbon covalent bond of the TB is lysine, and the non-α-carbon covalent bond is an isopeptide bond between the γ-carboxamide group of the glutamine and the ε-amino group of the lysine side chain.

[0322] 59. The method of item 49, wherein the amino acid forming the α-carbon covalent bond of the MM is lysine and the amino acid forming the α-carbon covalent bond of the TB is glutamine, and the non-α-carbon covalent bond is an isopeptide bond between the γ-carboxamide group of the glutamine and the ε-amino group of the lysine side chain.

[0323] 60. A method of making a dual-anchored activatable macromolecule, the method comprising providing a MM comprising a cysteine, the cysteine ​​being configured to form a non-alpha carbon covalent bond with a non-alpha carbon covalent bond forming amino acid in a TB, the TB being coupled to the MM.

[0324] Example

[0325] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims and provide proof-of-concept demonstrations of the advantages of the dual anchor structures of activatable macromolecules described in this disclosure.

[0326] Example 1: Generation of a single anchored BC2T masked nanobody (BC2T-Nb)

[0327] This example demonstrates the generation of an exemplary single anchored activatable antibody. The single anchored activatable antibody comprises a nanobody (Nb) capable of binding to β-catenin and an MM (BC2T) comprising a β-catenin sequence as an Nb epitope. The MM is single anchored to the Nb, i.e., the MM and the Nb are coupled via the CM but not tethered by a disulfide bond. The single anchored BC2T-Nb ( Figure 4B As shown). The single anchored BC2T-Nb (SEQ ID NO: 2 or 3) comprises a signal peptide from SP5 (SEQ ID NO: 12), a leader sequence (SEQ ID NO: 13), a BC2T MM (SEQ ID NO: 14), a GS linker (SEQ ID NO: 15), a CM (SEQ ID NO: 16 or 17), a GS linker (SEQ ID NO: 18), a Nb (SEQ ID NO: 19), a GGS linker and a His tag (SEQ ID NO: 21) for purification from the N-terminus to the C-terminus. The polypeptide is prepared by transforming a host cell with a polynucleotide encoding a polypeptide sequence of SEQ ID NO: 2 or 3, culturing the resulting recombinant host cell, and purifying the protein from the supernatant using standard immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC) methods. The resulting protein is a monomer (ProC653 or ProC654).

[0328] The control molecule (i.e., unmasked Nanobody without BC2T attached) was also prepared by recombinant methods. ProC649 (PP073) (SEQ ID NO: 1) comprises a signal peptide from SP5 (SEQ ID NO: 12), Nb (SEQ ID NO: 19), a GGS linker and a His tag for purification (SEQ ID NO: 21) from N-terminus to C-terminus. The polypeptide is prepared by transforming a host cell with a polynucleotide encoding the polypeptide sequence of SEQ ID NO: 1, then culturing the resulting recombinant host cells, and purifying using standard IMAC and SEC methods. The resulting protein (ProC649) is a monomer.

[0329] Example 2. Protease treatment of single anchored BC2T-Nb

[0330] To release the MM, unmasked nanobodies and single anchored BC2T-Nb molecules were treated with recombinant human proteases such as matrix metalloproteinases (MMPs) at 37°C overnight. Complete protease treatment was tested by non-reducing SDS-PAGE. Protein aliquots (2 μg) were denatured in sample buffer at 75°C for 10 minutes (reducing agent was added if necessary) and subjected to 4-12% NuPAGE at 175V in MOPS buffer. TMSeparation was performed on Bis-Tris gels (Thermo Fisher Scientific, Waltham, MA, catalog number NP0321) for 1 hour and eluted with InstantBlue TM The staining was developed after 1 hour and then destained in water for at least 4 hours.

[0331] Confirm that the protein is monomeric and has the expected molecular weight ( Figure 6 ). Protease treatment did not affect the integrity of the unmasked molecule ( Figure 6 , lanes 1, 2, and 3). The molecular weight of the BC2T and nanobody constructs expressed as polypeptides and linked to CM changed ( Figure 6 , lanes 4, 6). After protease treatment, the protein ran at the same level as the unmasked molecule ( Figure 6 , lanes 5 and 7).

