Activatable multispecific molecules and methods of use thereof

Abstract

An activatable protein comprising: a first target binding protein (TB1) that specifically binds to a first target; a second target binding protein (TB2) that specifically binds to a second target, wherein the TB2 is directly or indirectly coupled to the TB1; a first masking moiety (MM1) that inhibits the binding of the TB1 to the first target and is coupled to the TB1 directly or indirectly via a cleavable moiety, for example, via one or more linkers or other components; a second masking moiety (MM2) that inhibits the binding of the TB2 to the second target and is coupled directly or indirectly to either the TB1 or the TB2 via a cleavable moiety; and a half-life extending moiety (EM) that is coupled directly or indirectly to either the TB1 or the TB2 via a cleavable moiety.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. US63 / 326,692, filed April 1, 2022, which is incorporated by reference in its entirety.

[0002] Sequence Listing The Sequence Listing submitted with this application by EFS, entitled "4862-122PCT.xml", was created on March 30, 2023, is 639,463 bytes in size, and is incorporated by reference herein in its entirety.

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

[0004] Antibody-based therapies provide proven and effective treatments for a variety of diseases. However, in some cases, their therapeutic efficacy is limited by toxicity due to broad target expression. Furthermore, antibody-based therapeutics exhibit other limitations, such as rapid clearance from the circulation after administration.

[0005] Activatable antibodies are an attempt to expand the therapeutic index of antibody-based therapies. These molecules are administered as activatable prodrugs that are activated in vivo at or near the desired site of action. This mechanism of action can lead to an increase in the therapeutic index of the parent antibody. However, there is a continuing need for other strategies to increase the therapeutic index of antibody-based therapeutics. Summary of the Invention

[0006] The present disclosure provides activatable proteins, as well as related compositions and methods.

[0007] In one aspect, the present disclosure provides an activatable protein comprising: a first target binding domain (TB1) that specifically binds to a first target; a second target binding domain (TB2) that specifically binds to a second target, where TB2 is coupled to TB1; a first masking moiety (MM1) coupled to TB1 via a first cleavable moiety (CM1), where MM1 inhibits binding of TB1 to the first target; a second masking moiety (MM2) that inhibits binding of TB2 to the second target; a second cleavable moiety (CM2); a half-life extending moiety (EM) coupled either directly or indirectly to MM1 or MM2; wherein the activatable protein components are configured such that upon cleavage of CM1 and CM2, the resulting activated protein comprises TB1 and TB2, but does not comprise MM1, MM2 or EM. As used herein, and unless otherwise specified, components of an activatable molecule that are "coupled" can be coupled either by direct covalent bonds or indirect covalent bonds, for example, via one or more connecting peptides (also called "linkers"), cleavable moieties, or other components of the activatable protein.

[0008] In one aspect, the disclosure provides a first antigen binding domain (AB1) that specifically binds to a first target, where AB1 comprises a first heavy chain variable domain (HVD1) and a first light chain variable domain (LVD1); a second antigen binding domain (AB2) that specifically binds to a second target, where AB2 comprises a second heavy chain variable domain (HVD2) and a second light chain variable domain (LVD2), where AB2 is directly or indirectly coupled to the C-terminus of HVD1 or the C-terminus of LVD1; The present invention provides an activatable protein comprising: a first masking moiety (MM1) coupled to AB1 (either directly or indirectly, e.g., via one or more linkers or other components of the activatable protein) through a second cleavable moiety (CM2), where MM1 inhibits binding of AB1 to a first target; a half-life extending moiety (EM) coupled directly or indirectly to a second masking moiety (MM2), where EM is coupled to AB1 or AB2 (either directly or indirectly, e.g., via one or more linkers or other components of the activatable protein) through a second cleavable moiety (CM2), where MM2 inhibits binding of AB2 to a second target.

[0009] In one aspect, the disclosure provides a polypeptide comprising a first antigen binding domain (AB1) that specifically binds to a first target, wherein AB1 comprises a first heavy chain variable domain (HVD1) and a first light chain variable domain (LVD1); a second antigen binding domain (AB2) that specifically binds to a second target, wherein AB2 comprises a second heavy chain variable domain (HVD2) and a second light chain variable domain (LVD2), wherein AB2 is directly or indirectly coupled to the C-terminus of HVD1 or LVD1; and a second antigen binding domain (AB2) that specifically binds to a second target, wherein AB2 comprises a second heavy chain variable domain (HVD2) and a second light chain variable domain (LVD2), wherein AB2 is directly or indirectly coupled to the C-terminus of HVD1 or LVD1 via a first cleavable moiety (CM1) (directly or indirectly). the first masking moiety (MM1) coupled (either directly, e.g., via one or more linkers) to AB1, where MM1 inhibits binding of AB1 to a first target; a half-life extending moiety (EM) coupled (directly or indirectly) to a second masking moiety (MM2), where EM and MM2 are coupled (directly or indirectly) to either AB1 or AB2 via a second cleavable moiety (CM2), where MM2 inhibits binding of AB2 to a second target.

[0010] In one aspect, the disclosure provides a polypeptide comprising: a first antigen binding domain (AB1) that specifically binds to a first target, where AB1 comprises a first heavy chain variable domain (HVD1) and a first light chain variable domain (LVD1); a second antigen binding domain (AB2) that specifically binds to a second target, where AB2 comprises a second heavy chain variable domain (HVD2) and a second light chain variable domain (LVD2), where AB2 is directly or indirectly coupled to the C-terminus of HVD1 or LVD1 via a first cleavable moiety (CM1) (either directly or indirectly, e.g., via one or more linkers); The present invention provides an activatable protein comprising: a first masking moiety (MM1) coupled to AB1, where MM1 inhibits binding of AB1 to a first target; a half-life extending moiety (EM) comprising a dimer of a first half-life extending moiety (EM1) and a second half-life extending moiety (EM2), where EM1 is coupled (either directly or indirectly, e.g., via one or more linkers) to AB1 via a second cleavable moiety (CM2), and EM2 is coupled directly or indirectly to the second masking moiety (MM2), where MM2 inhibits binding of AB2 to a second target.

[0011] In one aspect, the disclosure provides a polypeptide comprising a first target binding domain (TB1) that specifically binds to a first target; a second target binding domain (TB2) that specifically binds to a second target, where TB2 is directly or indirectly coupled to TB1; a first masking moiety (MM1) coupled (either directly or indirectly, e.g., via one or more linkers) to TB1 via a first cleavable moiety (CM1), where MM1 inhibits binding of TB1 to the first target; a half-life extending moiety (EM) and a second masking moiety (MM2) coupled to TB1 or TB2 (either via one or more linkers), wherein MM2 inhibits binding of TB2 to a second target; wherein the activatable molecule components are configured such that cleavage of CM1 and CM2 releases MM1, MM2 and EM from TB1 and TB2 (if applicable), and optionally TB1 is an antigen binding molecule (AB1) comprising HVD1 and LVD1, and optionally TB2 is an antigen binding molecule (AB2) comprising HVD2 and LVD2.

[0012] In one aspect, the disclosure provides a polypeptide comprising a first antigen-binding domain (AB1) that specifically binds to a first target, where AB1 comprises a first heavy chain variable domain (HVD1) and a light chain variable domain (LVD1); a second antigen-binding domain (AB2) that specifically binds to a second target, where AB2 comprises a second heavy chain variable domain (HVD2) and a second light chain variable domain (LVD2), where AB2 is coupled either directly or indirectly (e.g., via a linker) to the C-terminus of HVD1 or LVD1; the first masking moiety (MM1) coupled to AB1 (either directly or indirectly, e.g., via one or more linkers), where MM1 inhibits binding of AB1 to a first target; the second masking moiety (MM2) coupled to AB2 (either directly or indirectly, e.g., via one or more linkers), via a second cleavable moiety (CM2), where MM2 inhibits binding of AB2 to a second target; the second masking moiety (MM2) coupled to a half-life extending moiety (EM) either directly or indirectly to MM1 or MM2.

[0013] In another aspect, the disclosure provides a polypeptide comprising a first antigen binding domain (AB1) that specifically binds to a first target, wherein AB1 comprises a first heavy chain variable domain (HVD1) and a first light chain variable domain (LVD1); a second antigen binding domain (AB2) that specifically binds to a second target, wherein AB2 comprises a second heavy chain variable domain (HVD2) and a second light chain variable domain (LVD2), wherein AB2 is directly or indirectly coupled to the N-terminus of HVD1 or the N-terminus of LVD1; a first cleavable moiety (CM1) and optionally one or more phosphorylation sites; The present invention provides an activatable protein comprising: a first masking moiety (MM1) coupled to AB1 via a C-terminus, where MM1 inhibits binding of AB1 to a first target; a second masking moiety (MM2) coupled to AB2 via a second cleavable moiety (CM2) and optionally one or more linkers, where MM2 inhibits binding of AB2 to a second target; a half-life extending moiety (EM) coupled to the C-terminus of HVD1 or the C-terminus of LVD1 via a third cleavable moiety (CM3) and optionally one or more linkers.

[0014] In some embodiments, the EM is a dimer formed by a first fragment crystallizable (Fc) domain and a second Fc domain. In some embodiments, the protein comprises at least a first polypeptide and a second polypeptide.

[0015] In some embodiments, the first polypeptide comprises, in N-terminal to C-terminal order, MM1, CM1, and VLD1 (including one or more optional linkers between the elements). In some embodiments, the second polypeptide comprises VHD1, VHD2, VLD2, CM2, MM2, and a first Fc domain, and the activatable protein further comprises a third polypeptide comprising a second Fc domain. In some embodiments, the second polypeptide comprises, in N-terminal to C-terminal order, VHD1, VHD2, VLD2, CM2, MM2, and a first Fc domain. In some embodiments, the second polypeptide comprises, in N-terminal to C-terminal order, VHD1, CM2, MM2, and a first Fc domain. In some embodiments, the second polypeptide comprises, in N-terminal to C-terminal order, VHD1, CM2, MM2, and a first Fc domain. In some embodiments, the second polypeptide comprises, in N-terminal to C-terminal order, VHD1, CM2, and a first Fc domain. In some embodiments, the first polypeptide comprises MM1, CM1, and VLD1, VHD2, and VLD2. In some embodiments, the first polypeptide comprises, in order from N-terminus to C-terminus, MM1, CM1, VLD1, VHD2, and VLD2. In some embodiments, the first polypeptide comprises, in order from N-terminus to C-terminus, MM1, CM1, VLD1, VLD2, and VHD2. In some embodiments, the protein comprises a third polypeptide, the third polypeptide comprises a second Fc domain and MM2. In each of the foregoing embodiments, and unless otherwise indicated, the polypeptide may include one or more optional linkers, for example, between each of the recited elements.

[0016] In some embodiments, MM2 is linked to the C-terminus of the second Fc domain via a connecting peptide. In some embodiments, MM2 is linked to the N-terminus of the second Fc domain via a connecting peptide (also referred to as a "linker"). In some embodiments, the second polypeptide further comprises a linker (L1) between MM2 and the first Fc domain. In some embodiments, L1 is a peptide having a length of 5-30, 6-29, 7-28, 8-27, 9-26, 10-25, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 amino acids. In the structural configurations disclosed in the preceding paragraph and throughout this disclosure, one or more linkers may optionally be present between elements. Additionally, the present disclosure also contemplates and includes activatable proteins in which any one or more of the disclosed elements are optionally in direct abutment with one another, such that there are no linkers or other amino acid sequences between the elements.

[0017] In some embodiments, the first Fc domain is a whole mutant of an Fc domain and the second Fc domain is a knob mutant of an Fc domain, in some embodiments, the whole mutant of an Fc domain comprises the sequence of SEQ ID NO:2 and the knob mutant of an Fc domain comprises the sequence of SEQ ID NO:1.

[0018] In some embodiments, the first target or epitope is a tumor-associated antigen. In some embodiments, the tumor-associated antigen is human epidermal growth factor receptor 2 (HER2). In some embodiments, AB1 is a Fab of trastuzumab. In some embodiments, HVD1 comprises the sequence of SEQ ID NO:27 and LVD1 comprises the sequence of SEQ ID NO:17. In some embodiments, AB2 is an immune effector cell associated with the scFv, a leukocyte associated with the scFv, a T cell associated with the scFv, a NK cell associated with the scFv, a macrophage associated with the scFv, or a monocyte associated with the scFv. In some embodiments, AB2 is or is derived from an anti-CD3 epsilon scFv or an anti-CTLA-4 scFv. In some embodiments, AB2 is or is derived from an anti-CD3 epsilon scFv. In some embodiments, HVD2 comprises the sequence of SEQ ID NO:30 and LVD2 comprises the sequence of SEQ ID NO:31.

[0019] In some embodiments, AB1 is or is derived from an anti-HER2 antibody. In some embodiments, AB1 is an scFv and the activatable protein is an activatable bispecific T cell engager (BiTE) or a dual affinity retargeting antibody (DART). In some embodiments, AB1 is a fragment antigen binding (Fab). In some embodiments, the second target is a costimulatory molecule. In some embodiments, the costimulatory molecule is CD3.

[0020] In some embodiments, CM1 and CM2 each comprise a substrate for the same protease. In some embodiments, CM1 and CM2 each comprise a substrate for a different protease. In some embodiments, CM1 and CM2 each independently comprise a substrate for ADAMS, ADAMTS, ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAMDEC1, ADAMTS1, ADAMTS4, ADAMTS5, aspartic protease, BACE, renin, aspartic cathepsin, cathepsin D, cathepsin E, caspase, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11, caspase 12, caspase 13, caspase 14, caspase 15, caspase 16, caspase 17, caspase 18, caspase 19, caspase 20, caspase 21, caspase 22, caspase 23, caspase 24, caspase 25, caspase 26, caspase 27, caspase 28, caspase 29, caspase 30, caspase 31, caspase 32, caspase 33, caspase 34, caspase 35, caspase 36, caspase 37, caspase 38, caspase 39 ...9, caspase 39, caspase 31, caspase 3 caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 14, cysteine ​​cathepsin, cathepsin B, cathepsin C, cathepsin K, cathepsin L, cathepsin S, cathepsin V / L2, cathepsin X / Z / P, cysteine ​​proteinase, cruzipain, legumain, otubein-2, KLK, KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, KLK14, metalloproteinase, meprin, neprila Isin, PSMA, BMP-1, MMP, MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP23, MMP24, MMP26, MMP27, serine protease, activated protein C, cathepsin A, cathepsin G, chymase, coagulation factor protease, FVIIa, FIXa, FXa, FXIa, FXIIa, elastase, The substrate comprises a substrate for a protease selected from the group consisting of granzyme B, guanidinobenzoatase, HtrA1, human neutrophil elastase, lactoferrin, marapsin, NS3 / 4A, PACE4, plasmin, PSA, tPA, thrombin, tryptase, uPA, type II transmembrane, serine protease, TTSP, DESC1, DPP-4, FAP, hepsin, matriptase-2, MT-SP1 / matriptase, TMPRSS2, TMPRSS3, and TMPRSS4.

[0021] In some embodiments, MM1 and MM2 are each independently 2-40 amino acids in length. In some embodiments, MM1 and MM2 are each independently 4-30 amino acids in length. In some embodiments, the heavy chain variable region of AB2 is directly or indirectly coupled to the C-terminus of the heavy chain fragment of AB1, the N-terminus of MM2 is coupled to the C-terminus of the light chain variable region of AB2 via CM2 (either directly or indirectly, e.g., via one or more linkers), and EM comprises a dimer of a first Fc domain and a second Fc domain, and the C-terminus of MM2 is directly or indirectly coupled to the N-terminus of the first Fc domain of EM. In some embodiments, the heavy chain variable region of AB2 is directly or indirectly coupled to the C-terminus of the light chain fragment of AB1, the N-terminus of MM2 is coupled to the C-terminus of the heavy chain fragment of AB1 via CM2 (either directly or indirectly, e.g., via one or more linkers), and the EM comprises a dimer of a first Fc domain and a second Fc domain, and the C-terminus of MM2 is directly or indirectly coupled to the N-terminus of the first Fc domain. In some embodiments, the heavy chain variable region of AB2 is directly or indirectly coupled to the C-terminus of the light chain fragment of AB1, the EM comprises a dimer of a first Fc domain and a second Fc domain, and the N-terminus of the first Fc domain is coupled to the C-terminus of the heavy chain fragment of AB1 via CM2 (either directly or indirectly, e.g., via one or more linkers), and the N-terminus of MM2 is directly or indirectly coupled to the C-terminus of the second Fc domain. In some embodiments, the heavy chain variable region of AB2 is directly or indirectly coupled to the C-terminus of the light chain fragment of AB1, the EM comprises a dimer of a first Fc domain and a second Fc domain, the N-terminus of the first Fc domain is coupled (either directly or indirectly, e.g., via one or more linkers) to the C-terminus of the heavy chain fragment of AB1 via CM2, and the C-terminus of MM2 is directly or indirectly coupled to the N-terminus of the second Fc domain.

[0022] In some embodiments, the activatable protein further comprises a linker between MM2 and a first or second Fc domain directly or indirectly coupled to MM2. In some embodiments, MM1 comprises the sequence of SEQ ID NO: 40, and MM2 comprises the sequence of any one of SEQ ID NOs: 34-37 or 66-70. In some embodiments, MM1 has a dissociation constant for binding to AB1 that is greater than the dissociation constant of AB1 for binding to a first target or epitope, and MM2 has a dissociation constant for binding to AB2 that is greater than the dissociation constant of AB2 for binding to a second target or epitope. In some embodiments, the activating molecule has a shorter half-life compared to a corresponding molecule that is the same as the activating molecule but includes an EM. In some embodiments, the activating molecule has a higher target binding activity compared to a corresponding molecule that is the same as the activating molecule but includes an EM. In some embodiments, the activating molecule has a higher target binding activity compared to an activatable molecule.

[0023] In some embodiments, the second polypeptide further comprises a linker (L2) between MM2 and AB2. In some embodiments, L2 is 5-30, 6-29, 7-28, 8-27, 9-26, 10-25, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 amino acids in length. In some embodiments, the second polypeptide further comprises a linker (L3) between AB2 and AB1. In some embodiments, L3 is 5-30, 6-29, 7-28, 8-27, 9-26, 10-25, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 amino acids in length. Generally, in each embodiment herein, unless otherwise specified, the polypeptide may include one or more optional linkers between each of the recited elements, and such linkers may be 1-30, 6-29, 7-28, 8-27, 9-26, 10-25, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 amino acids in length.

[0024] In another aspect, the disclosure provides a composition comprising an activatable protein herein and a carrier, in some embodiments, the composition is a pharmaceutical composition and the carrier is a pharma- ceutically acceptable carrier.

[0025] In another aspect, the disclosure provides a container, vial, syringe, injector pen, or kit comprising at least one dose of a composition herein.

[0026] In another aspect, the disclosure provides a nucleic acid comprising a sequence encoding the second polypeptide herein.

[0027] In another aspect, the disclosure provides a vector comprising a nucleic acid herein.

[0028] In another aspect, the disclosure provides a cell comprising a nucleic acid or vector herein.

[0029] In another aspect, the present disclosure provides a conjugated activatable protein, comprising the activatable protein herein conjugated to an agent. In some embodiments, the agent is a therapeutic agent, an anti-tumor agent, a toxin, a diagnostic agent, a therapeutic macromolecule, a targeting moiety, or a detectable moiety. In some embodiments, the agent is conjugated to the antibody via a linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker is a non-cleavable linker.

[0030] In another aspect, the disclosure provides a method of treating a subject in need of treatment comprising administering to the subject a therapeutically effective amount of an activatable protein, composition, or conjugated activatable protein herein, hi some embodiments, the subject has been identified or diagnosed as having cancer.

[0031] In another aspect, the disclosure provides a method of producing an activatable protein, the method comprising culturing a cell herein in a medium under conditions sufficient to produce the activatable protein, and recovering the activatable protein from the cell or medium. In some embodiments, the method further comprises isolating the activatable protein recovered from the cell or medium. In some embodiments, isolating the activatable protein is performed using protein purification tags and / or size exclusion chromatography. In some embodiments, the method further comprises formulating the activatable protein into a pharmaceutical composition.

