Activatable multispecific molecules and methods of use thereof

Abstract

An activatable protein comprising a first target binding protein (TB1) that specifically binds to the first target; a second target binding protein (TB2) specifically binding to a second target, wherein TB2 is directly or indirectly coupled to TB1; a first masking moiety (MM1) that inhibits binding of TB1 to a first target and is coupled to TB1 directly or indirectly via a cleavable moiety, such as via one or more linkers or other components; a second masking moiety (MM2) that inhibits binding of TB2 to a second target and is coupled directly or indirectly to TB1 or to TB2 via a cleavable moiety; and a half-life extending moiety (EM) coupled directly or indirectly to the TB1 or to the TB2 via a cleavable moiety. The activatable protein is configured such that when the cleavable moiety cleaves, the resulting activatable protein has a shorter half-life, a higher binding affinity for the first target and a higher binding affinity for the second target than the intact activatable protein.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application No. US63 / 326,692, filed on April 1, 2022, which is incorporated herein by reference in its entirety.

[0003] Sequence Listing

[0004] The sequence listing submitted by EFS together with this application is named "4862-122PCT.xml", created on March 30, 2023, with a size of 639,463 bytes, and is incorporated herein by reference in its entirety. Technical Field

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

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

[0007] Activatable antibodies advance efforts to expand the therapeutic index of antibody-based therapies. These molecules are administered in the form of activatable prodrugs that are activated at or near the desired site of action in vivo. This mechanism of action can increase the therapeutic index of the parent antibody.

[0008] However, additional strategies are still needed to improve the therapeutic index of antibody-based therapies. Summary of the invention

[0009] The present disclosure provides an activatable protein and related compositions and methods.

[0010] 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, wherein the TB2 is coupled to the TB1; a first masking portion (MM1), which is coupled to the TB1 via a first cleavable portion (CM1), wherein the MM1 inhibits the binding of the TB1 to the first target; a second masking portion (MM2), which inhibits the binding of the TB2 to the second target; a second cleavable portion (CM2); and a half-life extending portion (EM), which is directly or indirectly coupled to the MM1 or the MM2, wherein the components of the activatable protein are configured such that after cleavage of CM1 and CM2, the resulting activated protein comprises TB1 and TB2, but does not comprise MM1, MM2 and EM. As used herein and unless otherwise indicated, a "coupled" component of an activatable molecule can be coupled via a direct covalent bond or an indirect covalent bond, e.g., via one or more connecting peptides (also referred to as "linkers"), cleavable moieties, or other components of the activatable protein.

[0011] In one aspect, the present disclosure provides an activatable protein, the activatable protein comprising: a first antigen-binding domain (AB1) that specifically binds to a first target, wherein the 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 the AB2 comprises a second heavy chain variable domain (HVD2) and a second light chain variable domain (LVD2), and the AB2 is directly or indirectly coupled to the C-terminus of the HVD1 or to the C-terminus of the LVD1; a first masking portion (MM1 ), the first masking moiety is coupled to the AB1 via a first cleavable moiety (CM1) (directly or indirectly, e.g., via one or more linkers), wherein the MM1 inhibits the binding of the AB1 to the first target; a half-life extending moiety (EM), the half-life extending moiety is coupled directly or indirectly to a second masking moiety (MM2), wherein the EM is coupled to the AB1 or to the AB2 via a second cleavable moiety (CM2) (directly or indirectly, e.g., via one or more linkers or other components of an activatable protein), and wherein the MM2 inhibits the binding of the AB2 to the second target.

[0012] In one aspect, the present disclosure provides an activatable protein, the activatable protein comprising: a first antigen-binding domain (AB1) that specifically binds to a first target, wherein the 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 the AB2 comprises a second heavy chain variable domain (HVD2) and a second light chain variable domain (LVD2), and the AB2 is directly or indirectly coupled to the C-terminus of the HVD1 or the LVD1; a first shielding portion The invention relates to a method for preparing an antibody-binding protein comprising: comprising: a first masking moiety (MM1) coupled to said AB1 via a first cleavable moiety (CM1) (directly or indirectly, e.g., via one or more linkers), wherein said MM1 inhibits binding of said AB1 to said first target; and a half-life extending moiety (EM) coupled directly or indirectly to a second masking moiety (MM2), wherein said EM and said MM2 are coupled to said AB1 or to said AB2 via a second cleavable moiety (CM2) (directly or indirectly), and wherein said MM2 inhibits binding of said AB2 to said second target.

[0013] In one aspect, the present disclosure provides an activatable protein, the activatable protein comprising: a first antigen-binding domain (AB1) that specifically binds to a first target, wherein the 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 the AB2 comprises a second heavy chain variable domain (HVD2) and a second light chain variable domain (LVD2), and the AB2 is directly or indirectly coupled to the C-terminus of the HVD1 or the LVD1; a first masking portion (MM1), the first masking portion being cleavable via a first a cleavable moiety (CM1) coupled to the AB1 (directly or indirectly, e.g., via one or more linkers), wherein the MM1 inhibits the binding of the AB1 to the first target; and a half-life extending moiety (EM), the half-life extending moiety comprising a dimer of a first half-life extending moiety (EM1) and a second half-life extending moiety (EM2), wherein the EM1 is coupled to the AB1 via a second cleavable moiety (CM2) (directly or indirectly, e.g., via one or more linkers), and wherein the EM2 is coupled directly or indirectly to a second masking moiety (MM2), wherein the MM2 inhibits the binding of the AB2 to the second target.

[0014] In one aspect, the present disclosure provides an activatable protein, the 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, wherein the TB2 is directly or indirectly coupled to the TB1; a first masking moiety (MM1), the first masking moiety is coupled to the TB1 via a first cleavable moiety (CM1) (directly or indirectly, such as via one or more linkers), wherein the MM1 inhibits the binding of the TB1 to the first target; a half-life extending moiety (EM) and a second masking moiety (MM2), The half-life extending portion and the second masking portion are coupled to the TB1 or to the TB2 via a second cleavable portion (CM2) (directly or indirectly, for example via one or more linkers), wherein the MM2 inhibits the binding of the TB2 to the second target, wherein the components of the activatable molecule are configured such that cleavage of CM1 and CM2 releases MM1, MM2 and EM (where applicable) from TB1 and TB2, and wherein optionally the TB1 is an antigen binding molecule (AB1) comprising HVD1 and LVD1, and optionally the TB2 is an antigen binding molecule (AB2) comprising HVD2 and LVD2.

[0015] In one aspect, the present disclosure provides an activatable protein, the activatable protein comprising: a first antigen-binding domain (AB1) that specifically binds to a first target, wherein the 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, wherein the AB2 comprises a second heavy chain variable domain (HVD2) and a second light chain variable domain (LVD2), and the AB2 is coupled directly or indirectly (e.g., via a linker) to the C-terminus of the HVD1 or the LVD1; a first a masking moiety (MM1) coupled to the AB1 via a first cleavable moiety (CM1) (directly or indirectly, e.g., via a linker), wherein the MM1 inhibits the binding of the AB1 to the first target; a second masking moiety (MM2) coupled to the AB2 via a second cleavable moiety (CM2) (directly or indirectly, e.g., via a linker), wherein the MM2 inhibits the binding of the AB2 to the second target; and a half-life extending moiety (EM) coupled directly or indirectly to the MM1 or the MM2.

[0016] On the other hand, the present disclosure provides an activatable protein, the activatable protein comprising: a first antigen-binding domain (AB1) that specifically binds to a first target, wherein the 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 the AB2 comprises a second heavy chain variable domain (HVD2) and a second light chain variable domain (LVD2), and the AB2 is directly or indirectly coupled to the N-terminus of the HVD1 or to the N-terminus of the LVD1; a first masking portion (MM1), the A first masking moiety is coupled to the AB1 via a first cleavable moiety (CM1) and optionally one or more linkers, wherein the MM1 inhibits the binding of the AB1 to the first target; a second masking moiety (MM2), wherein the second masking moiety is coupled to the AB2 via a second cleavable moiety (CM2) and optionally one or more linkers, wherein the MM2 inhibits the binding of the AB2 to the second target; a half-life extending moiety (EM), wherein the half-life extending moiety is coupled to the C-terminus of the HVD1 or to the C-terminus of the LVD1 via a third cleavable moiety (CM3) and optionally one or more linkers.

[0017] 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.

[0018] In some embodiments, the first polypeptide comprises MM1, CM1 and VLD1 in sequence from N-terminus to C-terminus (with 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 wherein the activatable protein further comprises a third polypeptide, the third polypeptide comprising a second Fc domain. In some embodiments, the second polypeptide comprises VHD1, VHD2, VLD2, CM2, MM2 and a first Fc domain in sequence from N-terminus to C-terminus. In some embodiments, the second polypeptide comprises VHD1, CM2, MM2 and a first Fc domain in sequence from N-terminus to C-terminus. In some embodiments, the second polypeptide comprises VHD1, CM2 and a first Fc domain in sequence from N-terminus to C-terminus. In some embodiments, the first polypeptide comprises MM1, CM1 and VLD1, VHD2 and VLD2. In some embodiments, the first polypeptide comprises MM1, CM1, VLD1, VHD2 and VLD2 in sequence from N-terminus to C-terminus. In some embodiments, the first polypeptide comprises MM1, CM1, VLD1, VLD2 and VHD2 in order from N-terminus to C-terminus. In some embodiments, the protein comprises a third polypeptide, and wherein the third polypeptide comprises a second Fc domain and MM2. In each of the foregoing embodiments, and unless otherwise stated, the polypeptide may comprise, for example, one or more optional linkers between each of the listed elements.

[0019] In some embodiments, MM2 is connected to the C-terminus of the second Fc domain via a connecting peptide. In some embodiments, MM2 is connected to the N-terminus of the second Fc domain via a connecting peptide (also referred to as a "joint"). In some embodiments, the second polypeptide also includes a joint (L1) between MM2 and the first Fc domain. In some embodiments, L1 is a peptide of 5 to 30, 6 to 29, 7 to 28, 8 to 27, 9 to 26, 10 to 25, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 amino acids in length. In the aforementioned paragraphs and the structural arrangements disclosed in the present disclosure, one or more joints may optionally be present between the elements. In addition, the present disclosure also contemplates and includes activatable proteins, wherein any one or more disclosed elements are optionally directly adjacent to each other, so that there is no joint or other amino acid sequence between the elements.

[0020] In some embodiments, the first Fc domain is an Fc domain hole mutant and the second Fc domain is an Fc domain knob mutant. In some embodiments, the Fc domain hole mutant comprises the sequence of SEQ ID NO:2 and the Fc domain knob mutant comprises the sequence of SEQ ID NO:1.

[0021] 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 a sequence of SEQ ID NO: 27 and LVD1 comprises a sequence of SEQ ID NO: 17. In some embodiments, AB2 is: a scFv that engages immune effector cells; a scFv that engages leukocytes; a scFv that engages T cells; a scFv that engages NK cells; a scFv that engages macrophages; or a scFv that engages mononuclear cells. In some embodiments, AB2 is or is derived from an anti-CD3εscFv or an anti-CTLA-4scFv. In some embodiments, AB2 is or is derived from an anti-CD3εscFv. In some embodiments, HVD2 comprises a sequence of SEQ ID NO: 30 and LVD2 comprises a sequence of SEQ ID NO: 31.

[0022] In some embodiments, AB1 is or is derived from an anti-HER2 antibody. In some embodiments, AB1 is a 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 an antigen binding fragment (Fab). In some embodiments, the second target is a co-stimulatory molecule. In some embodiments, the co-stimulatory molecule is CD3.

[0023] In some embodiments, each of CM1 and CM2 comprises a substrate for the same protease. In some embodiments, CM1 and CM2 comprise substrates for different proteases. In some embodiments, each of CM1 and CM2 independently comprises a substrate for a protease selected from the group consisting of ADAMS, ADAMTS, ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAMDEC1, ADAMTS1, ADAMTS4, ADAMTS5, aspartic protease, BACE, renin, aspartic cathepsin, cathepsin D, cathepsin E, apoptotic protease, apoptotic protease 1, apoptotic protease 2, apoptotic protease 3, apoptotic protein Cathepsin 4, Cathepsin 5, Cathepsin 6, Cathepsin 7, Cathepsin 8, Cathepsin 9, Cathepsin 10, Cathepsin 14, Cysteine ​​cathepsins, Cathepsin B, Cathepsin C, Cathepsin K, Cathepsin L, Cathepsin S, Cathepsin V / L2, Cathepsin X / Z / P, Cysteine ​​proteases, Cruzipain, Otubain-2, KLK, KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK1 3. KLK14, metalloproteinases, transmembrane peptidase (Meprin), enkephalinase, 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, chymotrypsin, coagulation factor protease, FVIIa , FIXa, FXa, FXIa, FXIIa, Elastase, Granzyme B, Guanidinobenzoic Acidase, 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, Interstitial Proteinase-2, MT-SP1 / Interstitial Proteinase, TMPRSS2, TMPRSS3 and TMPRSS4.

[0024] In some embodiments, MM1 and MM2 are each independently 2 to 40 amino acids in length. In some embodiments, MM1 and MM2 are each independently 4 to 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 (directly or indirectly, for example, via one or more joints), and the EM comprises a dimer of the first Fc domain and the 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 (directly or indirectly, for example, via one or more joints), and the EM comprises a dimer of the first Fc domain and the 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 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 (directly or indirectly, for example, 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, and the N-terminus of the first Fc domain is coupled to the C-terminus of the heavy chain fragment of AB1 via CM2 (directly or indirectly, for example, via one or more linkers), and the C-terminus of MM2 is directly or indirectly coupled to the N-terminus of the second Fc domain.

[0025] In some embodiments, the activatable protein further comprises a linker between MM2 and a first Fc domain or a second Fc domain coupled directly or indirectly 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 NO: 34-37 or 66-70. In some embodiments, the dissociation constant of MM1 binding to AB1 exceeds the dissociation constant of AB1 binding to a first target or epitope, and the dissociation constant of MM2 binding to AB2 exceeds the dissociation constant of AB2 binding to a second target or epitope. In some embodiments, the activating molecule has a shorter half-life than a corresponding molecule identical to the activating molecule but comprising an EM. In some embodiments, the activating molecule has a higher target binding activity than a corresponding molecule identical to the activating molecule but comprising an EM. In some embodiments, the activating molecule has a higher target binding activity than an activatable molecule.

[0026] In some embodiments, the second polypeptide further comprises a linker (L2) between AB2 and AB2. In some embodiments, L2 is 5 to 30, 6 to 29, 7 to 28, 8 to 27, 9 to 26, 10 to 25, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 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 to 30, 6 to 29, 7 to 28, 8 to 27, 9 to 26, 10 to 25, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 amino acids in length. In general, in each embodiment herein, unless otherwise stated, the polypeptide may comprise one or more optional linkers between each of the listed elements, and such linkers may be 1 to 30, 6 to 29, 7 to 28, 8 to 27, 9 to 26, 10 to 25, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 amino acids in length.

[0027] In another aspect, the present disclosure provides a composition comprising an activatable protein as described herein and a carrier. In some embodiments, the composition is a pharmaceutical composition, wherein the carrier is a pharmaceutically acceptable carrier.

[0028] In another aspect, the present disclosure provides a container, a vial, a syringe, an injection pen or a kit comprising at least one dose of a composition herein.

[0029] In another aspect, the present disclosure provides a nucleic acid comprising a sequence encoding the second polypeptide herein.

[0030] In another aspect, the present disclosure provides a vector comprising a nucleic acid herein.

[0031] In another aspect, the disclosure provides a cell comprising a nucleic acid or a vector herein.

[0032] In another aspect, the present disclosure provides a conjugated activatable protein comprising an 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.

[0033] In another aspect, the disclosure provides a method of treating a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an activatable protein, composition, or conjugated activatable protein herein. In some embodiments, the subject has been identified or diagnosed as having cancer.

[0034] In another aspect, the present disclosure provides a method for producing an activatable protein, the method comprising: culturing the cells herein in a culture medium under conditions sufficient to produce the activatable protein; and recovering the activatable protein from the cells or culture medium. In some embodiments, the method further comprises isolating the recovered activatable protein from the cells or culture medium. In some embodiments, isolating the activatable protein is performed using a protein purification tag and / or size exclusion chromatography. In some embodiments, the method further comprises formulating the activatable protein into a pharmaceutical composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description which sets forth illustrative embodiments utilizing the principles of the invention and the accompanying drawings, in which:

[0036] Figure 1-4 The configuration of an exemplary activatable molecule is shown. The molecule is designed such that the activated molecule produced by activation of the activatable molecule does not contain a half-life extending moiety and therefore has a shorter half-life than a corresponding molecule that is the same as the activation molecule but contains a half-life extending moiety.

[0037] Figure 5 Schematic diagram of an exemplary activatable (double masked) bispecific antibody according to some embodiments of the present disclosure before and after activation by a protease. The left side schematically shows an activatable double masked protein. The dotted line between elements 501 and 505 and between elements 502 and 503 represents a cleavable portion. The right side schematically shows an activating protein. The activated bispecific antibody does not include a masking portion, so the activated bispecific antibody has an increased binding affinity to its target relative to the activatable bispecific antibody. The activated bispecific antibody also does not include a half-life extension portion and therefore has a shorter half-life than the activatable bispecific antibody.

[0038] Fig. 6A Schematic diagram of the components of an exemplary doubly masked bispecific activatable antibody having a masked Fab fragment that recognizes Her2, a masked scFV component that recognizes CD3, and a half-life extending portion comprising a pair of knob and hole Fc domains. Figure 6B Is the display code such as Fig. 6A Schematic diagram of the components of the three polypeptides of an exemplary doubly masked, bispecific, activatable antibody shown. The dotted line represents the cleavable portion.

[0039] Fig. 7A is an image of an SDS-PAGE gel run under reducing conditions. The gel was loaded as follows: (1) a doubly masked bispecific activatable antibody with a 20GG CD3 mask (ProC1446, SEQ ID NO: 21); (2) the product of ProC1446 and uPA (ProC1446+uPA); (3) a doubly masked bispecific activatable antibody with a MN15a CD3 mask (ProC1447, SEQ ID NO: 22); (4) the product of ProC1447 and uPA (ProC1447+uPA); (5) a doubly masked bispecific activatable antibody with a MN15b CD3 mask (ProC1448, SEQ ID NO: 23); and (6) the product of ProC1448 and uPA (ProC1448+uPA). Figure 7B is a table summarizing the expected molecular weight of the components of each activatable antibody construct before and after protease activation.

[0040] Figure 8 The results of an ELISA binding assay are provided, which determines the ability of activatable molecules and activating molecules to bind to plate-bound CD3 antigen: unmasked reference bispecific molecule (ProC531), doubly masked activatable bispecific molecule with 20GG CD3 mask (ProC1446, SEQ ID NO: 21), the product of ProC1446 and uPA (ProC1446 + uPA), doubly masked molecule with MN15a mask (ProC1447, SEQ ID NO: 22), the product of ProC1447 and uPA (ProC1447 + uPA), doubly masked molecule with MN15b mask (ProC1448, SEQ ID NO: 23), the product of ProC1448 and uPA (ProC1448 + uPA). The results show that the corresponding CD3 mask between the anti-CD3 scFv and the Fc region shows reduced binding of the anti-CD3 scFv to the CD3 antigen coated on the plate. Treatment of the doubly masked activatable bispecific antibody with the protease uPA resulted in CD3 antigen binding comparable to that of the unmasked reference bispecific molecule ProC531.

[0041] Figures 9A-9C Results of a HER2-dependent cytotoxicity assay determining the in vitro potency of a dual-masked, activatable bispecific antibody are provided. Fig. 9A :ProC1446; Fig. 9B :ProC1447; Fig. 9CThe results show that the protease-treated (activated) bispecific molecules of the present disclosure are more active than a monovalent, unmasked bispecific antibody control ("ProC306") having the same HER2 and CD3 binding domains but arranged in a different format.

[0042] Figure 10-11 The configuration of an exemplary activatable molecule is shown. The molecule comprises a doubly masked activatable bispecific antibody having an EM coupled to the C-terminus via a third cleavable moiety. The molecule is designed such that the activated molecule produced by activation of the activatable molecule does not comprise a half-life extending moiety and therefore has a shorter half-life than a corresponding molecule that is the same as the activation molecule but comprises a half-life extending moiety.

[0043] Fig.12 Schematic diagram of an exemplary activatable (dual masked) bispecific antibody before and after activation by a protease according to some embodiments of the present disclosure. The left side schematically shows an activatable dual masked protein. The right side schematically shows an activated protein. The activated bispecific antibody does not include a masking portion, so the activated bispecific antibody has an increased binding affinity to its target relative to the activatable bispecific antibody. The activated bispecific antibody also does not include a half-life extension portion and therefore has a shorter half-life than the activatable bispecific antibody. The dotted line represents a cleavable portion.

[0044] Figures 13A-13B The binding results of masked activatable short half-life antibodies ProC1446 (SHL1), ProC3007 (SHL2), ProC3008 (SHL2) and masked antibody ProC1441 (1 / 2TCB, not an activatable short half-life antibody) and unmasked (ProC1963 (SHL1, no mask or Fc), ProC1965 (SHL2, no mask or Fc) and ProC306) anti-CD3, anti-HER2 bispecific antibodies and secondary antibodies ("secondary antibody only", negative control) to NCI-N87 and SKOV3 cells (i.e., HER2 binding) are provided respectively. Fig. 13C Results for binding of the same molecules to Jurkat cells (ie, CD3 binding) are provided.

[0045] Figures 14A-14B Provided using NCI-N87 cells ( Fig.14A ) and SKOV3 cells ( Fig. 14B ) showing the dose response of ProC1963 (SHL1), ProC1965 (SHL2) and ProC306 at the indicated concentrations.

[0046] Figures 15A-15BProvided using NCI-N87 cells ( Fig.15A ) and SKOV3 cells ( Fig. 15B ) showing the dose response of ProC1963, ProC1965, ProC1446, ProC3007 and ProC3008 at the indicated concentrations.

[0047] Figures 16A-16D Results of cytotoxicity assays are presented. Figures 16A-16B The use of NCI-N87 cells ( Fig.16A ) and SKOV3 cells ( Fig. 16B ), dose response to ProC1963, ProC1965, ProC3007, ProC3008, ProC306 and ProC1441. Figures 16C-16D The use of NCI-N87 cells ( Fig. 16C ) and SKOV3 cells ( Fig.16D ), dose response to ProC1963, ProC1965, ProC1446, ProC306 and ProC1441.

[0048] Fig.17 Results of an in vivo tumor growth assay using the NCI-N87 xenograft model are provided. The graph shows tumor volume versus days after initial treatment with ProC1965, ProC3007, ProC3008, and ProC1441 in milligrams per kilogram (mpk).

[0049] The figures provided herein are for illustration purposes only and are not necessarily drawn to scale. DETAILED DESCRIPTION

[0050] Provided herein are activatable molecules (e.g., activatable proteins, such as activatable antibodies and other activatable therapeutic or activatable diagnostic proteins) having relatively low binding activity and structures comprising a half-life extension portion (EM). When activated by exposure to certain activation conditions (e.g., when the activatable molecule is delivered to a tumor), the resulting activated molecule has greater binding activity and a shorter half-life than 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, such as a therapeutic protein.