[0332] Example 3: Determination of the binding of monomeric single-anchored BC2T-Nb by biolayer interferometry (BLI)

[0333] The binding of unmasked nanobodies and single anchored BC2T-Nb to free BC2T peptides was assessed by BLI using the ForteBio Octet device. Although BLI is typically used to calculate the binding and dissociation kinetics of binding interactions, it is used herein to detect binding. The peptide ELIM Biopharmaceuticals corresponding to the following sequence was synthesized with biotin at its N-terminus: QGQSGQPDRKAAVSHWQ (SEQ ID NO: 567). The peptide was loaded into the SSA biosensor tip (Pall / ForteBio catalog number 18-5117) at 100nM in binding buffer (BB: 1X PBS pH 7.2, 5% glycerol, 0.1% Tween-20, 2% BSA) for 10 minutes. A 1-minute baseline incubation was performed in the BB before and after the capture step to confirm protein capture. The loaded tip was then incubated in 100nM Nb in the BB for 6 minutes, and then incubated in the BB for 6 minutes. The sensor tip interference distance (nm) was plotted against time to track the binding / dissociation of soluble proteins to the immobilized ligand on the tip ( Figure 7 ). The results showed that ProC653 and ProC654 were effectively masked, as they could bind the peptide like the unmasked control ProC649 only after protease (MMP9 or MMP14) treatment.

[0334] Example 4: Generation of Monomeric Dual-Anchored BC2T-Nb

[0335] This example demonstrates the production of an exemplary monomeric dual-anchored activatable antibody. The dual-anchored activatable antibody comprises a nanobody (Nb) capable of binding to β-catenin and an MM (BC2T) comprising a β-catenin sequence as an Nb epitope. MM is dual-anchored to Nb, i.e., MM and Nb are coupled via CM and also tethered via a disulfide bond. The dual-anchored BC2T-Nb ( Figure 4A As shown). ProC994 (PP123) (SEQ ID NO: 4) and ProC995 (PP124) (SEQ ID NO: 5) comprise, from N-terminus to C-terminus, a signal peptide from SP5 (SEQ ID NO: 12), a leader sequence (SEQ ID NO: 22) in which the third amino acid is mutated to cysteine, BC2T MM (SEQ ID NO: 14), a GS linker (SEQ ID NO: 15), CM (SEQ ID NO: 16), a GS linker (SEQ ID NO: 18), Nb (SEQ ID NO: 24 or 25) in which one amino acid is mutated to cysteine, a GGS linker, and a His tag (SEQ ID NO: 21) for purification. The polypeptide is prepared by transforming a host cell with a polynucleotide encoding the polypeptide sequence of SEQ ID NO: 4 or 5, and then culturing the resulting recombinant host cell. The resulting protein (ProC994 or ProC995) is a monomer.

[0336] ProC996 (PP125) (SEQ ID NO: 6) comprises, from N-terminus to C-terminus, a signal peptide from SP5 (SEQ ID NO: 12), a leader sequence (SEQ ID NO: 13), BC2T MM (SEQ ID NO: 23) in which the second amino acid is mutated to cysteine, a GS linker (SEQ ID NO: 15), a CM (SEQ ID NO: 16), a GS linker (SEQ ID NO: 18), Nb (SEQ ID NO: 26) in which one amino acid is mutated to cysteine, a GGS linker, and a His tag (SEQ ID NO: 21) for purification. The polypeptide is prepared by transforming a host cell with a polynucleotide encoding the polypeptide sequence of SEQ ID NO: 6, culturing the resulting recombinant host cell, and purifying the expressed protein using standard IMAC and SEC methods. The resulting protein (ProC996) is a monomer.

