Activatable anti-CTLA4 antibodies for treatment of cancer

Abstract

The present application provides compositions and methods for treating cancer using anti-cytotoxic T lymphocyte protein 4 (CTLA4) antibodies (e.g., activatable anti-CTLA4 antibodies). In some embodiments, the combination therapy comprises an anti-CTLA4 antibody and a PD-1 inhibitor.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 405,293, filed on September 9, 2022, and U.S. Provisional Patent Application No. 63 / 495,965, filed on April 13, 2023, the disclosures of which are incorporated herein by reference in their entirety for all purposes.

[0003] Sequence Listing Submission Using ASCII Text Files

[0004] The entire text of the electronic sequence listing (6954020024xxSEQLIST.xml; size: 364,433 bytes; and creation date: September 5, 2023) is incorporated herein by reference. Technical Field

[0005] The present application belongs to the field of cancer treatment and relates to antibodies that bind to human cytotoxic T lymphocyte protein 4 (CTLA4). Background Art

[0006] Cytotoxic T lymphocyte protein 4 (CTLA4) is a member of the immunoglobulin (Ig) protein superfamily that downregulates T cell activation and maintains homeostasis of immunogenicity. It has been shown that in a syngeneic mouse prostate cancer model, in vivo antibody-mediated CTLA4 blockade enhances anti-cancer immune responses (Kwon et al. (1997) Proc Natl Acad Sci USA, 94 (15): 8099-103). In addition, it has been shown that blocking CTLA4 function enhances anti-tumor T cell responses in tumor-bearing mice at various tumor growth stages (Yang et al. (1997) Cancer Res 57 (18): 4036-41; Hurwitz et al. (1998) Proc Natl Acad Sci USA 95 (17): 10067-7). However, the development of antibody-based therapeutics suitable for human use remains difficult because the translation from preclinical animal models to human safety is often poor. Therefore, there is a need for anti-CTLA4 antibodies that are cross-reactive between different species, such as humans and experimental animals (e.g., mice, monkeys, rats, etc.), to enable animal model studies and provide suitable human therapeutic candidates in parallel. In addition, there is a need to develop safer anti-CTLA4 antibodies that are active only in certain situations, such as in protease-rich tumor microenvironments. Summary of the Invention

[0007] The present application provides methods for treating cancer using anti-CTLA4 antibodies and activatable anti-CTLA4 antibodies. The present application also provides methods for treating cancer using anti-CTLA4 antibodies and a second therapeutic agent. The present application also provides methods for treating cancer using activatable anti-CTLA4 antibodies and a second therapeutic agent. The present application further provides methods for treating cancer using anti-CTLA4 antibodies and at least two (e.g., two or three) additional therapeutic agents.

[0008] In one aspect, provided herein are methods for treating cancer in a subject, comprising administering to the subject: (a) an effective amount of an activatable antibody, wherein the activatable antibody comprises: a polypeptide comprising, from N-terminus to C-terminus, a shielding moiety (MM), a cleavable moiety (CM), and a target binding moiety (TBM), wherein the MM comprises an amino acid sequence selected from the group consisting of: X m CPDHPYPCXX (SEQ ID NO: 181), X m CDAFYPYCXX (SEQ ID NO: 182), X m CDSHYPYCXX (SEQ ID NO: 183) and X m CVPYYYACXX (SEQ ID NO: 184), and wherein m is 2-10, and wherein each X is independently selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y amino acids; wherein when the CM is not cleaved, the MM inhibits binding of the activatable antibody to human CTLA4; wherein the CM comprises at least a first cleavage site; wherein: a) the TBM comprises an antibody light chain variable region (VL), and the activatable antibody further comprises a second polypeptide comprising an antibody heavy chain Variable region (VH); b) the TBM comprises an antibody heavy chain variable region (VH), and the activatable antibody further comprises a second polypeptide, the second polypeptide comprising an antibody light chain variable region (VL); c) the TBM comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH) from the N-terminus to the C-terminus; or d) the TBM comprises an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) from the N-terminus to the C-terminus; wherein when the CM is cleaved, the activatable antibody binds to human CTLA4 through the VH and VL. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal or a non-human mammal.

[0009] In some embodiments, the activatable antibody comprises a polypeptide comprising, from the N-terminus to the C-terminus, a blocking moiety (MM), a cleavable moiety (CM), and an anti-CTLA4 antibody described herein, wherein the MM comprises the amino acid sequence EVGSYPNPSSDCVPYYYACAY (SEQ ID NO: 192) and the cleavable moiety comprises the amino acid sequence SGRSAGGGGTPLGLAGSGGS (SEQ ID NO: 221). The MM and CM comprise, from the N-terminus to the C-terminus, the amino acid sequence EVGSYPNPSSDCVPYYYACAYSGRSAGGGGTPLGLAGSGGS (SEQ ID NO: 200). In specific embodiments, the MM and CM are covalently linked to the N-terminus of the anti-CTLA4 antibody light chain. In some embodiments, the MM and CM comprise, from the N-terminus to the C-terminus, an amino acid sequence having at least 90% or at least 95% sequence identity to SEQ ID NO: 200.

[0010] In some embodiments, the activatable anti-CTLA4 antibody can be administered as a monotherapy to a patient in need thereof. In other embodiments, the activatable anti-CTLA4 antibody can be administered in combination with one or more additional agents described herein. In some embodiments, the cancer is ovarian cancer, pancreatic cancer, bile duct cancer, lung cancer, breast cancer, hepatocellular carcinoma, glioblastoma, renal cell carcinoma, head and neck squamous cell carcinoma, colorectal cancer, gastrointestinal stromal tumor (GIST) or endometrial cancer. In some embodiments, the lung cancer is non-small cell lung cancer (NSCLC). In some embodiments, the colorectal cancer is microsatellite stability (MSS), microsatellite high instability (MSI-H) or mispairing repair deficiency (dMMR+) colorectal cancer. In some embodiments, the melanoma is uveal (UV) melanoma. In some embodiments, the cancer is squamous cell carcinoma (SCC) (e.g., anus, anorectum, penis or skin). In some embodiments, the cancer is anal squamous cell carcinoma or penile squamous cell carcinoma.

[0011] In some embodiments, the MM that can activate an anti-CTLA4 antibody further comprises an additional amino acid sequence at its N-terminus. In some embodiments, the additional amino acid sequence comprises the amino acid sequence of SEQ ID NO: 148.

[0012] In some embodiments, the first cleavage site of the activatable anti-CTLA4 antibody is a protease cleavage site of a protease selected from the group consisting of urokinase-type plasminogen activator (uPA), matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, Tobacco Etch Virus (TEV) protease, plasmin, thrombin, factor X, PSA, PSMA, cathepsin D, D), cathepsin K, cathepsin S, ADAM10, ADAM12, ADAMTS, Caspase-1, Caspase-2, Caspase-3, Caspase-4, Caspase-5, Caspase-6, Caspase-7, Caspase-8, Caspase-9, Caspase-10, Caspase-11, Caspase-12, Caspase-13, Caspase-14, and TACE.

[0013] In some embodiments, the CM further comprises a first linker (L1) at the C-terminus of the first cleavage site. In some embodiments, L1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 156-163.

[0014] In some embodiments, the CM further comprises a second cleavage site. In some embodiments, the second cleavage site is at the C-terminus of L1. In some embodiments, the second cleavage site is a protease cleavage site of a protease selected from the group consisting of urokinase-type plasminogen activator (uPA), matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, tobacco etch virus (TEV) protease, plasmin, thrombin, factor X, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAM10, ADAM12, ADAMTS, casparase-1, casparase-2, casparase-3, casparase-4, casparase-5, casparase-6, casparase-7, casparase-8, casparase-9, casparase-10, casparase-11, casparase-12, casparase-13, casparase-14, and TACE. In some embodiments, the first and second cleavage sites are different.

[0015] In some embodiments, the CM further comprises a second linker (L2) at the C-terminus of the second cleavage site. In some embodiments, L2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 156-163. In some embodiments, the CM further comprises a third linker (L3) at the N-terminus of the first cleavage site.

[0016] In some embodiments, the CM comprises at least a first protease cleavage site and is cleaved by one or more proteases selected from the group consisting of urokinase-type plasminogen activator (uPA), matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, tobacco etch virus (TEV) protease, plasmin, thrombin, Factor X, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAM10, ADAM12, ADAMTS, caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, caspase-14, and TACE.

[0017] In some embodiments, the activatable anti-CTLA4 antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 165-179.

[0018] In some embodiments, the activatable anti-CTLA4 antibody has a MM amino acid sequence selected from the group consisting of SEQ ID NOS: 189-196. In other embodiments, the activatable anti-CTLA4 antibody comprises a MM amino acid sequence selected from the group consisting of SEQ ID NOS: 213-216. In other embodiments, the activatable anti-CTLA4 antibody comprises a MM amino acid sequence selected from the group consisting of SEQ ID NOS: 141-147. In a specific embodiment, the activatable anti-CTLA4 antibody comprises the MM amino acid sequence of SEQ ID NO: 200.

[0019] In some embodiments, the activatable anti-CTLA4 antibody has an MM / CM combination amino acid sequence selected from the group consisting of SEQ ID NOS: 197-209. In a specific embodiment, the activatable anti-CTLA4 antibody has an MM / CM combination amino acid sequence of SEQ ID NO: 192.

[0020] In some embodiments, after MM cleavage, the activatable anti-CTLA4 antibody specifically binds to an epitope comprising amino acid residues Y105 and L106 of human CTLA4 but not residue I108, wherein the numbering of the amino acid residues is according to SEQ ID NO: 207.

[0021] In some embodiments, after MM cleavage, a) the cleaved anti-CTLA4 antibody dissociates with a dissociation constant (K) of about 350 nM or less. D ) binds to human CTLA4, cynomolgus monkey CTLA4, mouse CTLA4, rat CTLA4, and dog CTLA4; b) binding of the anti-CTLA4 antibody induces antibody-dependent cellular cytotoxicity (ADCC) against human cells expressing CTLA4 or human Treg cells, wherein the ADCC activity of the anti-CTLA4 antibody is greater than the ADCC activity of ipilimumab; and / or c) the anti-CTLA4 antibody has a higher IC50 for blocking binding of CD80 and / or CD86 to human CTLA4 than the IC50 of ipilimumab in an assay where CD80 and / or CD86 are coated on a plate or when human CTLA4 is present on the surface of cells.

[0022] In some embodiments, the activatable anti-CTLA4 antibody comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO:23, HVR-H2 comprising the amino acid sequence of SEQ ID NO:35, HVR-H3 comprising the amino acid sequence of SEQ ID NO:45, HVR-L1 comprising the amino acid sequence of SEQ ID NO:58, HVR-L2 comprising the amino acid sequence of SEQ ID NO:66, and HVR-L3 comprising the amino acid sequence of SEQ ID NO:75.

[0023] In some such embodiments, the activatable anti-CTLA4 antibody, upon cleavage, comprises: a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 87, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 100. In some of the foregoing embodiments, the anti-CTLA4 antibody comprises a heavy chain variable region comprising an amino acid sequence that has at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 87, and / or a light chain variable region comprising an amino acid sequence that has at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 100.

[0024] In some embodiments, the activatable anti-CTLA4 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 320 and a light chain comprising the amino acid sequence of SEQ ID NO: 322. The activatable antibody having a heavy chain of SEQ ID NO: 320 and a light chain of SEQ ID NO: 322 is referred to as TY21580. In some embodiments, the activatable antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 320 and a light chain comprising the amino acid sequence of SEQ ID NO: 322. In some embodiments, the activatable anti-CTLA4 antibody comprises a heavy chain having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 320. In some embodiments, the activatable anti-CTLA4 antibody comprises a light chain having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 321. In some embodiments, the activatable anti-CTLA4 antibody comprises a heavy chain having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 320. In some embodiments, the activatable anti-CTLA4 antibody comprises a light chain having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 322. In some embodiments, the activatable antibody is TY22404.

[0025] In another aspect, an activatable anti-CTLA4 antibody of the present disclosure is administered to a subject in combination with a PD-1 inhibitor (e.g., an anti-PD-1 antibody). In other such embodiments, the PD1 antibody is toripalimab. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal or a non-human mammal. In some embodiments, when administered in combination, the activatable anti-CTLA4 antibody and the PD-1 inhibitor (e.g., an anti-PD-1 antibody) exhibit a synergistic effect in combination. In some of the foregoing embodiments, the anti-CTLA4 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 87, or an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 87, and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 100, or an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 100. In some of the foregoing embodiments, the anti-CTLA4 antibody is a cleaved, activatable anti-CTLA4 antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 87, or an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 87, and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 100, or an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 100.

[0026] In some embodiments, provided herein is a method for treating cancer in a subject, comprising administering to the subject: (a) an effective amount of an activatable antibody, wherein the activatable antibody comprises: a polypeptide comprising, from the N-terminus to the C-terminus, a masking moiety (MM), a cleavable moiety (CM), and an anti-CTLA4 antibody described herein, wherein the MM comprises the amino acid sequence EVGSYPNPSSDCVPYYYACAY (SEQ ID NO: 192), and the cleavable moiety comprises the amino acid sequence SGRSAGGGGTPLGLAGSGGS (SEQ ID NO: 221); and (b) an effective amount of toripalimab. The MM and CM comprise, from the N-terminus to the C-terminus, the amino acid sequence EVGSYPNPSSDCVPYYYACAYSGRSAGGGGTPLGLAGSGGS (SEQ ID NO: 200). In specific embodiments, the MM and CM are covalently linked to the N-terminus of the anti-CTLA4 antibody light chain. In some embodiments, the MM and CM comprise, from the N-terminus to the C-terminus, an amino acid sequence having at least 90% or at least 95% sequence identity to SEQ ID NO: 200.

[0027] In some embodiments according to any one of the methods described above, cancer is resistant or refractory to previous therapy, wherein the previous therapy is CTLA4, PD-1 or PD-1 ligand inhibitor. In some embodiments, the subject is resistant to previous therapy or has relapsed from previous therapy, wherein the previous therapy is CTLA4, PD-1 or PD-1 ligand inhibitor. In some embodiments, the previous therapy is a CTLA4 inhibitor, such as an anti-CTLA4 antibody, such as ipilimumab. In some embodiments, the previous therapy is a PD-1 inhibitor, such as an anti-PD-1 antibody, such as nivolumab or toripalimab. In some embodiments, the previous therapy is a PD-1 ligand (e.g., PD-L1) inhibitor, such as an anti-PD-L1 antibody. In some embodiments, the previous therapy includes a CTLA4 inhibitor and a PD-1 inhibitor. In some embodiments, the previous therapy includes a CTLA4 inhibitor and a PD-L1 inhibitor. In some embodiments of the foregoing, the activatable anti-CTLA4 antibody comprises, after MM cleavage, a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 87, or an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 87, and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 100, or an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 100. In some embodiments of the foregoing, the activatable anti-CTLA4 antibody comprises a human IgG1 Fc region, e.g., a wild-type IgG1 Fc region or a variant with enhanced ADCC activity. In some of the foregoing embodiments, the activatable anti-CTLA4 antibody comprises a human IgG1 Fc region, such as a wild-type IgG1 Fc region or a variant with enhanced ADCC activity. In some of the foregoing embodiments, the activatable antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 320 or 321, or an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 320 or 321, and a light chain comprising the amino acid sequence of SEQ ID NO: 322, or an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 322.

[0028] In any of the foregoing embodiments, the activatable anti-CTLA4 antibody (e.g., TY22404), when administered as a monotherapy or in combination with another agent, can provide a steady-state concentration of the cleaved antibody (e.g., activated antibody after cleavage of the masking moiety (MM) and the cleavable moiety (CM)) that is above the EC50 value for the cleaved antibody. In some embodiments, the activatable anti-CTLA4 antibody (e.g., TY22404), when administered as a monotherapy or in combination with another agent, can provide a steady-state concentration of the cleaved antibody that is above the EC50 value for the cleaved antibody. In some of the foregoing embodiments, the concentration of the cleaved antibody is measured at a trough level of the anti-CTLA4 antibody.

[0029] In any of the foregoing embodiments, the activatable anti-CTLA4 antibody (e.g., TY22404), when administered as a monotherapy or in combination with another agent, can provide a dose administration of a cleaved antibody (e.g., activated antibody after cleavage of the masking moiety (MM) and the cleavable moiety (CM)) at a steady-state concentration of about 100 nM to about 600 nM. In some embodiments, the activatable anti-CTLA4 antibody can provide a dose administration of a cleaved antibody at a steady-state concentration of about 100 nM to about 175 nM. In some embodiments, the activatable anti-CTLA4 antibody can provide a dose administration of a cleaved antibody at a steady-state concentration of about 100 nM to about 200 nM. In some embodiments, the activatable anti-CTLA4 antibody can provide a dose administration of a cleaved antibody at a steady-state concentration of about 125 nM to about 200 nM. In some embodiments, the activatable anti-CTLA4 antibody can provide a dose administration of a cleaved antibody at a steady-state concentration of about 100 nM to about 150 nM. In some embodiments, the activatable anti-CTLA4 antibody can provide a steady-state concentration of cleaved antibody of about 125 nM to about 175 nM. In some embodiments, the activatable anti-CTLA4 antibody can provide a steady-state concentration of cleaved antibody of about 125 nM to about 150 nM. In some embodiments, the activatable anti-CTLA4 antibody can provide a steady-state concentration of cleaved antibody of about 150 nM to about 200 nM. In some embodiments, the activatable anti-CTLA4 antibody can provide a steady-state concentration of cleaved antibody of about 200 nM to about 600 nM. In some embodiments, the activatable anti-CTLA4 antibody can provide a steady-state concentration of cleaved antibody of about 200 nM to about 400 nM. In some embodiments, the activatable anti-CTLA4 antibody can provide a steady-state concentration of cleaved antibody of about 300 nM to about 500 nM. In some embodiments, the activatable anti-CTLA4 antibody can provide a steady-state concentration of cleaved antibody of about 400 nM to about 600 nM. In some of the aforementioned embodiments, the plasma concentration can be measured at the trough level of the anti-CTLA4 antibody (e.g., the minimum concentration of each dosing cycle). For example, the plasma concentration of a particular cycle can be measured immediately before the next cycle dose is administered.