[0032] A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments in which the principles of the invention may be utilized, and the accompanying drawings of which: [Brief description of the drawings]

[0033] [Figure 1]1 shows the configuration of an exemplary activatable molecule. The molecules are designed such that the activated molecule resulting from activation of the activatable molecule does not include a half-life extending moiety and therefore has a shorter half-life than the corresponding molecule that is the same as the activated molecule but includes a half-life extending moiety. [Diagram 2] 1 shows the configuration of an exemplary activatable molecule. The molecules are designed such that the activated molecule resulting from activation of the activatable molecule does not include a half-life extending moiety and therefore has a shorter half-life than the corresponding molecule that is the same as the activated molecule but includes a half-life extending moiety. [Diagram 3] 1 shows the configuration of an exemplary activatable molecule. The molecules are designed such that the activated molecule resulting from activation of the activatable molecule does not include a half-life extending moiety and therefore has a shorter half-life than the corresponding molecule that is the same as the activated molecule but includes a half-life extending moiety. [Figure 4] 1 shows the configuration of an exemplary activatable molecule. The molecules are designed such that the activated molecule resulting from activation of the activatable molecule does not include a half-life extending moiety and therefore has a shorter half-life than the corresponding molecule that is the same as the activated molecule but includes a half-life extending moiety. [Diagram 5] FIG. 1 is a schematic diagram of an exemplary activatable (doubly masked) bispecific antibody according to some embodiments of the present disclosure, before and after activation by a protease. On the left, an activatable doubly masked protein is illustrated. The dashed lines between elements 501 and 505, and between elements 502 and 503 indicate the cleavable moieties. On the right, an activated protein is illustrated. The activated bispecific antibody does not contain a masking moiety and therefore has increased binding affinity to its target compared to the activatable bispecific antibody. The activated bispecific antibody also does not contain a half-life extending moiety and therefore has a shorter half-life than the activatable bispecific antibody. [Figure 6A]FIG. 1 is a schematic diagram of an exemplary doubly masked bispecific activatable antibody component having a masked Fab fragment recognizing Her2, a masked scFV component recognizing CD3, and a half-life extending moiety comprising a knob and hole Fc domain pair. [Figure 6B] 6B is a schematic diagram showing the three polypeptide components encoding the exemplary double-masked bispecific activatable antibody shown in Figure 6A. The dashed lines indicate the cleavable moieties. [Figure 7A] 1 is an image of an SDS-PAGE gel run under reducing conditions. The gel was loaded with the following: (1) bispecific activatable antibody doubly masked with a 20GG CD3 mask (ProC1446, SEQ ID NO:21), (2) the product of ProC1446 and uPA (ProC1446+uPA), (3) bispecific activatable antibody doubly masked with an MN15a CD3 mask (ProC1447, SEQ ID NO:22), (4) the product of ProC1447 and uPA (ProC1447+uPA), (5) bispecific activatable antibody doubly masked with an MN15b CD3 mask (ProC1448, SEQ ID NO:23), (6) the product of ProC1448 and uPA (ProC1448+uPA). [Figure 7B] 1 is a table summarizing the predicted molecular weights of the components of each activatable antibody construct before and after protease activation. [Figure 8]1 provides the results of an ELISA binding assay to determine the ability of activatable and activating molecules to bind to plate-bound CD3 antigen: unmasked reference bispecific molecule (ProC531), activatable bispecific molecule doubly masked with a 20GG CD3 mask (ProC1446, SEQ ID NO: 21), the product of ProC1446 and uPA (ProC1446+uPA), the molecule doubly masked with an MN15a mask (ProC1447, SEQ ID NO: 22), the product of ProC1447 and uPA (ProC1447+uPA), the molecule doubly masked with an MN15b mask (ProC1448, SEQ ID NO: 23), the product of ProC1448 and uPA (ProC1448+uPA). The results show that each CD3 mask between the anti-CD3 scFv and the Fc region showed attenuation of binding of the anti-CD3 scFv to the plate-coated CD3 antigen. Treatment of the doubly masked activatable bispecific antibody with the protease uPA resulted in CD3 antigen binding comparable to the unmasked reference bispecific molecule ProC531. [Figure 9A] provides the results of a HER2-dependent cytotoxicity assay to determine the in vitro potency of a dual-masked, activatable bispecific antibody ( FIG. 9A : ProC1446). The results show that the protease-treated (activated) bispecific molecule of the present disclosure was more active than a monovalent, unmasked bispecific antibody control ("ProC306") with the same HER2 and CD3 binding domains but arranged in a different configuration. [Figure 9B] provides the results of a HER2-dependent cytotoxicity assay to determine the in vitro potency of a dual-masked, activatable bispecific antibody ( FIG. 9B : ProC1447). The results show that the protease-treated (activated) bispecific molecule of the present disclosure was more active than a monovalent unmasked bispecific antibody control ("ProC306") with the same HER2 and CD3 binding domains but arranged in a different configuration. [Figure 9C]provides the results of a HER2-dependent cytotoxicity assay to determine the in vitro potency of a dual-masked, activatable bispecific antibody (FIG. 9C: ProC1448). The results show that the protease-treated (activated) bispecific molecule of the present disclosure was more active than a monovalent, unmasked bispecific antibody control ("ProC306") with the same HER2 and CD3 binding domains but arranged in a different configuration. [Figure 10] 1 shows an exemplary configuration of an activatable molecule. The molecule comprises a double-masked activatable bispecific antibody with an EM coupled to the C-terminus via a third cleavable moiety. The molecule is designed such that the activated molecule resulting from activation of the activatable molecule does not contain a half-life extension moiety and therefore has a shorter half-life than the corresponding molecule that is the same as the activated molecule but contains a half-life extension moiety. [Figure 11] 1 shows an exemplary configuration of an activatable molecule. The molecule comprises a double-masked activatable bispecific antibody with an EM coupled to the C-terminus via a third cleavable moiety. The molecule is designed such that the activated molecule resulting from activation of the activatable molecule does not contain a half-life extension moiety and therefore has a shorter half-life than the corresponding molecule that is the same as the activated molecule but contains a half-life extension moiety. [Figure 12] FIG. 1 is a schematic diagram of an exemplary activatable (doubly masked) bispecific antibody according to some embodiments of the present disclosure, before and after activation by a protease. On the left, the activatable doubly masked protein is illustrated generally. On the right, the activated protein is illustrated generally. The activated bispecific antibody does not contain a masking moiety and therefore has increased binding affinity to its target compared to the activatable bispecific antibody. The activated bispecific antibody also does not contain a half-life extending moiety and therefore has a shorter half-life than the activatable bispecific antibody. The dashed lines indicate the cleavable moiety. [Figure 13A]Binding results (i.e., HER2 binding) are provided for masked activatable short half-life antibodies, ProC1446 (SHL1), ProC3007 (SHL2), ProC3008 (SHL2), and masked antibodies, ProC1441 (1 / 2 TCB, non-activatable, short half-life antibody), and unmasked (ProC1963 (SHL1, unmasked, no Fc), ProC1965 (SHL2, unmasked, no Fc), and ProC306) anti-CD3, anti-HER2 bispecific antibodies, and secondary antibodies ("Sec only", negative control) to NCI-N87 and SKOV3 cells, respectively. [Figure 13B] Binding results (i.e., HER2 binding) are provided for masked activatable short half-life antibodies, ProC1446 (SHL1), ProC3007 (SHL2), ProC3008 (SHL2), and masked antibodies, ProC1441 (1 / 2 TCB, non-activatable, short half-life antibody), and unmasked (ProC1963 (SHL1, unmasked, no Fc), ProC1965 (SHL2, unmasked, no Fc), and ProC306) anti-CD3, anti-HER2 bispecific antibodies, and secondary antibodies ("Sec only", negative control) to NCI-N87 and SKOV3 cells, respectively. [Figure 13C] Results of binding of the same molecules to Jurkat cells (ie, CD3 binding) are provided. [Figure 14A] 14A provides the results of a cytotoxicity assay showing the dose response of ProC1963 (SHL1), ProC1965 (SHL2), and ProC306 at the indicated concentrations using NCI-N87 cells (FIG. 14A). [Figure 14B] 14B provides the results of a cytotoxicity assay showing the dose response of ProC1963 (SHL1), ProC1965 (SHL2), and ProC306 at the indicated concentrations using SKOV3 cells (FIG. 14B). [Figure 15A] 15A provides the results of a cytotoxicity assay showing the dose response of ProC1963, ProC1965, ProC1446, ProC3007, and ProC3008 at the indicated concentrations using NCI-N87 cells (FIG. 15A). [Figure 15B] 15B provides the results of a cytotoxicity assay showing the dose response of ProC1963, ProC1965, ProC1446, ProC3007, and ProC3008 at the indicated concentrations using SKOV3 cells (FIG. 15B). [Figure 16A] The results of the cytotoxicity assay are provided. A shows the dose response of ProC1963, ProC1965, ProC3007, ProC3008, ProC306, and ProC1441 using NCI-N87 cells (FIG. 16A). [Figure 16B] The results of the cytotoxicity assay are provided in Fig. 16B, which shows the dose response of ProC1963, ProC1965, ProC3007, ProC3008, ProC306, and ProC1441 using SKOV3 cells (Fig. 16B). [Figure 16C] The results of the cytotoxicity assay are provided in Fig. 16C, which shows the dose response of ProC1963, ProC1965, ProC1446, ProC306, and ProC1441 using NCI-N87 cells (Fig. 16C). [Figure 16D] The results of the cytotoxicity assay are provided in Fig. 16D, which shows the dose response of ProC1963, ProC1965, ProC1446, ProC306, and ProC1441 using SKOV3 cells (Fig. 16D). [Figure 17] Provides the results of an in vivo tumor growth assay using the NCI-N87 xenograft model. The plot shows tumor volume versus days after initial treatment with ProC1965, ProC3007, ProC3008, and ProC1441 administered at the doses shown in milligrams per kilogram (mpk). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] The figures provided herein are for illustrative purposes only and are not necessarily drawn to scale.

[0035] Provided herein are activatable molecules (e.g., activatable proteins, such as activatable antibodies and other activatable therapeutic or activatable diagnostic proteins) that have a relatively low binding activity and a structure that includes a half-life extending moiety (EM). When activated by exposure to a specific activation condition (e.g., when the activatable molecule is delivered to a tumor), the resulting activated molecule has a higher binding activity and a shorter half-life compared to the activatable molecule. In one aspect, the activatable molecule can be an activatable therapeutic macromolecule. In some aspects, the activatable therapeutic macromolecule can be an activatable antibody or any other desired protein, e.g., a therapeutic protein.

[0036] In general, the activatable molecules herein may include one or more target binding domains (TBs), one or more masking moieties (MMs) that reduce, inhibit, or prevent binding of the TBs to their targets, one or more cleavable moieties (CMs) that couple one or more MMs to one or more TBs, and one or more half-life extending moieties (EMs) that are coupled to the TBs via one or more CMs. The coupling of two components in a polypeptide may be direct or indirect. When two components are directly coupled, the amino acid residue at the C-terminus of one component forms a peptide bond with the amino acid residue at the N-terminus of the other component. When two components are indirectly coupled, there is a stretch of amino acids between the two components. In some examples, two components of a polypeptide may be indirectly coupled via one or more other components in the polypeptide. That is, the one or more other components are between the two coupled components. When indirectly coupled or linked via another component, the one or more other components may be a linker, TB(s) (e.g., AB(s)), CM(s), MM(s), or any combination thereof.

[0037] CMs are polypeptides that contain substrates for sequence-specific proteases, e.g., proteases that are present in greater amounts (or in greater amounts in an active state) in the environment of diseased tissues, such as tumors, than in healthy tissues. The MMs and EMs of the activatable molecules described herein can be released from TB by cleaving the CM to create an activated molecule. The activated molecule exhibits higher binding affinity to its target compared to a corresponding activatable molecule that includes MM(s). In some embodiments, the activated molecule has a shorter half-life compared to a corresponding molecule that is the same as the activated molecule but includes an EM. The activated molecule can have reduced toxicity and reduced off-target effects compared to a corresponding molecule that is the same as the activated molecule but includes an EM.

[0038] In some embodiments, the activatable molecule may be a double-masked bispecific target binding molecule. In some aspects, such a molecule may comprise at least two target binding proteins and at least two masking moieties, each of which inhibits the binding of the target binding protein to its target. For example, the activatable molecule may comprise a first target binding protein (TB1) that specifically binds to a first target, a first masking moiety (MM1) that inhibits the binding of TB1 to the first target, a cleavable moiety (CM1) disposed between MM1 and TB1, a second target binding protein (TB2) that specifically binds to a second target, a second masking moiety (MM2) that inhibits the binding of TB2 to the second target, a cleavable moiety (CM2) disposed between MM2 and TB2, and an EM coupled to TB1 or TB2 via a cleavable moiety (CM). In some embodiments, the EM may be coupled to the TB via a CM that also binds the MM to the TB. In some embodiments, the EM may be coupled to the TB via a CM that is different from CM1 and CM2 (e.g., a third CM or "CM3"). In the activated state, EM can be released from the activated molecule. Thus, the activated molecule (comprising TB1 and TB2 but not including MM1, MM2 or EM) has a shorter half-life compared to the reference molecule containing TB1, TB2 and EM but not including MM1 or MM2. The activated molecule (comprising TB1 and TB2 but not including MM1, MM2 and EM) has a higher target binding activity compared to the reference molecule containing TB1, TB2 and EM but not including MM1 or MM2.

[0039] Also provided herein are related compositions, kits, nucleic acids and recombinant cells, as well as related methods, including methods of using and making any of the activatable molecules (e.g., activatable antibodies and other proteins) described herein.

[0040] 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. Methods and materials for use in this disclosure are described herein. Other suitable methods and materials known in the art can also be used. 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.

[0041] 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.

[0042] 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 one of ordinary skill in the art. For example, ±20%, ±10% or ±5%, where appropriate, within the intended meaning of the recited value.

[0043] 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 numerical 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 on a weight basis unless otherwise specified.

[0044] 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 of similar meaning, such as the terms "including" and "having" and their derivatives. The term "comprising" and its derivatives, as used herein, are intended to be closed-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. As used herein, the term "consisting essentially of" is intended to define the presence of the described features, elements, components, groups, integers and / or steps, as well as the presence of those that do not substantially 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") 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" finds direct support for any element disclosed throughout this disclosure to be so defined. Based on this definition, any element disclosed herein or incorporated by reference may be included or excluded from the claimed invention.

[0045] As used herein, for convenience, a plurality of compounds, elements, or steps may be presented in general lists. However, these lists should be construed as though each member of the list is 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 general group, unless otherwise indicated.

[0046] The term "exemplary" is used herein to mean serving as an example, instance, or illustration. Any aspect or design described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other aspects or designs. Rather, use of the word exemplary is intended to present concepts in a more specific manner.

[0047] Furthermore, 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 this 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 this disclosure and claims, all of which form the present application and claimed invention as of the filing date. For example, a listing of a composition, molecule, method step, kit, or composition described in connection with a composition, composition, or method is intended to find direct support for, and finds direct support for, embodiments related to the compositions, compositions, formulations, and methods described in any other part of this disclosure, even if those method steps, active agents, kits, or compositions are not re-enumerated in the context or section of that embodiment or aspect.

[0048] Activatable molecules In one aspect, the activatable molecule provided herein can be an activatable target binding protein (TB), such as an activatable antibody, or another protein that specifically binds to a target. In some embodiments, the activatable molecule comprises a TB that specifically binds to a target (e.g., an antigen binding protein (AB)); a cleavable moiety (CM) that is directly covalently bound to the TB (e.g., AB) (also referred to as "directly coupled") or indirectly covalently bound to the TB (e.g., AB) (also referred to as "indirectly coupled"), the CM being located between the TB and a masking moiety (MM) that reduces, inhibits, or prevents the binding of the TB (e.g., AB) to its target(s); and one or more half-life extending moieties (EMs) that are coupled to the TB (e.g., AB) via one or more CMs. The MMs and EMs can be released from the TB by cleaving the CM to generate an activated molecule. In some embodiments, the activatable molecule may include a first antigen binding protein (AB1) that specifically binds to a first target; a first masking moiety (MM1) that inhibits binding of AB1 to the first target and is coupled to AB1 via a first cleavable moiety (CM1); a second antigen binding protein (AB2) that specifically binds to a second target (AB2); a second masking moiety (MM2) that inhibits binding of AB2 to the second target and is coupled to either AB1 or AB1 via a second cleavable moiety (CM2); and an EM that is coupled to either AB1 or AB2 via a cleavable moiety. In some aspects, the EM may be coupled to AB1 via the same cleavable moiety (CM1) that couples MM1 to AB1. In some aspects, the EM may be coupled to AB2 via the same cleavable moiety (CM2) that couples MM2 to AB2. In some aspects, the EM may be coupled to AB1 via the same cleavable moiety (CM2) that couples MM2 to AB1. In some embodiments, the EM can be coupled to AB1 or AB2 via a third cleavable moiety (CM3).In each of the foregoing aspects, the elements of the activatable molecule may be directly coupled or indirectly coupled via one or more optional linkers between the elements. In an activated state, the EM may be released from the activatable protein, resulting in an activated protein that comprises AB1 and AB2 but does not comprise MM1, MM2, or EM, and the activated protein has a shorter half-life compared to a reference antibody that comprises AB1, AB2, and EM but does not comprise MM1 or MM2.

[0049] In some embodiments, the activatable protein provides for reduced toxicity and / or off-target side effects that may result from binding of TB (e.g., AB) at non-therapeutic sites if TB did not mask or otherwise inhibit binding to its target. In the activatable state, MM can interfere with the binding of TB to its target molecule.

[0050] In some embodiments, the activatable protein comprises: a first antigen binding protein (AB1) that specifically binds to a first target, where AB1 comprises an antibody or fragment thereof comprising a heavy chain fragment and a light chain fragment; a second antigen binding protein (AB2) that specifically binds to a second target, where AB2 comprises a single chain fragment variable (scFv) comprising a heavy chain variable region and a light chain variable region, where AB2 is coupled to the C-terminus of the heavy chain fragment or the light chain fragment of AB1; a first masking moiety (MM1) coupled to AB1 via a first cleavable moiety (CM1) and inhibits binding of AB1 to the first target when the activatable protein is in an uncleaved state; a second masking moiety (MM2) coupled to AB2 and inhibits binding of AB2 to the second target when the activatable protein is in an uncleaved state; and a half-life extending moiety (EM) coupled to a component of AB1 or AB2 via a second cleavable moiety (CM2). In some examples, AB1 can be a Fab. In some examples, AB1 can be a scFv. In some embodiments, EM is coupled to AB1 or AB2 by a masking moiety, e.g., EM-MM-CM-AB or AB-CM-MM-EM structures, optionally via one or more linkers between one or more components. As used herein, the symbol "-" in a structural formula indicates direct or indirect coupling of two components (e.g., an optional linker can be present between the components). The structural arrangements of the molecules of the present disclosure are described in detail below and are shown, for example, in Figures 1-6.

[0051] As used herein, the terms "activatable protein" and "activatable target binding protein" (e.g., "activatable antibody"), as well as any of the foregoing plus the terms "intact," "uncleaved," and / or "inactive," are used interchangeably to refer to a protein that comprises at least one set of MM, CM, and TB and exhibits attenuated binding to a biological target compared to the binding of a counterpart "activated" protein (e.g., an activated antibody, etc.) that comprises the same TB to the same biological target. It will be apparent to one of skill in the art that exposing an activatable protein to a CM-specific protease can produce an "activated" protein in which the MM does not reduce, inhibit, or interfere with binding between the TB (e.g., AB) and its target. In some embodiments, cleavage of the CM with a suitable protease can result in the release of the MM. In some embodiments, cleavage of the CM with a suitable protease can result in the release of the EM. The terms "activator protein," "activated target binding protein" (e.g., "activating antibody"), "truncated activatable protein," and "truncated activatable target binding protein" (e.g., "truncated activatable antibody") are used herein to refer interchangeably to the TB-containing cleavage products generated following exposure of an activatable protein to a CM-specific protease. As used throughout this disclosure, descriptions relating to activatable antibodies shall be construed as also applicable to activatable target binding proteins.

[0052] As used herein, the terms "masking moiety" and "MM" are used interchangeably to refer to a peptide or protein that, when placed proximal to a TB (e.g., AB), prevents the binding of the TB to a biological target. The terms "cleavable moiety" and "CM" are used interchangeably herein to refer to a peptide that contains a substrate for a sequence-specific protease. In an activatable protein, the CM is placed relative to the MM and the TB such that cleavage results in a molecule that can bind to the biological target of the TB. Thus, the activatable protein exhibits reduced binding to the biological target compared to the activated protein. In some embodiments, an activatable protein can be designed such that the MM provides masking of the TB or reduced binding of the TB to its target by selecting a TB of interest and constructing the remaining portion of the activatable protein. To provide this functional feature, structural design criteria can be considered.

[0053] The activatable protein may be a multispecific (e.g., bispecific, trispecific, tetraspecific, and other multispecific activatable proteins) activatable protein that is capable of binding to multiple distinct antigens when activated. In some embodiments, the multispecific activatable protein may be multivalent, e.g., comprising multiple target binding sites, whether the binding sites recognize the same or different antigens or epitopes. In some embodiments, the activatable protein may be monospecific, e.g., capable of binding to only one antigen when activated.

[0054] In some embodiments, the activatable protein is bispecific. The term "bispecific" means that the activatable protein can specifically bind to two different targets when activated. Usually, an activatable bispecific activatable protein comprises two TBs, i.e., a first TB and a second TB, each of which can specifically bind to a different target (i.e., a first target and a second target, respectively) after activation. In some embodiments, after activation, the resulting bispecific target binding molecule may be capable of simultaneously binding to two targets, for example, two target proteins expressed on two separate cells.

[0055] In some embodiments, the activatable protein may include AB1, which can bind to a molecule on the surface of a disease-associated cell (e.g., a tumor cell), and AB2, which can bind to a molecule on the surface of an immune cell. Upon activation, such a bispecific activatable protein may simultaneously bind to an immune cell and a disease-associated cell (e.g., a tumor cell), thus activating the immune cell and crosslinking the activated immune cell to the disease-associated cell. In some embodiments, the activatable protein may be formulated as part of a pro-bispecific T cell engager (pro-BiTE) molecule, a pro-chimeric antigen receptor (pro-CAR) modified T cell, or other engineered receptor or other immune effector cell, e.g., a CAR modified NK cell.

[0056] In some examples, the activatable protein can be an activatable T cell-associated bispecific antibody (TCB) or a fragment thereof. For example, the activatable protein can include AB1, which targets disease-associated cells, and AB2, which targets the T cell receptor.

[0057] The present disclosure includes activatable proteins in various structural configurations described herein. Exemplary configurations of activatable proteins are provided below. The N-terminal to C-terminal order of TB, MM, CM, and EM may be reversed in the activatable protein. CM and MM may overlap in amino acid sequence, for example, such that the CM sequence recognized by the sequence-specific protease is at least partially contained within the MM. Various structural configurations of activatable antigen-binding proteins are contemplated, for example, where AB1 is an antigen-binding fragment (Fab) and AB2 is a single-chain fragment variable, and may be represented by the following formula (in order from amino (N)-terminal region to carboxyl (C)-terminal region): In the formula below, a ":" separates two different polypeptides, "Fab_L" and "Fab_H" are the light and heavy chain fragments of Fab, respectively (where "Fab_L" comprises the variable light region (VL) and the light chain constant region, and "Fab_H" comprises the variable heavy region (VH) and the CH1 region), and "VL*" and "VH*" are the light and heavy chain variable regions of an scFv. Additionally, as used herein and unless otherwise specified, each dash (-) between components of an activatable molecule represents either a direct bond or an indirect bond via one or more linkers. (MM1-CM1-Fab_L):(Fab_H-VH*-VL*-CM2-MM2-EM) (MM1-CM1-Fab_L):(Fab_H-VH*-VL*-CM2-EM-MM2) Fab_L:(MM1-CM1-Fab_H-VH*-VL*-CM2-MM2-EM) Fab_L:(MM1-CM1-Fab_H-VH*-VL*-CM2-EM-MM2) (MM1-CM1-Fab_L-VH*-VL*-CM2-MM2-EM):Fab_H (MM1-CM1-Fab_L-VH*-VL*-CM2-EM-MM2):Fab_H (Fab_L-VH*-VL*-CM2-MM2-EM):(MM1-CM1-Fab_H) (Fab_L-VH*-VL*-CM2-EM-MM2):(MM1-CM1-Fab_H) (MM1-CM1-Fab_L-VH*-VL*):(Fab_H-CM2-MM2-EM) (MM1-CM1-Fab_L-VH*-VL*):(Fab_H-CM2-EM-MM2) (Fab_L-VH*-VL*):(MM1-CM1-Fab_H-CM2-MM2-EM) (Fab_L-VH*-VL*):(MM1-CM1-Fab_H-CM2-EM-MM2) (MM1-CM1-Fab_L-CM2-MM2-EM):(Fab_H-VH*-VL*) (MM1-CM1-Fab_L-CM2-EM-MM2):(Fab_H-VH*-VL*) (Fab_L-CM2-MM2-EM):(MM1-CM1-Fab_H-VH*-VL*) (Fab_L-CM2-EM-MM2):(MM1-CM1-Fab_H-VH*-VL*) (MM1-CM1-Fab_L):(MM2-CM2-VH*-VL*-Fab_H-CM3-EM) (MM1-CM1-Fab_H):(MM2-CM2-VH*-VL*-Fab_L-CM3-EM) (MM2-CM2-VH*-VL*-Fab_L):(MM1-CM1-Fab_H-CM3-EM) (MM2-CM2-VH*-VL*-Fab_H):(MM1-CM1-Fab_L-CM3-EM)

[0058] In any of the configurations, the activatable protein may include one or more linkers between any two of the components. For example, the activatable protein may include a linker between MM1 and CM1, a linker between CM1 and Fab_L, a linker between Fab_H and VH*, a linker between VH* and VL*, a linker between VL* and CM2, a linker between CM2 and MM2, a linker between MM2 and EM, a linker between Fab_L and VH*, a linker between CM1 and Fab_H, or any combination thereof.

