Activatable dual anchored masked molecules and methods of use thereof - Patents.com

Abstract

Provided herein is an activatable target binding protein comprising a target binding protein (TB) that specifically binds to a target, a masking moiety (MM) bound to the TB, wherein the MM and TB are tethered together by a non-alpha carbon covalent bond, such that when the activatable target binding protein is in an inactive state, the MM inhibits binding of the TB to the target, and a cleavable moiety (CM) bound to the TB, wherein the CM is a polypeptide that functions as a substrate for a protease and is positioned between the TB and the MM.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 323,718, filed March 25, 2022, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing The Sequence Listing submitted with this application by EFS is entitled "4862-121PCT.xml", was created on March 22, 2023, is 500,152 bytes in size, and is incorporated by reference herein in its entirety.

[0003] The present disclosure relates to the field of biotechnology, and more particularly to activatable molecules. [Background technology]

[0004] Antibody-based therapeutics provide proven effective treatments for a variety of diseases. However, in some cases, toxicity due to widespread target expression can limit their therapeutic effectiveness. In addition, antibody-based therapeutics have other limitations, such as rapid clearance from the circulation after administration.

[0005] Activatable antibodies that contain a mask peptide that binds to the antibody are therapeutic agents that are less toxic than normal antibodies. The mask can inhibit the activity of the antibody by preventing the antibody from binding to its target molecule. In an activation environment (e.g., when the activatable antibody is delivered to a tumor), the mask is removed so that the antibody can bind to its target molecule and regain its function. However, if a portion of the activatable antibody is left unmasked and allows target binding outside of the activation environment, toxicity can result.

[0006] Thus, there is a continuing need for activatable molecules that minimize target binding outside of the activation environment. Summary of the Invention

[0007] The present disclosure provides dual-anchored activatable target binding proteins and related compositions and methods.

[0008] In one aspect, the 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) bound to the TB, where the MM inhibits binding of the TB to the target, and a cleavable moiety (CM) bound to the TB and positioned between the TB and the MM, where the CM is a polypeptide that functions as a substrate for a protease, the activatable target binding protein further comprising a non-alpha carbon covalent bond tethering the MM to the TB.

[0009] In some embodiments, the TB is an antigen binding protein (AB). In some embodiments, the activatable target binding protein has a reduced target binding activity compared to a single-anchor activatable target binding protein lacking a non-alpha carbon covalent bond. In some embodiments, the non-alpha 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-alpha carbon covalent bond is between a functional group substituted at the alpha carbon in MM and AB. In some embodiments, the isopeptide bond is between the gamma carboxyamide group of glutamine and the epsilon amino group of a lysine side chain. In some embodiments, the non-alpha carbon covalent bond is an ester bond between threonine and glutamine. In some embodiments, the non-alpha carbon covalent bond is a thioester bond between cysteine ​​and glutamine. In some embodiments, the non-alpha carbon covalent bond is a thioether bond between cysteine ​​and tyrosine. In some embodiments, the non-alpha carbon covalent bond is formed by a crosslink between histidine and tyrosine (e.g., this type of histidine-tyrosine crosslink is known to exist in the cytochrome c oxidase enzyme). In some embodiments, the non-alpha carbon covalent bond is a nitrogen-oxygen-sulfur (NOS) bond formed between a lysine and a 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, where the first cysteine ​​is in the MM and the second cysteine ​​is in the TB, where the first cysteine ​​is in the peptide bound to the MM and the second cysteine ​​is in the TB, or where the first cysteine ​​is in the MM and the second cysteine ​​is in the peptide bound to the TB.

[0010] In some embodiments, the activatable target binding protein further comprises a second CM, the second CM being located between the MM and the non-alpha carbon covalent bond, the second CM being within the MM and up to 5 amino acids away from the cysteine ​​that forms the non-alpha carbon covalent bond, or the second CM being within the TB and up to 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.

[0011] In some embodiments, the protease is produced by the subject's tumor. In some embodiments, the AB is an antibody, a Fab fragment, a F(ab')2 fragment, a scFv, a scAb, a dAb, or a single domain antibody. In some embodiments, the AB is a single domain antibody. In some embodiments, the AB is an Fc-tagged single domain antibody. In some embodiments, the 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, the AB is a multispecific antibody. In some embodiments, the non-alpha carbon covalent bond is between the MM and the single domain antibody. The present disclosure includes a dual-anchored activatable macromolecule comprising a bispecific AB or a multispecific AB, each AB in the bispecific AB or multispecific AB having a dual-anchored MM. The present disclosure also includes dual anchored activatable macromolecules comprising bispecific or multispecific ABs, where at least one AB in the bispecific or multispecific ABs has a dual anchored MM, and at least one AB in the bispecific or multispecific ABs has a single anchored MM. The present disclosure also includes dual anchored activatable macromolecules comprising bispecific or multispecific ABs, where at least one AB in the bispecific or multispecific ABs has a dual anchored MM, and at least one AB in the bispecific or multispecific ABs has no MM.

[0012] In some embodiments, the non-alpha carbon covalent bond is between the MM and a region or domain of the fragment crystallizable region (Fc) that is bound to the TB. In some embodiments, the MM may include an epitope of the TB. In some embodiments, the MM does not include a subsequence of four or more consecutive amino acid residues of native TB. In some embodiments, the MM does not include a subsequence of four or more consecutive amino acid residues of a target that is bound by the TB. In some embodiments, the MM may include a subsequence of fewer than four consecutive amino acid residues of native TB. In some embodiments, the MM does not include an epitope of the TB. In some embodiments, the MM has a dissociation constant for binding to the TB that is greater than the dissociation constant of the TB for binding to the target. In some embodiments, the MM is a polypeptide between 2 and 40 amino acids in length.

[0013] In some embodiments, the activatable target binding protein comprises a linker between the MM and the CM. In some embodiments, the activatable target binding protein comprises a linker between the CM and the TB. In some embodiments, 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.

[0014] In another aspect, the disclosure provides a composition comprising an activatable target binding protein herein. In some embodiments, the composition is a pharmaceutical composition.

[0015] In another aspect, the disclosure provides a container, vial, syringe, injection pen, or kit comprising at least one dose of a composition herein.

[0016] In another aspect, the disclosure provides a nucleic acid comprising a sequence encoding an activatable target binding protein herein.

[0017] In another aspect, the disclosure provides a vector comprising the nucleic acid herein.

[0018] In another aspect, the disclosure provides a cell comprising a nucleic acid or vector herein.

[0019] In another aspect, the disclosure provides a conjugated activatable target binding protein comprising an activatable target binding protein herein conjugated to an agent, hi some embodiments, the agent is a therapeutic agent, a targeting moiety, or a detectable moiety.

[0020] In another aspect, the disclosure provides methods of treating a subject in need of treatment, comprising administering to the subject a therapeutically effective amount of an activatable target binding protein, composition, or conjugated activatable target binding protein herein, hi some embodiments, the subject has been identified or diagnosed as having cancer.

[0021] In another aspect, the 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.

[0022] 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.

[0023] In another aspect, the present disclosure provides a method for producing a dual anchored activatable protein, comprising: engineering a cysteine ​​residue at a disulfide bond site in a masking portion (MM) of the dual anchored activatable protein; engineering a cysteine ​​residue at a disulfide bond site in a target binding protein (TB) of the dual anchored activatable protein, where the MM and TB are linked and a cleavable portion (CM) is located between the MM and TB; expressing the dual anchored activatable protein; and recovering the dual anchored activatable protein, where the MM and TB are tethered at their disulfide bond sites in the recovered dual anchored activatable protein. In the present disclosure, the terms dual anchored activatable protein and dual anchored activatable macromolecule are used interchangeably.

[0024] In another aspect, the disclosure provides a method of producing a dual anchored activatable macromolecule, comprising engineering an arginine or lysine residue at an isopeptide bond site in a masking moiety (MM) of the dual anchored activatable macromolecule and / or engineering an aspartic acid or glutamic acid residue at an isopeptide bond site in a target binding protein (TB) of the dual anchored activatable macromolecule, where the MM and TB are linked and a cleavable moiety (CM) is positioned between the MM and TB, expressing the dual anchored activatable macromolecule, and recovering the dual anchored activatable macromolecule, where the MM and TB are tethered at their isopeptide bond sites in the recovered dual anchored activatable macromolecule. In another aspect, the disclosure provides a method of producing a dual anchored activatable macromolecule, comprising engineering an isopeptide bond site such that the gamma carboxyamide group of glutamine is available and configured to form an isopeptide bond with the epsilon amino group of a lysine side chain.

[0025] In another aspect, the disclosure provides a method of producing a dual anchored activatable polymer, comprising: engineering an aspartic acid or glutamic acid residue at an isopeptide binding site in a masking moiety (MM) of the dual anchored activatable polymer and / or engineering an arginine or lysine residue at an isopeptide binding site in a target binding protein (TB) of the dual anchored activatable polymer, wherein the MM and TB are linked and a cleavable moiety (CM) is positioned between the MM and TB; expressing the dual anchored activatable polymer; and recovering the dual anchored activatable polymer, wherein the MM and TB are tethered at their isopeptide binding sites in the recovered dual anchored activatable polymer.

[0026] In another aspect, the disclosure provides a method of making a dual-anchored activatable polymer, comprising providing a MM comprising a non-alpha carbon covalent bond forming amino acid configured to form a non-alpha carbon covalent bond with a non-alpha carbon covalent bond forming amino acid in a TB bound to the MM.

[0027] In another aspect, the disclosure provides a method of making a dual-anchored activatable polymer, comprising providing a MM comprising a cysteine ​​configured to form a non-alpha carbon covalent bond with a non-alpha carbon covalent bond forming amino acid in a TB bound to the MM.

[0028] An understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments in which the principles of the invention may be utilized, and the accompanying drawings of which: [Brief description of the drawings]

[0029] [Figure 1A]Schematic representation of the dynamic equilibrium between a fully mask-bound conformational state (left), an intermediate conformational state in which one mask is dynamically dissociated from the binding site of the target-binding protein (or antibody) (center), and a conformational state in which two masks are dynamically dissociated from the binding site of the target-binding protein (or antibody) (right). [Figure 1B] 1 shows the arrangement of components in an exemplary activatable antibody. The line connecting MM and AB indicates a non-alpha carbon covalent bond. Although AB is illustrated, the disclosure includes the use of any desired protein and is not limited to antibodies, but can include any target binding protein (TB). [Diagram 2] AD show schematic diagrams of exemplary activatable molecules. [Figure 3A] Three exemplary activatable molecules are shown. [Figure 3B] Three exemplary activatable molecules are shown. [Figure 3C] Three exemplary activatable molecules are shown. [Figure 4A] FIG. 1 is a schematic diagram of an exemplary dual-anchored BC2T-Nb in which the N-terminus of the MM is covalently linked to the Nb with 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 with a CM. [Figure 4B] FIG. 1 is a schematic diagram of a single anchored BC2T-Nb. [Figure 4C] 1 illustrates the cleavage reaction of a dual-anchored BC2T-Nb in which the MM is singly 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 of skill in the art will understand 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. [Figure 5A]FIG. 1 is a schematic diagram of an exemplary dimeric Fc-tagged version of a dual-anchored BC2T-Nb in which the N-terminus of the MM is covalently linked to the Nb with 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 with a CM. [Figure 5B] FIG. 13 is a schematic diagram of a dimeric Fc-tagged control molecule (i.e., the MM and Nb are not tethered by a non-alpha carbon covalent bond, e.g., a disulfide bond) to show the difference in binding between dual-anchored and single-anchored BC2T-Nbs. [Figure 5C] Illustrates the cleavage reaction of the Fc-tagged dual-anchored BC2T-Nb resulting in the MM being singly 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 of skill in the art would understand 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. [Figure 6] 1 is an image of an SDS-PAGE gel run under non-reducing conditions. The gel was loaded as follows: (1) single domain antibody with no MM conjugated (ProC649; SEQ ID NO:1); (2) product of ProC649 and MMP14 (ProC649+MMP14); (3) product of ProC649 and MMP9 (ProC649+MMP9); (4) single domain antibody with MM conjugated with CM 1490DNI (ProC653; SEQ ID NO:2); (5) product of ProC653 and MMP14 (ProC653+MMP14); (6) single domain antibody with MM conjugated with CM PLGLAG (SEQ ID NO:17) (ProC654; SEQ ID NO:3); (7) product of ProC654 and MMP9 (ProC654+MMP9); (8) MMP14; and (9) MMP9. [Figure 7-1]Sensorgram trace binding of intact and activated ProC653 and ProC654 are shown along with the ProC649 control. ProC649 with no MM bound bound to the biotinylated BC2 tag peptide on the biosensor tip. Intact ProC653 and ProC654 show no binding to the biotinylated BC2 tag peptide, but binding was restored upon activation with either MMP14 or MMP9, respectively. [Figure 7-2] Sensorgram trace binding of intact and activated ProC653 and ProC654 are shown along with the ProC649 control. ProC649 with no MM bound bound to the biotinylated BC2 tag peptide on the biosensor tip. Intact ProC653 and ProC654 show no binding to the biotinylated BC2 tag peptide, but binding was restored upon activation with either MMP14 or MMP9, respectively. [Figure 8] Images of SDS-PAGE gels run under non-reducing (top) and reducing (bottom) conditions. Gels were loaded as follows: (1) single domain antibody with no MM attached (ProC649); (2) single domain antibody with MM attached at CM 1490DNI (ProC653); (3) product of ProC653 with uPA (ProC653+uPA); (4) single domain antibody with engineered cysteines Q3C and Q157C and dual MM anchored at CM 1490DNI (ProC994; SEQ ID NO:4); (5) product of ProC994 with uPA (ProC994+uPA); (6) single domain antibody with engineered cysteines Q3C and W155C and dual MM anchored at CM 1490DNI (ProC995; SEQ ID NO:5); (7) product of ProC995 with uPA (ProC995+uPA); (8) engineered cysteines D3C and F154C and dual MM anchored at CM 1490DNI MM dual anchored single domain antibody (ProC996; SEQ ID NO:6); (9) Product of ProC996 and uPA (ProC996+uPA). [Figure 9]1 shows images of SDS-PAGE gels run under non-reducing (top) and reducing (bottom) conditions. Gels were loaded as follows: (1) Fc-tagged single domain antibody with no MM attached (ProC1283; SEQ ID NO: 7); (2) product of ProC1283 with uPA (ProC1283+uPA); (3) Fc-tagged single domain antibody with MM attached at CM 1490DNI (ProC1284; SEQ ID NO: 8); (4) product of ProC1284 with uPA (ProC1284+uPA); (5) Fc-tagged single domain antibody with engineered cysteines Q3C and Q157C and dual anchored MM at CM 1490DNI (ProC1285; SEQ ID NO: 9); (6) product of ProC1285 with uPA (ProC1285+uPA); (7) engineered cysteines D3C and F154C and CM 1490DNI MM dual anchored Fc-tagged single domain antibody (ProC1287; SEQ ID NO: 11); (8) product of ProC1287 and uPA (ProC1287+uPA); (9) uPA. [Figure 10]Results of ELISA binding assays to determine the shift in the ability of molecules to bind to plate-bound free masking peptide are provided: Fc-tagged single domain antibody with no MM attached (ProC1283), product of ProC1283 and uPA (ProC1283+uPA), Fc-tagged single domain antibody with MM attached with CM 1490DNI (ProC1284), product of ProC1284 and uPA (ProC1284+uPA), Fc-tagged single domain antibody with MM dual anchored with engineered cysteines Q3C and Q157C and CM 1490DNI (ProC1285), product of ProC1285 and uPA (ProC1285+uPA), engineered cysteines D3C and F154C and CM Fc-tagged single domain antibody with dual MM anchored at 1490DNI (ProC1287) and the product of ProC1287 and uPA (ProC1287+uPA). The results show that the single domain antibody with dual anchor mask was unable to bind to the peptide on the plate. However, treatment of the dual anchored molecule with protease restored the binding of the molecule, similar to the single anchored molecule (ProC1283). [Figure 11A] Illustrates an exemplary dual-anchor multispecific activatable antibody. The ovals represent the AB components, which may be the heavy chain variable region (VH), light chain variable region (VL), heavy chain constant region (CH), light chain constant region (CL), single variable domain on the heavy chain (VHH), or single chain variable fragment (scFv). The triangles represent the MM. The MM is linked to the AB components via the CM (with optional linker) and tethered to the AB by a disulfide bond (or non-alpha carbon covalent bond) (with optional linker(s) and / or CM(s) between the MM and a residue that forms a non-alpha carbon covalent bond with AB). [Figure 11B]Illustrates an exemplary dual-anchor multispecific activatable antibody. The ovals represent the AB components, which may be the heavy chain variable region (VH), light chain variable region (VL), heavy chain constant region (CH), light chain constant region (CL), single variable domain on the heavy chain (VHH), or single chain variable fragment (scFv). The triangles represent the MM. The MM is linked to the AB components via the CM (with optional linker) and tethered to the AB by a disulfide bond (or non-alpha carbon covalent bond) (with optional linker(s) and / or CM(s) between the MM and a residue that forms a non-alpha carbon covalent bond with AB). [Figure 11C] Illustrates an exemplary dual-anchor multispecific activatable antibody. The ovals represent the AB components, which may be the heavy chain variable region (VH), light chain variable region (VL), heavy chain constant region (CH), light chain constant region (CL), single variable domain on the heavy chain (VHH), or single chain variable fragment (scFv). The triangles represent the MM. The MM is linked to the AB components via the CM (with optional linker) and tethered to the AB by a disulfide bond (or non-alpha carbon covalent bond) (with optional linker(s) and / or CM(s) between the MM and a residue that forms a non-alpha carbon covalent bond with AB). [Figure 11D] Illustrates an exemplary dual-anchor multispecific activatable antibody. The ovals represent the AB components, which may be the heavy chain variable region (VH), light chain variable region (VL), heavy chain constant region (CH), light chain constant region (CL), single variable domain on the heavy chain (VHH), or single chain variable fragment (scFv). The triangles represent the MM. The MM is linked to the AB components via the CM (with optional linker) and tethered to the AB by a disulfide bond (or non-alpha carbon covalent bond) (with optional linker(s) and / or CM(s) between the MM and a residue that forms a non-alpha carbon covalent bond with AB). [Figure 11E]Illustrates an exemplary dual-anchor multispecific activatable antibody. The ovals represent the AB components, which may be the heavy chain variable region (VH), light chain variable region (VL), heavy chain constant region (CH), light chain constant region (CL), single variable domain on the heavy chain (VHH), or single chain variable fragment (scFv). The triangles represent the MM. The MM is linked to the AB components via the CM (with optional linker) and tethered to the AB by a disulfide bond (or non-alpha carbon covalent bond) (with optional linker(s) and / or CM(s) between the MM and a residue that forms a non-alpha carbon covalent bond with AB). [Figure 11F] Illustrates an exemplary dual-anchor multispecific activatable antibody. The ovals represent the AB components, which may be the heavy chain variable region (VH), light chain variable region (VL), heavy chain constant region (CH), light chain constant region (CL), single variable domain on the heavy chain (VHH), or single chain variable fragment (scFv). The triangles represent the MM. The MM is linked to the AB components via the CM (with optional linker) and tethered to the AB by a disulfide bond (or non-alpha carbon covalent bond) (with optional linker(s) and / or CM(s) between the MM and a residue that forms a non-alpha carbon covalent bond with AB). [Figure 11G] Illustrates an exemplary dual-anchor multispecific activatable antibody. The ovals represent the AB components, which may be the heavy chain variable region (VH), light chain variable region (VL), heavy chain constant region (CH), light chain constant region (CL), single variable domain on the heavy chain (VHH), or single chain variable fragment (scFv). The triangles represent the MM. The MM is linked to the AB components via the CM (with optional linker) and tethered to the AB by a disulfide bond (or non-alpha carbon covalent bond) (with optional linker(s) and / or CM(s) between the MM and a residue that forms a non-alpha carbon covalent bond with AB). [Figure 11H]Illustrates an exemplary dual-anchor multispecific activatable antibody. The ovals represent the AB components, which may be the heavy chain variable region (VH), light chain variable region (VL), heavy chain constant region (CH), light chain constant region (CL), single variable domain on the heavy chain (VHH), or single chain variable fragment (scFv). The triangles represent the MM. The MM is linked to the AB components via the CM (with optional linker) and tethered to the AB by a disulfide bond (or non-alpha carbon covalent bond) (with optional linker(s) and / or CM(s) between the MM and a residue that forms a non-alpha carbon covalent bond with AB). [Figure 11I] Illustrates an exemplary dual-anchor multispecific activatable antibody. The ovals represent the AB components, which may be the heavy chain variable region (VH), light chain variable region (VL), heavy chain constant region (CH), light chain constant region (CL), single variable domain on the heavy chain (VHH), or single chain variable fragment (scFv). The triangles represent the MM. The MM is linked to the AB components via the CM (with optional linker) and tethered to the AB by a disulfide bond (or non-alpha carbon covalent bond) (with optional linker(s) and / or CM(s) between the MM and a residue that forms a non-alpha carbon covalent bond with AB). [Figure 12A-1] Illustrates the three-dimensional structure of an activatable target binding protein (activatable anti-PDL1 antibody; SEQ ID NOs: 562-563) obtained using BIOVIA Discovery Studios from Dessault Systemes software. A zoomed-in region of the structure is shown in which solvent-accessible residues are within 2-5 angstroms of residues in the header region or N-terminus of the masked portion, with a second view of the structure rotated 90 degrees. Residues identified by mutagenesis that interact with the mask are indicated with an asterisk. [Figure 12A-2]Illustrates the three-dimensional structure of an activatable target binding protein (activatable anti-PDL1 antibody; SEQ ID NOs: 562-563) obtained using BIOVIA Discovery Studios from Dessault Systemes software. A zoomed-in region of the structure is shown in which solvent-accessible residues are within 2-5 angstroms of residues in the header region or N-terminus of the masked portion, with a second view of the structure rotated 90 degrees. Residues identified by mutagenesis that interact with the mask are indicated with an asterisk. [Figure 12B] FIG. 1 illustrates the three-dimensional structure of an activatable target binding protein (activatable anti-PDL1 antibody; SEQ ID NOs: 562-563) obtained using BIOVIA Discovery Studios from Dessault Systemes software. The three-dimensional structures of the Fab and pro domains (including labeled masked portions) are shown. The Fab domain is rendered in space-filling form and the pro domain is rendered in Cα framework form. [Figure 13A] Illustrated are homology-based three-dimensional models of antibody structures corresponding to J43v2 / anti-mouse PD1 Fab (FIG. 13A, SEQ ID NOs: 568-569), anti-CD166 (FIG. 13B, SEQ ID NOs: 572-573), and anti-PD1 (FIGS. 13C-13D, SEQ ID NOs: 570-571). FIGs. 13A-13C were modeled using BIOVIA Discovery Studio, and FIG. 13D was modeled using AlphaFold2 and rendered in BIOVIA Discovery Studio. CDRs are shown in dark grey. [Figure 13B] Illustrated are homology-based three-dimensional models of antibody structures corresponding to J43v2 / anti-mouse PD1 Fab (FIG. 13A, SEQ ID NOs: 568-569), anti-CD166 (FIG. 13B, SEQ ID NOs: 572-573), and anti-PD1 (FIGS. 13C-13D, SEQ ID NOs: 570-571). FIGs. 13A-13C were modeled using BIOVIA Discovery Studio, and FIG. 13D was modeled using AlphaFold2 and rendered in BIOVIA Discovery Studio. CDRs are shown in dark grey. [Figure 13C] Illustrated are homology-based three-dimensional models of antibody structures corresponding to J43v2 / anti-mouse PD1 Fab (FIG. 13A, SEQ ID NOs: 568-569), anti-CD166 (FIG. 13B, SEQ ID NOs: 572-573), and anti-PD1 (FIGS. 13C-13D, SEQ ID NOs: 570-571). FIGs. 13A-13C were modeled using BIOVIA Discovery Studio, and FIG. 13D was modeled using AlphaFold2 and rendered in BIOVIA Discovery Studio. CDRs are shown in dark grey. [Figure 13D] Illustrated are homology-based three-dimensional models of antibody structures corresponding to J43v2 / anti-mouse PD1 Fab (FIG. 13A, SEQ ID NOs: 568-569), anti-CD166 (FIG. 13B, SEQ ID NOs: 572-573), and anti-PD1 (FIGS. 13C-13D, SEQ ID NOs: 570-571). FIGs. 13A-13C were modeled using BIOVIA Discovery Studio, and FIG. 13D was modeled using AlphaFold2 and rendered in BIOVIA Discovery Studio. CDRs are shown in dark grey.