[0030] In some embodiments, the activatable anti-CTLA4 antibody is administered in combination with two or more therapeutic agents. In some embodiments, at least one therapeutic agent is an anti-PD1 antibody. In some such embodiments, the activatable anti-CTLA4 antibody is administered at a dose that provides a steady-state concentration of the cleaved antibody of about 50 nM to about 150 nM. In other such embodiments, the activatable anti-CTLA4 antibody is administered at a dose that provides a steady-state concentration of the cleaved antibody of about 50 nM to about 100 nM. In other such embodiments, the activatable anti-CTLA4 antibody is administered at a dose that provides a steady-state concentration of the cleaved antibody of about 50 nM to about 75 nM. In other such embodiments, the activatable anti-CTLA4 antibody is administered at a dose that provides a steady-state concentration of the cleaved antibody of about 75 nM to about 100 nM.

[0031] In any of the foregoing embodiments, the activatable anti-CTLA4 antibody (e.g., TY22404), when administered as a monotherapy or in combination with another agent, can be administered at a dose that provides a ratio of cleaved antibody to uncleaved antibody at steady-state plasma concentration of about 0.3 to about 1.0 at trough levels for a particular dosing cycle. In some embodiments, the activatable anti-CTLA4 antibody can be administered at a dose that provides a ratio of cleaved antibody to uncleaved antibody at steady-state plasma concentration of about 0.3 to about 1.0 at trough levels for a particular dosing cycle. In some embodiments, the activatable anti-CTLA4 antibody can be administered at a dose that provides a ratio of cleaved antibody to uncleaved antibody at steady-state plasma concentration of about 0.5 to about 0.8 at trough levels for a particular dosing cycle. In some embodiments, the activatable anti-CTLA4 antibody can be administered at a dose that provides a ratio of cleaved antibody to uncleaved antibody at steady-state plasma concentration of about 0.7 to about 1.0 at trough levels for a particular dosing cycle.

[0032] In any of the foregoing embodiments, the activatable anti-CTLA4 antibody can be administered to the subject at a dose between about 3 mg / kg and about 20 mg / kg, for example, about 3 mg / kg, about 6 mg / kg, about 10 mg / kg, about 15 mg / kg, or about 20 mg / kg. In some embodiments, the activatable antibody is administered once every three to six weeks. In some such embodiments, the activatable anti-CTLA4 antibody is administered once every three weeks. In some such embodiments, the activatable anti-CTLA4 antibody is administered once every six weeks. In some embodiments, the activatable antibody is administered at a dose of about 10 mg / kg once every three weeks. In some embodiments, the activatable antibody is administered at a dose of about 20 mg / kg once every three weeks.

[0033] In some embodiments, the activatable anti-CTLA4 antibody is administered at a first higher dose (e.g., between about 10 mg / kg and about 20 mg) for at least one treatment cycle (as defined herein) and in subsequent cycles, is administered at a lower dose (e.g., between about 3 mg / kg and about 10 mg / kg). In one embodiment, the activatable anti-CTLA4 antibody is administered at a dose of about 20 mg / kg for at least one treatment cycle (e.g., one to three treatment cycles) and at a dose of about 10 mg / kg in subsequent treatment cycles (e.g., once every three weeks). In one embodiment, the activatable anti-CTLA4 antibody is administered at a dose of about 20 mg / kg for at least one treatment cycle and at a dose of about 10 mg / kg in subsequent treatment cycles (e.g., once every three weeks).

[0034] Based on non-compartmental analysis (NCA) and population PK modeling, intact drug (e.g., uncleaved drug) showed a dose-dependent increase in plasma. However, surprisingly, body weight was not modeled as a significant (e.g., index values ​​> 0.8 in the abnormal scale were considered significant) covariate for key PK parameters such as clearance (CL) and volume of distribution (V). As described below, simulations of virtual patients showed that fixed dosing regimens are easy to implement when the anti-CTLA4 is used as a monotherapy or as part of a combination. Specifically, in some embodiments, the fixed dosing of an activatable anti-CTLA4 antibody (e.g., TY22404) may be about 500 mg to about 1000 mg once every three weeks. In other embodiments, the fixed dosing schedule of an activatable anti-CTLA4 antibody (e.g., TY22404) may be about 700 mg to about 1000 mg once every three weeks. In other embodiments, the fixed dosing schedule of an activatable anti-CTLA4 antibody (e.g., TY22404) may be about 500 mg to about 750 mg once every three weeks. In other embodiments, the fixed dosing schedule of an activatable anti-CTLA4 antibody (e.g., TY22404) may be about 750 mg to about 1000 mg once every three weeks. In other embodiments, the fixed dosing schedule of an activatable anti-CTLA4 antibody (e.g., TY22404) may be about 500 mg to about 1200 mg once every six weeks. In other embodiments, the fixed dosing schedule of an activatable anti-CTLA4 antibody (e.g., TY22404) may be about 500 mg to about 1000 mg once every six weeks. In other embodiments, the fixed dosing schedule of an activatable anti-CTLA4 antibody (e.g., TY22404) may be about 500 mg to about 750 mg once every six weeks. In other embodiments, the fixed dosing schedule of an activatable anti-CTLA4 antibody (e.g., TY22404) may be about 750 mg to about 1000 mg once every six weeks. In other embodiments, the fixed dosing schedule of the activatable anti-CTLA4 antibody (e.g., TY22404) may be about 500 mg once every three weeks or once every six weeks. In other embodiments, the fixed dosing schedule of the activatable anti-CTLA4 antibody (e.g., TY22404) may be about 700 mg once every three weeks or once every six weeks. In other embodiments, the fixed dosing schedule of the activatable anti-CTLA4 antibody (e.g., TY22404) may be about 1000 mg once every three weeks or once every six weeks.

[0035] It should be understood that one, some, or all of the properties of the various embodiments described above and herein may be combined to form other embodiments of the present disclosure. These and other aspects of the present disclosure will become readily apparent to those skilled in the art. These and other embodiments of the present disclosure are further described in detail below. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The CA125 response of a patient with ovarian serous carcinoma at the end of cycle 16 was shown, with a 90% decrease in CA125 from 303 to 31 U / ml (normal < 35 U / ml).

[0037] Figure 2 Clinical activity evaluation of TY22404 monotherapy is shown. (A) Maximum tumor burden reduction in patients at different dose levels (0.1 to 20 mg / kg); (B) Duration of treatment in patients at different dose levels (0.1 to 20 mg / kg).

[0038] Figure 3 The plasma PK of total and cleaved TY22404 over the first 4 cycles is shown in (A) 20 patients with available PK data and (B) a patient with ovarian serous carcinoma (case study). Total and intact TY22404 were measured by LC-MS using a signature peptide. Cleaved TY22404 was calculated as total TY22404 minus intact TY22404. C = treatment cycle

[0039] Figure 4 A-4C shows the efficacy of TY22404 monotherapy in a 39-year-old male patient with stage IIIB hepatocellular carcinoma. Figure 4 A shows the increased Teff / Treg ratio observed in paired tumor biopsies from patients. Figure 4 B shows Treg depletion observed in paired tumor biopsies from patients. Figure 4 C shows the increase in CD8+ T cells observed in paired tumor biopsies from patients.

[0040] Figures 5A-5B Shown are 18 evaluable patients who received TY22404 (6 mg / kg Q3W and 10 mg / kg Q3W or Q6W) plus toripalimab (240 mg Q3W) * Responses to TY22404 in combination with toripalimab in three dose-escalation cohorts. Figure 5A Shows a swim lane diagram. Figure 5B Waterfall plots are shown. *Evaluable patients had at least one valid post-treatment tumor assessment. For swim-lane plots, bars end at end of treatment (EOT), last dosing day, or study day of last tumor assessment, whichever occurs later.

[0041] Figure 6 A-6B shows the response of TY22404 + toripalimab in selected gastrointestinal “cold tumors” including MSS CRC with liver metastases and PDAC at baseline. Figure 6 A shows a lane diagram. Figure 6B shows a waterfall plot. DETAILED DESCRIPTION

[0042] I. Definition

[0043] Unless otherwise defined herein, the scientific and technical terms used in connection with the present application will have the meaning commonly understood by those of ordinary skill in the art. In addition, unless the context otherwise requires, singular terms should include plural terms, and plural terms should include singular terms. In general, the nomenclature and techniques used in antibody engineering, immunotherapy, cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry described herein are well known and commonly used in the art.

[0044] The term "antibody" is used herein in the broadest sense and specifically covers monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies), and antibody fragments (e.g., Fab, Fab', Fab'-SH, F(ab')2, Fv and / or single-chain variable fragments or scFv), so long as they exhibit the desired biological activity.

[0045] V H and V L The V domain can also be further subdivided based on structural and sequence analysis into regions of high variability, termed hypervariable regions (HVRs). HVRs are interspersed with more conserved regions, termed framework regions (FWs) (see, e.g., Chen et al. (1999) J. Mol. Biol. (1999) 293, 865-881). Each V domain is a highly conserved region. H and V L It consists of three HVRs and four FWs, arranged from amino terminus to carboxyl terminus in the following order: FW-1_HVR-1_FW-2_HVR-2_FW-3_HVR-3_FW4. Throughout this disclosure, the three HVRs of the heavy chain are referred to as HVR-H1, HVR-H2, and HVR-H3. Similarly, the three HVRs of the light chain are referred to as HVR-L1, HVR-L2, and HVR-L3.

[0046] As used herein, the term "CDR" or "complementarity determining region" is intended to refer to the non-contiguous antigen binding sites found within the variable regions of heavy and light chain polypeptides. These specific regions have been described in Kabat et al., J. Biol. Chem. 252: 6609-6616 (1977); Kabat et al., US Pat. of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al., J. Mol. Biol. 196: 901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., 273: 927-948 (1997); MacCallum et al., J. Mol. Biol. 262: 732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008); Lefranc et al., J. Mol. Biol. 196: 901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., 273: 927-948 (1997); MacCallum et al., J. Mol. Biol. 262: 732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008); Lefranc et al., J. Mol. MP et al., Dev. Comp. Immunol., 27:55-77 (2003); and Honegger and Plückthun, J. Mol. Biol., 309:657-670 (2001), wherein the definitions include overlapping or subsets of amino acid residues when compared to each other.

[0047] The term "antigen-binding fragment" or "antigen-binding portion" of an antibody refers to one or more portions of an antibody that retain the ability to bind to the antigen to which the antibody binds (eg, CTLA4).

[0048] The term "CTLA4" is used in this application and includes human CTLA4 (e.g., UniProt Accession No. P16410) as well as variants, isoforms, and species homologs thereof (e.g., mouse CTLA4 (UniProt Accession No. P09793), rat CTLA4 (UniProt Accession No. Q9Z1A7), dog CTLA4 (UniProt Accession No. Q9XSI1), cynomolgus monkey CTLA4 (UniProt Accession No. G7PL88), etc.). Thus, anti-CTLA4 antibodies (e.g., activatable antibodies) as defined and disclosed herein may also bind to CTLA4 from species other than human. In other cases, the anti-CTLA4 antibody may be completely specific for human CTLA4 and may not exhibit species cross-reactivity or other types of cross-reactivity.

[0049] The term "CTLA4 antibody" refers to an antibody as defined herein that is capable of binding to human CTLA4 (eg, an activatable anti-CTLA4 antibody).

[0050] The term "epitope" refers to the portion of an antigen to which an antibody (or antigen-binding fragment thereof) binds. An epitope can be formed by contiguous or discontinuous amino acids juxtaposed by the tertiary folding of a protein.

[0051] A "human antibody" is an antibody that possesses an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human or a human cell, or derived from a non-human source that utilizes a human antibody repertoire or other human antibody encoding sequences.

[0052] The term "humanized antibody" refers to a chimeric antibody comprising amino acid residues derived from human antibody sequences.

[0053] The term "illustrative antibodies" refers to any of the antibodies described in this disclosure and designated as those listed in Tables A and B, as well as any antibodies comprising the six HVRs and / or the VH and VL of the antibodies listed in Tables A and B. These antibodies can be of any class (e.g., IgA, IgD, IgE, IgG, and IgM).

[0054] The term "mammal" refers to any animal species of the class Mammalia.

[0055] As used herein, "sequence identity" between two polypeptide sequences indicates the percentage of amino acids that are identical between the sequences.

[0056] The term "treat," "treating," or "treatment" with respect to a disease condition in a mammal refers to causing a desired or beneficial effect in a mammal suffering from the disease condition. The desired or beneficial effect may include a reduction in the frequency or severity of one or more symptoms of the disease (i.e., tumor growth and / or metastasis, or other effects mediated by the number and / or activity of immune cells, etc.), or a cessation or inhibition of further development of the disease, condition, or illness. In the context of treating cancer in a mammal, the desired or beneficial effect may include inhibition of further growth or spread of cancer cells, death of cancer cells, inhibition of cancer recurrence, reduction of cancer-related pain, or improvement in mammalian survival. The effect may be subjective or objective. For example, if the mammal is a human, improvement in energy or vitality or reduction in pain may be recorded as an improved symptom or response to therapy. Alternatively, a clinician may notice a decrease in tumor size or tumor burden based on physical examination, laboratory parameters, tumor markers, or imaging findings. For response to treatment, some laboratory signs that clinicians can observe include standardized tests such as white blood cell count, red blood cell count, platelet count, erythrocyte sedimentation rate, and various enzyme levels. In addition, clinicians can observe a decrease in detectable tumor markers. Alternatively, other tests can be used to assess objective improvement, such as sonograms, magnetic resonance imaging, and positron emission tomography.

[0057] The terms "prevent" or "preventing" with respect to a disease condition in a mammal means preventing or delaying the onset of the disease, or preventing the appearance of clinical or subclinical symptoms thereof.

[0058] As used herein, "subject," "patient," or "individual" may refer to a human or non-human animal. "Non-human animal" may refer to any animal that is not classified as a human, such as domestic, farm, or zoo animals, sports animals, pet animals (such as dogs, horses, cats, cows, etc.), and animals used in research. Research animals may refer to, without limitation, nematodes, arthropods, vertebrates, mammals, frogs, rodents (e.g., mice or rats), fish (e.g., zebrafish or pufferfish), birds (e.g., chickens), dogs, cats, and non-human primates (e.g., rhesus monkeys, cynomolgus monkeys, chimpanzees, etc.). In some embodiments, the subject, patient, or individual is a human.

[0059] "Effective amount" refers to at least the following quantity: at the necessary dose and for the necessary period, the quantity effectively achieves one or more desired or indicated effects including treatment or prevention results. The effective amount can be provided by one or more administrations. For the purposes of this application, the effective amount of an antibody, drug, compound or pharmaceutical composition is an amount sufficient to directly or indirectly achieve preventive or therapeutic treatment. As understood in clinical situations, the effective amount of a drug, compound or pharmaceutical composition may or may not be combined with another drug, compound or pharmaceutical composition to achieve (for example, as an effective amount administered in the form of a monotherapy or combination therapy). Therefore, "effective amount" can be considered in the context of administering one or more therapeutic agents, and if combined with one or more other agents, a desirable result can be achieved or achieved, then a single agent can be considered to be given in an effective amount.

[0060] The terms "relapse," "recurrent relapse," or "recurrent" refer to the return of disease or cancer after it has disappeared as assessed clinically. The diagnosis of distant metastasis or local recurrence may be considered a relapse.

[0061] The term "refractory" or "drug-resistant" refers to a cancer or disease that does not respond to treatment.

[0062] As used herein, "complete response" or "CR" means the disappearance of all target lesions; "partial response" or "PR" means a decrease of at least 30% in the sum of the longest diameters of the target lesions (SLD), using the baseline SLD as a reference; and "stable disease" or "SD" means that the target lesions have not shrunk sufficiently to qualify as a PR, nor increased sufficiently to qualify as a PD, using the lowest SLD as a reference since the start of treatment.

[0063] As used herein, "progressive disease" or "PD" refers to an increase in the SLD of a target lesion by at least 20%, using the nadir SLD as a reference, since the start of treatment or the presence of one or more new lesions.

[0064] As used herein, "progression-free survival" (PFS) refers to the length of time during and after treatment that a disease (such as cancer) does not worsen. Progression-free survival can include the time a patient experiences a complete remission or partial remission, as well as the time a patient experiences stable disease.

[0065] As used herein, "overall response rate" (ORR) refers to the sum of the complete response (CR) rate and the partial response (PR) rate.

[0066] As used herein, "overall survival" refers to the percentage of individuals in a cohort who are likely to be alive after a specified duration of time.

[0067] As used herein, "baseline level" or "baseline value" refers to a level or value in a subject before starting treatment (eg, anti-CTLA4 antibody treatment).

[0068] As used herein, "reference sample," "reference cell," "reference tissue," "control sample," "control cell," or "control tissue" refers to a sample, cell, tissue, standard, or level used for comparison purposes.

[0069] "Correlate" or "correlating" means comparing in any way the performance and / or results of a first analysis or protocol with the performance and / or results of a second analysis or protocol. For example, the results of a first analysis or protocol can be used to initiate a second protocol and / or the results of a first analysis or protocol can be used to determine whether a second analysis or protocol should be initiated.

[0070] An "effective response" of a patient or "responsiveness" of a patient to a drug treatment, and similar expressions, refers to a clinical or therapeutic benefit conferred on a patient at risk for or suffering from a disease or condition (e.g., cancer). In one embodiment, such benefit includes any one or more of: prolonged survival (including overall survival and progression-free survival); resulting in an objective response (including complete or partial response); or ameliorating signs or symptoms of cancer.

[0071] Patients who "did not respond effectively" to treatment were those for whom none of the treatments prolonged survival (including overall survival and progression-free survival); resulted in an objective response (including complete response or partial response); or improved signs or symptoms of cancer.