[0337] Example 5. Protease treatment of monomeric dual-anchored BC2T-Nb

[0338] To release MM (see Figure 4C), single-anchored and dual-anchored BC2T-Nb molecules were treated with recombinant human proteases such as urokinase-type plasminogen activator (uPA) at 37°C overnight. Complete protease treatment and disulfide bond analysis were tested by reducing and non-reducing SDS-PAGE. Protein aliquots (2 μg) were denatured in sample buffer at 75°C for 10 minutes (reducing agent was added if necessary) and subjected to 4-12% NuPAGE in MOPS buffer at 175V. TM Separation was performed on a Bis-Tris gel (ThermoFisher Scientific, Waltham, MA, catalog number NP0321) for 1 hour and the samples were analyzed using InstantBlue TM The staining was developed after 1 hour and then destained in water for at least 4 hours.

[0339] The protein was confirmed to be monomeric and of the expected molecular weight ( Figure 8 , lower panel). This confirmed that engineered disulfide bonds had been formed in the non-reducing gel before protease treatment ( Figure 8 , upper panel, lanes 4, 6, and 8), and after protease treatment, the protein ran at the same level as the single-anchored masking molecule ( Figure 8 , upper panel, lanes 2, 5, 7, 9). The formation of the engineered disulfide bonds was confirmed by comparing the migration of the protease-treated dual-anchored masking protein in non-reducing and reducing gels.

[0340] Example 6: Generation of dimer dual-anchored macromolecules

[0341] This example demonstrates the generation of an exemplary dimer dual-anchored activatable antibody. The dual-anchored activatable molecule includes two nanobodies (Nb) capable of binding to β-catenin, an Fc dimer comprising two Fcs (each Fc coupled to one Nb), and two MMs (e.g., BC2T in this illustrative example) comprising a β-catenin sequence as an Nb epitope. Each MM is dual-anchored to the Nb, i.e., the MM and the Nb are coupled via the CM and are also tethered via a disulfide bond ( Figure 5A ).

[0342] The dimer double-anchored BC2T-Nb was prepared by a recombinant method. ProC1285 (HC699) (SEQ ID NO: 9) comprises a signal peptide from SP5 (SEQ ID NO: 12), a leader sequence with the third amino acid mutated to cysteine ​​(SEQ ID NO: 22), BC2T MM (SEQ ID NO: 14), a GS linker (SEQ ID NO: 15), a CM (SEQ ID NO: 16), a GS linker (SEQ ID NO: 18), Nb with one amino acid mutated to cysteine ​​(SEQ ID NO: 24), a GGGG linker (SEQ ID NO: 27) and human IgG1 Fc (SEQ ID NO: 28) from N-terminus to C-terminus. The polypeptide is prepared by transforming a host cell with a polynucleotide encoding the polypeptide sequence of SEQ ID NO: 9, culturing the resulting recombinant host cell, and purifying the expressed protein using standard IMAC and SEC methods. The resulting protein (ProC1285) is a dimer ( Figure 5A shown).

[0343] ProC1287 (HC701) (SEQ ID NO: 11) comprises, from N-terminus to C-terminus, a signal peptide from SP5 (SEQ ID NO: 12), a leader sequence (SEQ ID NO: 13), BC2T MM (SEQ ID NO: 23) in which the second amino acid is mutated to cysteine, a GS linker (SEQ ID NO: 15), a CM (SEQ ID NO: 16), a GS linker (SEQ ID NO: 18), Nb (SEQ ID NO: 26) in which one amino acid is mutated to cysteine, a GGGG linker (SEQ ID NO: 27), and human IgG1 Fc (SEQ ID NO: 28). The polypeptide is prepared by transforming a host cell with a polynucleotide encoding the polypeptide sequence of SEQ ID NO: 11, culturing the resulting recombinant host cell, and purifying the expressed protein using standard IMAC and SEC methods. The resulting protein (ProC1287) is a dimer ( Figure 5A shown).