[0030] The drawings herein are for illustrative purposes only and are not necessarily drawn to scale. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] overview Provided herein is an activatable molecule ("activatable dual-anchored masked target binding protein" or "activatable target binding protein") that includes at least one mask that is anchored at two points, usually directly or indirectly, to at least one polypeptide of an active binding moiety. In some embodiments detailed and illustrated in the present disclosure, the activatable target binding protein comprises a complex of multiple polypeptides. In some embodiments detailed and illustrated in the present disclosure, the mask is covalently attached to one polypeptide of the activatable target binding protein at two anchor positions. In some embodiments detailed and illustrated in the present disclosure, the mask is covalently attached to two polypeptides of the activatable target binding protein, e.g., anchored to a first polypeptide at one anchor position and anchored to a second polypeptide at a second anchor position. In solution, a masked activatable target binding protein molecule lacking the dual-anchored structure described herein exists in dynamic equilibrium between a conformational state in which one or more of the masks are actively bound to the binding site of the target binding protein, as shown in FIG. 1A, and a conformational state in which one or more of the masks are not actively bound to the binding site of the target binding protein. This dynamic equilibrium state may be referred to herein as "breathing" of the masked activatable target binding protein molecule, in which one or more binding surfaces of one or more target binding proteins in the masked activatable target binding protein molecule become available for binding to a target or other epitope, including target binding outside of the activation environment. Breathing occurs when the antigen binding site is briefly exposed on a portion of the intact molecule due to equilibrium binding of the tethered mask, as illustrated in FIG. 1A. The dual-anchored mask structure described herein reduces or inhibits the dynamic dissociation between the mask and the target binding protein, i.e., "breathing" of the activatable target binding protein molecule. Dual-anchored masks are believed to mask proteins that would otherwise bind to a target by increasing masking efficiency and / or reducing the population of molecules in which one or more masks are dynamically dissociated from their corresponding target-binding proteins.

[0032] In one embodiment, the activatable molecule can be an activatable therapeutic macromolecule ("activatable target binding protein"). In some embodiments, the activatable therapeutic macromolecule can be an activatable antibody or any other desired protein, e.g., a therapeutic protein. The activatable molecule can include a target binding protein (TB), a masking moiety (MM), and a cleavable moiety (CM) located between the MM and the TB. In some embodiments, the activatable molecule can include multiple CMs, e.g., as shown in Figures 2C and 2D. For example, the activatable molecule can include a first cleavable moiety (CM1) and a second cleavable moiety (CM2). In some embodiments, the activatable molecule can have a structure that includes a CM1 between the MM and the TB, and a CM2 between the MM and a residue that forms a non-alpha carbon covalent bond with the activatable molecule. Thus, in some embodiments, the present disclosure includes a TB-CM1-MM-CM2 construct, where cleavage of CM1 and CM2 completely cleaves the MM from the activatable molecule at both anchoring sites. In some embodiments, cleavage of both CM1 and CM2 results in full activation of the activatable target binding protein. In some embodiments, cleavage of both CM1 and CM2 is required for full activation of the activatable target binding protein. In some embodiments, cleavage of one of CM1 and CM2 is sufficient for activation of the activatable target binding protein.

[0033] In some aspects, an activatable antibody used in the context of the activatable dual-anchored masked antibodies of the present disclosure may comprise an antigen binding protein (AB), a masking moiety (MM), and one or more cleavable moieties (CM) located between the MM and the AB. In general, the activatable molecules herein may be dual-anchored, i.e., the MM and TB (e.g., AB) are linked via a CM (or CM1 and CM2) and are also tethered by one or more non-alpha carbon covalent bonds. Such activatable molecules may have reduced target binding activity compared to comparable activatable molecules lacking the non-alpha carbon covalent bond (i.e., a "single-anchored activatable molecule" or "comparative activatable target binding protein"). The enhanced masking efficiency of the MM in the dual-anchored activatable molecules described herein may result in an improved safety profile, e.g., reduced toxicity and reduced target binding outside of the activation environment, compared to single-anchored activatable molecules lacking a non-alpha carbon covalent bond (e.g., a disulfide bond) tethering the MM and the TB.

[0034] Also provided herein are related compositions, kits, nucleic acids, vectors, and recombinant cells, as well as related methods, including methods of using and producing any of the activatable molecules (e.g., activatable macromolecules, e.g., antibodies and other proteins) described herein.

[0035] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. This specification describes methods and materials for use in this disclosure. Other suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0036] The terms "a" and "an" refer to one or more (i.e., to at least one) of the grammatical object of the article. As an example, "a cell" includes one or more cells.

[0037] As used herein, the terms "about" and "approximately," when used to modify a quantity specified in a numerical value or range, refer not only to the numerical value but also to reasonable deviations from that value known to those of skill in the art. For example, where appropriate, ±20%, ±10% or ±5% can be within the intended meaning of the recited value.

[0038] Concentrations, amounts, and other numerical data may be expressed or presented in a range format herein. It should be understood that such range formats are used for convenience and brevity only, and thus should be interpreted flexibly to include not only the numerical values ​​explicitly recited as range limitations, but also all individual numerical values ​​or subranges subsumed within the range, as if each numerical value and subrange were explicitly recited. As an illustration, a numerical range of "about 0.01 to 2.0" should be interpreted to include not only the explicitly recited values ​​of about 0.01 to about 2.0, but also the individual values ​​and subranges within the stated range. Thus, within this numerical range, individual values, such as 0.5, 0.7, and 1.5, as well as subranges, such as 0.5 to 1.7, 0.7 to 1.5, and 1.0 to 1.5, are included. Moreover, such interpretation should be applied regardless of the breadth or characteristics of the range described. In addition, it should be noted that all percentages are calculated by weight unless otherwise specified.

[0039] In understanding the scope of the present disclosure, the terms "including" or "comprising" and their derivatives, as used herein, are intended to be open-ended terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers, and / or steps. The above also applies to words with similar meanings, such as the terms "including" and "having" and their derivatives. The term "consisting" and its derivatives, as used herein, are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but exclude the presence of other unstated features, elements, components, groups, integers, and / or steps. As used herein, the term "consisting essentially of" is intended to specify the presence of the described features, elements, components, groups, integers, and / or steps, as well as those that do not materially affect the basic and novel characteristic(s) of the features, elements, components, groups, integers, and / or steps. Reference to any one of these transitional phrases (i.e., "comprising," "consisting," or "consisting essentially of") is understood to provide direct support for the replacement of any of the other transitional phrases not specifically used. For example, the modification of the term "comprising" to "consisting essentially of" or "consisting of" will find direct support from this definition for any element disclosed throughout this disclosure. Based on this definition, any element disclosed or incorporated by reference herein may be included or excluded from the claimed invention.

[0040] As used herein, for convenience, a plurality of compounds, elements, or steps may be presented in common lists. However, these lists should be construed as if each member of the list were individually identified as a separate and unique member. Thus, the individual members of such lists should not be construed as being de facto equivalents to any other members of the same list solely based on their presentation in a common group, unless indicated to the contrary.

[0041] Additionally, certain molecules, constructs, compositions, elements, moieties, excipients, diseases, conditions, properties, steps, etc. may be discussed in the context of a particular embodiment or aspect of the disclosure, or in a separate paragraph or section. This is merely for convenience and brevity, and it is understood that any such disclosure is equally applicable to and intended to be combined with any other embodiment or aspect found anywhere in the disclosure and claims, all of which form the present application and claimed invention as of the filing date. For example, a listing of constructs, molecules, method steps, kits, or compositions described with respect to a construct, composition, or method is intended to, and does, find direct support for the related embodiments of the constructs, compositions, formulations, and methods described anywhere else in the disclosure, even if those method steps, active agents, kits, or compositions are not re-listed in the context or section of that embodiment or aspect.

[0042] The term "exemplary" is used herein to mean serving as an example, instance, or illustration. Any aspect or design described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other aspects or designs. Rather, use of the word exemplary is intended to present concepts in a concrete manner.

[0043] Activatable target-binding proteins In one aspect, the disclosure provides an activatable target binding protein (TB), e.g., 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, and a cleavable moiety (CM) directly or indirectly covalently attached (also referred to as "coupled" or "fused") to the TB (e.g., AB), the CM being a polypeptide that functions as a substrate for a protease and positioned between the TB and the masking moiety (MM), where the MM and TB are tethered by a non-alpha carbon covalent bond, and the MM inhibits binding of the TB to a target. As used herein, the term "fused" and grammatical variations thereof refer to the covalent attachment of the alpha carbon backbones of the same polypeptide, e.g., by recombinant fusion. As used herein, the term "tethering" and grammatical variations thereof refer to the attachment of two moieties (e.g., MM and AB in an activatable antibody) by a non-alpha carbon covalent bond (e.g., a disulfide bond between functional groups substituted at the alpha carbon in MM and AB, an amide / isopeptide bond, or other bond not involving an alpha carbon backbone bond). As used herein, the term "anchor" and grammatical variations thereof can include a moiety that is directly or indirectly covalently attached to another moiety (e.g., TB and CM, or MM and CM). In some embodiments, an isopeptide bond can be between a lysine residue and an aspartic acid or glutamic acid residue, or between the gamma carboxyamide group of glutamine and the epsilon amino group of a lysine side chain.

[0044] In some embodiments, the activatable antibody provides a reduction in toxicity and / or side effects that may result from TB (e.g., AB) binding at non-therapeutic sites if the TB did not mask or otherwise inhibit target binding. In an activatable TB, the MM may prevent TB from binding to its target molecule. In a single-anchor activatable antibody (e.g., a single-anchor activatable antibody in which the MM and AB are not tethered by a non-alpha carbon covalent bond), the masking effect of the MM on the target binding surface of the AB may be dynamic. Thus, in a given formulation containing single-anchor activatable TB molecules, some of the activatable TB molecules in the formulation may be unmasked for a short period of time due to breathing. At a sufficiently high concentration of single-anchor activatable TB, such unmasked binding of activatable TB to target molecules may have a large impact and cause undesirable toxicity or effects caused by target binding outside the activation environment.

[0045] As used herein, the term "activatable antibody" or "activatable target binding protein" refers to an activatable antibody or activatable target binding protein, respectively, in its inactive (uncleaved or native) form. It will be apparent to one skilled in the art that modification of the CM of an activatable antibody or activatable target binding protein by at least one protease may result in a cleaved protein in which the MM does not prevent binding between the TB or AB and its target. In some embodiments, cleavage of the CM by a protease may result in the release of the MM. The term "uncleaved" or "inactive" refers to the activatable TB in the absence of cleavage of the CM by a protease, i.e., the activatable TB in its native form. As used throughout this disclosure, the description of an activatable antibody should be construed as being applicable to an activatable target binding protein as well. Thus, in one aspect, the dual-anchor activatable target binding protein of the present disclosure may include an MM that inhibits binding of the AB to the target when the activatable target binding protein is in an inactive state.

[0046] As used herein, following modification of the CM by at least one protease, the activatable TB is "cleaved" or "activated." 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 generated by exposing the activatable protein to a cleavage agent (e.g., a protease).

[0047] As used herein, the terms "masking moiety" and "MM" are used interchangeably to refer to a peptide or protein that, when placed in proximity to a TB (e.g., an AB), prevents the TB from binding to its target.

[0048] The terms "cleavable moiety" and "CM" are used interchangeably herein to refer to a peptide of an amino acid sequence that comprises a substrate for a sequence-specific protease. In an activatable protein, the CM is positioned relative to the MM and TB such that cleavage results in a molecule that can bind to the biological target of the TB. Thus, the activatable protein shows reduced binding to the biological target compared to the activated protein.

[0049] In some embodiments, an activatable TB can be designed by selecting a TB of interest and constructing the remainder of the activatable TB such 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.

[0050] Activatable antibodies may be provided in a variety of structural configurations. Exemplary formulas of activatable antibodies are provided below. It is contemplated that the N-terminal to C-terminal order of AB, MM, and CM may be reversed in an activatable antibody. It is also contemplated that the amino acid sequences of CM and MM may overlap, e.g., a CM sequence recognized by a sequence-specific protease is at least partially contained within the MM. For example, an activatable antibody may be represented by the formula of FIG. 1B (ordered from amino (N)-terminal region to carboxyl (C)-terminal region). An activatable antibody may further include one or more linkers (L) between the MM and CM and / or between the CM and AB. The line connecting the MM and AB indicates a non-alpha carbon covalent bond.

[0051] Exemplary configurations of activatable antibodies are shown in Figures 2A-2D. Figure 2A shows an exemplary activatable antibody 210 that includes, from amino (N)-terminal region to carboxyl (C)-terminal region, MM211, optional linker 212, CM213, optional linker 214 having the same or different sequence as optional linker 212, and AB215. MM211 and AB215 are tethered together by a non-alpha carbon covalent bond 216. Figure 2B shows an exemplary activatable antibody 220 that includes, from amino (N)-terminal region to carboxyl (C)-terminal region, AB221, optional linker 222, CM223, optional linker 224 having the same or different sequence as optional linker 222, and MM225. AB221 and MM225 are tethered together by a non-alpha carbon covalent bond 226. 2C shows an exemplary activatable antibody 230 including, from amino (N) terminal region to carboxyl (C) terminal region, optional linker 231, CM232, optional linker 233, MM234, optional linker 235, CM236, optional linker 237, and AB238. MM234 and AB238 are tethered by a non-alpha carbon covalent bond 239. FIG 2D shows an exemplary activatable antibody 241 including, from amino (N) terminal region to carboxyl (C) terminal region, AB241, optional linker 242, CM243, optional linker 244, MM245, optional linker 246, CM247, and optional linker 248. AB241 and MM255 are tethered by a non-alpha carbon covalent bond 249.

[0052] In some examples, the AB (e.g., AB in Figures 2A-2D) may comprise only one polypeptide. In such cases, the MM may be coupled to the polypeptide via a CM and tethered to the polypeptide. In some embodiments, the first polypeptide comprises a MM, a CM, and at least one antibody variable domain selected from the group selected from a light chain variable domain ("LVD" or "VL") and a heavy chain variable domain ("HVD" or "VH"). In some examples, the AB (e.g., AB in Figures 2A-2D) may comprise multiple polypeptides (e.g., AB is a complex formed by multiple polypeptides). In some embodiments, the AB comprises at least two polypeptides, at least three polypeptides, at least four polypeptides, or more. In such cases, the MM may be coupled to a polypeptide of the AB via a CM and tethered to the same polypeptide via a non-alpha carbon covalent bond. Alternatively, the MM may be coupled to a first polypeptide of the AB via a CM and tethered to a second polypeptide of the AB via a non-alpha carbon covalent bond. An activatable antibody can have one or more polypeptides of the sequence 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, unless otherwise indicated, a dash (-) between ACC moieties represents either a direct linkage or a linkage via one or more linkers.

[0053] In some embodiments, an activatable antibody may have two polypeptides. In some examples, an activatable antibody may include a first polypeptide that includes an HVD and a second polypeptide that includes, from N-terminal to C-terminal, any one of 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, an activatable antibody may include a first polypeptide comprising LVD and a second polypeptide comprising, in the N-terminal to C-terminal direction, any one of 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 such cases, the MM may be tethered to the second polypeptide (i.e., the polypeptide comprising the MM) via a non-alpha carbon covalent bond. Alternatively or additionally, the MM may be tethered to the first polypeptide (i.e., the polypeptide not comprising the MM) via a non-alpha carbon covalent bond.

[0054] In some embodiments, an activatable antibody may have more than two polypeptides. Such an activatable antibody may include any combination of the above-mentioned polypeptides. In some embodiments, an activatable antibody may include four polypeptides. In some examples, two of the polypeptides may each include HVD, and the other two polypeptides may each include, 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, two of the polypeptides may each include an LVD, and the other two polypeptides may each include, from N-terminus to C-terminus, 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 MM-containing polypeptide via a non-alpha carbon covalent bond. Alternatively or additionally, the MM may be tethered to the MM-free polypeptide via a non-alpha carbon covalent bond.

[0055] In the examples described herein, the HVD and LVD can be comprised in an antibody or a fragment thereof (e.g., a Fab). An activatable antibody can further comprise one or more additional components of an antibody, such as, for example, a heavy chain constant region (CH), a light chain constant region (CL), a hinge, an Fc domain, or a combination thereof.

[0056] Figures 3A-C show three exemplary configurations of activatable antibodies. Figure 3A shows an exemplary activatable antibody comprising a nanobody (i.e., an exemplary antibody or single domain antibody as the TB) and an MM bound thereto via a CM. The nanobody and MM are tethered by a non-alpha carbon covalent bond between the mask and the nanobody.

[0057] FIG. 3B shows the heavy chain variable region (V H ) and the light chain variable region (V L ) and V via CM L The activatable single chain fragment variable (scFv) comprises an MM linked to a V. The MM and scFv also comprise an MM linked to a V. H The activatable antibody examples also include several alternative configurations of the activatable scFv of FIG. 3B. In one example, the MM is tethered to the V of the scFv. L The MM and scFv are also linked to the mask and V L In another example, the MM is also tethered to the V of the scFv by a non-alpha carbon covalent bond between the H MM and scFv are also linked to V via CM. H In another example, the MM is also tethered to the V of the scFv by a non-alpha carbon covalent bond between the H MM and scFv are also linked to V via CM. L It is also tethered by a non-alpha carbon covalent bond between

[0058] FIG. 3C shows an activatable full-length antibody comprising a dimer, where each monomer of the dimer comprises a heavy chain, a light chain, and a MM linked to the light chain via a CM. The MM and the activatable full-length antibody are tethered by a non-alpha carbon covalent bond between the MM and the heavy chain. Examples of activatable antibodies also include several alternative configurations of the activatable full-length antibody of FIG. 3C. In one example, the MM is linked to the light chain via a 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 linked to the heavy chain via a 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 linked to the heavy chain via a CM. The MM and the full-length antibody are tethered by a non-alpha carbon covalent bond between the MM and the heavy chain.