[0059] In some embodiments, an EM may comprise two or more moieties (e.g., a pair of Fc domains). For example, an EM may be a protein complex comprising two moieties, EM1 and EM2. In such a case, an example of such an activatable protein may be represented by the following formula (in order from the amino (N)-terminal region to the carboxyl (C)-terminal region): (MM1-CM1-Fab_L):(Fab_H-VH*-VL*-CM2-MM2-EM1):EM2 (MM1-CM1-Fab_L):(Fab_H-VH*-VL*-CM2-EM1-MM2):EM2 Fab_L:(MM1-CM1-Fab_H-VH*-VL*-CM2-MM2-EM1):EM2 Fab_L:(MM1-CM1-Fab_H-VH*-VL*-CM2-EM1-MM2):EM2 (MM1-CM1-Fab_L):(Fab_H-VH*-VL*-CM2-EM1):(MM2-EM2) (MM1-CM1-Fab_L):(Fab_H-VH*-VL*-CM2-EM1):(EM2-MM2) Fab_L:(MM1-CM1-Fab_H-VH*-VL*-CM2-EM1):(MM2-EM2) Fab_L:(MM1-CM1-Fab_H-VH*-VL*-CM2-EM1):(EM2-MM2) (MM1-CM1-Fab_L-VH*-VL*-CM2-MM2-EM1):Fab_H:EM2 (MM1-CM1-Fab_L-VH*-VL*-CM2-EM1-MM2):Fab_H:EM2 (Fab_L-VH*-VL*-CM2-MM2-EM1):(MM1-CM1-Fab_H):EM2 (Fab_L-VH*-VL*-CM2-EM1-MM2):(MM1-CM1-Fab_H):EM2 (MM1-CM1-Fab_L-VH*-VL*-CM2-EM1):Fab_H:MM2-EM2 (MM1-CM1-Fab_L-VH*-VL*-CM2-EM1):Fab_H:EM2-MM2 (Fab_L-VH*-VL*-CM2-EM1):(MM1-CM1-Fab_H):MM2-EM2 (Fab_L-VH*-VL*-CM2-EM1):(MM1-CM1-Fab_H):EM2-MM2 (MM1-CM1-Fab_L-VH*-VL*):(Fab_H-CM2-MM2-EM1):EM2 (MM1-CM1-Fab_L-VH*-VL*):(Fab_H-CM2-EM1-MM2):EM2 (Fab_L-VH*-VL*):(MM1-CM1-Fab_H-CM2-MM2-EM1):EM2 (Fab_L-VH*-VL*):(MM1-CM1-Fab_H-CM2-EM1-MM2):EM2 (MM1-CM1-Fab_L-VH*-VL*):(Fab_H-CM2-EM1):MM2-EM2 (MM1-CM1-Fab_L-VH*-VL*):(Fab_H-CM2-EM1):EM2-MM2 (Fab_L-VH*-VL*):(MM1-CM1-Fab_H-CM2-EM1):MM2-EM2 (Fab_L-VH*-VL*):(MM1-CM1-Fab_H-CM2-EM1):EM2-MM2 (MM1-CM1-Fab_L-CM2-MM2-EM1):(Fab_H-VH*-VL*):EM2 (MM1-CM1-Fab_L-CM2-EM1-MM2):(Fab_H-VH*-VL*):EM2 (Fab_L-CM2-MM2-EM1):(MM1-CM1-Fab_H-VH*-VL*):EM2 (Fab_L-CM2-EM1-MM2):(MM1-CM1-Fab_H-VH*-VL*):EM2 (MM1-CM1-Fab_L-CM2-EM1):(Fab_H-VH*-VL*):MM2-EM2 (MM1-CM1-Fab_L-CM2-EM1):(Fab_H-VH*-VL*):EM2-MM2 (Fab_L-CM2-EM1):(MM1-CM1-Fab_H-VH*-VL*):MM2-EM2 (Fab_L-CM2-EM1):(MM1-CM1-Fab_H-VH*-VL*):EM2-MM2 (MM1-CM1-Fab_L):(MM2-CM2-VH*-VL*-Fab_H-CM3-EM1):EM2 (MM1-CM1-Fab_H):(MM2-CM2-VH*-VL*-Fab_L-CM3-EM1):EM2 (MM2-CM2-VH*-VL*-Fab_L):(MM1-CM1-Fab_H-CM3-EM1):EM2 (MM2-CM2-VH*-VL*-Fab_H):(MM1-CM1-Fab_L-CM3-EM):EM2

[0060] In some embodiments, EM1 and EM2 can be two fragment crystallizable (Fc) domains. The two Fc domains can form a dimer as a half-life extender. In some examples, EM1 and EM2 can be two identical Fc domains, thus forming a homodimer. In some constructs, EM1 and EM2 comprise Fc domains with two different amino acid sequences that together form a heterodimer. In some examples, the two Fc domains can be a hole mutant of the Fc domain and a knob mutant of the Fc domain, forming a heterodimer. In any of these configurations, the activatable protein can include one or more linkers between any two of the components. For example, the activatable protein may comprise a linker between MM1 and CM1, a linker between CM1 and Fab_L, a linker between Fab_H and VH*, a linker between VH* and VL*, a linker between VL* and CM2, a linker between CM2 and MM2, a linker between MM2 and EM, a linker between Fab_L and VH*, a linker between CM1 and Fab_H, a linker between CM2 and EM1, a linker between MM2 and EM1, a linker between MM2 and EM2, or any combination thereof.

[0061] 1-4 show exemplary configurations of activatable molecules disclosed herein. In these non-limiting examples, the activatable molecules include AB1, which may be a Fab; AB2, which may be an scFv; EM, which is a dimer formed by two Fc domains; MM1, which is coupled to AB1 via CM1 and can interfere with the binding of AB1 to its target; MM2, which can interfere with the binding of AB2 to its target; and CM2 between the Fc domain of EM and AB1 or AB2. It will be understood that the activatable molecule structures illustrated in FIGS. 1-4 can be similarly applied to molecules in which AB1 and AB2 are antigen-binding proteins other than Fab and scFv. Similarly, the activatable molecule structures illustrated in FIGS. 1-4 can be similarly applied to molecules in which AB1 and AB2 are replaced by TB1 and TB2, respectively, which may be target-binding proteins that do not necessarily include an antigen-binding domain.

[0062] 1 shows an exemplary activatable protein 100 that includes three polypeptides. The first polypeptide includes, from amino (N)-terminal region to carboxyl (C)-terminal region, MM1 101, optional linker 102, CM1 103, optional linker 104, and light chain fragment of AB1 105. The second polypeptide includes, from amino (N)-terminal region to carboxyl (C)-terminal region, heavy chain fragment of AB1 121, linker 122, heavy chain variable region of AB2 123, linker 124, light chain variable region of AB2 125, linker 126, CM2 127, optional linker 128, MM2 129, linker 130, and first Fc domain of EM 131. The third polypeptide includes second Fc domain of EM 141. In an alternative exemplary configuration, in FIG. 1, 105 is a heavy chain fragment of AB1 and 121 is a light chain fragment of AB1. In an alternative exemplary configuration, in FIG. 1, 123 is a light chain variable region of AB2 and 125 is a heavy chain variable region of AB2. In an alternative exemplary configuration, in FIG. 1, the first Fc domain of the EM, 131, is a whole mutant of the Fc domain and the second Fc domain of the EM, 141, is a knob mutant of the Fc domain. In an alternative exemplary configuration, in FIG. 1, the first Fc domain of the EM, 131, is a knob mutant of the Fc domain and the second Fc domain of the EM, 141, is a whole mutant of the Fc domain.

[0063] 2 shows another exemplary activatable protein 200 that includes three polypeptides. The first polypeptide includes, from amino (N)-terminal region to carboxyl (C)-terminal region, MM1 201, optional linker 202, CM1 203, optional linker 204, light chain fragment of AB1 205, linker 206, heavy chain variable region of AB2 207, linker 208, and light chain variable region of AB2 209. The second polypeptide includes, from amino (N)-terminal region to carboxyl (C)-terminal region, heavy chain fragment of AB1 221, optional linker 222, CM2 223, optional linker 224, MM2 225, linker 226, and first Fc domain of EM 227. The third polypeptide includes second Fc domain of EM 241. In an alternative exemplary configuration, in FIG. 2, 205 is a heavy chain fragment of AB1 and 221 is a light chain fragment of AB1. In an alternative exemplary configuration, in FIG. 2, 207 is a light chain variable region of AB2 and 209 is a heavy chain variable region of AB2. In an alternative exemplary configuration, in FIG. 2, the first Fc domain of the EM 227 is a whole mutant of the Fc domain and the second Fc domain of the EM 241 is a knob mutant of the Fc domain. In an alternative exemplary configuration, in FIG. 2, the first Fc domain of the EM 227 is a knob mutant of the Fc domain and the second Fc domain of the EM 241 is a whole mutant of the Fc domain.

[0064] 3 shows another exemplary activatable protein 300 that includes three polypeptides. The first polypeptide includes, from amino (N)-terminal region to carboxyl (C)-terminal region, MM1 301, optional linker 302, CM1 303, optional linker 304, light chain fragment of AB1 305, linker 306, heavy chain variable region of AB2 307, linker 308, and light chain variable region of AB2 309. The second polypeptide includes, from amino (N)-terminal region to carboxyl (C)-terminal region, heavy chain fragment of AB1 321, optional linker 322, CM2 323, optional linker 324, and first Fc domain of EM 325. The third polypeptide includes, from amino (N)-terminal region to carboxyl (C)-terminal region, second Fc domain of EM 341, linker 342, and MM2 343. In an alternative exemplary configuration, in FIG. 3, 305 is a heavy chain fragment of AB1 and 321 is a light chain fragment of AB1. In an alternative exemplary configuration, in FIG. 3, 307 is a light chain variable region of AB2 and 309 is a heavy chain variable region of AB2. In an alternative exemplary configuration, in FIG. 3, the first Fc domain of the EM 325 is a whole mutant of the Fc domain and the second Fc domain of the EM 341 is a knob mutant of the Fc domain. In an alternative exemplary configuration, in FIG. 3, the first Fc domain of the EM 325 is a knob mutant of the Fc domain and the second Fc domain of the EM 341 is a whole mutant of the Fc domain.

[0065] 4 shows another exemplary activatable protein 400 that includes three polypeptides. The first polypeptide includes, from amino (N)-terminal region to carboxyl (C)-terminal region, MM1 401, optional linker 402, CM1 403, optional linker 404, light chain fragment of AB1 405, linker 406, heavy chain variable region of AB2 407, linker 408, and light chain variable region of AB2 409. The second polypeptide includes, from amino (N)-terminal region to carboxyl (C)-terminal region, heavy chain fragment of AB1 421, optional linker 422, CM2 423, optional linker 424, and first Fc domain of EM 425. The third polypeptide includes, from amino (N)-terminal region to carboxyl (C)-terminal region, MM2 441, linker 442, and second Fc domain of EM 443. In an alternative exemplary configuration, in FIG. 4, 405 is a heavy chain fragment of AB1 and 421 is a light chain fragment of AB1. In an alternative exemplary configuration, in FIG. 4, 407 is a light chain variable region of AB2 and 409 is a heavy chain variable region of AB2. In an alternative exemplary configuration, in FIG. 4, the first Fc domain of the EM 425 is a hole mutant of the Fc domain and the second Fc domain of the EM 443 is a knob mutant of the Fc domain. In an alternative exemplary configuration, in FIG. 4, the first Fc domain of the EM 425 is a knob mutant of the Fc domain and the second Fc domain of the EM 443 is a hole mutant of the Fc domain.

[0066] 5 shows an exemplary activatable bispecific antibody comprising a Fab component (501) that binds a first target; a first prodomain comprising CM1 and MM1 that masks the Fab component (505); an scFv component (502) that binds a second target; a second prodomain comprising CM2 and MM2 that masks the scFv component (503); and an EM (504) that comprises a knob and hole Fc domain pair. Upon activation, CM1 is cleaved from the activated bispecific antibody to release MM1, and CM2 is cleaved to release both MM2 and the Fc domain (504). An activated bispecific antibody lacking an Fc domain has a relatively short half-life compared to its parent activatable bispecific antibody.

[0067] FIG. 6A shows an exemplary activatable bispecific antibody targeting Her2 and CD3. In this example, the activatable bispecific antibody comprises three polypeptides. The first polypeptide comprises, in order from N-terminus to C-terminus, a heavy chain fragment of Fab of trastuzumab (anti-HER2 antibody); a linker having 25 amino acids; an anti-CD3 scFv; a GSAT linker having 27 amino acids; CM1; MM1 masking the anti-CD3 scFv; a GS linker having 24 amino acids; and a hole mutant of the Fc domain. The second polypeptide comprises MM2, CM2, and a light chain fragment of the Fab to generate a Fab. The third polypeptide comprises a knob mutant of the Fc domain. An example of an activatable bispecific antibody having the configuration of FIG. 6 may comprise a first polypeptide comprising any one of the sequences of SEQ ID NOs: 21-24, a second polypeptide comprising the sequence of SEQ ID NO: 18, and a third polypeptide comprising the sequence of SEQ ID NO: 1. FIG. 6B is a schematic diagram of the three polypeptides forming the activatable bispecific antibody shown in FIG. 6A.

[0068] 10 shows another exemplary activatable protein 1000 that includes three polypeptides. The first polypeptide includes, from amino (N) terminal region to carboxyl (C) terminal region, MM1 1001, optional linker 1002, CM1 1003, optional linker 1004, and light chain fragment of AB1 1005. The second polypeptide includes, from amino (N) terminal region to carboxyl (C) terminal region, MM2 1021, optional linker 1022, CM2 1023, optional linker 1024, heavy chain variable region of AB2 1025, linker 1026, light chain variable region of AB2 1027, linker 1028, heavy chain fragment of AB1 1029, optional linker 1030, third cleavable portion (CM3) 1031, optional linker 1032, and first domain of EM (EM1) 1033. The third polypeptide comprises a second domain of EM (EM2) 1041. In an alternative exemplary configuration, in FIG. 10, 1005 is a heavy chain fragment of AB1 and 1029 is a light chain fragment of AB1. In an alternative exemplary configuration, in FIG. 10, 1025 is a light chain variable region of AB2 and 1027 is a heavy chain variable region of AB2. In an alternative exemplary configuration, in FIG. 10, the first domain of EM (EM1) 1033 is a hole mutant of the Fc domain and the second domain of EM (EM2) 1041 is a knob mutant of the Fc domain. In an alternative exemplary configuration, in FIG. 10, EM1 1033 is a knob mutant of the Fc domain and EM2 1041 is a hole mutant of the Fc domain.

[0069] 11 shows another exemplary activatable protein 1100 that includes three polypeptides. The first polypeptide includes, from amino (N) terminal region to carboxyl (C) terminal region, MM2 1101, optional linker 1102, CM2 1103, optional linker 1104, heavy chain variable region of AB2 1105, linker 1106, light chain variable region of AB2 1107, linker 1108, and light chain fragment of AB1 1109. The second polypeptide includes, from amino (N) terminal region to carboxyl (C) terminal region, MM1 1121, optional linker 1122, CM1 1123, optional linker 1124, heavy chain fragment of AB1 1125, optional linker 1126, third cleavable portion (CM3) 1127, optional 1128, and first domain of EM (EM1) 1129. The third polypeptide comprises a second domain of EM (EM2) 1141. In an alternative exemplary configuration, in FIG. 11, 1109 is a heavy chain fragment of AB1 and 1125 is a light chain fragment of AB1. In an alternative exemplary configuration, in FIG. 11, 1105 is a light chain variable region of AB2 and 1107 is a heavy chain variable region of AB2. In an alternative exemplary configuration, in FIG. 11, EM1 1129 is a hole mutant of the Fc domain and EM2 1141 is a knob mutant of the Fc domain. In an alternative exemplary configuration, in FIG. 11, EM1 1129 is a knob mutant of the Fc domain and EM2 1141 is a hole mutant of the Fc domain.

[0070] FIG. 12 shows an exemplary activatable bispecific antibody comprising a Fab component (1204) that binds to a first target; a first prodomain (1203) comprising CM1 (dashed line) and MM1 (triangle) that masks the Fab component; an scFv component (1202) that binds to a second target; a second prodomain (1201) comprising CM2 (dashed line) and MM2 (triangle) that masks the scFv component; an EM (1206) that comprises a knob and hole Fc domain pair; and a third cleavable moiety (CM3) (1205) between the EM and the Fab. Upon activation, CM1 is cleaved to release MM1, CM2 is cleaved to release MM2, and CM3 is cleaved to release EM (1206) from the activated bispecific antibody. An activated bispecific antibody lacking an EM has a relatively short half-life compared to its parent activatable bispecific antibody.

[0071] In some embodiments, the activated protein resulting from the activation of the activatable protein of the present disclosure is not attached to an EM. Such an activated protein may have a shorter half-life compared to the activatable protein. Such an activated protein may have a shorter half-life compared to a corresponding protein that is the same as the activated protein but includes an EM. As used herein, the term "half-life" is the time it takes for the concentration of a molecule or molecular complex to reach 50% of its original concentration in an environment. In some examples, the environment may be serum, and the half-life is the serum half-life, which is the time it takes for the concentration of a molecule or molecular complex to reach 50% of its original concentration in serum (e.g., in the circulation of a subject). In some examples, an activated protein that includes AB1 and AB2 but does not include MM1, MM2, or EM (i.e., a protein resulting from the activation of an activatable protein) may have a shorter half-life compared to a corresponding protein that is the same as the activated protein but includes an EM. That is, the half-life of the activated molecule (AB1-AB2) is shorter than the half-life of the corresponding protein (AB1-AB2-EM).

[0072] For example, an activated protein resulting from activation of an activatable protein herein may have a half-life (e.g., serum half-life) of less than 15 days, 14 days, 13 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, 23 hours, 22 hours, 21 hours, 20 hours, 19 hours, 18 hours, 17 hours, 16 hours, 15 hours, 14 hours, 13 hours, 12 hours, 11 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, or 3 hours. In one example, an activated protein resulting from activation of an activatable protein herein may have a half-life (e.g., serum half-life) of 5, 4, 3, or 2 days or less. In some examples, the activated protein resulting from activation of an activatable protein herein may have a half-life (e.g., serum half-life) that is up to 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5% of the corresponding protein that is the same as the activated protein but contains an EM.

[0073] In some embodiments, an activated protein resulting from activation of an activatable protein herein (i.e., an activated protein that is not attached to an EM or MM) may have a higher target binding activity compared to a corresponding protein that is the same as the activated protein but has an EM attached thereto. In some examples, an activated protein that includes TB1 and TB2 but does not include MM1, MM2 or an EM has a level of target binding activity that is greater than a corresponding protein that is the same as the activated protein but includes an EM (i.e., TB1-TB2-EM). For example, an activated protein resulting from activation of an activatable protein disclosed herein may have a target binding activity that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 3-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, or 500-fold greater than the target binding activity of a corresponding protein that is the same as the activated protein but contains an EM.

[0074] In some embodiments, the activatable protein (prior to activation) may be characterized, either directly or indirectly, by a target binding activity that is lower than a control level of target binding activity of a TB not coupled to it with a MM. For example, in some embodiments, the activatable protein is characterized by at least 2, 4, 6, 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 5000, or 10000 times lower target binding activity compared to the control level of target binding activity of a TB not coupled to it with a MM.

[0075] Target-binding proteins An activatable protein according to the present disclosure may include one or more target binding proteins (TBs). In some examples, an activatable protein may be multispecific. For example, an activatable protein may include multiple TBs, each with specificity for a different epitope on the same target. In some examples, the TBs of an activatable protein herein may bind to different targets, e.g., targets on different cell types. Thus, in an activated protein resulting from activation of an activatable protein disclosed herein, the TBs may co-localize with different cell types. In some examples of a multispecific activatable protein of the present disclosure, one of the TBs binds to a target on an immune cell, and another of the TBs binds to a cell associated with a disease. By targeting and co-localizing immune cells and cells associated with a disease, the activated protein may provide targeted treatment of the disease.

[0076] In some embodiments, the target binding protein (TB) can be an antigen binding protein (AB). In some embodiments, the AB can 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 (nanobody, e.g., single domain heavy chain antibody, single domain light chain antibody). A single domain antibody can be an antibody fragment that is a single monomeric variable antibody domain. A single domain antibody can have an affinity for an antigen similar to a corresponding full-length antibody. In some embodiments, the AB can be a full-length antibody. In some embodiments, the AB can be an immunologically active fragment. In some embodiments, the AB can be an antigen binding fragment ("Fab"). In one example, the activatable protein includes a Fab as the first AB and a scFv as the second AB. In some embodiments, the AB can be a scFv. 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 above domains, such as 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.

[0077] The term "antibody" is used herein in its broadest sense and includes a specific type of immunoglobulin molecule that contains one or more antigen binding domains that specifically bind to an antigen or epitope. The term "antibody" specifically includes, for example, intact antibodies (e.g., intact immunoglobulins), antibody fragments, bispecific, and multispecific antibodies. An example of an antibody is V H -V L It is the antigen-binding domain formed by a dimer. Further examples of antibodies are described herein. Further examples of antibodies are known in the art.

[0078] 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.

[0079] The "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 (in humans, IgG1, 2, 3, and 4).

[0080] A "fragment antigen binding" (Fab) contains an intact light chain paired with the VH and CH1 domains of a heavy chain. An AF(ab')2 fragment is formed when an antibody is cleaved below the hinge region with pepsin, in which case the two fragment antigen binding domains (Fab) of the antibody molecule remain linked. An AF(ab')2 fragment contains two intact light chains paired with the two domains VH and CH1 of the heavy chain joined together by the hinge region. A "fragment crystallizable" (Fc) fragment (referred to herein as Fc) is a fragment that is formed when an antibody is cleaved below the hinge region with pepsin, in which case the two fragment antigen binding domains (Fab) of the antibody molecule remain linked. An AF(ab')2 fragment contains two intact light chains paired with the two domains VH and CH1 of the heavy chain joined together by the hinge region. 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. The "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 stretch of synthetic peptide. The name single-chain Fv comes from the fragment variable. The "hinge region" or "interdomain" is a flexible stretch of amino acids that joins or links the Fab fragment to the Fc domain. The "synthetic hinge region" is the amino acid sequence that joins or links the Fab fragment to the Fc domain.

[0081] A "prodomain" has a portion that inhibits antigen binding, called the masking portion (MM), and a portion that contains a protease-cleavable substrate, called the cleavable peptide (CM), and when linked to a target binding protein (TB) (e.g., an antigen binding protein (AB), such as an antibody or antigen-binding fragment thereof), 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.

[0082] 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 smaller K d represents a greater affinity. The immunological binding properties of a selected polypeptide 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 off Both the concentration and the actual rates of association and dissociation can be determined by calculating K off / K on The ratio ofd (See generally, Davies et al. (1990) Annual Rev Biochem 59:439-473). The TB or antibody binding domain (AB) of the present disclosure has a dissociation constant (K d In some embodiments, an antibody is said to "specifically bind" or "immunospecifically bind" to a target when the binding affinity of the antibody is 100 μM or less, in some embodiments 1 μM or less, in some embodiments 100 nM or less, in some embodiments 10 nM or less, and in some embodiments 100 pM or less to about 1 pM.

[0083] The target of TB (e.g., AB) can be a protein or other type of molecule. Exemplary 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.