[0072] As used herein, the 20 conventional amino acids and their abbreviations follow conventional usage. See Immunology—A Synthesis (2nd edition, ES Golub and DR Gren, eds., Sinauer Associates, Sunderland, Mass. (1991)).

[0073] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a molecule" optionally includes a combination of two or more of those molecules, and so on.

[0074] As used herein, the term "about" refers to the usual error range for the corresponding value that is readily known to those skilled in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments related to that value or parameter itself.

[0075] It should be understood that aspects and embodiments of the present disclosure described herein include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0076] As used herein, reference to "other than" a value or parameter generally refers to describing "other than" a value or parameter. For example, "the method is not used to treat type X cancer" means that the method is used to treat cancers other than type X.

[0077] As used herein, the term "about XY" has the same meaning as "about X to about Y."

[0078] As used herein, the term "and / or," phrases such as "A and / or B," are intended to include both A and B; A or B; A (alone); and B (alone). Similarly, as used herein, the term "and / or," phrases such as "A, B, and / or C," are intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0079] II. Treatment Methods

[0080] The present application provides methods for treating cancer in a subject using an activatable anti-CTLA4 antibody that specifically binds to human CTLA4. The activatable anti-CTLA4 antibody can be administered alone as a monotherapy or in combination with one or more additional therapeutic agents or therapies.

[0081] The methods described herein can be used to treat a variety of cancers. In some embodiments, the cancer is a solid cancer. In some embodiments, the cancer is a liquid cancer. A variety of cancers involving CTLA4, whether malignant or benign and whether primary or secondary, can be treated or prevented using the methods provided by this disclosure. Exemplary cancers include, but are not limited to, liver cancer, digestive system cancers (e.g., colon cancer, colorectal cancer (CRC), gastrointestinal stromal tumors (GIST), cecal adenocarcinoma), lung cancer, bone cancer, heart cancer, brain cancer, kidney cancer, bladder cancer, blood cancers (e.g., leukemia), skin cancer, breast cancer, thyroid cancer, neuroendocrine cancer, pancreatic cancer (e.g., pancreatic ductal adenocarcinoma (PDAC)), head and / or neck cancer, eye-related cancers, male reproductive system cancers (e.g., prostate cancer, testicular cancer), or female reproductive system cancers (e.g., uterine cancer, cervical cancer (e.g., cervical squamous cell carcinoma and adenocarcinoma (CESC), endometrioid adenocarcinoma, endometrial cancer). In some embodiments, the cancer is squamous cell carcinoma (SCC) (e.g., anal, anorectal, penis, or skin). In some embodiments, the cancer is anal squamous cell carcinoma or penile squamous cell carcinoma. Cancer. In some embodiments, the cancer is adenocarcinoma. In some embodiments, the cancer is kidney cancer, such as renal cell carcinoma or urothelial carcinoma. In some embodiments, the cancer is ovarian cancer (e.g., ovarian serous cystadenocarcinoma (OV), pancreatic cancer, bile duct cancer, lung cancer (e.g., NSCLC), breast cancer, melanoma, hepatocellular carcinoma, glioblastoma, renal cell carcinoma, head and neck squamous cell carcinoma, or colorectal cancer (e.g., MSI-H or dMMR+ colorectal cancer, microsatellite stable (MSS) colorectal cancer)). In some embodiments, the cancer is melanoma, NSCLC, hepatocellular carcinoma, renal cell carcinoma, head and neck squamous cell carcinoma, pancreatic cancer, anal squamous cell carcinoma, penile squamous cell carcinoma, or colorectal cancer (e.g., MSI-H or dMMR+ colorectal cancer, microsatellite stable (MSS) colorectal cancer).

[0082] In some embodiments, the activatable anti-CTLA4 antibody is administered to a cancer patient as a monotherapy. In some embodiments, the activatable anti-CTLA4 antibody is administered to a cancer patient as a combination therapy. In some embodiments, the cancer is squamous cell carcinoma (e.g., anus, anorectal, penis, or skin). In some embodiments, the cancer is adenocarcinoma. In some embodiments, the cancer is melanoma. In other embodiments, the cancer is ovarian cancer, pancreatic cancer, bile duct cancer, lung cancer, breast cancer, hepatocellular carcinoma, glioblastoma, renal cell carcinoma, head and neck squamous cell carcinoma, colorectal cancer, gastrointestinal stromal tumor (GIST), endometrial cancer. In some embodiments, the lung cancer is non-small cell lung cancer (NSCLC). In some embodiments, the colorectal cancer is MSS CRC. In some embodiments, the cancer is anal squamous cell carcinoma or penile squamous cell carcinoma.

[0083] Cancer treatment can be assessed, for example, by tumor regression, reduction in tumor weight or size, time to progression, duration of survival, progression-free survival, overall response rate, duration of response, quality of life, protein expression and / or activity. Methods for determining efficacy of therapy can be employed, including, for example, measuring response by radiological imaging.

[0084] The activatable anti-CTLA4 antibodies and one or more additional therapeutic agents and compositions provided by the present disclosure can be administered by any suitable enteral or parenteral route of administration. The term "enteral route" of administration refers to administration via any part of the gastrointestinal tract. Examples of enteral routes include oral, mucosal, buccal and rectal routes, or intragastric routes. "Parenteral route" of administration refers to a route of administration other than the enteral route. Examples of parenteral routes of administration include intravenous, intramuscular, intradermal, intraperitoneal, intratumoral, intravesical, intraarterial, intrathecal, intracapsular, intraorbital, intracardial, transtracheal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal, subcutaneous or topical administration. Any suitable method can be used to administer the antibodies and compositions of the present disclosure, such as by oral ingestion, nasogastric tube, gastrostomy tube, injection, infusion, implantable infusion pump, and osmotic pump. The appropriate route and method of administration may vary depending on a variety of factors, such as the specific antibody used, the desired rate of absorption, the specific formulation or dosage form used, the type or severity of the condition being treated, the specific site of action, and the condition of the patient, and can be readily selected by one skilled in the art. In some embodiments, the anti-CTLA4 antibody is administered intravenously.

[0085] An effective amount of an activatable anti-CTLA4 antibody and / or one or more additional therapeutic agents can be administered in a single dose or multiple doses. For methods comprising administering an anti-CTLA4 antibody in multiple doses, exemplary dosing frequencies include, but are not limited to, once a week, weekly, two weeks out of three weeks, once a week out of three weeks, once every three weeks, once every two weeks, monthly, once every six months, annually, and the like. In some embodiments, the anti-CTLA4 antibody is administered approximately once a week, once every two weeks, once every three weeks, once every six weeks, or once every 12 weeks. In some embodiments, the interval between each administration is less than about any one of 3 years, 2 years, 12 months, 11 months, 10 months, 9 months, 8 months, 7 months, 6 months, 5 months, 4 months, 3 months, 2 months, 1 month, 4 weeks, 3 weeks, 2 weeks, or 1 week. In some embodiments, the interval between each administration is greater than about any of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, or 3 years. In some embodiments, there is no interruption in the dosing regimen.

[0086] In some embodiments, the activatable anti-CTLA4 antibody and / or one or more additional therapeutic agents are administered infrequently, for example, no more than once a week, once every other week, once every three weeks, once a month, once every two months, once every three months, once every four months, once every five months, once every six months, once every seven months, once every eight months, once every nine months, once every ten months, once every eleven months, once a year, or less. In some embodiments, the anti-CTLA4 antibody and / or one or more additional therapeutic agents are administered in a single dose. In some embodiments, the activatable anti-CTLA4 antibody and / or one or more additional therapeutic agents are administered approximately once every three weeks. In some embodiments, the activatable anti-CTLA4 antibody and / or one or more additional therapeutic agents are administered approximately once every six weeks.

[0087] In some embodiments, the activatable anti-CTLA4 antibody and / or one or more additional therapeutic agents are administered for 2 or more cycles, such as about any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles. In some embodiments, the anti-CTLA4 antibody is administered for at least 4 cycles.

[0088] In some embodiments, the treatment comprises an initial phase and a subsequent maintenance phase. In some embodiments, the activatable anti-CTLA4 antibody and / or one or more additional therapeutic agents are administered less frequently in the maintenance phase than in the initial phase. In some embodiments, the anti-CTLA4 antibody and / or one or more additional therapeutic agents are administered at the same frequency in the maintenance phase as in the initial phase. In some embodiments, the treatment comprises an initial phase wherein the anti-CTLA4 antibody and / or one or more additional therapeutic agents are administered approximately once every three weeks for at least four cycles, and a maintenance phase wherein the anti-CTLA4 antibody and / or one or more additional therapeutic agents are administered approximately once every four weeks to once every 12 weeks, for example, once every four weeks, once every six weeks, once every eight weeks, once every 10 weeks, or once every 12 weeks. In some embodiments, the dosing frequency in the maintenance phase is adjusted based on one or more biomarkers, for example, T reg cells, CD8+T em cells, CD4+T em cells, CD8+T em T cells and reg The ratio of cells, CD4+T em Cells and T reg For example, if a subject receives an anti-CTLA4 antibody and shows CD8+ T cells, CD25+ T cells and / or NK cells em Cells and T reg If the ratio of T cells increases, the subject can be further administered anti-CTLA4 antibody approximately every 4 weeks.

[0089] Administration of the anti-CTLA4 antibody and / or one or more additional therapeutic agents can be extended over a period of time, for example, from about one week to about one month, from about one month to about one year, from about one year to about several years. In some embodiments, the anti-CTLA4 antibody and / or one or more additional therapeutic agents are administered for at least about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, or longer.

[0090] In some embodiments, the activatable anti-CTLA4 antibody, upon MM cleavage, comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 87 or an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 87, and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 100 or an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 101. The amino acid sequence of NO: 100 has an amino acid sequence with at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity, and the activatable anti-CTLA4 antibody is administered once every three weeks at a dose of about 3 mg / kg to about 20 mg / kg, once every three weeks at a dose of about 3 mg / kg to about 15 mg / kg, once every three weeks at a dose of about 6 mg / kg to about 15 mg / kg, or once every three weeks at a dose of about 6 mg / kg to about 10 mg / kg. In some such embodiments, the activatable anti-CTLA4 antibody is administered once every three weeks at a dose of about 3 mg / kg. In other such embodiments, the activatable anti-CTLA4 antibody is administered once every three weeks at a dose of about 6 mg / kg. In other such embodiments, the activatable anti-CTLA4 antibody is administered once every three weeks at a dose of about 10 mg / kg. In some of the foregoing embodiments, the activatable antibody comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 23, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 35, an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 45, an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 58, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 66, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 75. In some of the foregoing embodiments, the activatable anti-CTLA4 antibody comprises a human IgG1 Fc region, such as a wild-type IgG1 Fc region or a variant with enhanced ADCC activity. In some of the foregoing embodiments, the activatable anti-CTLA4 antibody comprises an MM amino acid sequence selected from the group consisting of SEQ ID NOS: 189-196. In some of the foregoing embodiments, the activatable anti-CTLA4 antibody comprises an MM amino acid sequence selected from the group consisting of SEQ ID NOS: 213-216. In some of the foregoing embodiments, the activatable anti-CTLA4 antibody comprises an MM amino acid sequence selected from the group consisting of SEQ ID NOS: 141-147.In some embodiments of the foregoing, the activatable anti-CTLA4 antibody comprises the MM amino acid sequence of SEQ ID NO: 200. In some embodiments of the foregoing, the activatable anti-CTLA4 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 320 or 321, or an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 320 or 321, and a light chain comprising the amino acid sequence of SEQ ID NO: 322, or an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 322. In some embodiments of the foregoing, the activatable anti-CTLA4 antibody is administered to a patient with melanoma, non-small cell lung cancer, renal cell carcinoma or hepatocellular carcinoma. In other embodiments of the foregoing, the activatable anti-CTLA4 antibody is administered to a patient with MSI-H or dMMR cancer. In other embodiments of the foregoing, the activatable anti-CTLA4 antibody is administered to a patient whose cancer has metastasized. In some embodiments of the foregoing, the activatable anti-CTLA4 antibody is administered to a patient who is resistant or refractory to a previous cancer therapy (including other anti-CTLA4 antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, or a combination thereof).

[0091] In some embodiments, the activatable anti-CTLA4 antibody, upon MM cleavage, comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 87 or an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 87, and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 100 or an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 101. The amino acid sequence of NO: 100 has an amino acid sequence with at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity, and the activatable anti-CTLA4 antibody is administered at a first higher dose (e.g., between about 10 mg / kg and about 20 mg) for at least one treatment cycle (as defined herein), and in subsequent cycles, at a lower dose (e.g., between about 3 mg / kg and about 10 mg / kg). In some such embodiments, the activatable anti-CTLA4 antibody is administered at a dose of about 10 mg / kg for at least one treatment cycle (e.g., one to three treatment cycles) and at a dose of about 6 mg / kg in subsequent treatment cycles. In other such embodiments, the activatable anti-CTLA4 antibody is administered at a dose of about 10 mg / kg for at least one treatment cycle (e.g., one to three treatment cycles) and at a dose of about 3 mg / kg in subsequent treatment cycles. In other such embodiments, the activatable anti-CTLA4 antibody is administered at a dose of about 15 mg / kg for at least one treatment cycle (e.g., one to three treatment cycles) and at a dose of about 10 mg / kg in subsequent treatment cycles. In other such embodiments, the activatable anti-CTLA4 antibody is administered at a dose of about 20 mg / kg for at least one treatment cycle (e.g., one to three treatment cycles) and at a dose of about 10 mg / kg in subsequent treatment cycles. In some of the foregoing embodiments, the activatable antibody comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 23, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 35, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 45, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 58, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 66, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 75.In some of the foregoing embodiments, the activatable anti-CTLA4 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 320 or 321, or an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 320 or 321, and a light chain comprising the amino acid sequence of SEQ ID NO: 322, or an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 322. In some of the foregoing embodiments, the activatable anti-CTLA4 antibody is administered to a patient with melanoma, non-small cell lung cancer, renal cell carcinoma or hepatocellular carcinoma. In other embodiments of the foregoing, the activatable anti-CTLA4 antibody is administered to a patient with MSI-H or dMMR cancer. In other embodiments of the foregoing, the activatable anti-CTLA4 antibody is administered to a patient whose cancer has metastasized. In some embodiments of the foregoing, the activatable anti-CTLA4 antibody is administered to a patient who is resistant or refractory to a previous cancer therapy including other anti-CTLA4 antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, or a combination thereof.

[0092] In some embodiments, the activatable anti-CTLA4 antibody is administered at a dose of about 20 mg / kg for one treatment cycle and at a dose of about 10 mg / kg in subsequent treatment cycles (e.g., once every three weeks). For example, in one embodiment, the anti-CTLA4 antibody is administered at an initial (loading) dose of 20 mg / kg and subsequently at an additional dose (maintenance dose) of 10 mg / kg once every three weeks. The maintenance dose may begin at a predetermined time after the loading dose is administered. For example, in some embodiments, the first maintenance dose may be administered three weeks after the loading dose is administered.

[0093] It has been found that, as described above, administration of a single loading dose followed by subsequent maintenance doses allows the cleaved antibody to establish steady-state concentrations more quickly than when the activatable antibody is administered without a loading dose. For example, in some embodiments, steady-state concentrations of the cleaved antibody can be established within 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, or 7 weeks after the initial administration of the loading dose.

[0094] In any of the foregoing, the activatable anti-CTLA4 antibody is administered at a dose that provides a steady-state concentration of cleaved antibody of about 100 nM to about 200 nM. In some embodiments, the activatable anti-CTLA4 antibody is administered at a dose that provides a steady-state concentration of cleaved antibody of about 125 nM to about 200 nM. In some embodiments, the activatable anti-CTLA4 antibody is administered at a dose that provides a steady-state concentration of cleaved antibody of about 100 nM to about 175 nM. In some embodiments, the activatable anti-CTLA4 antibody is administered at a dose that provides a steady-state concentration of cleaved antibody of about 125 nM to about 175 nM. In some embodiments, the activatable anti-CTLA4 antibody is administered at a dose that provides a steady-state concentration of cleaved antibody of about 100 nM to about 150 nM. In some embodiments, the activatable anti-CTLA4 antibody is administered at a dose that provides a steady-state concentration of cleaved antibody of about 125 nM to about 150 nM. In some embodiments, the activatable anti-CTLA4 antibody is administered at a dosage that provides a steady-state concentration of cleavage antibodies of about 150nM to about 200nM. In some embodiments, the activatable anti-CTLA4 antibody is administered at a dosage that provides a steady-state concentration of cleavage antibodies of about 200nM to about 600nM. In some embodiments, the activatable anti-CTLA4 antibody is administered at a dosage that provides a steady-state concentration of cleavage antibodies of about 200nM to about 400nM. In some embodiments, the activatable anti-CTLA4 antibody is administered at a dosage that provides a steady-state concentration of cleavage antibodies of about 300nM to about 500nM. In some embodiments, the activatable anti-CTLA4 antibody is administered at a dosage that provides a steady-state concentration of cleavage antibodies of about 400nM to about 600nM. In some of the aforementioned embodiments, plasma concentration can be measured at the trough level (e.g., the minimum concentration of each dosing cycle) of the anti-CTLA4 antibody. For example, the plasma concentration of a particular cycle can be measured immediately before the next cycle dose is administered.

[0095] In some embodiments, an activatable anti-CTLA4 antibody is administered in combination with two or more therapeutic agents. In some embodiments, at least one therapeutic agent is an anti-PD1 antibody. In some such embodiments, the activatable anti-CTLA4 antibody is administered at a dose that provides a steady-state concentration of the cleavage antibody of about 50 nM to about 150 nM. In other such embodiments, the activatable anti-CTLA4 antibody is administered at a dose that provides a steady-state concentration of the cleavage antibody of about 50 nM to about 100 nM. In other such embodiments, the activatable anti-CTLA4 antibody is administered at a dose that provides a steady-state concentration of the cleavage antibody of about 50 nM to about 75 nM. In other such embodiments, the activatable anti-CTLA4 antibody is administered at a dose that provides a steady-state concentration of the cleavage antibody of about 75 nM to about 100 nM.