[0344] Single anchored BC2T-Nb-IgG1 (i.e., MM and Nb are not tethered by disulfide bonds, Figure 5BAs shown). ProC1284 (HC698) (SEQ ID NO: 8) comprises a signal peptide from SP5 (SEQ ID NO: 12), a leader sequence (SEQ ID NO: 13), BC2T MM (SEQ ID NO: 14), a GS linker (SEQ ID NO: 15), CM (SEQ ID NO: 16), another GS linker (SEQ ID NO: 18), Nb (SEQ ID NO: 19), a GGGG linker (SEQ ID NO: 27) and human IgG1 Fc (SEQ ID NO: 28) from N-terminus to C-terminus. The polypeptide is prepared by transforming a host cell with a polynucleotide encoding the polypeptide sequence of SEQ ID NO: 8, culturing the resulting recombinant host cell, and purifying the expressed protein using standard IMAC and SEC methods. The resulting protein (ProC1284) is a dimer.

[0345] Example 7: Protease treatment of the dimer double-anchored BC2T-Nb of Example 6

[0346] To release the MM in the molecule of Example 6, the single-anchored and dual-anchored BC2T-Nb molecules were treated with a recombinant human protease (such as urokinase-type plasminogen activator (uPA)) at 37°C overnight, as Figure 5C Schematic diagram. Complete protease treatment and disulfide bond analysis were tested by reducing and non-reducing SDS-PAGE. Protein aliquots (2 μg) were denatured in sample buffer at 75°C for 10 min (reducing agent added if necessary) and subjected to 4-12% NuPAGE at 175 V in MOPS buffer. TM Separation was performed on Bis-Tris gels (Thermo Fisher Scientific, Waltham, MA, catalog number NP0321) for 1 hour and eluted with InstantBlue TM The staining was developed after 1 hour and then destained in water for at least 4 hours.

[0347] The protein was confirmed to be monomeric on reducing gels with the expected molecular weight of approximately 19 and 16 kDa, respectively, before and after protease treatment. Fig. 9 , lower panel). This confirmed that engineered disulfide bonds had been formed in the non-reducing gel before protease treatment ( Fig. 9 , upper panel, lanes 4, 6, and 8), and after protease treatment, the protein ran at the same level as the single-anchored masking molecule ( Fig. 9 , upper panel, lanes 2, 5, 7, 9). The formation of the engineered disulfide bonds was confirmed by comparing the migration of the protease-treated dual-anchored masking protein in non-reducing and reducing gels.

[0348] Example 8: Peptide Binding ELISA

[0349] BC2T peptide was synthesized by ELIM Biopharmaceuticals. The peptide sequence contained a leader sequence (SEQ ID NO: 13) and BC2T MM (SEQ ID NO: 14) from N-terminus to C-terminus.

[0350] 5 μM BC2T peptide dissolved in 0.05 M carbonate-bicarbonate buffer was adsorbed to the wells of a 96-well microtiter plate overnight at 4°C. The plates were washed and blocked with blocking buffer (1X PBS pH 7.4, 0.05% Tween-20, 1% BSA). The dimeric dual-anchored masking molecules (ProC1285 and ProC1287) were serially diluted threefold with unmasked control protein (ProC1283) and dimeric single-anchored BC2T-Nb (ProC1284) without or after protease treatment and applied to the peptide-coated plates. The degree of protein-peptide binding was measured by anti-human IgG immunodetection. The A450 absorbance was measured on a plate reader. Dose-response curves were generated by sigmoid fit nonlinear regression and EC50 values ​​were obtained using Graph Pad Prism software. The results are presented in Fig.10 As shown in Fig.10 As shown, the dimeric dual-anchored masking molecules (ProC1285 and ProC1287) exhibited greater masking than the comparative dimeric single-anchored BC2T-Nb (ProC1284), and also showed an unexpectedly large degree of recovery of binding activity after cleavage compared to the recovery exhibited by the positive control ProC1283 (unmasked antibody). Thus, despite the antibody being tethered to the masker at one end after cleavage, it still showed a significant recovery of binding activity.