[0084] In some embodiments, the TB in the activatable protein may bind to a target that is a molecule on or in a cell associated with a disease. For example, the TB in the activatable protein may bind to a tumor cell. In such a case, the TB may bind to a tumor-associated antigen. As used herein, the term "tumor-associated antigen" refers to any antigen, including proteins, glycoproteins, gangliosides, carbohydrates, lipids, associated with cancer. Such antigens may be expressed on tumor cells (e.g., malignant cells) or in the tumor microenvironment, for example, on tumor-associated blood vessels, extracellular matrix, mesenchymal stroma, or immune infiltrates. In some embodiments, the tumor-associated antigen that is the target of the AB may be human epidermal growth factor receptor 2 (HER2). For example, the AB may be trastuzumab or a fragment thereof, for example, the Fab of trastuzumab.

[0085] In some embodiments, the AB in the activatable protein may bind to a target that is a molecule on an immune cell and / or that can activate an immune cell. In some examples, the target of the AB may be a costimulatory molecule, which is a cell surface molecule other than an antigen receptor or its ligand that is required for a highly efficient immune response. Examples of costimulatory molecules that may be targets of the AB include components of the T cell receptor (TCR), CD3 zeta, CD3 gamma, CD3 delta, and CD3 epsilon.

[0086] In some examples, the AB may bind to a costimulatory molecule expressed on the surface of a T lymphocyte, e.g., a cytotoxic T lymphocyte, which can interact with the antigen binding molecule to induce activation of the T cell. The interaction of the antigen binding molecule with an activating T cell antigen can induce activation of the T cell by triggering a signaling cascade of the T cell receptor complex. Once activated, the AB may bind to such a costimulatory molecule to activate the T cell. As used herein, "T cell activation" refers to one or more cellular responses of T lymphocytes, particularly cytotoxic T lymphocytes, selected from proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, and expression of activation markers. The T cell activating bispecific antigen binding molecules of the present invention can induce activation of the T cell.

[0087] In some examples, AB can bind to CD3. For example, CD3 can be the epsilon subunit of CD3, e.g., NCBI RefSeq number NP_000724.1. In some examples, AB can be an anti-CD3 scFv. The anti-CD3 scFv can include one or more of the sequences of SEQ ID NOs: 1-9, 143-145, 149, and 150 of US20190135943, which is incorporated herein by reference in its entirety. Such sequences include, for example, the following: [Table 6-1] [Table 6-2] [Table 6-3]

[0088] Exemplary CDR sequences for CD3 binding antibodies include the following: [Table 7]

[0089] Additional examples of anti-CD3 AB include: [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4] [Table 8-5] [Table 8-6]

[0090] In some embodiments, the activatable protein herein may comprise AB1 that binds to a tumor-associated antigen and AB2 that binds to a costimulatory molecule. In one example, the activatable protein may comprise AB1 that binds to HER2 and AB2 that binds to CD3. In a particular example, the activatable protein may comprise AB1 that is an anti-HER2 Fab (e.g., a Fab of trastuzumab) and AB2 that is an anti-CD3 scFv.

[0091] In some embodiments, AB2 can bind to a target that is an antigen on any immune effector cell. Examples of immune effector cells include leukocytes, T cells, natural killer (NK) cells, macrophages, monocytes, and myelomonocytic cells. In some examples, the activatable protein can include an immune effector cell associated with a bispecific activatable antibody that crosslinks the immune effector cell with another cell (e.g., a cell associated with a disease such as cancer or an infectious disease).

[0092] The activatable protein may comprise a bispecific activatable antibody associated with a white blood cell, a bispecific activatable antibody associated with a T cell, a bispecific activatable antibody associated with a NK cell, a bispecific activatable antibody associated with a macrophage cell, a bispecific activatable antibody associated with a mononuclear cell, or a bispecific activatable antibody associated with a myelomononuclear cell. In one example, the activatable antibody may comprise a bispecific antibody associated with a T cell.

[0093] Half-life extension moiety The activatable protein may include a half-life extending moiety (EM). In the activatable protein, the EM may be coupled to the TB or a component thereof in the activatable protein via the CM. Upon activation of the activatable protein, the EM may be cleaved from the TB. In some embodiments, for example, the CM is located at a position between the C-terminus of the TB and the N-terminus of the EM. In certain of these embodiments, the CM is located at a position between the C-terminus of the TB and the N-terminus of the EM, and the MM is located at a position that is C-terminal to the CM and either N-terminal or C-terminal to the EM (e.g., from N-terminus to C-terminus: TB-CM-EM, TB-CM-EM-MM, TB-CM-MM-EM, etc., where each "-" independently indicates direct or indirect (e.g., via a linker) coupling) (see, e.g., Figures 1 and 2). In some embodiments, the EM may be a dimer, e.g., a pair of Fc domains of immunoglobulins. In such an embodiment, the first polypeptide may comprise a TB, a CM, and a first Fc domain, and the second polypeptide may comprise a MM and a second Fc domain, and the two polypeptides are covalently linked via one or more disulfide bonds between the first Fc domain and the second Fc domain. In such an embodiment, the MM may be located either at the N-terminus or C-terminus of the second Fc domain (see, e.g., Figures 3 and 4), and cleavage of the CM on the first polypeptide releases the MM and EM (e.g., both Fc domains) from the activated protein. Thus, in some embodiments, the activated protein resulting from activation of the activatable protein does not contain an EM. In some examples, the half-life extending moiety may be a serum half-life extending moiety, i.e., it can extend the half-life of a molecule bound to the EM in serum.

[0094] In some examples, the EM may comprise a fragment crystallizable region (Fc domain) of an antibody. For example, the EM may be an Fc domain of an IgG (e.g., IgG1, IgG2, or IgG4). In some examples, the EM may comprise a dimer formed by two Fc domains. The Fc domain may be a wild-type Fc domain, or a mutant thereof. For example, the EM may comprise a dimer formed by two Fc domain mutants. In such a case, the two Fc domain mutants may comprise a hole mutant of the Fc domain and a knob mutant of the Fc domain. The knob and hole mutants may interact with each other to promote dimerization of the two Fc domains. In some embodiments, the knob and hole mutants may comprise one or more amino acid modifications within the interface between the two Fc domains (e.g., in the CH3 domain). In one example, the modification includes the amino acid substitution T366W, and optionally the amino acid substitution S354C, in one of the antibody heavy chains, and the amino acid substitutions T366S, L368A, Y407V, and optionally Y349C in the other of the antibody heavy chains (numbered according to the EU numbering system). An example of a knob mutant of the Fc domain includes the sequence of SEQ ID NO: 1. An example of a hole mutant of the Fc domain includes the sequence of SEQ ID NO: 2.

[0095] Examples of Fc domain variants also include those described in U.S. Patent No. 7,695,936, which is incorporated herein by reference in its entirety. In one example, the modification comprises the amino acid substitution T366Y in one IgG Fc domain and the amino acid substitution Y407T in the other IgG Fc domain. In one example, the modification comprises the amino acid substitution T366W in one IgG Fc domain and the amino acid substitution Y407A in the other IgG Fc domain. In one example, the modification comprises the amino acid substitution F405A in one IgG Fc domain and the amino acid substitution T394W in the other IgG Fc domain. In one example, the modification comprises the amino acid substitutions T366Y and F405A in one IgG Fc domain and the amino acid substitutions T394W and Y407T in the other IgG Fc domain. In one example, the modification comprises the amino acid substitutions T366W and F405W in one IgG Fc domain and the amino acid substitutions T394S and Y407A in the other IgG Fc domain. In one example, the modification comprises the amino acid substitutions F405W and Y407A in one IgG Fc domain and the amino acid substitutions T366W and T394S in the other IgG Fc domain. In one example, the modification comprises the amino acid substitution F405W in one IgG Fc domain and the amino acid substitution T394S in the other IgG Fc domain. The mutation positions within the Fc domain are numbered according to the EU numbering system. The IgG Fc domain may comprise the sequences of SEQ ID NOs: 3-6 (IgG1, IgG2, IgG3 or IgG4). In these sequences, amino acids 1-107 correspond to EU numbering 341-447.

[0096] In some examples, the Fc domain variants may have reduced effector function. Examples of such Fc domains include those disclosed in US20190135943, which is incorporated herein by reference in its entirety.

[0097] Further examples of EMs include immunoglobulins (e.g., IgG), serum albumins (e.g., human serum albumin (HSA), hexa-hat GST (glutathione S-transferase) glutathione affinity, calmodulin binding peptide (CBP), strep tag, cellulose binding domain, maltose binding protein, S-peptide tag, chitin binding tag, immunoreactive epitopes, epitope tags, E2 Tag, HA epitope tag, Myc epitope, FLAG epitope, AU1 and AU5 epitopes, Glu-Glu epitope, KT3 epitope, IRS epitope, Btag epitope, protein kinase-C epitope, and VSV epitope.

[0098] In some embodiments, the serum half-life of the activatable protein may be longer than that of a corresponding protein that is the same as the activatable protein but does not have a half-life extending moiety. In some embodiments, the serum half-life of the activatable protein may be longer than the serum half-life of the activated protein. In some embodiments, the serum half-life of the activatable protein is at least 15 days, 12 days, 11 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, 20 hours, 18 hours, 16 hours, 14 hours, 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, 3 hours, 2 hours, or 1 hour when administered to an organism.

[0099] Masking part (MM) The activatable proteins herein may contain one or more masking moieties (MM) that can interfere with the binding of the TB to a target. A masking moiety in an activatable molecule "masks" or reduces or otherwise inhibits the binding of the activatable macromolecule to its target and / or epitope. In some embodiments, coupling or modification of a target binding protein (TB) (e.g., AB or other therapeutic or diagnostic protein) with a MM may inhibit the ability of the TB to specifically bind to its target and / or epitope by inhibitions known in the art (e.g., conformational changes, competition for antigen binding domains, etc.). In some embodiments, coupling or modification of a TB with a MM may result in a conformational change that reduces or inhibits the ability of the TB to specifically bind to its target and / or epitope. In some embodiments, coupling or modification of a protein that includes an antigen binding domain with a MM sterically blocks, reduces or inhibits the ability of the antigen binding domain to specifically bind to its target and / or epitope. The MM can be coupled to the TB (eg, AB) either directly or indirectly (eg, via one or more linkers described herein) by the CM.

[0100] Alternatively, the MM that interferes with target binding of the TB can be coupled to a component of the activatable protein that is not the TB. For example, as illustrated in Figure 2, the activatable protein may include TB1 and TB2, and the MM that interferes with TB2 can be coupled to TB1. In another example, as illustrated in Figures 3 and 4, the activatable protein may include TB1, TB2, and EM, and the MM that interferes with TB2 can be coupled to EM. In either case, in the tertiary or quaternary structure of the activatable structure, the MM can be in a position that allows the MM to mask the TB (e.g., proximal to the TB to be masked).

[0101] In some embodiments, the MM may interact with TB, thereby reducing or inhibiting the interaction between TB 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 of TB. 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 polypeptide of TB (e.g., AB). For example, the MM may comprise the sequence of an epitope of TB or a fragment thereof. As used herein, the term "naturally occurring" as applied to an object refers to the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence present in an organism (including a virus) that can be isolated from a natural source and has not been intentionally modified by humans in the laboratory or is otherwise naturally occurring.

[0102] 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 TB. In some embodiments, the MM does not contain a subsequence of more than 4, 5, 6, 7, 8, 9, or 10 consecutive amino acid residues of a natural binding partner of TB. The MM may be a modified binding partner of TB that contains amino acid changes that reduce binding affinity and / or activity to TB. In some embodiments, the MM may not contain, or may not substantially contain, nucleic acid or amino acid homology with a natural binding partner of TB. In other embodiments, the MM is no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% similar to a natural binding partner of TB.

[0103] In some embodiments, the MM may not specifically bind to the TB (or other activatable protein), but prevents binding of the target binding protein (e.g., AB) to its binding partner through non-specific interactions, e.g., steric hindrance. For example, the MM may be positioned in the activatable protein such that the tertiary or quaternary structure of the activatable protein allows the MM to mask the AB through charge-based interactions, thereby holding the MM in place and preventing the binding partner from approaching the TB.

[0104] 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 TB for the target. In some embodiments, the MM may not interfere with or compete with the TB for binding to the target in a cleaved state.

[0105] The structural characteristics of the MM can be selected according to factors such as the minimum amino acid sequence required to prevent a protein from binding to a target, the target protein-protein binding pair of interest, the size of the TB, the presence or absence of a linker, and the like.

[0106] In some embodiments, the MM may be specific to the coupled TB. Examples of MM include MMs that have been specifically screened to bind to a binding domain of a TB, e.g., an AB, or a fragment thereof (e.g., an affinity mask). Methods for screening MMs to obtain MMs that are specific to a TB and that specifically and / or selectively bind to a binding domain of a binding partner / target are provided herein and can include protein display methods.

[0107] As used herein, the term "masking efficiency" or "ME" refers to the activity (e.g., EC50) of an activatable protein divided by the activity of a control target binding protein (e.g., an antibody), which can be either a cleavage product of the activatable protein (i.e., an activated protein) or a target binding protein (e.g., an antibody or fragment thereof) used as the TB of the activatable protein. An activatable protein having reduced levels of target binding activity can have a masking efficiency of greater than 10. In some embodiments, the activatable proteins described herein can have a masking efficiency of greater than 10, 100, 1000, or 5000.

[0108] In some embodiments, the MM can be a peptide of about 2-50 amino acids in length. For example, the MM can be a peptide of 2-40, 2-30, 2-20, 2-10, 5-15, 10-20, 15-25, 20-30, 25-35, 30-40, 35-45, 40-50 amino acids in length. For example, the MM can be a peptide of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length. In some instances, the MM can be a polypeptide greater than 50 amino acids in length, for example, a polypeptide of 100, 200, 300, 400, 500, 600, 700, 800, or more amino acids.

[0109] In some embodiments, the activatable protein exhibits a TB and blocking MM in the presence of a TB target, as measured in vivo, or in a masking efficiency assay, as described in, for example, US 20200308243 A1. There is no or substantially no binding of TB to the target, or 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% binding of TB to its target, compared to the binding of the corresponding antibody without the interfering MM, for at least 0.1, 0.5, 1, 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, 96 hours, or 5, 10, 15, 30, 45, 60, 90, 120, 150, 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months, as measured by an in vitro immunosorbent assay. For example, the ability of MM to inhibit binding of an activatable protein to its binding partner at therapeutically relevant concentrations and times can be measured, in which an immunoabsorption assay (MEA, Mask Efficiency Assay) for measuring time-dependent binding of an activatable protein to its binding partner has been developed and described in US20200308243A1, the entirety of which is incorporated herein by reference.

[0110] The binding affinity of the TB for a target or binding partner with an interfering MM is 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, 50,000,000-fold, or 50,000,000-fold lower than the binding affinity of the TB for that binding partner in the absence of the interfering MM, or is at least 5-fold, 10-100-fold, 10-1,000-fold, 10-1,000-fold, or 10-1,000-fold lower than the binding affinity of the TB for that binding partner in the absence of the interfering MM. 0,000x, 10~100,000x, 10~1,000,000x, 10~10,000,000x, 100~1,000x, 100~10,000 times, 100~100,000 times, 100~1,000,000 times, 100~10,000,000 times, 1,000~10,000 times, 1,000~100, 000 times, 1,000-1,000,000 times, 1000-10,000,000 times, 10,000-100,000 times, 10,000-1,000,000 times, 10,000-10,000,000 times, 100,000-1,000,000 times, or 100,000-10,000,000 times lower.

[0111] The dissociation constant (K d ) is the K of the TB (e.g., AB) against the target d K of MM for masked TB may be larger than d is the K of TB against the target dThe binding affinity of the MM to the masked TB 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 to the target. Conversely, the binding affinity of the MM to the masked TB may be lower than the binding affinity of the TB to the target. The binding affinity of the MM to the TB 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 less than the binding affinity of the TB to the target.

[0112] In some embodiments, MM may contain genetically encoded 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, MM contains no more than 50%, 40%, 30%, 20%, 15%, 10%, 5%, or 1% non-genetically encoded amino acids.

[0113] In some embodiments, MM may have biological activity or therapeutic effect, e.g., binding ability, when released from activatable protein and in a free state. For example, free MM may bind to the same or different binding partners. In certain embodiments, free MM may exert therapeutic effect and provide secondary function to the compositions disclosed herein. In some embodiments, MM may advantageously not exhibit biological activity when released from activatable protein and in a free state. For example, in some embodiments, free MM does not induce immune response in a subject.

[0114] Suitable MMs can be identified and / or further optimized from a library of candidate activatable proteins having various MMs through screening procedures. 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 through 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.

[0115] In some embodiments, MMs with a particular binding affinity to TB (e.g., AB) can be identified through a screening procedure that includes providing a library of peptide scaffolds containing 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 with 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 then determining the masking efficiency, for example, as described in WO2009025846 and US20200308243A1, which are incorporated herein by reference in their entirety.

[0116] In some embodiments, the MM may be selected for use with a particular protein, antibody, or antibody fragment. For example, a suitable MM for use with an AB that binds to an epitope may include the sequence of the epitope. In one example, if the activatable comprises AB1, an anti-HER2 Fab, and AB2, an anti-CD3 scFv, MM1 (to mask AB1) may include the sequence of HER2 to which AB1 binds, and MM2 (to mask AB2) may include the sequence of CD3 to which AB2 binds. In other embodiments, the MM may not include the sequence of the natural binding partner of TB. In some examples, a suitable MM1 for masking an anti-HER2 Fab comprises the sequence of ALICCSDVSGLCRWC (SEQ ID NO: 40). In some examples, suitable MM2 for masking anti-CD3 scFv includes MM comprising the sequence of GYLWGCEWNCGGITT (SEQ ID NO: 34), NAFRCWWDPPCQPMT (SEQ ID NO: 35), ARGLCWWDPPCTHDL (SEQ ID NO: 36), or NHSLCYWDPPCEPST (SEQ ID NO: 37). In some examples, suitable MM2 for masking anti-CD3 scFv includes MM comprising the sequence of MMYCGGNEVLCGPRV (SEQ ID NO: 66), GYRWGCEWNCGGITT (SEQ ID NO: 67), MMYCGGNEIFCEPRG (SEQ ID NO: 68), GYGWGCEWNCGGSSP (SEQ ID NO: 69), or MMYCGGNEIFCGPRG (SEQ ID NO: 70).

[0117] Further suitable MMs are WO2021207657, WO2021142029, WO2021061867, WO2020252349, WO2020252358, WO2020236679, WO2020176672, WO2020118109, WO2020092881, WO2020086665, WO2019213444, WO2019183218, WO2019173771, WO2019165143, WO201907 No. 5405, WO2019046652, WO2019018828, WO2019014586, WO2018222949, WO2018165619, WO2018085555, WO2017011580, WO2016179335, WO2016179285, WO2016179257, WO2016149201, and WO2016014974, which are incorporated by reference in their entireties.

[0118] Cuttable Part (CM) The activatable protein may contain one or more cleavable moieties (CM) as defined above.

[0119] In some embodiments, the activatable protein may include a CM between the TB (e.g., AB) and the MM. The activatable protein may further include a CM between the TB and the EM. In some instances, the CM between the TB and the MM is also between the TB and the EM (see, e.g., FIG. 1, where CM127 is between TB123 / 125 and MM129, and CM127 is also between TB123 / 125 and MM131 / 141). In such cases, cleavage of the CM may release both the MM and the EM from the TB. In some examples, the CM is disposed between a first TB (TB1) and a MM (MM2) that binds to a second TB (TB2) (see, e.g., FIG. 2, where CM223 is disposed between the first TB (TB221) and MM225, which is a masking moiety that inhibits binding of the second TB (TB207 / 209). See also FIG. 3, where CM323 is disposed between the first TB (TB321) and MM343, which is a masking moiety that inhibits binding of the second TB (TB307 / 309) to its target). In certain examples, the CM between the TB and MM is not between the TB and EM. In such cases, the activatable protein may include a first CM between the TB and MM, and a second CM between the TB and EM. In some instances, an activatable protein may have three CMs: a first CM between the first TB and the first MM, a second CM between the second TB and the second MM, and a third CM between the EM and the first or second TB (see, e.g., Figures 10 and 11). Activation of the activatable protein may cleave both CMs, such that both the MM and EM are released from the EM.

[0120] The CM and TB of an activatable protein may be selected such that the TB comprises a binding moiety for a given target, the CM comprises a substrate for one or more proteases, and the one or more proteases co-localize with the target within a tissue (e.g., at a therapeutic or diagnostic site of a subject). In some embodiments, an activatable protein may find particular use when, for example, one or more proteases capable of cleaving a moiety in the CM are present at relatively higher levels (or are more active) in target-containing tissue at a therapeutic or diagnostic site than in tissue at a non-therapeutic site (e.g., healthy tissue).

[0121] 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 prevalently found in cancer cells and tissues. Thus, in certain embodiments, the CM may include substrates of proteases that are more prevalently found 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, malignant 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) can be produced by a tumor in a subject. In some embodiments, the activatable protein may comprise a first CM between the MM and the TB (e.g., AB) and a second CM between the EM and the same or different TB. In an activated state, both CMs may be cleaved, such that the MM and EM are released from the TB(s). In some examples, the first and second CM may comprise a substrate for the same protease. In some examples, the first and second CM may comprise a substrate for a different protease. In some examples, the first and second CM may comprise or consist of the same sequence. In some examples, the first and second CM may comprise or consist of different sequences.

[0122] The second CM may be at a location within the activatable protein whose cleavage promotes dissociation of the EM from the TB. In some examples, the second CM may be between the C-terminus of the TB (or its components, if the TB comprises multiple polypeptides) and the N-terminus of the MM, and the C-terminus of the MM is coupled to the N-terminus of the EM (or its components, if the EM comprises multiple polypeptides). In some examples, the second CM may be between the N-terminus of the TB (or its components, if the TB comprises multiple polypeptides) and the C-terminus of the MM, and the N-terminus of the MM is coupled to the C-terminus of the EM (or its components, if the EM comprises multiple polypeptides). In some examples, the second CM may be between the C-terminus of the TB (or its components, if the TB comprises multiple polypeptides) and the N-terminus of the EM (or its components, if the EM comprises multiple polypeptides), and the C-terminus of the EM (or its components, if the EM comprises multiple polypeptides) is coupled to the N-terminus of the MM. In some examples, the second CM may be between the N-terminus of the TB (or a component thereof if the TB comprises multiple polypeptides) and the C-terminus of the EM (or a component thereof if the EM comprises multiple polypeptides), and the N-terminus of the EM (or a component thereof if the EM comprises multiple polypeptides) is coupled to the C-terminus of the MM. In these examples, the MM may be a masking moiety of the TB or a different TB (e.g., on the same or another polypeptide) in the activatable protein.

[0123] Suitable CMs for use with the activatable proteins 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).

[0124] 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 B), cysteine ​​proteases (e.g., cruzipain, legumain, otubain, etc.), caspases (e.g., caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 14), 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, otubain, etc.), -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), metalloproteinase (e.g., meprin, neprilysin, PSMA, BMP-1), lactoferrin, marapsin, matros The proteases may include substrates for tryptase-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.

[0125] In some embodiments, a protease substrate of a CM can comprise a polypeptide sequence that is not substantially identical (e.g., less than 90%, 80%, 70%, 60%, or 50% identical) to any polypeptide sequence that is naturally cleaved by the same protease.