[0051] In general, the activatable molecules herein may include one or more target binding domains (TB), one or more masking moieties (MM) that reduce, inhibit or interfere with the binding of TB to its target, one or more cleavable moieties (CM) that couple one or more MMs to one or more TBs, and one or more half-life extension moieties (EM) that couple one or more CMs to TBs. 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 another component. When two components are indirectly coupled, there will be a stretch of amino acids between the two components. In some instances, the two components of a polypeptide may be indirectly coupled via one or more other components in the polypeptide, i.e., one or more other components are between two coupling components. For indirect coupling or connection via another component, one or more other components may be a linker, TB (e.g., AB), CM, MM, or any combination thereof.

[0052] CM is a polypeptide comprising a substrate for a sequence-specific protease, such as a protease that is present in greater amounts (or in greater amounts in an active state) in the environment of a diseased tissue (e.g., a tumor) than in a healthy tissue. The MM and EM of the activatable molecules described herein can be released from the TB by cleaving the CM, thereby generating an activation molecule. Compared to the corresponding activatable molecule comprising the MM, the activation molecule exhibits greater binding affinity to its target. Compared to the corresponding molecule identical to the activation molecule but comprising the EM, the activation molecule without the EM may have a shorter half-life. Compared to the corresponding molecule identical to the activation molecule but comprising the EM, the activation molecule may have reduced toxicity and reduced off-target effects.

[0053] In some embodiments, the activatable molecule can be a dual-masked bispecific target binding molecule. In some aspects, such molecules can include at least two target binding proteins and at least two masking parts, each of which inhibits the binding of the target binding protein to its target. For example, the activatable molecule can include a first target binding protein (TB1) that specifically binds to a first target, a first masking part (MM1) that inhibits the binding of TB1 to the first target, a cleavable part (CM1) between MM1 and TB1, a second target binding protein (TB2) that specifically binds to a second target, a second masking part (MM2) that inhibits the binding of TB2 to the second target, a cleavable part (CM2) between MM2 and TB2, and an EM coupled to TB1 or TB2 via a cleavable part (CM). In some embodiments, EM can be coupled to TB via CM, which also couples MM to TB. In some embodiments, EM can be coupled to TB via a CM different from CM1 and CM2 (e.g., a third CM or "CM3"). In the activated state, EM can be released from the activation molecule. Therefore, compared with the reference molecule comprising TB1, TB2 and EM but not comprising MM1 or MM2, the activation molecule (comprising TB1 and TB2 but not comprising MM1, MM2 or EM) has a shorter half-life. Compared with the reference molecule comprising TB1, TB2 and EM but not comprising MM1 or MM2, the activation molecule (comprising TB1 and TB2 but not comprising MM1, MM2 and EM) has a higher target binding activity.

[0054] Also provided herein are related compositions, kits, nucleic acids, vectors and recombinant cells, as well as related methods, including methods of using any of the activatable molecules (e.g., activatable antibodies and other proteins) described herein and methods of producing any of the activatable molecules (e.g., activatable antibodies and other proteins) described herein.

[0055] definition

[0056] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Methods and materials for use in the present disclosure are described herein; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In the event of a conflict, the present specification, including definitions, will prevail.

[0057] The terms "a" or "an" refer to one or more (ie, at least one) grammatical object of the article. By way of example, "a cell" encompasses one or more cells.

[0058] As used herein, the terms "about" and "approximately" when used to modify a numerical value or an amount specified in a range, represent the numerical value and reasonable deviations from the value known to those skilled in the art. For example, ±20%, ±10% or ±5% are within the intended meaning of the stated value, where appropriate.

[0059] Concentration, amount and other numerical data can be expressed or presented in range format herein.It should be understood that this type of range format is used only for convenience and simplicity, and should therefore be flexibly interpreted as not only including the numerical value of the limit value of the range explicitly stated, but also including all individual numerical values ​​or sub-ranges contained within the range, as if each numerical value and sub-range were explicitly stated.As an illustration, the numerical range of "about 0.01 to 2.0" should be interpreted as not only including the value of about 0.01 to about 2.0 explicitly stated, but also including individual values ​​and sub-ranges within the indicated range.Therefore, individual values, such as 0.5, 0.7 and 1.5, and sub-ranges, such as 0.5 to 1.7, 0.7 to 1.5 and 1.0 to 1.5, etc., are included in this numerical range.In addition, no matter how the width of the range or the features described, this explanation should apply.In addition, it should be noted that, unless otherwise specified, all percentages are calculated by weight.

[0060] In understanding the scope of the present disclosure, the term "comprising" or "including" and their derivatives as used herein are intended to specify the existence of stated features, elements, components, groups, integers and / or steps, but do not exclude the open-ended terms of the existence of other unstated features, elements, components, groups, integers and / or steps. The foregoing also applies to words with similar meanings, such as the terms "comprising", "having" and their derivatives. The term "consisting of" and its derivatives as used herein are intended to specify the existence of stated features, elements, components, groups, integers and / or steps, but exclude the closed terms of the existence of other unstated features, elements, components, groups, integers and / or steps. The term "consisting essentially of" as used herein is intended to specify the existence of stated features, elements, components, groups, integers and / or steps and those features, elements, components, groups, integers and / or steps that do not substantially affect one or more basic and novel features of features, elements, components, groups, integers and / or steps. It should be understood that reference to any of these transitional terms (i.e., "comprising," "consisting of," or "consisting essentially of") provides direct support for substitution with any other transitional term not specifically used. For example, since this definition is directed to any element disclosed throughout this disclosure, modifying the term from "comprising" to "consisting essentially of" or "consisting of" will be directly supported. Based on this definition, any element disclosed or incorporated by reference herein may be included in or excluded from the claimed invention.

[0061] As used herein, for convenience, multiple compounds, elements or steps may be presented in a common list. However, these lists should be interpreted as if each member of the list is individually identified as a separate and unique member. Therefore, individual members of such a list should not be interpreted as de facto equivalents of any other members of the same list based solely on their presentation in a common group without contrary indication.

[0062] The term "exemplary" is used herein to mean used as an example, instance or illustration. Any aspect or design described herein as "exemplary" is not necessarily to be construed as being better or superior to other aspects or designs. On the contrary, the word exemplary is used to present concepts in a more specific way.

[0063] In addition, certain molecules, constructs, compositions, elements, parts, excipients, diseases, illnesses, properties, steps, etc. may be discussed in the context of a specific embodiment or aspect or in a separate paragraph or chapter of the disclosure. It should be understood that this is only for convenience and brevity, and any such disclosure is equally applicable to any other embodiment or aspect found anywhere in the disclosure and claims, and is intended to be combined with any other embodiment or aspect described, all of which form the present application and the invention required on the filing date. For example, the list of constructs, molecules, method steps, kits, or compositions described with respect to constructs, compositions, or methods is intended to and does find direct support for the embodiments related to the constructs, compositions, preparations, and methods described in any other part of the disclosure, even if those method steps, activating agents, kits, or compositions are not re-listed in the context or chapter of the embodiment or aspect.

[0064] Activatable molecules

[0065] In one aspect, the activatable molecules provided herein can be activatable target binding proteins (TBs), such as activatable antibodies or another protein that specifically binds to a target. In some embodiments, the activatable molecules include: a TB that specifically binds to a target (e.g., an antigen binding protein (AB)); a cleavable portion (CM) that is directly covalently attached to (also referred to as "directly coupled to") or indirectly covalently attached to (also referred to as "indirectly coupled to") TB (e.g., AB), wherein the CM is located between the TB and a masking portion (MM), which reduces, inhibits, or interferes with the binding of the TB (e.g., AB) to its target; and one or more half-life extension portions (EMs) that are coupled to the TB (e.g., AB) via one or more CMs. By cleaving the CM, the MM and EM can be released from the TB to produce an activation 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 the 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 the binding of AB2 to the second target and is coupled to AB1 or to AB2 via a second cleavable moiety (CM2); and an EM that is coupled to AB1 or to 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 (see, e.g., Figure 2 ). In some aspects, the EM can be coupled to AB1 or AB2 via a third cleavable moiety (CM3). In each of the aforementioned aspects, the elements of the activatable molecule can be coupled directly, or indirectly via one or more optional linkers between the elements. In the activated state, the EM can be released from the activatable protein to produce an activated protein comprising AB1 and AB2 but not MM1, MM2 or EM, wherein the activated protein has a shorter half-life compared to a reference antibody comprising AB1, AB2 and EM but not MM1 or MM2.

[0066] In some embodiments, the activatable protein provides reduced toxicity and / or off-target side effects that could result from the binding of the TB (e.g., AB) at non-therapeutic sites if the TB is not masked or otherwise inhibited from binding to the target. In the activatable state, the MM may interfere with the binding of the TB to its target molecule.

[0067] In some embodiments, the activatable protein comprises: a first antigen binding protein (AB1) that specifically binds to a first target, wherein AB1 comprises an antibody or a 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, wherein AB2 comprises a single chain variable fragment (scFv) comprising a heavy chain variable region and a light chain variable region, and AB2 is coupled to the C-terminus of the heavy chain fragment or the light chain fragment of AB1; a first masking portion (MM1), when the activatable protein is in an inactivated state, the first masking portion is coupled to AB1 via a first cleavable portion (CM1) and inhibits the binding of AB1 to the first target; a second masking portion (MM2), when the activatable protein is in an uncleaved state, the second masking portion is coupled to AB2 and inhibits the binding of AB2 to the second target; and a half-life extending portion (EM), the half-life extending portion is coupled to a component of AB1 or AB2 via a second cleavable portion (CM2). In some examples, AB1 can be Fab. In some examples, AB1 can be scFv. In some aspects, EM is coupled to AB1 or to AB2 via a masking moiety, such as an EM-MM-CM-AB or AB-CM-MM-EM structure, optionally with one or more linkers between one or more components. As used herein, the symbol "-" in the structural formula indicates that the two components are coupled directly or indirectly (e.g., there may be an optional linker between the components). The structural configuration of the molecules disclosed herein is described in detail below, and for example in Figure 1 -Depicted in 6.

[0068] As used herein, the terms "activatable protein" and "activatable target binding protein" (e.g., "activatable antibody"), as well as any of the foregoing together with the terms "intact," "uncleaved," and / or "inactive," are used interchangeably to refer to a protein comprising at least one set of MM, CM, and TB, and having reduced binding to a biological target compared to the binding of a corresponding "activating" protein (e.g., activating antibody) comprising the same TB to the same biological target. It will be apparent to one of ordinary skill that exposure of an activatable protein to a CM-specific protease can produce an "activating" protein in which the MM does not reduce, inhibit, or interfere with the binding between the TB (e.g., AB) and its target. In some embodiments, cleavage of the CM by an appropriate protease may result in the release of the MM. In some embodiments, cleavage of the CM by an appropriate protease may result in the release of the EM. The terms "activating protein," "activating target binding protein" (e.g., "activating antibody"), "cleaved activatable protein," and "cleaved activatable target binding protein" (e.g., "cleaved activatable antibody") are used interchangeably herein to refer to a cleavage product containing TB produced after exposure of an activatable protein to a CM-specific protease. As used throughout this disclosure, descriptions referring to activatable antibodies should be interpreted as also applying to activatable target binding proteins.

[0069] As used herein, the terms "masking moiety" and "MM" are used interchangeably herein and refer to a peptide or protein that interferes with the binding of TB to a biological target when located near TB (e.g., AB). The terms "cleavable moiety" and "CM" are used interchangeably herein and refer to a peptide that comprises a substrate for a sequence-specific protease. In the activatable protein, the CM is positioned relative to the MM and TB so that cleavage produces a molecule that can bind to the biological target of TB. Therefore, the binding of the activatable protein to the biological target is reduced compared to the activating protein. In some embodiments, the activatable protein can be designed by selecting a TB of interest and constructing the remainder of the activatable protein so that the MM provides masking of the TB or a reduction in the binding of the TB to its target. Structural design criteria can be considered to provide this functional feature.

[0070] The activatable protein can be a multispecific (e.g., bispecific, trispecific, tetraspecific, and other multispecific activatable proteins) activatable protein that is capable of binding to multiple different antigens when activated. In some embodiments, the multispecific target binding protein can 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 can be monospecific, e.g., capable of binding to only one antigen when activated.

[0071] 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. Typically, the 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 can simultaneously bind to two targets, such as two target proteins expressed on two different cells.

[0072] In some embodiments, the activatable protein may include AB1 that can bind to a molecule on the surface of a cell associated with the disease (e.g., a tumor cell) and AB2 that can bind to a molecule on the surface of an immune cell. This type of bispecific activatable protein can bind to immune cells and disease-associated cells (e.g., tumor cells) at the same time when activated, thereby activating the immune cells and cross-linking the activated immune cells with the disease-associated cells. In some embodiments, the activatable protein can be formulated as a part of a former bispecific T cell engager (former BiTE) molecule, a former chimeric antigen receptor (former CAR) modified T cell or other engineered receptor or other immune effector cell (e.g., CAR-modified NK cell).

[0073] In some examples, the activatable protein can be an activatable T cell engaging bispecific antibody (TCB) or a fragment thereof. For example, the activatable protein can include AB1 targeting cells associated with a disease and AB2 targeting a T cell receptor.

[0074] The present disclosure includes activatable proteins in various structural configurations described herein. Exemplary configurations of activatable proteins are provided below. Within the activatable protein, the N-terminal to C-terminal order of TB, MM, CM, and EM may be reversed. CM and MM may overlap in the amino acid sequence, for example, so that the CM sequence recognized by the sequence-specific protease is at least partially contained in the MM. For example, various structural configurations of activatable antigen-binding proteins are contemplated, wherein AB1 is an antigen-binding fragment (Fab), AB2 is a single-chain variable fragment, and can be represented by the following formula (in the order of the amino (N) terminal region to the carboxyl (C) terminal region). In the following formula, ":" separates two different polypeptides; "Fab_L" and "Fab_H" are the light chain and heavy chain fragments of Fab, respectively ("Fab_L" includes a variable light chain region (VL) and a light chain constant region; "Fab_H" includes a variable heavy chain region (VH) and a CH1 region); "VL*" and "VH*" are the light chain and heavy chain variable regions of scFv. Furthermore, as used herein, and unless otherwise indicated, each dash (-) between components of an activatable molecule represents a direct connection or an indirect connection via one or more linkers.

[0075] (MM1-CM1-Fab_L):(Fab_H-VH*-VL*-CM2-MM2-EM)

[0076] (MM1-CM1-Fab_L):(Fab_H-VH*-VL*-CM2-EM-MM2)

[0077] Fab_L:(MM1-CM1-Fab_H-VH*-VL*-CM2-MM2-EM)

[0078] Fab_L:(MM1-CM1-Fab_H-VH*-VL*-CM2-EM-MM2)

[0079] (MM1-CM1-Fab_L-VH*-VL*-CM2-MM2-EM):Fab_H

[0080] (MM1-CM1-Fab_L-VH*-VL*-CM2-EM-MM2):Fab_H

[0081] (Fab_L-VH*-VL*-CM2-MM2-EM):(MM1-CM1-Fab_H)

[0082] (Fab_L-VH*-VL*-CM2-EM-MM2):(MM1-CM1-Fab_H)

[0083] (MM1-CM1-Fab_L-VH*-VL*):(Fab_H-CM2-MM2-EM)

[0084] (MM1-CM1-Fab_L-VH*-VL*):(Fab_H-CM2-EM-MM2)

[0085] (Fab_L-VH*-VL*):(MM1-CM1-Fab_H-CM2-MM2-EM)

[0086] (Fab_L-VH*-VL*):(MM1-CM1-Fab_H-CM2-EM-MM2)

[0087] (MM1-CM1-Fab_L-CM2-MM2-EM):(Fab_H-VH*-VL*)

[0088] (MM1-CM1-Fab_L-CM2-EM-MM2):(Fab_H-VH*-VL*)

[0089] (Fab_L-CM2-MM2-EM):(MM1-CM1-Fab_H-VH*-VL*)

[0090] (Fab_L-CM2-EM-MM2):(MM1-CM1-Fab_H-VH*-VL*)

[0091] (MM1-CM1-Fab_L):(MM2-CM2-VH*-VL*-Fab_H-CM3-EM)

[0092] (MM1-CM1-Fab_H):(MM2-CM2-VH*-VL*-Fab_L-CM3-EM)

[0093] (MM2-CM2-VH*-VL*-Fab_L):(MM1-CM1-Fab_H-CM3-EM)

[0094] (MM2-CM2-VH*-VL*-Fab_H):(MM1-CM1-Fab_L-CM3-EM)

[0095] In any configuration, the activatable protein may include one or more linkers between any two 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.

[0096] In some embodiments, the EM may comprise two or more parts (e.g., a pair of Fc domains). For example, the EM may be a protein complex comprising two parts, EM1 and EM2. In this 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):

[0097] (MM1-CM1-Fab_L):(Fab_H-VH*-VL*-CM2-MM2-EM1):EM2

[0098] (MM1-CM1-Fab_L):(Fab_H-VH*-VL*-CM2-EM1-MM2):EM2

[0099] Fab_L:(MM1-CM1-Fab_H-VH*-VL*-CM2-MM2-EM1):EM2

[0100] Fab_L:(MM1-CM1-Fab_H-VH*-VL*-CM2-EM1-MM2):EM2

[0101] (MM1-CM1-Fab_L):(Fab_H-VH*-VL*-CM2-EM1):(MM2-EM2)

[0102] (MM1-CM1-Fab_L):(Fab_H-VH*-VL*-CM2-EM1):(EM2-MM2)

[0103] Fab_L:(MM1-CM1-Fab_H-VH*-VL*-CM2-EM1):(MM2-EM2)

[0104] Fab_L:(MM1-CM1-Fab_H-VH*-VL*-CM2-EM1):(EM2-MM2)

[0105] (MM1-CM1-Fab_L-VH*-VL*-CM2-MM2-EM1):Fab_H:EM2

[0106] (MM1-CM1-Fab_L-VH*-VL*-CM2-EM1-MM2):Fab_H:EM2

[0107] (Fab_L-VH*-VL*-CM2-MM2-EM1):(MM1-CM1-Fab_H):EM2

[0108] (Fab_L-VH*-VL*-CM2-EM1-MM2):(MM1-CM1-Fab_H):EM2

[0109] (MM1-CM1-Fab_L-VH*-VL*-CM2-EM1):Fab_H:MM2-EM2

[0110] (MM1-CM1-Fab_L-VH*-VL*-CM2-EM1):Fab_H:EM2-MM2

[0111] (Fab_L-VH*-VL*-CM2-EM1):(MM1-CM1-Fab_H):MM2-EM2

[0112] (Fab_L-VH*-VL*-CM2-EM1):(MM1-CM1-Fab_H):EM2-MM2

[0113] (MM1-CM1-Fab_L-VH*-VL*):(Fab_H-CM2-MM2-EM1):EM2

[0114] (MM1-CM1-Fab_L-VH*-VL*):(Fab_H-CM2-EM1-MM2):EM2

[0115] (Fab_L-VH*-VL*):(MM1-CM1-Fab_H-CM2-MM2-EM1):EM2

[0116] (Fab_L-VH*-VL*):(MM1-CM1-Fab_H-CM2-EM1-MM2):EM2

[0117] (MM1-CM1-Fab_L-VH*-VL*):(Fab_H-CM2-EM1):MM2-EM2

[0118] (MM1-CM1-Fab_L-VH*-VL*):(Fab_H-CM2-EM1):EM2-MM2

[0119] (Fab_L-VH*-VL*):(MM1-CM1-Fab_H-CM2-EM1):MM2-EM2

[0120] (Fab_L-VH*-VL*):(MM1-CM1-Fab_H-CM2-EM1):EM2-MM2

[0121] (MM1-CM1-Fab_L-CM2-MM2-EM1):(Fab_H-VH*-VL*):EM2

[0122] (MM1-CM1-Fab_L-CM2-EM1-MM2):(Fab_H-VH*-VL*):EM2

[0123] (Fab_L-CM2-MM2-EM1):(MM1-CM1-Fab_H-VH*-VL*):EM2

[0124] (Fab_L-CM2-EM1-MM2):(MM1-CM1-Fab_H-VH*-VL*):EM2

[0125] (MM1-CM1-Fab_L-CM2-EM1):(Fab_H-VH*-VL*):MM2-EM2

[0126] (MM1-CM1-Fab_L-CM2-EM1):(Fab_H-VH*-VL*):EM2-MM2

[0127] (Fab_L-CM2-EM1):(MM1-CM1-Fab_H-VH*-VL*):MM2-EM2

[0128] (Fab_L-CM2-EM1):(MM1-CM1-Fab_H-VH*-VL*):EM2-MM2

[0129] (MM1-CM1-Fab_L):(MM2-CM2-VH*-VL*-Fab_H-CM3-EM1):EM2

[0130] (MM1-CM1-Fab_H):(MM2-CM2-VH*-VL*-Fab_L-CM3-EM1):EM2

[0131] (MM2-CM2-VH*-VL*-Fab_L):(MM1-CM1-Fab_H-CM3-EM1):EM2

[0132] (MM2-CM2-VH*-VL*-Fab_H):(MM1-CM1-Fab_L-CM3-EM):EM2

[0133] In some embodiments, EM1 and EM2 can be two crystallizable fragment (Fc) domains. Two Fc domains can form a dimer as a half-life extension portion. In some instances, EM1 and EM2 can be two identical Fc domains, so homodimers can be formed. In some constructs, EM1 and EM2 include Fc domains with two different amino acid sequences, which together form heterodimers. In some instances, two Fc domains can be Fc domain hole mutants and Fc domain knob mutants and can form heterodimers. In any of these configurations, the activatable protein can include one or more joints between any two 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, a linker between CM2 and EM1, a linker between MM2 and EM1, a linker between MM2 and EM2, or any combination thereof.

[0134] Figure 1-4 Exemplary configurations of activatable molecules disclosed herein are shown. In these non-limiting examples, the activatable molecules include: AB1, which can be a Fab; AB2, which can be a scFv; EM, which is a dimer formed by two Fc domains; MM1, which is coupled to AB1 via CM1 and is capable of interfering with the binding of AB1 to its target; MM2, which is capable of interfering with the binding of AB2 to its target; and CM2 between the Fc domain of EM and AB1 or AB2. It should be understood that Figure 1-4 The activatable molecular structures exemplified in can be similarly applied to molecules in which AB1 and AB2 are antigen binding proteins other than Fab and scFv. Similarly, Figure 1-4 The activatable molecular structures exemplified in can be similarly applied to molecules in which AB1 and AB2 are replaced by TB1 and TB2, respectively, which molecules can be target binding proteins that do not necessarily contain an antigen binding domain.

[0135] Figure 1An exemplary activatable protein 100 comprising three polypeptides is shown. The first polypeptide comprises, from the amino (N) terminal region to the carboxyl (C) terminal region, MM1 101, an optional linker 102, CM1 103, an optional linker 104, and a light chain fragment 105 of AB1. The second polypeptide comprises, from the amino (N) terminal region to the carboxyl (C) terminal region, a heavy chain fragment 121 of AB1, a linker 122, a heavy chain variable region 123 of AB2, a linker 124, a light chain variable region 125 of AB2, a linker 126, CM2 127, an optional linker 128, MM2 129, a linker 130, and a first Fc domain 131 of EM. The third polypeptide comprises a second Fc domain 141 of EM. In an alternative exemplary configuration, in Figure 1 In one alternative exemplary configuration, 105 is a heavy chain fragment of AB1 and 121 is a light chain fragment of AB1. Figure 1 In one alternative exemplary configuration, 123 is the light chain variable region of AB2 and 125 is the heavy chain variable region of AB2. Figure 1 In one alternative exemplary configuration, the first Fc domain 131 of the EM is a Fc domain hole mutant and the second Fc domain 141 of the EM is a Fc domain knob mutant. Figure 1 In the embodiment, the first Fc domain 131 of the EM is an Fc domain knob mutant and the second Fc domain 141 of the EM is an Fc domain hole mutant.