[0059] The schematics in Figures 2A-2D and 3A-3B are illustrated as non-limiting proof-of-concept examples. Activatable antibodies in which the MM is dual-anchored (e.g., via a CM as well as a non-alpha carbon covalent bond) to the AB broadly include any type of activatable antibody, including activatable full-length antibodies, activatable multispecific antibodies (e.g., bispecific and trispecific antibodies, including multispecific antibodies that can cross-link two cells, such as bispecific T cell engagers (BiTEs)), and activatable antibody fragments (e.g., scFvs, diabodies, nanobodies, Fabs, etc.). Examples of dual-anchored multispecific activatable antibodies are shown in Figures 11A-11I. Although Figures 11A-11I illustrate dual anchors at all mask positions, the present disclosure also includes constructs that include one or more masks that are dual-anchored and one or more other masks that are not dual-anchored (i.e., anchored only to the TB via a CM (single-anchored)). The present disclosure also includes constructs that include one or more masks that are dual-anchored and one or more TBs that are unmasked. FIG. 11A shows an exemplary bispecific nanobody tandem. FIG. 11B shows an exemplary diabody comprising two scFvs. In some examples, the two scFvs can be linked by a peptide linker. In alternative examples, the two scFvs are not linked by any linker. FIG. 11C shows an exemplary bispecific antibody comprising an scFv and a Fab. In some examples, the bispecific antibody can be a bispecific T cell engager (BiTE). The antibody of FIG. 11C can further comprise one or more Fc domains. FIG. 11D shows an exemplary bispecific F(ab')2. FIG. 11D shows an exemplary bispecific antibody. FIGs. 11E-11I show further examples of dual-anchored trispecific and other multispecific activatable antibodies. In dual-anchored multispecific activatable antibodies, the MM can be linked to the AB via the CM. The MM can be tethered by one or more non-alpha carbon covalent bonds to the AB linked to the MM or to another component of the activatable antibody. In some examples of multispecific activatable antibodies, all of the MMs are of the dual anchor type.In certain multispecifically activatable instances, only some, but not all, MMs are dual-anchored. The TBs in Figures 2A-2D can be any antigen binding protein, including antibodies and those described in the Target Binding Proteins section below.

[0060] In some embodiments, an activatable target binding protein (e.g., an activatable antibody) may be characterized by a reduction in its target binding activity compared to a control level of target binding activity of AB without MM. For example, an activatable TB may be characterized by at least a 1-fold, 2-fold, 4-fold, 6-fold, 8-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 2000-fold, 5000-fold, or 10000-fold reduction in target binding activity compared to a control level of target binding activity of TB without MM.

[0061] In some embodiments, an activatable TB (e.g., an activatable antibody) may be characterized by a decrease in its target binding activity compared to a control level of target binding activity of a TB bound to an MM, but where the TB and MM are not tethered by a non-alpha carbon covalent bond (i.e., a single-anchor activatable antibody). For example, in some embodiments, the activatable TB is characterized by at least a 2-fold, 4-fold, 6-fold, 8-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 2000-fold, 5000-fold, 10,000-fold, 15,000-fold, 20,000-fold, 30,000-fold, 40,000-fold, or 50,000-fold reduction in target binding activity compared to a control level of target binding activity of TB bound to MM, wherein the TB and MM are not tethered by a non-alpha carbon covalent bond. In some embodiments, the activatable TB is characterized by at least a two-fold decrease in target binding activity. In some embodiments, the activatable TB is characterized by at least a four-fold decrease in target binding activity. In some embodiments, the activatable TB is characterized by at least a ten-fold decrease in target binding activity. In some embodiments, the activatable TB is characterized by at least a five-fold decrease in target binding activity. In some embodiments, the activatable TB is characterized by at least a hundred-fold decrease in target binding activity. In some embodiments, the activatable TB is characterized by at least a two-hundredth decrease in target binding activity. In some embodiments, the activatable TB is characterized by at least a three-hundredth decrease in target binding activity. In some embodiments, the activatable TB is characterized by at least a five-hundredth decrease in target binding activity. In some embodiments, the activatable TB is characterized by at least a thousand-fold decrease in target binding activity.In some embodiments, the activatable TB is characterized by at least a 5000-fold decrease in target binding activity. In some embodiments, the activatable TB is characterized by at least a 10000-fold decrease in target binding activity. In some embodiments, the activatable TB is characterized by at least a 15000-fold decrease in target binding activity. In some embodiments, the activatable TB is characterized by at least a 20000-fold decrease in target binding activity. In some embodiments, the activatable TB is characterized by at least a 30000-fold decrease in target binding activity.

[0062] Target-binding proteins The activatable target binding proteins (e.g., activatable antibodies) disclosed herein may comprise one or more target binding proteins, i.e., proteins capable of binding to a target molecule. In some embodiments, the target binding protein (TB) may be a cytokine, hormone, growth factor, or agonist. In some embodiments, the target binding protein (TB) may be an antigen binding protein (AB). In some embodiments, the 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 fragment, a single chain variable fragment (scFv), a diabody (a non-covalent dimer of scFv), a single chain antibody (scab), a VHH, a domain antibody (dAb), or a single domain antibody (SDA) (nanobody, e.g., single domain heavy chain antibody, single domain light chain antibody). In some embodiments, the AB may be a full-length antibody. In some embodiments, the AB may be an immunologically active fragment. In some embodiments, the AB is an antigen binding fragment ("Fab"). In some embodiments, the AB may be a murine, other rodent, chimeric, humanized or fully human monoclonal antibody. The present disclosure includes structures having one or more polypeptides comprising any of the domains listed above, e.g., one or more of SDA, Fv, ScFv, Fab, scFab, VHH, and dAb, in combination with one or more selected from SDA, Fv, scFv, Fab, VHH, scFab, and dAb.

[0063] The term "antibody" is used herein in its broadest sense and includes certain types of immunoglobulin molecules that contain one or more antigen-binding domains that specifically bind to an antigen or epitope. Antibodies specifically include, for example, intact antibodies (e.g., intact immunoglobulins), antibody fragments, bispecific, and multispecific antibodies. One example of an antigen-binding domain is the antigen-binding domain formed by a VH-VL dimer. Further examples of antibodies are described herein. Further examples of antibodies are known in the art.

[0064] In some examples, the AB may be a single domain antibody (also called a nanobody). A single domain antibody may be an antibody fragment that is a single monomeric variable antibody domain. A single domain antibody may have an affinity for an antigen similar to the corresponding full-length antibody. A single domain antibody may be an Fc-tagged single domain antibody that comprises an Fc dimer, with each Fc monomer binding to a single domain antibody.

[0065] In some embodiments, the AB may be monospecific, e.g., capable of binding only one antigen. In some embodiments, the AB may be multispecific (e.g., bispecific or trispecific), e.g., capable of binding multiple antigens. In some embodiments, the activatable antibody may be formulated as part of a pro-bispecific T cell engager (pro-BITE) molecule or a dual affinity retargeting antibody (DART). In some embodiments, the activatable antibody may be formulated as part of a pro-chimeric antigen receptor (pro-CAR) modified T cell, or other engineered receptor or other immune effector cell, such as a CAR modified NK cell. In some embodiments, the activatable antibody may be formulated as part of a pro-chimeric antigen receptor (CAR) modified T cell. In some embodiments, the activatable antibody may be formulated as part of a pro-chimeric antigen receptor (CAR) modified NK cell. In some embodiments, the activatable antibody may be formulated as part of a T cell bispecific antibody (TCB).

[0066] A "light chain" consists of one variable domain (VL) and one constant domain (CL). There are two different light chain types or classes, called kappa or lambda.

[0067] A "heavy chain" consists of one variable domain (VH) and three constant region domains (CH1, CH2, CH3). There are five major heavy chain classes or isotypes, some of which have several subtypes, which 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, 2, 3, and 4 in humans).

[0068] "Fragment antigen binding" (Fab) contains a complete light chain paired with the VH and CH1 domains of the heavy chain.

[0069] F(ab')2 fragments are formed when an antibody is cleaved below the hinge region by pepsin, in which case the two fragment antigen-binding domains (Fab) of the antibody molecule remain linked. The F(ab')2 fragment contains two complete light chains paired with two domains, VH and CH1, of the heavy chains joined together by the hinge region.

[0070] "Fragment crystallizable" (Fc) fragments (referred to herein as F C The Fc domain (also called the Fc domain) corresponds to the paired CH2 and CH3 domains and is the part of the antibody molecule that interacts with effector molecules and cells. The functional differences between the heavy chain isotypes are mainly in the Fc fragment.

[0071] A "single-chain Fv" (scFv) contains only the variable domain of the light chain (VL) linked to the variable domain of the heavy chain (VH) by a synthetic peptide extension. The name single-chain Fv comes from the fragment variable region.

[0072] The "hinge region" or "interdomain" is a flexible stretch of amino acids that joins or links the Fab fragment to the Fc domain.

[0073] A "synthetic hinge region" is an amino acid sequence that joins or links the Fab fragment to the Fc domain.

[0074] A "prodomain" refers to a polypeptide having a portion that inhibits antigen binding, called the masking peptide (MM), and a portion that contains a protease-cleavable substrate, called the 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), functions to inhibit antigen binding by the TB or AB. The prodomain may include a linker peptide (L1) between the MM and the CM. The prodomain may also include a linker peptide (L2) at the carboxyl terminus of the prodomain to facilitate binding of the prodomain to an antibody. In certain embodiments, the prodomain comprises one of the following formulas (the formulas below represent the amino acid sequence from the N-terminus to the C-terminus): (MM)-(CM), (MM)-L1-(CM), (MM)-(CM)-L2, or (MM)-L1-(CM)-L2.

[0075] The 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 the antigen for which the immunoglobulin is specific. The strength or affinity of an immunological binding interaction is determined by the dissociation constant (K d ) and can be expressed in units of smaller K d represents a greater affinity. The immunological binding properties of selected polypeptides can be quantified using methods well known in the art. One such method involves measuring the rates of antigen-binding site / antigen complex formation and dissociation, which depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that affect the rate equally in both directions. Thus, the "on-rate constant" (K on ) and "off rate constant" (K offBoth the K and the KD can be determined by calculating the concentration and the actual rates of association and dissociation. (See Nature 361:186-87 (1993)). off / K on The ratio of d (See generally Davies et al. (1990) Annual Rev Biochem 59:439-473). The TB of this disclosure is equal to the binding constant (K d In some embodiments, the TBs of the present disclosure can specifically bind to a target with a binding constant (K d In some embodiments, the TBs of the present disclosure can specifically bind to a target with a binding constant (K d In some embodiments, the TBs of the present disclosure can specifically bind to a target with a binding constant (K) < 10 nM, as measured by an assay such as a radioligand binding assay or similar assay known to one of skill in the art. d )≦100 pM to about 1 pM and can specifically bind to the target.

[0076] The target of TB (e.g., AB) can be a protein or other type of molecule. Examples of classes 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 examples, the target of TB can be a protein associated with a disease (e.g., cancer) in a subject.

[0077] 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 to 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.

[0078] In some examples, the half-life extending moiety can be the crystallizable region (Fc) region of a fragment of an antibody. Other examples of half-life extending moieties include hexa-hat GST (glutathione S-transferase) glutathione affinity, calmodulin binding peptide (CBP), strep tag, cellulose binding domain, maltose binding protein, S-peptide tag, chitin binding tag, immunoreactive epitope, epitope tag, E2Tag, HA epitope tag, Myc epitope, FLAG epitope, AU1 and AU5 epitope, Glu-Glu epitope, KT3 epitope, IRS epitope, Btag epitope, protein kinase-C epitope, and VSV epitope.

[0079] In some embodiments, the serum half-life of an activatable target binding protein or activatable antibody may be longer than the serum half-life of the corresponding protein (e.g., the activatable antibody does not have a half-life extending moiety), e.g., the pK of the activatable antibody is longer than the pK of the corresponding antibody. In some embodiments, the serum half-life of an activatable target binding protein or activatable antibody is similar to the serum half-life of the corresponding antibody. In some embodiments, the serum half-life of an activatable target binding protein (e.g., an activatable antibody) when administered to an organism 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, 18 hours, 16 hours, 14 hours, 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, 3 hours, 2 hours, or 1 hour.

[0080] Masking part (MM) A "masking moiety" within an activatable macromolecule "masks" or weakens or otherwise inhibits the binding of the activatable macromolecule to its target and / or epitope. In some embodiments, binding or modification of a target binding protein (TB) (e.g., AB or other therapeutic or diagnostic protein) with MM can inhibit the ability of TB to specifically bind to its target and / or epitope by inhibitions known in the art (e.g., but not limited to, conformational changes and competition of the antigen binding domain). In some embodiments, binding or modification of TB with MM can result in conformational changes that reduce or inhibit the ability of TB to specifically bind to its target and / or epitope. In some embodiments, binding or modification of a protein that includes an antigen binding domain with MM sterically blocks, reduces, or inhibits the ability of the antigen binding domain to specifically bind to its target and / or epitope.

[0081] The activatable target binding proteins (e.g., activatable antibodies) herein may contain one or more masking moieties (MMs) that can prevent the target binding protein (e.g., AB) from binding to a target. In general, the MMs can be attached to the target binding protein (e.g., AB) by a CM as described herein and optionally one or more linkers. Furthermore, the MMs may be additionally tethered to the activatable target binding proteins (or antibodies) as described herein to form activatable dual-anchored masked target binding proteins. In some embodiments, the MMs block the activatable TB from binding to the target, but once the molecule is activated (the CM is cleaved by a protease), the MMs do not substantially or significantly prevent the target binding protein from binding to the target.

[0082] In some embodiments, the MM may interact with 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 a partial or complete amino acid sequence of a naturally occurring binding partner (e.g., AB) of the target binding protein. For example, the MM may be a fragment of the naturally occurring binding partner. The fragment may retain no more than 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 cognate peptide of the target binding protein (e.g., AB). For example, the MM may comprise the sequence of an epitope of the target binding protein (e.g., AB) or a fragment thereof. 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 viruses) that can be isolated from a natural source and has not been intentionally modified by man in the laboratory or otherwise occurs in nature.

[0083] In some embodiments, the MM may comprise an amino acid sequence that is not naturally occurring or does not contain the amino acid sequence of a naturally occurring binding partner or target protein. In certain embodiments, the MM is not a natural binding partner of the target binding protein (e.g., AB). The MM may be a modified binding partner for the target binding protein (e.g., AB) that contains amino acid changes that reduce the affinity and / or avidity of binding to the target binding protein (e.g., AB). In some embodiments, the MM may contain no, or substantially no, nucleic acid or amino acid homology with the natural binding partner of AB. In other embodiments, the MM has no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% similarity to the natural binding partner of the target binding protein (e.g., AB).

[0084] In some embodiments, the MM does not specifically bind to AB (or other activatable proteins), but may interfere with the binding of the target binding protein (e.g., AB) to a binding partner through non-specific interactions such as steric hindrance. For example, placement of the MM within an activatable target binding protein may allow the tertiary or quaternary structure of the activatable target binding protein to allow the MM to mask the target binding protein through charge-based interactions, thereby holding the MM in place and preventing access of the binding partner to the target binding protein.

[0085] In some embodiments, the MM may have a dissociation constant for binding to a target binding protein (e.g., AB) that is equal to or less than the dissociation constant of the target binding protein for its target. In some embodiments, the MM may not interfere with or compete with the target binding protein to bind to the target after cleavage of the CM.

[0086] The structural characteristics of the MM can be selected according to factors such as the minimum amino acid sequence required to prevent the protein from 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.

[0087] In some embodiments, the MM can be unique to the bound target binding protein. Examples of MMs include MMs (e.g., affinity masked, etc.) that are specifically screened to bind to the binding domain of the target binding protein or a fragment thereof. Methods for screening MMs to obtain MMs that are unique to the target binding protein and that specifically and / or selectively bind to the binding domain of the binding partner / target are provided herein and can include protein display methods.

[0088] As used herein, the term "masking efficiency" or "ME" refers to the activity (e.g., EC50) of an activatable target binding protein (e.g., an activatable AB) divided by the activity of a control target binding protein (e.g., an antibody), which can be either a cleavage product of an activatable target binding protein (e.g., an activatable antibody) or a target binding protein (e.g., an antibody) or a fragment thereof used as the target binding protein of an activatable target binding protein. An activatable target binding protein having a reduced level of targeted binding (or antibody) activity can have a masking efficiency of greater than 10. In some embodiments, an activatable target binding protein (e.g., an activatable antibody) described herein can have a masking efficiency of greater than 10, 100, 1000, 5000, 10,000, or 15,000.

[0089] In some embodiments, the MM can be a polypeptide between about 2 and 50 amino acids in length. For example, the MM can be a polypeptide between 2 and 40, 2 and 30, 2 and 20, 2 and 10, 5 and 15, 10 and 20, 15 and 25, 20 and 30, 25 and 35, 30 and 40, 35 and 45, 40 and 50 amino acids in length. For example, the MM can be a polypeptide between 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 of more than 50 amino acids in length, for example, 100, 200, 300, 400, 500, 600, 700, 800, or more amino acids.

[0090] In some embodiments, when the MM is tethered to a target binding protein (e.g., AB) to which it is bound, it remains active for at least 0.1, 0.5, 1, 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours, or for 5, 10, 15, 30, 45, 60, 90, 120, 150, or 180 hours, as measured in the presence of the target of the target binding protein, in vivo, or in a masking efficiency assay, or in an in vitro immunosorbent assay, e.g., as described in US20200308243A1. The target binding protein may not bind or substantially not bind to the target, or may bind less than 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 50% of the target binding protein bound to the MM but not tethered to the MM (i.e., not dual anchored) for a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days or more.

[0091] The binding affinity of the TB (e.g., AB) to a target or binding partner when the TB (e.g., AB) is tethered to a MM bound to the TB (e.g., AB) is at least 5-fold, 10-fold, 25-fold, 50-fold, 100-fold, 250-fold, 500-fold, 1,000-fold, 2,500-fold, or more than the binding affinity of the TB (e.g., AB) to a binding partner when the TB (e.g., AB) is not bound to the MM. MM, or by a factor of 5-10, 10-10, 150,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, or 50,000,000, or by a factor of 5-10, 10-100, compared to the binding affinity of the TB (e.g., AB) for its binding partner when the TB (e.g., AB) is not bound to the MM. 1 / 10th, 1 / 10th to 1 / 1,000th, 1 / 10th to 1 / 10,000th, 1 / 10th to 1 / 100,000th, 1 / 10th to 1 / 1,000,000th, 1 / 10th to 1 / 10,000,000th, 1 / 100th to 1 / 1,000,000th, 1 / 100th to 1 / 100,000th, 1 / 100th to 1 / 1,000,000th, 1 / 100th to 1 / 10,000,000th, 1 / 1,000 1 / 1000, 1 / 1,000 to 1 / 100,000, 1 / 1,000 to 1 / 1,000,000, 1 / 1000 to 1 / 10,000,000, 1 / 10,000 to 1 / 100,000, 1 / 10,000 to 1 / 1,000,000, 1 / 10,000 to 1 / 10,000,000, 1 / 100,000 to 1 / 1,000,000, or 1 / 100,000 to 1 / 10,000,000.

[0092] The binding affinity of the TB (e.g., AB) to a target or binding partner when the TB (e.g., AB) is tethered to the MM bound to the TB (e.g., AB) may be reduced by at least 5-fold, 10-fold, 25-fold, 50-fold, 100-fold, 250-fold, 500-fold, 1,000-fold, 2,500-fold, 5,000-fold, 10,000-fold, 50,000-fold, 100,000-fold, 500,000-fold, 1,000,000-fold, 5,000,000-fold, 10,000,000-fold, or 50,000,000-fold, compared to the binding affinity of the TB (e.g., AB) to a binding partner when the TB (e.g., AB) is cleaved from the MM by a protease.

[0093] The binding affinity of the TB (e.g., AB) to a target or binding partner when the TB (e.g., AB) is tethered to a MM bound to the TB (e.g., AB) is at least 5-fold, 10-fold, 25-fold, 50-fold, 100-fold, 250-fold, 500-fold, or 1,000-fold lower than the binding affinity of the TB (e.g., AB) to a binding partner when the TB (e.g., AB) is not bound and is not tethered to a MM. , 2,500-fold, 5,000-fold, 10,000-fold, 50,000-fold, 100,000-fold, 500,000-fold, 1,000,000-fold, 5,000,000-fold, 10,000,000-fold, or 50,000,000-fold, or 5-10-fold lower than the binding affinity of the TB (e.g., AB) for its binding partner when the TB (e.g., AB) is unbound and untethered to the MM. 1 / 10th to 1 / 100th, 1 / 10th to 1 / 10,000th, 1 / 10th to 1 / 100,000th, 1 / 10th to 1 / 100,000th, 1 / 10th to 1 / 1,000,000th, 1 / 10th to 1 / 10,000,000th, 1 / 100th to 1 / 1,000, 1 / 100th to 1 / 100,000th, 1 / 100th to 1 / 1,000,000th, 1 / 100th to 1 / 10,000,000th, 1 / 100th to 1 / 10,000,000th, 1 / 100th to 1 / 10,000,000th, 1 / 100th to 1 / 10 1 / 10,000, 1 / 1,000, 1 / 1000, 1 / 1,000, 1 / 1000, 1 / 1000, 1 / 1000, 1 / 1000, 1 / 1000, 1 / 10, ...