[0351] Example 9: Design of disulfide bond-based dual-anchored masked antibodies:

[0352] This example describes how to engineer non-alpha carbon linkages, such as cysteine-disulfide bonds, between residues in the prodomain (the N-terminus of the masking peptide) and residues in the variable domain of the antibody using an activatable anti-PDL1 antibody as an example. The sequence of the activatable anti-PDL1 antibody (SEQ ID NOs: 562 and 563) consists of a prodomain connected to a typical antibody light chain. Using the antibody primary sequence, a homology-based antibody 3D model ( Figures 12A-12B The masker sequence from the prodomain was used to generate a homology-based masker model and docked into the antibody structure ( Figures 12A-12B ). Fig. 12AThe three-dimensional structure of the activatable anti-PDL1 antibody obtained using BIOVIA Discovery Studios by Dassault Systemes software is shown, showing the solvent accessible residues within 2-5 angstroms of the head region or N-terminal residues located in the masking portion. Fig. 12B The interface between the Fab domain of the activatable anti-PDL1 antibody (space-filling format) and the prodomain with masking moieties (shown as Cα backbone) is shown.

[0353] A variety of experimental techniques, including mutagenesis, hydrogen-deuterium exchange (HDX), and XL-MS (crosslinker MS), can optionally be used to confirm the structural model. After obtaining a three-dimensional model of the MM bound to the TB, an energy minimization algorithm is used to provide information about the location of the head and linker (including CM) regions of the prodomain. The user can then use software such as BIOVIA DiscoveryStudio to identify residue pairs, one in the head region of the prodomain and the other in the antibody variable domain, whose Cα atoms are in the position of a typical disulfide-linked cysteine. The distance between the Cα of the first amino acid and the Cα of the second amino acid can be reduced to a more detailed list of residues that are most likely to form the designed disulfide bond using a software application such as SSBondPre. The residues are then mutated to cysteine ​​residues, either singly or in pairs, to identify a pair of residues that do not affect the binding of the antibody or masker when alone, but when present together form a disulfide bond that improves masking. The presence of properly formed disulfide bonds can be confirmed by mass spectrometry techniques such as disulfide bond mapping.

[0354] Example 10: Design of disulfide bond-based dual-anchored masked antibodies or other TBs:

[0355] This example provides further examples of how to prepare and use homology-based three-dimensional models of antibody structures to engineer non-alpha carbon linkages, such as cysteine ​​disulfide bonds, between residues in the prodomain (masking the N-terminus of the peptide) and residues in the antibody variable domain.

[0356] The J43v2 / anti-mouse PD1 ( Fig.13A ; SEQ ID NO: 568-569), anti-CD166 ( Fig. 13B ; SEQ ID NO: 572-573) and human anti-PD1 ( Fig. 13C; SEQ ID NO: 570-571) sequence. Anti-PD1 human anti-PD1 (SEQ ID NO: 570-571) was additionally modeled in AlphaFold2 and presented in BIOVIA Discovery Studio, as shown in Fig.13D shown.

[0357] A variety of experimental techniques, including mutagenesis, hydrogen-deuterium exchange (HDX), and XL-MS (crosslinker MS), can optionally be used to confirm the structural model. Modeled human anti-PD1 MMs are disclosed in WO2017 / 011580, and modeled anti-CD166 MMs are disclosed in WO2016 / 179285, both of which are incorporated herein by reference. A three-dimensional model of the corresponding MM bound to the corresponding TB can be obtained, and software such as BIOVIA Discovery Studio can be used to identify residue pairs, one of which is located in the head region of the prodomain and the other in the antibody variable domain, whose Cα atoms are in The distance between the Cα of the first amino acid and the Cα of the second amino acid can be reduced to a more detailed list of residues that are most likely to form the desired bond using a software application such as SSBondPre. The residues are then mutated individually or in pairs to the desired residues to form the desired bond to identify a pair of residues that do not affect the binding of the antibody or masker when alone, but when present together form a bond that improves masking as described herein. Correctly formed bonds, such as the presence of disulfide bonds, can be confirmed by mass spectrometry techniques such as disulfide bond mapping.