[0136] Figure 2 Another exemplary activatable protein 200 comprising three polypeptides is shown. The first polypeptide comprises, from the amino (N) terminal region to the carboxyl (C) terminal region, MM1 201, an optional linker 202, CM1 203, an optional linker 204, a light chain fragment 205 of AB1, a linker 206, a heavy chain variable region 207 of AB2, a linker 208, and a light chain variable region 209 of AB2. The second polypeptide comprises, from the amino (N) terminal region to the carboxyl (C) terminal region, a heavy chain fragment 221 of AB1, an optional linker 222, CM2 223, an optional linker 224, MM2 225, a linker 226, and a first Fc domain 227 of EM. The third polypeptide comprises a second Fc domain 241 of EM. In an alternative exemplary configuration, in Figure 2 In one alternative exemplary formulation, 205 is a heavy chain fragment of AB1 and 221 is a light chain fragment of AB1. Figure 2 In one alternative exemplary formulation, 207 is the light chain variable region of AB2 and 209 is the heavy chain variable region of AB2. Figure 2In an alternative exemplary formulation, the first Fc domain 227 of the EM is a Fc domain hole mutant and the second Fc domain 241 of the EM is a Fc domain knob mutant. Figure 2 In the embodiment, the first Fc domain 227 of EM is an Fc domain knob mutant and the second Fc domain 241 of EM is an Fc domain hole mutant.

[0137] Figure 3 Another exemplary activatable protein 300 comprising three polypeptides is shown. The first polypeptide comprises, in order from the amino (N) terminal region to the carboxyl (C) terminal region, MM1 301, an optional linker 302, CM1 303, an optional linker 304, a light chain fragment 305 of AB1, a linker 306, a heavy chain variable region 307 of AB2, a linker 308, and a light chain variable region 309 of AB2. The second polypeptide comprises, in order from the amino (N) terminal region to the carboxyl (C) terminal region, a heavy chain fragment 321 of AB1, an optional linker 322, CM2 323, an optional linker 324, and a first Fc domain 325 of EM. The third polypeptide comprises, in order from the amino (N) terminal region to the carboxyl (C) terminal region, a second Fc domain 341 of EM, a linker 342, and MM2 343. In an alternative exemplary configuration, in Figure 3 In one alternative exemplary configuration, 305 is a heavy chain fragment of AB1 and 321 is a light chain fragment of AB1. Figure 3 , 307 is the light chain variable region of AB2 and 309 is the heavy chain variable region of AB2. In an alternative exemplary configuration, Figure 3 In one alternative exemplary configuration, the first Fc domain 325 of the EM is a Fc domain hole mutant and the second Fc domain 341 of the EM is a Fc domain knob mutant. Figure 3 In the embodiment, the first Fc domain 325 of EM is an Fc domain knob mutant and the second Fc domain 341 of EM is an Fc domain hole mutant.

[0138] Figure 4Another exemplary activatable protein 400 comprising three polypeptides is shown. The first polypeptide comprises, in order from the amino (N) terminal region to the carboxyl (C) terminal region, MM1 401, an optional linker 402, CM1 403, an optional linker 404, a light chain fragment 405 of AB1, a linker 406, a heavy chain variable region 407 of AB2, a linker 408, and a light chain variable region 409 of AB2. The second polypeptide comprises, in order from the amino (N) terminal region to the carboxyl (C) terminal region, a heavy chain fragment 421 of AB1, an optional linker 422, CM2 423, an optional linker 424, and a first Fc domain 425 of EM. The third polypeptide comprises, in order from the amino (N) terminal region to the carboxyl (C) terminal region, MM2 441, a linker 442, and a second Fc domain 443 of EM. In an alternative exemplary configuration, in Figure 4 In one alternative exemplary configuration, 405 is a heavy chain fragment of AB1 and 421 is a light chain fragment of AB1. Figure 4 In one alternative exemplary configuration, 407 is the light chain variable region of AB2 and 409 is the heavy chain variable region of AB2. Figure 4 In one alternative exemplary configuration, the first Fc domain 425 of the EM is a Fc domain hole mutant and the second Fc domain 443 of the EM is a Fc domain knob mutant. Figure 4 In the embodiment, the first Fc domain 425 of EM is an Fc domain knob mutant and the second Fc domain 443 of EM is an Fc domain hole mutant.

[0139] Figure 5 An exemplary activatable bispecific antibody is shown, comprising: a Fab component (501) that binds to a first target; a first prodomain (505) that comprises CM1 and MM1 that masks the Fab component; a scFv component (502) that binds to a second target; a second prodomain (503) that comprises CM2 and MM2 that masks the scFv component; and an EM (504) that comprises a pair of knob and hole Fc domains. Upon activation, CM1 is cleaved, releasing MM1 from the activated bispecific antibody, and CM2 is cleaved, releasing MM2 and the Fc domain (504). Compared to the parent activatable bispecific antibody, the activated bispecific antibody lacking the Fc domain has a relatively shorter half-life.

[0140] Fig. 6AAn exemplary activatable bispecific antibody targeting Her2 and CD3 is shown. In this example, the activatable bispecific antibody comprises three polypeptides. The first polypeptide comprises, from N to C terminus, a heavy chain fragment of Fab of trastuzumab (anti-HER2 antibody), a linker with 25 amino acids, an anti-CD3 scFv, a GSAT linker with 27 amino acids, CM1, MM1 for masking anti-CD3 scFv, a GS linker with 24 amino acids, and an Fc domain hole mutant. The second polypeptide comprises MM2, CM2, and a light chain fragment of Fab for masking Fab. The third polypeptide comprises an Fc domain knob mutant. An example of an activatable bispecific antibody having the configuration in Figure 6 may comprise: a first polypeptide comprising the sequence of any one 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. Figure 6B is formed Fig. 6A Schematic diagram of the three polypeptides that can activate the bispecific antibody shown in .

[0141] Fig.10 Another exemplary activatable protein 1000 comprising three polypeptides is shown. The first polypeptide comprises, from the amino (N) terminal region to the carboxyl (C) terminal region, MM1 1001, an optional linker 1002, CM1 1003, an optional linker 1004, and a light chain fragment 1005 of AB1. The second polypeptide comprises, from the amino (N) terminal region to the carboxyl (C) terminal region, MM2 1021, an optional linker 1022, CM2 1023, an optional linker 1024, a heavy chain variable region 1025 of AB2, a linker 1026, a light chain variable region 1027 of AB2, a linker 1028, a heavy chain fragment 1029 of AB1, an optional linker 1030, a third cleavable moiety (CM3) 1031, an optional linker 1032, and a first domain 1033 of EM (EM1). The third polypeptide comprises a second domain 1041 of EM (EM2). 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, Fig.10 , 1025 is the light chain variable region of AB2 and 1027 is the heavy chain variable region of AB2. In an alternative exemplary configuration, Fig.10 In one alternative exemplary configuration, the first domain 1033 of the EM (EM1) is a Fc domain hole mutant and the second domain 1041 of the EM (EM2) is a Fc domain knob mutant. Fig.10 Among them, EM1 1033 is an Fc domain knob mutant and EM2 1041 is an Fc domain hole mutant.

[0142] Fig.11 Another exemplary activatable protein 1100 comprising three polypeptides is shown. The first polypeptide comprises, from the amino (N) terminal region to the carboxyl (C) terminal region, MM2 1101, an optional linker 1102, CM2 1103, an optional linker 1104, a heavy chain variable region 1105 of AB2, a linker 1106, a light chain variable region 1107 of AB2, a linker 1108, and a light chain fragment 1109 of AB1. The second polypeptide comprises, from the amino (N) terminal region to the carboxyl (C) terminal region, MM11121, an optional linker 1122, CM11123, an optional linker 1124, a heavy chain fragment 1125 of AB1, an optional linker 1126, a third cleavable portion (CM3) 1127, an optional linker 1128, and a first domain 1129 of EM (EM1). The third polypeptide comprises a second domain 1141 of EM (EM2). In an alternative exemplary configuration, in Fig.11 In one alternative exemplary configuration, 1109 is a heavy chain fragment of AB1 and 1125 is a light chain fragment of AB1. Fig.11 , 1105 is the light chain variable region of AB2 and 1107 is the heavy chain variable region of AB2. In an alternative exemplary configuration, Fig.11 In one alternative exemplary configuration, EM1 1129 is an Fc domain hole mutant and EM2 1141 is an Fc domain knob mutant. Fig.11 Among them, EM1 1129 is an Fc domain knob mutant and EM2 1141 is an Fc domain hole mutant.

[0143] Fig.12 An exemplary activatable bispecific antibody is shown, which comprises 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; a 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) comprising a pair of knob and hole Fc domains; and a third cleavable portion (CM3) (1205) between the EM and the Fab. Upon activation, CM1 is cleaved to release MM1 from the activated bispecific antibody, CM2 is cleaved to release MM2, and CM3 is cleaved to release EM (1206). Compared to the parent activatable bispecific antibody, the activated bispecific antibody lacking the EM has a relatively shorter half-life.

[0144] In some embodiments, the activation protein produced by the activation of the activatable protein disclosed herein is not attached to the EM. Compared with the activatable protein, the half-life of such an activating protein may be shorter. Such activating proteins may have a shorter half-life than the corresponding protein that is the same as the activating protein but contains EM. As used herein, the term "half-life" is the time required for the concentration of a molecule or a molecular complex in the environment to reach 50% of its original concentration. In some examples, the environment can be serum, and the half-life is the serum half-life, that is, the time required for the concentration of a molecule or a molecular complex in serum (e.g., in the circulation of a subject) to reach 50% of its original concentration. In some examples, an activating protein comprising AB1 and AB2 but not containing MM1, MM2 or EM (i.e., caused by the activation of the activating protein) may have a shorter half-life than the corresponding protein that is the same as the activating protein but contains EM, i.e., the half-life of the activating molecule (AB1-AB2) is shorter than the half-life of the corresponding protein (AB1-AB2-EM).

[0145] For example, the activated protein produced by the activation of the activatable protein herein can 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, the activated protein produced by the activation of the activatable protein herein can have a half-life (e.g., serum half-life) of less than or equal to 5, 4, 3, or 2 days. In some examples, the half-life (e.g., serum half-life) of the activated protein resulting from activation of the activatable protein herein is up to 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5% of the half-life (e.g., serum half-life) of a corresponding protein that is the same as the activated protein but comprises an EM.

[0146] In some embodiments, the activated protein produced by activation of the activatable protein herein (i.e., the activated protein not attached to an EM or MM) may have a higher target binding activity than a corresponding protein identical to the activated protein but comprising an EM attached thereto. In some examples, an activated protein comprising TB1 and TB2 but without MM1, MM2 or EM has a higher target binding activity level than a corresponding protein identical to the activated protein but comprising an EM (i.e., TB1-TB2-EM). For example, the activated protein produced by activation of the activatable protein disclosed herein can have a target binding activity that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 3 times, 4 times, 6 times, 8 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 200 times, 300 times, 400 times, or 500 times greater than the target binding activity of a corresponding protein that is the same as the activated protein but comprises an EM.

[0147] In some embodiments, the activatable protein (before activation) may be characterized in that the target binding activity is less than a control level of target binding activity of TB not coupled to MM (directly or indirectly). For example, in some embodiments, the activatable protein is characterized in that its target binding activity is 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 than a control level of target binding activity of TB not coupled to MM.

[0148] Target binding protein

[0149] According to the activatable protein disclosed herein, one or more target binding proteins (TB) may be included. In some examples, the activatable protein may be multi-specific. For example, the activatable protein may include multiple TBs, each TB being specific to different epitopes on the same target. In some examples, the TBs in the activatable protein herein may bind to different targets, such as targets on different types of cells. In this way, in the activated protein produced by the activation of the activatable protein disclosed herein, the TBs may co-localize different types of cells. In some examples of the multi-specific activatable protein disclosed herein, one of the TBs binds to a target on an immune cell, and another TB binds to a cell associated with the disease. By targeting and co-localizing immune cells and cells associated with the disease, the activating protein can provide targeted treatment for the disease.

[0150] In some embodiments, the target binding protein (TB) can be an antigen binding protein (AB). In some embodiments, 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 Fragments, single-chain variable fragments (scFv), diabodies (non-covalent dimers of scFv), single-chain antibodies (scab), VHH, domain antibodies (dAb) or single domain antibodies (nanoantibodies, such as single domain heavy chain antibodies, single domain light chain antibodies). Single domain antibodies can be antibody fragments, which are single monomer variable antibody domains. Single domain antibodies can have antigen affinity similar to that of corresponding full-length antibodies. In some embodiments, AB can be a full-length antibody. In some embodiments, AB can be an immunologically active fragment. In some embodiments, AB can be an antigen binding fragment ("Fab"). In one example, the activatable protein comprises a Fab of a first AB and a scFv as a second AB. In some embodiments, AB can be a scFv. In some embodiments, AB can be a mouse antibody, other rodent antibodies, chimeric antibodies, humanized antibodies, or fully human monoclonal antibodies. The present disclosure includes structures having one or more polypeptides comprising any of the domains listed above, such as one or more of SDA, Fv, ScFv, Fab, scFab, VHH, and dAb in combination with one or more selected from SDA, Fv, scFv, Fab, VHH, scFab, and dAb.

[0151] The term "antibody" is used herein in its broadest sense to include certain types of immunoglobulin molecules that include one or more antigen binding domains that specifically bind to an antigen or epitope. The term "antibody" specifically includes, for example, complete antibodies (e.g., complete immunoglobulins), antibody fragments, bispecific antibodies, and multispecific antibodies. An example of an antibody is a V H -V L Dimers form antigen binding domains. Additional examples of antibodies are described herein. Additional examples of antibodies are known in the art.

[0152] A "light chain" is composed of a variable domain (VL) and a constant domain (CL). There are two different types or classes of light chains, called kappa or lambda.

[0153] The "heavy chain" consists of a variable domain (VH) and three constant region domains (CH1, CH2, CH3). There are five main classes or isotypes of heavy chains, some of which have several subtypes, and these determine the functional activity of the antibody molecule. The five major classes of immunoglobulins are immunoglobulin M (IgM), immunoglobulin D (IgD), immunoglobulin G (IgG), immunoglobulin A (IgA), and immunoglobulin E (IgE). IgG is by far the most abundant immunoglobulin and has several subclasses (IgG1, IgG2, IgG3, and IgG4 in humans).

[0154] "Fragment antigen binding" (Fab) contains a complete light chain paired with the VH domain and CH1 domain of the heavy chain. F(ab') is formed when the antibody is cleaved by pepsin below the hinge region. 2 Fragments, in which case the two fragment antigen binding domains (Fab) of an antibody molecule remain linked. F(ab') 2 The fragment contains two complete light chains paired with two VH and CH1 domains of the heavy chain joined together by a hinge region. The "crystallizable fragment" (Fc) fragment (also referred to herein as F C The "single-chain Fv" (scFv) contains only the light chain variable domain (VL) connected to the heavy chain variable domain (VH) by a synthetic peptide. The name single-chain Fv comes from the variable fragment. The "hinge region" or "interdomain" is a flexible amino acid segment that joins or connects the Fab fragment to the Fc domain. The "synthetic hinge region" is an amino acid sequence that joins or connects the Fab fragment to the Fc domain.

[0155] "Prodomain" refers to a polypeptide having a portion that inhibits antigen binding, referred to as a masking portion (MM), and a portion that contains a protease-cleavable substrate, referred to as a cleavable portion (CM), which, when linked to a target binding protein (TB) (e.g., an antigen binding protein (AB), such as an antibody or an antigen-binding fragment thereof), is used to inhibit the binding of TB or AB to an antigen. The prodomain may include a connecting peptide (L1) between the MM and the CM. The prodomain may also include a connecting peptide (L2) at the carboxyl terminus of the prodomain to facilitate the linkage of the prodomain to the antibody. In certain embodiments, the prodomain comprises one of the following formulae (wherein the following formulae represent amino acid sequences in the direction from the N-terminus to the C-terminus): (MM)-(CM), (MM)-L1-(CM), (MM)-(CM)-L2, or (MM)-L1-(CM)-L2.

[0156] TB (e.g., AB) specifically binds to a target. As used herein, the terms "specific binding," "immunological binding," and "immunological binding properties" refer to the type of non-covalent interaction that occurs between an immunoglobulin molecule and an antigen for which the immunoglobulin has specificity. The strength or affinity of the immunological binding interaction can be measured based on the dissociation constant (K) of the interaction. d ) is used to represent the d The smaller the value, the greater the affinity. Methods well known in the art can be used to quantify the immunological binding properties of a selected polypeptide. One such method requires measuring the rates of formation and dissociation of the antigen binding site / antigen complex, where those rates depend on the concentration of the complex partners, the affinity of the interaction, and geometric parameters that affect the rates equally in both directions. Thus, the "association rate constant" (K) can be determined by calculating the concentration and the actual rates of association and dissociation. on ) and the “dissociation rate constant” (K off (See Nature 361: 186-87 (1993)). off / K on The ratio of 2 can eliminate all parameters not related to affinity and is equal to the dissociation constant K. d (See generally Davies et al. (1990) Annual Rev Biochem 59:439-473). When the dissociation constant (K) is measured as measured by, for example, a radioligand binding assay or similar assays known to those skilled in the art, d )≤100 μM, in some embodiments ≤1 μM, in some embodiments ≤100 nM, in some embodiments ≤10 nM, and in some embodiments ≤100 pM to about 1 pM, a TB or antibody binding domain (AB) of the present disclosure is said to “specifically bind” or “immunospecifically bind” to a target.

[0157] The target of TB (e.g., AB) can be a protein or other type of molecule. Example 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.

[0158] In some embodiments, TB in an activatable protein can be targeted to a molecule on or in a cell associated with a disease. For example, TB in an activatable protein can bind to a tumor cell. In this case, TB can bind to a tumor-associated antigen. As used herein, the term "tumor-associated antigen" means any antigen associated with cancer, including proteins, glycoproteins, gangliosides, carbohydrates, lipids. Such antigens may be expressed on tumor cells (e.g., malignant cells) or in a tumor microenvironment (e.g., tumor-associated blood vessels, extracellular matrix, mesenchymal matrix, or immune infiltrates). In some embodiments, a tumor-associated antigen as a target of AB may be human epidermal growth factor receptor 2 (HER2). For example, AB may be trastuzumab or a fragment thereof, such as Fab of trastuzumab.

[0159] In some embodiments, the AB in the activatable protein can 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 can be a co-stimulatory molecule, which is a cell surface molecule other than an antigen receptor or its ligand required for an efficient immune response. Examples of co-stimulatory molecules that can be targets of the AB include components of the T cell receptor (TCR), CD3ζ, CD3γ, CD3δ, and CD3ε.

[0160] In some examples, AB can bind to a co-stimulatory molecule expressed on the surface of a T lymphocyte, such as a cytotoxic T lymphocyte, which can induce T cell activation after interacting with an antigen binding molecule. The interaction between the antigen binding molecule and the activated T cell antigen can induce T cell activation by triggering the signal cascade of the T cell receptor complex. When activated, AB can bind to this co-stimulatory molecule to activate T cells. 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, cytotoxic effector molecule release, cytotoxic activity, and expression of activation markers. The T cell activation bispecific antigen binding molecules of the present invention can induce T cell activation.

[0161] In some examples, AB can bind to CD3. For example, CD3 can be the epsilon subunit of CD3, such as the sequence of NCBI RefSeqno.NP_000724.1. In some examples, AB can be an anti-CD3 scFv. The anti-CD3 scFv may include one or more sequences in SEQ ID NO: 1-9, 143-145, 149 and 150 in US20190135943, which is incorporated herein by reference in its entirety. Such sequences include, for example, the following:

[0162]

[0163]

[0164] Exemplary CDR sequences for CD3 binding antibodies include the following:

[0165] name CD3 Ab CDR sequences SEQ ID NO: SP34L1 RSSTGAVTTSNYAN SEQ ID NO:7 SP34L2 GTNKRAP SEQ ID NO:8 SP34L3 ALWYSNLWV SEQ ID NO:9 SP34H1 TYAMN SEQ ID NO:10 SP34H2 RIRSKYNNYATYYADSVKD SEQ ID NO:11 SP34H3 HGNFGNSYVSWFAY SEQ ID NO:12

[0166] Additional examples of anti-CD3 ABs include the following:

[0167] v619_HLc

[0168]

[0169]

[0170] v619_HLh

[0171] v619-HLh-CC4

[0172]

[0173] v619_Hh8

[0174]

[0175]

[0176] v619_HLp2

[0177]

[0178] v619_Hh13

[0179]

[0180]

[0181] In some embodiments, the activatable protein herein may include AB1 that binds to a tumor-associated antigen and AB2 that binds to a co-stimulatory molecule. In one example, the activatable protein may include AB1 that binds to HER2 and AB2 that binds to CD3. In a specific example, the activatable protein may include AB1 that is an anti-HER2 Fab (e.g., Fab of trastuzumab) and AB2 that is an anti-CD3 scFv.

[0182] 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, mononuclear cells, and myeloid mononuclear cells. In some examples, the activatable protein may include a bispecific activatable antibody that engages an immune effector cell, which crosslinks the immune effector cell with another cell (e.g., a cell associated with a disease such as cancer or infection).

[0183] The activatable protein may include a bispecific activatable antibody that engages leukocytes, a bispecific activatable antibody that engages T cells, a bispecific activatable antibody that engages NK cells, a bispecific activatable antibody that engages macrophages, a bispecific activatable antibody that engages mononuclear cells, or a bispecific activatable antibody that engages myeloid mononuclear cells. In one example, the activatable antibody may include a bispecific antibody that engages T cells.

[0184] Half-life extension

[0185] The activatable protein may include a half-life extension portion (EM). In the activatable protein, the EM may be coupled to the TB or its component in the activatable protein via the CM. Once the activatable protein is activated, the EM will be cleaved from the TB. In some embodiments, for example, the CM is located between the C-terminus of the TB and the N-terminus of the EM. In certain embodiments of these embodiments, the CM is located 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 relative to the CM and N-terminal or C-terminal relative to the EM (e.g., from N-terminal to C-terminal, TB-CM-EM, TB-CM-EM-MM, TB-CM-MM-EM, etc., where each "-" independently represents direct or indirect (e.g., via a linker) coupling) (see, e.g. Figure 1 and Figure 2 ). In some embodiments, the EM may be a dimer, such as a pair of Fc domains of an immunoglobulin. In such embodiments, the first polypeptide may comprise TB, CM, and a first Fc domain, and the second polypeptide may comprise MM and a second Fc domain, and the two polypeptides may be covalently linked via one or more disulfide bonds between the first Fc domain and the second Fc domain. In such embodiments, the MM may be located at the N-terminus or C-terminus of the second Fc domain (see, e.g. Figure 3 and Figure 4 ), and cleavage of the CM on the first polypeptide causes the MM and EM (e.g., two Fc domains) to be released from the activator protein. Thus, in some embodiments, the activator protein produced by activation of the activatable protein does not contain the EM. In some examples, the half-life extending moiety can be a serum half-life extending moiety, i.e., one that is capable of extending the half-life of the molecule attached to the EM in serum.