[0094] The dissociation constant (K d ) is the K d The K of MM against TB (e.g., AB) can be greater than d is the K of TB (e.g. AB) against the targetd The binding affinity of the MM to the TB (e.g., AB) may be at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 100,000, 1,000,000, or even 10,000,000 times greater than the binding affinity of the TB (e.g., AB) to the target. Conversely, the binding affinity of the MM to the TB (e.g., AB) may be lower than the binding affinity of the TB (e.g., AB) to the target. The binding affinity of MM to TB (e.g., AB) can be at least 5-fold, 10-fold, 25-fold, 50-fold, 100-fold, 250-fold, 500-fold, 1,000-fold, 2,500-fold, 5,000-fold, 10,000-fold, 100,000-fold, 1,000,000-fold, or even 10,000,000-fold lower than the binding affinity of TB (e.g., AB) to the target.

[0095] In some embodiments, the MM may include genetically encoded amino acids or non-genetically encoded amino acids. Examples of non-genetically encoded amino acids include, but are not limited to, D-amino acids, β-amino acids, and γ-amino acids. In certain embodiments, the MM contains no more than 50%, 40%, 30%, 20%, 15%, 10%, 5%, or 1% non-genetically encoded amino acids.

[0096] In some embodiments, once cleaved from the TB and in a free state, the MM may have biological activity or therapeutic effect, e.g., binding ability. For example, the free peptide may bind to the same or different binding partners. In certain embodiments, the free MM (e.g., MM that is not bound or tethered to the TB) may exert a therapeutic effect and provide a secondary function to the compositions disclosed herein. In some embodiments, once cleaved from the TB and in a free state, the MM may advantageously not exhibit biological activity. For example, in some embodiments, the free MM does not induce an immune response in a subject.

[0097] The TB and MM may contain one or more cysteine ​​residues capable of forming a non-alpha carbon covalent bond(s) between the TB and the MM. In some embodiments, the one or more non-alpha carbon covalent bonds may be formed between the sulfur atoms of cysteines or other amino acid residues that contain sulfur atoms. Such residues may be naturally present in the activatable TB (e.g., TB and MM) or may be incorporated into the activatable TB by site-directed mutagenesis, chemical conversion, or misincorporation of a non-natural amino acid.

[0098] In some embodiments, the cysteine ​​residues may be in a position that provides a conformationally constrained activatable TB, but following CM cleavage, the MM may be in a position that does not substantially or significantly interfere with target binding of the activated TB.

[0099] The location of the 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., a crystal structure or other structural model 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 the three-dimensional structure of the target binding protein with or without the MM. These include, for example, Rosetta modeling software (rosettacommons.org), Discovery Studio (Dassault Systemes BIOVIA), BioLuminate, PIPER, Prime (Schrodinger, Inc.), AlphaFold Colab (Google, Inc.), SWISSMODELER, and the like. An example of a three-dimensional structure obtained using Discovery Studio software is the structure of the activatable target binding protein (activatable anti-PDL1 antibody) shown in Figures 12A-12B. For example, the region where the MM interacts with the TB can be determined, and a cysteine ​​residue (naturally occurring or introduced) in that region can be used to form a non-alpha carbon covalent bond tethering the TB and the MM. Generally, the Cα atoms of disulfide-bonded cysteine ​​residues are in the range of 3.0-7.5 Å. Therefore, as a first approximation, MM and TB residues with Cα distances within that range are good candidates for cysteine ​​mutations. For example, various disulfide prediction programs can be used to further identify MM and TB residues that are likely to form disulfide bonds, including MODIP (Dani, Ramakrishnan, Varadarajan 2003), Disulfide by Design (Craig & Dombkowski, 2013), and SSbondPre (Gao, Dong, Li, Liu & Liu, 2020).

[0100] In some embodiments, a non-alpha carbon covalent bond, such as a disulfide bond tethering the MM and TB (e.g., AB), can be formed by a first cysteine ​​and a second cysteine. In one example, the first cysteine ​​is in the MM and the second cysteine ​​is in the TB. In another example, the first cysteine ​​is in the peptide bound to the MM and the second cysteine ​​is in the TB. In another example, the first cysteine ​​is in the MM and the second cysteine ​​is in the peptide bound to the TB. In such an example, the peptide bound to the MM or TB can be a linker or peptide. In one example, the peptide bound to the MM or TB can be a leader peptide, which is a peptide position adjacent to the terminus (e.g., N-terminus or C-terminus) of the MM or TB. The leader peptide can be located between the signal peptide and the MM or TB.

[0101] In some embodiments, one or more of the cysteine(s) that form a disulfide bond with the MM can be a cysteine(s) that is naturally present in the activatable target binding protein. In some embodiments, one or more of the cysteines that form a disulfide bond can be engineered into the activatable TB (e.g., AB).

[0102] Suitable MMs can be identified and / or further optimized through screening procedures from a library of candidate activatable TBs with regulatable MMs. 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 screening procedures described below to identify MMs that provide an activatable phenotype. For example, random peptide libraries (e.g., peptide libraries containing 2-40 or more amino acids) may be used in the screening methods disclosed herein to identify suitable MMs.

[0103] In some embodiments, MMs having specific binding affinity to TB (e.g., AB) can be identified through a screening procedure that includes providing a library of peptide scaffolds consisting of candidate MMs, each scaffold being composed of a transmembrane protein and a candidate MM. The library can then be contacted with a whole or part of a protein, such as a full-length protein, a naturally occurring protein fragment, or a non-naturally occurring fragment containing the protein (capable of binding to a binding partner of interest), to identify one or more candidate MMs having detectably bound proteins. Screening can be performed by one or more rounds of magnetic activated sorting (MACS) or fluorescence activated sorting (FACS), as well as determining the binding affinity of the MM to AB and subsequent masking efficiency, for example, as described in WO2009025846 and US20200308243A1 (incorporated herein by reference in their entirety).

[0104] In some embodiments, the MM may be selected for use with a particular antibody or antibody fragment. Additional suitable MMs are described in WO2021207657, WO2021142029, WO2021061867, WO2020252349, WO2020252358, WO2020236679, WO2020176672, WO2020118109, WO2020092881, WO2020086665, WO2019213444, WO2019183218, WO2019173771, WO2019165143, These are disclosed in WO2019075405, WO2019046652, WO2019018828, WO2019014586, WO2018222949, WO2018165619, WO2018085555, WO2017011580, WO2016179335, WO2016179285, WO2016179257, WO2016149201, WO2016014974, and WO2016118629.

[0105] Cuttable Part (CM) The activatable target binding protein may comprise one or more cleavable moieties (CM) as defined above.

[0106] In some embodiments, an activatable TB may include a CM between the TB (e.g., AB) and the MM. The CM and TB of an activatable target binding protein (e.g., AB) may be selected such that the TB includes a binding moiety for a given target and the CM includes a substrate for one or more proteases, where the one or more proteases are co-localized with the target in a tissue (e.g., a treatment or diagnostic site of a subject). In some embodiments, an activatable TB may be particularly useful when, for example, one or more proteases capable of cleaving a site of the CM are present at relatively higher levels in target-containing tissue at a treatment or diagnostic site than in tissue at a non-treatment site (e.g., healthy tissue).

[0107] In some embodiments, the CM herein may include substrates of proteases that have been reported in cancer or some cancers. See, e.g., La Roca et al., British J.Cancer 90(7):1414-1421, 2004. Substrates suitable for use in the CM components used herein include substrates that are more prevalent in cancer cells and tissues. Thus, in certain embodiments, the CM may include substrates of proteases that are more prevalent in diseased tissues associated with cancer. Examples of cancer include gastric cancer, breast cancer, osteosarcoma, esophageal cancer, breast cancer, HER2-positive cancer, Kaposi's sarcoma, hairy cell leukemia, chronic myelogenous 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), colon cancer, esophageal cancer, gallbladder cancer, glioma, head and neck cancer, uterine cancer, cervical cancer, or testicular cancer. In some embodiments, the CM component comprises a substrate for a protease(s) that is more prevalent in tumor tissue. For example, the protease(s) may be produced by a tumor in the subject.

[0108] In some embodiments, an activatable TB may include a first CM (CM1) between the MM and the TB (e.g., AB) and a second CM (CM2) that can completely separate the MM from the TB by cleavage of the CM. In some examples, the first CM and the second CM may include a substrate for the same protease. In some examples, the first CM and the second CM may include a substrate for a different protease. 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.

[0109] The second CM (CM2) may be at a location within the activatable TB whose cleavage promotes dissociation of the MM from the TB. In some embodiments, the CM2 may be located between the MM and the non-alpha carbon covalent bond. In some embodiments, the second CM may be within the MM and up to 10 amino acids away from the non-alpha carbon covalent bond. In some embodiments, the second CM may be within the TB and up to 10 amino acids away from the non-alpha carbon covalent bond. The second CM may be located adjacent to the cysteine ​​that forms the non-alpha carbon covalent bond (e.g., 0 amino acids between the second CM and the cysteine). Alternatively, the second CM may be located up to 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid(s) away from the cysteine ​​that forms the non-alpha carbon covalent bond, e.g., a disulfide bond. The activatable target binding protein may have enhanced masking efficiency due to tethering between TB and MM to minimize toxicity and effects caused by target binding outside the activation environment, and may be sufficiently activated by a protease to provide the desired activity (e.g., therapeutic effect, target detection, etc.).

[0110] Suitable CMs for use in the activatable TBs herein include any of the protease substrates known in the art. In some examples, the CM may include a substrate for a serine protease (e.g., u-type plasminogen activator (uPA, also referred to as urokinase), matriptase (also referred to herein as MT-SP1 or MTSP1). In some examples, the CM may include a substrate for a matrix metalloprotease (MMP). In some examples, the CM may include a substrate for a cysteine ​​protease (CP) (e.g., legumain).

[0111] In some embodiments, the CM is a disintegrin and metalloprotease (ADAM) or a disintegrin and metalloprotease with thrombospondin motifs (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), caspases (e.g., caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 14), cysteine ​​cathepsins (e.g., 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, legumain, otubaiin, n)-2), chymase, DESC1, DPP-4, FAP, elastase, FVIIa, FiXA, FXa, FXIa, FXIIa, granzyme B, guanidinobenzoatase, hepsin, HtrA1, human neutrophil elastase, KLK (e.g., KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, KLK14), metalloproteases (e.g., meprin, neprilysin, PSMA, BMP-1), lactoferrin, marapsin, Substrates for matriptase-2, MT-SP1 / matriptase, 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.

[0112] In some embodiments, a protease substrate in a CM can include a polypeptide sequence that is substantially non-identical (e.g., no more than 90%, 80%, 70%, 60%, or 50% identical) to any polypeptide sequence that is naturally cleaved by the same protease.

[0113] In some embodiments, the CM may be or include the sequence of LSGRSDNH (SEQ ID NO: 214) or the sequence of PLGLAG (SEQ ID NO: 17). In some embodiments, the CM may be or include the sequence encompassed by 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 WO2015048329, the entirety of which is incorporated herein by reference, and SEQ ID NOs: 1-162, 268-306 disclosed in WO2015116933, the entirety of which is incorporated herein by reference.

[0114] In some embodiments, the CM is selected from the group consisting of 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, and 484 disclosed in WO2015048329, SEQ ID NOs: 163-267, 307-384, 402-445, and 665-683 disclosed in WO2015116933, and SEQ ID NOs: 163-267, 307-384, 402-445, and 665-683 disclosed in WO201611, which are incorporated by reference in their entirety. 8629, and any one of SEQ ID NOs: 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 disclosed in WO2020118109, the entirety of which is incorporated herein by reference. 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 include WO2010 / 081173, WO2021207669, WO2021207657, WO2021142029, WO2021061867, WO2020252349, WO2020252358, WO2020236679, WO2020176672, WO2020118109, WO2020092881, WO2020086665, WO2019213444, WO2019183218, WO2019173771, WO2019165 143, WO2019075405, WO2019046652, WO2019018828, WO2019014586, WO2018222949, WO2018165619, WO2018085555, WO2017011580, WO2016179335, WO2016179285, WO2016179257, WO2016149201, and WO2016014974, which are incorporated by reference in their entireties.

[0115] In some embodiments, the CM can be or include any one of the sequences in the tables below, or can be encompassed by a consensus sequence of any of the sequences in the tables below. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9]

[0116] In some embodiments, the CM may be or include a combination, C-terminal truncation variant, or N-terminal truncation variant of the above exemplary sequences. Truncation variants of the above amino acid sequences suitable for use in the CM may be any that retain the recognition site of the corresponding protease. These include C-terminal and / or N-terminal truncation variants that contain at least three consecutive amino acids of the above amino acid sequences, or at least 4, 5, 6, 7, 8, 9, or 10 amino acids of the aforementioned amino acid sequences that retain the recognition site of the protease. In certain embodiments, truncation variants of the above amino acid sequences may be amino acid sequences that correspond to any of the above but are truncated at the C-terminus and / or N-terminus by 1-10 amino acids, 1-9 amino acids, 1-8 amino acids, 1-7 amino acids, 1-6 amino acids, 1-5 amino acids, 1-4 amino acids, or 1-3 amino acids, which (1) have at least three amino acid residues; and (2) retain the recognition site of the protease. In some of the foregoing embodiments, the truncated CM is a CM truncated at the N-terminus. In some embodiments, the truncated CM is a CM truncated at the C-terminus. In some embodiments, the truncated C is a CM truncated at both the C-terminus and the N-terminus.

[0117] 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 3-25, 3-20, 3-15, 3-10, 3-5, 5-25, 5-20, 5-15, 5-10, 10-25, 10-20, 10-15, 15-25, 15-20, or 20-25 amino acids.

[0118] In some embodiments, the CM is digested by at least a protease at a concentration of about 0.001 to 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 x 10 4 M -1 S -1 This rate is determined by the substrate cleavage rate (k cat / K m ) can be measured.

[0119] Linker (L) An activatable TB (e.g., an activatable antibody or other therapeutic protein) may include one or more linkers (L). A linker may include an extension of amino acid sequence that connects two components in the activatable TB. The linker cannot be cleaved by any protease. In some embodiments, one or more linkers (e.g., flexible linkers) can be introduced into the activatable TB to provide flexibility at one or more of the junctions between domains, between moieties, between moieties and domains, or at any other junctions where a linker would be beneficial. In some embodiments, when the activatable TB is provided as a conformationally constrained construct, a flexible linker can be inserted to facilitate the formation and maintenance of structure in the uncleaved activatable TB. Any of the linkers described herein can provide the desired flexibility to facilitate inhibition of target binding or facilitate cleavage of the CM by a protease. In some embodiments, the linker included in the activatable TB is flexible in whole or in part, such that the linker can include, in addition to a flexible linker, one or more moieties that provide a less flexible structure to provide a desired activatable TB. Some linkers may contain cysteine ​​residues, which may form non-alpha carbon covalent bonds and reduce the flexibility of the construct.

[0120] In most cases, the length of a linker can be determined by counting the number of amino acids in the N-terminal to C-terminal direction, from the N-terminus of the linker adjacent to the C-terminal amino acid of the previous component to the C-terminus of the linker adjacent to the N-terminal amino acid of the next component (i.e., where the length of the linker does not include either the C-terminal amino acid of the previous component or the N-terminal amino acid of the next component).

[0121] In some embodiments, the linkers include a total of 1 to 50, 1 to 40, 1 to 30, 1 to 25 (e.g., 1 to 24, 1 to 22, 1 to 20, 1 to 18, 1 to 16, 1 to 15, 1 to 14, 1 to 12, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 25, 2 to 24, 2 to 22, 2 to 20, 2 to 18, 2 to 16, 2 to 15, 2 to 14, 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 8, 2 to 8, 2 to 8, 2 to 8, 2 to 8, 2 to 8, 2 to 8, 2 to 8, 2 to 8, 2 to 8, 2 to 8, 2 to 8, 2 to 8, 2 to 8, 2 to 8, 2 to 8, 2 to 8, 2 to 2 ... ~5, 2~4, 2~3, 4~25, 4~24, 4~22, 4~20, 4~18, 4~16, 4~15, 4~14, 4~12, 4~10, 4~8, 4~6, 4~5, 5~25, 5~24, 5~22, 5~20, 5~18, 5~16, 5~15, 5~14, 5~12, 5~10, 5~8, 5~6, 6~25, 6~24, 6~22, 6~20, 6~18, 6~16, 6~15, 6~14, 6~ 12, 6~10, 6~8, 8~25, 8~24, 8~22, 8~20, 8~18, 8~16, 8~15, 8~14, 8~12, 8~10, 10~25, 10~24, 10~22, 10~20, 10~18, 10~16, 10~15, 10~14, 10~12, 12~25, 12~24, 12~22, 12~20, 12~18, 12~16, 12~15, 12~14, 14~25, 14~ In some embodiments, the linker may comprise a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids.

[0122] In some embodiments, the linker can be rich in glycine (Gly or G) residues. In some embodiments, the linker can be rich in serine (Ser or S) residues. In some embodiments, the linker can be rich in glycine and serine residues. In some embodiments, the linker can have one or more glycine-serine residue pairs (GS) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GS pairs).

[0123] 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). Exemplary linkers may include glycine polymers (G)n, glycine-serine polymers (e.g., (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 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured and can therefore function as neutral links between components. Glycine has access to a much larger phi-psi space than alanine and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)).Exemplary flexible linkers include GGSG (SEQ ID NO: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), GGGGSGGGGSGGGGGS (SEQ ID NO:546), GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:547), GGGGSGGGGS (SEQ ID NO:548), GGGGS (SEQ ID NO:535), GS, GGGGSGS (SEQ ID NO:549), GGGGSGGGGSGGGGSGS (SEQ ID NO:550), G and one or more combinations of GSLDPKGGGGS (SEQ ID NO:551), PKSCDKTHTCPPCPAPELLG (SEQ ID NO:552), SKYGPPCPPCPAPEFLG (SEQ ID NO:553), GKSSGSGSESKS (SEQ ID NO:554), GSTGSSGKSSEGKG (SEQ ID NO:555), GSTGSSGKSSEGSGSTKG (SEQ ID NO:556), GSTGSSGKPGSGEGSTKG (SEQ ID NO:557), GSTGSSGKPGSSEGST (SEQ ID NO:558), GSTGSSGKPGSSEGST (SEQ ID NO:559), GGGSSGGS (SEQ ID NO:15), GGGGSGGGGSS (SEQ ID NO:560), and GGGSSGGSGGSSGGS (SEQ ID NO:561). Exemplary linkers may further include sequences that are at least 70% (e.g., at least 72%, at least 74%, at least 75%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the exemplary linkers described herein.Those skilled in the art will recognize that the design of an activatable TB can include linkers that are all or partially flexible, such that the linker can include one or more moieties that impart a less flexible structure, as well as flexible linkers, to provide the desired activatable TB structure.

[0124] In some embodiments, the activatable TB can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 linker sequence(s) (e.g., linker sequences the same or different from any of the exemplary linker sequences described herein or known in the art). In some embodiments, the linker can include sulfo-SIAB (sulfosuccinimidyl (4-iodoacetyl) aminobenzoate), SMPB (succinimidyl 4-(N-maleimidophenyl) butyrate), and sulfo-SMPB (sulfosuccinimidyl 4-(N-maleimidophenyl) butyrate), which reacts with sulfhydryls of primary amines.

[0125] Conjugation Agents In some embodiments, the activatable TB (e.g., activatable AB) can further comprise one or more additional agents, such as a targeting moiety that facilitates delivery to a cell or tissue of a subject, a therapeutic agent (e.g., an anti-tumor agent such as a chemotherapeutic agent or an anti-neoplastic agent), a toxin, or a fragment thereof. The additional agent can be conjugated to the activatable TB. The term "agent" is used herein to refer to a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract extracted from biological material.

[0126] 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 radioisotope.