[0096] In any of the embodiments of the preceding two paragraphs, the anti-CTLA4 antibody can be administered as a monotherapy or in combination with one or more anti-cancer agents disclosed herein. For example, the anti-CTLA4 antibody can be administered in combination with an anti-PD-1 antibody. In some embodiments, the combination of the anti-CTLA4 antibody and the anti-PD-1 antibody exhibits a synergistic effect.

[0097] In some embodiments, treatment with an anti-CTLA4 antibody and / or one or more additional therapeutic agents depletes Treg cells in the tumor. In some embodiments, treatment with an anti-CTLA4 antibody and / or one or more additional therapeutic agents does not deplete Treg cells in peripheral tissues.

[0098] In some embodiments, the subject has previously been treated with a prior therapy. In some embodiments, the subject has previously received any of 1, 2, 3, 4 or more prior therapies. In some embodiments, the subject has exhausted all other available therapies. In some embodiments, the subject is unresponsive to or resistant to the prior therapy. In some embodiments, the subject has a relapse of the disease after the prior therapy. In some embodiments, the subject is refractory to the prior therapy. In some embodiments, the subject fails to respond to the prior therapy within about 1 year, 6 months, 3 months or less. In some embodiments, the subject has not previously received prior therapy.

[0099] In some embodiments, the subject has been previously treated with standard therapy for cancer. In some embodiments, the subject is unresponsive to or resistant to standard therapy. In some embodiments, the subject has a relapse of disease after standard therapy. In some embodiments, the subject is refractory to standard therapy. In some embodiments, the subject fails to respond to standard therapy in about 1 year, 6 months, 3 months, or less. In some embodiments, the subject has not previously received standard therapy. In some embodiments, the subject refuses or is not a candidate for standard therapy.

[0100] In some embodiments, the previous therapy (e.g., standard therapy) is selected from the group consisting of viral gene therapy, immunotherapy, targeted therapy, radiotherapy and chemotherapy. In some embodiments, the previous therapy is an immune checkpoint inhibitor. In some embodiments, the previous therapy is a CTLA4, PD-1 or PD-1 ligand (e.g., PD-L1 or PD-L2) inhibitor. In some embodiments, the previous therapy is a CTLA4 inhibitor, for example, an anti-CTLA4 antibody different from the anti-CTLA4 antibodies described herein. In some embodiments, the previous therapy is ipilimumab. In some embodiments, the previous therapy is ipilimumab and / or nivolumab.

[0101] In some embodiments, the prior therapy is a PD-1 or PD-1 ligand inhibitor, including a PD-1 binding antagonist, a PDL1 binding antagonist, and a PDL2 binding antagonist. Other names for "PD-1" include CD279 and SLEB2. Other names for "PDL1" include B7-H1, B7-4, CD274, and B7-H. Other names for "PDL2" include B7-DC, Btdc, and CD273. In some embodiments, PD-1, PDL1, and PDL2 are human PD-1, PDL1, and PDL2.

[0102] In some embodiments, a PD-1 inhibitor is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In some embodiments, a PD-1 ligand inhibitor is a PD-L1 and / or PD-L2 inhibitor. In some embodiments, a PD-L1 inhibitor is a molecule that inhibits the binding of PDL1 to its binding partner. In some embodiments, the PD-L2 binding partner is PD-1 and / or B7-1. In some embodiments, a PD-1 ligand inhibitor is a molecule that inhibits the binding of PD-L2 to its binding partner. In some embodiments, the PD-L2 binding partner is PD-1. The inhibitor can be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide.

[0103] In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab and CT-011. In some embodiments, the PD-1 inhibitor is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular domain or a PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In some embodiments, the PD-1 inhibitor is AMP-224. Nivolumab, also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and is an anti-PD-1 antibody described in WO2006 / 121168. CT-011, also known as hBAT or hBAT-1, is an anti-PD-1 antibody described in WO2009 / 101611. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in WO2010 / 027827 and WO2011 / 066342. In some embodiments, the anti-PD-1 antibody is nivolumab (CAS Reg. No. 946414-94-4).

[0104] In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab and CT-011. In some embodiments, the PD-1 inhibitor is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular domain or a PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In some embodiments, the PD-1 inhibitor is AMP-224. Nivolumab, also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and is an anti-PD-1 antibody described in WO2006 / 121168. CT-011, also known as hBAT or hBAT-1, is an anti-PD-1 antibody described in WO2009 / 101611. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in WO2010 / 027827 and WO2011 / 066342. In some embodiments, the anti-PD-1 antibody is nivolumab (CAS Reg. No. 946414-94-4).

[0105] Prior treatment (e.g., standard treatment) also encompasses surgery and radiation therapy to remove the tumor. Exemplary radiation therapy includes, but is not limited to, a combination of ionizing (electromagnetic) radiation therapy (e.g., X-rays or gamma rays) and particle beam radiation therapy (e.g., high linear energy radiation). The radiation source may be external or internal to the subject.

[0106] The methods described herein can be used in various aspects of cancer treatment. In some embodiments, a method of inhibiting cell proliferation (e.g., tumor growth) in an individual is provided, comprising administering to the individual an effective amount of any of the anti-CTLA4 antibodies described herein. In some embodiments, at least about 10% (including, for example, at least about 20%, about 30%, about 40%, about 60%, about 70%, about 80%, about 90%, about 95% or more) of cell proliferation is inhibited.

[0107] In some embodiments, a method of inhibiting tumor metastasis in an individual is provided, comprising administering to the individual an effective amount of any of the anti-CTLA4 antibodies described herein. In some embodiments, metastasis is inhibited by at least about 10% (including, for example, at least about 20%, about 30%, about 40%, about 60%, about 70%, about 80%, about 90%, about 95% or more).

[0108] In some embodiments, a method of reducing (e.g., eliminating) pre-existing tumor metastasis (e.g., metastasis to lymph nodes) in an individual is provided, comprising administering to the individual an effective amount of any of the anti-CTLA4 antibodies described herein. In some embodiments, metastasis is reduced by at least about 10% (including, for example, at least about 20%, about 30%, about 40%, about 60%, about 70%, about 80%, about 90%, about 95%, or more).

[0109] In some embodiments, methods are provided for reducing the incidence or burden of pre-existing tumor metastases (eg, metastases to lymph nodes) in a subject, comprising administering to the subject an effective amount of any one of the anti-CTLA4 antibodies described herein.

[0110] In some embodiments, a method of reducing tumor size in an individual is provided, comprising administering to the individual an effective amount of any of the anti-CTLA4 antibodies described herein. In some embodiments, the method reduces tumor size by at least about 10% (including, for example, at least about 20%, about 30%, about 40%, about 60%, about 70%, about 80%, about 90%, about 95%, or more).

[0111] In some embodiments, a method of extending time to disease progression in a subject comprising administering to the subject an effective amount of any of the anti-CTLA4 antibodies described herein is provided. In some embodiments, the method extends time to disease progression by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 16, 20, 24, 28, 32, 36, or more weeks.

[0112] In some embodiments, a method of extending the survival (e.g., overall survival or progression-free survival) of an individual with cancer is provided, comprising administering to the individual an effective amount of any of the anti-CTLA4 antibodies described herein. In some embodiments, the method extends the survival of the individual by at least any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, or 24 months.

[0113] In some embodiments, a method of alleviating one or more symptoms in a subject suffering from cancer is provided, comprising administering to the subject an effective amount of any of the anti-CTLA4 antibodies described herein. In some embodiments, the anti-CTLA4 antibody is administered in combination with one or more additional therapeutic agents.

[0114] In some embodiments, a method of improving the quality of life of an individual having cancer is provided, comprising administering to the individual an effective amount of any one of the anti-CTLA4 antibodies described herein.

[0115] III. Activatable Binding Polypeptides Targeting CTLA4

[0116] The present disclosure also relates, in part, to precision / context-dependent activatable binding polypeptides (i.e., activatable antibodies) that bind to human CTLA4, including activatable antibodies comprising any of the anti-CTLA4 antibodies described herein (e.g., anti-CTLA4 antibodies, anti-CTLA4 antibody binding fragments, and / or anti-CTLA4 antibody derivatives); antigen-binding fragments of activatable anti-CTLA4 antibodies and / or derivatives of activatable anti-CTLA4 antibodies. In some embodiments, the activatable anti-CTLA4 antibodies described herein may have an improved safety profile. For example, the anti-CTLA4 antibodies described herein may have a better safety margin, as assessed by changes in spleen weight. Changes in spleen size with increasing doses of administered drug are used as a benchmark for assessing the safety margin of drug candidates. The activatable anti-CTLA4 antibodies described herein have a better safety margin relative to the parent antibody (an antibody without a shielding moiety). In some embodiments, the activatable antibody is TY22404.

[0117] In some embodiments, the activatable antibodies of the present disclosure comprise: (a) a shielding moiety (MM); (b) a cleavable moiety (CM); and (c) a target binding moiety (TBM). In some embodiments, the MM is any shielding moiety described herein. In some embodiments, the CM is any cleavable moiety described herein. In some embodiments, the TBM is any target binding moiety described herein (e.g., a target binding moiety (TBM) comprising an antibody light chain variable region and / or an antibody heavy chain variable region, such as the VH and / or VL of any anti-CTLA4 antibody described herein).

[0118] In some embodiments, the activatable antibody comprises: (a) a polypeptide comprising, from N-terminus to C-terminus, a masking moiety (MM), a cleavable moiety (CM), and a target binding moiety (TBM), wherein the MM is any masking moiety described herein, the CM is any cleavable moiety described herein, and wherein the TBM comprises an antibody light chain variable region (VL); and (b) an antibody heavy chain variable region (VH).

[0119] In some embodiments, the activatable antibody comprises: (a) a polypeptide comprising, from N-terminus to C-terminus, a blocking moiety (MM), a cleavable moiety (CM), and a target binding moiety (TBM), wherein the MM is any blocking moiety described herein, the CM is any cleavable moiety described herein, and wherein the TBM comprises an antibody heavy chain variable region (VH); and (b) an antibody light chain variable region (VL).

[0120] In some embodiments, the activatable antibody comprises: a polypeptide comprising, from N-terminus to C-terminus, a masking moiety (MM), a cleavable moiety (CM), and a target binding moiety (TBM), wherein the MM is any masking moiety described herein, the CM is any cleavable moiety described herein, and wherein the TBM comprises an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL).

[0121] The terms "activatable binding polypeptide," "ABP," or "activatable antibody" include polypeptides comprising a target binding portion (TBM), a cleavable portion (CM), and a masking portion (MM). In some embodiments, the TBM comprises an amino acid sequence that binds to a target. In some embodiments, the TBM comprises an antigen binding domain (ABD) of an antibody or antibody fragment thereof (e.g., any antibody or antigen binding fragment described herein). In some embodiments, the antigen binding domain comprises a heavy chain variable region comprising one, two, or three of the heavy chain variable regions HVR described herein, and a light chain variable region comprising one, two, or three of the light chain variable regions HVR described herein (e.g., one, two, or three of the heavy chain variable region HVR sequences shown in Table A, and / or one, two, or three of the light chain variable region HVR sequences, including all six HVRs of any exemplary antibody shown in Table A). In some embodiments, the antigen binding domain comprises a heavy chain variable region containing any heavy chain variable region sequence described herein, and a light chain variable region containing any light chain variable region sequence described herein (e.g., a heavy chain variable region sequence and / or a light chain variable region sequence as shown in Table B). In some embodiments, the TBM (e.g., comprising an ABD) comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), wherein the VH and the VL form a binding domain that binds to a target in the absence of a MM. In some embodiments, VH and VL are covalently linked, for example, in scFv. In some embodiments, VH and VL are not covalently linked. In some embodiments, VH and VL form a Fab fragment. In some embodiments, VH is connected to an antibody heavy chain constant region, and VL is connected to an antibody light chain constant region.

[0122] Table A. Anti-CTLA4 HVR sequences

[0123]

[0124]

[0125] Table B. Anti-CTLA4 variable region amino acid sequences

[0126]

[0127]

[0128] In some embodiments, the activatable antibody comprises a polypeptide comprising a structure of a shielding moiety (MM)-cleavable moiety (CM)-VL from the N-terminus to the C-terminus, and the activatable antibody further comprises a second polypeptide comprising VH (e.g., a Fab fragment). In some embodiments, the activatable antibody comprises a polypeptide comprising a structure of a shielding moiety (MM)-cleavable moiety (CM)-VL-VH (e.g., scFv) from the N-terminus to the C-terminus. In some embodiments, the activatable antibody comprises a polypeptide comprising a structure of a shielding moiety (MM)-cleavable moiety (CM)-VH from the N-terminus to the C-terminus, and the activatable antibody further comprises a second polypeptide comprising VL (e.g., a Fab fragment). In some embodiments, the activatable antibody comprises a polypeptide comprising a structure of a shielding moiety (MM)-cleavable moiety (CM)-VH-VL (e.g., scFv) from the N-terminus to the C-terminus.

[0129] CMs typically include amino acid sequences that are cleavable, e.g., serve as substrates for enzymes, and / or cysteine-cysteine ​​pairs capable of forming reducible disulfide bonds. Thus, when the terms "cleavage," "cleavable," "cleaved," and the like are used in connection with CMs, the terms encompass enzymatic cleavage, e.g., by proteases, as well as disruption of disulfide bonds between cysteine-cysteine ​​pairs by reduction of the disulfide bonds, which can be caused by exposure to a reducing agent.

[0130] The MM refers to the amino acid sequence of the MM that interferes with or inhibits the binding of the TBM to its target when the CM of the activatable antibody is intact (e.g., not cleaved by the corresponding enzyme and / or contains unreduced cysteine-cysteine ​​disulfide bonds). In some embodiments, the MM interferes with or inhibits the binding of the TBM to its target so effectively that the binding of the TBM to its target is extremely low and / or below the limit of detection (e.g., binding cannot be detected in an ELISA or flow cytometry assay). The amino acid sequence of the CM may overlap with the MM or be included within the MM. It should be noted that, for convenience, "ABP" or "activatable antibody" is used herein to refer to the ABP or activatable antibody in its uncleaved (or "native") state as well as in its cleaved state. It will be apparent to one of ordinary skill that, in some embodiments, the cleaved ABP may lack the MM due to, for example, cleavage of the CM by a protease, which results in the release of at least the MM (e.g., when the MM is not bound to the ABP by a covalent bond (e.g., a disulfide bond between cysteine ​​residues)). Exemplary ABPs are described in more detail below.

[0131] In some embodiments, the masking moiety (MM) interferes with, hinders, reduces the ability of the target binding moiety to bind to its target, prevents, inhibits the target binding moiety from binding to its target, or competes with the target binding moiety for binding to its target (e.g., an "inactive" activatable antibody). In some embodiments, the masking moiety (MM) interferes with, hinders, reduces, prevents, inhibits the target binding moiety from binding to its target, or competes with the target binding moiety for binding to its target only when the polypeptide has not yet been activated (e.g., activated by a pH change (increase or decrease), activated by a temperature change (increase or decrease), activated after contact with a second molecule (such as a small molecule or protein ligand), etc.). In some embodiments, activation induces cleavage of the polypeptide within the cleavage moiety. In some embodiments, activation induces a conformational change in the polypeptide (e.g., a shift in the masking moiety (MM)), resulting in the masking moiety no longer preventing the activatable antibody from binding to its target. In some embodiments, the masking moiety (MM) interferes with, hinders, reduces the ability of the target binding moiety to bind to its target, prevents, inhibits, or competes with the target binding moiety for binding to its target only when the cleavable moiety (CM) has not been cleaved by one or more proteases that cleave within the cleavable moiety (CM). In some embodiments, prior to activation, the masking moiety (MM) has a masking moiety of at least about 2.0 (e.g., at least about 2.0, at least about 3.0, at least about 4.0, at least about 5.0, at least about 6.0, at least about 7.0, at least about 8.0, at least about 9.0, at least about 10, at least about 25, at least about 50, at least about 75, at least about 100, at least about 150, at least about 200, at least about 300, at least about 400, at least about 500, etc.). In some embodiments, the masking moiety is measured as the difference in affinity with which an activatable antibody comprising the masking moiety (MM) binds to its target (before activation) relative to the affinity with which a polypeptide lacking the masking moiety binds to its target (e.g., the difference in affinity for a target antigen (such as CTLA4) of an activatable antibody comprising the masking moiety (MM) (before activation) relative to a parent antibody lacking the masking moiety (MM), or the difference in affinity for a target antigen (such as CTLA4) of an activatable antibody comprising the masking moiety (MM) (before activation) relative to the affinity of the activatable antibody for the target antigen after activation). In some embodiments, the masking moiety is detected by binding of an activatable antibody comprising the masking moiety (MM) to an EC 50 (before activation) divided by the EC of the parent antibody 50 to measure (e.g. by measuring EC using ELISA 50In some embodiments, the shielding moiety is measured as the difference in affinity with which an activatable antibody comprising the shielding moiety (MM) binds to its target before activation relative to the affinity with which an activatable antibody comprising the shielding moiety (MM) binds to its target after activation (e.g., the difference in affinity for a target antigen (such as CTLA4) before activation relative to the activatable antibody after activation). In some embodiments, the shielding moiety (MM) binds to the target binding moiety (TBM) and prevents the activatable antibody from binding to its target (e.g., an "inactive" activatable antibody). In some embodiments, the shielding moiety (MM) has a dissociation constant for binding to the target binding moiety (TBM) that is greater than the dissociation constant of the target binding moiety (TBM) for its target.