[0358] The sequences of the molecules in the Examples are listed in Table 4 below.

[0359] Table 4

[0360]

[0361]

[0362]

[0363]

[0364]

[0365]

[0366] Other Implementations

[0367] It will be appreciated that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the following claims.

[0368] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict, the present specification, including definitions, will prevail. The materials, methods, and examples are illustrative only and are not intended to be limiting.

Claims

1. A dual-anchored activatable target binding protein, the dual-anchored activatable target binding protein comprising: a target binding protein (TB) that specifically binds to the target; a masking moiety (MM) coupled to the TB, wherein the MM inhibits binding of the AB to the target; and a cleavable moiety (CM) coupled to the TB and positioned between the TB and the MM, wherein the CM is a polypeptide that serves as a protease substrate, and Also included are non-alpha carbon covalent bonds tethering the MM and the TB.

2. The activatable target binding protein of claim 1, wherein the TB is an antigen binding protein (AB).

3. The activatable target binding protein of claim 1 or claim 2, wherein the activatable target binding protein has lower target binding activity compared to a single anchored activatable target binding protein lacking the non-alpha carbon covalent bond.

4. The activatable target binding protein of any one of claims 1-3, wherein the non-α carbon covalent bond is an ester bond or a thioester bond.

5. The activatable target binding protein of claim 4, wherein the ester bond is between threonine and glutamine.

6. The activatable target binding protein of claim 4, wherein the thioester bond is between cysteine ​​and glutamine or tyrosine.

7. The activatable target binding protein of any one of claims 1-3, wherein the non-alpha carbon covalent bond is a cross-link between histidine and tyrosine or a cross-link between lysine and cysteine.

8. The activatable target binding protein of any one of claims 1-3, wherein the non-alpha carbon covalent bond is an isopeptide bond.

9. The activatable target binding protein of claim 8, wherein the isopeptide bond is between a lysine and a glutamic acid or aspartic acid residue.

10. The activatable target binding protein of claim 8, wherein the isopeptide bond is between the γ-carboxamide group of glutamine and the ε-amino group of the lysine side chain.

11. The activatable target binding protein of any one of claims 1-3, wherein the non-alpha carbon covalent bond is a disulfide bond.

12. The activatable target binding protein of claim 11, wherein the disulfide bond is formed between the first cysteine ​​and the second cysteine, wherein The first cysteine ​​is within the MM and the second cysteine ​​is within the TB; The first cysteine ​​is within a peptide coupled to the MM and the second cysteine ​​is within the TB, or The first cysteine ​​is within the MM and the second cysteine ​​is within a peptide coupled to the TB.

13. The activatable target binding protein of any one of claims 1 to 12, further comprising a second CM, wherein The second CM is located between the MM and the non-α carbon covalent bond; The second CM is within the MM and is at most 5 amino acids away from the cysteine ​​forming the non-alpha carbon covalent bond, or The second CM is within the TB and is at most 5 amino acids away from the cysteine ​​forming the non-alpha carbon covalent bond.

14. The activatable target binding protein of any one of claims 1-13, wherein the first CM and the second CM are substrates for different proteases.

15. The activatable target binding protein of any one of claims 1-13, wherein the first CM and the second CM are substrates for the same protease.

16. The activatable target binding protein of any one of claims 1-12, wherein the protease is produced by a tumor in a subject.

17. The activatable target binding protein of any one or combination of claims 2-16, wherein the AB is an antibody, a Fab fragment, a F(ab')2 fragment, a scFv, a scAb, a dAb, a VHH, or a single domain antibody.

18. The activatable target binding protein of any one of claims 2-17, wherein the AB is a single domain antibody.

19. The activatable target binding protein of any one of claims 2-18, wherein the AB is an Fc-tagged single domain antibody.

20. The activatable target binding protein of any one of claims 2-17, wherein the AB is a bispecific antibody.

21. The activatable target binding protein of claim 20, wherein the bispecific antibody is a bispecific T cell engager (BiTE) or a dual affinity retargeting antibody (DART).