[0186] In some instances, 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 instances, the EM may comprise a dimer formed by two Fc domains. The Fc domain may be a wild-type Fc domain or a mutant. For example, the EM may comprise a dimer formed by two Fc domain mutants. In this case, the two Fc domain mutants may comprise an Fc domain hole mutant and an Fc domain knob mutant. The knob mutant and the hole mutant may interact with each other to promote the dimerization of the two Fc domains. In some embodiments, the knob mutant and the hole mutant 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 comprises the amino acid substitution T366W and optionally the amino acid substitution S354C in one antibody heavy chain, and the amino acid substitutions T366S, L368A, Y407V, and optionally Y349C in the other antibody heavy chain (numbered according to the EU numbering system). An example of an Fc domain knob mutant comprises the sequence of SEQ ID NO: 1. An example of an Fc domain hole mutant comprises the sequence of SEQ ID NO: 2.

[0187] Examples of Fc domain mutants 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 is contained in an amino acid substitution T366Y in an IgG Fc domain, and an amino acid substitution Y407T in another IgG Fc domain. In one example, the modification is contained in an amino acid substitution T366W in an IgG Fc domain, and an amino acid substitution Y407A in another IgG Fc domain. In one example, the modification is contained in an amino acid substitution F405A in an IgG Fc domain, and an amino acid substitution T394W in another IgG Fc domain. In one example, the modification is contained in an amino acid substitution T366Y and F405A in an IgG Fc domain, and an amino acid substitution T394W and Y407T in another IgG Fc domain. In one example, the modification is contained in an amino acid substitution T366W and F405W in an IgG Fc domain, and an amino acid substitution T394S and Y407A in another IgG Fc domain. In one example, modification is included in the amino acid replacement F405W and Y407A in an IgG Fc domain, and the amino acid replacement T366W and T394S in another IgG Fc domain. In one example, modification is included in the amino acid replacement F405W in an IgG Fc domain, and the amino acid replacement T394S in another IgG Fc domain. The mutation position in the Fc domain is numbered according to the EU numbering system. The IgG Fc domain can include the sequence (IgG1, IgG2, IgG3 or IgG4) of SEQ ID NO:3-6. In these sequences, amino acid 1-107 corresponds to EU numbering 341-447.

[0188] In some examples, the Fc domain mutant may have reduced effector function. Examples of such Fc domains include those disclosed in US20190135943, which is incorporated herein by reference in its entirety.

[0189] Other examples of EMs include immunoglobulins (e.g., IgG), serum albumins (e.g., human serum albumin (HSA), hexacapped GST (glutathione S-transferase) glutathione affinity, calmodulin (CBP) binding peptide (CBP), Strep-tag, cellulose binding domain, maltose binding protein, S peptide tag, chitin binding tag, immunoreactive epitope, epitope tag, E2Tag, HA epitope tag, Myc epitope, FLAG epitope, AU1 and AU5 epitopes, Glu-Glu epitope, KT3 epitope, IRS epitope, Btag epitope, protein kinase-C epitope and VSV epitope.

[0190] In some embodiments, the serum half-life of the activatable protein may be longer than the serum half-life of a corresponding protein that is the same as the activatable protein but does not have a half-life extending portion. 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, when administered to an organism, 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.

[0191] Masking part(MM)

[0192] The activatable protein herein may include one or more masking moieties (MM) that can interfere with the binding of TB to the target. The masking moiety in the activatable molecule "masks" or reduces or otherwise inhibits the binding of the activatable macromolecule to its target and / or epitope. In some embodiments, the coupling or modification of the target binding protein (TB) (e.g., AB or other therapeutic or diagnostic proteins) with MM can inhibit the ability of TB to specifically bind to its target and / or epitope by inhibition methods known in the art (e.g., structural changes, competitive antigen binding domains, etc.). In some embodiments, the coupling or modification of TB with MM can cause structural changes, thereby reducing or inhibiting the ability of TB to specifically bind to its target and / or epitope. In some embodiments, the coupling or modification of a protein comprising an antigen binding domain to MM spatially blocks, reduces or inhibits the ability of the antigen binding domain to specifically bind to its target and / or epitope. MM can be coupled to TB (e.g., AB) directly or indirectly (e.g., via one or more linkers described herein) through CM.

[0193] Alternatively, the MM that interferes with TB target binding can be coupled to a component of a non-TB activatable protein. Figure 2 As exemplified, the activatable protein may include TB1 and TB2, and the MM interfering with TB2 may be coupled to TB1. Figure 3 and Figure 4 As exemplified, the activatable protein may comprise TB1, TB2 and EM, and the MM interfering with TB2 may be coupled to the EM. In either case, in the tertiary or quaternary structure of the activatable structure, the MM may be in a position that allows the MM to mask the TB (e.g., close to the TB to be masked).

[0194] In some embodiments, MM may interact with TB, thereby reducing or inhibiting the interaction between TB and its binding partner. In some embodiments, MM may comprise at least part or all of the amino acid sequence of a naturally occurring binding partner of TB. For example, MM may be a fragment of a naturally occurring binding partner. The fragment may retain up to 95%, 90%, 80%, 75%, 70%, 60%, 50%, 40%, 30%, 25% or 20% nucleic acid or amino acid sequence homology with a naturally occurring binding partner. In some embodiments, MM may be a homologous polypeptide of TB. For example, MM may comprise the sequence of a TB epitope or a fragment thereof. As used herein, the term "naturally occurring" when applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence present in an organism (including a virus) that can be isolated from a source in nature and is not intentionally modified by a person in a laboratory or otherwise is naturally occurring.

[0195] In some embodiments, MM may comprise non-naturally occurring amino acid sequences, or amino acid sequences that do not contain naturally occurring binding partners or target proteins. In certain embodiments, MM is not a natural binding partner of TB. In some embodiments, MM does not comprise a subsequence of more than 4, 5, 6, 7, 8, 9 or 10 consecutive amino acid residues of a natural binding partner of TB. MM may be a modified binding partner of TB that contains amino acid changes that reduce binding affinity and / or avidity to TB. In some embodiments, MM does not contain or substantially does not contain nucleic acid or amino acid homology with a natural binding partner of TB. In other embodiments, MM is at most 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or 80% similar to a natural binding partner of TB.

[0196] In some embodiments, the MM may not specifically bind to the TB (or other activatable protein), but rather interfere with the binding of the target binding protein (e.g., AB) to its binding partner through non-specific interactions such as steric hindrance. For example, the MM may be located in the activatable 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 to interfere with the binding partner's access to the TB.

[0197] In some embodiments, the dissociation constant of MM binding to the target binding protein (eg, AB) does not exceed the dissociation constant of TB binding to the target. In some embodiments, in the cleaved state, MM does not interfere with the binding of TB to the target or compete with TB for binding to the target.

[0198] The structural properties of the MM can be selected based on factors such as the minimal amino acid sequence required to interfere with protein binding to the target, the target protein-protein binding pair of interest, the size of the TB, the presence or absence of a linker, etc.

[0199] In some embodiments, the MM is unique to the coupled TB. Examples of MMs include MMs that are specifically screened to bind to a binding domain of a TB (e.g., AB) or a fragment thereof (e.g., affinity masking). Methods for screening MMs to obtain MMs that are unique to TB and those that specifically and / or selectively bind to a binding domain of a binding partner / target are provided herein and may include protein display methods.

[0200] 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., antibody), wherein the control target binding protein (e.g., antibody) may be a cleavage product of an activatable protein (i.e., an activating protein), or a target binding protein (e.g., an antibody or fragment thereof) used as a TB for an activatable protein. An activatable protein with a reduced level of target binding activity may have a masking efficiency greater than 10. In some embodiments, the activatable protein described herein may have a masking efficiency greater than 10, 100, 1000, or 5000.

[0201] In some embodiments, the MM can be a peptide of about 2 to 50 amino acids in length. For example, the MM can be a peptide of 2 to 40, 2 to 30, 2 to 20, 2 to 10, 5 to 15, 10 to 20, 15 to 25, 20 to 30, 25 to 35, 30 to 40, 35 to 45, 40 to 50 amino acids in length. For example, the MM can be a 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 examples, the MM can be a polypeptide that is greater than 50 amino acids in length, such as 100, 200, 300, 400, 500, 600, 700, 800 or more amino acids.

[0202] In some embodiments, when measured in vivo or in a masking efficiency assay, or, for example, as described in US20200308243A1, in an in vitro immunosorbent assay, an activatable protein having TB and an interfering MM, in the presence of a target of TB, TB has no binding or substantially no binding to the target, or TB has binding to its target of at most 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4% compared to the binding of the corresponding antibody without the interfering MM. , 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 50%, 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. For example, the ability of MM to inhibit the binding of an activatable protein to its binding partner at therapeutically relevant concentrations and times can be measured. To perform this measurement, an immunosorbent assay (MEA, masking efficiency assay) has been developed to measure the time-dependent binding of activatable proteins to their binding partners and is described in US20200308243A1, which is incorporated herein by reference in its entirety.

[0203] The binding affinity of TB to the target or binding partner under the interfering MM may be at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 times lower than the binding affinity of TB to its binding partner when the interfering MM is not present, or 5-10, 10-100, 10-1,000, 10-10 ... 10-1,000,000, 10-10,000,000, 100-1,000, 100-10,000, 100-1,000,000, 100-10,000,000, 1,000-10,000,000, 1,000-10,000,000, 1,000-10,000,000, 1000-10,000,000, 10,000-1,000,000, 10,000-10,000,000, 100,000-1,000,000 or 100,000-10,000,000 times.

[0204] The dissociation constant (K) of MM to its masked TB (e.g. AB) d ) may be greater than the K of TB (e.g., AB) for the target d MM K of masked TB d Comparable TB K for target d MM may have a binding affinity for TB that is 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 TB for the target. In contrast, the binding affinity of MM for the masked TB may be lower than the binding affinity of TB for the target. The binding affinity of MM for 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 lower than the binding affinity of TB for the target.

[0205] In some embodiments, the MM may contain genetically encoded amino acids or genetically non-encoded amino acids. Examples of genetically non-encoded amino acids are, but are not limited to, D-amino acids, β-amino acids, and γ-amino acids. In specific embodiments, the MM contains up to 50%, 40%, 30%, 20%, 15%, 10%, 5%, or 1% of genetically non-encoded amino acids.

[0206] In some embodiments, once released from the activatable protein and in a free state, the MM may have a biological activity or therapeutic effect, such as binding ability. For example, the free MM may bind to the same or different binding partners. In certain embodiments, the free MM may exert a therapeutic effect, thereby providing a secondary function for the compositions disclosed herein. In some embodiments, once uncoupled from the activatable protein and in a free state, the MM may advantageously not exhibit biological activity. For example, in some embodiments, the MM in a free state does not induce an immune response in a subject.

[0207] Suitable MMs can be identified and / or further optimized from a library of candidate activatable proteins with variable MMs by a screening procedure. For example, TB and CM can be selected to provide a desired enzyme / target combination, and the amino acid sequence of the MM can be identified by a screening procedure described below to identify MMs that provide an activatable phenotype. For example, a random peptide library (e.g., a random peptide library comprising peptides of 2 to 40 amino acids or more) can be used to identify suitable MMs in the screening methods disclosed herein.

[0208] In some embodiments, MMs with specific binding affinity for TB (e.g., AB) can be identified by a screening procedure comprising providing a library of peptide scaffolds comprising candidate MMs, wherein each scaffold consists of a transmembrane protein and a candidate MM. The library can then be contacted with the entire protein or a portion (e.g., a full-length protein, a naturally occurring protein fragment, or a non-naturally occurring fragment containing the protein (also capable of binding the binding partner of interest)), and one or more candidate MMs with detectably bound protein are identified. Screening can be performed by one or more rounds of magnetic activated sorting (MACS) or fluorescence activated sorting (FACS), and determining the binding affinity of the MMs for the AB and subsequently determining the masking efficiency, such as described in WO2009025846 and US20200308243A1, which are incorporated herein by reference in their entirety.

[0209] In some embodiments, MM can be selected for use with specific proteins, antibodies or antibody fragments. For example, MM suitable for use with ABs that bind to epitopes may include the sequence of the epitope. In one example, when the activatable substance includes AB1 as an anti-HER 2 Fab and AB2 as an anti-CD3 scFv, MM1 (for masking AB1) may include a sequence in HER2 bound by AB1, and MM2 (for masking AB2) may include a sequence in CD3 bound by AB2. In other embodiments, MM may not include the sequence of the natural binding partner of TB. In some examples, MM1 suitable for masking anti-HER2 Fab includes the sequence of ALICCSDVSGLCRWC (SEQ ID NO: 40). In some examples, MM2 suitable for masking anti-CD3 scFv includes MMs 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, MM2 suitable for masking anti-CD3 scFv includes MMs 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).

[0210] Additional suitable MMs are disclosed in WO2021207657, WO2021142029, WO2021061867, WO2020252349, WO2020252358, WO2020236679, WO2020176672, WO2020118109, WO2020092881, WO2020086665, WO2019213444, WO2019183218, WO2019173771, WO2019 165143, WO2019075405, WO2019046652, WO2019018828, WO2019014586, WO2018222949, WO2018165619, WO2018085555, WO2017011580, WO2016179335, WO2016179285, WO2016179257, WO2016149201 and WO2016014974, are incorporated herein by reference in their entirety.

[0211] Cleavable fraction (CM)

[0212] The activatable protein may comprise one or more cleavable moieties (CM) as defined above.

[0213] In some embodiments, the activatable protein may include a CM between TB (e.g., AB) and MM. The activatable protein may also include a CM between TB and EM. In some examples, the CM between TB and MM is also between TB and EM (see, e.g., Figure 1 , wherein CM 127 is between TB 123 / 125 and MM 129, and CM 127 is also between TB 123 / 125 and EM 131 / 141). In this case, cleavage of the CM can release the MM and EM from the TB. In some examples, the CM is located between a first TB (TB1) and an MM (MM2) bound to a second TB (TB2) (see, e.g. Figure 2 , wherein CM 223 is between the first TB (TB221) and MM 225, wherein MM 225 is a masking moiety (TB 207 / 209) that inhibits the binding of the second TB; see also Figure 3, wherein CM 323 is located between the first TB (TB 321) and MM 343, and MM 343 is a masking moiety (TB 307 / 309) that inhibits the binding of the second TB to its target. In some examples, the CM between the TB and the MM is not between the TB and the EM. In this case, the activatable protein may include a first CM between the TB and the MM and a second CM between the TB and the EM. In some examples, the 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. Fig.10 and 11 ). Activation of the activatable protein may cleave both CMs, thereby releasing both the MM and the EM from the EM.

[0214] The CM and TB of the activatable protein can be selected such that the TB comprises a binding moiety for a given target, while the CM comprises a substrate for one or more proteases, wherein the one or more proteases are co-localized with the target in a tissue (e.g., at a treatment site or a diagnostic site in a subject). In some embodiments, the activatable protein can have a particular use where, for example, one or more proteases capable of cleaving a site in the CM are present at a relatively higher level (or greater activity) in target-containing tissue at a treatment site or a diagnostic site than in tissue at a non-treatment site (e.g., in healthy tissue).

[0215] In some embodiments, the CM herein may include substrates for proteases that have been reported in one or more cancers. See, for example, La Roca et al., British J. Cancer 90(7):1414-1421, 2004. Substrates suitable for use in the CM components employed herein include those more commonly found in cancerous cells and tissues. Thus, in certain embodiments, the CM may include substrates for proteases that are more commonly found in diseased tissues associated with cancer. Examples of cancer include gastric cancer, breast cancer, osteosarcoma, esophageal cancer, breast cancer, HER2-positive cancer, Kaposi sarcoma, hairy cell leukemia, chronic myeloid leukemia (CML), follicular lymphoma, renal cell carcinoma (RCC), melanoma, neuroblastoma, basal cell carcinoma, cutaneous T-cell lymphoma, nasopharyngeal adenocarcinoma, ovarian cancer, bladder cancer, BCG-resistant non-muscle invasive bladder cancer (NMIBC), endometrial cancer, pancreatic cancer, non-small cell lung cancer (NSCLC), colorectal cancer, esophageal cancer, gallbladder cancer, glioma, head and neck cancer, uterine cancer, cervical cancer or testicular cancer, etc. In some embodiments, the CM component comprises a substrate for a protease that is more prevalent in tumor tissue. For example, the protease can be produced by a tumor of a subject.

[0216] In some embodiments, the activatable protein may comprise a first CM between an MM and a TB (e.g., AB), and a second CM between an EM and the same or different TB. In the activated state, the two CMs may be cleaved, so that the MM and EM are released from the TB. In some examples, the first CM and the second CM may comprise a substrate for the same protease. In some examples, the first CM and the second CM may comprise substrates for different proteases. In some examples, the first CM and the second CM may comprise the same sequence or consist of the same sequence. In some examples, the first CM and the second CM may comprise different sequences or consist of different sequences.

[0217] The second CM may be located at a position in the activatable protein where its cleavage facilitates the dissociation of the EM from the TB. In some instances, the second CM may be between the C-terminus of the TB (or if the TB comprises a plurality of polypeptides, it is a component thereof) and the N-terminus of the MM, wherein the C-terminus of the MM is coupled to the N-terminus of the EM (or if the EM comprises a plurality of polypeptides, it is a component thereof). In some instances, the second CM may be between the N-terminus of the TB (or if the TB comprises a plurality of polypeptides, it is a component thereof) and the C-terminus of the MM, wherein the N-terminus of the MM is coupled to the C-terminus of the EM (or if the EM comprises a plurality of polypeptides, it is a component thereof). In some instances, the second CM may be between the C-terminus of the TB (or if the TB comprises a plurality of polypeptides, it is a component thereof) and the N-terminus of the EM (or if the EM comprises a plurality of polypeptides, it is a component thereof), wherein the C-terminus of the EM (or if the EM comprises a plurality of polypeptides, it is a component thereof) 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), wherein 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 portion of a TB or a different TB (e.g., on the same or another polypeptide) in the activatable protein.

[0218] Suitable CMs for the activatable proteins herein include any protease substrate known in the art. In some examples, the CM may include substrates for serine proteases (e.g., u-type plasminogen activator (uPA, also known as urokinase)), matriptase (also referred to herein as MT-SP1 or MTSP1). In some examples, the CM may include substrates for matrix metalloproteinases (MMPs). In some examples, the CM may include substrates for cysteine ​​proteases (CPs) (e.g., legumin).

[0219] In some embodiments, the CM may comprise substrates for a disintegrin and metalloproteinase (ADAM) or a disintegrin and metalloproteinase with a thrombospondin motif (ADAMTS) (e.g., ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADEMDEC1, ADAMTS1, ADAMTS4, ADAMTS5), an aspartic protease (e.g., BACE, renin), an aspartic cathepsin (e.g., cathepsin D, cathepsin E), an apoptotic protease (e.g., apoptotic cathepsin A, cathepsin B, cathepsin C, cathepsin G, cathepsin K, cathepsin L, cathepsin S, cathepsin V / L2, cathepsin X / Z / P), cysteine ​​proteases (e.g., Cruzipain, legumin, Otubain-2), Protease, DESC1, DPP-4, FAP, Elastase, FVIIa, FiXA, FXa, FXIa, FXIIa, Granzyme B, Guanidinobenzoic acid enzyme, Hepsin, HtrA1, Human neutrophil elastase, KLK (e.g., KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, KLK14), Metalloproteinase (e.g., Meprin, Enkephalinase, PSMA, BMP-1), Lactoferrin, Marapsin , interstitial proteinase-2, MT-SP1 / interstitial proteinase, NS3 / 4A, PACE4, plasmin, PSA, MMPs (e.g., MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP23, MMP24, MMP26, MMP27), TMPRSS2, TMPRSS3, TMPRSS4, tPA, thrombin, tryptase, and uPA.

[0220] In some embodiments, the protease substrate in the CM may comprise a polypeptide sequence that is substantially non-identical (e.g., no more than 90%, 80%, 70%, 60%, or 50% identical) to any polypeptide sequence naturally cleaved by the same protease. In some embodiments, the CM may be or comprise the sequence of LSGRSDDH (SEQ ID NO: 33) or ISSGLLSGRSDNH (SEQ ID NO: 41). In some embodiments, the CM may be or comprise the sequence of any one of the sequences in the following table or be encompassed by the consensus sequence of the sequences.

[0221]

[0222]

[0223]

[0224]

[0225]

[0226]

[0227]

[0228] Examples of CM also include WO 2010 / 081173, WO2021207669, WO2021207657, WO2021142029, WO2021061867, WO2020252349, WO2020252358, WO2020236679, WO2020176672, WO2020118109, WO2020092881, WO2020086665, WO2019213444, WO2019183218, WO2019173771, WO201 9165143, WO2019075405, WO2019046652, WO2019018828, WO2019014586, WO2018222949, WO2018165619, WO2018085555, WO2017011580, WO2016179335, WO2016179285, WO2016179257, WO2016149201, WO2016014974 (which are incorporated herein by reference in their entirety).

[0229] In some embodiments, the CM may be or include a combination of the above-mentioned exemplary sequences, a C-terminal truncated variant, or an N-terminal truncated variant. The truncated variants of the above-mentioned amino acid sequences suitable for use in the CM may be any truncated variants that retain the recognition site of the corresponding protease. These include C-terminal and / or N-terminal truncated variants that retain the recognition site of the protease and contain at least 3 consecutive amino acids of the above-mentioned amino acid sequence or at least 4, 5, 6, 7, 8, 9 or 10 amino acids of the aforementioned amino acid sequence. In certain embodiments, the truncated variants of the above-mentioned amino acid sequence may correspond to any of the above-mentioned amino acid sequences, but the C-terminus and / or N-terminus are truncated by 1 to 10 amino acids, 1 to 9 amino acids, 1 to 8 amino acids, 1 to 7 amino acids, 1 to 6 amino acids, 1 to 5 amino acids, 1 to 4 amino acids or 1 to 3 amino acids, and: (1) have at least three amino acid residues; and (2) retain the recognition site of the protease. In some of the aforementioned embodiments, the truncated CM is an N-terminal truncated CM. In some embodiments, the truncated CM is a C-terminal truncated CM. In some embodiments, the truncated C is a C-terminally and N-terminally truncated CM.

[0230] In some embodiments, the CM may comprise a total of 3 amino acids to 25 amino acids. In some embodiments, the CM may comprise a total of 3 to 25, 3 to 20, 3 to 15, 3 to 10, 3 to 5, 5 to 25, 5 to 20, 5 to 15, 5 to 10, 10 to 25, 10 to 20, 10 to 15, 15 to 25, 15 to 20, or 20 to 25 amino acids.

[0231] In some embodiments, CM can be prepared by at least one protease at a concentration of about 0.001-1500×10 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 of specific cleavage is determined by the substrate cleavage kinetics (k cat / K m ) is measured as disclosed in WO2016118629.