[0132] In some embodiments, the masking moiety (MM) does not interfere with, hinder, reduce the ability of the target binding moiety (TBM) to bind to its target, prevent, inhibit, or compete with the target binding moiety (TBM) for binding to its target after the activatable antibody has been activated (e.g., by treatment with one or more proteases that cleave within the cleavable moiety (CM), by a change in pH (increase or decrease), by a shift in temperature (increase or decrease), upon contact with a second molecule (such as an enzyme or protein ligand), etc.). In some embodiments, the masking moiety (MM) does not interfere with, hinder, reduce the ability of the target binding moiety (TBM) to bind to its target, prevent, inhibit, or compete with the target binding moiety (TBM) for binding to its target after the cleavable moiety (CM) has been cleaved by one or more proteases that cleave within the cleavable moiety (CM). In some embodiments, after activation, the masking moiety (MM) has a masking moiety of at most about 1.75 (e.g., at most about 1.75, at most about 1.5, at most about 1.4, at most about 1.3, at most about 1.2, at most about 1.1, at most about 1.0, at most about 0.9, at most about 0.8, at most about 0.7, at most about 0.6, or at most about 0.5, etc.).

[0133] In some embodiments, the activatable antibodies of the present disclosure contain a shielding moiety (MM) comprising a pair of cysteine ​​residues at fixed positions to ensure that the activatable antibody has a constrained conformation, and / or has few or no chemically labile residues (such as methionine or tryptophan). Advantageously, the inclusion of a pair of cysteine ​​residues at fixed positions ensures that the activatable antibody has a constrained conformation, thereby tending to exhibit increased binding affinity and / or specificity. In addition, the activatable antibodies of the present disclosure include shielding moieties that have few or no residues that are detrimental to the manufacturing process, such as methionine or tryptophan.

[0134] In some embodiments, the activatable antibodies of the present disclosure are context-dependent (e.g., they are activated (only able to bind to their targets) under certain circumstances, such as in a protease-rich tumor microenvironment). In some embodiments, the activatable antibodies of the present disclosure provide improved safety over more traditional non-activatable antibodies (e.g., exhibit reduced toxicity, do not induce significant changes in the weight of many organs, do not alter liver tissue pathology, hematology, and / or blood biochemistry, etc.). In some embodiments, the activatable antibodies of the present disclosure have improved pharmacokinetic properties (e.g., have a longer in vivo half-life) compared to more traditional non-activatable antibodies.

[0135] Anti-CTLA4 activatable antibody activity

[0136] In some embodiments, the present disclosure relates to activatable antibodies that bind to human CTLA4 when in active form (e.g., the activatable antibodies are active after cleavage in the cleavable portion (e.g., by one or more proteases), but are inactive before cleavage in the cleavable portion (e.g., by one or more proteases). In some embodiments, when in active form, the activatable antibodies have at least one (e.g., at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or all nine) of the following functional properties: (a) with a K of 500 nM or less, e.g., about 10 nM or less; (b) with a K of 500 nM or less, e.g., about 10 nM or less; (c) with a K of 500 nM or less, e.g., about 10 nM or less; (d) with a K of 500 nM or less, e.g., about 10 nM or less; (e) with a K of 500 nM or less, e.g., about 10 nM or less; DBinds to human, cynomolgus monkey, mouse, rat and / or dog CTLA4; (b) has antagonistic activity against human CTLA4; (c) does not bind to human PD-1, PD-L1, PD-L2, LAG3, TIM3, B7-H3, CD95, CD120a, OX40, CD40, BTLA, VISTA, ICOS and / or B7-H4 at concentrations up to 100 nM; (d) has cross-reactivity with monkey, mouse, rat and / or dog CTLA4; (e) induces ADCC effects (e.g., on Tregs); (f) activates human PBMCs (e.g., stimulates the secretion of IL-2 and / or IFNγ); (g) is capable of inhibiting tumor cell growth; (h) has a therapeutic effect on cancer; and (i) inhibits the binding of human CTLA4 to human CD80 and / or human CD86. Also provided herein are one or more activatable antibodies that compete or cross-compete for binding to human CTLA4 with one or more of the activatable antibodies targeting CTLA4 and / or anti-CTLA4 antibodies described herein.

[0137] In some embodiments, when in an inactive form, the activatable antibody can be activated with a K of about 500 nM or greater. D Binds to human, cynomolgus monkey, mouse, rat and / or dog CTLA4. In some embodiments, when in active form, the activatable antibody has a K of about 500 nM or less (e.g., about 500 nM or less, about 450 nM or less, about 400 nM or less, about 350 nM or less, about 300 nM or less, about 250 nM or less, about 200 nM or less, about 150 nM or less, about 100 nM or less, about 90 nM or less, about 80 nM or less, about 70 nM or less, about 60 nM or less, about 50 nM or less, about 40 nM or less, about 30 nM or less, about 25 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less, about 0.1 nM or less, etc.). D Binds to human, cynomolgus monkey, mouse, rat and / or dog CTLA4. In some embodiments, when in active form, the activatable antibody has a K of about 350 nM or less. D Binds to human, cynomolgus monkey, mouse, rat and / or dog CTLA4. In some embodiments, when in active form, the activatable antibody has a K of about 100 nM or less. D Binds to human CTLA4. In some embodiments, when in active form, the activatable antibody binds to human CTLA4 with a K of about 50 nM or less. D Binds to human CTLA4. In some embodiments, when in active form, the activatable antibody binds to human CTLA4 with a K of about 10 nM or less. D Binds to human CTLA4. Measure K of activatable antibodies DThe method can be performed using any method known in the art, including, for example, by surface plasmon resonance, ELISA, isothermal titration calorimetry, filter binding assay, EMSA, etc. In some embodiments, K D Measured by ELISA (see, e.g., the Examples below).

[0138] In some embodiments, when in an inactive form, the activatable antibody does not have antagonistic activity against human CTLA4. In some embodiments, when in an active form, the activatable antibody has antagonistic activity against human CTLA4 (e.g., inducing ADCC effects (such as for Treg), activating PBMC (such as by activating, inducing and / or stimulating IL-2 and / or IFNγ secretion), blocking the binding of human CTLA4 to human CD80 and / or human CD86, etc.). In some embodiments, when in an active form, the activatable antibody suppresses one or more activities of human CTLA4 (e.g., when a cell expressing human CTLA4 (such as a human cell) is contacted by an activatable antibody, one or more activities of human CTLA4 are suppressed).

[0139] In some embodiments, when in an inactive form, the activatable antibody does not cross-react with monkey (e.g., cynomolgus monkey), mouse, rat, and / or dog CTLA4. In some embodiments, when in an active form, the activatable antibody has cross-reactivity with monkey (e.g., cynomolgus monkey), mouse, rat, and / or dog CTLA4. In some embodiments, when in an active form, the activatable antibody has cross-reactivity with monkey CTLA4. In some embodiments, when in an active form, the activatable antibody has cross-reactivity with mouse CTLA4. In some embodiments, when in an active form, the activatable antibody has cross-reactivity with rat CTLA4. In some embodiments, when in an active form, the activatable antibody has cross-reactivity with dog CTLA4. In some embodiments, when in active form, the activatable antibody cross-reacts with monkey and mouse CTLA4; monkey and rat CTLA4; monkey and dog CTLA4; mouse and rat CTLA4; mouse and dog CTLA4; rat and dog CTLA4; monkey, mouse, and rat CTLA4; monkey, mouse, and dog CTLA4; monkey, rat, and dog CTLA4; mouse, rat, and dog CTLA4; or monkey, mouse, rat, and dog CTLA4. In some embodiments, when in active form, the activatable binding polypeptide cross-reacts at about 350 nM (e.g., at about 1 nM, at about 10 nM, at about 25 nM, at about 50 nM, at about 75 nM, at about 100 nM, at about 150 nM, at about 200 nM, at about 250 nM, at about 300 nM, at about 350 nM). Methods for measuring cross-reactivity are known in the art and include, but are not limited to, surface plasmon resonance, ELISA, isothermal titration calorimetry, filter binding assays, EMSA, and the like.

[0140] In some embodiments, when in an inactive form, the activatable antibody does not induce ADCC effects (e.g., for human cells expressing CTLA4 such as Treg). In some embodiments, compared to a control binding polypeptide (e.g., a parent antibody), when in an inactive form, the activatable antibody has a reduced ADCC effect (e.g., for human cells expressing CTLA4 such as Treg). In some embodiments, when in an active form, the activatable antibody induces ADCC effects (e.g., for cells expressing CTLA4 such as Treg). Methods for measuring ADCC effects (e.g., in vitro methods) are known in the art, including but not limited to the methods described in the following examples. In some embodiments, relative to a control (e.g., a parent antibody), when in an inactive form, the activatable antibody induces an ADCC effect of less than about 10% (e.g., induces ADCC less than about 10%, less than about 5%, less than about 1%, etc.). In some embodiments, the activatable antibody induces an ADCC effect greater than about 10% (e.g., induces ADCC greater than about 10%, greater than about 15%, greater than about 20%, greater than about 25%, greater than about 30%, greater than about 35%, greater than about 40%, etc.) when in active form relative to a control (e.g., an isotype control).

[0141] In some embodiments, the activatable antibody is capable of inhibiting tumor cell growth and / or proliferation. In some embodiments, relative to corresponding tumor cells not contacted with the activatable antibody (or relative to corresponding tumor cells contacted with an isotype control antibody), when contacted with the activatable antibody, tumor cell growth and / or proliferation is inhibited by at least about 5% (e.g., at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or at least about 99%). In some embodiments, when an activatable antibody is administered to a subject, the activatable antibody is capable of reducing the tumor volume in the subject. In some embodiments, the activatable antibody is capable of reducing the volume of a tumor in the subject by at least about 5% (e.g., at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 99%) relative to the initial tumor volume in the subject (e.g., before administration of the activatable antibody; compared to a corresponding tumor in a subject administered an isotype control antibody). Methods for monitoring tumor cell growth / proliferation, tumor volume, and / or tumor inhibition are known in the art, including, for example, by the methods described in the Examples below.

[0142] In some embodiments, the activatable antibody has a therapeutic effect on cancer. In some embodiments, the activatable antibody alleviates one or more signs or symptoms of cancer. In some embodiments, when the activatable antibody is administered, the subject suffering from cancer obtains partial or complete remission.

[0143] In some embodiments, the present disclosure provides isolated, activatable antibodies that, when in active form, compete or cross-compete for binding to human CTLA4 with an antibody comprising: a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 23; HVR-H2 comprising the amino acid sequence of SEQ ID NO: 35; and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 45; and / or b) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 58; HVR-L2 comprising the amino acid sequence of SEQ ID NO: 66; and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 75. In some embodiments, the present disclosure provides isolated, activatable antibodies that, when in active form, compete or cross-compete for binding to human CTLA4 with an antibody comprising: a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 87; and / or b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 100. The ability of an activatable antibody to compete or cross-compete with an antibody can be determined using standard binding assays known in the art such as BIAcore analysis, ELISA assays, or flow cytometry. For example, an antibody (e.g., as described above) can be allowed to bind to human CTLA4 under saturation conditions, followed by measurement of the ability of the test activatable antibody (when in active form) to bind to CTLA4. If the test activatable antibody is able to bind to CTLA4 simultaneously with the antibody, then the test activatable antibody is tested for binding to different epitopes from the antibody. However, if the test activatable antibody cannot bind to CTLA4 simultaneously, then the test activatable antibody is tested for binding to the same epitope, overlapping epitopes, or an epitope that is closely adjacent to the epitope bound by the antibody. This experiment can be performed using various methods such as ELISA, RIA, FACS, or surface plasmon resonance.

[0144] In some embodiments, the activatable antibody (when in an inactive form) does not inhibit the binding between CTLA4 and one or more of its binding partners (e.g., human CTLA4 and human CD80, human CTLA4 and human CD86). In some embodiments, the activatable antibody (when in an active form) inhibits the binding between CTLA4 and one or more of its binding partners (e.g., human CTLA4 and human CD80, human CTLA4 and human CD86). In some embodiments, the activatable antibody inhibits the binding between CTLA4 and its ligand in vitro. In some embodiments, the activatable antibody has a half-maximal inhibitory concentration (IC) of about 500 nM or less (e.g., about 500 nM or less, about 400 nM or less, about 300 nM or less, about 200 nM or less, about 100 nM or less, about 50 nM or less, about 25 nM or less, about 10 nM or less, about 1 nM or less, etc.) for inhibiting the binding of CTLA4 to CD80 and / or CD86. 50 In some embodiments, the activatable antibody has a half-maximal inhibitory concentration (IC) of about 100 nM or less for inhibiting the binding of CTLA4 to CD80 and / or CD86. 50 ). In some embodiments, when provided at a concentration of about 100 nM or greater (e.g., about 100 nM or greater, about 500 nM or greater, about 1 μM or greater, about 10 μM or greater, etc.), the activatable antibody completely inhibits the binding of human CTLA4 to CD80 and / or CD86. As used herein, the term "completely inhibiting / completely inhibits" means that the activatable antibody is able to reduce the binding between the first protein and the second protein by at least about 80% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, etc.). Methods for measuring the ability of a polypeptide to inhibit the binding of a first protein (e.g., human CTLA4) and a second protein (e.g., human CD80 or human CD86) are known in the art, including but not limited to BIAcore analysis, ELISA assays, and flow cytometry.

[0145] Shielding part (MM)

[0146] In some embodiments, the present disclosure relates to an activatable antibody comprising a masking moiety (MM). In some embodiments, the masking moiety (MM) comprises a moiety according to formula (XIX): Z m CZ n CZ o(SEQ ID NO: 135), wherein m is 2 to 10, n is 3 to 10, and o is 1 to 10, and each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P. In some embodiments, m is 6 to 8. In some embodiments, m is 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, m is 6. In some embodiments, n is 6 to 8. In some embodiments, n is 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, n is 6. In some embodiments, n is 8. In some embodiments, o is 1 to 2. In some embodiments, o is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, o is 2.

[0147] In some embodiments, the masking moiety (MM) comprises an amino acid sequence according to formula (XXI): Z6CX6CZ2 (SEQ ID NO: 137), wherein each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, and wherein each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P.

[0148] In some embodiments, the masking moiety (MM) comprises an amino acid sequence according to formula (XXII): Z6CX8CZ2 (SEQ ID NO: 138), wherein each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, and wherein each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P.

[0149] In some embodiments, the first peptide (FP) comprises an amino acid sequence according to formula (XXIII): (Z6)C(Z6)C(Z2) (SEQ ID NO: 139), wherein each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H and P.

[0150] In some embodiments, the masking moiety (MM) comprises an amino acid sequence according to Formula (XXIV): (Z6)C(Z8)C(Z2) (SEQ ID NO: 140), wherein each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P. In some embodiments, the activatable antibody comprises an amino acid selected from the group consisting of X m CPDHPYPCXX (SEQ ID NO: 181), Xm CDAFYPYCXX (SEQ ID NO: 182), X m CDSHYPYCXX (SEQ ID NO: 183) and X m CVPYYYACXX (SEQ ID NO: 184), wherein m is 2-10, and wherein each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y. In some embodiments, the activatable antibody comprises a masking moiety (MM) comprising the sequence EVGSYNFVADSCPDHPYPCSA (SEQ ID NO: 189), EVGSYIVHHSDCDAFYPYCDS (SEQ ID NO: 190), EVGSYYSAYPACDSHYPYCNS (SEQ ID NO: 191), EVGSYPNPSSDCVPYYYACAY (SEQ ID NO: 192), EVGSYYSAYPACDSHYPYCQS (SEQ ID NO: 193), EVGSYPQPSSDCVPYYYACAY (SEQ ID NO: 195), or EVGSYPNPASDCVPYYYACAY (SEQ ID NO: 196). In some embodiments, the MM comprises the sequence EDCVPYYYACAY (SEQ ID NO: 213), EVGSSDCVPYYYACAY (SEQ ID NO: 214), EDCDAFYPYCDS (SEQ ID NO: 215), or EVGHSDCDAFYPYCDS (SEQ ID NO: 216).

[0151] In some embodiments, the masking moiety (MM) comprises an amino acid sequence selected from NFVADSCPDHPYPCSA (SEQ ID NO: 141), IVHHSDCDAFYPYCDS (SEQ ID NO: 142), YSAYPACDSHYPYCNS (SEQ ID NO: 143), PNPSDCVPYYYACAY (SEQ ID NO: 144), YSAYPACDSHYPYCQS (SEQ ID NO: 145), PQPSSDCVPYYYACAY (SEQ ID NO: 146), and PNPASDCVPYYYACAY (SEQ ID NO: 147).

[0152] In some embodiments, any masking moiety (MM) described herein may further comprise one or more additional amino acid sequences (e.g., one or more polypeptide tags). Examples of suitable additional amino acid sequences may include, but are not limited to, purification tags (such as his tags, flag tags, maltose binding protein, and glutathione-S-transferase tags), detection tags (such as tags detectable photometrically (e.g., red or green fluorescent proteins, etc.), tags with detectable enzymatic activity (e.g., alkaline phosphatase, etc.), tags containing secretion sequences, leader sequences, and / or stabilization sequences, protease cleavage sites (e.g., furin cleavage sites, TEV cleavage sites, thrombin cleavage sites), and the like. In some embodiments, the one or more additional amino acid sequences are at the N-terminus of the masking moiety (MM). In some embodiments, the additional amino acid sequence comprises or consists of the sequence EVGSY (SEQ ID NO: 148).

[0153] In some embodiments, the masking moiety binds to the target binding moiety (TBM) and inhibits the activatable antibody from binding to its target before activation (e.g., before treatment with one or more proteases that cleave within the cleavable moiety (CM), before subjecting to a (local) pH change (increase or decrease), before subjecting to a temperature shift (increase or decrease), before contacting with a second molecule (such as a small molecule or protein ligand), etc.), but does not bind to the TBM and / or inhibit the activatable antibody from binding to its target after activation (e.g., after treatment with one or more proteases that cleave within the cleavable moiety (CM), after subjecting to a (local) pH change (increase or decrease), after subjecting to a temperature shift (increase or decrease), after contacting with a second molecule (such as a small molecule or protein ligand), etc.). In some embodiments, the masking moiety (MM) inhibits the binding of the activatable antibody to its target when the CM is not cleaved, but does not inhibit the binding of the activatable antibody to its target when the CM is cleaved. In some embodiments, the masking moiety (MM) has a dissociation constant for binding to the TBM that is greater than the dissociation constant of the activatable antibody for its target (when in active form) (e.g., at least about 1.5-fold greater, at least about 2-fold greater, at least about 2.5-fold greater, at least about 3-fold greater, at least about 3.5-fold greater, at least about 4-fold greater, at least about 4.5-fold greater, at least about 5-fold greater, at least about 10-fold greater, at least about 100-fold greater, at least about 500-fold greater, etc.).