[0127] Examples of cytotoxic agents that can be conjugated to activatable TBs include dolastatins and derivatives thereof (e.g., auristatin E, AFP, monomethylauristatin D (MMAD), monomethylauristatin F (MMAF), monomethylauristatin E (MMAE), desmethylauristatin E (DMAE), auristatin F, desmethylauristatin F (DMAF), dolastatin 16 (DmJ), dolastatin 16 (Dpv), auristatin derivatives (e.g., auristatin tyramine, auristatin quinolones), maytansinoids (e.g., DM-1, DM-4), maytansinoid derivatives, duocarmycins, alpha-amanitin, turbostatin, phenstatin, hydroxyphenstatin, sponges, and the like). statin 5, spongiostatin 7, halistatin 1, halistatin 2, halistatin 3, halocomstatin, pyrrolobenzimidazole (PBI), cibrostatin 6, doxaliform, cemadotin analog (CemCH2-SH), Pseudomonas toxin A (PES8) variant, Pseudomonas toxin A (ZZ-PE38) variant, ZJ-101, anthracyclines, doxorubicin, daunorubicin, bryostatin, camptothecin, 7-substituted campothecins, 10,11-difluoromethylenedioxycamptothecin, combretastatin, debromoaplysiatoxin, KahaMide-F, discodermolide, and ecteinascidin.

[0128] 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, dianfhin protein, Phytoiaca Americana protein (e.g., PAPI, PAPII, and PAP-8), momordica charantia inhibitor, curcin, crotirs, sapaonaria officinalis inhibitor, geionin, mitogeliin, restrictocin, phenomycin, neomycin, and trichothecin.

[0129] Examples of anti-tumor drugs that can be conjugated to activatable TB include adriamycin, cerbidine, bleomycin, alkeran, velban, oncovin, fluorouracil, methotrexate, thiotepa, bisantrene, novantrone, thioguanine, procarabidine, and cytarabine.

[0130] Examples of antiviral agents that can be conjugated to activatable TB include acyclovir, Bira A, and Symmetrel. Examples of antifungal agents that can be conjugated to activatable TB include nystatin. Examples of detection reagents 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. Examples of 3beta,16beta,17alpha-trihydroxycholest-5-en-22-one 16-O-(2-O-4-methoxybenzoyl-beta-D-xylopyranosyl)-(1-->3)-(2-O-acetyl-alpha-L-arabinopyranoside) (OSW-1) that can be conjugated to activatable TB include s-nitrobenzyloxycarbonyl derivatives of O6-benzylguanine, topoisomerase inhibitors, hemiasterin, cephalotaxine, homoharringionine, pyrrol obenzodiazepine dimers (PBDs), functionalized pyrrolobenzodiazepenes, calcicheamicin, podophyiitoxin, taxanes, and vinca alkoids. Examples of radiopharmaceuticals that can be conjugated to activatable TBs 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 99Examples 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 tylosin, spectinomycin, streptomycin B, ampicillin, sulfanilamide, polymyxin, and chloramphenicol.

[0131] In some embodiments, the activatable TB may include a signal peptide. The signal peptide may be a peptide (e.g., 10-30 amino acids in length) that is present at the end (e.g., N-terminus or C-terminus) of a newly synthesized protein that is directed toward the secretory pathway. In some embodiments, the signal peptide may be conjugated to the activatable TB via a spacer. In some embodiments, the spacer may be conjugated to the activatable TB in the absence of a signal peptide.

[0132] One of skill in the art will recognize that a wide variety of possible drugs can be conjugated to any of the activatable TBs described herein. Drugs may be conjugated to another component of the activatable TB by a conjugating moiety. Conjugation can include any chemical reaction that links two molecules together, so long as the activatable TB and the other moiety retain their respective activities. Conjugation can include many chemical reaction mechanisms, such as covalent bonding, affinity binding, intercalation, coordinate bonding, and complexation. In some embodiments, the bond can be covalent. Covalent bonding can be achieved either by direct condensation of existing side chains or by incorporation of an external bridging molecule. Many bivalent or multivalent linking agents can be useful in conjugating any of the activatable TBs described herein. For example, conjugates can contain organic compounds such as thioesters, carbodiimides, succinimide esters, glutaraldehyde, diazobenzene, and hexamethylenediamine. In some embodiments, the activatable TB can include or otherwise introduce one or more non-natural amino acid residues to provide suitable sites for conjugation.

[0133] In some embodiments, the agent and / or conjugate may be attached to the antigen-binding domain by a non-alpha carbon covalent bond (e.g., a disulfide bond on a cysteine ​​molecule). Because many cancers naturally release high levels of glutathione, a reducing agent, glutathione present in the cancer tissue microenvironment can reduce the non-alpha carbon covalent bond, followed by release of the agent and / or conjugate at the delivery site.

[0134] In some embodiments, when the conjugate binds to the target in the presence of complement within the target site (e.g., diseased tissue (e.g., cancer tissue)), the amide or ester bond attaching the conjugate and / or agent to the linker is cleaved, resulting in release of the conjugate and / or agent in an activated state. These conjugates and / or agents, when administered to a subject, can achieve delivery and release of the conjugate and / or agent at the target site (e.g., diseased tissue (e.g., cancer tissue)). These conjugates and / or agents can be effective for in vivo delivery of any of the conjugates and / or agents described herein.

[0135] In some embodiments, the conjugate moiety is not cleavable by the enzymes of the complement system. For example, the conjugate and / or agent is released without complement activation, which ultimately lyses the target cell. In such embodiments, the conjugate and / or agent is to be delivered to the target cell (e.g., a hormone, enzyme, corticosteroid, neurotransmitter, or gene). Additionally, the conjugate moiety may be susceptible to gentle cleavage by serum proteases, such that the conjugate and / or agent is slowly released at the target site.

[0136] In some embodiments of either, the conjugate and / or agent may be designed such that the conjugate and / or agent is delivered to a target site (e.g., diseased tissue (e.g., cancerous tissue)) but the conjugate and / or agent is not released.

[0137] In some embodiments, the conjugate and / or agent may be attached directly to the antigen binding domain or via an amino acid (e.g., D-amino acid), peptide, thiol-containing moiety, or other organic compound that can be modified by the methods described herein to contain a functional group that can then be utilized for attachment to the antigen binding domain.

[0138] In some embodiments, the activatable TB may include at least one conjugation point of an agent. In some embodiments, all available conjugation points are available for conjugation to an agent. In some embodiments, the one or more conjugation points may include sulfur atoms involved in non-alpha carbon covalent bonds, sulfur atoms involved in interchain non-alpha carbon covalent bonds, sulfur atoms involved in interchain sulfide bonds but not intrachain non-alpha carbon covalent bonds, and / or sulfur atoms of cysteine ​​or other amino acid residues that contain sulfur atoms. In such cases, the residue may be naturally occurring in the protein construct structure or may be incorporated into the protein construct using methods such as site-directed mutagenesis, chemical conversion, or misincorporation of non-natural amino acids.

[0139] The present disclosure also provides methods and materials for preparing activatable TB with one or more conjugated agents. In some embodiments, activatable TB may 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, reduction of disulfide bonds may only be partial. As used herein, the term partial reduction refers to a situation in which activatable TB is contacted with a reducing agent and a portion of all possible conjugation sites undergo reduction (e.g., not all disulfide bonds are reduced). In some embodiments, an activatable TB may be partially reduced if, after contact with a reducing agent, all available sites on the conjugate are reduced by less than 99% (e.g., less than 98%, less than 97%, less than 96%, less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, or less than 5%). In some embodiments, an activatable TB having reduction in one or more interchain disulfide bonds may be conjugated to an agent that is reactive with free thiols.

[0140] The present disclosure also provides methods and materials for conjugating therapeutic agents to specific positions on the activatable TB. In some embodiments, the activatable TB may be modified to allow for therapeutic agents to be conjugated to the activatable TB at specific positions on the activatable TB. For example, the activatable TB may be partially reduced in a manner that facilitates conjugation to the activatable TB. In such cases, partial reduction of the activatable TB may occur in a manner in which the conjugation site within the activatable TB is not reduced. In some embodiments, the conjugation site(s) on the activatable TB may be selected to facilitate conjugation of an agent at a specific position on the protein construct. Upon treatment with a reducing agent, various factors may affect the "reduction level" of the activatable TB. For example, to achieve partial reduction of the activatable TB using the methods and materials described herein, optimization of, but is not limited to, the ratio of reducing agent to activatable TB, the length of incubation, the incubation temperature, and / or the pH of the reduction reaction solution may be required. Any suitable combination of factors (e.g., the ratio of reducing agent to activatable TB, the length and temperature of incubation with the reducing agent, and / or the pH of the reducing agent) can be used to achieve partial reduction of the activatable TB (e.g., total reduction of available conjugation sites, or reduction of specific conjugation sites).

[0141] An 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 for conjugation to a drug (e.g., overall reduction of available conjugation sites, or reduction of specific conjugation sites). In some embodiments, the ratio of reducing agent to activatable TB may 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.

[0142] Effective incubation times and temperatures for treating activatable TB with a reducing agent can be any time and temperature that at least partially reduces the activatable TB (e.g., overall reduction of available conjugation sites, or reduction of specific conjugation sites) in a manner that allows for conjugation of an agent to the activatable TB. In some embodiments, incubation times and temperatures for treating activatable TB can range from about 1 hour at 37° C. to about 12 hours at 37° C. (or any subrange therein).

[0143] The effective pH of the reduction reaction for treating activatable TB with a reducing agent can be any pH that at least partially reduces the activatable TB (e.g., overall reduction of available conjugation sites, or reduction of specific conjugation sites) in a manner that allows for conjugation of the activatable TB to a drug.

[0144] When the partially reduced activatable TB is contacted with a thiol-containing drug, the drug can be conjugated to the interchain thiol in the activatable TB. The drug can be modified to contain a thiol using a thiol-containing reagent (e.g., cysteine ​​or N-acetylcysteine). 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. An 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 a thiol-containing drug (e.g., overall reduction of available conjugation sites, or reduction of specific conjugation sites).

[0145] 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.

[0146] In some embodiments, the agent (e.g., the agent conjugated to the activatable TB) may be a detectable moiety, such as, for example, a label or other marker. For example, the agent may be or may include a radiolabeled amino acid, one or more biotinyl moieties that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or calorimetric methods), one or more radioisotopes or radionuclides, one or more fluorescent labels, one or more enzymatic labels, and / or one or more chemiluminescent agents. In some embodiments, the detectable moiety may be attached by a spacer molecule. In some embodiments, the detectable label may include an imaging agent, a contrast agent, an enzyme, a fluorescent label, a chromophore, a dye, one or more metal ions, or a ligand-based label. In some embodiments, the imaging agent may include a radioisotope. In some embodiments, the radioisotope may be indium or technetium. In some embodiments, the contrast agent may include iodine, gadolinium, or iron oxide. In some embodiments, the enzyme may include horseradish peroxidase, alkaline phosphatase, or β-galactosidase. In some embodiments, the fluorescent label may include yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), green fluorescent protein (GFP), modified red fluorescent protein (mRFP), red fluorescent protein t dimer 2 (RFP t dimer 2), HCRED, or europium derivatives. In some embodiments, the luminescent label may include N-methylacridium derivatives. In some embodiments, the label may include an Alexa Fluor® label, such as Alex Fluor® 680 or Alexa Fluor® 750. In some embodiments, the ligand-based label may include biotin, avidin, streptavidin, or one or more haptens.

[0147] In some embodiments, an agent may be conjugated to an activatable TB using a carbohydrate moiety, a sulfhydryl group, an amino group, or a carboxylate group. In some embodiments, an agent may be conjugated to an activatable TB via a linker and / or CM as described herein. In some embodiments, an agent may be conjugated to a cysteine ​​or lysine in the activatable TB. In some embodiments, an agent may be conjugated to another residue of the activatable TB, such as those disclosed herein.

[0148] In some embodiments, a variety of bifunctional protein coupling agents can be used to conjugate agents to activatable TBs, including N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyladipimidate HCL), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)ethylenediamine), diisocyanates (e.g., 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, radionucleotides can be conjugated to activatable TBs using carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) chelating agents. (See, e.g., WO94 / 11026).

[0149] Suitable conjugation 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 acetylhydrazide derivatives coupled to activatable antibodies via oligopeptides. In some embodiments, suitable conjugation moieties include (i) EDC (1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride, (ii) SMPT (4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)-toluene (Pierce Chem. Co., catalog (21558G)), (iii) SPDP (succinimidyl-6[3-(2-pyridyldithio)propionamido]hexanoate (Pierce (iv) sulfo-LC-SPDP (sulfosuccinimidyl 6[3-(2-pyridyldithio)-propianamido]hexanoate (Pierce Chem. Co. catalog number 2165-G), and (v) sulfo-NHS (N-hydroxysulfo-succinimide: Pierce Chem. Co. catalog number 24510) conjugated with EDC. Additional exemplary conjugation moieties include SMCC, sulfo-SMCC (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate), SPDB, and sulfo-SPDB.

[0150] The conjugation moieties may contain components with different attributes, thus resulting in conjugates with different physicochemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates. NHS ester-containing linkers are less soluble than sulfo-NHS esters. In addition, SMPT contains a sterically hindered disulfide bond, which can form highly stable conjugates. Disulfide bonds are generally less stable than other bonds, as disulfide bonds are cleaved in vitro, resulting in less usable conjugates. Sulfo-NHS can particularly increase the stability of carbodiimide coupling. When carbodiimide coupling (e.g., EDC) is used in conjunction with sulfo-NHS, it forms esters that are more resistant to hydrolysis than carbodiimide coupling reactions alone.

[0151] Those of skill in the art will appreciate that a wide variety of possible moieties can be linked to the activatable TBs 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 a drug (e.g., a cytotoxic agent) to an activatable TB can be achieved by any chemical reaction that conjugates the drug to the activatable TB while also allowing the drug and activatable TB to retain functionality.

[0152] Nucleic Acids and Vectors In some aspects, the disclosure further 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 coding sequences for the TB, CM, MM, and linker in the activatable TB. When the activatable TB comprises multiple peptides, for example, when the activatable TB comprises multiple peptides, the nucleic acid may comprise coding sequences for multiple peptides. In some examples, the coding sequence for one of the peptides is comprised in a nucleic acid and the coding sequence for another one of the peptides is comprised in another nucleic acid. In some examples, the coding sequences for two or more of the multiple peptides are comprised in the same nucleic acid.

[0153] Unless otherwise specified, a "nucleic acid sequence encoding a protein" includes all nucleotide sequences that are degenerate versions of each other and therefore code for the same amino acid sequence. The term "nucleic acid" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) or combinations thereof, in single-stranded or double-stranded form. Unless otherwise limited, the term encompasses nucleic acids that contain known analogs of natural nucleotides that have similar binding properties as the referenced nucleotides. Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses complementary sequences in addition to the sequence explicitly indicated. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is RNA.

[0154] The term "located at the N-terminus," when referring to the position of a first domain or sequence in the primary amino acid sequence of a polypeptide relative to a second domain or sequence, means that the first domain is located near the N-terminus of the primary amino acid sequence of the polypeptide. In some embodiments, there may be additional sequences and / or domains between the first domain or sequence and the second domain or sequence.

[0155] The term "located at the C-terminus," when referring to the position of a first domain or sequence in the primary amino acid sequence of a polypeptide relative to a second domain or sequence, means that the first domain is located near the C-terminus of the primary amino acid sequence of the polypeptide. In some embodiments, there may be additional sequences and / or domains between the first domain or sequence and the second domain or sequence.

[0156] Modifications to the nucleotide sequence can be introduced by standard techniques known in the art, such as site-directed mutagenesis and polymerase chain reaction (PCR)-mediated mutagenesis. Conservative amino acid substitutions are those in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with acidic side chains (e.g., 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 families of amino acids include the aliphatic hydroxyamino acids (e.g., serine and threonine), the amide family (e.g., asparagine and glutamine), the aliphatic family (e.g., alanine, valine, leucine and isoleucine), and the aromatic family (e.g., phenylalanine, tryptophan and tyrosine).

[0157] The present disclosure further provides vectors and sets of vectors comprising any of the nucleic acids described herein. One of skill in the art would be able to select a suitable vector or set of vectors (e.g., expression vectors) to generate any of the activatable TBs described herein, and to use the vector or set of vectors to express any of the activatable TBs described herein. For example, when selecting a vector or set of vectors, a cell type may be selected that requires that the vector(s) be integrated into the chromosome of the cell and / or be able to replicate therein. Examples of vectors that can be used to generate activatable TBs are also described herein. As used herein, the term "vector" refers to a polynucleotide that can induce expression of a recombinant protein (e.g., a first or second monomer) in a cell (e.g., any of the cells described herein). A "vector" is capable of delivering nucleic acids and fragments thereof into a host cell and contains control sequences (e.g., promoters, enhancers, poly(A) signals). An exogenous polynucleotide may be inserted into an expression vector for expression. The term "vector" also encompasses artificial chromosomes, plasmids, retroviruses, and baculovirus vectors.

[0158] Methods for making vectors containing any of the nucleic acids described herein and suitable for transforming cells (e.g., mammalian cells) are well known in the art. See, for example, Sambrook et al., Eds., "Molecular Cloning: A Laboratory Manual," 2 nd Ed., Cold Spring Harbor Press, 1989 and Ausubel et al., Eds. "Current Protocols in Molecular Biology," Current Protocols, 1993.

[0159] Examples of vectors include plasmids, transposons, cosmids, and viral vectors (e.g., any of the adenoviral vectors (e.g., pSV or pCMV vectors), adeno-associated viral (AAV) vectors, lentiviral vectors, and retroviral vectors), as well as any of the Gateway® vectors. A vector can, for example, contain sufficient cis-acting elements for expression, and other elements for expression can be supplied by the host mammalian cell or in an in vitro expression system. One of skill in the art would be able to select suitable vectors and mammalian cells for making any of the activatable TBs described herein.

[0160] In some embodiments, activatable TB may be made biosynthetically using recombinant DNA technology and expression in eukaryotic or prokaryotic species.

[0161] cell In some aspects, the disclosure provides a recombinant host cell comprising any of the vectors or nucleic acids described herein. The cell can be used to produce an activatable TB (e.g., an activatable antibody) 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-bound nucleus. Such cells can include, for example, mammalian (e.g., rodent, non-human primate, or human), insect, fungal, or plant cells. In some embodiments, the eukaryotic cell is a yeast cell, such as Saccharomyces cerevisiae. In some embodiments, the eukaryotic cell is a higher eukaryotic cell, such as a mammalian, avian, plant, or insect cell. Non-limiting examples of mammalian cells include Chinese hamster ovary (CHO) cells and human embryonic kidney cells (e.g., HEK293 cells). In some embodiments, the cell may be a prokaryotic cell.

[0162] Methods for introducing nucleic acids and vectors (e.g., any of the vectors or sets of vectors described herein) into cells are known in the art. Examples of methods that can be used to introduce nucleic acids into cells include lipofection, transfection, calcium phosphate transfection, cationic polymer transfection, viral transfection (e.g., adenoviral transduction, lentiviral transduction), nanoparticle transfection, and electroporation.

[0163] In some embodiments, the introduction step includes introducing into the cell a vector (e.g., any of the vectors or sets of vectors described herein) containing a nucleic acid encoding any of the activatable TB constituent monomers described herein.

[0164] Compositions and kits The present disclosure also provides compositions and kits that include the activatable TB described herein, and may further include one or more excipients, carriers, reagents, and instructions required for use of the activatable TB.

[0165] In some embodiments, the composition may be a pharmaceutical composition comprising an activatable TB, an antibody, derivatives, fragments, analogs and homologs thereof. The pharmaceutical composition may 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 and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Suitable examples of such carriers or diluents include water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles such as liposomes and fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Use of any conventional media or agent in the composition is contemplated except insofar as it is incompatible with the active compound. Supplementary active compounds may also be incorporated into the composition.

[0166] Pharmaceutical compositions can be formulated to be compatible with the intended route of administration. Examples of routes of administration include parenteral (e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (e.g., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous administration can include one or more of the following components: a sterile diluent (e.g., water for injection, saline, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents), an antibacterial agent (such as benzyl alcohol or methylparaben), an antioxidant (such as ascorbic acid or sodium bisulfite), a chelating agent (such as ethylenediaminetetraacetic acid (EDTA)), a buffer (such as acetates, citrates, or phosphates), and an agent for adjusting the tonicity (such as sodium chloride or dextrose). pH The concentration can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be placed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic. In some cases, any of the activatable TBs described herein are prepared with carriers that protect against rapid elimination from the body, such as slow and controlled release formulations, such as 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-glycolic acid copolymers. Methods for preparing such pharmaceutical compositions and formulations are apparent to those skilled in the art.

[0167] In some embodiments, pharmaceutical compositions suitable for injection include sterile aqueous solutions (if water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate buffered saline (PBS). The composition may be sterile, and must be liquid and of a viscosity that is easy to inject. It may be stable under the conditions of manufacture and storage, and preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier may be, for example, a solvent or dispersion medium containing water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. For dispersed particle compositions, proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, on the particles, and by maintaining the required particle size in the case of dispersion, and by the use of surfactants. In some embodiments, the pharmaceutical composition may further include one or more antibacterial and / or antibacterial antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In some embodiments, isotonic agents, such as sugars, polyalcohols such as mannitol, sorbitol, and salts such as sodium chloride, may be included in the composition. Prolonged absorption of the injectable composition can be achieved by including in the composition an agent that delays absorption, such as aluminum monostearate and gelatin.