[0232] Connectors

[0233] The activatable protein may include one or more joints. The joint may include an amino acid sequence connecting two components in the activatable protein. The joint may not be cleaved by any protease. In some embodiments, one or more joints (e.g., flexible joints) may be introduced into the activatable protein to provide flexibility at one or more junctions between domains, at junctions between parts, at junctions between parts and domains, or at any other junctions where the joint will be beneficial. In some embodiments, when the activatable protein is provided in the form of a conformationally restricted construct, a flexible joint may be inserted to facilitate the formation and maintenance of the structure in the uncleaved activatable protein. Any joint described herein can provide the required flexibility to help inhibit the binding of the target, or to facilitate the cleavage of the CM by the protease. In some embodiments, the joint included in the activatable protein may be flexible in whole or in part, so that the joint may include a flexible joint and one or more parts of a structure that imparts less flexibility to provide the desired activatable protein. Some joints may include cysteine ​​residues that can form disulfide bonds and reduce the flexibility of the construct.

[0234] In some embodiments, the linker coupled to the MM may have a length that enables the MM to be in a tertiary or quaternary position to effectively mask the TB (eg, close to the TB to be masked), thereby enabling the MM to mask the TB.

[0235] In most cases, the linker length is determined by counting the number of amino acids from the N-terminus of the linker adjacent to the C-terminal amino acid of the preceding component to the C-terminus of the linker adjacent to the N-terminal amino acid of the following component in the N to C direction (i.e., wherein the linker length does not include the C-terminal amino acid of the preceding component or the N-terminal amino acid of the following component).

[0236] In some embodiments, the linkers may include 1 to 50, 1 to 40, 1 to 30, 1 to 25 total (e.g., 1 to 24, 1 to 22, 1 to 20, 1 to 18, 1 to 16, 1 to 15, 1 to 14, 1 to 12, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 25, 2 to 24, 2 to 22, 2 to 20, 2 to 18, 2 to 16, 2 to 15, 2 to 14, 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 4 to 25, 4 to 24, 4 to 22, 4 to 20, 4 to 18, 4 to 16, 4 to 15, 4 to 14, 4 to 12, 4 to 10, 4 to 8, 4 to 6, 4 to 5, 5 to 25, 5 to 24, 5 to 22, 5 to 20, 5 to 18, 5 to 16, 5 to 15, 5 to 14, 5 to 12, 5 to 10, 5 to 8, 5 to 6, 6 to 25, 6 to 24, 6 to 22, 6 to 20, 6 to 18, 6 to 16, 6 to 15, 6 to 14, 6 to 12, 6 to 10, 6 to 8, 8 to 25, 8 to 24, 8 to 22, 8 to 20, 8 to 18, 8 to 16, 8 to 15, 8 to 14, 8 to 12, 8 to 10, 10 to 25, 10 to 24, 10 to 22, 10 to 20, 10 to 18, 10 to 16, 10 to 15, 10 to 14, 10 to 12, 12 to 25, 12 to 24, 12 to 22, 12 to 20, 12 to 18, 12 to 16, 12 to 15, 12 to 14, 14 to 25, 20 to 24, 20 to 22, 22 to 25, 22 to 24, or 24 to 25 amino acids). In some embodiments, the linker may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids total.

[0237] In some embodiments, the linker may be rich in glycine (Gly or G) residues. In some embodiments, the linker may be rich in serine (Ser or S) residues. In some embodiments, the linker may be rich in glycine and serine residues. In some embodiments, the linker may have one or more pairs of glycine-serine residues (GS) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more pairs of GS).

[0238] 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 linkers may include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, (GGS)n, (GSGGS)n, and (GGGS)n, where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured and therefore may be able to act as neutral links between components. Glycine even approaches significantly greater affinity than alanine. sterically and are much less constrained than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Exemplary flexible linkers include one or a combination of one or more of the following: GGSG (SEQ ID NO: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), GGGGSGGGGSGGGGS (SEQ ID NO:81), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:82), GGGGSGGGGS (SEQ ID NO:83), GGGGS (SEQ ID NO:84), GS, GGGGSGS (SEQ ID NO:85). NO:85), GGGGSGGGSGGGGSGS (SEQ ID NO:86), GGSLDPKGGGGS (SEQ ID NO:87), PKSCDKTHTCPPCPAPELLG (SEQ ID NO:88), SKYGPPCPPCPAPEFLG (SEQ ID NO:89), GKSSGGSESKS (SEQ ID NO:90), GSTSGSGKSSEGKG (SEQ ID NO:91),GSTSSGSGKSSEGSGSTKG(SEQ ID NO:92),GSTSGSGKPGSGEGSTKG(SEQ ID NO:93),GSTSGSGKPGSSEGST(SEQ ID NO:94),GGGSSGGS(SEQ ID NO:95),GGGGSGGGGSS(SEQ ID NO:96),GGGSSGGSGGSSGGS(SEQ ID NO:97) and GSTSGSGKPGSSEGST (SEQ ID NO:98).

[0239] Examples of linkers can also include sequences that are at least 70% identical (e.g., at least 72%, at least 74%, at least 75%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the exemplary linkers described herein. One of ordinary skill will recognize that the design of an activatable protein can include a fully or partially flexible linker, such that a linker can include a flexible linker and one or more portions that impart a less flexible structure to provide the desired activatable protein structure.

[0240] In some embodiments, the activatable protein can include one, two, three, four, five, six, seven, eight, nine, or ten linker sequences (e.g., the same or different linker sequences of any exemplary linker sequences described herein or known in the art). In some embodiments, the linker can include Sulfo-SIAB, SMPB, and Sulfo-SMPB, wherein the linker reacts with primary amine thiol groups.

[0241] Conjugating agent

[0242] In some aspects, the activatable molecule (e.g., an activatable protein, such as an activatable antibody) can also include one or more additional agents, such as a targeting moiety that facilitates delivery to a cell or tissue of interest, a therapeutic agent (e.g., an anti-tumor agent such as a chemotherapeutic agent or an anti-tumor agent), a toxin or a fragment thereof. The additional agent can be conjugated to the activatable TB. The term "agent" is used herein to refer to a chemical compound, a mixture of chemical compounds, a biomacromolecule, or an extract made from a biological material.

[0243] In some embodiments, the activatable protein can be conjugated to a cytotoxic agent, such as a toxin (eg, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof) or a radioactive isotope.

[0244] Examples of cytotoxic agents that can be conjugated to activatable proteins include dolastatin and its derivatives (e.g., auristatin E, AFP, monomethyl auristatin D (MMAD), monomethyl auristatin F (MMAF), monomethyl auristatin E (MMAE), demethyl auristatin E (DMAE), auristatin F, demethyl auristatin F (DMAF), dolastatin 16 (DmJ), dolastatin 16 (Dpv), auristatin derivatives (e.g., auristatin tyramine, auristatin quinolone (auristatin quinolone), maytansinoids (e.g., DM-1, DM-4), maytansinoid derivatives, duocarmycin, alpha-amanitin, turbostatin, phenstatin, hydroxyphenstatin, spongistatin 5, spongistatin 7, halistatin 1 1), halocomstatin 2, halocomstatin 3, halocomstatin, pyrrolobenzimidazole (PBI), cibrostatin 6, doxaliform, cemadotin analog (CemCH2-SH), Pseudomonas toxin A (PES8) variant, Pseudomonas toxin A (ZZ-PE38) variant, ZJ-101, anthracycline, doxorubicin, daunorubicin, bryostatin, camptothecin, 7-substituted camptothecin, 10,11-difluoromethylenedioxycamptothecin, combretastatin, debromoaplysiatoxin, KahaMide-F, discodermolide and ecteinascidin.

[0245] Examples of enzymatically active toxins that can be conjugated to the activatable protein include: diphtheria toxin, exotoxin A chain from Pseudomonas aeruginosa, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleuriies fordii protein, dianthin protein, Phytoiaca Americana proteins (e.g., PAPI, PAPII, and PAP-8), momordica charantia inhibitors, curcin, crotirs, sapaonaria officinalis inhibitors, gelonin, mitogeliin, restrictocin, phenomycin, neomycin, and tricothecene.

[0246] Examples of anti-tumor agents that can be conjugated to the activatable protein include: adriamycin, cerubidine, bleomycin, alkeran, velban, oncovin, fluorouracil, methotrexate, thiotepa, bisantrene, novantrone, thioguanine, procarabizine, and cytarabine.

[0247] Examples of antiviral agents that can be conjugated to activatable proteins include acyclovir, vira A, and symmetrel. Examples of antifungal agents that can be conjugated to activatable proteins include nystatin. Examples of detection agents that can be conjugated to activatable proteins include fluorescein and its derivatives, fluorescein isothiocyanate (FITC). Examples of antibacterial agents that can be conjugated to activatable proteins include aminoglycosides, streptomycin, neomycin, kanamycin, amikacin, gentamicin, and tobramycin. 3β,16β,17α-trihydroxycholest-5-en-22-one 16-O-(2-O-4-methoxybenzoyl-β-D-xylopyranosyl)-(1-->3)-(2-O-acetyl-α-L-arabinopyranoside) (OSW-1) that can be conjugated to activatable proteins include: s-nitrobenzyloxycarbonyl derivatives of O6-benzylguanine, topoisomerase inhibitors, hemiasterlin, cephalotaxine, homoharringionine, pyrrolobenzodiazepines Dimer (PBD), functionalized pyrrolobenzodiazepine calcicheamicin, podophyllotoxin, taxanes, and vinca alkaloids. Examples of radiopharmaceuticals that can be conjugated to activatable 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 99mTc (technetium). Examples of heavy metals that can be conjugated to the activatable protein include: barium, gold, and platinum. Examples of anti-mycoplasma agents that can be conjugated to the activatable protein include: tylosine, spectinomycin, streptomycin B, ampicillin, sulfanilamide, polymyxin, and chloramphenicol.

[0248] In some embodiments, the activatable protein may include a signal peptide. If multiple polypeptides are included, the activatable protein may include multiple signal peptides, such as one signal peptide for each polypeptide in the multiple polypeptides. The signal peptide can be a peptide (e.g., 10-30 amino acids long) located at the end (e.g., N-terminus or C-terminus) of a newly synthesized protein, the destination of which is the secretory pathway. In some embodiments, the signal peptide can be conjugated to the activatable protein via a spacer. In some embodiments, the spacer is conjugated to the activatable protein in the absence of a signal peptide.

[0249] Those of ordinary skill in the art will recognize that a large number of possible agents can be conjugated to any activatable protein described herein. The agent can be conjugated to another component of the activatable protein through a conjugated portion. Conjugation may include any chemical reaction that will bind the two molecules, as long as the activatable protein and the other parts retain their corresponding activities. Conjugation may include many chemical mechanisms, such as covalent binding, affinity binding, embedding, coordination binding and complexation. In some embodiments, the binding may be covalent binding. Covalent binding can be achieved by direct condensation of existing side chains or by incorporation of external bridging molecules. Many divalent or multivalent linkers can be used to conjugate any activatable protein described herein. For example, conjugation may include organic compounds such as thioesters, carbodiimides, succinimide esters, glutaraldehyde, diazobenzene and hexamethylenediamine. In some embodiments, the activatable protein may include or otherwise introduce one or more non-natural amino acid residues to provide a suitable conjugation site.

[0250] In some embodiments, the agent and / or conjugate can be attached to the antigen binding domain via a disulfide bond (e.g., a disulfide bond on a cysteine ​​molecule). Because many cancers naturally release high levels of the reducing agent glutathione, the glutathione present in the cancerous tissue microenvironment can reduce the disulfide bonds and subsequently release the agent and / or conjugate at the delivery site.

[0251] In some embodiments, when the conjugate binds to its target in the presence of complement at the target site (e.g., diseased tissue (e.g., cancerous tissue)), the amide bond or ester bond attaching the conjugate and / or agent to the linker is cleaved, thereby releasing the conjugate and / or agent in its activated form. When administered to a subject, these conjugates and / or agents can achieve delivery and release of the conjugate and / or agent at the target site (e.g., diseased tissue (e.g., cancerous tissue). These conjugates and / or agents can effectively deliver any conjugate and / or agent described herein in vivo.

[0252] In some embodiments, the conjugated moiety cannot be cleaved by enzymes of the complement system. For example, the conjugate and / or agent is released without complement activation, because complement activation will eventually dissolve the target cell. In such embodiments, the conjugate and / or agent will be delivered to the target cell (e.g., hormone, enzyme, corticosteroid, neurotransmitter or gene). In addition, the conjugated moiety is slightly susceptible to cleavage by serum proteases, and the conjugate and / or agent is slowly released at the target site.

[0253] In some embodiments, the conjugate and / or agent can be designed such that the conjugate and / or agent is delivered to a target site (eg, diseased tissue (eg, cancerous tissue)), but the conjugate and / or agent is not released.

[0254] In some embodiments, the conjugate and / or agent can be attached directly to the antigen binding domain, or via amino acids (e.g., D-amino acids), peptides, thiol-containing moieties, or other organic compounds that can be modified to include functional groups that can then be used to attach to the antigen binding domain by the methods described herein.

[0255] In some embodiments, the activatable protein may include at least one conjugation point of the agent. In some embodiments, all possible conjugation points can be used for conjugation with the agent. In some embodiments, one or more conjugation points may include sulfur atoms involved in disulfide bonds, sulfur atoms involved in interchain disulfide bonds, sulfur atoms involved in interchain sulfide bonds but not sulfur atoms involved in intrachain disulfide bonds, and / or sulfur atoms of cysteine ​​or other amino acid residues containing sulfur atoms. In such cases, the residues may be naturally present in the protein construct structure, or may be incorporated into the protein construct using methods including site-directed mutagenesis, chemical conversion, or misincorporation of non-natural amino acids.

[0256] The present disclosure also provides methods and materials for preparing activatable proteins with one or more conjugating agents. In some embodiments, the activatable protein can be modified to include one or more interchain disulfide bonds. For example, the disulfide bond can be reduced after exposure to a reducing agent, such as but not limited to TCEP, DTT or β-mercaptoethanol. In some cases, the reduction of the disulfide bond is only partial. As used herein, the term partial reduction refers to a situation in which the activatable protein is contacted with a reducing agent and a portion of all possible conjugation sites are reduced (e.g., not all disulfide bonds are reduced). In some embodiments, if less than 99% (e.g., less than 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5%) of all possible conjugation sites are reduced after contact with a reducing agent, then the activatable protein is partially reduced. In some embodiments, an activatable protein having one or more interchain disulfide bonds reduced can be conjugated to a drug reactive with a free thiol.

[0257] The present disclosure also provides methods and materials for conjugating therapeutic agents to specific positions on activatable proteins. In some embodiments, activatable proteins can be modified so that therapeutic agents can be conjugated to activatable proteins at specific positions on activatable proteins. For example, activatable proteins can be partially reduced in a manner that is conducive to binding to activatable proteins. In such cases, partial reduction of activatable proteins can occur in a manner where the conjugation site in the activatable protein is not reduced. In some embodiments, one or more conjugation sites on the activatable protein are selected to facilitate conjugation of the agent at a specific position on the protein construct. Various factors can affect the "reduction level" of the activatable protein after treatment with a reducing agent. For example, without limitation, the ratio of the reducing agent to the activatable protein, the length of incubation, the incubation temperature, and / or the pH value of the reduction reaction solution may need to be optimized to achieve partial reduction of the activatable protein using the methods and materials described herein. Any appropriate combination of factors (e.g., the ratio of the reducing agent to the activatable protein, the length of incubation with the reducing agent and the temperature, and / or the pH value of the reducing agent) can be used to achieve partial reduction of the activatable protein (e.g., general reduction of possible conjugation sites or reduction at specific conjugation sites).

[0258] The 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 conjugation to the agent (eg, general reduction of possible conjugation sites or reduction at a specific conjugation site). In some embodiments, the ratio of reducing agent to activatable 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.

[0259] The effective incubation time and temperature for treating the 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 the agent to conjugate to the activatable protein (e.g., general reduction of possible conjugation sites or reduction at specific conjugation sites). In some embodiments, the incubation time and temperature for treating the activatable protein can be in the range of about 1 hour at 37° C. to about 12 hours at 37° C. (or any sub-range therein).

[0260] The effective pH of the reduction reaction for treating the activatable protein with the reducing agent can be any pH that at least partially reduces the activatable protein in a manner that allows conjugation of the activatable protein to the agent (e.g., general reduction of possible conjugation sites or reduction at a specific conjugation site).

[0261] When the partially reduced activatable protein is contacted with a thiol-containing agent, the agent can be conjugated to an interchain thiol in the activatable protein. The agent can be modified using a thiol-containing agent (e.g., cysteine ​​or N-acetylcysteine) to include a thiol. 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 reducing agent to activatable protein ratio can be any ratio that partially reduces at least two interchain disulfide bonds located in the activatable protein in a manner that allows the thiol-containing agent to conjugate (e.g., general reduction of possible conjugation sites or reduction at specific conjugation sites).

[0262] In some embodiments, the activatable protein can be reduced by a reducing agent in a manner that avoids reduction of any intrachain disulfide bonds. In some embodiments, the activatable protein can be reduced by a reducing agent in a manner that avoids reduction of any intrachain disulfide bonds and reduces at least one interchain disulfide bond.

[0263] In some embodiments, the agent (e.g., an agent conjugated to an activatable protein) is a detectable portion, such as an image tag or other marker. For example, the agent may be or include a radiolabeled amino acid, one or more biotinyl moieties detectable by labeled avidin (e.g., streptavidin containing a fluorescent marker or enzyme activity that can be detected by optical or calorimetric methods), one or more radioisotopes or radionuclides, one or more fluorescent labels, one or more enzyme labels, and / or one or more chemiluminescent agents. In some embodiments, the detectable portion 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 beta-galactosidase. In some embodiments, the fluorescent label may include yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), green fluorescent protein (GFP), modified red fluorescent protein (mRFP), red fluorescent protein tdimer2 (RFP tdimer2), HCRED or europium derivatives. In some embodiments, the luminescent label may include N-methyl acridine derivatives. In some embodiments, the label may include Alexa Fluor. Tags, such as Alex 680 or Alexa 750. In some embodiments, the ligand-based label may comprise biotin, avidin, streptavidin, or one or more haptens.

[0264] Other 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, dichlorotriazinamine fluorescein, dansyl chloride, or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive materials include 125 I. 131 I. 35 S or 3 H.

[0265] In some embodiments, the agent can be conjugated to the activatable protein using a carbohydrate moiety, a sulfhydryl group, an amino group, or a carboxylate group. In some embodiments, the agent can be conjugated to the activatable protein via a linker and / or CM as described herein. In some embodiments, the agent can be conjugated to a cysteine ​​or lysine in the activatable protein. In some embodiments, the agent can be conjugated to another residue of the activatable protein (e.g., those disclosed herein).

[0266] In some embodiments, a variety of bifunctional protein coupling agents can be used to conjugate the agent to the activatable protein, including but not limited to N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl diimidoadipate hydrochloride), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). In some embodiments, carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) chelator can be used to conjugate radionucleotides to activatable proteins (see, e.g., WO94 / 11026).

[0267] Suitable conjugated moieties include those described in the literature. (See, e.g., Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984), which describes the use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester). See also U.S. Pat. No. 5,030,719, which describes the use of halogenated acetyl hydrazide derivatives coupled to activatable proteins via oligopeptides. In some embodiments, suitable conjugated moieties include: (i) EDC (1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride; (ii) SMPT (4-succinimidyloxycarbonyl-α-methyl-α-(2-pyridyl-disulfide)-toluene (Pierce Chem., Catalog No. (21558G); (iii) SPDP (succinimidyl-6-[3-(2-pyridyldisulfide) propionamido] hexanoate (Pierce Chem., Catalog No. 21651G); (iv) sulfo-LC-SPDP (6-[3-(2-pyridyldisulfide)-propionamido] hexanoic acid sulfosuccinimidyl ester (Pierce Chem., Catalog No. 2165-G); and (v) sulfo-NHS (N-hydroxysulfo-succinimide: Pierce Chem.) conjugated to EDC. Chem., Cat. No. 24510). Additional exemplary conjugated moieties include SMCC, Sulfo-SMCC, SPDB, and Sulfo-SPDB.

[0268] The conjugated moieties described above may contain components with different properties, thus making the conjugates have different physicochemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates. The solubility of the linker containing NHS esters is less than that of sulfo-NHS esters. In addition, SMPT contains a disulfide bond with steric hindrance and can form a conjugate with increased stability. In general, the stability of disulfide bonds is less than that of other bonds because disulfide bonds are cleaved in vitro, resulting in less available conjugates. In particular, sulfo-NHS can enhance the stability of carbodiimide coupling. Compared to a single carbodiimide coupling reaction, carbodiimide coupling (e.g., EDC) forms an ester that is more resistant to hydrolysis when used in conjunction with sulfo-NHS.

[0269] One of ordinary skill in the art will recognize a variety of possible moieties that 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 binds the agent to the activatable protein while also allowing the agent and the activatable protein to retain functionality.

[0270] Nucleic acids and vectors

[0271] In some aspects, the present disclosure also provides nucleic acids comprising sequences encoding activatable molecules (e.g., activatable antibodies) or components or fragments thereof herein. Nucleic acids may include coding sequences of TB, CM, MM, EM and linkers in activatable proteins. In the case where the activatable protein comprises multiple polypeptides (e.g., multiple TBs on different polypeptides, or TB comprises multiple polypeptides), the nucleic acid may comprise coding sequences of multiple polypeptides. In some examples, the coding sequence of one polypeptide is contained in one nucleic acid, and the coding sequence of another polypeptide is contained in another nucleic acid. In some examples, the coding sequences of two or more polypeptides in multiple polypeptides are contained in the same nucleic acid. The present disclosure includes polynucleotides encoding proteins or parts thereof as described herein, and the use of such polynucleotides for producing proteins and / or for therapeutic purposes. Such polynucleotides may include DNA and RNA molecules (e.g., mRNA, self-replicating RNA, self-amplifying mRNA, etc.) encoding proteins as defined herein. The present disclosure includes compositions comprising such polynucleotides. In some aspects, such compositions can be used therapeutically or prophylactically.

[0272] Unless otherwise specified, "protein encoding nucleic acid sequence" includes all nucleotide sequences that are degenerate forms of each other and therefore encode the same amino acid sequence. The term "nucleic acid" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in single-stranded or double-stranded form, or a combination thereof. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides with similar binding properties to the reference nucleotides. Unless otherwise indicated, a specific nucleic acid sequence also implicitly encompasses complementary sequences as well as sequences explicitly indicated. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is RNA.

[0273] When referring to the position of a first domain or sequence relative to a second domain or sequence in a polypeptide primary amino acid sequence, the term "N-terminal positioning" means that the first domain is positioned closer to the N-terminus of the polypeptide primary amino acid sequence. In some embodiments, there may be additional sequences and / or domains between the first domain or sequence and the second domain or sequence. When referring to the position of a first domain or sequence relative to a second domain or sequence in a polypeptide primary amino acid sequence, the term "C-terminal positioning" means that the first domain is positioned closer to the C-terminus of the polypeptide primary amino acid sequence. In some embodiments, there may be additional sequences and / or domains between the first domain or sequence and the second domain or sequence.