[0154] Cleavable portion (CM)

[0155] In some embodiments, the present disclosure relates to activatable antibodies comprising a cleavable moiety (CM). In some embodiments, the cleavable moiety (CM) is cleaved and / or destroyed by treatment with one or more proteases that cleave within the cleavable moiety (CM), by a change in pH (increase or decrease), by a shift in temperature (increase or decrease), and / or by contact with a second molecule (such as a small molecule or protein ligand).

[0156] In some embodiments, the cleavable moiety (CM) comprises at least a first cleavage site (CS1) (e.g., a first protease cleavage site). In some embodiments, the first cleavage site is a first protease cleavage site. Any suitable protease cleavage site that is recognized and / or cleaved by any protease known in the art (e.g., a protease known to co-localize with a target of an activatable antibody comprising a CM) can be used, including, for example, protease cleavage sites recognized and / or cleaved by urokinase-type plasminogen activator (uPA); matrix metalloproteinases (e.g., MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-16, MMP-17, MMP-19, MMP-20, MMP-21, MMP-22, MMP-23, MMP-24, MMP-25, MMP-26, MMP-27, MMP-28, MMP-29, MMP-30, MMP-31, MMP-32, MMP-33, MMP-34, MMP-35, MMP-36, MMP-37, MMP-38, MMP-39, MMP-40, MMP-41, MMP-42, MMP-43, MMP-44, MMP-45, MMP-46, MMP-47, MMP-48, MMP-49, MMP-50, MMP-51, MMP-52, MMP-53, MMP-54, MMP-55, MMP-56, MMP-57, MMP-58, MMP-59, MMP-6 3, MMP-24, MMP-26 and / or MMP-27); tobacco etch virus (TEV) protease; plasmin; thrombin; PSA; PSMA; ADAMS / ADAMTS (e.g., ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAMDEC1, ADAMTS1, ADAMTS4 and / or ADAMTS5); caspers (e.g., caspers-1, caspers-2, caspers-3, caspers-4, caspers-5, caspers-6, caspers-7, caspers-8, caspers-9, caspers , caspase-10, caspase-11, caspase-12, caspase-13 and / or caspase-14); aspartic proteases (e.g., RACE and / or Renin); aspartic cathepsins (e.g., cathepsin D and / or cathepsin E); cysteine ​​cathepsins (e.g., cathepsin B, cathepsin C, cathepsin K, cathepsin L, cathepsin S, cathepsin V / L2 and / or cathepsin X / Z / P); cysteine ​​proteases (e.g., Cruzipain, Legumain and / or Otubain-2); KLKs (e.g., KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13 and / or KLK14); metalloproteinases (e.g., meprin, neprilysin, PSMA and / or BMP-1); serine proteases (e.g., activated protein C, cathepsin A, cathepsin G, chymase and / or coagulation factor proteases (such as FVIIa, FIXa, FXa, FXIa, FXIIa)); elastase; granzyme B; guanidinobenzoic acidase; HtrA1; human neutrophil elastase;Lactoferrin; marapsin; NS3 / 4A; PACE4; tPA; tryptase; type II transmembrane serine protease (TTSP) (e.g., DESCl, DPP-4, FAP, hepsin, matriptase-2, MT-SP1 / matriptase, TMPRSS2, TMPRSS3 and / or TMPRSS4); etc. In some embodiments, the first protease cleavage site is a cleavage site of a protease selected from the group consisting of uPA, MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, TEV protease, plasmin, thrombin, Factor X, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAM10, ADAM12, ADAMTS, Caspase-1, Caspase-2, Caspase-3, Caspase-4, Caspase-5, Caspase-6, Caspase-7, Caspase-8, Caspase-9, Caspase-10, Caspase-11, Caspase-12, Caspase-13, Caspase-14, and TACE. In some embodiments, the first protease cleavage site is a cleavage site of a protease selected from the group consisting of uPA, MMP-2, and / or MMP-9. In some embodiments, the protease cleavage site comprises an amino acid sequence selected from SGRSA (SEQ ID NO: 149), PLGLAG (SEQ ID NO: 150).

[0157] Any suitable linker known in the art (e.g., a flexible linker) can be used, including, for example: a glycine polymer (G)n, wherein n is an integer of at least 1 (e.g., at least one, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, etc.); a glycine-serine polymer (GS)n, wherein n is an integer of at least 1 (e.g., at least one, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, etc.), such as GGGGS (SEQ ID NO: 156), SGGS (SEQ ID NO: 157), GGSG (SEQ ID NO: 158), GGSGG (SEQ ID NO: 159), GSGSG (SEQ ID NO: 160), GSGGG (SEQ ID NO: 161), GGGSG (SEQ ID NO: 162), and / or GSSSG (SEQ ID NO: 163); a glycine-alanine polymer; an alanine-serine polymer; etc. The linker sequence can be of any length, such as from about 1 amino acid (e.g., glycine or serine) to about 20 amino acids (e.g., a 20 amino acid glycine polymer or a glycine-serine polymer), from about 4 amino acids to about 10 amino acids, from about 5 amino acids to about 9 amino acids, from about 6 amino acids to about 8 amino acids, etc. In some embodiments, the length of the linker is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In some embodiments, the linker comprises an amino acid sequence selected from SEQ ID NOs: 159-163. In some embodiments, the linker comprises an amino acid sequence of SEQ ID NOs: 156 or 157.

[0158] In some embodiments, the cleavable moiety (CM) further comprises at least a second cleavage site (e.g., at least a second, at least a third, at least a fourth, at least a fifth, etc.). In some embodiments, the cleavable moiety (CM) further comprises a second cleavage site (CS2). In some embodiments, the second cleavage site is a second protease cleavage site. The second protease cleavage site can be any suitable protease cleavage site recognized and / or cleaved by any of the proteases described above. In some embodiments, the first (CS1) and second (CS2) cleavage sites are protease cleavage sites recognized and / or cleaved by the same protease. In some embodiments, the first (CS1) and second (CS2) cleavage sites are protease cleavage sites that are recognized and / or cleaved by different proteases (e.g., the first protease cleavage site is recognized and / or cleaved by uPA, while the second protease cleavage site is recognized and / or cleaved by MMP-2; the first protease cleavage site is recognized and / or cleaved by uPA, while the second protease cleavage site is recognized and / or cleaved by MMP-9; in some embodiments, at least the second cleavage site (CS2) is at the C-terminus of the first linker (L1). In some embodiments, the cleavable moiety (CM) comprises the following structure from N-terminus to C-terminus: (CS1)-L1-(CS2).

[0159] In some embodiments, the cleavable portion (CM) further comprises at least a second linker (e.g., at least a second, at least a third, at least a fourth, at least a fifth, etc.). In some embodiments, the cleavable portion (CM) further comprises a second linker (L2). The second linker (L2) can be any suitable linker described above. In some embodiments, the second linker comprises an amino acid sequence selected from SEQ ID NOs: 156 to 163. In some embodiments, the first (L1) and second (L2) linkers are the same (e.g., both linkers comprise the sequence of SEQ ID NOs: 156 or 157). In some embodiments, the first (L1) and second (L2) linkers are different (e.g., the first linker (L1) comprises the amino acid sequence of SEQ ID NO: 156, and the second linker (L2) comprises the amino acid sequence of SEQ ID NO: 157, etc.). In some embodiments, the second linker (L2) is at least the C-terminus of the second cleavage site (CS2). In some embodiments, the cleavable portion (CM) comprises the structure of (CS1)-L1-(CS2)-L2 from N-terminus to C-terminus.

[0160] In some embodiments, the cleavable portion (CM) further comprises at least a second linker (e.g., at least a second, at least a third, at least a fourth, at least a fifth, etc.). In some embodiments, the cleavable portion (CM) further comprises a second linker (L2). The second linker (L2) can be any suitable linker as described above. In some embodiments, the second linker comprises an amino acid sequence selected from SEQ ID NOs: 156-163. In some embodiments, the first (L1) and second (L2) linkers are identical (e.g., both linkers comprise a sequence of SEQ ID NOs: 156 or 157). In some embodiments, the first (L1) and second (L2) linkers are different (e.g., the first linker (L1) comprises an amino acid sequence of SEQ ID NO: 156, and the second linker (L2) comprises an amino acid sequence of SEQ ID NO: 157, etc.). In some embodiments, at least the second linker (L2) is at the C-terminus of the second cleavage site (CS2). In some embodiments, the cleavable portion (CM) comprises the following structure from N-terminus to C-terminus: (CS1)-L1-(CS2)-L2.

[0161] Exemplary MM-CM sequences

[0162] In some embodiments, an activatable antibody of the present disclosure comprises the following structure from N-terminus to C-terminus: (FP)-(PCS1)-L1-(PCS2)-L2. In some embodiments, an activatable antibody of the invention comprises the following amino acid sequence: EVGSYNFVADSCPDHPYPCSASGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 168); EVGSYIVHHSDCDAFYPYCDSSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 170); EVGSYYSAYPACDSHYPYCNSSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 172); EVGSYPNPSSDCVPYYYACAYSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 174); EVGSYYSAYPACDSHYPYCQSSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 176); EVGSYYSAYPACDSHYPYCNSAGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 177). In some embodiments, the polypeptide of the present invention comprises a structure of (FP)-(PCS1)-L1-(PCS2)-L2-(TBM) from the N-terminus to the C-terminus.

[0163] In some embodiments, the activatable antibody comprises the amino acid sequence SGRSAGGGGTENLYFQGSGGS (SEQ ID NO: 220), SGRSAGGGGTPLGLAGSGGS (SEQ ID NO: 221), or SGRSAPLGLA (SEQ ID NO: 222). In some embodiments, the activatable antibody comprises the sequence EV(Zn)C(X8)C(Z2)SGRSA (SEQ ID NO: 217), EDC(Z6)C(Z2)SGRSA (SEQ ID NO: 218), or EDC(Z6)C(Z2)PLGLA (SEQ ID NO: 219), wherein each X is independently an amino acid selected from the group consisting of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, wherein n is 1-11, and wherein each Z is independently an amino acid selected from the group consisting of D, A, Y, S, T, N, I, L, F, V, H, and P.

[0164] In certain embodiments, the MM comprises the amino acid sequence EVGSYPNPSSDCVPYYYACAY (SEQ ID NO: 192), and the cleavable moiety comprises the amino acid sequence SGRSAGGGGTPLGLAGSGGS (SEQ ID NO: 221). In such embodiments, the MM and CM comprise, from the N-terminus to the C-terminus, the amino acid sequence EVGSYPNPSSDCVPYYYACAYSGRSAGGGGTPLGLAGSGGS (SEQ ID NO: 200). In some embodiments, the MM and CM are covalently linked to the N-terminus of the anti-CTLA4 antibody light chain. In some embodiments, the MM and CM comprise, from the N-terminus to the C-terminus, an amino acid sequence that has at least 90% or at least 95% sequence identity to SEQ ID NO: 200.

[0165] Target Binding Moiety (TBM)

[0166] In some embodiments, the present disclosure relates to an activatable antibody comprising a target binding portion (TBM). In some embodiments, the target binding portion (TBM) comprises an antibody light chain variable region and / or an antibody heavy chain variable region. In some embodiments, the target binding portion (TBM) comprises an antibody light chain variable region. In some embodiments, the target binding portion (TBM) comprises an antibody heavy chain variable region. In some embodiments, the target binding portion (TBM) comprises an antibody light chain variable region and an antibody heavy chain variable region.

[0167] In some embodiments, the target binding moiety (TBM) comprises a full-length antibody light chain and / or a full-length antibody heavy chain. The antibody light chain may be a kappa or lambda light chain. The antibody heavy chain may be in any class, such as IgG, IgM, IgE, IgA, or IgD. In some embodiments, the antibody heavy chain is in the IgG class, such as IgG1, IgG2, IgG3, or IgG4 subclass. The antibody heavy chains described herein can be converted from one class or subclass to another class or subclass using methods known in the art.

[0168] Any one or more of the target binding moieties (TBMs) described herein may be combined with: any HVR sequence described herein (e.g., one, two, or three of the heavy chain variable region HVR sequences, and / or one, two, or three of the light chain variable region HVR sequences, as shown in Table A above); any heavy chain variable region sequence and / or light chain variable region sequence described herein (e.g., a heavy chain variable region sequence and / or a light chain variable region sequence, as shown in Table B above); and / or any antibody described herein.

[0169] In some embodiments, the target binding moiety (TBM) comprises the sequence of one or more of the anti-CTLA4 antibodies described herein, including the antibodies described with respect to the specific amino acid sequences of the HVRs, variable regions (VL, VH), and / or light and heavy chains (e.g., IgG1, IgG2, IgG4). In some embodiments, the target binding moiety (TBM) comprises an antibody light chain variable region comprising an HVR-L1 comprising the amino acid sequence RASQSVRGRFLA (SEQ ID NO: 58), an HVR-L2 comprising the amino acid sequence DASNRATGI (SEQ ID NO: 66), and / or an HVR-L3 comprising the amino acid sequence YCQQSSSWPPT (SEQ ID NO: 75). In some embodiments, the target binding moiety (TBM) comprises an antibody light chain variable region comprising the amino acid sequence of SEQ ID NO: 100, or a sequence having at least 90% (e.g., 95%, 96%, 97%, 98%, or 99%) sequence identity to the sequence of SEQ ID NO: 100. In some embodiments, the target binding moiety (TBM) comprises an antibody heavy chain variable region comprising HVR-H1 comprising the amino acid sequence of YSISSGYHWSWI (SEQ ID NO: 23), HVR-H2 comprising the amino acid sequence of LARIDWDDDKYYSTSLKSRL (SEQ ID NO: 35), and / or HVR-H3 comprising the amino acid sequence of ARSYVYFDY (SEQ ID NO: 45). In some embodiments, the target binding moiety (TBM) comprises an antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO:87 or a sequence having at least 90% (e.g., 95%, 96%, 97%, 98% or 99%) sequence identity to the sequence of SEQ ID NO:87. In some embodiments, the target binding moiety (TBM) comprises: a) an antibody light chain variable region comprising HVR-L1 comprising the amino acid sequence of RASQSVRGRFLA (SEQ ID NO:58), HVR-L2 comprising the amino acid sequence of DASNRATGI (SEQ ID NO:66), and / or HVR-L3 comprising the amino acid sequence of YCQQSSSWPPT (SEQ ID NO:75); and b) an antibody heavy chain variable region comprising HVR-H1 comprising the amino acid sequence of YSISSGYHWSWI (SEQ ID NO:23), HVR-H2 comprising the amino acid sequence of LARIDWDDDKYYSTSLKSRL (SEQ ID NO:35), and / or HVR-H3 comprising the amino acid sequence of ARSYVYFDY (SEQ ID NO:45).In some embodiments, the target binding moiety (TBM) comprises an antibody light chain variable region comprising the amino acid sequence of SEQ ID NO: 100, and an antibody heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 87.

[0170] Activatable binding peptide properties

[0171] In some embodiments, the activatable binding polypeptides (i.e., activatable antibodies) of the present disclosure comprise: (a) a masking moiety (MM), (b) a cleavable moiety, and (c) a target binding moiety. In some embodiments, the masking moiety (MM) binds to the target binding moiety (TBM) of the activatable antibody and reduces or inhibits the binding of the activatable binding moiety to CTLA4 (e.g., human CTLA4) compared to the binding of the corresponding binding polypeptide lacking the masking moiety to CTLA4 (e.g., human CTLA4) and / or compared to the binding of the parent antibody to CTLA4 (e.g., human CTLA4).

[0172] In some embodiments, an "activatable" binding polypeptide refers to a binding polypeptide that exhibits a first level of binding to CTLA4 when in an inhibited, masked, and / or uncleaved state, and exhibits a second level of binding to CTLA4 when in an uninhibited, unmasked, and / or cleaved state, wherein the second level of CTLA4 binding is greater than the first level of CTLA4 binding. In some embodiments, upon cleavage within the cleavable moiety (e.g., by one or more proteases), the accessibility of the activatable binding polypeptide to CTLA4 is increased.

[0173] In some embodiments, the activatable anti-CTLA4 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 320 and a light chain comprising the amino acid sequence of SEQ ID NO: 322. In some embodiments, the activatable anti-CTLA4 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 321 and a light chain comprising the amino acid sequence of SEQ ID NO: 322. The activatable antibody having a heavy chain of SEQ ID NO: 320 and a light chain of SEQ ID NO: 322 is referred to as TY22404. In some embodiments, the activatable antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 320 and a light chain comprising the amino acid sequence of SEQ ID NO: 322. In some embodiments, the activatable anti-CTLA4 antibody comprises a heavy chain having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 320. In some embodiments, the activatable anti-CTLA4 antibody comprises a light chain having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 321. In some embodiments, the activatable anti-CTLA4 antibody comprises a heavy chain having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 320. In some embodiments, the activatable anti-CTLA4 antibody comprises a light chain having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 322. In some embodiments, the activatable antibody is TY22404.