22. The activatable target binding protein of any one of claims 2-17, wherein the AB is a multispecific antibody.

23. The activatable target binding protein of any one of claims 18-19, wherein the non-alpha carbon covalent bond is between the MM and the single domain antibody.

24. The activatable target binding protein of any one of claims 1-22, wherein the non-alpha carbon covalent bond is between the MM and the Fc domain.

25. The activatable target binding protein of any one of claims 1-24, wherein the MM comprises an epitope of the TB.

26. The activatable target binding protein of any one of claims 1-24, wherein the MM does not comprise four or more consecutive amino acids of an epitope of the TB.

27. The activatable target binding protein of any one of claims 1-26, wherein the dissociation constant of the binding of the MM to the TB exceeds the dissociation parameter of the binding of the TB to the target.

28. The activatable target binding protein of any one of claims 1-27, wherein the MM is a polypeptide of 2 to 40 amino acids in length.

29. The activatable target binding protein of any one of claims 1-28, wherein the activatable target binding protein comprises a linker between the MM and the CM.

30. The activatable target binding protein of any one of claims 1-29, wherein the activatable target binding protein comprises a linker between the CM and the TB.

31. The activatable target binding protein of any one of claims 1-29, wherein the activatable target binding protein comprises a first linker between the MM and the CM and a second linker between the CM and the TB.

32. A composition comprising the activatable target binding protein of any one of claims 1 to 31 and a carrier.

33. The composition of claim 32, wherein the composition is a pharmaceutical composition.

34. A container, vial, syringe, injection pen or kit comprising at least one dose of the composition of claim 32 or 33.

35. A nucleic acid comprising a sequence encoding the activatable target binding protein of any one of claims 1 to 31.

36. A vector comprising the nucleic acid of claim 35.

37. A cell comprising the nucleic acid of claim 35 or the vector of claim 36.

38. A conjugated activatable target binding protein comprising the activatable target binding protein of any one of claims 1 to 31 conjugated to an agent.

39. The conjugated, activatable target binding protein of claim 38, wherein the agent is a therapeutic agent, a targeting moiety, or a detectable moiety.

40. A method of treating a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an activatable target binding protein as described in any one of claims 1 to 31, a composition as described in claim 32 or 33, or a conjugated activatable target binding protein as described in claim 38 or 39.

41. The method of claim 40, wherein the subject has been identified or diagnosed as having cancer, an inflammatory disorder, condition or disease, or an autoimmune disorder, condition or disease.

42. A method for producing an activatable target binding protein, the method comprising: Culturing the cell of claim 37 in a culture medium under conditions sufficient to produce the activatable target binding protein; as well as The activatable target binding protein is recovered from the cells or the culture medium.

43. The method of claim 42, further comprising isolating the activatable target binding protein recovered from the cells or the culture medium.

44. The method of claim 43, wherein the separation is performed using a protein purification tag and / or size exclusion chromatography.

45. The method of claim 43 or claim 44, further comprising formulating the activatable target binding protein into a pharmaceutical composition.

46. ​​A method for producing a dual-anchored activatable macromolecule, the method comprising: engineering a cysteine ​​residue at a disulfide bonding site in the masking portion (MM) of the dual-anchored activatable macromolecule; Engineering a cysteine ​​residue at a disulfide bonding site in a target binding protein (TB) of the dual-anchored activatable macromolecule, wherein the MM is coupled to the TB and a cleavable moiety (CM) is located between the MM and the TB; expressing the dual-anchored activatable macromolecule; and recovering the dual-anchored activatable macromolecule, wherein in the recovered dual-anchored activatable macromolecule the MM and the TB are tethered at their disulfide bonding sites.