[0274] Modifications can be introduced into the nucleotide sequence by standard techniques known in the art, such as site-directed mutagenesis and polymerase chain reaction (PCR)-mediated mutagenesis. Conservative amino acid substitutions are amino acid substitutions in which the amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include: amino acids with acidic side chains (e.g., aspartic acid and glutamic acid), amino acids with basic side chains (e.g., lysine, arginine, and histidine), nonpolar amino acids (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), uncharged polar amino acids (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine), hydrophilic amino acids (e.g., arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine, and threonine), hydrophobic amino acids (e.g., alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine). Other amino acid families include: aliphatic-hydroxy amino acids (e.g., serine and threonine), amide family (e.g., asparagine and glutamine), aliphatic family (e.g., alanine, valine, leucine, and isoleucine), and aromatic family (e.g., phenylalanine, tryptophan, and tyrosine).

[0275] The present disclosure also provides vectors and vector groups comprising any nucleic acid described herein. Those skilled in the art will be able to select a vector or vector group (e.g., an expression vector) suitable for preparing any activatable protein described herein, and use the vector or vector group to express any activatable protein described herein. For example, when selecting a vector or vector group, a cell type can be selected so that one or more vectors may need to be able to be integrated into the chromosome of the cell and / or replicated in the cell. Exemplary vectors that can be used to produce activatable proteins are also described herein. As used herein, the term "vector" refers to a polynucleotide capable of inducing expression of a recombinant protein (e.g., a first or second monomer) in a cell (e.g., any cell described herein). "Vector" can deliver nucleic acids and fragments thereof to a host cell, and includes regulatory sequences (e.g., promoters, enhancers, polyadenylic acid (poly (A)) signals). Exogenous polynucleotides can be inserted into an expression vector for expression. The term "vector" also includes artificial chromosomes, plasmids, retroviruses, and baculovirus vectors.

[0276] Methods for constructing suitable vectors comprising any of the nucleic acids described herein and suitable for transforming cells (e.g., mammalian cells) are well known in the art. See, e.g., Sambrook et al., ed., "Molecular Cloning: A Laboratory Manual," 2nd ed., Cold Spring Harbor Press, 1989 and Ausubel et al., ed., "Current Protocols in Molecular Biology," Current Protocols, 1993.

[0277] Examples of vectors include plasmids, transposons, cosmids, and viral vectors (e.g., any adenoviral vector (e.g., pSV or pCMV vector), adeno-associated virus (AAV) vector, lentiviral vector, and retroviral vector), as well as any Vector. The vector may, for example, include cis-acting elements sufficient to achieve expression; other elements for expression may be provided by the host mammalian cell or in an in vitro expression system. A skilled practitioner will be able to select vectors and mammalian cells suitable for preparing any activatable protein described herein.

[0278] In some embodiments, the activatable protein can be produced biosynthetically using recombinant DNA technology and expression in eukaryotic or prokaryotic species.

[0279] cell

[0280] In some aspects, the disclosure provides a recombinant host cell comprising any vector or nucleic acid described herein. The cell can be used to produce an activatable molecule (e.g., an activatable antibody) as described herein. In some embodiments, the cell can be an animal cell, a mammalian cell (e.g., a human cell), a rodent cell (e.g., a mouse cell, a rat cell, a hamster cell, or a guinea pig cell), a non-human primate cell, an insect cell, a bacterial cell, a fungal cell, or a plant cell. In some embodiments, the cell can be a eukaryotic cell. As used herein, the term "eukaryotic cell" refers to a cell with a distinct, membrane-surrounded nucleus. Such cells may include, for example, mammals (e.g., rodents, non-human primates, or humans), insects, fungi, or plant cells. In some embodiments, the eukaryotic cell is a yeast cell, such as Saccharomyces cerevisiae. In some embodiments, the eukaryotic cell is a higher eukaryotic cell, such as a mammal, a bird, a plant, or an insect cell. Non-limiting examples of mammalian cells include Chinese hamster ovary (CHO) cells and human embryonic kidney cells (e.g., HEK293 cells). In some embodiments, the cell is a prokaryotic cell.

[0281] Methods for introducing nucleic acids and vectors (e.g., any vector or any vector group described herein) into cells are known in the art. Examples of methods that can be used to introduce nucleic acids into cells include: liposome transfection, transfection, calcium phosphate transfection, cationic polymer transfection, viral transduction (e.g., adenoviral transduction, lentiviral transduction), nanoparticle transfection, and electroporation.

[0282] In some embodiments, the introducing step comprises introducing into the cell a vector (eg, any vector or set of vectors described herein) comprising a nucleic acid encoding a monomer that makes up any of the activatable proteins described herein.

[0283] Compositions and kits

[0284] The present disclosure also provides compositions and kits comprising the activatable molecules (eg, activatable antibodies) described herein. The compositions and kits may also include one or more excipients, carriers, reagents, instructions for using the activatable protein.

[0285] In some embodiments, the composition can be a pharmaceutical composition comprising an activatable protein, its derivatives, fragments, analogs and homologues. The pharmaceutical composition may comprise an activatable protein and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delay agents compatible with drug administration. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, which is a standard reference text in the art and is incorporated herein by reference. Suitable examples of such carriers or diluents include water, saline, Ringer's solution, dextrose solution and 5% human serum albumin. Liposomes and non-aqueous vehicles, such as fixed oils, can also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agents are incompatible with the active compound, it is expected to be used in the composition. Supplementary active compounds may also be incorporated into the composition.

[0286] The pharmaceutical composition can be formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (e.g., topical), transmucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous applications may include one or more of the following components: sterile diluents, such as water for injection, saline solutions, fixed oils, polyethylene glycols, glycerol, propylene glycol, or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetates, citrates, or phosphates, and agents for adjusting tension, such as sodium chloride or dextrose. pH values ​​may be adjusted with acids or bases (e.g., hydrochloric acid or sodium hydroxide). Parenteral preparations may be packaged in ampoules, disposable syringes, or multidose vials made of glass or plastic. In some, any activatable protein described herein is prepared with a carrier that prevents rapid elimination from the body, such as sustained and controlled release formulations, including implants and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid-co-glycolic acid and polylactic acid can be used. The preparation methods of such pharmaceutical compositions and preparations are obvious to those skilled in the art. For example, the activatable protein can be embedded in microcapsules (e.g., hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules, respectively) prepared by coacervation technology or by interfacial polymerization, embedded in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or embedded in coarse emulsions.

[0287] Sustained release preparations can be prepared.Sustained release preparations include semipermeable matrixes of solid hydrophobic polymers containing antibodies, and the matrix is ​​in the form of formed articles, such as films or microcapsules.Examples of sustained release matrixes include polyesters, hydrogels (such as poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol)), polylactic acid, copolymers of L-glutamic acid and L-glutamic acid γ ethyl ester, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers (such as injectable microspheres composed of lactic acid-glycolic acid copolymers and leuprolide acetate (leuprolide acetate)) and poly-D-(-)-3-hydroxybutyric acid.Although polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid can release molecules for more than 100 days, the time for some hydrogels to release proteins is shorter.

[0288] In some embodiments, pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where soluble in water) or dispersions and sterile powders for preparing sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL TM (BASF, Parsippany, NJ), phosphate buffered saline (PBS), etc. The composition can be sterile and should be fluid, and have a viscosity that is convenient for injection. It must be stable under the conditions of manufacture and storage and must be preserved to prevent the contamination of microorganisms such as bacteria and fungi. The carrier can be a solvent or a dispersion medium, which contains, for example, water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol, etc.) and their suitable mixtures. For dispersed particulate compositions, for example, by using a coating such as lecithin on the particles, and by maintaining the required particle size in the case of dispersions, and by using a surfactant, appropriate fluidity can be maintained. In some embodiments, the pharmaceutical composition may also include one or more antibacterial agents and / or antibacterial agents and antifungal agents (such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.). In some embodiments, isotonic agents, such as sugars, polyols (such as mannitol, sorbitol), etc., and salts, such as sodium chloride, etc., may be included in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0289] In some embodiments, the pharmaceutical composition may include a sterile injection solution. A sterile injection solution may be prepared by incorporating the required amount of active compound and a composition listed above or a combination thereof into an appropriate solvent as required, followed by filtration sterilization. Typically, dispersions may be prepared by incorporating the active compound into a sterile vehicle containing other components required in an alkaline dispersion medium and those components listed above. In the case of a sterile powder for the preparation of a sterile injection solution, the preparation method is vacuum drying and freeze drying to obtain a powder of the active ingredient plus any other required components from a previously sterile filtered solution.

[0290] In some embodiments, the pharmaceutical composition may include an oral composition. The oral composition may include an inert diluent or an edible carrier. They may be encapsulated in a gelatin capsule or compressed into a tablet. For the purpose of oral therapeutic administration, the active compound may be blended with an excipient and used in the form of a tablet, lozenge or capsule. Oral compositions may also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is taken orally and gargled, coughed out or swallowed. Pharmaceutically compatible binders and / or adjuvant materials may be included as part of the composition. Tablets, pills, capsules, lozenges, etc. may contain any of the following ingredients or compounds of similar nature: a binder, such as microcrystalline cellulose, tragacanth or gelatin; an excipient, such as starch or lactose; a disintegrant such as alginic acid, Primogel or corn starch; a lubricant, such as magnesium stearate or Sterotes; a glidant, such as colloidal silicon dioxide; a sweetener, such as sucrose or saccharin; or a flavoring agent, such as mint, methyl salicylate or orange flavoring.

[0291] In one embodiment, the pharmaceutical compositions may be formulated for administration by inhalation. For example, the compounds may be delivered in the form of an aerosol spray from a pressured container or dispenser that contains a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer.

[0292] In one embodiment, the pharmaceutical composition can be formulated for systemic administration. For example, systemic administration can be intravenous, or it can be transmucosal or transdermal. For transmucosal or transdermal administration, a penetrant suitable for the barrier to be penetrated can be used in the formulation. Such penetrants are generally known in the art, and include, for example, detergents, bile salts, and fusidic acid derivatives for transmucosal administration. Transmucosal administration can be achieved by using nasal sprays or suppositories. For transdermal administration, the active compound can be formulated into an ointment, salves, gels, or creams, as generally known in the art.

[0293] In some embodiments, the pharmaceutical compositions may be prepared in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0294] In one embodiment, the pharmaceutical composition can be prepared with a carrier that prevents rapid elimination from the body, such as sustained and controlled release formulations, including implants and microencapsulated delivery systems. Biodegradable biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid-co-glycolic acid and polylactic acid. Methods for preparing such preparations will be apparent to those skilled in the art.

[0295] Oral or parenteral compositions are preferably formulated in unit dosage form for ease of administration and uniformity of dosage. Unit dosage form as used herein refers to physically discrete units suitable as unitary dosages for subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the unit dosage forms of the present disclosure are dictated by and directly dependent on the unique characteristics of the active compound and the specific therapeutic effect to be achieved, and the inherent limitations in the art of compounding such active compounds for treating individuals.

[0296] In some embodiments, a composition (eg, a pharmaceutical composition) may be included in a container, vial, syringe, pen, pack, or dispenser, optionally together with instructions for administration.

[0297] Also provided herein are kits comprising any activatable protein described herein, any composition comprising any activatable protein described herein, or any pharmaceutical composition comprising any activatable protein described herein. Also provided are kits comprising one or more second therapeutic agents in addition to the activatable protein described herein. The one or more second therapeutic agents may be provided in a dosage administration form separate from the activatable protein. Alternatively, the one or more second therapeutic agents may be formulated together with the activatable protein.

[0298] Any kit described herein may include instructions for using any composition (e.g., pharmaceutical composition) and / or any activatable protein described herein. In some embodiments, the kit may include instructions for performing any method described herein. In some embodiments, the kit may include at least one dose of any composition (e.g., pharmaceutical composition) described herein. In some embodiments, the kit may provide a syringe for administering any pharmaceutical composition described herein.

[0299] Also provided herein are activatable proteins produced by any of the methods described herein. Also provided 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 comprising at least one dose of any of the compositions (e.g., pharmaceutical compositions) described herein.

[0300] Methods for producing activatable molecules

[0301] Provided herein are methods for producing any activatable molecule (e.g., an activatable protein) described herein, the method comprising: (a) culturing any recombinant host cell described herein in a liquid culture medium under conditions sufficient to produce the activatable molecule; and (b) recovering the activatable molecule from the host cell and / or the liquid culture medium.

[0302] Methods for culturing cells are well known in the art. In some embodiments, cells can be maintained in vitro under conditions that are conducive to cell proliferation, cell differentiation, and cell growth. For example, recombinant cells can be cultured by contacting cells (e.g., any cells described herein) with a cell culture medium that includes necessary growth factors and supplements sufficient to support cell viability and growth.

[0303] In some embodiments, the method may further include separating the recovered activatable protein. The separation of the activatable protein can be performed using any separation or purification technique for separating protein species, such as affinity tag-based protein separation (e.g., polyhistidine (His) tag, glutathione-S-transferase tag, etc.), ammonium sulfate precipitation, polyethylene glycol precipitation, size exclusion chromatography, ligand affinity chromatography (e.g., protein A chromatography), ion exchange chromatography (e.g., anion or cation), hydrophobic interaction chromatography, etc.

[0304] The compositions and methods described herein may involve the use of non-reducing or partially reducing conditions that allow for the formation of a disulfide bond between the MM and TB of an activatable protein.

[0305] In some embodiments, the method further comprises formulating the isolated activatable protein into a pharmaceutical composition. Various formulations are known in the art and are described herein. Any isolated activatable protein described herein can be formulated for any route of administration (e.g., intravenous, intratumoral, subcutaneous, intradermal, oral (e.g., inhalation), transdermal (e.g., topical), transmucosal, or intramuscular).

[0306] How to use activatable molecules

[0307] In some aspects, the present disclosure also provides methods of using the activatable molecules (e.g., activatable antibodies) herein. In some embodiments, the present disclosure provides methods of treating a disease (e.g., cancer (e.g., any cancer described herein)) in a subject, the method comprising administering a therapeutically effective amount of any activatable protein described herein to the subject. In some embodiments, the present disclosure provides methods of preventing a disease in a subject, delaying the progression of a disease, treating a disease, alleviating symptoms of a disease, or otherwise improving a disease, the method being performed by administering a therapeutically effective amount of an activatable protein described herein to a subject in need. The term "treatment" refers to improving at least one symptom of a condition. In some embodiments, the condition treated may be cancer or an autoimmune disease or may be to improve at least one symptom of a cancer or autoimmune disease. As used herein, the term "subject" refers to any mammal. In some embodiments, the subject is a feline (e.g., a cat), a canine (e.g., a dog), an equine (e.g., a horse), a rabbit, a pig, a rodent (e.g., a mouse, a rat, a hamster, or a guinea pig), a non-human primate (e.g., a monkey (e.g., a baboon, a marmoset) or an ape (e.g., a chimpanzee, a gorilla, an orangutan, or a gibbon)), or a human. In some embodiments, the subject is a human. The terms subject and patient are used interchangeably herein. In some embodiments, the subject has been previously identified or diagnosed as having a disease (e.g., a cancer (e.g., any of the cancers described herein)).

[0308] In some embodiments, the subject may be identified as having an increase in the expression and / or active HER2 gene mutation of HER2 in mammalian cells (for example, any mammalian cells described herein).For example, the HER2 gene mutation increasing the expression and / or active HER2 in mammalian cells can be gene repetition, and the mutation causes the HER2 expression with one or more amino acid substitutions (for example, 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) (compared to wild-type protein).See, for example, Weigelt and Reis-Filho, Cancer Discov.2013, 3 (2): 145-147.

[0309] Non-limiting examples of methods for detecting HER2-related diseases in a subject include: immunohistochemistry, fluorescent in situ hybridization (FISH), chromogenic in situ hybridization (CISH). See, for example, Yan et al., Cancer Metastasis Rev. 2015, 34: 157-164.

[0310] The therapeutically effective amount of the activatable protein disclosed herein generally relates to the amount required to achieve the therapeutic goal. As described above, this may be a binding interaction between the antibody and its target antigen, which in some cases interferes with the function of the target. The amount required for administration will also depend on the binding affinity of the activatable protein to its specific target, and will also depend on the rate at which the administered activatable protein is consumed from the free volume of other subjects to which it is administered. The common range of therapeutically effective doses of the activatable protein disclosed herein may be (by way of non-limiting example) about 0.001, 0.01, 0.1, 0.3, 0.5, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 mg / kg body weight or more. The structure of the activatable protein disclosed herein makes it possible to reduce the dose of the activatable protein administered to the subject compared to conventional activatable antibodies and compared to conventional antibodies. For example, the administered dose per unit dose or the total dose during one dosing regimen may be reduced by 10%, 20%, 30%, 40% or 50% compared to the corresponding dose of a corresponding conventional activatable protein or a corresponding conventional antibody.

[0311] Typical dosing frequencies may range, for example, from once or twice daily, weekly, biweekly, or monthly.

[0312] The effectiveness of the treatment is determined in conjunction with any known method for diagnosing or treating the particular condition.Methods for screening for activatable proteins with the desired specificity include, but are not limited to, enzyme-linked immunosorbent assays (ELISA) and other immune-mediated techniques known in the art.

[0313] In another embodiment, the activatable protein against two or more targets is used in methods known in the art related to target localization and / or quantification (e.g., for measuring the level of one or more targets in an appropriate physiological sample, for diagnostic methods, for imaging proteins, etc.). In a given embodiment, the activatable protein against two or more targets, or its derivative, fragment, analog or homolog containing an antibody-derived antigen binding domain, is used as a pharmacologically active compound (hereinafter referred to as a "therapeutic agent").

[0314] The activatable proteins used in any of the embodiments of these methods and uses can be administered at any stage of the disease. For example, such activatable proteins can be administered to cancer patients at any stage, from early stage to metastatic. In some embodiments, activatable proteins and formulations thereof can be administered to subjects suffering from or susceptible to a disease or condition associated with aberrant target expression and / or activity.

[0315] Any of a variety of methods known in the art can be used to identify subjects suffering from or susceptible to a disease or condition associated with abnormal target expression and / or activity. For example, any of a variety of clinical and / or laboratory tests (e.g., physical examination and blood, urine and / or stool analysis) can be used to assess health status to identify subjects suffering from cancer or other tumor disorders. For example, any of a variety of clinical and / or laboratory tests (e.g., physical examination and / or body fluid analysis, such as blood, urine and / or stool analysis) can be used to assess health status to identify subjects suffering from inflammation and / or inflammatory conditions.

[0316] In some embodiments, if any of a variety of laboratory or clinical goals are achieved, then the administration of an activatable protein to a patient with a disease or condition associated with abnormal target expression and / or activity is considered successful. For example, if one or more symptoms associated with the disease or condition are alleviated, reduced, suppressed, or do not progress to a further (ie, worse) state, then the administration of an activatable protein to a patient with a disease or condition associated with abnormal target expression and / or activity is considered successful. If the disease or condition enters remission or does not progress to a further (ie, worse) state, then the administration of an activatable protein to a patient with a disease or condition associated with abnormal target expression and / or activity is considered successful.

[0317] As used herein, the term "treating" includes reducing the severity, frequency, or number of one or more (e.g., 1, 2, 3, 4, or 5) symptoms or signs of a disease (e.g., cancer (e.g., any cancer described herein)) in a subject (e.g., any subject described herein). In some embodiments where the disease is cancer, treatment reduces cancer growth, inhibits cancer progression, inhibits cancer metastasis, or reduces the risk of cancer recurrence in a subject with cancer.