[0174] In some embodiments, the binding affinity of the polypeptide for CTLA4 (e.g., human CTLA4) is increased by at least about 2-fold (e.g., at least about 2-fold, at least about 2.5-fold, at least about 3-fold, or at least about 5-fold) after activation of the activatable antibody (e.g., after activation by treatment with one or more proteases that cleave within the cleavable moiety (CM), after activation by a pH change (increase or decrease), after activation by a temperature shift (increase or decrease), after activation by contact with a second molecule (such as a small molecule), etc.) as compared to before activation of the activatable antibody. 3, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold, at least about 9.5-fold, at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 250-fold, at least about 500-fold, at least about 750-fold, or at least about 1000-fold or more), the activatable antibodies of the present disclosure are generally considered to be "activatable" binding polypeptides if, after "activation," the EC of the activatable antibody is 50 An activatable antibody of the present disclosure is generally considered "activatable" if its activity is reduced by at least about 2-fold (e.g., at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold, at least about 9.5-fold, at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 250-fold, at least about 500-fold, at least about 750-fold, or at least about 1000-fold, or less) (e.g., as measured by an ELISA or FACS assay; see the Examples below). In some embodiments, if the EC of the polypeptide is 50 An activatable antibody of the present disclosure is generally considered "activatable" if its expression level is reduced by at least about 1 / 2 (e.g., as measured by ELISA or FACS assay; see Examples below).

[0175] In some embodiments, when the masking moiety (MM) is bound to the target binding moiety (TBM) of the activatable antibody, the K of the activatable antibody for CTLA4 is Dis when the masking moiety (MM) is not bound to the target binding moiety (TBM) (e.g., after "activation" of the activatable antibody (such as after protease treatment to cleave within the cleavable moiety (CM))) and / or is the K of the parent antibody with respect to CTLA4 D Methods of measuring affinity are known in the art and include, for example, the methods described in the examples below.

[0176] In some embodiments, the K of the parent antibody for CTLA4 is relative to when the shielding moiety is not bound to the target binding moiety (e.g., after "activation" of the activatable antibody (such as after protease treatment to cleave within the cleavable moiety (CM))), and / or relative to the K of the parent antibody for CTLA4. D When the shielding portion is bound to the target binding portion of the activatable antibody, the K of the activatable antibody with respect to CTLA4 is D By at least about 25% (e.g., at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%). Methods of measuring affinity are known in the art and include, for example, the methods described in the Examples below.

[0177] In some embodiments, the blocking moiety sterically hinders the binding of the activatable antibody to CTLA4 and / or allosterically hinders the binding of the activatable antibody to CTLA4. In some embodiments, the blocking moiety does not comprise the amino acid sequence of the natural binding partner of the activatable antibody and / or parent antibody.

[0178] In some embodiments, the dissociation constant of the shielding portion with respect to the target binding portion is greater than the dissociation constant of the activatable antibody with respect to CTLA4 (when activated). In some embodiments, the dissociation constant of the shielding portion with respect to the target binding portion is about 2 (e.g., about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 25, about 50, about 75, about 100, about 250, about 500, about 750, or about 1000 or more) times greater than the dissociation constant of the activatable antibody with respect to CTLA4 (when activated). In some embodiments, the dissociation constant of the shielding portion with respect to the target binding portion is approximately equal to the dissociation constant of the activatable antibody with respect to CTLA4 (when activated).

[0179] The activatable antibodies described herein can be further modified. In some embodiments, the activatable antibodies are linked to additional molecular entities. Examples of additional molecular entities include pharmaceutical agents, peptides or proteins, detection agents or labels, and antibodies.

[0180] In some embodiments, the activatable antibodies of the present disclosure are linked to pharmaceutical agents. Examples of pharmaceutical agents include cytotoxic agents or other cancer therapeutic agents and radioactive isotopes. Specific examples of cytotoxic agents include paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, miramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin, and analogs or homologs thereof. Therapeutic agents also include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, dacarbazine), alkylating agents (e.g., dichloromethyldiethylamine, thiotepa, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, milramycin, and anthramycin (AMC)), and antimitotics (e.g., vincristine and vinblastine). Examples of radioactive isotopes that can be conjugated to antibodies for diagnostic or therapeutic use include, but are not limited to, iodine. 131 ,indium 111 ,yttrium 90 and lutetium 177Methods for linking polypeptides to pharmaceutical agents are known in the art, such as using various linker technologies. Examples of linker types include hydrazones, thioethers, esters, disulfide, and peptide-containing linkers. For further discussion of linkers and methods for attaching therapeutic agents to antibodies, see, e.g., Saito et al., Adv. Drug Deliv. Rev. 55: 199-215 (2003); Trail et al., Cancer Immunol. Immunother. 52: 328-337 (2003); Payne, Cancer Cell 3: 207-212 (2003); Allen, Nat. Rev. Cancer 2: 750-763 (2002); Pastan and Kreitman, Curr. Opin. Investig. Drugs 3: 1089-1091 (2002); Senter and Springer (2001) Adv. Drug Deliv. Rev. 53: 247-264.

[0181] IV. Combination Therapy with PD-1 Antagonists

[0182] In some embodiments, the activatable antibodies of the present disclosure are administered in combination with PD-1 antagonists. In one embodiment, the PD-1 antagonists used in the treatment, medicaments and uses of the present invention include monoclonal antibodies (mAbs) or antigen-binding fragments thereof that specifically bind to PD-1 or PD-L1, and preferably specifically bind to human PD-1 or human PD-L1. The monoclonal antibody may be a human antibody, a humanized antibody or a chimeric antibody, and may include a human constant region. In some embodiments, the human constant region is selected from the group consisting of IgG1, IgG2, IgG3 and IgG4 constant regions, and in some embodiments, the human constant region is IgG1 or IgG4 constant region. In some embodiments, the antigen-binding fragment is selected from the group consisting of Fab, Fab'-SH, F(ab')2, scFv and Fv fragments.

[0183] Examples of monoclonal antibodies that bind to human PD-1 for use in the treatment methods, medicaments, and uses of the present invention are described in U.S. Pat. Nos. 7,488,802, 7,521,051, 8,008,449, 8,354,509, and 8,168,757, and International Application Publication Nos. WO 2004 / 004771, WO 2004 / 072286, WO 2004 / 056875, US 2011 / 0271358, and WO 2008 / 156712. Specific anti-human PD-1 monoclonal antibodies for use as PD-1 antagonists in the treatment methods, medicaments, and uses of the present invention include: a humanized IgG4 monoclonal antibody, the structure of which is described in WHO Drug Information, Vol. 27, No. 2, pp. 161-162 (2013), comprising the heavy and light chain amino acid sequences shown in Table B; nivolumab (BMS-936558), a human IgG4 monoclonal antibody, the structure of which is described in WHO Drug Information, Vol. 27, No. 1, pp. 68-69 (2013); humanized antibodies h409A11, h409A16, and h409A17, described in WO2008 / 156712, and AMP-514, being developed by MedImmune; cemiplizumab; camrelizumab; sintilimab; tislelizumab, and toripalimab. Other anti-PD-1 antibodies contemplated for use herein include MEDI0680 (U.S. Patent No. 8,609,089), BGB-A317 (U.S. Patent Publication No. 2015 / 0079109), INCSHR1210 (SHR-1210) (PCT International Application Publication No. WO2015 / 085847), REGN-2810 (PCT International Application Publication No. WO2015 / 112800), PDR001 (PCT International Application Publication No. WO2015 / 112900), TSR-042 (ANB011) (PCT International Application Publication No. WO2014 / 179664), and STI-1110 (PCT International Application Publication No. WO2014 / 194302).

[0184] In certain embodiments, the activatable antibodies of the present disclosure are administered in combination with toripalimab. In some embodiments, provided herein is a method for treating cancer in a subject, comprising administering to the subject: (a) an effective amount of an activatable antibody, wherein the activatable antibody of the present disclosure comprises a polypeptide comprising a shielding portion (MM), a cleavable portion (CM) and an anti-CTLA4 antibody described herein from the N-terminus to the C-terminus, wherein the MM comprises the amino acid sequence EVGSYPNPSSDCVPYYYACAY (SEQ ID NO: 192), and the cleavable portion comprises the amino acid sequence SGRSAGGGGTPLGLAGSGGS (SEQ ID NO: 221); and (b) an effective amount of toripalimab. The MM and CM comprise the amino acid sequence EVGSYPNPSSDCVPYYYACAYSGRSAGGGGTPLGLAGSGGS (SEQ ID NO: 200) from the N-terminus to the C-terminus. In certain embodiments, the MM and the CM are covalently linked to the N-terminus of the anti-CTLA4 antibody light chain. In some embodiments, the MM and CM comprise, from N-terminus to C-terminus, an amino acid sequence having at least 90% or at least 95% sequence identity to SEQ ID NO: 200. In some embodiments, TY22404 is administered in combination with toripalimab.

[0185] In some embodiments, the activatable antibody (e.g., TY22404) is administered at a dose of about 3 mg / kg to about 20 mg / kg or about 6 mg / kg to about 10 mg / kg every three weeks to every six weeks (e.g., once every three weeks, once every four weeks, once every five weeks, or once every six weeks). In some such embodiments, toripalimab is administered at a dose of about 200 mg to about 400 mg once every three weeks. In another embodiment, toripalimab is administered at a dose of about 240 mg once every three weeks. In another embodiment, toripalimab is administered at a dose of about 300 mg to about 600 mg once every six weeks. In another embodiment, toripalimab is administered at a dose of about 480 mg once every six weeks.

[0186] In one embodiment, the present disclosure provides a method of treating cancer in a subject, comprising administering to the subject an effective amount of an activatable anti-CTLA4 antibody (e.g., TY22404) as described above in combination with Teplizumab, wherein the anti-CTLA4 antibody is administered at a dose of about 3 mg / kg to about 10 mg / kg. In some embodiments, the anti-CTLA4 antibody (e.g., TY22404) is administered at a dose of about 3 mg / kg. In some embodiments, the anti-CTLA4 antibody (e.g., TY22404) is administered at a dose of about 5 mg / kg. In some embodiments, the anti-CTLA4 antibody (e.g., TY22404) is administered at a dose of about 6 mg / kg. In some embodiments, the anti-CTLA4 antibody (e.g., TY22404) is administered at a dose of about 8 mg / kg. In some embodiments, the anti-CTLA4 antibody (e.g., TY22404) is administered at a dose of about 10 mg / kg. In any of the foregoing embodiments, toripalimab can be administered at a dose of about 100 mg to about 400 mg, or about 200 mg to about 300 mg, or about 230 mg to about 250 mg. In some embodiments, toripalimab is administered at a dose of about 240 mg. In a specific embodiment, the activatable CTLA4 antibody (e.g., TY22404) and 240 mg of toripalimab are administered once every three weeks or once every six weeks. In any of the foregoing embodiments, toripalimab can be administered in combination with an activatable anti-CTLA4 antibody on the same day of a specific dosing regimen or on different days of a specific dosing regimen. In some embodiments, both the activatable anti-CTLA4 antibody and toripalimab are administered on the first day of three or six weeks of the dosing regimen.

[0187] In some embodiments, an activatable anti-CTLA4 antibody (e.g., TY22404) and toripalimab are administered to a patient in need thereof, wherein the activatable anti-CTLA4 antibody (e.g., TY22404) is administered at a dose of 6 mg / kg once every three weeks and the toripalimab is administered at a dose of 240 mg / kg once every three weeks. In some embodiments, the activatable anti-CTLA4 antibody and toripalimab are administered simultaneously.

[0188] In some embodiments, an activatable anti-CTLA4 antibody (e.g., TY22404) and toripalimab are administered to a patient in need thereof, wherein the activatable anti-CTLA4 antibody (e.g., TY22404) is administered at a dose of 10 mg / kg once every three weeks and the toripalimab is administered at a dose of 200 mg / kg once every three weeks. In some embodiments, the activatable anti-CTLA4 antibody and toripalimab are administered simultaneously.

[0189] In some embodiments, an activatable anti-CTLA4 antibody (e.g., TY22404) and toripalimab are administered to a patient in need thereof, wherein the activatable anti-CTLA4 antibody (e.g., TY22404) is administered at a dose of 20 mg / kg once every three weeks and the toripalimab is administered at a dose of 200 mg / kg once every three weeks. In some embodiments, the activatable anti-CTLA4 antibody and toripalimab are administered simultaneously.

[0190] In some embodiments, an activatable anti-CTLA4 antibody (e.g., TY22404) and toripalimab are administered to a patient in need thereof, wherein the activatable anti-CTLA4 antibody (e.g., TY22404) is administered at a dose of 6 mg / kg once every six weeks and the toripalimab is administered at a dose of 240 mg / kg once every six weeks. In some embodiments, the activatable anti-CTLA4 antibody and toripalimab are administered simultaneously.

[0191] In some embodiments, an activatable anti-CTLA4 antibody (e.g., TY22404) and toripalimab are administered to a patient in need thereof, wherein the activatable anti-CTLA4 antibody (e.g., TY22404) is administered at a dose of 10 mg / kg once every six weeks and the toripalimab is administered at a dose of 200 mg / kg once every six weeks. In some embodiments, the activatable anti-CTLA4 antibody and toripalimab are administered simultaneously.

[0192] In some embodiments, an activatable anti-CTLA4 antibody (e.g., TY22404) and toripalimab are administered to a patient in need thereof, wherein the activatable anti-CTLA4 antibody (e.g., TY22404) is administered at a dose of 20 mg / kg once every six weeks and the toripalimab is administered at a dose of 200 mg / kg once every six weeks. In some embodiments, the activatable anti-CTLA4 antibody and toripalimab are administered simultaneously.

[0193] In some embodiments, the activatable anti-CTLA4 antibody is administered intravenously. In some embodiments, the activatable anti-CTLA4 antibody and toripalimab are both administered intravenously. In some embodiments, the activatable anti-CTLA4 antibody is administered subcutaneously. In some embodiments, the activatable anti-CTLA4 antibody and toripalimab are administered intravenously or subcutaneously once every three weeks. In some embodiments, the activatable anti-CTLA4 antibody and toripalimab are administered intravenously or subcutaneously once every six weeks. In some embodiments, the subject receives at least 4 cycles of activatable anti-CTLA4 antibody and toripalimab treatment. In some embodiments, the subject further receives maintenance therapy, which comprises administering an effective amount of activatable anti-CTLA4 antibody to the subject approximately once every four weeks to approximately once every twelve weeks (e.g., once every 4, 6, 8, 10, or 12 weeks). In some embodiments, a certain dose of activatable anti-CTLA4 antibody and toripalimab may be administered simultaneously. In other embodiments, a certain dose of an activatable anti-CTLA4 antibody and toripalimab may be administered. For example, in some embodiments, the activatable anti-CTLA4 antibody and toripalimab are both administered intravenously. In some embodiments, the activatable anti-CTLA4 antibody is administered subcutaneously. In some embodiments, the activatable anti-CTLA4 antibody and toripalimab are administered intravenously or subcutaneously once every three weeks. In some embodiments, the activatable anti-CTLA4 antibody and toripalimab are administered intravenously or subcutaneously once every six weeks. In some embodiments, the subject receives at least 4 cycles of activatable anti-CTLA4 antibody and toripalimab treatment. In some embodiments, the subject further receives maintenance therapy, which comprises administering an effective amount of an activatable anti-CTLA4 antibody to the subject approximately once every four weeks to approximately once every twelve weeks (e.g., once every 4, 6, 8, 10, or 12 weeks). In some embodiments, a certain dose of an activatable anti-CTLA4 antibody and toripalimab may be administered simultaneously. In other embodiments, a certain dose of an activatable anti-CTLA4 antibody and toripalimab may be administered. For example, toripalimab may be administered about 0.5 hours to about 5 hours before or after the first day of administration of the activatable anti-CTLA4 antibody dosing schedule (e.g., a three-week dosing schedule). It may be administered about 0.5 hours to about 5 hours before or after the first day of administration of the activatable anti-CTLA4 antibody dosing schedule (e.g., a three-week dosing schedule). Both are administered intravenously. In some embodiments, the activatable anti-CTLA4 antibody is administered subcutaneously. In some embodiments, the activatable anti-CTLA4 antibody and. In some embodiments, the activatable anti-CTLA4 antibody and toripalimab are both administered intravenously. In some embodiments, the activatable anti-CTLA4 antibody is administered subcutaneously.In some embodiments, the activatable anti-CTLA4 antibody and toripalimab are administered intravenously or subcutaneously once every three weeks. In some embodiments, the activatable anti-CTLA4 antibody and toripalimab are administered intravenously or subcutaneously once every six weeks. In some embodiments, the subject receives at least 4 cycles of activatable anti-CTLA4 antibody and toripalimab treatment. In some embodiments, the subject further receives maintenance therapy, which comprises administering an effective amount of activatable anti-CTLA4 antibody to the subject about once every four weeks to about once every twelve weeks (e.g., once every 4, 6, 8, 10, or 12 weeks). In some embodiments, a certain dose of activatable anti-CTLA4 antibody and toripalimab can be administered simultaneously. In other embodiments, a certain dose of activatable anti-CTLA4 antibody and toripalimab can be administered. For example, toripalimab can be administered about 0.5 hours to about 5 hours before or after the first day of the activatable anti-CTLA4 antibody dosing schedule (e.g., a three-week dosing schedule). Administered intravenously or subcutaneously once every three weeks. In some embodiments, the activatable anti-CTLA4 antibody and toripalimab are administered intravenously or subcutaneously once every six weeks. In some embodiments, the subject receives at least 4 cycles of activatable anti-CTLA4 antibody and toripalimab treatment. In some embodiments, the subject further receives maintenance therapy, which comprises administering an effective amount of activatable anti-CTLA4 antibody to the subject about once every four weeks to about once every twelve weeks (e.g., once every 4, 6, 8, 10 or 12 weeks). In some embodiments, a certain dose of activatable anti-CTLA4 antibody and toripalimab can be administered simultaneously. In other embodiments, a certain dose of activatable anti-CTLA4 antibody and toripalimab can be administered. For example, toripalimab can be administered about 0.5 hours to about 5 hours before or after the first day of the activatable anti-CTLA4 antibody dosing schedule (e.g., a three-week dosing schedule).