47. A method for producing a dual-anchored activatable macromolecule, the method comprising: Engineering an arginine or lysine residue at the isopeptide bonding site in the masking moiety (MM) of the dual-anchored activatable macromolecule and / or engineering an aspartic acid or glutamic acid residue at the isopeptide bonding site in the target binding protein (TB) of the dual-anchored activatable macromolecule, wherein the MM is coupled to the TB and the cleavable moiety (CM) is located between the MM and the TB; expressing the dual-anchored activatable macromolecule; and recovering the dual-anchored activatable macromolecule, wherein in the recovered dual-anchored activatable macromolecule the MM and the TB are tethered at their isopeptide bonding sites.

48. A method for producing a dual-anchored activatable macromolecule, the method comprising: Engineering an aspartic acid or glutamic acid residue at the isopeptide bonding site in the masking moiety (MM) of the dual-anchored activatable macromolecule and / or engineering an arginine or lysine residue at the isopeptide bonding site in the target binding protein (TB) of the dual-anchored activatable macromolecule, wherein the MM is coupled to the TB and the cleavable moiety (CM) is located between the MM and the TB; expressing the dual-anchored activatable macromolecule; and recovering the dual-anchored activatable macromolecule, wherein in the recovered dual-anchored activatable macromolecule the MM and the TB are tethered at their isopeptide bonding sites.

49. A method of making a dual-anchored activatable macromolecule, the method comprising providing a MM comprising a non-α carbon covalent bond forming amino acid, wherein the non-α carbon covalent bond forming amino acid is configured to form a non-α carbon covalent bond with a non-α carbon covalent bond forming amino acid in a TB, wherein the TB is coupled to the MM.

50. The method of claim 49, wherein the alpha-carbon covalent bond-forming amino acid of the MM is threonine and the alpha-carbon covalent bond-forming amino acid of the TB is glutamine.

51. The method of claim 49, wherein the alpha-carbon covalent bond-forming amino acid of the MM is glutamine and the alpha-carbon covalent bond-forming amino acid of the TB is threonine.

52. The method of claim 49, wherein the alpha-carbon covalent bond-forming amino acid of the MM is cysteine ​​and the alpha-carbon covalent bond-forming amino acid of the TB is glutamine or tyrosine.

53. The method of claim 49, wherein the alpha-carbon covalent bond-forming amino acid of the MM is glutamine or tyrosine and the alpha-carbon covalent bond-forming amino acid of the TB is cysteine.

54. The method of claim 49, wherein the alpha-carbon covalent bond-forming amino acid of the MM is histidine and the alpha-carbon covalent bond-forming amino acid of the TB is tyrosine.

55. The method of claim 49, wherein the alpha-carbon covalent bond-forming amino acid of the MM is tyrosine and the alpha-carbon covalent bond-forming amino acid of the TB is histidine.

56. The method of claim 49, wherein the alpha-carbon covalent bond-forming amino acid of the MM is lysine and the alpha-carbon covalent bond-forming amino acid of the TB is cysteine, glutamic acid, or aspartic acid.

57. The method of claim 49, wherein the alpha-carbon covalent bond-forming amino acid of the MM is cysteine, glutamic acid, or aspartic acid and the alpha-carbon covalent bond-forming amino acid of the TB is lysine.

58. The method of claim 49, wherein the alpha-carbon covalent bond-forming amino acid of the MM is glutamine and the alpha-carbon covalent bond-forming amino acid of the TB is lysine, and the non-alpha-carbon covalent bond is an isopeptide bond between the γ-carboxamide group of the glutamine and the ε-amino group of the lysine side chain.

59. The method of claim 49, wherein the alpha-carbon covalent bond-forming amino acid of the MM is lysine and the alpha-carbon covalent bond-forming amino acid of the TB is glutamine, and the non-alpha-carbon covalent bond is an isopeptide bond between the γ-carboxamide group of the glutamine and the ε-amino group of the lysine side chain.

60. A method of making a dual-anchored activatable macromolecule, the method comprising providing a MM comprising a cysteine, the cysteine ​​being configured to form a non-alpha carbon covalent bond with a non-alpha carbon covalent bond forming amino acid in a TB, the TB being coupled to the MM.

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