[0168] In some embodiments, pharmaceutical compositions may contain sterile injectable solutions.Sterile injectable solutions can be prepared by incorporating active compound in the required amount in suitable solvent with one or a combination of the above-listed components, if necessary, and then sterilizing by filtration.Generally, dispersions can be prepared by incorporating active compound in a sterile vehicle that contains a basic dispersion medium and other components required from the above-listed components.In the case of sterile powders for preparing sterile injectable solutions, the preparation method is vacuum drying and freeze-drying, which produces a powder of active ingredient and any additional desired ingredients from the solution that has been previously sterilized and filtered.

[0169] In some embodiments, the pharmaceutical composition may include an oral composition. The oral composition may include an inert diluent or an edible carrier. They may be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound may be mixed with an excipient and used in the form of tablets, troches or capsules. Oral compositions may also be prepared using a liquid carrier for use as a mouthwash, where the compound in the liquid carrier is taken orally and expectorated or swallowed. Pharmaceutically compatible binding agents and / or adjuvant materials may be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients: binders (such as microcrystalline cellulose, gum tragacanth or gelatin), excipients (such as starch or lactose), disintegrants (such as alginic acid, Primogel or corn starch), lubricants (such as magnesium stearate or Sterotes), flow agents (such as colloidal silicon dioxide), sweeteners (such as sucrose or saccharin), or flavoring agents (such as peppermint, methyl salicylate or orange flavoring), or compounds of a similar nature.

[0170] In some embodiments, 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 which contains a suitable propellant, such as a gas such as carbon dioxide, or a nebulizer.

[0171] In some embodiments, the pharmaceutical composition may be formulated for systemic administration. For example, systemic administration may be intravenous, transmucosal or transdermal. For transmucosal or transdermal administration, a penetrant suitable for the barrier to be permeated may be used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts and fusidic acid derivatives. Transmucosal administration may be carried out through the use of nasal sprays or suppositories. For transdermal administration, the active compound may be formulated into ointments, salves, gels or creams, as generally known in the art.

[0172] In some embodiments, pharmaceutical compositions may be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0173] In one embodiment, pharmaceutical compositions may be prepared with carriers that protect the compound from rapid elimination from the body, such as controlled release formulations, including implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers may be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid, and polylactic-glycolic acid copolymers.Methods for preparing such formulations will be clear to those skilled in the art.

[0174] For ease of administration and uniformity of dosage, it may be advantageous to formulate oral or parenteral compositions in dosage unit form.Dosage unit form as used herein refers to a physically separate unit suitable as a unitary dosage of the subject to be treated.Each unit contains a predetermined amount of active compound calculated to produce a desired therapeutic effect in association with the required pharmaceutical carrier.The specification of dosage unit form of the present disclosure is determined by and can directly depend on the unique characteristics of active compound and the specific therapeutic effect to be achieved, as well as the inherent limitations of the technical field of compounding such active compound for the treatment of individuals.

[0175] In some embodiments, the compositions (eg, pharmaceutical compositions) may be included in a container, vial, syringe, injection pen, pack, or dispenser, optionally with instructions for administration.

[0176] Also provided herein is a kit containing any of the activatable TBs described herein, any of the compositions containing any of the activatable TBs described herein, or any of the pharmaceutical compositions containing any of the activatable TBs described herein. Also provided is a kit containing one or more second therapeutic agent(s) in addition to the activatable TBs described herein. The second therapeutic agent(s) may be provided in a dosage form separate from the activatable TB. Alternatively, the second therapeutic agent(s) may be prepared together with the activatable TB.

[0177] Any of the kits described herein can include any of the compositions (e.g., pharmaceutical compositions) and / or instructions for using any of the activatable TBs described herein. In some embodiments, the kits can include instructions for practicing any of the methods described herein. In some embodiments, the kits can include at least one dose of any of the compositions (e.g., pharmaceutical compositions) described herein. In some embodiments, the kits can provide a syringe for administering any of the pharmaceutical compositions described herein.

[0178] Also provided herein is an activatable TB made by any of the methods described herein. Also provided is a composition (e.g., pharmaceutical composition) containing any of the activatable TB made by any of the methods described herein. Also provided herein is a kit containing at least one dose of any of the compositions (e.g., pharmaceutical compositions) described herein.

[0179] Methods for Producing Activatable Target-Binding Proteins - Patent application Provided herein is a method of producing any of the activatable TB described herein, the method comprising: (a) culturing any of the recombinant host cells described herein in a liquid medium under conditions sufficient to produce activatable TB; and (b) recovering the activatable TB from the host cells and / or the liquid medium.

[0180] Methods for culturing cells are well known in the art. In some embodiments, cells can be maintained in vitro under conditions that favor cell proliferation, cell differentiation, and cell growth. For example, recombinant cells can be cultured by contacting cells (e.g., any of the cells described herein) with a cell culture medium containing the necessary growth factors and supplements sufficient to support cell viability and growth.

[0181] In some embodiments, the method may further comprise isolating the recovered activatable TB. Isolation of activatable TB can be performed using any separation or purification technique for separating protein species, such as affinity tag-based protein purification (e.g., polyhistidine (His) tag, glutathione-S-transferase tag, etc.), ammonium sulfate precipitation, polyethylene glycol precipitation, size exclusion chromatography, ligand affinity chromatography (e.g., Protein A chromatography), ion exchange chromatography (e.g., anion or cation), hydrophobic interaction chromatography, etc.

[0182] The compositions and methods described herein may involve the use of non-reducing or partially reducing conditions that allow the formation of a non-alpha carbon covalent bond, such as a disulfide bond, between the MM and AB of an activatable TB.

[0183] In some embodiments, a dual anchored activatable macromolecule (e.g., a dual anchored activatable TB or dual anchored activatable antibody) of the disclosure is prepared by a method comprising engineering a cysteine ​​residue at a disulfide bond site in the MM of the dual anchored activatable macromolecule and engineering a cysteine ​​residue at a disulfide bond site in the TB of the dual anchored activatable macromolecule, wherein the MM and TB are linked and the CM is located between the MM and TB. In some aspects, the disclosure comprises expressing an activatable macromolecule having an engineered cysteine ​​residue at a disulfide bond site in the TB of the dual anchored activatable macromolecule and recovering the dual anchored activatable macromolecule, wherein the MM and TB are tethered at the disulfide bond site in the recovered dual anchored activatable macromolecule.

[0184] In some embodiments, a dual anchored activatable macromolecule (e.g., a dual anchored activatable TB or a dual anchored activatable antibody) of the present disclosure is prepared by a method comprising attaching a cysteine-containing MM to a TB, where the cysteine ​​forms a non-alpha carbon covalent bond with a non-alpha carbon covalent bond-forming amino acid in the TB.

[0185] In some embodiments, a 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 binding a TB to a MM comprising a non-alpha carbon covalent bond forming amino acid, where the MM forms a non-alpha carbon covalent bond with the non-alpha carbon covalent bond forming amino acid in the TB.

[0186] In some embodiments, the disclosure includes a method of making a dual anchored activatable polymer comprising providing a MM comprising a non-alpha carbon covalent bond forming amino acid configured to form a non-alpha carbon covalent bond with a non-alpha carbon covalent bond forming amino acid in the TB bound to the MM. In some embodiments, the disclosure includes a method of making a dual anchored activatable polymer comprising providing a MM comprising a cysteine ​​configured to form a non-alpha carbon covalent bond with a non-alpha carbon covalent bond forming amino acid in the TB bound to the MM.

[0187] In some embodiments, the disclosure includes a method of identifying a position for inserting a non-alpha carbon covalent bond forming amino acid in a dual anchored activatable polymer. In some embodiments, the method includes analyzing the structural arrangement of the TB and MM in a dual anchored activatable polymer and identifying a position in the TB for inserting a non-alpha carbon covalent bond forming amino acid. In some embodiments, the method includes analyzing the structural arrangement of the TB and MM in a dual anchored activatable polymer and identifying a position in the MM for inserting a non-alpha carbon covalent bond forming amino acid. In some embodiments, the method includes analyzing the structural arrangement of the TB and MM in a dual anchored activatable polymer and identifying a position in a peptide bound to the TB for inserting a non-alpha carbon covalent bond forming amino acid. In some embodiments, the method includes analyzing the structural arrangement of the TB and MM in a dual anchored activatable polymer and identifying a position in a peptide bound to the MM for inserting a non-alpha carbon covalent bond forming amino acid. In some embodiments, one or more of the non-alpha carbon covalent bond forming amino acids is cysteine.

[0188] In some embodiments, the disclosure includes a method of identifying a location for inserting a non-alpha carbon covalent bond forming amino acid in a dual anchored activatable polymer, the method including mapping a three-dimensional structure of TB bound to MM and identifying a first amino acid of TB located proximate to a second amino acid of MM, the first amino acid being a location for inserting a non-alpha carbon covalent bond forming amino acid in TB. In some embodiments, the second amino acid is a location for inserting a non-alpha carbon covalent bond forming amino acid in MM. In some embodiments, the alpha carbon atom of the first amino acid is located within 2-15 angstroms of the alpha carbon atom of the second amino acid in the three-dimensional structure of TB bound to MM. In some embodiments, the alpha carbon atom of the first amino acid is located within 15, 10, 9, 8, 7, 6, 5, 4, 3, or 2 angstroms of the alpha carbon atom of the second amino acid in the three-dimensional structure of TB bound to MM. If one or both of the amino acids are in a flexible loop, the above distance between the Cαs applies when the two amino acids are closest to each other. In some embodiments, non-alpha carbon covalent bonds include, but are not limited to, the following: 1. A disulfide bond between two Cys residues. 2. Isopeptide bond between lys and glutamic acid or glutamine residue. 3. An ester bond between Thr and glutamine. 4. Thioester bond between Cys and Gln. 5. Thioether bond between Cys and Tyr. 6. His-Tyr is a bridge between His and Tyr (known to exist, for example, in cytochrome c oxidase-type proteins). 7. Lys-Cys NOS (nitrogen-oxygen-sulfur) bridge.

[0189] In some embodiments, the disclosure includes a method for producing a dual-anchored activatable polymer comprising attaching a cysteine-containing MM to a TB, where the cysteine ​​forms a non-alpha carbon covalent bond with a non-alpha carbon covalent bond-forming amino acid in the TB.

[0190] In some embodiments, the disclosure includes a method for making a dual-anchored activatable polymer comprising attaching a TB to a MM comprising a non-alpha carbon covalent bond forming amino acid, where the MM forms a non-alpha carbon covalent bond with the non-alpha carbon covalent bond forming amino acid in the TB.

[0191] In some embodiments, the method further comprises formulating the isolated activatable TB into a pharmaceutical composition. Various formulations are known in the art and described herein. The isolated activatable TB described herein can be formulated for any route of administration (e.g., intravenous, intratumoral, subcutaneous, intradermal, oral, inhalation, intranasal, intrapulmonary, intrathecal, injection, transdermal, topical, transmucosal, or intramuscular).

[0192] Treatment method In some aspects, the disclosure further provides a method of treating a disease in a subject (e.g., a cancer (e.g., any of the cancers described herein), an inflammatory condition, disorder or disease, or an autoimmune condition, disorder or disease), the method comprising administering to the subject a therapeutically effective amount of any of the activatable TBs described herein. In some embodiments, the disclosure provides a method of preventing, slowing the progression of, treating, alleviating a symptom, or otherwise ameliorating a disease in a subject by administering to a subject in need thereof a therapeutically effective amount of an activatable TB described herein. The term "treatment" means improving at least one symptom of the disease. In some embodiments, the disease treatment is for cancer and is to alleviate at least one symptom of the cancer. As used herein, the term "subject" means any mammal. In some embodiments, the subject is a feline (e.g., a cat), a canine (e.g., a dog), an equine (e.g., a horse), a rabbit, a pig, a rodent (e.g., a mouse, a rat, a hamster, or a guinea pig), a non-human primate (e.g., an anthropoid (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. 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)).

[0193] The activatable TB used in any of these method and use embodiments can be administered at any stage of disease. For example, such activatable TB can be administered to patients suffering from any stage of cancer, from early to metastatic. In some embodiments, activatable TB and formulations thereof can be administered to subjects suffering from or susceptible to a disease or disorder associated with aberrant target expression and / or activity.

[0194] Subjects suffering from or susceptible to diseases or disorders associated with abnormal target expression and / or activity can be identified using any of a variety of methods known in the art. For example, subjects suffering from inflammatory conditions, disorders or diseases, or autoimmune conditions, disorders or diseases, cancer or other neoplastic conditions may be identified using any of a variety of clinical and / or laboratory tests to assess health status, such as physical examination and blood, urine, and / or stool analysis. For example, subjects suffering from inflammation and / or inflammatory disorders can be identified using any of a variety of clinical and / or laboratory tests to assess health status, such as physical examination and / or body fluid analysis (e.g., blood, urine, and / or stool analysis).

[0195] In some embodiments, administration of activatable TB to a patient suffering from a disease or disorder associated with aberrant target expression and / or activity may be considered successful if any of a variety of laboratory or clinical objectives are achieved. For example, administration of activatable TB to a patient suffering from a disease or disorder associated with aberrant target expression and / or activity may be considered successful if one or more of the symptoms associated with the disease or disorder are alleviated, reduced, suppressed, or do not progress to a more severe or worse state. Administration of activatable TB to a patient suffering from a disease or disorder associated with aberrant target expression and / or activity may be considered successful if the disease or disorder goes into remission or does not progress to a more severe or worse state.

[0196] 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 of the cancers described herein)) in a subject (e.g., any of the subjects described herein). In some embodiments where the disease is cancer, treating results in a reduction in cancer growth, inhibition of cancer progression, inhibition of cancer metastasis, or a reduction in the risk of cancer recurrence in a subject with cancer.

[0197] In some embodiments, the disease may be cancer. In some embodiments, the subject may have been identified or diagnosed with cancer. Examples of cancer include solid tumors, hematological tumors, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing's sarcoma, osteosarcoma, B cell neoplasms, multiple myeloma, lymphomas (e.g., B cell lymphoma, B cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, cutaneous T cell lymphoma), leukemias (e.g., hairy cell leukemia, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloma, myeloma, leukemia ... The cancers include myeloma, myelodysplastic syndrome (MDS), Kaposi's sarcoma, retinoblastoma, gastric cancer, urothelial cancer, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, brain cancer, colon cancer, bone cancer, lung cancer, breast cancer, colorectal cancer, ovarian cancer, 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 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-ovarian cancer (BRCA1 or BRAC2 mutation) syndrome, and the like. The disclosed methods are also useful for treating non-solid cancers. Exemplary solid tumors include malignant tumors (e.g., sarcomas, adenocarcinomas, and carcinomas) of various organ systems, such as the lung, breast, lymphatic system, gastrointestinal tract (e.g., colon), genitourinary tract (e.g., renal, urothelial, or testicular tumors), pharynx, prostate, and ovary. Exemplary adenocarcinomas include colorectal carcinoma, renal cell carcinoma, liver cancer, non-small cell carcinoma of the lung, and cancer of the small intestine. Further examples of cancers that may be treated by the compositions and methods herein include acute lymphocytic leukemia, adult; acute lymphocytic leukemia, pediatric; acute myeloid leukemia, adult; adrenal cortical carcinoma; adrenal cortical carcinoma, pediatric; AIDS-related lymphoma; AIDS-related malignancies; anal cancer; astrocytoma, pediatric cerebellum; astrocytoma, pediatric cerebral; cholangiocarcinoma, extrahepatic; bladder cancer; bladder cancer, pediatric; bone cancer, osteosarcoma / malignant fibrous histiocytoma; brain stem glioma, pediatric; brain tumor, adult; brain tumor, brain stem glioma, pediatric; brain tumor, cerebellar astrocytoma, pediatric; brain tumor, cerebral astrocytoma / malignant glioma, pediatric; brain tumor, ependymoma, pediatric; brain tumor, medulloblastoma, pediatric;Brain tumors, supratentorial primitive neuroectodermal tumor, children;Brain tumors, visual pathway and hypothalamic glioma, children;Brain tumors, children (other);Breast cancer;Breast cancer and pregnancy;Breast cancer, children;Breast cancer, male;Bronchial adenoma / carcinoid, children;Carcinoid tumors, children;Carcinoid tumors, gastrointestinal;Cytoma, adrenal cortical;Cytoma, islet cell;Carcinoma of unknown primary;Central nervous system lymphoma, primary;Cerebellar astrocytoma, children;Cerebral astrocytoma / malignant glioma, children;Cervical cancer;Childhood cancer;Chronic lymphocytic leukemia;Chronic myeloid leukemia;Chronic myeloproliferative disorder;Clear cell sarcoma of tendon sheath;Colon cancer;Colorectal cancer, children;Cutaneous T-cell lymphoma; Endometrial cancer;Ependymoma, pediatric;Epithelial carcinoma, ovarian;Esophageal cancer;Esophageal cancer, pediatric;Ewing's family of tumors;Extracranial germ cell tumors, pediatric;Extragonadal germ cell tumors;Extrahepatic bile duct cancer;Eye cancer, intraocular melanoma;Eye cancer, retinoblastoma;Gallbladder cancer;Gastric (Stomach) cancer;Gastric (stomach) cancer, pediatric;Gastrointestinal carcinoid tumors;Germ cell tumors, extracranial, pediatric;Germ cell tumors, extragonadal;Germ cell tumors, ovarian;Gestational trophoblastic tumors;Glioma, pediatric brain stem;Glioma, pediatric visual pathway and hypothalamus;Hairy cell leukemia;Head and neck cancer;Hepatocellular (liver) cancer, adult (primary);Hepatocellular (liver) cancer Cancer, childhood (primary);Hodgkin's lymphoma, adult;Hodgkin's lymphoma, childhood;Hodgkin's lymphoma in pregnancy;Hypopharyngeal cancer;Hypothalamic and visual pathway glioma, childhood;Intraocular melanoma;Islet cell carcinoma (islet);Kaposi's sarcoma;Kidney cancer;Laryngeal cancer;Laryngeal cancer, childhood;Leukemia, acute lymphocytic leukemia, adult;Leukemia, acute lymphocytic leukemia, childhood;Leukemia, acute myeloid leukemia, adult;Leukemia, acute myeloid leukemia, childhood;Leukemia, chronic lymphocytic;Leukemia, chronic myeloid;Leukemia, hairy cell;Lip and oral cavity cancer;Liver cancer, adult (primary);Liver cancer, childhood (primary);Lung cancer, non-small cell;Lung cancer, small cell; Lymphocytic leukemia, adult acute;Lymphoblastic leukemia, childhood acute;Lymphocytic leukemia, chronic;Lymphoma, AIDS-related;Lymphoma, central nervous system (primary);Lymphoma, cutaneous T-cell;Lymphoma, Hodgkin's lymphoma, adult;Lymphoma, Hodgkin's lymphoma, childhood;Lymphoma during pregnancy, Hodgkin's lymphoma;Lymphoma, non-Hodgkin's lymphoma, adult;Lymphoma, non-Hodgkin's lymphoma, childhood;Lymphoma during pregnancy, non-Hodgkin's lymphoma;Lymphoma, primary central nervous system;Macroglobulinemia, Waldenström's disease, male breast cancer;Malignant mesothelioma, adult;Malignant mesothelioma, childhood;Malignant thymoma;Medulloblastoma, childhood;Malignant melanoma;Malignant melanoma, intraocular;Merkel cell carcinoma;Mesothelioma, malignant;Metastatic squamous cell carcinoma of the neck of unknown primary;Multiple endocrine neoplasia syndrome, childhood;Multiple myeloma / plasma cell neoplasm;Mycosis fungoides;Myelodysplastic syndrome;Myeloid leukemia, chronic;Myeloid leukemia, childhood acute;Myeloma, multiple;Myeloproliferative disorders, chronic;Nasal cavity and paranasal sinus cancer;Nasopharyngeal carcinoma;Nasopharyngeal carcinoma, childhood;Neuroblastoma;Non-Hodgkin's lymphoma, adult;Non-Hodgkin's lymphoma, childhood;Non-Hodgkin's lymphoma in pregnancy;Non-small cell lung cancer;Oral cancer, childhood;Oral and lip cancer ;Oropharyngeal cancer;Osteosarcoma / malignant fibrous histiocytoma of bone;Ovarian cancer, children;Ovarian epithelial cancer;Ovarian germ cell tumors;Ovarian low malignant potential tumors;Pancreatic cancer;Pancreatic cancer, children;Pancreatic cancer, islet cell;Sino-nasal and nasal cancer;Parathyroid cancer;Penile cancer;Pheochromocytoma;Pineal and supratentorial primitive neuroectodermal tumors, children;Pituitary tumors;Plasma cell neoplasms / multiple myeloma;Pleuropulmonary blastoma;Pregnancy and breast cancer;Pregnancy and Hodgkin's lymphoma;Pregnancy and non-Hodgkin's lymphoma;Primary central nervous system lymphoma;Primary liver cancer, adults;Primary liver cancer, children;Prostate cancer;Rectal cancer;Renal cell (kidney) cancer;Kidney Cell carcinoma, children;Renal pelvis and ureter, transitional cell carcinoma;Retinoblastoma;Rhabdomyosarcoma, children;Salivary gland carcinoma;Salivary gland carcinoma, children;Sarcoma, Ewing's family of tumors;Sarcoma, Kaposi's sarcoma;Sarcoma (osteosarcoma) / malignant fibrous histiocytoma of bone;Sarcoma, rhabdomyosarcoma, children;Sarcoma, soft tissue, adults;Sarcoma, soft tissue, children;Sezary syndrome;Skin cancer;Skin cancer, children;Skin cancer (melanoma);Skin cancer, Merkel cell;Small cell lung cancer;Small intestine cancer;Soft tissue sarcoma, adults;Soft tissue sarcoma, children;Cervical squamous cell carcinoma of unknown primary, metastatic;Gastric (stomach) cancer;Gastric (stomach) cancer, children ;supratentorial primitive neuroectodermal tumor, pediatric;T-cell lymphoma, skin;testicular cancer;thymoma, pediatric;thymoma, malignant;thyroid cancer;thyroid cancer, pediatric;transitional cell carcinoma of the renal pelvis and ureter;trophoblastic tumor, gestational;unknown primary site, pediatric cancer;rare cancer of childhood;ureter and renal pelvis, transitional cell carcinoma;urethral cancer;uterine sarcoma;vaginal cancer;visual pathway and hypothalamic glioma, pediatric;vulvar cancer;Waldenstrom's macroglobulinemia;Wilm's tumor;diffuse large B-cell lymphoma (DLBCL);and mantle cell lymphoma (MCL).Metastasis of the aforementioned cancers can also be treated or prevented according to the methods described herein.;

[0198] In some embodiments, the methods herein can result in a reduction in the number, severity, or frequency of one or more symptoms of cancer in a subject (e.g., compared to the number, severity, or frequency of one or more symptoms of cancer in the subject before treatment).