[0126] In some embodiments, the CM can be or include the sequence LSGRSDDH (SEQ ID NO: 33) or the sequence ISSGLLSGRSDNH (SEQ ID NO: 41). In some embodiments, the CM can be or include any one of the sequences in the following table, or can be included in the consensus of sequences: [Table 9-1] [Table 9-2] [Table 9-3] [Table 9-4] [Table 9-5] [Table 9-6] [Table 9-7] [Table 9-8] [Table 9-9]

[0127] Examples of CM are also described in WO2010 / 081173, WO2021207669, WO2021207657, WO2021142029, WO2021061867, WO2020252349, WO2020252358, WO2020236679, WO2020176672, WO2020118109, WO2020092881, WO2020086665, WO2019213444, WO2019183218, WO2019173771, WO2019165143, W WO2019075405, WO2019046652, WO2019018828, WO2019014586, WO2018222949, WO2018165619, WO2018085555, WO2017011580, WO2016179335, WO2016179285, WO2016179257, WO2016149201, WO2016014974, which are incorporated by reference in their entireties for all purposes.

[0128] In some embodiments, the CM may be or include a combination, C-terminal truncated variant, or N-terminal truncated variant of the above exemplary sequences. The truncated variants of the above amino acid sequences suitable for use in the CM may be any truncated variant that retains the recognition site of the corresponding protease. These include C-terminal and / or N-terminal truncated variants that contain at least 3 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, truncated variants of the amino acid sequences described above may be amino acid sequences corresponding to any of the above but 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 3 amino acid residues; and (2) retain a recognition site for a 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 CM is a CM truncated at the C-terminus and at the N-terminus.

[0129] In some embodiments, the CM can include between 3 amino acids and 25 amino acids, in some embodiments, the CM can include between 3 and 25, 3 and 20, 3 and 15, 3 and 10, 3 and 5, 5 and 25, 5 and 20, 5 and 15, 5 and 10, 10 and 25, 10 and 20, 10 and 15, 15 and 25, 15 and 20, or 20 and 25 amino acids in total. In some embodiments, the CM is about 0.001 to 1500 x 10 4 M -1 S -1, or at least 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, 7.5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 200, 250, 500, 750, 1000, 1250, or 1500 × 10 4 M -1 S -1 The rate can be determined by the reaction rate of substrate cleavage (k cat / K m ) can be measured.

[0130] Linker The activatable protein may include one or more linkers. A linker may include a stretch of amino acid sequence that connects two components in the activatable protein. 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 protein 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 protein is provided as a conformationally constrained construct, a flexible linker can be inserted to facilitate the formation and maintenance of a structure in the uncleaved activatable protein. Any of the linkers described herein can provide the desired flexibility to facilitate the inhibition of target binding or facilitate the cleavage of the CM by a protease. In some embodiments, the linker contained in the activatable protein can be fully or partially flexible, such that the linker can contain, in addition to the flexible linker, one or more moieties that impart a less flexible structure to provide the desired activatable protein. Some linkers may contain cysteine ​​residues, which may form disulfide bonds and reduce the flexibility of the construct.

[0131] In some embodiments, the linker coupled to the MM may have a length that allows the MM to be in a tertiary or quaternary position that effectively masks the TB, e.g., proximal to the TB to be masked.

[0132] 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).

[0133] In some embodiments, the linkers are 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, 2 to 2, 2 to 2, 2 to 2, 2 to 2, 2 to 2, 2 to 2, 2 to 2, 2 to 2, 2 to 2, 2 to 2, 2 to 2, 2 to 2, 2 to 2, 2 to 2, 2 to 2, 2 to 2, 2 to 2, 2 to 2, 2 to ~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.

[0134] 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).

[0135] In some embodiments, the linker may have one or more Gly-Gly-Gly-Ser (GGGS) 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) 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) sequences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGSG sequences). Examples of flexible linkers include glycine polymers (G), glycine-serine polymers (e.g., (GS), (GGS), (GSGGS), and (GGGS), 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 may be relatively unstructured and therefore may be able to function as neutral links between building blocks. Glycine has significant access to more phi-psi space than alanine and is not as restricted as residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)).Exemplary flexible linkers include GGSG (SEQ ID NO:71), GGSGG (SEQ ID NO:72), GSGSG (SEQ ID NO:73), GSGGG (SEQ ID NO:74), GGGSG (SEQ ID NO:75), GSSSG (SEQ ID NO:76), GSSGGSGGSGG (SEQ ID NO:77), GGGS (SEQ ID NO:78), GGGSGGGS (SEQ ID NO:79), GGGSGGGSGGGS (SEQ ID NO:80), GGGGSGGGGSGGGGGS (SEQ ID NO:81), GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:82), GGGGSGGGGS (SEQ ID NO:83), GGGGS (SEQ ID NO:84), GS,GGGGSGS (SEQ ID NO:85), GGGGSGGGGSGGGGSGS (SEQ ID NO:86), GGS and combinations of one or more of LDPKGGGGS (SEQ ID NO:87), PKSCDKTHTCPPCPAPELLG (SEQ ID NO:88), SKYGPPCPPCPAPEFLG (SEQ ID NO:89), GKSSGSGSESKS (SEQ ID NO:90), GSTGSSGKSSEGKG (SEQ ID NO:91), GSTGSSGKSSEGSGSTKG (SEQ ID NO:92), GSTGSSGKPGSGEGSTKG (SEQ ID NO:93), GSTGSSGKPGSSEGST (SEQ ID NO:94), GGGGSGGS (SEQ ID NO:95), GGGGSGGGGSS (SEQ ID NO:96), GGGGSSGGSGGSSGGS (SEQ ID NO:97), and GSTGSSGKPGSEGST (SEQ ID NO:98).

[0136] Exemplary linkers may further include sequences that are at least 70% identical (e.g., at least 72%, at least 74%, at least 75%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 96%, 97%, at least 98%, at least 99%, or 100% identical) to the exemplary linkers described herein. Those skilled in the art will recognize that the design of activatable proteins can include linkers that are all or partially flexible, such that the linker can include one or more moieties that confer a less flexible structure, as well as flexible linkers, to provide the desired activatable protein structure.

[0137] In some embodiments, the activatable protein 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 linkers can include sulfo-SIAB, SMPB, and sulfo-SMPB, which react with primary amine sulfhydryls.

[0138] Conjugate Agents In some embodiments, the activatable molecule (e.g., an activatable protein such as an activatable antibody) can further comprise one or more additional agents, such as a targeting moiety to facilitate delivery to a cell or tissue of interest, a therapeutic agent (e.g., an anti-neoplastic agent such as a chemotherapeutic agent or an anti-tumor agent), a toxin, or a fragment thereof. The additional agent can be conjugated to the activatable antibody. 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.

[0139] In some embodiments, the activatable protein can be conjugated to a cytotoxic agent, e.g., a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof) or a radioisotope.

[0140] Examples of cytotoxic agents that can be conjugated to the activatable proteins include dolastatins and their derivatives (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, α-amanitin, turbostatin, phenstatin, hydroxylase ... These include ciphenstatin, spongistatin 5, spongistatin 7, halistatin 1, halistatin 2, halistatin 3, halocompstatin, pyrrolobenzimidazole (PBI), cibrostatin 6, doxaliform, cemadotin analog (CemCH2-SH), Pseudomonas toxin A (PES8) mutant, Pseudomonas toxin A (ZZ-PE38) mutant, ZJ-101, anthracyclines, doxorubicin, daunorubicin, bryostatin, camptothecin, 7-substituted camptothecins, 10,11-difluoromethylenedioxycamptothecin, combretastatin, debromoaplysiatoxin, KahaMide-F, discodermolide, and ecteinascidin.

[0141] Examples of enzymatically active toxins that can be conjugated to an activatable protein include diphtheria toxin, exotoxin A chain from Pseudomonas aeruginosa, ricin A chain, abrin A chain, modeccin A chain, α-sarcin, Aleuriies fordii proteins, dianfhin proteins, Phytoiaca Americana proteins (e.g., PAPI, PAPII, and PAP-8), momordica charantia inhibitor, curcin, crotirs, sapaonaria officinalis inhibitor, geoionin, mitogeliin, restrictocin, phenomycin, neomycin, and the trichothecenes.

[0142] Examples of antineoplastic drugs that can be conjugated to the activatable proteins include adriamycin, cerbidine, bleomycin, alkeran, velban, oncovin, fluorouracil, methotrexate, thiotepa, bisantrene, novantrone, thioguanine, procarbazine, and cytarabine.

[0143] Examples of antiviral drugs that can be conjugated to activatable proteins include acyclovir, virus A, and symmerel. Examples of antifungal agents that can be conjugated to activatable proteins include nystatin. Examples of detection reagents that can be conjugated to activatable proteins include fluorescein and its derivatives, fluorescein isothiocyanate (FITC). Examples of antibacterial drugs that can be conjugated to activatable proteins include aminoglycosides, streptomycin, neomycin, kanamycin, amikacin, gentamicin, and tobramycin. Examples of 3β,16β,17α-trihydroxycholest-5-en-22-one 16-O-(2-O-4-methoxybenzoyl-β-D-xylopyranosyl)-(1-->3)-(2-O-acetyl-α-L-arabinopyranoside) (OSW-1) that can be conjugated to activatable proteins include s-nitrobenzyloxycarbonyl derivatives of O6-benzylguanine, topoisomerase inhibitors, hemiasterlin, cephalotaxine, homoharringionine, pyrrolobenzodiazepine dimers (PBD), functionalized pyrrolobenzodiazepines, calicheamicins, podophyiitoxins, taxanes, and vinca alkaloids. Examples of radiopharmaceuticals that can be conjugated to activatable proteins 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 proteins include barium, gold, and platinum. Examples of anti-mycoplasma agents that can be conjugated to activatable proteins include tylosin, spectinomycin, streptomycin B, ampicillin, sulfanilamide, polymyxin, and chloramphenicol.

[0144] In some embodiments, the activatable protein may include a signal peptide. When including multiple polypeptides, the activatable protein may include multiple signal peptides, e.g., one signal peptide for each of the multiple polypeptides. The signal peptide may be a peptide (e.g., 10-30 amino acids long) 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 protein via a spacer. In some embodiments, the spacer may be conjugated to the activatable protein if the signal peptide is not present.

[0145] Those skilled in the art will appreciate that a wide variety of possible agents can be conjugated to any of the activatable proteins described herein. An agent can be conjugated to another component of the activatable protein by a conjugation moiety. Conjugation can include any chemical reaction that binds two molecules together, so long as the activatable protein and the other moiety retain their respective activities. Conjugation can include many chemical reaction mechanisms, such as covalent bonding, affinity bonding, intercalation, coordinate bonding, and complexation. In some embodiments, the bond can be a covalent bond. 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 proteins described herein. For example, conjugates can contain organic compounds, such as thioesters, carbodiimides, succinimide esters, glutaraldehyde, diazobenzene, and hexamethylenediamine. In some embodiments, the activatable protein can contain or otherwise incorporate one or more unnatural amino acid residues to provide suitable sites for conjugation.

[0146] In some embodiments, the agent and / or conjugate may be attached to the antigen-binding domain by a disulfide bond (e.g., a disulfide bond on a cysteine ​​molecule). Since many cancers naturally release high levels of glutathione, a reducing agent, glutathione present in the cancer tissue microenvironment can reduce the disulfide bond, followed by release of the agent and / or conjugate at the delivery site.

[0147] 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.

[0148] In some embodiments, the conjugate moiety is not cleavable by 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 is susceptible to gentle cleavage by serum proteases, allowing the conjugate and / or agent to be slowly released at the target site.

[0149] In some embodiments, the conjugate and / or agent can 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.

[0150] In some embodiments, the conjugate and / or agent may be attached directly to the antigen binding domain or may be attached via an amino acid (e.g., a D-amino acid), a peptide, a thiol-containing moiety, or other organic compound that can be modified by the methods described herein to contain a functional group that is then available for attachment to the antigen binding domain.

[0151] In some embodiments, the activatable protein may include at least one conjugation point to an agent. In some embodiments, all possible conjugation points are available for conjugation to an agent. In some embodiments, the one or more conjugation points may include sulfur atoms involved in disulfide bonds, sulfur atoms involved in interchain disulfide bonds, sulfur atoms involved in interchain sulfide bonds but not intrachain disulfide 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.

[0152] The present disclosure also provides methods and materials for preparing activatable proteins with one or more conjugated agents. In some embodiments, activatable proteins can be modified to include one or more interchain disulfide bonds. For example, disulfide bonds can be reduced after exposure to a reducing agent, such as, but not limited to, TCEP, DTT, or β-mercaptoethanol. In some cases, reduction of disulfide bonds may only be partial. As used herein, the term partial reduction refers to the situation where an activatable protein 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 protein may be partially reduced after contact with a reducing agent if less than 99% (e.g., less than 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%) of all available conjugation sites are reduced. In some embodiments, an activatable protein having reduction in one or more interchain disulfide bonds may be conjugated to an agent that is reactive with free thiols.

[0153] The present disclosure also provides methods and materials for conjugating therapeutic agents to specific positions on an activatable protein. In some embodiments, an activatable protein may be modified such that a therapeutic agent may be conjugated to the activatable protein at a specific position on the activatable protein. For example, the activatable protein may be partially reduced to facilitate conjugation to the activatable protein. In such cases, partial reduction of the activatable protein may occur such that the conjugation site of the activatable protein is not reduced. In some embodiments, the conjugation site(s) on the activatable protein may be selected to facilitate conjugation of the agent at a specific position on the protein construct. In treating with a reducing agent, various factors may affect the "reduction level" of the activatable protein. For example, to achieve partial reduction of an activatable protein using the methods and materials described herein, optimization of, but is not limited to, the ratio of reducing agent to activatable protein, 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 protein, 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 protein (e.g., total reduction of available conjugation sites, or reduction of specific conjugation sites).

[0154] An effective ratio of reducing agent to activatable protein can be any ratio that at least partially reduces the activatable protein in a manner that allows for conjugation to an agent (e.g., overall reduction of available conjugation sites, or reduction of specific conjugation sites). In some embodiments, the ratio of reducing agent to activatable protein can be in the range of about 20:1 to 1:1, 10:1 to 1:1, 9:1 to 1:1, 8:1 to 1:1, 7:1 to 1:1, 6:1 to 1:1, 5:1 to 1:1, 4:1 to 1:1, 3:1 to 1:1, 2:1 to 1:1, 20:1 to 1:1.5, 10:1 to 1:1.5, 9:1 to 1:1.5, 8:1 to 1:1.5, 7:1 to 1:1.5, 6:1 to 1:1.5, 5:1 to 1:1.5, 4:1 to 1:1.5, 3:1 to 1:1.5, 2:1 to 1:1.5, 1.5:1 to 1:1.5, or 1:1 to 1:1.5.

[0155] Effective incubation times and temperatures for treating an activatable protein with a reducing agent can be any time and temperature that at least partially reduces the activatable protein in a manner that allows for conjugation of an agent to the activatable protein (e.g., overall reduction of available conjugation sites, or reduction of specific conjugation sites). In some embodiments, incubation times and temperatures for treating an activatable protein can range from about 1 hour at 37° C. to about 12 hours at 37° C. (or any subrange therein).

[0156] An effective pH for a reduction reaction for treating an activatable protein with a reducing agent can be any pH that at least partially reduces the activatable protein (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 protein.

[0157] When the partially reduced activatable protein is contacted with a thiol-containing agent, the agent can be conjugated to the interchain thiol in the activatable protein. The agent can be modified to contain a thiol using a thiol-containing reagent (e.g., cysteine ​​or N-acetylcysteine). For example, the activatable protein can be partially reduced after incubation with a reducing agent (e.g., TEPC) at a desired ratio of reducing agent to activatable protein for about 1 hour at about 37°C. The effective ratio of reducing agent to activatable protein can be any ratio that partially reduces at least two interchain disulfide bonds in the activatable protein in a manner that allows conjugation to a thiol-containing agent (e.g., global reduction of available conjugation sites, or reduction of specific conjugation sites).

[0158] In some embodiments, the activatable protein can be reduced by a reducing agent in a manner that avoids reduction of any intrachain disulfide bonds, hi some embodiments, the activatable protein is reduced by a reducing agent in a manner that avoids reduction of any intrachain disulfide bonds and reduces at least one interchain disulfide bond.

[0159] In some embodiments, the agent (e.g., an agent conjugated to an activatable protein) may be a detectable moiety, such as 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 labeled avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected optically or calorimetrically), 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 tdimer2 (RFP tdimer2), 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. Further examples of detectable labels include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase. Examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin. Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin. An example of a luminescent material includes luminol. Examples of bioluminescent materials include luciferase, luciferin, and aequorin. Examples of suitable radioactive materials include 125 I, 131 I, 35 S, or 3 Examples include H.

[0160] In some embodiments, an agent may be conjugated to an activatable protein 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 protein via a linker and / or CM described herein. In some embodiments, an agent may be conjugated to a cysteine ​​or lysine in an activatable protein. In some embodiments, an agent may be conjugated to a residue of an activatable protein, such as a residue disclosed herein.

[0161] In some embodiments, a variety of bifunctional protein coupling agents can be used to conjugate drugs to activatable proteins, including N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCL), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., triene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). In some embodiments, radionucleotides can be conjugated to activatable proteins using carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) chelating agents (see, e.g., WO 94 / 11026).

[0162] Suitable conjugation moieties include those described in the literature (see, for example, 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. Patent No. 5,030,719, which describes the use of halogenated acetylhydrazide derivatives coupled to activatable proteins 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 Chem. Co., catalog number 21651G), (iv) Sulfo-LC-SPDP (Sulfosuccinimidyl-6[3-(2-pyridyldithio)-propionamido]hexanoate (Pierce Chem. Co. catalog #2165-G), and (v) Sulfo-NHS (N-hydroxysulfo-succinimide: Pierce Chem. Co. catalog #2165-G) conjugated to EDC. Chem. Co., catalog number 24510. Additional exemplary conjugation moieties include SMCC, sulfo-SMCC, SPDB, and sulfo-SPDB.

[0163] 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. Furthermore, 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 carboimidide coupling (e.g., EDC) is used in conjugation with sulfo-NHS, it forms esters that are more resistant to hydrolysis than the carboimidide coupling reaction alone.

[0164] Those of skill in the art will appreciate that a wide variety of possible moieties can be coupled to the activatable proteins of the present disclosure (see, e.g., "Conjugate Vaccines", Contributions to Microbiology and Immunology, JM Cruse and RE Lewis, Jr (eds), Carger Press, New York, (1989), the entire contents of which are incorporated herein by reference). In general, effective conjugation of an agent (e.g., a cytotoxic agent) to an activatable protein can be achieved by any chemical reaction that bonds the agent to the activatable protein while also allowing the agent and activatable protein to retain functionality.

[0165] Nucleic Acids and Vectors In some aspects, the disclosure further provides a nucleic acid comprising a sequence encoding an activatable molecule herein (e.g., an activatable antibody), or a component or fragment thereof. The nucleic acid may comprise coding sequences for the TB, CM, MM, EM, and linker(s) in the activatable protein. When the activatable protein comprises multiple polypeptides (e.g., multiple TBs on different polypeptides, or one TB comprises multiple polypeptides), the nucleic acid may comprise coding sequences for multiple polypeptides. In some examples, the coding sequence for one of the polypeptides is comprised in a nucleic acid and the coding sequence for another of the polypeptides is comprised in another nucleic acid. In some examples, the coding sequences for two or more of the multiple polypeptides are comprised in the same nucleic acid. The disclosure includes polynucleotides encoding the proteins or portions thereof described herein, as well as the use of such polynucleotides to generate proteins and / or for therapeutic purposes. Such polynucleotides may include DNA and RNA molecules (e.g., mRNA, self-replicating RNA, self-amplifying mRNA, etc.) that encode the proteins defined herein. The disclosure includes compositions comprising such polynucleotides. In some aspects, such compositions may be used therapeutically or prophylactically.

[0166] 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.

[0167] The term "located at the N-terminus", when referring to the position of a first domain or sequence relative to a second domain or sequence in the primary amino acid sequence of a polypeptide, means that the first domain is located towards 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. The term "located at the C-terminus", when referring to the position of a first domain or sequence relative to a second domain or sequence in the primary amino acid sequence of a polypeptide, means that the first domain is located towards 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.

[0168] 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., aspartate and glutamate), amino acids with basic side chains (e.g., lysine, arginine and histidine), nonpolar amino acids (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine and tryptophan), uncharged polar amino acids (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine and tyrosine), hydrophilic amino acids (e.g., arginine, asparagine, aspartate, glutamine, glutamate, histidine, lysine, serine and threonine), 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).

[0169] The present disclosure further provides vectors and vector sets 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 proteins described herein, and to use the vector or set of vectors to express any of the activatable proteins described herein. For example, in selecting a vector or set of vectors, a cell type may be selected, such that the vector(s) may need to be integrated into and / or replicate in the chromosome of the cell. Exemplary vectors that can be used to generate activatable proteins 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" can deliver nucleic acids and fragments thereof into a host cell, and includes control sequences (e.g., promoters, enhancers, poly(A) signals). An exogenous polynucleotide can be inserted into an expression vector for expression. The term "vector" also includes artificial chromosomes, plasmids, retroviruses, and baculovirus vectors.

[0170] Methods for making vectors containing any one of the nucleic acids described herein and suitable for transforming a cell (e.g., a mammalian cell) are well known in the art. See, e.g., Sambrook et al., Eds. "Molecular Cloning: A Laboratory Manual," 2nd Ed., Cold Spring Harbor Press, 1989 and Ausubel et al., Eds. "Current Protocols in Molecular Biology," Current Protocols, 1993.

[0171] 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. 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 proteins described herein.

[0172] In some embodiments, the activatable protein may be made biosynthetically using recombinant DNA technology and expression in eukaryotic or prokaryotic species.

[0173] 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 molecule (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, e.g., Saccharomyces cerevisiae. In some embodiments, the eukaryotic cell is a higher eukaryotic cell, e.g., 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.

[0174] 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.

[0175] In some embodiments, the introducing step comprises introducing into the cell a vector (e.g., any of the vectors or sets of vectors described herein) containing nucleic acids encoding monomers that make up any of the activatable proteins described herein.

[0176] Compositions and kits The present disclosure also provides compositions and kits that include the activatable molecules (e.g., activatable antibodies) described herein. The compositions and kits may further include one or more excipients, carriers, reagents, and instructions necessary for use of the activatable proteins.

[0177] In some embodiments, the composition may be a pharmaceutical composition comprising the activatable protein, its derivatives, fragments, analogs, and homologs. The pharmaceutical composition may comprise the activatable protein and a pharma- ceutically acceptable carrier. As used herein, the term "pharma- ceutically acceptable carrier" is intended to include any solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are 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 pharma- ceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the composition is contemplated. Supplementary active ingredients may also be incorporated into the composition.

[0178] A pharmaceutical composition can be formulated to suit its 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 contain one or more of the following components: sterile diluents such as water for injection, saline, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates, or phosphates; and agents for adjusting isotonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic. In some cases, any of the activatable proteins described herein are prepared with carriers that protect against rapid elimination from the body, such as sustained and controlled release formulations, such as implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid-glycolic acid copolymers, and polylactic acid can be used. Methods for preparing such pharmaceutical compositions and formulations are clear to those skilled in the art. For example, the activatable protein may be incorporated into microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules, colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or macroemulsions.