[0318] In some embodiments, the disease can be cancer. In some embodiments, the subject can have been identified or diagnosed as having cancer. Examples of cancer include: solid tumors, hematological tumors, sarcomas, osteosarcomas, glioblastomas, neuroblastomas, melanomas, rhabdomyosarcomas, Ewing sarcomas, sarcoma), osteosarcoma, B cell tumors, multiple myeloma, lymphoma (e.g., B cell lymphoma, B cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, cutaneous T cell lymphoma), leukemia (e.g., hairy cell leukemia, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL)), myelodysplastic syndrome (MDS), Kaposi's sarcoma, retinoblastoma, gastric cancer, urothelial cancer, lung cancer, renal cell carcinoma, gastric cancer and esophageal cancer, pancreatic cancer, prostate cancer, brain cancer, colon cancer, bone cancer, lung cancer, breast cancer, colorectal cancer, ovarian cancer, nasopharyngeal adenocarcinoma, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma, endometrial cancer, bladder cancer, cervical cancer, liver cancer, and hepatocellular carcinoma. In some embodiments, the cancer is a lymphoma. In some embodiments, the lymphoma is Burkitt's lymphoma. In some aspects, the subject has been identified or diagnosed as having a familial cancer syndrome, such as Li-Fraumeni Syndrome, familial breast cancer-ovarian cancer (BRCA1 or BRAC2 mutation) syndrome, etc. The disclosed method can also be used to treat non-solid cancers. Exemplary solid tumors include malignancies (e.g., sarcomas, adenocarcinomas, and carcinomas) of various organ systems, such as lung, breast, lymph, gastrointestinal (e.g., colon) and urogenital (e.g., kidney, urothelial or testicular tumors) tracts, pharynx, prostate, and ovarian malignancies. Exemplary adenocarcinomas include colorectal cancer, renal cell carcinoma, liver cancer, non-small cell lung cancer, and small intestinal cancer. Other examples of cancers treatable by the compositions and methods herein include: acute lymphoblastic leukemia in adults; acute lymphoblastic leukemia in children; acute myeloid leukemia in adults; adrenocortical carcinoma; adrenocortical carcinoma in children; AIDS-related lymphoma; AIDS-related malignancies; anal cancer; cerebellar astrocytoma in children; cerebral astrocytoma in children; extrahepatic bile duct cancer; bladder cancer; bladder cancer in children; osteosarcoma / malignant fibrous histiocytoma bone cancer; brain stem glioma in children; brain tumors in adults; brain stem glioma brain tumors in children; cerebellar astrocytoma brain tumors in children; cerebral astrocytoma / malignant glioma brain tumors in children; ependymoma brain tumors in children; medulloblastoma brain tumors in children; supratentorial primitive neuroectodermal tumor brain tumors in children; optic pathway and hypothalamic glioma brain tumors in children; brain tumors in children (other); breast cancer; breast cancer and pregnancy; breast cancer in children; breast cancer in men; bronchial adenoma / carcinoid in children; carcinoid tumors in children; gastrointestinal carcinoid tumors;Adrenocortical carcinoma; Islet cell carcinoma; Carcinoma of unknown primary; Primary central nervous system lymphoma; Cerebellar astrocytoma, childhood; Cerebral astrocytoma / glioblastoma, childhood; Cervical cancer; Childhood cancer; Chronic lymphocytic leukemia; Chronic myeloid leukemia; Chronic myeloproliferative disorder; Clear cell sarcoma of the tendon sheath; Colon cancer; Childhood colorectal cancer; Cutaneous T-cell lymphoma; Endometrial carcinoma; Childhood ependymoma; Ovarian epithelial carcinoma; Esophageal cancer; Childhood esophageal cancer; Ewing family tumors; Childhood extracranial germ cell tumors; Extragonadal germ cell tumors tumors; extrahepatic bile duct cancer; intraocular melanoma eye cancer; retinoblastoma eye cancer; gallbladder cancer; stomach (gastric) cancer; childhood stomach (gastric) cancer; gastrointestinal carcinoid tumors; childhood extracranial germ cell tumors; extragonadal germ cell tumors; ovarian germ cell tumors; gestational trophoblastic tumors; childhood brain stem gliomas; childhood optic pathway and hypothalamic gliomas; hairy cell leukemia; head and neck cancer; adult (primary) hepatocellular (liver) cancer; childhood (primary) hepatocellular (liver) cancer; adult Hodgkin's lymphoma (Hodgkin's Lymphoma; Hodgkin's lymphoma in children; Hodgkin's lymphoma in pregnancy; Hypopharyngeal cancer; Hypothalamic and optic pathway gliomas in children; Intraocular melanoma; Islet cell carcinoma (endocrine pancreas); Kaposi's sarcoma; Kidney cancer; Laryngeal cancer; Laryngeal cancer in children; Acute lymphoblastic leukemia in adults; Acute lymphoblastic leukemia in children; Acute myeloid leukemia in adults; Acute myeloid leukemia in children; Chronic lymphocytic leukemia; Chronic myeloid leukemia; Hairy cell leukemia; Lip and oral cancer; Liver cancer (primary) in adults; Liver cancer (primary) in children; Non-small cell lung cancer; Small cell lung cancer; Acute lymphoblastic leukemia in adults; Acute lymphoblastic leukemia in children ; Chronic lymphocytic leukemia; AIDS-related lymphoma; Central nervous system (primary) lymphoma; Cutaneous T-cell lymphoma; Hodgkin's lymphoma, adult; Hodgkin's lymphoma, childhood; Hodgkin's lymphoma, pregnancy; Non-Hodgkin's lymphoma, adult; Non-Hodgkin's lymphoma, childhood; Non-Hodgkin's lymphoma, pregnancy; Primary central nervous system lymphoma; Macroglobulinemia, Waldenstrom's; Male breast cancer; Malignant mesothelioma, adult; Malignant mesothelioma, childhood; Malignant thymoma; Medulloblastoma, childhood; Melanoma; Intraocular melanoma; Merkel cell carcinoma CellCarcinoma); Malignant Mesothelioma; Metastatic Squamous Cell Carcinoma of the Neck with Occult Primary; Multiple Endocrine Neoplasia Syndrome in Children; Multiple Myeloma / Plasma Cell Neoplasm; Mycosis Fungoides; Myelodysplastic Syndrome; Chronic Myeloid Leukemia; Acute Myeloid Leukemia in Children; Multiple Myeloma; Chronic Myeloproliferative Disorders; Nasal and Paranasal Sinus Cancer; Nasopharyngeal Carcinoma; Nasopharyngeal Carcinoma in Children; Neuroblastoma; Non-Hodgkin's Lymphoma in Adults; Non-Hodgkin's Lymphoma in Children; Non-Hodgkin's Lymphoma in Pregnancy; Non-Small Cell Lung Cancer;Oral cancer, childhood; oral cavity and lip cancer; oropharyngeal cancer; osteosarcoma / malignant fibrous histiocytoma of bone; ovarian cancer, childhood; ovarian epithelial cancer; ovarian germ cell tumors; ovarian low-grade malignant potential tumors; pancreatic cancer; pancreatic cancer, childhood; islet cell pancreatic cancer; paranasal sinus and nasal cavity cancer; parathyroid cancer; penile cancer; pheochromocytoma; pineal and supratentorial primitive neuroectodermal tumors, childhood; pituitary tumors; plasma cell neoplasms / multiple myeloma; pleuropulmonary blastoma; pregnancy and breast cancer; pregnancy and Hodgkin lymphoma; pregnancy and non Hodgkin lymphoma; primary central nervous system lymphoma; primary liver cancer, adults; primary liver cancer, children; prostate cancer; colorectal cancer; renal cell (kidney) cancer; renal cell cancer, children; transitional cell carcinoma of the renal pelvis and ureter; retinoblastoma; rhabdomyosarcoma, children; salivary gland cancer; salivary gland cancer, children; Ewing family tumor sarcoma; Kaposi sarcoma; sarcoma (osteosarcoma) / malignant fibrous histiocytoma of bone; rhabdomyosarcoma, children; soft tissue sarcoma, adults; soft tissue sarcoma, children; Sezary syndrome Syndrome); skin cancer; childhood skin cancer; skin cancer (melanoma); Merkel cell skin cancer; small cell lung cancer; small intestinal cancer; adult soft tissue sarcoma; childhood soft tissue sarcoma; metastatic squamous cell carcinoma of the neck with occult primary site; stomach (gastric) cancer; childhood stomach (gastric) cancer; childhood supratentorial primitive neuroectodermal tumor; cutaneous T-cell lymphoma; testicular cancer; childhood thymoma; malignant thymoma; thyroid cancer; childhood thyroid cancer; transitional cell carcinoma of the renal pelvis and ureter; gestational trophoblastic tumor; childhood cancer of unknown primary site; unusual childhood cancer; transitional cell carcinoma of the ureter and renal pelvis; urethral cancer; uterine sarcoma; vaginal cancer; childhood optic pathway and hypothalamic glioma; vulvar cancer; Waldenstrom's macroglobulinemia; Wilms' Tumor; diffuse large B-cell lymphoma (DLBCL); and mantle cell lymphoma (MCL). Metastasis of the above-mentioned cancers can also be treated or prevented according to the methods described herein. ;

[0319] In some embodiments, the disease can be an autoimmune disease or disorder. In some embodiments, the subject may have been identified or diagnosed as having an autoimmune disease or disorder, or is at high risk of developing an autoimmune disease or disorder. Examples of autoimmune diseases include cancer, 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, Sjögren's syndrome ( 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 (flu), Lyme disease, malaria, measles, meningitis, mumps, poliomyelitis (polio), pneumonia, Rocky mountain spotted fever, rubella (German measles), Salmonella infections, severe acute respiratory syndrome (SARS), sexually transmitted diseases, shingles (herpes zoster), tetanus, toxic shock syndrome, tuberculosis, viral hepatitis, West Nile virus, whooping cough (whooping cough), cough) (pertussis), chronic inflammation or transplant rejection (e.g., kidney, liver, or heart transplants), autoimmune diseases, infectious diseases, chronic inflammation or transplant rejection.

[0320] In some embodiments, the methods herein may result in a decrease in the number, severity, or frequency of one or more symptoms of cancer in a subject (eg, compared to the number, severity, or frequency of one or more symptoms of cancer in the subject prior to treatment).

[0321] The method may further comprise administering to the subject one or more additional agents.

[0322] In some embodiments, the activatable protein can be administered in combination with one or more additional agents during and / or after treatment. In some embodiments, the activatable protein can be formulated into a single therapeutic composition, and the activatable protein and one or more additional agents can be administered simultaneously. Alternatively, the activatable protein and one or more additional agents can be separated from each other, for example, each formulated into a separate therapeutic composition, and the activatable protein and the additional agent are administered simultaneously, or the activatable protein and the additional agent are administered at different times during the treatment regimen. For example, the activatable protein can be administered before the administration of the additional agent, after the administration of the additional agent, or in an alternating manner. The activatable protein and one or more additional agents can be administered in a single dose or multiple doses.

[0323] One or more activatable proteins herein can 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 activatable proteins herein can be combined with one or more other types of activatable proteins (e.g., activatable proteins without EM or activatable proteins whose activated form includes EM).

[0324] The present disclosure also provides methods for detecting the presence or absence of a cleavage agent and / or a target in a subject or sample. Such methods may include: (i) contacting a subject or biological sample with an activatable protein, wherein the activatable protein includes a detectable label located on a portion of the activatable protein that is released after CM cleavage; and (ii) measuring the level of activated protein in the subject or biological sample, wherein a detectable level of activated protein in the subject or biological sample indicates that a cleavage agent, a target, or both a cleavage agent and a target are not present and / or insufficiently present in the subject or biological sample, such that target binding and / or protease cleavage of the activatable protein cannot be detected in the subject or biological sample, and wherein a decrease in the detectable level of activated protein in the subject or biological sample indicates the presence of a cleavage agent and a target in the subject or biological sample.

[0325] The reduction in the level of the detectable marker can be, for example, a reduction of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or a substantial reduction of 100%. In some embodiments, the detectable marker can be conjugated to a component of the activatable protein (e.g., TB). In some embodiments, a secondary reagent that specifically binds to the activatable protein can be used to measure the level of the activatable protein in a subject or sample, wherein the reagent comprises a detectable marker. The secondary reagent can be an antibody comprising a detectable marker.

[0326] In some embodiments, the activatable protein can also be used to detect targets in patient samples and can therefore be used as a diagnostic agent. For example, the activatable protein can be used in in vitro assays, such as ELISA, to detect target levels in patient samples. For example, the activatable TB can be fixed on a solid support (e.g., a well of a microtiter plate). The fixed activatable protein can be used as a capture protein for any target that may be present in a test sample. Before contacting the fixed activatable protein with the patient sample, the solid support can be rinsed and treated with a blocking agent (e.g., milk protein or albumin) to prevent nonspecific adsorption of the analyte.

[0327] In some embodiments, based on the results obtained in an in vitro diagnostic assay using an activatable protein, the subject's disease can be staged based on the expression level of the target protein (e.g., antigen). For a given disease, blood samples are drawn from subjects diagnosed as being at various stages of disease progression and / or at various time points of disease therapeutic treatment. Using a sample population that provides statistically significant results for each stage of progression or treatment, a target protein (e.g., antigen) concentration range that can be considered as a characteristic of each stage is specified.

[0328] The activatable proteins herein can also be used for diagnostic and / or imaging methods. In some embodiments, such methods can be in vitro methods. In some embodiments, such methods can be in vivo methods. In some embodiments, such methods can be in situ methods. In some embodiments, such methods can be ex vivo methods. For example, an activatable protein with a CM can be used to detect the presence or absence of an enzyme that can cleave the CM. Such activatable proteins can be used in diagnosis, which may include detecting (e.g., qualitatively or quantitatively) enzyme activity in vivo (or in some embodiments, an environment with an increased reduction potential, such as an environment that can provide disulfide bond reduction) by measuring the accumulation of activated proteins (i.e., antibodies produced by cleavage of activatable proteins) in a given cell or tissue of a given host organism. Such accumulation of activated proteins not only indicates that the tissue expresses enzyme activity (or an increased reduction potential depending on the nature of the CM substrate), but also indicates that the tissue expresses the target to which the activated protein binds.

[0329] For example, CM can be selected as a protease substrate for a protease found at a tumor site, a site of viral or bacterial infection at a biologically restricted site (e.g., such as an abscess, an organ, etc.). The TB can be a TB that binds a target protein (e.g., an antigen). Using methods familiar to those skilled in the art, a detectable label (e.g., a fluorescent label or a radioactive label or a radioactive tracer) can be conjugated to the TB or other regions of the activatable protein. Suitable detectable labels can be discussed in the context of the above screening methods, and additional specific examples are provided below. Using a TB specific for a protein or peptide of a disease state and a protease with increased activity in the disease tissue of interest, the activatable protein will exhibit an increased binding rate to the disease tissue relative to tissues in which the CM-specific enzyme is not present at a detectable level or is present at a level lower than that in the disease tissue or is inactive (e.g., in a zymogen form or in a complex with an inhibitor). Because the kidney filtration system rapidly clears small proteins and polypeptides from the blood, and because the enzyme specific for CM is not present at a detectable level (or is present at a lower level in non-disease tissue or in an inactive conformation), the accumulation of activated proteins in disease tissues relative to non-disease tissues is increased.

[0330] In some embodiments, the activatable protein can be used for in vivo imaging, wherein the detection of a fluorescent signal in a subject (e.g., a mammal, including a human) indicates that the disease site contains the target and contains a protease specific to the CM of the activatable protein. In vivo imaging can be used to identify or otherwise refine a patient population suitable for treatment with the activatable protein of the present disclosure. For example, a patient who tests positive for both the target and the protease that cleaves the substrate in the CM of the activatable protein being tested (e.g., the activated protein accumulates at the disease site) is identified as a candidate suitable for treatment with such an activatable protein comprising such CM. Similarly, a patient who tests negative can be identified as a suitable candidate for another therapy (i.e., not suitable for treatment with the activatable protein being tested). In some embodiments, such patients who test negative for the first activatable protein can be tested with other activatable proteins containing different CMs until an activatable protein suitable for treatment is identified (e.g., an activatable protein containing CM cleaved by the patient at the disease site).

[0331] In some embodiments, in situ imaging can be used to identify a method for treating a patient. For example, in in situ imaging, an activatable protein can be used to screen patient samples to identify those patients who have appropriate proteases and targets at appropriate locations, such as tumor sites. In some embodiments, in situ imaging is used to identify or otherwise refine patient populations suitable for treatment with the activatable protein disclosed herein. For example, patients who test positive for both the target and the protease that cleaves the substrate in the CM of the activatable protein being tested (e.g., the activated antibody accumulates at the disease site) are identified as candidates suitable for treatment with such an activatable protein containing such CM. Similarly, patients who test negative for one or both of the proteases of the CM used in the activatable protein being tested using these methods for the target and the cleavage are identified as suitable candidates for another therapy (i.e., not suitable for treatment with the tested activatable protein). In some embodiments, such patients who test negative for the first activatable protein can be tested with other activatable proteins containing different CMs until an activatable protein suitable for treatment is identified (e.g., an activatable protein containing CM cleaved by the patient at the disease site).

[0332] Example

[0333] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims and provide proof-of-concept demonstrations of the advantageous structures of the activatable macromolecules described in this disclosure.

[0334] Example 1: Generation of activatable bispecific molecules

[0335] This example shows the production of an exemplary activatable bispecific protein, wherein the activating protein does not comprise a half-life extending portion (e.g., an Fc domain). The dual masked activatable bispecific molecule was prepared by a recombinant method. The protein was prepared by transforming a host cell with three polynucleotides: one polynucleotide having the sequence of SEQ ID NO: 21 (for ProC1446), 22 (for ProC1447), or 23 (for ProC1448); one polynucleotide having the sequence of SEQ ID NO: 1; and one polynucleotide having the sequence of SEQ ID NO: 18, and then culturing the resulting recombinant host cells. These proteins comprise 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 pair of knob and hole mutant Fc domains (EM). Fig. 6A The structures of these activatable proteins are depicted in .

[0336] Reference molecules ProC306 and ProC531 (unmasked bispecific molecules comprising a Fab that specifically binds HER2; a scFv that specifically binds CD3; and a pair of knob and hole Fc domains, which are configured differently from the exemplary activatable bispecific molecules described above) were also prepared by recombinant methods.

[0337] Example 2. Protease Treatment of Activatable Bispecific Molecules

[0338] To release the masked peptides, the dual-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 at 75°C for 10 minutes (reducing agent was added if necessary) and subjected to 4-12% NuPAGE at 175V in MOPS buffer. TM Separation was performed on Bis-Tris gels (Thermo Fisher Scientific, Waltham, MA, catalog number NP0321) for 1 hour and the samples were analyzed using InstantBlue TM The staining was developed after 1 hour and then destained in water for at least 4 hours.

[0339] The untreated protein was confirmed to have all three chains in the reducing gel ( Fig. 7A and Figure 7B ). After overnight protease treatment, activation was incomplete, but most of the protease products had the expected molecular weight.

[0340] Example 3: CD3 antigen binding ELISA

[0341] The ability of the doubly 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 (ACROBiosystems) 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 (1X PBS pH 7.4, 0.05% Tween-20, 1% BSA). The doubly masked activatable bispecific molecules were serially diluted fourfold with an unmasked reference protein (ProC531) without or after protease treatment and applied to the antigen-coated plate. The degree of protein binding to the peptide was measured by anti-human IgG (Fab-specific) immunodetection. The A450 absorbance was measured on a plate reader. Dose-response curves were generated by sigmoid fit nonlinear regression using Graph Pad Prism software and EC50 values ​​were obtained. The results are in Figure 8 These results show that each of the three dual-masked activatable bispecific molecules (ProC1446, ProC1447 and ProC1448) showed reduced binding to CD3 compared to the reference bispecific molecule without a mask (ProC531) and relative to the corresponding protease-treated activating molecule. After treatment with uPA, the activity of the dual-masked activatable bispecific molecules was restored to the same or nearly the same level as the corresponding reference bispecific molecule (ProC531).

[0342] Example 4: HER2-dependent cytotoxicity of dual-masked activatable bispecific molecules

[0343] The in vitro potency of the dual masked activatable bispecific molecules prepared in Example 1 was determined in a cytotoxicity assay. SKOV3-luc2 target cells and human PBMC effector cells (Stemcell technologies) were plated together for co-culture at a target cell to effector cell ratio of 1:10 in RPMI medium (Gibco catalog number 22400071) supplemented with 5% human serum (MP Bio catalog number 2930949). To this co-culture was added intact ProC1446, ProC1447 and ProC1448 and their protease activated versions (uPA treated ProC1446, uPA treated ProC1447, uPA treated ProC1448) and unmasked reference ProC306. The plates were incubated at 37°C and 5% CO. 2 After incubation, use ONE-Glo TMCytotoxicity was assessed using a luciferase assay system (Promega catalog number E6130) and luminescence was measured on a plate reader (TECAN). Percent cytotoxicity was calculated as follows: (1-(experimental RLU / average RLU of untreated)) × 100. Percent cytotoxicity data were plotted using GraphPad PRISM and EC50 values ​​were calculated. Results are presented in Figures 9A-9C These results show that each of the three doubly masked activatable bispecific molecules (ProC1446, ProC1447 and ProC1448) showed reduced cytotoxicity compared to the reference bispecific molecule without a mask (ProC306) and relative to the corresponding protease-treated activation molecule. After treatment with uPA, the cytotoxic activity of the activation molecules was stronger than that of the reference bispecific molecule without a mask (ProC306).

[0344] Example 5: Binding of double-masked bispecific antibodies to Her2+NCI-N87, SKOV3 and CD3ε+Jurkat cells

[0345] To determine whether the described Her2 and CD3ε masking peptides were able to inhibit binding in the context of a doubly masked bispecific antibody, a flow cytometry-based binding assay was performed.

[0346] NCI-N87 (ATCC), SKOV3 (ATCC) and Jurkat (clone E6-1, ATCC, TIB-152) cells were cultured in RPMI-1640 + glutamax (Life Technologies, catalog 72400-047), 10% heat-inactivated fetal bovine serum (HI-FBS, Life Technologies, catalog 10438-026) and puromycin (for NCI-N87 cells, Gibco, catalog A11138-03, 2ug / ml). The following bispecific antibodies were tested: recombinantly produced activating SHL1-ProC1963, SHL2-ProC1965, 1 / 2TCB ProC306 and their respective dual masked activatable bispecific antibodies ProC1446 (SHL1), ProC3007 (SHL2), ProC3008 (SHL2) and ProC1441 (1 / 2TCB). The cleavable moiety (CM) present between the EM (half-life extending moiety) and the C-terminus utilized two types, namely CM1 in ProC3007 and CM2 in ProC3008.

[0347] With Versene TMNCI-N87 and SKOV3 cells were isolated by ELISA (Life Technologies, catalog 15040-066), washed, plated at 150,000 cells / well in a 96-well plate, and resuspended in 50 μL of primary antibody (bispecific antibody). Jurkat cells were counted and plated as described for NCI-N87 and SKOV3. Figures 13A-13B The primary antibody was titrated starting at the concentration indicated in , and then serially diluted 3-fold in FACS staining buffer + 2% FBS (BD Pharmingen, catalog 554656) and added to the cells. The cells were incubated with shaking at 4°C for about 1 hour, harvested and washed with 2×200 μL FACS staining buffer. The cells were resuspended in 50 μL Alexa 647 conjugated anti-human IgG, F(ab') 2 Fragment-specific antibody (1.5 μg / ml, Jackson ImmunoResearch, catalog 109-605-097) was added and incubated with shaking at 4°C for about 1 hour. The cells were harvested, washed, and resuspended in a final volume of 200 μL of FACS staining buffer containing 2.5 μg / ml 7-AAD (BD Biosciences, catalog 559925). Cells stained with secondary antibody alone served as negative controls. TM The data were acquired using the NxT flow cytometer and Median fluorescence intensity (MFI) of live cells was calculated using ELISA software v10.8.1. Raw MFI data were plotted in GraphPad Prism using curve fitting analysis.

[0348] Figures 13A-13B Binding of masked activatable short half-life antibodies ProC1446 (SHL1), ProC3007 (SHL2), ProC3008 (SHL2) and masked antibody ProC1441 (1 / 2TCB, not an activatable short half-life antibody) and unmasked (ProC1963 (SHL1, no masker or Fc), ProC1965 and ProC306) anti-CD3, anti-HER2 bispecific antibodies and secondary antibodies ("secondary antibody only", negative control) to NCI-N87 and SKOV3 cells (i.e., HER2 binding), respectively. Fig. 13C The binding of the same molecule to Jurkat cells (i.e., CD3 binding) is depicted. The results show that all masked molecules exhibit reduced binding to HER2 and CD3 relative to the corresponding unmasked form, as reflected in the right shift of the binding curves of the masked molecules (low or no binding even at the highest concentration). EC50 values ​​are determined by repeated experiments. Average EC50 values ​​are provided in Table 1 below.

[0349] Table 1. Average EC50

[0350]

[0351] Results show that unmasked anti-HER2, anti-CD3 TCBs in activatable short half-life formats (SHL1 and SHL2) exhibited comparable CD3 and HER2 binding to the corresponding unmasked 1 / 2TCB formats. A trend toward modestly increased HER2 binding was observed for the activatable short half-life formats relative to the 1 / 2TCB formats. Masked activatable short half-life molecules exhibited highly attenuated HER2 and CD3 binding that was comparable to that observed for the 1 / 2TCB molecules. The masked 1 / 2TCB molecule (ProC1441) is a Fig.12 The molecule depicted on the left, but lacks the third cleavable moiety (CM3) (1205) between the EM and the Fab, meaning that this molecule cannot be cleaved to release the EM.

[0352] Example 6: Biological activity of dual-masked activatable bispecific antibodies

[0353] Cytotoxicity assays were used to determine the biological activity of intact activatable bispecific antibodies and recombinantly produced activating bispecific antibodies. Human PBMCs were purchased from HemaCare (Van Nuys, CA) and co-cultured at a 10:1 ratio with Her2-expressing cancer cell lines NCI-N87 (ATCC) or SKOV3 (ATCC) in RPMI-1640 + glutamax (Sigma, catalog H3667) supplemented with 5% heat-inactivated human serum. Testing Figures 14A-14B 15A-15B, followed by 3-fold serial dilutions of intact ProC1446(SHL1), ProC3007(SHL2), ProC3008(SHL2), ProC1441(1 / 2TCB) and recombinantly produced activating bispecific antibodies ProC1963(SHL1), ProC1965(SHL2), and ProC306(1 / 2TCB) in co-culture. TM Cytotoxicity was assessed using the luciferase assay system (Promega, Madison, WI, catalog E6130). Luminescence was measured using a Tecan M200 Pro (Tecan Trading AG, Switzerland). Percent cytotoxicity was calculated and plotted using curve fitting analysis in GraphPad PRISM.