[0199] The method may further include administering one or more additional agents to the subject. In some embodiments, the activatable TB may be administered in combination with one or more additional agents during and / or after treatment. In some embodiments, the activatable TB may be formulated into a single therapeutic composition, and the activatable TB and the additional agent(s) may be administered simultaneously. Alternatively, the activatable TB and the additional agent(s) may be separate from each other, e.g., each formulated into a separate therapeutic composition, and the activatable TB and the additional agent(s) are administered simultaneously, or the activatable TB and the additional agent(s) are administered at different times during the treatment regimen. For example, the activatable TB may be administered before the administration of the additional agent, after the administration of the additional agent, or alternately. The activatable TB and the additional agent(s) may be administered in a single dose or multiple doses.

[0200] The disclosure also provides a method for detecting the presence or absence of a cleavage agent and a target in a subject or a sample, such a method comprising: (i) contacting a subject or biological sample with an activatable TB, where the activatable TB includes a detectable label located in a portion of the activatable TB that is released after cleavage of the CM; and (ii) measuring a level of activated (cleaved) TB in the subject or biological sample, where a detectable level of activated TB in the subject or biological sample indicates that the cleavage agent, the target, or both the cleavage agent and the target are absent and / or sufficiently absent in the subject or biological sample such that target binding and / or protease cleavage of the activatable TB cannot be detected in the subject or biological sample, and a decrease in the detectable level of activated (cleaved) TB in the subject or biological sample indicates that the cleavage agent and the target are present in the subject or biological sample.

[0201] The reduction in the level of detectable label can be, for example, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% reduction, or substantially 100% reduction. In some embodiments, the detectable label can be conjugated to a component of activatable TB, such as AB. In some embodiments, measuring the level of activatable TB in a subject or sample can be achieved using a secondary reagent that specifically binds to activated TB, which reagent comprises a detectable label. The secondary reagent can be a TB that comprises a detectable label.

[0202] In some embodiments, activatable TBs may also be useful for detecting targets in patient samples, and thus are useful as diagnostic agents. For example, activatable TBs may be used in in vitro assays, such as ELISA, to detect target levels in patient samples. For example, activatable TBs may be immobilized on a solid support (e.g., the well(s) of a microtiter plate). The immobilized activatable TBs may act as capture TBs for any targets that may be present in a test sample. Before contacting the immobilized TB with a patient sample, the solid support may be rinsed and treated with a blocking agent, such as milk protein or albumin, to prevent non-specific adsorption of analytes.

[0203] In some embodiments, based on the results obtained using activatable TB in an in vitro diagnostic assay, the stage of disease in a subject can be determined based on the expression level of the target antigen(s). For a given disease, blood samples can be taken from subjects diagnosed as being at different stages of disease progression and / or at different time points of the therapeutic treatment of the disease. A population of samples that provides statistically significant results for each stage of progression or treatment is used to specify a range of concentrations of antigens that can be considered characteristic of each stage.

[0204] The activatable TBs herein may also be used in diagnostic and / or imaging methods. In some embodiments, such methods may be in vitro methods. In some embodiments, such methods may be in vivo methods. In some embodiments, such methods may be in situ methods. In some embodiments, such methods may be ex vivo methods. For example, activatable TBs having CMs may be used to detect the presence or absence of an enzyme capable of cleaving the CM. Such activatable TBs may be used in diagnostics, which may include in vivo detection (e.g., qualitative or quantitative) of enzyme activity (or, in some embodiments, an environment of increased reduction potential that may result in reduction of disulfide bonds) through the measured accumulation of activated TB (i.e., TB resulting from cleavage of the activatable TB) in specific cells or tissues of a specific host organism. Such accumulation of activated TB indicates not only that the tissue expresses the enzyme activity (or increased reduction potential depending on the nature of the CM), but also that the tissue expresses a target to which the activated TB binds.

[0205] For example, the CM can be selected to be a protease substrate for proteases found at tumor sites, sites of viral or bacterial infection, biologically restricted sites (e.g., abscesses, within organs, etc.), and the like. The AB can be one that binds to a target antigen. A detectable label (e.g., a fluorescent label or a radioactive label or a radioactive tracer) can be conjugated to the AB or other region of the activatable TB using methods well known to those of skill in the art. Suitable detectable labels can be discussed in the context of the screening methods described above, with additional specific examples provided below. When a TB (e.g., an AB) specific for a disease state protein or peptide is used with a protease that has increased activity in the diseased tissue of interest, the activatable TB may have an increased rate of binding to the diseased tissue compared to tissue in which the CM-specific enzyme is not present at detectable levels, is present at lower levels than the diseased tissue, or is inactive (e.g., in a zymogen form or complexed with an inhibitor). Because small proteins and peptides are rapidly cleared from the blood by the renal filtration system and CM-specific enzymes are not present at detectable levels (or are present at lower levels or in an inactive conformation in non-diseased tissue), accumulation of activated TB in diseased tissue may be increased compared to non-diseased tissue.

[0206] In some embodiments, the activatable TB may be useful for in vivo imaging, where detection of a fluorescent signal in a subject, e.g., a mammal, including a human, indicates that a disease site contains a target and that the CM of the activatable TB contains a specific protease. In vivo imaging may be used to identify or narrow a patient population suitable for treatment with the activatable TB of the present disclosure. For example, a patient who tests positive for both a protease that cleaves a substrate in the CM of the activatable TB being tested and a target (e.g., accumulates activated TB at a disease site) may be identified as a suitable candidate for treatment with such activatable TB containing such CM. Similarly, a patient who tests negative may be identified as a suitable candidate for another treatment (i.e., not suitable for treatment with the activatable TB being tested). In some embodiments, a patient who tests negative for a first activatable TB may be tested with other activatable TB containing different CM until an activatable TB suitable for treatment (e.g., an activatable TB containing a CM that is cleaved at the patient's disease site) is identified.

[0207] In some embodiments, in situ imaging can be useful in methods to identify which patients to treat. For example, in situ imaging can use activatable TB to screen patient samples to identify patients with the appropriate protease(s) and target(s) in the appropriate location, e.g., tumor site. In some embodiments, in situ imaging is used to identify or narrow the patient population suitable for treatment with the activatable TB of the present disclosure. For example, patients who test positive for both the protease and target that cleaves a substrate in the CM of the activatable TB being tested (e.g., accumulate activated TB at disease sites) are identified as suitable candidates for treatment with such activatable TB containing such CM. Similarly, patients who test negative for either or both the protease and target that cleaves a substrate in the CM used for the activatable TB being tested using these methods are identified as suitable candidates for alternative treatment (i.e., not suitable for treatment with the activatable TB being tested). In some embodiments, a patient who tests negative for a first activatable TB may be tested with other activatable TBs containing different CMs until an activatable TB suitable for treatment is identified (e.g., an activatable TB containing a CM that is cleaved at the patient's disease site).

[0208] In some embodiments, the disclosure includes any one or any combination of the following non-limiting numbered clauses:

[0209] 1. A target binding protein (TB) that specifically binds to a target; a masking moiety (MM) attached to the TB, the MM inhibiting binding of the AB to the target; a cleavable moiety (CM) bound to the TB and located between the TB and the MM, the CM being a polypeptide that functions as a substrate for a protease; The activatable target binding protein further comprises a non-alpha carbon covalent bond tethering the MM and the TB.

[0210] 2. The activatable target binding protein of clause 1, wherein said TB is an antigen binding protein (AB).

[0211] 3. An activatable target binding protein according to clause 1 or clause 2, which has reduced target binding activity compared to a single anchor activatable target binding protein lacking said non-alpha carbon covalent bond.

[0212] 4. The activatable target binding protein of any one or any combination of clauses 1-3, wherein the non-alpha carbon covalent bond is an ester bond or a thioester bond.

[0213] 5. The activatable target binding protein of clause 4, wherein the ester bond is between threonine and glutamine.

[0214] 6. The activatable target binding protein of clause 4, wherein the thioester bond is between a cysteine ​​and a glutamine or a tyrosine.

[0215] 7. The activatable target binding protein of any one or any combination of clauses 1-3, wherein the non-alpha carbon covalent bond is a bridge between histidine and tyrosine, or a bridge between lysine and cysteine.

[0216] 8. The activatable target binding protein of any one or any combination of clauses 1-3, wherein the non-alpha carbon covalent bond is an isopeptide bond.

[0217] 9. The activatable target binding protein of clause 8, wherein the isopeptide bond is between a lysine and a glutamic acid or aspartic acid residue.

[0218] 10. The activatable target binding protein of clause 8, wherein the isopeptide bond is between a gamma-carboxyamide group of glutamine and an epsilon-amino group of a lysine side chain.

[0219] 11. The activatable target binding protein of any one or any combination of clauses 1-3, wherein the non-alpha carbon covalent bond is a disulfide bond.

[0220] 12. The disulfide bond is formed between a first cysteine ​​and a second cysteine; the first cysteine ​​is in the MM and the second cysteine ​​is in the TB; the first cysteine ​​is in a peptide bound to the MM and the second cysteine ​​is in the TB; or 12. The activatable target binding protein of clause 11, wherein the first cysteine ​​is in the MM and the second cysteine ​​is in a peptide bound to the TB.

[0221] 13. Furthermore, a second commercial is included. said second CM is located between said MM and said non-alpha carbon covalent bond; the second CM is within the MM and is a maximum of 5 amino acids away from the cysteine ​​that forms the non-alpha carbon covalent bond; or 13. The activatable target binding protein of any one or any combination of clauses 1-12, wherein the second CM is within the TB and is located up to 5 amino acids away from the cysteine ​​that forms the non-alpha carbon covalent bond.

[0222] 14. The activatable target binding protein of any one or any combination of clauses 1-13, wherein the first CM and the second CM are substrates for different proteases.

[0223] 15. The activatable target binding protein of any one or any combination of clauses 1-13, wherein the first CM and the second CM are substrates for the same protease.

[0224] 16. The activatable target binding protein of any one or any combination of clauses 1-12, wherein said protease is produced by a tumor of the subject.

[0225] 17. The activatable target binding protein of any one or any combination of clauses 2-16, wherein said 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.

[0226] 18. The activatable target binding protein of any one or any combination of clauses 2-17, wherein said AB is a single domain antibody.

[0227] 19. The activatable target binding protein of any one or any combination of clauses 2-18, wherein said AB is an Fc-tagged single domain antibody.

[0228] 20. The activatable target binding protein of any one or any combination of clauses 2-17, wherein said AB is a bispecific antibody.

[0229] 21. The activatable target binding protein of clause 20, wherein the bispecific antibody is a bispecific T cell engager (BiTE) or a dual affinity retargeting antibody (DART).

[0230] 22. The activatable target binding protein of any one or any combination of clauses 2-17, wherein said AB is a multispecific antibody.

[0231] 23. The activatable target binding protein of any one or any combination of clauses 18-19, wherein the non-alpha carbon covalent bond is between the MM and the single domain antibody.

[0232] 24. The activatable target binding protein of any one or any combination of clauses 1-22, wherein the non-alpha carbon covalent bond is between the MM and Fc domains.

[0233] 25. The activatable target binding protein of any one or any combination of clauses 1-24, wherein said MM comprises an epitope of said TB.

[0234] 26. The activatable target binding protein of any one or any combination of clauses 1-24, wherein said MM does not comprise four or more consecutive amino acids of an epitope of said TB.

[0235] 27. The activatable target binding protein of any one or any combination of clauses 1-26, wherein the MM has a dissociation constant for binding to the TB that is greater than the dissociation constant of the TB for binding to the target.

[0236] 28. The activatable target binding protein of any one or any combination of clauses 1-27, wherein the MM is a polypeptide of 2 to 40 amino acids in length.

[0237] 29. An activatable target binding protein according to any one or any combination of clauses 1 to 28, comprising a linker between the MM and the CM.

[0238] 30. An activatable target binding protein according to any one or any combination of clauses 1 to 29, comprising a linker between the CM and the TB.

[0239] 31. An activatable target binding protein according to any one or any combination of clauses 1 to 29, comprising a first linker between the MM and the CM, and a second linker between the CM and the TB.

[0240] 32. A composition comprising an activatable target binding protein according to any one or any combination of clauses 1 to 31 and a carrier.

[0241] 33. The composition according to claim 32, which is a pharmaceutical composition.

[0242] 34. A container, vial, syringe, injection pen, or kit comprising at least one dose of the composition of clause 32 or clause 33.

[0243] 35. A nucleic acid comprising a sequence encoding an activatable target binding protein according to any one or any combination of clauses 1 to 31.

[0244] 36. A vector comprising the nucleic acid according to clause 35.

[0245] 37. A cell comprising the nucleic acid of clause 35, or the vector of clause 36.

[0246] 38. A conjugated activatable target binding protein comprising an activatable target binding protein according to any one or any combination of clauses 1-31 conjugated to a drug.

[0247] 39. The conjugated activatable target binding protein of clause 38, wherein the agent is a therapeutic agent, a targeting moiety, or a detectable moiety.

[0248] 40. A method of treating a subject in need of treatment, comprising administering to the subject a therapeutically effective amount of an activatable target binding protein according to any one or any combination of clauses 1 to 31, a composition according to clause 32 or clause 33, or a conjugated activatable target binding protein according to clause 38 or clause 39.

[0249] 41. The method of clause 40, wherein the subject has been identified or diagnosed as having cancer, an inflammatory condition, disorder or disease, or an autoimmune condition, disorder or disease.

[0250] 42. A method for producing an activatable target binding protein, comprising: Culturing the cell of clause 37 in a culture medium under conditions sufficient to produce said activatable target binding protein; recovering said activatable target binding protein from said cells or said culture medium; The method comprising:

[0251] 43. The method of clause 42, further comprising isolating the activatable target binding protein recovered from the cells or the culture medium.

[0252] 44. The method of clause 43, wherein the isolating is carried out using a protein purification tag and / or size exclusion chromatography.

[0253] 45. The method of clause 43 or clause 44, further comprising formulating the activatable target binding protein into a pharmaceutical composition.

[0254] 46. ​​A method for producing a dual anchored activatable polymer, comprising: engineering a cysteine ​​residue at a disulfide bond site in a masking moiety (MM) of the dual anchored activatable polymer; engineering a cysteine ​​residue at a disulfide bond site in a target binding protein (TB) of the dual anchored activatable polymer, wherein the MM and the TB are linked and a cleavable moiety (CM) is positioned between the MM and the TB; expressing the dual anchored activatable polymer; and recovering the dual anchored activatable polymer, wherein the MM and the TB are tethered at their disulfide bond sites in the recovered dual anchored activatable polymer.

[0255] 47. A method for producing a dual anchored activatable polymer, comprising: engineering an arginine or lysine residue at an isopeptide binding site in a masking moiety (MM) of the dual anchored activatable polymer, and / or engineering an aspartic acid or glutamic acid residue at an isopeptide binding site in a target binding protein (TB) of the dual anchored activatable polymer, wherein the MM and the TB are linked and a cleavable moiety (CM) is positioned between the MM and the TB; expressing the dual anchored activatable polymer; and recovering the dual anchored activatable polymer, wherein the MM and the TB are tethered at their isopeptide binding sites in the recovered dual anchored activatable polymer.

[0256] 48. A method for producing a dual anchored activatable polymer, comprising: manipulating an aspartic acid or glutamic acid residue at an isopeptide binding site in a masking moiety (MM) of the dual anchored activatable polymer, and / or manipulating an arginine or lysine residue at an isopeptide binding site in a target binding protein (TB) of the dual anchored activatable polymer, wherein the MM and the TB are linked and a cleavable moiety (CM) is positioned between the MM and the TB; expressing the dual anchored activatable polymer; and recovering the dual anchored activatable polymer, wherein the MM and the TB are tethered at their isopeptide binding sites in the recovered dual anchored activatable polymer.

[0257] 49. A method for making a dual-anchored activatable polymer, comprising: providing a MM comprising a non-alpha carbon covalent bond-forming amino acid configured to form a non-alpha carbon covalent bond with a non-alpha carbon covalent bond-forming amino acid in a TB bound to the MM.

[0258] 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.

[0259] 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.

[0260] 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.

[0261] 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.

[0262] 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.

[0263] 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.

[0264] 56. The method of clause 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.

[0265] 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.

[0266] 58. The method of claim 49, wherein the alpha carbon covalent bond forming amino acid of the MM is glutamine, 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 gamma carboxyamide group of the glutamine and the epsilon amino group of the lysine side chain.

[0267] 59. The method of claim 49, wherein the alpha carbon covalent bond forming amino acid of the MM is lysine, 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 gamma carboxyamide group of the glutamine and the epsilon amino group of the lysine side chain.

[0268] 60. A method for making a dual-anchored activatable polymer, comprising: providing a MM containing a cysteine ​​configured to form a non-alpha carbon covalent bond with a non-alpha carbon covalent bond-forming amino acid in a TB bound to the MM. EXAMPLES

[0269] The present invention is further described in the following examples, which do not limit the scope of the invention as claimed, but rather demonstrate proof of concept of the advantages of the dual anchor structure of the activatable polymer described in this disclosure.

[0270] Example 1: Production of single anchored BC2T masked nanobodies (BC2T-Nbs) This example shows the production of an exemplary single-anchor activatable antibody. The single-anchor activatable antibody comprises a nanobody (Nb) capable of binding to beta-catenin and a MM (BC2T) that contains the sequence of beta-catenin, which is the epitope of the Nb. The MM is single-anchor with the Nb, i.e., the MM and the Nb are linked via the CM but are not tethered by a disulfide bond. The single-anchor BC2T-Nb (shown in FIG. 4B) was prepared by recombinant methods. The single-anchor BC2T-Nb (SEQ ID NO: 2 or 3) comprises, from the N-terminus to the C-terminus, 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 for purification (SEQ ID NO: 21). The polypeptides were prepared by transforming host cells with a polynucleotide encoding the polypeptide sequence of SEQ ID NO: 2 or 3, followed by culturing the resulting recombinant host cells and purifying the protein from the supernatant using standard immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC) techniques. The resulting proteins are monomeric (ProC653 or ProC654).

[0271] A control molecule (i.e. unmasked nanobody without BC2T binding) was also prepared by recombinant methods. ProC649 (PP073) (SEQ ID NO: 1) contains from N- to C-terminus a signal peptide from SP5 (SEQ ID NO: 12), an Nb (SEQ ID NO: 19), a GGS linker and a His tag for purification (SEQ ID NO: 21). The polypeptide was prepared by transforming a host cell with a polynucleotide encoding the polypeptide sequence of SEQ ID NO: 1, followed by culturing the resulting recombinant host cell and purifying it using standard IMAC and SEC techniques. The resulting protein (ProC649) is monomeric.

[0272] Example 2. Protease treatment of single anchored BC2T-Nb To release MM, unmasked nanobodies and single-anchored BC2T-Nb molecules were treated with recombinant human proteases, such as matrix metalloproteases (MMPs), overnight at 37°C. Complete protease treatment was tested by non-reducing SDS-PAGE. Protein aliquots (2 μg) were denatured in sample buffer (reducing agents added if necessary) for 10 min at 75°C, separated on 4-12% NuPAGE™ Bis-Tris gels (Thermo Fisher Scientific, Waltham, MA, Cat. No. NP0321) in MOPS buffer at 175 V for 1 h, and visualized by staining with InstantBlue™ for 1 h followed by destaining in water for at least 4 h.