[0179] Sustained release preparations may be prepared. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, such as films, or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactides, copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers (e.g., injectable microspheres composed of lactic acid-glycolic acid copolymers and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid allow the release of molecules for over 100 days, certain hydrogels release proteins for shorter periods of time.

[0180] In some embodiments, pharmaceutical compositions suitable for injection use 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 fluid and of a viscosity that allows easy injection. It may be stable under the conditions of manufacture and storage and may be 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 on the particles, such as lecithin, and by the maintenance of the required particle size in the case of dispersions, 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.

[0181] In some embodiments, pharmaceutical compositions may contain sterile injection.Sterile injection can be prepared by incorporating the required amount of active compound into a suitable solvent with one or a combination of the above-listed ingredients as required, followed by filtration sterilization.Generally, dispersion can be prepared by incorporating active compound into a sterile vehicle that contains a basic dispersion medium and other ingredients required from the above-listed ingredients.In the case of sterile powder for preparing sterile injection, the preparation method is vacuum drying and freeze-drying, which obtains a powder of active ingredient plus any additional desired ingredients from its solution that has been previously sterile-filtered.

[0182] 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 incorporated with an excipient and used in the form of a tablet, a troche, or a capsule. Oral compositions may also be prepared using a fluid carrier for use as a mouthwash, in which the compound 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, or compounds of a similar nature: a binder (such as microcrystalline cellulose, gum tragacanth or gelatin), a filler (such as starch or lactose), a disintegrating agent (such as alginic acid, Primogel or corn starch), a lubricant (such as magnesium stearate or Sterotes), a flow agent (such as colloidal silicon dioxide), a sweetening agent (such as sucrose or saccharin), or a flavoring agent (such as peppermint, methyl salicylate, or orange flavoring).

[0183] In some embodiments, the pharmaceutical composition can be formulated for administration by inhalation. For example, the compound can be delivered in the form of an aerosol spray from a pressured container or dispenser containing a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0184] In some embodiments, the pharmaceutical composition can be formulated for systemic administration. For example, systemic administration can be intravenous and by transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrant appropriate to the barrier to be permeated can 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 can be achieved through the use of nasal sprays or suppositories. For transdermal administration, the active compound can be formulated into ointments, salves, gels, or creams, as generally known in the art.

[0185] In some embodiments, pharmaceutical compositions can be prepared in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0186] In one embodiment, the pharmaceutical composition can be prepared with a carrier that protects the composition against rapid elimination from the body, for example, a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic-co-glycolic acid, and polylactic acid. Methods for preparing such formulations are clear to those skilled in the art.

[0187] For ease of administration and uniformity of dosage, it may be particularly advantageous to formulate oral or parenteral compositions in unitary dosage form.As used herein, unitary dosage form refers to a physically separate unit suitable as a unitary dosage for treating a subject, each unit containing a predetermined amount of active compound calculated to produce a desired therapeutic effect in association with the required pharmaceutical carrier.The specification of the unitary dosage form of the present disclosure can be determined and directly depend on the inherent 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 treating an individual.

[0188] In some embodiments, the compositions (eg, pharmaceutical compositions) may be included in a container, vial, syringe, injection pen, pack, or dispenser, optionally together with instructions for administration.

[0189] Also provided herein is a kit that contains any of the activatable proteins described herein, any of the compositions that contain any of the activatable proteins described herein, or any of the pharmaceutical compositions that contain any of the activatable proteins described herein.Also provided herein is a kit that contains one or more second therapeutic agent(s) in addition to the activatable proteins described herein.The second therapeutic agent(s) may be provided in a dosage form separate from the activatable protein.Alternatively, the second therapeutic agent(s) may be formulated together with the activatable protein.

[0190] Any of the kits described herein can include instructions for using any of the compositions (e.g., pharmaceutical compositions) and / or any of the activatable proteins 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.

[0191] Also provided herein are activatable proteins produced by any of the methods described herein. Also provided herein are compositions (e.g., pharmaceutical compositions) comprising any of the activatable proteins produced by any of the methods described herein. Also provided herein are kits containing at least one dose of any of the compositions (e.g., pharmaceutical compositions) described herein.

[0192] Methods for generating activatable molecules Provided herein are methods of producing any of the activatable molecules (e.g., activatable proteins) described herein, the methods comprising: (a) culturing any of the recombinant host cells described herein in a liquid medium under conditions sufficient to produce the activatable protein; and (b) recovering the activatable protein from the host cells and / or the liquid medium.

[0193] 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.

[0194] In some embodiments, the method may further include isolating the recovered activatable protein. Isolation of the activatable protein 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.

[0195] The compositions and methods described herein may involve the use of non-reducing or partially reducing conditions that allow for the formation of disulfide bonds between the MM and TB of an activatable protein.

[0196] In some embodiments, the method further comprises formulating the isolated activatable protein into a pharmaceutical composition. A variety of formulations are known in the art and described herein. Any of the isolated activatable proteins described herein can be prepared for any route of administration (e.g., intravenous, intratumoral, subcutaneous, intradermal, oral (e.g., inhalation), transdermal (e.g., topical), transmucosal, or intramuscular).

[0197] Methods of Using Activatable Molecules In some aspects, the disclosure further provides methods of using the activatable molecules (e.g., activatable antibodies) herein. In some embodiments, the disclosure provides methods of treating a disease in a subject (e.g., cancer (e.g., any of the cancers described herein)), comprising administering to the subject a therapeutically effective amount of any of the activatable proteins described herein. In some embodiments, the disclosure provides methods of preventing, delaying the progression of, treating, alleviating a symptom of, or ameliorating a disease in a subject by administering to a subject in need thereof a therapeutically effective amount of an activatable protein described herein. The term "treatment" means improving at least one symptom of a disease. In some embodiments, the disease being treated may be cancer or an autoimmune disease, and may be for alleviating at least one symptom of a cancer or an autoimmune disease. As used herein, the term "subject" means any mammal. In some embodiments, the subject is a feline (e.g., a cat), a canine (e.g., a dog), an equine (e.g., a horse), a rabbit, a pig, a rodent (e.g., a mouse, a rat, a hamster, or a guinea pig), a non-human primate (e.g., a simian (e.g., a monkey (e.g., a baboon, a marmoset), or an ape (e.g., a chimpanzee, a gorilla, an orangutan, or a gibbon)), or a human. In some 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)).

[0198] In some embodiments, the subject may be identified as having a mutation in the HER2 gene that increases the expression and / or activity of HER2 in a mammalian cell (e.g., any of the mammalian cells described herein). For example, a mutation in the HER2 gene that increases the expression and / or activity of HER2 in a mammalian cell may be a mutation that results in the expression of HER2 (compared to the wild-type protein) with gene duplication, one or more amino acid substitutions (e.g., one or more amino acid substitutions selected from the group consisting of G309A, G309E, S310F, R678Q, L755S, L755W, I767M, D769H, D769Y, V777L, Y835F, V842I, R896C, and G1201V). See, e.g., Weigelt and Reis-Filho, Cancer Discov. 2013, 3(2):145-147.

[0199] Non-limiting examples of the method for detecting HER2-related disease in a subject include immunohistochemistry, fluorescent in situ hybridization (FISH), and chromogenic in situ hybridization (CISH).See, for example, Yan et al., Cancer Metastasis Rev. 2015, 34:157-164.

[0200] The therapeutically effective amount of the activatable protein of the present disclosure generally relates to the amount necessary to achieve a therapeutic goal. As mentioned above, this may be a binding interaction between the antibody and its target antigen, which in certain cases prevents the function of the target. The amount that needs to be administered further depends on the binding affinity of the activatable protein for its particular target, and also on the rate at which the administered activatable protein is depleted from the free deposits of other subjects to which it is administered. The general range of therapeutically effective dosages of the activatable protein of the present disclosure may be, as a non-limiting example, about 0.001, 0.01, 0.1, 0.3, 0.5, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 mg / kg body weight or more. The structure of the activatable protein of the present disclosure allows for a reduced dose of the activatable protein to be administered to a subject, as compared to conventional activatable antibodies, and as compared to conventional antibodies. For example, the dose administered based on a unit dosage form, or the total dose administered over a dosing regimen, may be reduced by 10, 20, 30, 40, or 50% compared to the corresponding dose of a corresponding conventional activatable protein or corresponding conventional antibody.

[0201] Typical dosing frequencies may range, for example, from once or twice daily, once or twice weekly, once or twice every two weeks, or once or twice monthly.

[0202] The efficacy of treatment is determined in association with any known method for diagnosing or treating a particular disorder. Methods for screening for activatable proteins possessing the desired specificity include, but are not limited to, enzyme-linked immunosorbent assays (ELISAs) and other immunologically mediated techniques known within the art.

[0203] In another embodiment, activatable proteins directed to two or more targets are used in methods known in the art for localizing and / or quantitating the targets (e.g., for use in measuring the levels of one or more of the targets in an appropriate physiological sample, for use in diagnostic methods, for use in protein imaging, etc.). In a given embodiment, activatable proteins directed to two or more targets, or derivatives, fragments, analogs or homologs thereof, including antigen-binding domains derived from antibodies, are utilized as pharmacologically active compounds (hereinafter referred to as "therapeutic agents").

[0204] The activatable proteins used in any of these method and use embodiments can be administered at any stage of disease. For example, such activatable proteins can be administered to patients suffering from any stage of cancer, from early to metastatic. In some embodiments, the activatable proteins 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.

[0205] 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 cancer or other neoplastic conditions can be identified using any of a variety of clinical and / or laboratory tests, such as physical examination and blood, urine and / or fecal analysis to assess health status. For example, subjects suffering from inflammation and / or inflammatory disorders can be identified using any of a variety of clinical and / or laboratory tests, such as physical examination and body fluid analysis, such as blood, urine and / or fecal analysis to assess health status.

[0206] In some embodiments, administration of an activatable protein 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 experimental or clinical objectives are achieved. For example, administration of an activatable protein 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 further, i.e., to a worse state. Administration of an activatable protein 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 further, i.e., to a worse state.

[0207] 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 reducing cancer growth, inhibiting cancer progression, inhibiting cancer metastasis, or reducing the risk of cancer recurrence in a subject with cancer.

[0208] In some embodiments, the disease may be cancer. In some embodiments, the subject may have been identified or diagnosed as having 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, malignant cutaneous T cell lymphoma), leukemias (e.g., hairy cell leukemia, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic bone marrow leukemia (CRL), chronic myelogenous leukemia (CRM), chronic myelogenous leukemia (CHLL), chronic myelogenous leukemia (CRM ... The cancers include myeloid leukemia (CML), acute lymphocytic leukemia (ALL), 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), squamous cell head and neck cancer, uterine 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 and 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 carcinoma, non-small cell carcinoma of the lung, and carcinoma of the small intestine. Further examples of cancers that may be treated by the compositions and methods herein include acute lymphoblastic leukemia (adult); acute lymphoblastic leukemia (pediatric); acute myeloid leukemia (adult); adrenal cortical carcinoma; adrenal cortical carcinoma (pediatric); AIDS-related lymphoma; AIDS-related malignancies; anal cancer; pediatric cerebellar astrocytoma; pediatric cerebral astrocytoma; extrahepatic bile duct cancer; 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 tumors, medulloblastoma (childhood);Brain tumors, supratentorial primitive neuroectodermal tumors (childhood);Brain tumors, visual pathway and hypothalamic gliomas (childhood);Other brain tumors (childhood);Breast cancer;Breast cancer and pregnancy;Breast cancer (childhood);Breast cancer (male);Bronchial adenoma / carcinoid (childhood);Carcinoid tumors (childhood);Gastrointestinal carcinoid tumors;Adrenal cortical carcinoma;Pancreatic islet cell carcinoma;Cancer of unknown primary;Primary central nervous system lymphoma;Cerebellar astrocytoma (childhood);Brain astrocytoma / malignant glioma (childhood);Cervical cancer;Childhood cancer;Chronic lymphocytic leukemia;Chronic myeloid leukemia;Chronic myeloproliferative disorder;Clear cell sarcoma of the tendon sheath;Colon cancer;Colon cancer (small pediatric);Cutaneous T-cell lymphoma;Endometrial cancer;Ependymoma (pediatric);Ovarian epithelial carcinoma;Esophageal cancer;Esophageal cancer (pediatric);Ewing's sarcoid 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;Extracranial germ cell tumors (pediatric);Extracranial germ cell tumors;Ovarian germ cell tumors;Gestational trophoblastic tumor;Pediatric brain stem glioma;Pediatric visual pathway and hypothalamic glioma;Hairy cell leukemia;Head and neck Neck cancer;Primary hepatocellular (liver) carcinoma (adult);Primary hepatocellular (liver) carcinoma (childhood);Hodgkin's lymphoma (adult);Hodgkin's lymphoma (childhood);Hodgkin's lymphoma during pregnancy;Hypopharyngeal cancer;Hypothalamic and visual pathway glioma (childhood);Intraocular melanoma;Islet cell carcinoma (endocrine pancreas);Kaposi's sarcoma;Kidney cancer;Laryngeal cancer;Laryngeal cancer (childhood);Acute lymphoblastic leukemia (adult);Acute lymphoblastic leukemia (childhood);Acute myeloid leukemia (adult);Acute myeloid leukemia (childhood);Chronic lymphocytic leukemia;Chronic myeloid leukemia;Hairy cell leukemia;Lip and oral cavity cancer;Primary liver cancer (adult);Primary liver cancer (childhood) ;non-small cell lung cancer;small cell lung cancer;acute lymphoblastic leukemia (adult);acute lymphoblastic leukemia (childhood);chronic lymphocytic leukemia;AIDS-related lymphoma;primary central nervous system lymphoma;cutaneous T-cell lymphoma;Hodgkin lymphoma (adult);Hodgkin lymphoma (childhood);Hodgkin lymphoma during pregnancy;non-Hodgkin lymphoma (adult);Non-Hodgkin lymphoma (childhood);Non-Hodgkin lymphoma during pregnancy;primary central nervous system lymphoma;Waldenstrom's macroglobulinemia;male breast cancer;malignant mesothelioma (adult);malignant mesothelioma (childhood);malignant thymoma;medulloblastoma (childhood);melanoma;Intraocular melanoma;Merkel cell carcinoma;Malignant mesothelioma;Metastatic squamous cell carcinoma of unknown primary;Multiple endocrine neoplasia syndrome (childhood);Multiple myeloma / plasma cell neoplasm;Mycosis fungoides;Myelodysplastic syndrome;Myeloid leukemia (chronic);Acute myeloid leukemia (childhood);Multiple myeloma;Chronic myeloproliferative disorders;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 cavity cancer (childhood);Oral and lip cancer;Oropharyngeal cancer;Osteosarcoma / malignant fibrous bone Histiocytoma;Ovarian cancer (childhood);Ovarian epithelial cancer;Ovarian germ cell tumor;Ovarian low malignant potential tumor;Pancreatic cancer;Pancreatic cancer (childhood);Islet cell pancreatic cancer;Sino-nasal and nasal cancer;Parathyroid cancer;Penile cancer;Pheochromocytoma;Primary neuroectodermal tumors of the pineal gland and supraventricle (childhood);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 (adult);Primary liver cancer (childhood);Prostate cancer;Rectal cancer;Renal cell carcinoma;Renal cell carcinoma (childhood);Renal pelvic and ureteral junction Skin Cancer;Retinoblastoma;Rhabdomyosarcoma (Childhood);Salivary Gland Cancer;Salivary Gland Cancer (Childhood);Ewing Family of Tumors;Kaposi's Sarcoma;Sarcoma (Osteosarcoma) / Malignant Fibrous Histiocytoma;Rhabdomyosarcoma (Childhood);Soft Tissue Sarcoma (Adult);Soft Tissue Sarcoma (Childhood);Sezary Syndrome;Skin Cancer;Skin Cancer (Childhood);Skin Cancer (Melanoma);Merkel Cell Skin Cancer;Small Cell Lung Cancer;Small Intestine Cancer;Soft Tissue Sarcoma (Adult);Soft Tissue Sarcoma (Childhood);Primary and Metastatic Squamous Cell Carcinoma;Stomach (Gastric) Cancer;Stomach (Gastric) ) cancer (pediatric); primary supraventricular neuroectodermal tumor (pediatric); cutaneous T-cell lymphoma; testicular cancer; thymoma (pediatric); malignant thymoma; thyroid cancer; thyroid cancer (pediatric); transitional cell carcinoma of the renal pelvis and ureter; trophoblastic tumor during pregnancy; cancer of unknown primary site (pediatric); unusual cancers of childhood; transitional cell carcinoma of the ureter and renal pelvis; urethral cancer; uterine sarcoma; vaginal cancer; visual pathway and hypothalamic glioma (pediatric); vulvar cancer; Waldenstrom giant cell globulinemia; Wilms' tumor; diffuse large B-cell lymphoma (DLBCL); and mantle cell lymphoma (MCL). Metastasis of the above cancers can also be treated or prevented according to the methods described herein.;

[0209] In some embodiments, the disease may be an autoimmune disease or condition. In some embodiments, the subject may have been identified or diagnosed as having an autoimmune disease or condition, or may be at high risk of developing an autoimmune disease or condition. Examples of autoimmune diseases include type 1 diabetes, rheumatoid arthritis (RA), psoriasis / psoriatic arthritis, multiple sclerosis, systemic lupus erythematosus, inflammatory bowel disease (e.g., Crohn's disease, ulcerative colitis), Addison's disease, Graves' disease, Sjogren's syndrome, Hashimoto's thyroiditis, myasthenia gravis, autoimmune vasculitis, pernicious anemia, celiac disease), infectious diseases (e.g., chickenpox, common cold, diphtheria, E. coli, giardiasis, HIV / AIDS, infectious mononucleosis, influenza, and the like). , Lyme disease, malaria, measles, meningitis, mumps, polio, pneumonia, Rocky Mountain spotted fever, rubella (three-day measles), Salmonella infection, Severe Acute Respiratory Syndrome (SARS), sexually transmitted diseases, shingles, tetanus, toxic shock syndrome, tuberculosis, viral hepatitis, West Nile virus, whooping cough), chronic inflammation, transplant rejection (e.g., in kidney, liver, or heart transplants), autoimmune disease, infection, chronic inflammation, or transplant rejection.

[0210] 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 a subject prior to treatment).

[0211] The method may further include administering to the subject one or more additional agents.

[0212] In some embodiments, the activatable protein may be administered during and / or after treatment in combination with one or more additional agents. In some embodiments, the activatable protein may be formulated in a single therapeutic composition, and the activatable protein and the additional agent(s) may be administered simultaneously. Alternatively, the activatable protein and the additional agent(s) may be separate from each other, e.g., each formulated in a separate therapeutic composition, and the activatable protein and the additional agent(s) are administered simultaneously, or the activatable protein and the additional agent(s) are administered at different times during the treatment regimen. For example, the activatable protein may be administered prior to administration of the additional agent, subsequent to administration of the additional agent, or alternating. The activatable protein and the additional agent(s) may be administered in a single dose or multiple doses.

[0213] One or more of the activatable proteins herein may be co-formulated and / or co-administered with one or more anti-inflammatory drugs, immunosuppressants, or metabolic or enzyme inhibitors. In some embodiments, one or more of the activatable proteins herein may be combined with one or more other types of activatable proteins (e.g., activatable proteins that do not have an EM, or activatable proteins whose activated form includes an EM).

[0214] The disclosure also provides methods for detecting the presence or absence of a cleavage agent and / or target in a subject or sample. Such methods may include (i) contacting a subject or biological sample with an activatable protein, where the activatable protein comprises a detectable label disposed on a portion of the activatable protein that is released following cleavage of the CM, and (ii) measuring the level of the activator protein in the subject or biological sample, where a detectable level of the activator protein 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, and thus target binding and / or protease cleavage of the activatable protein cannot be detected in the subject or biological sample, and a decrease in the detectable level of the activator protein in the subject or biological sample indicates that the cleavage agent and the target are present in the subject or biological sample.

[0215] 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%, or substantially 100% reduction. In some embodiments, the detectable label can be conjugated to a component of the activatable protein, such as TB. In some embodiments, measuring the level of the activatable protein in a subject or sample can be accomplished using a secondary reagent that specifically binds to the activator protein, the reagent comprising a detectable label. The secondary reagent can be an antibody comprising a detectable label.

[0216] In some embodiments, the activatable protein may also be useful for detecting targets in patient samples, and thus is useful as a diagnostic agent. For example, the activatable protein may be used in in vitro assays, such as ELISA, to detect target levels in patient samples. For example, the activatable protein may be immobilized on a solid support (e.g., the well(s) of a microtiter plate). The immobilized activatable protein may function as a capture protein for any target that may be present in a test sample. Before contacting the immobilized activatable protein 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 the analyte.

[0217] In some embodiments, based on the results obtained using the activatable protein in an in vitro diagnostic assay, the stage of disease in a subject can be determined based on the expression level of a target protein (e.g., an antigen). For a given disease, blood samples can be taken from subjects diagnosed as being at various stages of disease progression and / or at various time points of 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 concentration range of the target protein (e.g., an antigen) that can be considered characteristic of each stage.

[0218] The activatable proteins 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, an activatable protein having a CM may be used to detect the presence or absence of an enzyme capable of cleaving the CM. Such activatable proteins may be used for diagnosis, which may include in vivo detection (e.g., qualitative or quantitative) of enzyme activity (or, in some embodiments, an environment of increased reduction potential, such as may provide for reduction of disulfide bonds) by measured accumulation of activating antibodies (i.e., antibodies resulting from cleavage of the activatable protein) in a given cell or tissue of a given host organism. Such accumulation of activating proteins indicates not only that the tissue expresses the enzyme activity (or an increase in reduction potential, depending on the nature of the CM), but also that the tissue expresses the target to which the activating protein binds.

[0219] For example, the CM can be selected to be a substrate for a protease found at a tumor site, a site of viral or bacterial infection, a biologically restricted site (e.g., abscess, within an organ, etc.), etc. The TB can be one that binds to a target protein (e.g., an antigen). A detectable label (e.g., a fluorescent label or a radioactive label or a radioactive tracer) can be conjugated to the TB or other region of the activatable protein using methods well known to those of skill in the art. Suitable detectable labels can be discussed in the context of the screening methods above, with additional specific examples provided below. Using a TB specific for a disease state protein or peptide in conjunction with a protease that has increased activity in the diseased tissue of interest, the activatable protein can show 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 tissues), accumulation of activated proteins in diseased tissues may be increased compared to non-diseased tissues.

[0220] In some embodiments, the activatable protein 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 a specific protease in the CM of the activatable protein. In vivo imaging may be used to identify or screen patient populations suitable for treatment with the activatable proteins of the present disclosure. For example, patients who test positive for both the target being tested and a protease that cleaves a substrate in the CM of the activatable protein (e.g., accumulate activator protein at disease sites) are identified as suitable candidates for treatment with such activatable protein that contains such CM. Similarly, patients who test negative may be identified as suitable candidates for another form of treatment (i.e., not suitable for treatment with the activatable protein being tested). In some embodiments, such patients who test negative for a first activatable protein may be tested with other activatable proteins that contain different CMs until an activatable protein suitable for treatment (e.g., an activatable protein that contains a CM that is cleaved by the patient at the disease site) is identified.