[0354] Fig.14A and14B The recombinantly produced activating bispecifics ProC1963 (SHL1) and ProC1965 (SHL2) showed increased potency compared to ProC306 (EC50 decreased by >1200-fold and 50-fold, respectively), as indicated by a leftward shift in the dose-response curves.

[0355] Fig.15A and 15B It was shown that in these assays the intact bispecifics ProC1446, ProC3007, ProC3008 and ProC1446 were strongly masked as indicated by the rightward shift in the dose response curve relative to their recombinantly produced activated forms ProC1963 and ProC1965.

[0356] Figures 16A-16D It was shown that in these assays the intact bispecifics ProC1446, ProC3007, ProC3008 and ProC1441 were strongly masked as indicated by the rightward shift in the dose response curve relative to their recombinantly produced activated forms ProC1963, ProC1965 and ProC306.

[0357] EC50 values ​​and masking efficiency (ME) were determined by repeated experiments. The results are provided in Tables 2A and 2B below.

[0358] Table 2A. Average EC50 and masking efficiency

[0359]

[0360] N / A = Not Applicable

[0361] ND = Not Determined

[0362] The EC50 of ProC1963 was about 1200-3000 times lower than that of ProC306. The EC50 of ProC1965 was 50 times lower than that of ProC306. The results show that the unmasked activatable short-lived TCB format exhibits higher potency compared to the control (ProC306) which is not activatable in terms of half-life, i.e., cleavable to release the EM.

[0363] Table 2B. Average EC50 and masking efficiency

[0364]

[0365]

[0366] N / A = Not Applicable

[0367] ND = Not Determined

[0368] The results showed that masking attenuated the activity of all 3 formats: SHL1, SHL2, and 1 / 2TCB.

[0369] Example 7: Dual-masked bispecific activatable antibody ProC3007 and its corresponding activated version ProC1965 induce regression of established NCI-N87 tumors in mice

[0370] In this example, the intact activatable bispecific antibodies ProC3007 (SHL2TCB), ProC3008 (SHL2 TCB), ProC1441 and the recombinantly produced activating bispecific antibody ProC1965 targeting Her2 and CD3ε were analyzed for their ability to induce regression or growth reduction of established NCI-N87 xenograft tumors in human PBMC-engrafted NOD scidγ (NSG) mice.

[0371] Human gastric cancer cell line NCI-N87 was obtained from ATCC and cultured in RPMI+Glutamax+10% FBS according to established procedures. Purified frozen human PBMCs were obtained from Hemacare, Van Nuys, CA (donor ID# D163477; lot number 22077550). NSG TM (NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ) mice were obtained from The Jackson Laboratories, Bar Harbor, ME.

[0372] On day 0, each mouse was inoculated subcutaneously on the right flank with 1 × 10 6 100 μL RPMI+Glutamax serum-free medium (containing ) in NCI-N87 cells. Previously frozen PBMCs from a single donor were thawed and administered (ip) on day 7 at a 1:1 CD3+ T cell to tumor cell ratio. When tumor volume reached approximately 125 mm 3 At 14:00, mice were randomized, assigned to treatment groups and dosed iv according to Table 3. Recombinant bispecific ProC1965 (SHL2 format, no masker and no Fc domain) was dosed 3 times per week to compensate for the expected increased clearance due to the lack of the half-life extension (Fc) domain. The dose level of ProC1965 was adjusted to accommodate the difference in molecular weight. Tumor volume was measured twice per week. One mouse in the 0.5 mpk group was euthanized prematurely. Subsequently, n=7 for this group at the 21st and 25th day time points.

[0373] Table 3. Groups and doses for NCI-N87 xenograft study C23-005.

[0374] Group count deal with Dosage (mg / kg) Dosing schedule 1 8 PBS + 6% sucrose N / A QW 2 8 ProC1965 0.15 3 times a week 3 8 ProC1965 0.5 3 times a week 4 8 ProC3007 0.3 QW 5 8 ProC3007 1.0 QW 6 8 ProC3008 0.3 QW 7 8 ProC3008 1 QW 8 8 ProC1441 1 QW

[0375] Fig.17 is a graph of tumor volume versus days after initial treatment dose. The results show that both the intact activatable bispecific antibody ProC3007 and the recombinantly produced activating bispecific antibody ProC1965 induced regression of NCI-N87 xenograft tumors at 1 and 0.5 mpk, respectively. The results show that ProC1965 and ProC3007 were more effective than ProC1441 in this study. ProC3008 had similar efficacy to ProC1441 at the same dose level.

[0376] The second in vivo study was conducted as described above, but using PBMCs from a different donor (Hemacare, donor ID# D327579; batch number 21070049). In this study, a panel of bispecific activatable antibodies included a recombinantly produced activatable short half-life bispecific antibody (ProC1446) and a corresponding recombinantly produced activated version of the activatable 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 growth reduction of established NCI-N87 xenograft tumors in human PBMC-transplanted NSG mice. Both ProC1963 and ProC1446 exhibited antitumor activity in this study, so ProC1963 retained the ability to induce tumor regression.

[0377] Table 4: Dose Levels of C22-014 in the NCI-N87 Xenograft Study

[0378] Group count deal with Dosage (mg / kg) Dosing schedule 1 8 PBS + 6% sucrose N / A QW 2 8 ProC1963 0.2 3 times a week 3 8 ProC1963 0.6 3 times a week 4 8 ProC1446 0.3 QW 5 8 ProC1446 1.0 QW

[0379] N / A = Not Applicable The sequences of the molecules in the Examples and other sequences disclosed herein are listed in Table 5 below.

[0380] Table 5

[0381]

[0382]

[0383]

[0384]

[0385]

[0386]

[0387]

[0388]

[0389]

[0390]

[0391]

[0392]

[0393]

[0394]

[0395]

[0396]

[0397]

[0398]

[0399]

[0400]

[0401]

[0402]

[0403]

[0404]

[0405]

[0406]

[0407]

[0408]

[0409]

[0410]

[0411]

[0412]

[0413]

[0414]

[0415]

[0416]

[0417] *Note: X is a lysine residue or is present at the C-terminus.

[0418] **Note: N is either adenine, forming a codon for lysine, or each N is absent, so a codon for lysine does not exist.

[0419] It will be appreciated that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the following claims.

[0420] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict, the present specification, including definitions, will prevail. The materials, methods, and examples are illustrative only and are not intended to be limiting.

Claims

1. An activatable protein, comprising: a first antigen binding domain (AB1) that specifically binds to a first target, wherein the 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 the AB2 comprises a second heavy chain variable domain (HVD2) and a second light chain variable domain (LVD2), and the AB2 is directly or indirectly coupled to the C-terminus of the HVD1 or to the C-terminus of the LVD1; a first masking moiety (MM1) coupled directly or indirectly to the AB1 via a first cleavable moiety (CM1), wherein the MM1 inhibits binding of the AB1 to the first target; a half-life extending moiety (EM) coupled directly or indirectly to a second masking moiety (MM2); wherein the EM is coupled directly or indirectly to the AB1 or to the AB2 via a second cleavable moiety (CM2), and wherein the MM2 inhibits binding of the AB2 to the second target.

2. The activatable protein of claim 1, wherein the EM is a dimer formed by a first fragment crystallizable (Fc) domain and a second Fc domain.

3. The activatable protein of claim 1 or claim 2, wherein the EM is at the C-terminus relative to the AB1 or the AB2 to which the EM is directly or indirectly coupled via the CM2, and wherein MM1 is at the N-terminus relative to the AB1.

4. An activatable protein, comprising: a first antigen binding domain (AB1) that specifically binds to a first target, wherein the 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 the AB2 comprises a second heavy chain variable domain (HVD2) and a second light chain variable domain (LVD2), and the AB2 is directly or indirectly coupled to the C-terminus of the HVD1 or the LVD1; a first masking moiety (MM1) coupled to the AB1 via a first cleavable moiety (CM1) and optionally one or more linkers, wherein the MM1 inhibits binding of the AB1 to the first target; and a half-life extending moiety (EM), said half-life extending moiety (EM) being coupled directly or indirectly to a second masking moiety (MM2), wherein said EM and said MM2 are coupled to said AB1 or to said AB2 via a second cleavable moiety (CM2) and optionally one or more linkers, and wherein the MM2 inhibits binding of the AB2 to the second target.

5. The activatable protein of claim 4, wherein the CM2 is at the N-terminus of the MM2 and the MM2 is at the N-terminus of the EM, and the CM2 is at the C-terminus of the AB1 or the AB2 to which the EM and the MM2 are coupled via the CM2 and optionally one or more linkers.

6. An activatable protein, comprising: a first antigen binding domain (AB1) that specifically binds to a first target, wherein the 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 the AB2 comprises a second heavy chain variable domain (HVD2) and a second light chain variable domain (LVD2), and the AB2 is directly or indirectly coupled to the C-terminus of the HVD1 or the LVD1; a first masking moiety (MM1) coupled to the AB1 via a first cleavable moiety (CM1) and optionally one or more linkers, wherein the MM1 inhibits binding of the AB1 to the first target; as well as a half-life extending moiety (EM), the half-life extending moiety comprising a dimer of a first half-life extending moiety (EM1) and a second half-life extending moiety (EM2); wherein the EM1 is coupled to the AB1 via a second cleavable moiety (CM2) and optionally one or more linkers, and wherein said EM2 is coupled directly or indirectly to a second masking moiety (MM2), wherein the MM2 inhibits binding of the AB2 to the second target. 7 . The activatable protein of claim 6 , wherein the CM2 is at the N-terminus of the EM1 and the CM2 is at the C-terminus of the AB1.

8. The activatable protein of any one of claims 1 to 7, wherein the activatable protein comprises at least a first polypeptide and a second polypeptide. 9 . The activatable protein of claim 8 , wherein the first polypeptide comprises the MM1, the CM1 and the VLD1 in order from the N-terminus to the C-terminus.

10. The activatable protein of any one of claims 8-9, wherein the second polypeptide comprises the VHD1, the VHD2, the VLD2, the CM2, the MM2, and the first Fc domain, and wherein the activatable protein further comprises a third polypeptide comprising a second Fc domain. 11 . The activatable protein of claim 10 , wherein the second polypeptide comprises the VHD1, the VHD2, the VLD2, the CM2, the MM2 and the first Fc domain in order from the N-terminus to the C-terminus. 12 . The activatable protein of claim 9 , wherein the second polypeptide comprises the VHD1, the CM2, the MM2, and the first Fc domain in order from the N-terminus to the C-terminus. 13 . The activatable protein of claim 9 , wherein the second polypeptide comprises the VHD1, the CM2, and the first Fc domain in sequence from the N-terminus to the C-terminus.

14. The activatable protein of any one of claims 9 or 12-13, wherein the first polypeptide comprises the MM1, the CM1, the VLD1, the VHD2, and the VLD2. 15 . The activatable protein of claim 13 , wherein the first polypeptide comprises the MM1, the CM1, the VLD1, the VHD2, and the VLD2 in order from the N-terminus to the C-terminus. 16 . The activatable protein of claim 13 , wherein the first polypeptide comprises the MM1, the CM1, the VLD1, the VLD2, and the VHD2 in order from the N-terminus to the C-terminus.

17. The activatable protein of any one of claims 13-16, wherein the protein comprises a third polypeptide, and wherein the third polypeptide comprises a second Fc domain and the MM2.

18. The activatable protein of claim 17, wherein the MM2 is linked to the C-terminus of the second Fc domain via a linker.

19. The activatable protein of claim 17, wherein the MM2 is linked to the N-terminus of the second Fc domain via a linker.

20. The activatable protein according to any one of claims 1 to 19, further comprising a linker coupled to: (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, (viii) CM2 and EM, (ix) EM and MM2, (x) VLD1 and VHD2, and / or (xi) VLD1 and VLD2.

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

22. The activatable protein of any one of claims 2 and 10-19, wherein the first Fc domain is an Fc domain hole mutant and the second Fc domain is an Fc domain knob mutant.

23. The activatable protein of claim 22, wherein the Fc domain hole mutant comprises the sequence of SEQ ID NO: 2 and the Fc domain knob mutant comprises the sequence of SEQ ID NO:

1. The activatable protein of claim 1 , wherein the CM2 is directly or indirectly coupled to the C-terminus of the AB1 or the C-terminus of the AB2.

25. The activatable protein of claim 24, wherein 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.

26. The activatable protein of claim 1 or claim 24, wherein the EM comprises a dimer of a first half-life extending moiety (EM1) and a second half-life extending moiety (EM2). The activatable protein of claim 26 , wherein the CM2 is directly or indirectly coupled to the N-terminus of the EM1.

28. The activatable protein of claim 26, wherein 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.

29. The activatable protein of any one of claims 26, wherein the MM2 is coupled directly or indirectly to the N-terminus of the EM2.

30. The activatable protein of any one of claims 1-29, wherein the first target or epitope is a tumor-associated antigen.

31. The activatable protein of claim 30, wherein the tumor-associated antigen is human epidermal growth factor receptor 2 (HER2).

32. The activatable protein of claim 31, wherein the AB1 is a Fab of trastuzumab.

33. The activatable protein of claim 31, wherein the HVD1 comprises the sequence of SEQ ID NO: 27 and the LVD1 comprises the sequence of SEQ ID NO:

17.

34. The activatable protein of any one of claims 1 to 33, wherein AB2 is: scFvs that engage immune effector cells; scFvs that bind to leukocytes; scFv that engages T cells; scFv that engages NK cells; A scFv that binds macrophages; or scFvs that engage mononuclear cells.

35. The activatable protein of any one of claims 1-34, wherein AB2 is or is derived from anti-CD3ε scFv or anti-CTLA-4 scFv. The activatable protein of claim 35 , wherein the AB2 is or is derived from an anti-CD3ε scFv.

37. The activatable protein of claim 36, wherein the HVD2 comprises the sequence of SEQ ID NO: 30 and the LVD2 comprises the sequence of SEQ ID NO:

31.

38. The activatable protein of any one of claims 1-37, wherein AB1 is or is derived from an anti-HER2 antibody.

39. The activatable protein of any one of claims 1-38, wherein CM1 and CM2 each independently comprise a substrate for a protease that is upregulated in a tumor microenvironment.

40. The activatable protein of any one of claims 1-39, wherein AB1 is an antigen binding fragment (Fab).

41. The activatable protein of any one of claims 1-40, wherein the second target is a co-stimulatory molecule.

42. The activatable protein of claim 41, wherein the co-stimulatory molecule is CD3.

43. The activatable protein of any one of claims 1-42, wherein each of the CM1 and the CM2 comprises a substrate for the same protease.

44. The activatable protein of any one of claims 1-42, wherein the CM1 and the CM2 comprise substrates for different proteases.

45. An activatable molecule, 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, wherein the TB2 is directly or indirectly coupled to the TB1; a first masking moiety (MM1) coupled to the TB1 directly or indirectly via a first cleavable moiety (CM1), wherein the MM1 inhibits the binding of the TB1 to the first target; a half-life extending moiety (EM) and a second masking moiety (MM2), wherein the half-life extending moiety and the second masking moiety are coupled directly or indirectly to the TB1 or to the TB2 via a second cleavable moiety (CM2), wherein the MM2 inhibits the binding of the TB2 to the second target, wherein the components of the activatable molecule are configured such that cleavage of the CM1 and the CM2 releases the MM1, the MM2 and the EM from the TB1 directly or indirectly coupled to the TB2, and Wherein optionally the TB1 is an antigen binding molecule (AB1) comprising HVD1 and LVD1, and optionally the TB2 is an antigen binding molecule (AB2) comprising HVD2 and LVD2.

46. ​​The activatable protein of any one of claims 1-45, wherein each of the CM1 and the CM2 independently comprises a substrate for a protease selected from the group consisting of ADAMS, ADAMTS, ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAMDEC1, ADAMTS1, ADAMTS4, ADAMTS5, aspartic proteases, BACE, renin, aspartic cathepsins, cathepsin D, cathepsin E, apoptotic proteases, apoptosis proteinase 1, apoptosis proteinase 2, apoptosis proteinase 3, apoptosis proteinase 4, apoptosis proteinase 5, apoptosis proteinase 6, apoptosis proteinase 7, apoptosis proteinase 8, apoptosis proteinase 9, apoptosis proteinase 10, apoptosis proteinase 14, cysteine ​​cathepsins, cathepsin B, cathepsin C, cathepsin K, cathepsin L, cathepsin S, cathepsin V / L2, cathepsin X / Z / P, cysteine ​​proteases, cruzi proteases, legumin, Otubain-2, KLK, KLK4, KLK5, KLK6, KLK7, KLK 8. KLK10, KLK11, KLK13, KLK14, metalloproteinases, transmembrane peptidases, enkephalinases, 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 proteases, activated protein C, cathepsin A, cathepsin G, chymotrypsin , coagulation factor protease, FVIIa, FIXa, FXa, FXIa, FXIIa, elastase, granzyme B, guanidinobenzoic acidase, HtrA1, human neutrophil elastase, lactoferrin, malathion, NS3 / 4A, PACE4, plasmin, PSA, tPA, thrombin, tryptase, uPA, type II transmembrane, serine protease, TTSP, DESC1, DPP-4, FAP, amber, interstitial proteinase-2, MT-SP1 / interstitial proteinase, TMPRSS2, TMPRSS3 and TMPRSS4.

47. The activatable protein of any one of claims 1-46, wherein the MM1 is a peptide of 2 to 40 amino acids in length.

48. The activatable protein of any one of claims 1-47, wherein the MM2 is a peptide of 2 to 40 amino acids in length.

49. The activatable protein of claim 47 or 48, wherein 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 the MM2 is coupled directly or indirectly to the C-terminus of the light chain variable region of the AB2 via the CM2, and 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.

50. The activatable protein of claim 47 or 48, wherein The heavy chain variable region of the AB2 is directly or indirectly coupled to the C-terminus of the light chain fragment of the AB1, The N-terminus of the MM2 is coupled directly or indirectly to the C-terminus of the heavy chain fragment of the AB1 via the CM2, and 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.

51. The activatable protein of claim 47 or 48, wherein The heavy chain variable region of the AB2 is directly or indirectly coupled to the C-terminus of the light chain fragment of the 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 directly or indirectly coupled to the C-terminus of the heavy chain fragment of the AB1 via the CM2, and The N-terminus of the MM2 is directly or indirectly coupled to the C-terminus of the second Fc domain.

52. The activatable protein of claim 47 or 48, wherein The heavy chain variable region of the AB2 is directly or indirectly coupled to the C-terminus of the light chain fragment of the 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 directly or indirectly coupled to the C-terminus of the heavy chain fragment of the AB1 via the CM2, and The C-terminus of the MM2 is directly or indirectly coupled to the N-terminus of the second Fc domain.

53. The activatable protein of any one of claims 49 to 52, further comprising a linker between the MM2 and the first Fc domain or the second Fc domain directly or indirectly coupled to the MM2.

54. The activatable protein of any one of claims 1 to 53, wherein the MM1 comprises the sequence of SEQ ID NO: 40 and the MM2 comprises the sequence of any one of SEQ ID NOs: 34-37 or 66-70.

55. The activatable protein of any one of claims 1 to 54, wherein the dissociation constant of the binding of MM1 to the AB1 exceeds the dissociation constant of the binding of AB1 to the first target or epitope, and the dissociation constant of the binding of MM2 to the AB2 exceeds the dissociation constant of the binding of AB2 to the second target or epitope.

56. The activatable protein of any one of claims 1-55, wherein the components of the activatable protein are configured such that cleavage of the CM1 and the CM2 separates the MM1, the MM2, and the EM from the activatable protein, thereby producing an activatable protein having a shorter half-life than a corresponding molecule comprising the TB1, the TB2, and the EM.

57. The activatable protein of any one of claims 1 to 55, wherein the components of the activatable protein are configured such that cleavage of the CM1 and the CM2 separates the MM1, the MM2, and the EM from the activatable protein, thereby producing an activatable protein having higher target binding activity than a corresponding molecule comprising the TB1, the TB2, and the EM.

58. The activatable protein of any one of claims 1-57, wherein the second polypeptide further comprises a linker (L2) between the MM2 and the AB2.

59. The activatable protein of claim 58, wherein L2 is a peptide of 5 to 30, 6 to 29, 7 to 28, 8 to 27, 9 to 26, 10 to 25, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 amino acids in length.

60. The activatable protein of any one of claims 1-59, wherein the second polypeptide further comprises a linker (L3) between the AB2 and the AB1.

61. The activatable protein of claim 60, wherein L3 is a peptide of 5 to 30, 6 to 29, 7 to 28, 8 to 27, 9 to 26, 10 to 25, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 amino acids in length.

62. The activatable molecule of claim 45, further comprising the features of any one or more of claims 1-44.

63. An activatable protein, comprising: a first antigen binding domain (AB1) that specifically binds to a first target, wherein the 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 the AB2 comprises a second heavy chain variable domain (HVD2) and a second light chain variable domain (LVD2), wherein the AB2 is directly or indirectly coupled to the N-terminus of the HVD1 or to the N-terminus of the LVD1; a first masking moiety (MM1) coupled to the AB1 via a first cleavable moiety (CM1) and optionally one or more linkers, wherein the MM1 inhibits binding of the AB1 to the first target; a second masking moiety (MM2) coupled to said AB1 via a second cleavable moiety (CM2) and optionally one or more linkers, wherein said MM2 inhibits binding of said AB2 to said second target; A half-life extending moiety (EM) which is coupled directly or indirectly to the C-terminus of the HVD1 or to the C-terminus of the LVD1 via a third cleavable moiety (CM3) and optionally one or more linkers.

64. The activatable molecule of claim 63, further comprising the features of any one or more of claims 1-61.

65. A composition comprising the activatable protein of any one of claims 1 to 64 and a carrier.

66. The composition of claim 65, wherein the composition is a pharmaceutical composition, wherein the carrier is a pharmaceutically acceptable carrier.

67. A container, vial, syringe, injection pen or kit comprising at least one dose of the composition of claim 66.

68. A nucleic acid comprising a sequence encoding the second polypeptide of any one of claims 8, 10-13, 58 or 60.

69. A vector comprising the nucleic acid of claim 68.

70. A cell comprising the nucleic acid of claim 68 or the vector of claim 69.

71. A conjugated activatable protein comprising the activatable protein of any one of claims 1 to 64 conjugated to an agent.

72. The conjugated, activatable protein of claim 71, wherein 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.

73. The conjugated activatable protein of claim 71 or claim 72, wherein the agent is conjugated to the antibody via a linker.

74. The conjugated, activatable protein of claim 72, wherein the linker is a cleavable linker.

75. The conjugated, activatable protein of claim 72, wherein the linker is a non-cleavable linker.

76. A method of treating a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an activatable protein as described in any one of claims 1 to 64, a composition as described in claim 65 or 66, or a conjugated activatable protein as described in any one of claims 71-75.

77. The method of claim 76, wherein the subject has been identified or diagnosed as having cancer.

78. A method for producing an activatable protein, the method comprising: Culturing the cell of claim 70 in a culture medium under conditions sufficient to produce the activatable protein; as well as The activatable protein is recovered from the cells or the culture medium.

79. The method of claim 78, further comprising isolating the activatable protein recovered from the cells or the culture medium.

80. The method of claim 79, wherein isolating the activatable protein is performed using a protein purification tag and / or size exclusion chromatography.

81. The method of any one of claims 78-80, further comprising formulating the activatable protein into a pharmaceutical composition.

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