[0273] The protein was confirmed to be monomeric and of the expected molecular weight (Figure 6). Protease treatment did not affect the integrity of the unmasked molecule (Figure 6, lanes 1, 2, 3). Constructs in which BC2T and nanobody were expressed as polypeptides and linked to CM showed a shift in molecular weight (Figure 6, lanes 4, 6). After protease treatment, the protein migrated at the same level as the unmasked molecule (Figure 6, lanes 5, 7).

[0274] Example 3: Binding of Monomeric Single Anchored BC2T-Nb by BioLayer Interferometry (BLI) Binding of unmasked nanobodies and single anchor BC2T-Nb to free BC2T peptide was assessed by BLI using a ForteBio Octet device. BLI is commonly used to calculate association and dissociation kinetics of binding interactions, but here it was used to detect binding. A peptide corresponding to the following sequence: QGQSGQPDRKAAVSHWQ (SEQ ID NO: 567) was synthesized at ELIM Biopharmaceuticals with biotin at the N-terminus. The peptide was loaded onto a SSA biosensor tip (Pall / ForteBio Cat. No. 18-5117) at 100 nM in binding buffer (BB: 1XPBS, pH 7.2, 5% glycerol, 0.1% Tween®-20, 2% BSA) for 10 min. Protein capture was confirmed by a 1 min baseline incubation in BB before and after this capture step. The loaded tip was then incubated with 100 nM Nb in BB for 6 min, followed by a 6 min incubation in BB. The change in interference distance (in nanometers) at the sensor tip is plotted against time to track the binding / dissociation of soluble proteins from the ligand immobilized at the tip (Figure 7). The results show that ProC653 and ProC654, as well as the unmasked control ProC649, are effectively masked as they can only bind to the peptide after protease (MMP9 or MMP14) treatment.

[0275] Example 4: Production of monomeric double-anchored BC2T-Nb This example shows the production of an exemplary monomeric dual-anchored activatable antibody. The dual-anchored activatable antibody comprises a nanobody (Nb) capable of binding to beta-catenin and a MM that contains the sequence of beta-catenin, which is the epitope of the Nb (BC2T). The MM is dual-anchored with the Nb, i.e., the MM and the Nb are linked via the CM and are also tethered by a disulfide bond. The dual-anchored BC2T-Nb (shown in FIG. 4A) was prepared by recombinant methods. ProC994 (PP123) (SEQ ID NO: 4) and ProC995 (PP124) (SEQ ID NO: 5) contain, from the N-terminus to the C-terminus, a signal peptide from SP5 (SEQ ID NO: 12), a leader sequence with the third amino acid mutated to cysteine ​​(SEQ ID NO: 22), a 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), an Nb with one amino acid mutated to cysteine ​​(SEQ ID NO: 24 or 25), a GGS linker, and a His tag for purification (SEQ ID NO: 21). The polypeptides were prepared by transforming a host cell with a polynucleotide encoding the polypeptide sequence of SEQ ID NO: 4 or 5 and culturing the resulting recombinant host cell. The resulting protein (ProC994 or ProC995) is a monomer.

[0276] 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), a BC2T MM with the second amino acid mutated to cysteine ​​(SEQ ID NO:23), a GS linker (SEQ ID NO:15), a CM (SEQ ID NO:16), a GS linker (SEQ ID NO:18), an Nb with one amino acid mutated to cysteine ​​(SEQ ID NO:26), a GGS linker, and a His tag for purification (SEQ ID NO:21). The polypeptide was 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 techniques. The resulting protein (ProC996) is monomeric.

[0277] Example 5. Protease treatment of monomeric double anchored BC2T-Nb To release MM (see Figure 4C), single- and double-anchored BC2T-Nb molecules were treated with recombinant human proteases such as urokinase-type plasminogen activator (uPA) overnight at 37°C. Complete protease treatment and disulfide bond analysis were examined by reducing and non-reducing SDS-PAGE. Protein aliquots (2 μg) were denatured in sample buffer (reducing agent added as needed) at 75°C for 10 min, separated on 4-12% NuPAGE™ Bis-Tris gels (Thermo Fisher Scientific, Waltham, MA, Cat. No. NP0321) in MOPS buffer at 175 V for 1 h, and visualized by staining with InstantBlue™ for 1 h followed by destaining in water for at least 4 h.

[0278] Before and after protease treatment, the protein was confirmed to be monomeric and of the expected molecular weight on a reducing gel (Figure 8, bottom panel). A non-reducing gel before protease treatment confirmed the formation of the engineered disulfide bond (Figure 8, top panel, lanes 4, 6, and 8), and after protease treatment, the protein migrated at the same level as the single-anchored masked molecule (Figure 8, top panel, lanes 2, 5, 7, and 9). Comparing the migration of the protease-treated doubly-anchored masked protein on non-reducing and reducing gels confirmed the formation of the engineered disulfide bond.

[0279] Example 6: Production of dimeric double anchored polymers This example shows the production of an exemplary dimeric dual-anchored activatable antibody. The dual-anchored activatable molecule includes two nanobodies (Nb) capable of binding to beta-catenin, an Fc dimer containing two Fcs each bound to an Nb, and two MMs (BC2T in this illustrative example) containing the sequence of beta-catenin, which is the epitope of the Nb. Each MM is dual-anchored with the Nb, i.e., the MM and Nb are linked via the CM and are also tethered by a disulfide bond (Figure 5A).

[0280] A dimeric double-anchored BC2T-Nb was prepared by recombinant methods. ProC1285 (HC699) (SEQ ID NO: 9) comprises, from N-terminus to C-terminus, a signal peptide from SP5 (SEQ ID NO: 12), a leader sequence with the third amino acid mutated to cysteine ​​(SEQ ID NO: 22), a 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), an Nb with one amino acid mutated to cysteine ​​(SEQ ID NO: 24), a GGGG linker (SEQ ID NO: 27), and a human IgG1 Fc (SEQ ID NO: 28). The polypeptide was 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 techniques. The resulting protein (ProC1285) is a dimer (shown in FIG. 5A).

[0281] 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), a BC2T MM with the second amino acid mutated to cysteine ​​(SEQ ID NO:23), a GS linker (SEQ ID NO:15), a CM (SEQ ID NO:16), a GS linker (SEQ ID NO:18), an Nb with one amino acid mutated to cysteine ​​(SEQ ID NO:26), a GGGG linker (SEQ ID NO:27), and a human IgG1 Fc (SEQ ID NO:28). The polypeptide was 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 techniques. The resulting protein (ProC1287) is a dimer (shown in FIG. 5A).

[0282] Single anchored BC2T-Nb-IgG1 (i.e., MM and Nb are not tethered by a disulfide bond, as shown in FIG. 5B) was also prepared by recombinant methods. ProC1284(HC698) (SEQ ID NO:8) contains, 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: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 a human IgG1 Fc (SEQ ID NO:28). The polypeptide was 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 techniques. The resulting protein (ProC1284) is a dimer.

[0283] Example 7: Protease treatment of dimeric double-anchored BC2T-Nb of Example 6 To release the MM in the molecules of Example 6, the single-anchored and double-anchored BC2T-Nb molecules were treated with recombinant human proteases such as urokinase-type plasminogen activator (uPA) at 37°C overnight, as shown diagrammatically in Figure 5C. Complete protease treatment and disulfide bond analysis were examined by reducing and non-reducing SDS-PAGE. Protein aliquots (2 μg) were denatured in sample buffer (reducing agent added as needed) at 75°C for 10 min, separated on 4-12% NuPAGE™ Bis-Tris gels (Thermo Fisher Scientific, Waltham, MA, Cat. No. NP0321) in MOPS buffer at 175 V for 1 h, and visualized by staining with InstantBlue™ for 1 h, followed by destaining in water for at least 4 h.

[0284] Before and after protease treatment, the protein was confirmed to be monomeric with the predicted molecular weight on a reducing gel, approximately 19 kDa and approximately 16 kDa, respectively (Figure 9, lower panel). A non-reducing gel before protease treatment confirmed that the engineered disulfide bond had been formed (Figure 9, upper panel, lanes 4, 6, and 8), and after protease treatment, the protein migrated at the same level as the single-anchored masked molecule (Figure 9, upper panel, lanes 2, 5, 7, and 9). Comparing the migration of the protease-treated doubly anchored masked protein on non-reducing and reducing gels confirmed that the engineered disulfide bond had been formed.

[0285] Example 8: Peptide binding ELISA The BC2T peptide was synthesized by ELIM Biopharmaceuticals. The peptide sequence comprises, from N- to C-terminus, a leader sequence (SEQ ID NO: 13) and the BC2T MM (SEQ ID NO: 14).

[0286] 5 μM BC2T peptide dissolved in 0.05 M carbonate-bicarbonate buffer was adsorbed overnight at 4° C. to wells of a 96-well microtiter plate. Plates were washed and blocked with blocking buffer (1×PBS, pH 7.4, 0.05% Tween®-20, 1% BSA). Three-fold serial dilutions of dimeric doubly anchored masked molecules (ProC1285 and ProC1287) and unmasked control protein (ProC1283), with or without protease treatment, and dimeric single anchored BC2T-Nb (ProC1284) were made and applied to the peptide-coated plates. The extent of protein binding to the peptide was measured by anti-human IgG immunodetection. A450 absorbance was measured on a plate reader. Dose-response curves were generated and EC50 values ​​obtained by sigmoidal fit nonlinear regression using Graph Pad Prism software. The results are shown in FIG. 10. As shown in Figure 10, the dimeric doubly anchored masked molecules (ProC1285 and ProC1287) showed much higher masking than the comparable dimeric single anchored BC2T-Nb (ProC1284) and also showed a surprisingly high recovery of binding activity upon cleavage compared to that shown by the positive control ProC1283 (unmasked antibody). Thus, despite being tethered to the mask at one end after cleavage, the antibody showed substantial recovery of binding activity.

[0287] Example 9: Design of disulfide-based dual anchor masked antibodies: In this example, an activatable anti-PDL1 antibody is used as an example to illustrate how to engineer non-alpha carbon bonds, such as cysteine-disulfide bonds, between residues in the prodomain (N-terminal to the mask peptide) and in the antibody variable domain. The sequence of the activatable anti-PDL1 antibody (SEQ ID NOs: 562 and 563) consists of a prodomain connected to a standard antibody light chain. The primary sequence of the antibody was used to generate a homology-based three-dimensional model of the antibody using software such as Discovery Studio (Figures 12A-12B). Using the mask sequence from the prodomain, a homology-based model of the mask was generated and docked to the antibody structure (Figures 12A-12B). Figure 12A shows the three-dimensional structure of the activatable anti-PDL1 antibody obtained using Dassault Systemes software BIOVIA Discovery Studios, showing the solvent accessible residues within 2-5 angstroms of the header region or N-terminal residues of the mask portion. FIG. 12B shows the interface between the Fab domain (space-filling format) of an activatable anti-PDL1 antibody and the prodomain with a masked portion (shown as a Cα backbone).

[0288] To confirm the structural model, various experimental techniques such as mutagenesis, hydrogen-deuterium exchange (HDX), and XL-MS (cross-linker MS) can be optionally used. After obtaining a three-dimensional model of the MM bound to the TB, an energy minimization algorithm is used to provide information on the location of the header and linker regions (including the CM) of the prodomain. The user can then use software such as BIOVIA Discovery Studio to identify pairs of residues, one in the header region of the prodomain and the other in the antibody variable domain, whose Cα atoms are within 3-7.5 Å (i.e., the distance between the Cα of the first amino acid and the Cα of the second amino acid) of a canonical disulfide bond cysteine. Using software applications such as SSBondPre, this list can be narrowed down to a smaller list of residues that are likely to form the designed disulfide bond. Residues are then mutated individually and in pairs to cysteine ​​residues to identify pairs of residues that individually do not affect antibody or mask binding, but when present together form a disulfide bond and improve masking. The presence of correctly formed disulfides can be confirmed by mass spectrometry techniques such as disulfide mapping.

[0289] Example 10: Design of disulfide-based dual anchored masked antibodies or other TBs: This example provides further illustration of how a homology-based three-dimensional model of an antibody structure can be prepared and used to engineer non-alpha carbon bonds, such as cysteine-disulfide bonds, between residues of the prodomain (N-terminal to the mask peptide) and residues of an antibody variable domain.

[0290] BIOVIA Discovery Studio was used to generate homology-based three-dimensional models of antibody structures corresponding to the sequences of J43v2 / anti-mouse PD1 (Figure 13A, SEQ ID NOs: 568-569), anti-CD166 (Figure 13B, SEQ ID NOs: 572-573), and human anti-PD1 (Figure 13C, SEQ ID NOs: 570-571). Anti-PD1 human anti-PD1 (SEQ ID NOs: 570-571) was further modeled in AlphaFold2 and rendered in BIOVIA Discovery Studio, as shown in Figure 13D.

[0291] To confirm the structural model, various experimental techniques such as mutagenesis, hydrogen-deuterium exchange (HDX), and XL-MS (cross-linker MS) can be optionally used. A modeled human anti-PD1 MM is disclosed in WO2017 / 011580 and a modeled anti-CD166 MM is disclosed in WO2016 / 179285, both of which are incorporated herein by reference. A three-dimensional model of each MM bound to each TB can be obtained and software such as BIOVIA Discovery Studio can be used to identify pairs of residues, one in the header region of the prodomain and the other in the antibody variable domain, whose Cα atoms are within the range of 3-7.5 Å (i.e., the distance between the Cα of the first amino acid and the Cα of the second amino acid). Using software applications such as SSBondPre, this list can be narrowed down to a smaller list of residues that are likely to form the desired bond. Residues are then mutated individually and in pairs to the desired residues to form the desired bond, identifying pairs of residues that individually do not affect antibody or mask binding, but that when present together form bonds that improve masking as described herein. The presence of correctly formed bonds (e.g., disulfide bonds) can be confirmed by mass spectrometry techniques such as disulfide mapping.

[0292] The sequences of the molecules in the examples are listed in Table 4 below. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6]

[0293] Other embodiments While the invention has been described in connection with the above detailed description, it is to be understood that the foregoing description is intended to be illustrative and not limiting of the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0294] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, section headings, materials, methods, and examples are illustrative only and are not intended to be limiting.

Claims

1. Target-binding proteins (TBs) that specifically bind to a target, A masking portion (MM) coupled to the TB, wherein the MM inhibits the binding of the AB to the target, A cleavable portion (CM) bound to the TB and located between the TB and the MM, wherein the CM is a polypeptide that functions as a substrate for a protease, and the CM and A double-anchored, activatable target-binding protein, The activatable target-binding protein further comprises a non-alpha carbon covalent bond tethering the MM and the TB.

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

3. An activatable target-binding protein according to claim 1, wherein the activatable target-binding protein has lower target-binding activity compared to a single-anchor type activatable target-binding protein lacking a non-alpha carbon covalent bond.

4. The activatable target-binding protein according to claim 1, wherein the non-alpha carbon covalent bond is an ester bond or a thioester bond.

5. The activatable target-binding protein according to claim 4, wherein the ester bond is located between threonine and glutamine.

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

7. The activatable target-binding protein according to claim 1, wherein the non-alpha carbon covalent bond is a crosslink between histidine and tyrosine, or a crosslink between lysine and cysteine.

8. The activatable target-binding protein according to claim 1, wherein the non-alpha carbon covalent bond is an isopeptide bond.

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

10. The activatable target-binding protein according to claim 8, wherein the isopeptide bond is located between the gammacarboxamide group of glutamine and the epsilonamino group of the lysine side chain.

11. The activatable target-binding protein according to claim 1, wherein the non-alpha carbon covalent bond is a disulfide bond.

12. The disulfide bond is formed between the first cysteine ​​and the second cysteine, The first cysteine ​​is located in the MM, and the second cysteine ​​is located in the TB. The first cysteine ​​is located within the peptide bound to the MM, and the second cysteine ​​is located within the TB, or The activatable target-binding protein according to claim 11, wherein the first cysteine ​​is located within the MM and the second cysteine ​​is located within the peptide bound to the TB.

13. Furthermore, including the second commercial, The second CM is located between the MM and the non-alpha carbon covalent bond. The second CM is located within the MM and is at a maximum of 5 amino acids away from the cysteine ​​that forms the non-alpha carbon covalent bond, or The activatable target-binding protein according to claim 1, wherein the second CM is located within the TB and is at a position up to 5 amino acids away from the cysteine ​​that forms the non-alpha carbon covalent bond.

14. The first commercial and the second commercial are, i) It is a substrate of a different protease, or ii) Whether they are substrates of the same protease, iii) The protease is produced by the target tumor, An activatable target-binding protein according to any one of claims 1 to 13.

15. a) The AB is an antibody, a Fab fragment, F(ab') ２ A fragment, scFv, scAb, dAb, VHH, or single-domain antibody, and / or b) i) The above AB is A) It is a single-domain antibody, and / or B) A single-domain antibody with an Fc tag, ii) The above AB is a bispecific antibody, and optionally the bispecific antibody is a bispecific T cell engager (BiTE) or a biaffinity retargeting antibody (DART), or iii) The above AB is a polyspecific antibody, The activatable target-binding protein according to claim 2.

16. The activatable target-binding protein according to claim 15, wherein the non-alpha carbon covalent bond is located between the MM and the single-domain antibody.

17. The activatable target-binding protein according to any one of claims 1 to 2, wherein the non-alpha carbon covalent bond is located between the MM and the Fc domain.

18. a) The MM is, i) containing the epitope of TB, or ii) The epitope of TB does not contain four or more consecutive amino acids, b) The MM has a dissociation constant for binding to the TB that is greater than the dissociation constant of the TB for binding to the target, c) The MM is a polypeptide with a length of 2 to 40 amino acids. d) The activatable target-binding protein includes a linker between the MM and the CM, and / or e) The activatable target-binding protein is i) A linker is included between the CM and the TB, or ii) Including a first linker between the MM and the CM, and a second linker between the CM and the TB, An activatable target-binding protein according to any one of claims 1 to 2.

19. A composition comprising an activatable target-binding protein as described in claim 1 and a carrier, wherein the composition is optionally a pharmaceutical composition.

20. A container, vial, syringe, injection pen, or kit comprising at least one dose of the composition according to claim 19.

21. A nucleic acid comprising a sequence encoding an activatable target-binding protein as described in claim 1.

22. A vector comprising the nucleic acid described in claim 21.

23. A cell comprising the nucleic acid according to claim 21, or the vector according to claim 22.

24. A conjugated activatable target-binding protein comprising an activatable target-binding protein according to claim 1, conjugated with a drug, wherein the drug is optionally a therapeutic agent, a target-directing moiety, or a detectable moiety.

25. A composition comprising the activatable target-binding protein according to claim 1 or the conjugated activatable target-binding protein according to claim 24, or the composition according to claim 19, for treating a subject in need of treatment, wherein the subject is identified or diagnosed with cancer, an inflammatory condition, disorder or disease, or an autoimmune condition, disorder or disease.

26. A method for producing an activatable target-binding protein, The cells according to claim 23 are cultured in a culture medium under conditions sufficient to produce the activatable target-binding protein, Recovering the activatable target-binding protein from the cells or culture medium, The method, including the method described above.

27. The process further comprises isolating the activatable target-binding protein recovered from the cells or culture medium, and optionally a) The isolation is performed using protein purification tags and / or size exclusion chromatography, and / or b) Further comprising formulating the activatable target-binding protein into a pharmaceutical composition, The method according to claim 26.

28. A method for producing a double-anchored, activatable polymer, a) Manipulating a cysteine ​​residue at the disulfide bond site in the masking portion (MM) of the double-anchored activatable polymer, and manipulating a cysteine ​​residue at the disulfide bond site in the target-binding protein (TB) of the double-anchored activatable polymer, wherein the MM and the TB are bound and a cleavable portion (CM) is positioned between the MM and the TB; expressing the double-anchored activatable polymer; and recovering the double-anchored activatable polymer, wherein the MM and the TB are tethered at their disulfide bond sites in the recovered double-anchored activatable polymer; b) Manipulating an arginine or lysine residue at the isopeptide binding site in the masking portion (MM) of the double-anchored activatable polymer, and / or manipulating an aspartic acid or glutamic acid residue at the isopeptide binding site in the target binding protein (TB) of the double-anchored activatable polymer, wherein the MM and the TB are bound and a cleavable portion (CM) is positioned between the MM and the TB; expressing the double-anchored activatable polymer; and recovering the double-anchored activatable polymer, wherein the MM and the TB are tethered at their isopeptide binding sites in the recovered double-anchored activatable polymer; or c) Manipulating an aspartic acid residue or glutamic acid residue at the isopeptide binding site in the masking portion (MM) of the double-anchored activatable polymer, and / or manipulating an arginine residue or lysine residue at the isopeptide binding site in the target binding protein (TB) of the double-anchored activatable polymer, wherein the MM and the TB are bound and a cleavable portion (CM) is positioned between the MM and the TB; expressing the double-anchored activatable polymer; and recovering the double-anchored activatable polymer, wherein the MM and the TB are tethered at their isopeptide binding sites in the recovered double-anchored activatable polymer; The method, including the method described above.

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