[0221] 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 proteins 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 refine patient populations suitable for treatment with the activatable proteins of the present disclosure. For example, patients who test positive for both the target being tested and a protease that cleaves a substrate within the CM of the activatable protein (e.g., accumulate activating antibodies at disease sites) are identified as suitable candidates for treatment with such activatable proteins that contain such CM. Similarly, patients who test negative for either or both the target and the protease that cleaves the CM used in the activatable proteins being tested using these methods are identified as suitable candidates for another form of treatment (i.e., not suitable for treatment with the activatable protein being tested). In some embodiments, such patients who test negative for a first activatable protein may be tested with other activatable proteins that contain different CMs until an activatable protein suitable for treatment is identified (e.g., an activatable protein that contains a CM that is cleaved by the patient at the site of disease). EXAMPLES

[0222] The invention is further described in the following examples, which do not limit the scope of the invention as described in the claims, but rather provide proof of concept for the advantageous structures of the activatable polymers described in this disclosure.

[0223] Example 1: Generation of activatable bispecific molecules This example illustrates the generation of an exemplary activatable bispecific protein in which the activating protein does not include a half-life extending moiety (e.g., Fc domain). Doubly masked activatable bispecific molecules were prepared by recombinant methods. The proteins were prepared by transforming host cells with three polynucleotides: one with the sequence of SEQ ID NO: 21 (for ProC1446), 22 (for ProC1447), or 23 (for ProC1448); one with the sequence of SEQ ID NO: 1; and one with the sequence of SEQ ID NO: 18; and then culturing the resulting recombinant host cells. These proteins include a masked Fab (AB1) that specifically binds to HER2 in the activated state, a masked scFv (AB2) that specifically binds to CD3 in the activated state, and a knob and hole mutated Fc domain pair (EM). The structures of these activatable proteins are shown in FIG. 6A.

[0224] The reference molecules ProC306 and ProC531 (unmasked bispecific molecules comprising a Fab that specifically binds HER2; an scFv that specifically binds CD3; and a knob and hole Fc domain pair in a different arrangement than the exemplary activatable bispecific molecules above) were also prepared by recombinant methods.

[0225] Example 2. Protease treatment of activatable bispecific molecules To release the masking peptides, the doubly masked activatable bispecific binding molecules prepared in Example 1 were treated with recombinant human proteases such as matrix metalloproteinases (MMPs) or uPA overnight at 37°C. Complete protease treatment was tested by reducing SDS-PAGE. Protein aliquots (2 μg) were denatured in sample buffer (reducing agent added, if necessary) for 10 min at 75°C, separated on 4-12% NuPAGE™ Bis-Tris gels (Thermo Fisher Scientific, Waltham, MA catalog no. NP0321) in MOPS buffer at 175 V for 1 h, stained with InstantBlue™ for 1 h, followed by destaining in water for at least 4 h before visualization.

[0226] The untreated protein was confirmed to have all three chains in reducing gels (Figures 7A and 7B). After overnight protease treatment, activation was incomplete, but the majority of the protease products were of the expected molecular weight.

[0227] Example 3: CD3 antigen binding ELISA The ability of the double-masked activatable bispecific molecules prepared in Example 1 to bind to the CD3 antigen was tested using a CD3 binding ELISA. 100 μg of CD3e-his antigen (ACRO Biosystems) dissolved in 0.05 M carbonate-bicarbonate buffer was adsorbed to the wells of a 96-well microtiter plate overnight at 4° C. The plate was washed and blocked with blocking buffer (1× PBS, pH 7.4, 0.05% Tween®-20, 1% BSA). The double-masked activatable bispecific molecules were serially diluted 4-fold with or without protease treatment together with an unmasked reference protein (ProC531) and applied to the antigen-coated plate. The extent of protein bound to the peptide was measured by anti-human IgG (Fab specific) immunodetection. The absorbance at A450 was measured on a plate reader. Dose-response curves were generated and EC50 values ​​obtained by nonlinear regression with a sigmoidal fit using Graph Pad Prism software. The results are shown in Figure 8. These results demonstrate that each of the three doubly masked activatable bispecific molecules (ProC1446, ProC1447, and ProC1448) exhibits reduced binding to CD3 compared to the unmasked reference bispecific molecule (ProC531) and compared to the corresponding protease-treated activated molecule. The activity of the doubly masked activatable bispecific molecules was restored to the same or nearly the same levels as the corresponding reference bispecific molecule (ProC531) upon treatment with uPA.

[0228] Example 4: HER2-dependent cytotoxicity of doubly masked activatable bispecific molecules The in vitro potency of the double-masked activatable bispecific molecules prepared in Example 1 was determined by a cytotoxicity assay. SKOV3-luc2 target cells and human PBMC effector cells (Stemcell technologies) were plated together in a co-culture in RPMI medium (Gibco catalog number 22400071) supplemented with 5% human serum (MP Bio catalog number 2930949) at a target to effector cell ratio of 1:10. Intact ProC1446, ProC1447, and ProC1448, as well as titrations of their protease-activated forms (uPA-treated ProC1446, uPA-treated ProC1447, uPA-treated ProC1448), and the unmasked reference ProC306 were added to the co-culture. Plates were incubated at 37° C. and 5% CO2 for approximately 48 hours. After incubation, cytotoxicity was assessed using the ONE-Glo™ Luciferase Assay System (Promega Cat. No. E6130) and luminescence was measured on a plate reader (TECAN). Percent cytotoxicity was calculated as follows: (1-(experimental RLU / mean untreated RLU))*100. Percent cytotoxicity data was plotted and EC50 values ​​were calculated using GraphPad PRISM. Results are shown in Figures 9A-9C. These results demonstrate that each of the three doubly masked activatable bispecific molecules (ProC1446, ProC1447, and ProC1448) exhibits reduced cytotoxicity compared to the unmasked reference bispecific molecule (ProC306) and compared to the corresponding protease-treated activated molecules. The cytotoxic activity of the activated molecules after treatment with uPA was more potent than the unmasked reference bispecific molecule (ProC306).

[0229] Example 5: Binding of double-masked bispecific antibodies to Her2+ NCI-N87, SKOV3 and CD3ε+ Jurkat cells To determine whether the described Her2 and CD3ε masking peptides could inhibit binding in the context of a doubly masked bispecific antibody, a flow cytometry-based binding assay was performed.

[0230] NCI-N87 (ATCC), SKOV3 (ATCC) and Jurkat (clone E6-1, ATCC, TIB-152) cells were cultured in RPMI-1640 + glutamax (Life Technologies, Cat. 72400-047), 10% heat inactivated-fetal bovine serum (HI-FBS, Life Technologies, Cat. 10438-026) and, in the case of NCI-N87 cells, puromycin (Gibco, Cat. A11138-03, @2ug / ml). The following bispecific antibodies were tested: recombinantly produced activated SHL1-ProC1963, SHL2-ProC1965, 1 / 2TCB ProC306 and their respective double masked activatable bispecific antibodies, ProC1446 (SHL1), ProC3007 (SHL2), ProC3008 (SHL2) and ProC1441 (1 / 2TCB). Two versions of the cleavable moiety (CM) present between the EM (half-life extending moiety) and the C-terminus were utilized, namely CM1 in ProC3007 versus CM2 in ProC3008.

[0231] NCI-N87 and SKOV3 cells were detached with Versene™ (Life Technologies, Catalog 15040-066), washed, plated at 150,000 cells / well in 96-well plates, and resuspended in 50 μL of primary antibody (bispecific antibody). Jurkat cells were counted and plated as described for NCI-N87 and SKOV3. A titration of primary antibody starting at the concentration shown in Figures 13A-B, followed by a titration of 3-fold serial dilutions in FACS stain buffer + 2% FBS (BD Pharmingen, Catalog 554656) was added to the cells. Cells were incubated with shaking for approximately 1 hour at 4°C, harvested, and washed with 2x200 μL FACS stain buffer. Cells were resuspended in 50 μL of Alexa Fluor® 647-conjugated anti-human IgG, F(ab')2 fragment-specific antibody (1.5 μg / ml, Jackson ImmunoResearch, Cat. 109-605-097) and incubated at 4° C. with shaking for approximately 1 hour. Cells were harvested, washed, and resuspended in a final volume of 200 μL of FACS staining buffer containing 2.5 μg / ml of 7-AAD (BD Biosciences, Cat. 559925). Cells stained with secondary antibody alone were used as a negative control. Data were acquired on an Attun™ NxT flow cytometer, and median fluorescence intensity (MFI) of live cells was calculated using FlowJo® V10.8.1. Raw MFI data was graphed using curve fit analysis in GraphPad Prism.

[0232] Figures 13A-B show binding (i.e., HER2 binding) of masked activatable short half-life antibodies, ProC1446 (SHL1), ProC3007 (SHL2), ProC3008 (SHL2), and masked antibodies, ProC1441 (1 / 2 TCB, non-activatable, short half-life antibody), and unmasked (ProC1963 (SHL1, unmasked, no Fc), ProC1965, and ProC306) anti-CD3, anti-HER2 bispecific antibodies, and secondary antibodies ("Sec only", negative control) to NCI-N87 and SKOV3 cells, respectively. Figure 13C shows binding (i.e., CD3 binding) of the same molecules to Jurkat cells. The results show that all of the masked molecules showed reduced binding to both HER2 and CD3 compared to their corresponding unmasked forms, as represented by a right shift in the binding curves of the masked molecules (very low / no binding even at the highest concentration). EC50 values ​​were determined from replicate experiments. The average EC50 values ​​are shown in Table 1 below. [Table 1]

[0233] The results show that the unmasked anti-HER2, anti-CD3 TCB in activatable short half-life forms (SHL1 and SHL2) exhibited CD3 and HER2 binding to the same extent as the corresponding unmasked 1 / 2TCB forms. A moderate trend towards increased HER2 binding was observed for the activatable short half-life forms compared to the 1 / 2TCB forms. The masked activatable short half-life molecules exhibited highly attenuated HER2 and CD3 binding to the same extent as that observed for the masked 1 / 2TCB molecules. The masked 1 / 2TCB molecule (ProC1441), the molecule shown on the left in Figure 12, lacks the third cleavable moiety (CM3) (1205) between the EM and Fab, meaning that this molecule is not cleavable to release the EM.

[0234] Example 6: Biological activity of doubly masked activatable bispecific antibodies The biological activity of intact and recombinantly produced activatable bispecific antibodies was assayed using a cytotoxicity assay. Human PBMCs were purchased from HemaCare Inc (Van Nuys, CA) and co-cultured with Her2-expressing cancer cell lines NCI-N87 (ATCC) or SKOV3 (ATCC) at a ratio of 10:1 in RPMI-1640 + glutamax supplemented with 5% heat-inactivated human serum (Sigma, catalog H3667). Dose response of intact ProC1446 (SHL1), ProC3007 (SHL2), ProC3008 (SHL2), ProC1441 (1 / 2TCB) with recombinantly produced activating bispecific antibodies ProC1963 (SHL1), ProC1965 (SHL2), and ProC306 (1 / 2TCB) in co-culture medium at starting concentrations shown in Figures 14A-14B and 15A-15B, followed by 3-fold serial dilutions, was tested. After 48 hours, cytotoxicity was assessed using the ONE-Glo™ Luciferase Assay System (Promega, Madison, WI Cat. E6130). Luminescence was measured on an Infinite® M200 Pro (Tecantting AG, Switzerland). Percent cytotoxicity was calculated and plotted using curve fit analysis in GraphPad PRISM.

[0235] Figures 14A and 14B show that recombinantly produced activated bispecific proteins ProC1963 (SHL1) and ProC1965 (SHL2) have increased potency compared to ProC306 (EC50 >1200-fold and 50-fold lower, respectively) as indicated by a left shift in the dose-response curves.

[0236] Figures 15A and 15B show that in these assays, the intact bispecifics ProC1446, ProC3007, ProC3008, and ProC1446 are strongly masked as shown by the right-shifted dose-response curves compared to their recombinantly produced activated versions ProC1963 and ProC1965.

[0237] Figures 16A and 16D show that in these assays, the intact bispecifics ProC1446, ProC3007, ProC3008 and ProC1441 are strongly masked as shown by the right-shifted dose-response curves compared to their recombinantly produced activated versions ProC1963, ProC1965 and ProC306, respectively.

[0238] EC50 values ​​and masking efficiencies (ME) were determined from replicate experiments, and the results are provided in Tables 2A and 2B below. [Table 2A]

[0239] The EC50 for ProC1963 was approximately 1200-3000-fold lower than that for ProC306. The EC50 for ProC1965 was 50-fold lower than that for ProC306. These results suggest that the unmasked, activatable, short-lived TCB forms exhibit higher potency compared to the control (ProC306) that is not activatable in terms of half-life, i.e., not cleavable to release EM. [Table 2B]

[0240] These results indicate that masking attenuated the activity of all three forms of SHL1, SHL2, and 1 / 2TCB.

[0241] Example 7: The double-masked, bispecific, activatable antibody ProC3007 and its corresponding activated form ProC1965 induced regression of established NCI-N87 tumors in mice

[0242] In this example, intact activatable bispecific antibodies ProC3007 (SHL2TCB), ProC3008 (SHL2TCB), ProC1441, and the recombinantly produced activating bispecific ProC1965 targeting Her2 and CD3ε were analyzed for their ability to induce regression or reduce the growth of established NCI-N87 xenograft tumors in NOD scid gamma (NSG) mice engrafted with human PBMCs.

[0243] Human gastric cancer cell line NCI-N87 was obtained from ATCC and cultured in RPMI+Glutamax+10%FBS according to established procedures. Purified and frozen human PBMCs were obtained from Hemacare Inc (Van Nuys, CA) (Donor ID number D163477; Lot number 22077550). NSG™ (NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ) mice were obtained from Theackson Laboratories (Bar Harbor, ME).

[0244] On day 0, 1x10 cells were cultured in 100 μL of RPMI+Glutamax serum-free medium containing Matrigel®. 6 Each mouse was inoculated subcutaneously with 100 mg of NCI-N87 cells in the right flank. Pre-frozen PBMCs from a single donor were thawed and administered (ip) at a 1:1 ratio of CD3+ T cells to tumor cells on day 7. Tumor volumes were approximately 125 mm 3 Upon reaching 100 mg / kg / day, mice were randomized, assigned to treatment groups, and administered iv according to Table 3. Recombinant bispecific ProC1965 (SHL2 form, unmasked and without Fc domain) was administered 3 times per week to compensate for the expected increased clearance rate due to the lack of a half-life extending (Fc) domain. Dose levels of ProC1965 were adjusted to account for the difference in molecular weight. Tumor volumes were measured twice weekly. One mouse from the 0.5mpk cohort was euthanized early. Thereafter, n=7 for that cohort at days 21 and 25. [Table 3]

[0245] Figure 17 is a plot of tumor volume versus days after first treatment administration. The results demonstrate that both intact activatable bispecific antibody ProC3007 and recombinantly produced activatable bispecific antibody ProC1965 induced regression of NCI-N87 xenograft tumors at 1 and 0.5 mpk, respectively. The results showed that ProC1965 and ProC3007 were more effective than ProC1441 in this study. ProC3008 had similar efficacy to ProC1441 at comparable dose levels.

[0246] A second in vivo experiment was performed as above, but using PBMCs from a different donor (Hemacare, donor ID number D327579, lot number 21070049). In this study, a panel of bispecific activating antibodies including a recombinantly produced activatable short half-life bispecific antibody (ProC1446) and the corresponding recombinantly produced activated version of this activated molecule (ProC1963, i.e., having the structure of ProC1446 but lacking the mask and Fc domain). ProC1446 and ProC1963 were administered as described in Table 4 and evaluated for their ability to induce regression or reduce the growth of established NCI-N87 xenograft tumors in human PBMC-engrafted NSG mice. Both ProC1963 and ProC1446 appeared to have antitumor activity in this experiment, and therefore ProC1963 retains the ability to induce tumor regression. [Table 4]

[0247] The sequences of the example molecules and other sequences disclosed herein are listed in Table 5 below. [Table 5-1] [Table 5-2]

Table 5-3

Table 5-4

Table 5-5

Table 5-6

Table 5-7

Table 5-8

Table 5-9

Table 5-10

Table 5-11

Table 5-12

Table 5-13

Table 5-14

Table 5-15

Table 5-16

Table 5-17

Table 5-18

Table 5-19

Table 5-20

Table 5-21

Table 5-22

Table 5-23

Table 5-24

Table 5-25

Table 5-26

Table 5-27

Table 5-28

Table 5-29

Table 5-30

Table 5-31

Table 5-32

Table 5-33

Table 5-34

Table 5-35

Table 5-36

Table 5-37

Table 5-38

Table 5-39

Table 5-40

Table 5-41

Table 5-42

Table 5-43

Table 5-44

Table 5-45

Table 5-46

Table 5-47

Table 5-48

Table 5-49

Table 5-50

Table 5-51

Table 5-52

Table 5-53

[0248] 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.

[0249] 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 to the section headings, the materials, methods, and examples are illustrative only and not intended to be limiting.

Claims

1. A protein that can be activated, a) A first antigen-binding domain (AB1) that specifically binds to a first target, wherein AB1 comprises a first heavy chain variable domain (HVD1) and a first light chain variable domain (LVD1); A second antigen-binding domain (AB2) that specifically binds to a second target, wherein AB2 comprises a second heavy chain variable domain (HVD2) and a second light chain variable domain (LVD2), and AB2 is directly or indirectly coupled to the C-terminus of HVD1 or the C-terminus of LVD1; A first masking portion (MM1) coupled to the AB1 either directly or indirectly via a first cleavable portion (CM1), wherein the MM1 inhibits the binding of the AB1 to the first target; A second masking portion (MM2) is coupled directly or indirectly to a half-life extension portion (EM); The EM is coupled to the AB1 or AB2 either directly or indirectly via a second detachable portion (CM2), The MM2 inhibits the binding of the AB2 to the second target, b) A first antigen-binding domain (AB1) that specifically binds to a first target, wherein AB1 comprises a first heavy chain variable domain (HVD1) and a first light chain variable domain (LVD1); A second antigen-binding domain (AB2) that specifically binds to a second target, wherein AB2 comprises a second heavy chain variable domain (HVD2) and a second light chain variable domain (LVD2), and AB2 is directly or indirectly coupled to the C-terminus of HVD1 or LVD1; A first cleavable portion (CM1) and a first masking portion (MM1) optionally coupled to the AB1 via one or more linkers, wherein the MM1 inhibits the binding of the AB1 to the first target; A half-life extension portion (EM) directly or indirectly coupled to a second masking portion (MM2), wherein the EM and the MM2 are coupled to a second cleavable portion (CM2) and optionally to the AB1 or the AB2 via one or more linkers; The MM2 inhibits the binding of the AB2 to the second target, or c) A first antigen-binding domain (AB1) that specifically binds to a first target, wherein AB1 comprises a first heavy chain variable domain (HVD1) and a first light chain variable domain (LVD1); A second antigen-binding domain (AB2) that specifically binds to a second target, wherein AB2 comprises a second heavy chain variable domain (HVD2) and a second light chain variable domain (LVD2), and AB2 is directly or indirectly coupled to the C-terminus of HVD1 or LVD1; A first cleavable portion (CM1) and a first masking portion (MM1) optionally coupled to the AB1 via one or more linkers, wherein the MM1 inhibits the binding of the AB1 to the first target; A half-life extension portion (EM) comprising a dimer of a first half-life extension portion (EM1) and a second half-life extension portion (EM2); The EM1 is coupled to the AB1 via a second detachable portion (CM2) and optionally one or more linkers. The EM2 is directly or indirectly coupled to the second masking portion (MM2). The MM2 inhibits the binding of the AB2 to the second target. The aforementioned activatable protein.

2. The activatable protein according to claim 1, wherein CM2 is the N-terminus of EM1 and CM2 is the C-terminus of AB1.

3. The activatable protein according to claim 1, wherein CM2 is the N-terminus of MM2, MM2 is the N-terminus of EM, and CM2 is the C-terminus of AB1 or AB2, to which EM and MM2 are coupled via CM2.

4. An activatable protein, a) A first target-binding domain (TB1) that specifically binds to the first target; A second target-binding domain (TB2) that specifically binds to a second target, wherein the TB2 is directly or indirectly coupled to the TB1; A first masking portion (MM1) directly or indirectly coupled to the TB1 via a first cleavable portion (CM1), wherein the MM1 inhibits the binding of the TB1 to the first target; A half-life extension portion (EM) and a second masking portion (MM2) directly or indirectly coupled to the TB1 or the TB2 via a second cleavable portion (CM2), wherein the MM2 inhibits the binding of the TB2 to the second target; The components of the activatable molecule are configured such that the cleavage of CM1 and CM2 releases MM1, MM2, and EM from TB1 which is directly or indirectly coupled to TB2, and / or b) A first antigen-binding domain (AB1) that specifically binds to a first target, wherein AB1 comprises a first heavy chain variable domain (HVD1) and a first light chain variable domain (LVD1); A second antigen-binding domain (AB2) that specifically binds to a second target, wherein AB2 comprises a second heavy chain variable domain (HVD2) and a second light chain variable domain (LVD2), and AB2 is directly or indirectly coupled to the N-terminus of HVD1 or the N-terminus of LVD1; A first cleavable portion (CM1) and a first masking portion (MM1) optionally coupled to the AB1 via one or more linkers, wherein the MM1 inhibits the binding of the AB1 to the first target; A second cleavable portion (CM2) and a second masking portion (MM2) optionally coupled to AB1 via one or more linkers, wherein the MM2 inhibits the binding of AB2 to the second target; Including a third cleavable portion (CM3) and a half-life extension portion (EM) that is optionally coupled directly or indirectly to the C-terminus of the HVD1 or the C-terminus of the LVD1 via one or more linkers, The aforementioned activatable protein.

5. The activatable protein according to claim 4, wherein TB1 is an antigen-binding molecule (AB1) containing HVD1 and LVD1, or TB2 is an antigen-binding molecule (AB2) containing HVD2 and LVD2.

6. a) The EM is a dimer formed by a first fragment crystallizable (Fc) domain and a second Fc domain, and / or b) The EM is the C-terminus of AB1 or AB2, which is coupled directly or indirectly via CM2, and the MM1 is the N-terminus of AB1. The activatable protein according to claim 1.

7. The activatable protein comprises at least a first polypeptide and a second polypeptide. a) The first polypeptide comprises, in order from the N-terminus to the C-terminus, MM1, CM1, and VLD1, and / or b) The second polypeptide comprises the VHD1, VHD2, VLD2, CM2, MM2, and a first Fc domain, and the activatable protein further comprises a third polypeptide comprising a second Fc domain, or the second polypeptide comprises the VHD1, VHD2, VLD2, CM2, MM2, and a first Fc domain in order from the N-terminus to the C-terminus, The activatable protein according to claim 1.

8. a) i) The second polypeptide comprises, in order from the N-terminus to the C-terminus, the VHD1, the CM2, the MM2, and the first Fc domain, or ii) The second polypeptide comprises, in order from the N-terminus to the C-terminus, the VHD1, the CM2, and the first Fc domain, and / or b) The first polypeptide comprises MM1, CM1, VLD1, VHD2, and VLD2, The activatable protein according to claim 7.

9. a) The first polypeptide comprises, in order from the N-terminus to the C-terminus, MM1, CM1, VLD1, VHD2, and VLD2, or b) The first polypeptide comprises, in order from the N-terminus to the C-terminus, MM1, CM1, VLD1, VLD2, and VHD2, The activatable protein according to claim 8.

10. The protein comprises a third polypeptide, the third polypeptide comprising a second Fc domain and the MM2, and / or a) The MM2 is linked to the C-terminus of the second Fc domain via a linker, or b) The MM2 is linked to the N-terminus of the second Fc domain via a linker. The activatable protein according to claim 8.

11. The protein comprises a third polypeptide, the third polypeptide comprising a second Fc domain and the MM2, and / or a) The MM2 is linked to the C-terminus of the second Fc domain via a linker, or b) The MM2 is linked to the N-terminus of the second Fc domain via a linker. The activatable protein according to claim 10.

12. (i) MM1 and CM1, (ii) CM1 and VLD1, (iii) VHD1 and VLD2, (iv) VHD1 and VHD2, (v) VHD1 and CM2, (vi) VLD2 and VHD2, (vii) CM2 and MM2, (viiii) CM2 and EM, (ix)EM and MM2, (x) VLD1 and VHD2, and / or (xi) Further comprising a linker for coupling VLD1 and VLD2, and / or The linker is a peptide having a length of 5-30, 6-29, 7-28, 8-27, 9-26, 10-25, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 amino acids. The activatable protein according to any one of claims 1, 10, or 11.

13. The activatable protein according to any one of claims 10 or 11, wherein the linker is a peptide having a length of 5-30, 6-29, 7-28, 8-27, 9-26, 10-25, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 amino acids.

14. The activatable protein according to claim 7, wherein the first Fc domain is a whole variant of the Fc domain, and the second Fc domain is a knob variant of the Fc domain.

15. The activatable protein according to claim 14, wherein the whole variant of the Fc domain comprises the sequence of SEQ ID NO: 2, and the knob variant of the Fc domain comprises the sequence of SEQ ID NO:

1.

16. a) The CM2 is directly or indirectly coupled to the C-terminus of the AB1 or the C-terminus of the AB2, or the MM2 is directly or indirectly coupled to the C-terminus of the CM2, and the MM2 is directly or indirectly coupled to the N-terminus of the EM, and / or b) The EM comprises a dimer of a first half-life extension portion (EM1) and a second half-life extension portion (EM2), or i) The CM2 is directly or indirectly coupled to the N-terminus of the EM1, ii) The CM2 is directly or indirectly coupled to the N-terminus of the MM2, and the MM2 is directly or indirectly coupled to the N-terminus of the EM1, or iii) The MM2 is directly or indirectly coupled to the N-terminus of the EM2, The activatable protein according to claim 1.

17. a) The first target or epitope is a tumor-associated antigen, and / or the tumor-associated antigen is human epidermal growth factor receptor 2 (HER2), or i) The AB1 is a fab of trastuzumab, or ii) The HVD1 includes the sequence of sequence number 27, and the LVD1 includes the sequence of sequence number 17. b) The above AB2 is, Immune effector cells that associate with scFv, White blood cells that associate with scFv, T cells that associate with scFv, NK cells that associate with scFv, Macrophages that associate with scFv, or It is a mononuclear cell that associates with scFv. c) The AB2 is anti-CD3 epsilon scFv or anti-CTLA-4 scFv, or derived therefrom. d) The HVD2 includes the sequence of sequence number 30, and the LVD2 includes the sequence of sequence number 31. e) The AB1 is an anti-HER2 antibody or derived therefrom. f) Each of the CM1 and CM2 independently contains a substrate for a protease that is upregulated within the tumor microenvironment. g) The AB1 is a fragment antigen binder (Fab), h) The second target is a co-stimulatory molecule, and / or i) 1) Each of CM1 and CM2 contains the same protease substrate, or 2) The CM1 and CM2 contain substrates for different proteases. The activatable protein according to claim 1.

18. The activatable protein according to claim 17, wherein the co-stimulatory molecule is CD3.

19. a) Each of the CM1 and CM2 independently contains ADAMS, ADAMTS, ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAMDEC1, ADAMTS1, ADAMTS4, ADAMTS5, aspartate protease, BACE, renin, aspartate cathepsin, cathepsin D, cathepsin E, caspase, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6 Caspase 7, Caspase 8, Caspase 9, Caspase 10, Caspase 14, Cysteine ​​Cathepsin, Cathepsin B, Cathepsin C, Cathepsin K, Cathepsin L, Cathepsin S, Cathepsin V / L2, Cathepsin X / Z / P, Cysteine ​​Proteinase, Crudipain, Regmain, Otsubine-2, KLK, KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, KLK14, Metalloproteinase, Meprin, Neprilysin PSMA, BMP-1, MMP, MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP23, MMP24, MMP26, MMP27, serine protease, activated protein C, cathepsin A, cathepsin G, chymase, coagulation factor protease, FVIIa, FIXa, FXa, FXIa, FXIIa, elastase, g The substrate contains a protease selected from the group consisting of Lanzyme B, guanidinobenzoate, HtrA1, human neutrophil elastase, lactoferrin, malapsin, NS3 / 4A, PACE4, plasmin, PSA, tPA, thrombin, tryptase, uPA, type II transmembrane, serine protease, TTSP, DESC1, DPP-4, FAP, hepsin, matryptase-2, MT-SP1 / matryptase, TMPRSS2, TMPRSS3, and TMPRSS4. b) The MM1 is a peptide with a length of 2 to 40 amino acids, and / or c) The MM2 is a peptide with a length of 2 to 40 amino acids. The activatable protein according to any one of claims 1 to 8.

20. The MM1 is a peptide with a length of 2 to 40 amino acids, and / or the MM2 is a peptide with a length of 2 to 40 amino acids. a) i) The heavy chain variable region of AB2 is directly or indirectly coupled to the C-terminus of the heavy chain fragment of AB1. The N-terminus of MM2 is directly or indirectly coupled to the C-terminus of the light chain variable region of AB2 via CM2. The EM comprises a dimer of a first Fc domain and a second Fc domain, and the C-terminus of the MM2 is directly or indirectly coupled to the N-terminus of the first Fc domain of the EM, ii) The heavy chain variable region of AB2 is directly or indirectly coupled to the C-terminus of the light chain fragment of AB1. The N-terminus of MM2 is directly or indirectly coupled to the C-terminus of the heavy chain fragment of AB1 via CM2. The EM comprises a dimer of a first Fc domain and a second Fc domain, and the C-terminus of the MM2 is directly or indirectly coupled to the N-terminus of the first Fc domain of the EM, iii) The heavy chain variable region of AB2 is directly or indirectly coupled to the C-terminus of the light chain fragment of AB1. The EM comprises a dimer of a first Fc domain and a second Fc domain, wherein the N-terminus of the first Fc domain is directly or indirectly coupled to the C-terminus of the heavy chain fragment of AB1 via the CM2. The N-terminus of MM2 is directly or indirectly coupled to the C-terminus of the second Fc domain, or iv) The heavy chain variable region of AB2 is directly or indirectly coupled to the C-terminus of the light chain fragment of AB1. The EM comprises a dimer of a first Fc domain and a second Fc domain, wherein the N-terminus of the first Fc domain is directly or indirectly coupled to the C-terminus of the heavy chain fragment of AB1 via the CM2. The C-terminus of MM2 is directly or indirectly coupled to the N-terminus of the second Fc domain. The activatable protein according to any one of claims 1 to 8.

21. The activatable protein according to claim 20, further comprising a linker between the MM2 and the first or second Fc domain directly or indirectly coupled to the MM2.

22. a) The MM1 includes the sequence of sequence number 40, and the MM2 includes one of the sequences of sequence numbers 34 to 37 or 66 to 70. b) The MM1 has a dissociation constant for binding to AB1 that is greater than the dissociation constant of AB1 for binding to the first target or epitope, and the MM2 has a dissociation constant for binding to AB2 that is greater than the dissociation constant of AB2 for binding to the second target or epitope. c) The components of the activatable protein are, i) The cleavage of CM1 and CM2 is configured to cleave MM1, MM2, and EM from the activatable protein, thereby producing an activated protein with a shorter half-life compared to the corresponding molecule containing TB1, TB2, and EM, or ii) The cleavage of CM1 and CM2 is configured to cleave MM1, MM2, and EM from the activatable protein, thereby producing an activated protein having higher target-binding activity compared to the corresponding molecule containing TB1, TB2, and EM. d) The second polypeptide further comprises a linker (L2) between the MM2 and the AB2, and / or e) The second polypeptide further comprises a linker (L3) between AB2 and AB1, L2 and / or L3 in d) or e) are peptides having a length of 5-30, 6-29, 7-28, 8-27, 9-26, 10-25, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 amino acids. An activatable protein according to any one of claims 1 to 2.

23. a) The following: (A) A first antigen-binding domain (AB1) that specifically binds to a first target, wherein AB1 comprises a first heavy chain variable domain (HVD1) and a first light chain variable domain (LVD1); (B) A second antigen-binding domain (AB2) that specifically binds to a second target, wherein AB2 comprises a second heavy chain variable domain (HVD2) and a second light chain variable domain (LVD2), and AB2 is directly or indirectly coupled to the C-terminus of HVD1 or the C-terminus of LVD1; (C) A first masking portion (MM1) coupled to the AB1 either directly or indirectly via a first cleavable portion (CM1), wherein the MM1 inhibits the binding of the AB1 to the first target; (D) A half-life extension portion (EM) directly or indirectly coupled to a second masking portion (MM2); A protein containing, (E) The EM is coupled to the AB1 or AB2 either directly or indirectly via a second severable portion (CM2), (F) The MM2 inhibits the binding of the AB2 to the second target, and / or (G) The EM is a dimer formed by a first fragment crystallizable (Fc) domain and a second Fc domain, and / or the EM is the C-terminus of AB1 or AB2, which is directly or indirectly coupled via CM2, and MM1 is the N-terminus of AB1, and / or the first Fc domain is a hole variant of the Fc domain, and the second Fc domain is a knob variant of the Fc domain, and / or (H) The activatable protein comprises at least a first polypeptide and a second polypeptide, i) The first polypeptide comprises MM1, CM1, and VLD1 in order from the N-terminus to the C-terminus, and the second polypeptide comprises VHD1, CM2, MM2, and the first Fc domain in order from the N-terminus to the C-terminus, and / or the first polypeptide comprises MM1, CM1, VLD1, VHD2, and VLD2, or ii) The second polypeptide comprises the VHD1, CM2, and the first Fc domain in order from the N-terminus to the C-terminus, and the first polypeptide comprises the MM1, CM1, VLD1, VHD2, and VLD2, further a) The first polypeptide comprises, in order from the N-terminus to the C-terminus, MM1, CM1, VLD1, VHD2, and VLD2, or the first polypeptide comprises, in order from the N-terminus to the C-terminus, MM1, CM1, VLD1, VLD2, and VHD2, and / or b) The protein comprises a third polypeptide, the third polypeptide comprising a second Fc domain and the MM2, and / or the MM2 is linked to the C-terminus of the second Fc domain via a linker, or the MM2 is linked to the N-terminus of the second Fc domain via a linker, and / or iii) The second polypeptide comprises the VHD1, VHD2, VLD2, CM2, MM2, and a first Fc domain, and the activatable protein further comprises a third polypeptide comprising a second Fc domain, for example, the second polypeptide comprises the VHD1, VHD2, VLD2, CM2, MM2, and a first Fc domain in order from the N-terminus to the C-terminus, protein, b) A first antigen-binding domain (AB1) that specifically binds to a first target, wherein AB1 comprises a first heavy chain variable domain (HVD1) and a first light chain variable domain (LVD1); A second antigen-binding domain (AB2) that specifically binds to a second target, wherein AB2 comprises a second heavy chain variable domain (HVD2) and a second light chain variable domain (LVD2), and AB2 is directly or indirectly coupled to the C-terminus of HVD1 or the C-terminus of LVD1; A first cleavable portion (CM1) and a first masking portion (MM1) optionally coupled to the AB1 via one or more linkers, wherein the MM1 inhibits the binding of the AB1 to the first target; A half-life extension portion (EM) directly or indirectly coupled to a second masking portion (MM2), wherein the EM and the MM2 are coupled to the second cleavable portion (CM2) and optionally one or more linkers to the AB1 or the AB2; A protein containing, The MM2 inhibits the binding of the AB2 to the second target, and / or the CM2 is the N-terminus of the MM2, the MM2 is the N-terminus of the EM, and the CM2 is the C-terminus of the AB1 or AB2, to which the EM and the MM2 are coupled via the CM2 and optionally one or more linkers. protein, c) Below A first antigen-binding domain (AB1) that specifically binds to a first target, wherein AB1 comprises a first heavy chain variable domain (HVD1) and a first light chain variable domain (LVD1); A second antigen-binding domain (AB2) that specifically binds to a second target, wherein AB2 comprises a second heavy chain variable domain (HVD2) and a second light chain variable domain (LVD2), and AB2 is directly or indirectly coupled to the C-terminus of HVD1 or the C-terminus of LVD1; A first cleavable portion (CM1) and a first masking portion (MM1) optionally coupled to the AB1 via one or more linkers, wherein the MM1 inhibits the binding of the AB1 to the first target; A half-life extension portion (EM) containing a dimer of a first half-life extension portion (EM1) and a second half-life extension portion (EM2); A protein containing, The EM1 is coupled to the AB1 via a second detachable portion (CM2) and optionally one or more linkers. The EM2 is directly or indirectly coupled to the second masking portion (MM2). The MM2 inhibits the binding of the AB2 to the second target, and / or the CM2 is the N-terminus of the EM1, and the CM2 is the C-terminus of the AB1. protein The activatable protein according to claim 2, further comprising the characteristics of the above.

24. (i) MM1 and CM1, (ii) CM1 and VLD1, (iii) VHD1 and VLD2, (iv) VHD1 and VHD2, (v) VHD1 and CM2, (vi) VLD2 and VHD2, (vii) CM2 and MM2, (viiii) CM2 and EM, (ix)EM and MM2, (x) VLD1 and VHD2, and / or (xi) VLD1 and VLD2 It further includes a linker that couples, The linker is a peptide having a length of 5-30, 6-29, 7-28, 8-27, 9-26, 10-25, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 amino acids. The activatable protein according to claim 23.

25. a) The CM2 is directly or indirectly coupled to the C-terminus of the AB1 or the C-terminus of the AB2, and optionally, the MM2 is directly or indirectly coupled to the C-terminus of the CM2, and the MM2 is directly or indirectly coupled to the N-terminus of the EM. b) The EM comprises a dimer of a first half-life extension portion (EM1) and a second half-life extension portion (EM2), and optionally, i) The CM2 is directly or indirectly coupled to the N-terminus of the EM1, ii) The CM2 is directly or indirectly coupled to the N-terminus of the MM2, and the MM2 is directly or indirectly coupled to the N-terminus of the EM1, or iii) The MM2 is directly or indirectly coupled to the N-terminus of the EM2, c) The first target or epitope is a tumor-associated antigen, and / or the tumor-associated antigen is human epidermal growth factor receptor 2 (HER2), and / or i) The AB1 is either a fab of trastuzumab or ii) The HVD1 includes the sequence of sequence number 27, and the LVD1 includes the sequence of sequence number 17. d) AB2 is, Immune effector cells that associate with scFv, White blood cells that associate with scFv, T cells that associate with scFv, NK cells that associate with scFv, Macrophages that associate with scFv, or It is a mononuclear cell that associates with scFv. e) The AB2 is anti-CD3 epsilon scFv or anti-CTLA-4 scFv, or derived therefrom. f) The HVD2 includes the sequence of sequence number 30, and the LVD2 includes the sequence of sequence number 31. g) The AB1 is an anti-HER2 antibody or derived therefrom. h) Each of the CM1 and CM2 independently contains a substrate for a protease that is upregulated within the tumor microenvironment. i) The AB1 is a fragment antigen binder (Fab), j) The second target is a co-stimulatory molecule, and / or k) i) Each of CM1 and CM2 contains the same protease substrate, or ii) The CM1 and CM2 contain substrates for different proteases, The activatable protein according to claim 23.

26. a) The first target or epitope is a tumor-associated antigen, and / or the tumor-associated antigen is human epidermal growth factor receptor 2 (HER2), or i) The AB1 is a fab of trastuzumab, or ii) The HVD1 includes the sequence of sequence number 27, and the LVD1 includes the sequence of sequence number 17. b) The above AB2 is, Immune effector cells that associate with scFv, White blood cells that associate with scFv, T cells that associate with scFv, NK cells that associate with scFv, Macrophages that associate with scFv, or It is a mononuclear cell that associates with scFv. c) The AB2 is anti-CD3 epsilon scFv or anti-CTLA-4 scFv, or derived therefrom. d) The HVD2 includes the sequence of sequence number 30, and the LVD2 includes the sequence of sequence number 31. e) The AB1 is an anti-HER2 antibody or derived therefrom. f) Each of the CM1 and CM2 independently contains a substrate for a protease that is upregulated within the tumor microenvironment. g) The AB1 is a fragment antigen binder (Fab), h) The second target is a co-stimulatory molecule, and / or i) 1) Each of CM1 and CM2 contains the same protease substrate, or 2) The CM1 and CM2 contain substrates for different proteases. The activatable protein according to claim 23.

27. ​​a) Each of the CM1 and CM2 independently contains ADAMS, ADAMTS, ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAMDEC1, ADAMTS1, ADAMTS4, ADAMTS5, aspartate protease, BACE, renin, aspartate cathepsin, cathepsin D, cathepsin E, caspase, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6 Caspase 7, Caspase 8, Caspase 9, Caspase 10, Caspase 14, Cysteine ​​Cathepsin, Cathepsin B, Cathepsin C, Cathepsin K, Cathepsin L, Cathepsin S, Cathepsin V / L2, Cathepsin X / Z / P, Cysteine ​​Proteinase, Crudipain, Regmain, Otsubine-2, KLK, KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, KLK14, Metalloproteinase, Meprin, Neprilysin PSMA, BMP-1, MMP, MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP23, MMP24, MMP26, MMP27, serine protease, activated protein C, cathepsin A, cathepsin G, chymase, coagulation factor protease, FVIIa, FIXa, FXa, FXIa, FXIIa, elastase, g The substrate contains a protease selected from the group consisting of Lanzyme B, guanidinobenzoate, HtrA1, human neutrophil elastase, lactoferrin, malapsin, NS3 / 4A, PACE4, plasmin, PSA, tPA, thrombin, tryptase, uPA, type II transmembrane, serine protease, TTSP, DESC1, DPP-4, FAP, hepsin, matryptase-2, MT-SP1 / matryptase, TMPRSS2, TMPRSS3, and TMPRSS4. b) The MM1 is a peptide with a length of 2 to 40 amino acids, and / or c) The MM2 is a peptide with a length of 2 to 40 amino acids. The activatable molecules in b) and c) are A) to G) A) i) The heavy chain variable region of AB2 is directly or indirectly coupled to the C-terminus of the heavy chain fragment of AB1, The N-terminus of MM2 is directly or indirectly coupled to the C-terminus of the light chain variable region of AB2 via CM2. The EM comprises a dimer of a first Fc domain and a second Fc domain, and the C-terminus of the MM2 is directly or indirectly coupled to the N-terminus of the first Fc domain of the EM, ii) The heavy chain variable region of AB2 is directly or indirectly coupled to the C-terminus of the light chain fragment of AB1, The N-terminus of MM2 is directly or indirectly coupled to the C-terminus of the heavy chain fragment of AB1 via CM2. The EM comprises a dimer of a first Fc domain and a second Fc domain, and the C-terminus of the MM2 is directly or indirectly coupled to the N-terminus of the first Fc domain of the EM, iii) The heavy chain variable region of AB2 is directly or indirectly coupled to the C-terminus of the light chain fragment of AB1, The EM comprises a dimer of a first Fc domain and a second Fc domain, wherein the N-terminus of the first Fc domain is directly or indirectly coupled to the C-terminus of the heavy chain fragment of AB1 via the CM2. The N-terminus of MM2 is directly or indirectly coupled to the C-terminus of the second Fc domain, or iv) The heavy chain variable region of AB2 is directly or indirectly coupled to the C-terminus of the light chain fragment of AB1. The EM comprises a dimer of a first Fc domain and a second Fc domain, wherein the N-terminus of the first Fc domain is directly or indirectly coupled to the C-terminus of the heavy chain fragment of AB1 via the CM2. The C-terminus of MM2 is directly or indirectly coupled to the N-terminus of the second Fc domain. B) A linker between the MM2 and the first or second Fc domain directly or indirectly coupled to the MM2, C) The MM1 includes the sequence of sequence number 40, and the MM2 includes one of the sequences of sequence numbers 34-37 or 66-70. D) The MM1 has a dissociation constant for binding to AB1 that is greater than the dissociation constant of AB1 for binding to the first target or epitope, and the MM2 has a dissociation constant for binding to AB2 that is greater than the dissociation constant of AB2 for binding to the second target or epitope. E) The components of the activatable protein are: i) The cleavage of CM1 and CM2 is configured to cleave MM1, MM2, and EM from the activatable protein, thereby producing an activated protein with a shorter half-life compared to the corresponding molecule containing TB1, TB2, and EM, or ii) The cleavage of CM1 and CM2 is configured to cleave MM1, MM2, and EM from the activatable protein, thereby producing an activated protein having higher target-binding activity compared to the corresponding molecule containing TB1, TB2, and EM. F) The second polypeptide further comprises a linker (L2) between MM2 and AB2, or L2 is a peptide having a length of 5-30, 6-29, 7-28, 8-27, 9-26, 10-25, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 amino acids, and G) The second polypeptide further comprises a linker (L3) between AB2 and AB1, or L3 is a peptide having a length of 5-30, 6-29, 7-28, 8-27, 9-26, 10-25, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 amino acids. The activatable protein according to claim 23, further comprising one or more of the above.

28. A composition comprising an activatable protein and a carrier according to any one of claims 1, 2, or 23.

29. A container, vial, syringe, injection pen, or kit comprising at least one dose of the composition according to claim 28.

30. A nucleic acid comprising a sequence encoding a second polypeptide according to any one of claims 7, 8, or 23.

31. A vector comprising the nucleic acid described in claim 30.

32. A cell comprising the nucleic acid described in claim 30.

33. A conjugated activatable protein comprising an activatable protein according to any one of claims 1, 2, or 23, conjugated with a drug.

34. a) The agent is a therapeutic agent, an antitumor agent, a toxin, a diagnostic agent, a therapeutic polymer, a targeting portion, or a detectable portion, and / or b) The drug is conjugated to the antibody via a linker. The conjugated, activatable protein according to claim 33.

35. i) The linker is a severable linker, or ii) The linker is a non-cutting type linker. The conjugated, activatable protein according to claim 33.

36. A composition for use in a method of treating a subject requiring treatment, comprising an activatable protein according to any one of claims 1, 2, and 23, or a conjugated activatable protein comprising an activatable protein according to any one of claims 1, 2, and 23, wherein the composition is administered to the subject.

37. The composition according to claim 36, characterized in that the subject is identified or diagnosed with cancer.

38. A method for generating an activatable protein, The cells described in claim 32 are cultured in a culture medium under conditions sufficient to produce the activatable protein, This includes recovering the activatable protein from the cells or the culture medium, or The process further includes isolating the activatable protein recovered from the cells or culture medium, The isolation of the activatable protein is performed using protein purification tags and / or size exclusion chromatography, and / or The further comprising incorporating the activatable protein into the pharmaceutical composition, The aforementioned method.

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