[0194] V. Pharmaceutical Compositions, Kits, and Articles of Manufacture

[0195] In other aspects, the present application provides a composition comprising any anti-CTLA4 antibody described herein (e.g., an activatable antibody). In some embodiments, the composition is a pharmaceutical composition comprising an anti-CTLA4 antibody (e.g., an activatable antibody) and a pharmaceutically acceptable carrier. In some embodiments, a composition is provided herein comprising one or more additional therapeutic agents (e.g., an anti-PD-1 antibody, an anti-PD-L1 antibody). The composition can be prepared by conventional methods known in the art.

[0196] The term "pharmaceutically acceptable carrier" refers to any inactive substance in a formulation suitable for delivering an active agent (e.g., an anti-CTLA4 antibody). A carrier can be an anti-adherent, a binder, a coating, a disintegrant, a filler or diluent, a preservative (such as an antioxidant, an antibacterial agent, or an antifungal agent), a sweetener, an absorption delaying agent, a wetting agent, an emulsifier, a buffer, and the like. Examples of suitable pharmaceutically acceptable carriers include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), dextrose, vegetable oils (such as olive oil), saline, buffer, buffered saline, and isotonic agents (such as sugars, polyols, sorbitol, and sodium chloride). The composition can be in any suitable form, such as liquid, semisolid, and solid dosage forms. Examples of liquid dosage forms include solutions (e.g., injectable and infusible solutions), microemulsions, liposomes, dispersions, or suspensions. Examples of solid dosage forms include tablets, pills, capsules, microcapsules, and powders. A specific form of composition suitable for delivery of anti-CTLA4 antibodies is an injectable or infusible sterile liquid, such as a solution, suspension or dispersion. Sterile solutions can be prepared by incorporating the desired amount of antibody into an appropriate carrier and then sterilizing by microfiltration. In general, dispersions are prepared by incorporating the antibody into a sterile vehicle comprising a basic dispersion medium and other carriers. For sterile powders for preparing sterile liquids, preparation methods include vacuum drying and freeze drying (lyophilization) to produce a powder of the active ingredient plus any additional desired ingredients from its previously sterile-filtered solution. Various dosages of the composition can be prepared by conventional techniques known in the art.

[0197] The relative amount of anti-CTLA4 antibody included in the composition will depend on a variety of factors, such as the specific anti-CTLA4 antibody and carrier used, the dosage form, and the desired release and pharmacodynamic characteristics. The amount of anti-CTLA4 antibody in a single dosage form is generally that amount that produces a therapeutic effect, but may be a lesser amount. Generally, this amount will be in the range of about 0.01% to about 99%, about 0.1% to about 70%, or about 1% to about 30% relative to the total weight of the dosage form.

[0198] In addition to the anti-CTLA4 antibody, one or more additional therapeutic agents may be included in the composition. Examples of additional therapeutic agents are described herein under "Methods of Treatment." Suitable amounts of additional therapeutic agents to be included in the composition can be readily selected by one skilled in the art and will vary depending on many factors, such as the specific agent and carrier used, the dosage form, and the desired release and pharmacodynamic characteristics. The amount of additional therapeutic agent included in a single dosage form will generally be that amount of the agent that produces a therapeutic effect, but may also be a smaller amount.

[0199] In some embodiments, a product is provided that includes a material that can be used to treat cancer. The product may include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container can be formed from a variety of materials, such as glass or plastic. Typically, the container holds the composition described herein for effectively treating cancer and may have a sterile access port (for example, the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). A package insert refers to instructions typically included in the commercial packaging of a therapeutic product that include information about indications, usage, dosage, administration, contraindications, and / or warnings about the use of such therapeutic products. In some embodiments, the package insert indicates that the composition is used to treat cancer. The label or package leaflet may further include instructions for administering the composition to the patient.

[0200] In addition, the article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may also include other materials desired from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0201] Also provided is a kit that can be used for various purposes, for example, for the treatment of cancer as described herein, optionally used in combination with products. The kit of the present application includes one or more containers, which include any one of the compositions (or unit dosage forms and / or products) described herein. In some embodiments, the kit also includes other agents (e.g., one or more additional therapeutic agents) and / or instructions for use according to any one of the methods described herein. The kit may also include a description of an individual selected for treatment. The instructions provided in the kit of the present application are typically written instructions on a label or package insert (e.g., paper included in the kit), but machine-readable instructions (e.g., instructions stored on a magnetic disk storage device or an optical disk storage device) are also acceptable.

[0202] For example, in some embodiments, a kit is provided comprising a pharmaceutical composition comprising any one of the anti-CTLA4 antibodies described herein and a pharmaceutically acceptable carrier, and instructions for administering the pharmaceutical composition to an individual with cancer. In some embodiments, the kit further comprises a pharmaceutical composition comprising an additional therapeutic agent, such as a chemotherapeutic agent. In some embodiments, the kit further comprises a pharmaceutical composition comprising an anti-PD-1 antibody. In some embodiments, the kit comprises a kit for determining the level of one or more biomarkers described herein (e.g., CD8+ T cells, CD4+ T cells, CD8+ T cells, em cells, CD4+Tem cells, T reg cells, CD8+T em Cells and T reg The ratio of cells, CD4+T em Cells and T reg One or more determination methods or reagents thereof for detecting the ratio of B cells, NK cells, and B cells.

[0203] The kits of the present application are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed polyester film or plastic bags), etc. The kits may optionally include other components, such as a buffer sheet and explanatory information. Therefore, the present application also provides articles including vials (e.g., sealed vials), bottles, jars, flexible packaging, etc.

[0204] The container can be a unit dose, bulk packaging (e.g., multi-dose packaging) or subunit dose. Kits can also include multiple unit doses of the pharmaceutical composition and instructions for use, and are packaged in quantities sufficient to be stored and used in pharmacies, such as hospital pharmacies and compounding pharmacies.

[0205] The foregoing written description is considered sufficient to enable those skilled in the art to practice the present disclosure. The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present disclosure in any way. Indeed, various modifications of the present disclosure, in addition to those shown and described herein, will become apparent to those skilled in the art based on the foregoing description and fall within the scope of the appended claims.

[0206] Example

[0207] The present invention may be further understood by reference to the following examples, which are provided by way of illustration and are not meant to be limiting.

[0208] Example 1. First-in-Human (FIH), Open-label, Phase I, Dose-Escalation Study of TY22404 and TY22404 in Combination with an Anti-PD-1 Antibody in Patients with Advanced / Metastatic Solid Tumors

[0209] method

[0210] This is a first-in-human, Phase I, open-label, multicenter, sequential dose-escalation and dose-expansion study to evaluate the safety, tolerability, PK, and preliminary efficacy of TY22404 monotherapy and the TY22404-toripalimab combination regimen in patients with advanced / metastatic solid tumors. The study consists of Part 1: TY22404 monotherapy dose escalation, followed by a biopsy cohort and expansion (concurrently); and Part 2: TY22404-toripalimab combination dose escalation and expansion. The objectives of this study are to evaluate the safety and tolerability of TY22404 monotherapy at escalating dose levels and in combination with toripalimab in adult patients with advanced / metastatic solid tumors who have exhausted their treatment options.

[0211] Dose escalation. TY22404 monotherapy dose escalation will follow a traditional 3+3 cohort design. Three to six patients will be enrolled in up to six consecutive ascending dose levels: 0.1, ≤0.3, ≤1, ≤3, ≤10, and ≤20 mg / kg, administered every three weeks (Q3W). The dose level or dosing interval may be adjusted during the study based on observed safety data, but will not exceed the predetermined MAD of 20 mg / kg.

[0212] Combination dose escalation. The combination therapy will start at a dose level that is lower than the defined dose of the monotherapy dose escalation arm and is approved by the SRC. Based on non-clinical data, the RP2D is expected to be 20 mg / kg once every three weeks (Q3W). In order to maximize patient safety, the starting dose of the combination dose escalation arm will be lower than the RP2D dose (e.g., 10 mg / kg Q3W). Enrollment in the combination therapy dose escalation arm (which can be enrolled at the same time) can occur before the RP2D / MAD of the monotherapy dose escalation arm is defined. A modified toxicity probability interval (mTPI) design, with a target DLT rate of approximately 30%, will be used for dose escalation and confirmation to determine the RP2D of the combination of TY22404 and toripalimab. Dose escalation includes dose escalation of the combination regimen and 3 dose escalation levels for each combination regimen as shown below:

[0213]

[0214] DL = dose level; mTPI = modified toxicity probability interval; SRC = safety review committee; Q3W = every 3 weeks

[0215] a DL3 denotes dose level 3. The number of dose levels will be determined based on data generated during dose escalation of the combination therapy.

[0216] Key inclusion criteria: Patients with advanced / metastatic solid tumors with ECOG ≤ 1 and at least one measurable lesion as measured by RECIST 1.1. Imaging was performed every 6 weeks for the first 4 cycles and every 9 weeks thereafter. Tumor response was determined by the investigator using RECIST 1.1 and iRECIST. Prior treatment with anti-PD-1 or anti-CTLA-4 therapy was permitted.

[0217] Endpoints. The primary endpoints were safety and tolerability, determination of the maximum administered dose (MAD), maximum tolerated dose (MTD), and recommended phase 2 dose (RP2D). Secondary endpoints were PK, anti-drug antibodies (ADA), ORR, DCR, DOR, PFS, and OS according to RECIST 1.1. Assessments. Imaging was performed every 6 weeks for the first 4 cycles and every 9 weeks thereafter.

[0218] Preliminary interim results of TY22404 monotherapy

[0219] Twenty-six heavily pretreated subjects received TY22404 monotherapy. Tumor types included breast cancer, bile duct cancer, colorectal cancer, epithelial ovarian cancer, glioblastoma, hepatocellular carcinoma, melanoma, non-small cell lung cancer, pancreatic adenocarcinoma, renal cell carcinoma, uveal melanoma, and others.

[0220] TY22404 monotherapy (N=26) was well tolerated, with no dose-limiting toxicities up to 20 mg / kg. The most common treatment-related adverse events (TRAEs) (>10%) were fatigue (12%), pruritus (12%), rash (12%), and diarrhea (12%) (Table 2). TY22404 demonstrated monotherapy antitumor efficacy in 23 evaluable patients, with a disease control rate of 39% ( Figure 1 AB).

[0221] A patient who had previously undergone radical salpingo-oophorectomy and 5 lines of systemic therapy showed a sustained response after treatment with 1 mg / kg TY22404, with a 90% decrease in CA125 from 303 to 31 U / ml (normal <35 U / ml) ( Figure 2 ), and target lesions were reduced by 22% at the end of cycle 16 (Table 3). Treatment is still ongoing at cycle 18.

[0222] Plasma pharmacokinetics (PK) of total and intact drug were approximately linear with dose as determined by LC-MS using their respective signature peptides, and calculated average accumulation of cleaved (i.e., activated) TY22404 was ~3-fold during repeated dosing (e.g., Cycle 4 vs. Cycle 1) ( Figure 3Non-compartmental PK analysis (NCA) demonstrated that the terminal half-life of circulating total TY22404 was prolonged by an average of approximately 1.5-fold compared to its parental monoclonal antibody, TY21580. Circulating cleaved drug PK reflects the prolonged exposure of activated TY22404 to the tumor microenvironment. PK modeling also predicted a target saturation of approximately 6-10 mg / kg at steady-state.

[0223] Table 1. Baseline Characteristics

[0224]

[0225]

[0226] Table 2. Summary of TRAEs in Patients Treated with TY22404 Monotherapy

[0227]

[0228] Table 3. Clinical responses in patients with ovarian serous carcinoma

[0229]

[0230] TY22404 interim results update

[0231] Across all dose levels, 30 patients (Table 4) received TY22404 monotherapy, and 20 patients received TY22404 (6 mg / kg Q3W and 10 mg / kg Q3W or Q6W) plus toripalimab (240 mg Q3W). Patients had received extensive prior therapy. Tumor types included breast cancer, bile duct cancer, colorectal cancer, epithelial ovarian cancer, glioblastoma, hepatocellular carcinoma, melanoma, non-small cell lung cancer, pancreatic adenocarcinoma, renal cell carcinoma, uveal melanoma, prostate cancer, endometrial cancer, gastrointestinal stromal tumor (GIST), squamous cell carcinoma (anal, anorectal, penile, or skin), adenocarcinoma, and others. These tumor types were included in the study, including "cold tumors" or immune-experienced "hot tumors."

[0232] Table 4. Patient Demographics, Characteristics, and Drug Exposures

[0233]

[0234] *Study treatment cycles included TY22404 and / or toripalimab

[0235] Safety – TY22404 monotherapy

[0236] As shown in Table 5, TY22404 monotherapy was well tolerated, with no dose-limiting toxicities observed at repeated doses up to 20 mg / kg. No treatment-related adverse events (TRAEs) of grade 2 or higher or any treatment-related serious adverse events (SAEs) were reported. No patient discontinued study treatment due to a TRAE.

[0237] Table 5. TRAEs reported with TY22404 monotherapy at any dose level

[0238]

[0239] Safety-TY22404+Toripalimab dose escalation

[0240] Three dose-escalation cohorts were explored, including 7 patients who received TY22404 6 mg / kg Q3W + toripalimab 240 mg Q3W, 7 patients who received TY22404 10 mg / kg Q6W + toripalimab 240 mg Q3W, and 9 patients who received TY22404 10 mg / kg Q3W + toripalimab 240 mg Q3W (Table 6). No significant differences in safety were observed among the three dose-escalation cohorts. No DLTs or grade >3 TRAEs were reported. Five patients (21%) experienced G3 TRAEs: most G3 TRAEs occurred no earlier than cycle 4. Three patients experienced treatment-related SAEs, including 1 case of G3 hepatitis§, 1 case of G3 sepsis and 1 case of G3 myocarditis (This resulted in the study being interrupted).

[0241] Table 6. TRAEs reported with dose escalation of TY22404 + toripalimab at any dose level

[0242]

[0243] Efficacy - TY22404 monotherapy

[0244] Across all dose levels, the disease control rate (DCR) was 37% in 27 evaluable patients with at least one valid post-baseline tumor assessment. Long-term stable disease was observed in 5 patients, including a 14-cycle NSCLC patient and a 22-cycle ovarian cancer patient. A 12% reduction in the sum of target lesions was observed in a NSCLC patient who had previously received pembrolizumab and docetaxel; the NSCLC patient completed 14 cycles of TY22404 20 mg / kg Q3W and had no TRAEs. A patient with ovarian serous carcinoma received TY22404 1 mg / kg Q3W and had stable disease, a 90% reduction in CA125, and a 22% reduction in the sum of target lesions.

[0245] A 39-year-old male patient with stage IIIB hepatocellular carcinoma. Baseline ECOGPS was 0. The patient had previously received first-line treatment with the anti-PD-L1 therapy atezolizumab in combination with bevacizumab (July 2021-February 2022, PD); the patient subsequently received second-line treatment with lenvatinib (March-September 2022, PD). The patient is receiving the sixth cycle of TY22404 10mg / kg Q3W, and tumor growth is controllable (stable disease). Figure 4 As shown, an increase in the Teff / Treg ratio was observed in paired tumor biopsies ( Figure 4 A) Treg depletion ( Figure 4 B) and CD8+ T cells increased ( Figure 4 C).

[0246] Efficacy – TY22404 + toripalimab dose escalation

[0247] As shown in Table 7 and Figure 5, responses were calculated for 18 evaluable patients treated with TY22404 (6 mg / kg Q3W and 10 mg / kg Q3W or Q6W) + toripalimab (240 mg Q3W) in three dose escalation cohorts. Among the 18 evaluable patients, the overall ORR = 11% and the DCR = 56%. In the 7 evaluable patients treated with TY22404 10 mg / kg Q3W + TORI 240 mg Q3W, ORR = 28% and DCR = 57% were reported. Confirmed partial responses (PRs) were observed in two patients with penile squamous cell carcinoma (SCC) and anal SCC who had previously received chemotherapy. Long-term stable disease was observed in PDAC patients, with a 5% reduction in the total number of target lesions. A 58% and 21% reduction in the total number of target lesions was observed at baseline in two patients with microsatellite stable colorectal cancer (MSS CRC) and liver metastases, respectively.

[0248] Table 7. Best responses to TY22404+toripalimab dose escalation

[0249]

[0250] Case study: confirmed PR in two patients with anal SCC and penile SCC

[0251] As shown in Table 8, a confirmed PR with a 36% reduction in the total number of target lesions was observed in a 51-year-old female patient with metastatic anal squamous cell carcinoma (SCC) who received TY22404 10 mg / kg Q3W plus toripalimab 240 mg Q3W (at the time of data cutoff, she was in the fifth cycle of treatment). Her baseline ECOGPS was 0. She had previously received fluorouracil (5FU) / mitomycin C plus radiation.

[0252] Table 8. Tumor Assessment in Patients with Metastatic Anal Squamous Cell Carcinoma

[0253]

[0254] As shown in Table 9, a confirmed PR with a 72% reduction in the total number of target lesions was observed in a 64-year-old male patient with metastatic penile SCC who received TY22404 10 mg / kg Q3W plus toripalimab 240 mg Q3W (at the time of data cutoff, the patient was in the sixth cycle of treatment). His baseline ECOGPS was 1. The patient had previously received partial penectomy, bilateral lymph node dissection, chemotherapy (paclitaxel, ifosfamide, cisplatin), and palliative RT.

[0255] Table 9. Tumor Assessment in Patients with Metastatic Penile SCC

[0256]

[0257] Case Study: The Promising Potential of TY22404 + Toripalimab in "Cold" Gastrointestinal Tumors

[0258] As shown in Table 10, a 53-year-old man with MSS sigmoid colon adenocarcinoma and liver metastases at baseline received TY22404 10 mg / kg every 6 weeks plus toripalimab 240 mg every 3 weeks (treatment was ongoing at the time of data cutoff). His baseline ECOGPS was 0. He had previously undergone radical and palliative surgery for liver metastases, as well as three lines of therapy: folinic acid + 5FU + irinotecan + oxaliplatin, bevacizumab + TAS102, and regorafenib. A mixed response was observed in cycle 17, with a 58% reduction in the total number of target lesions.

[0259] Table 10. Tumor Assessments in Patients with MSS CRC

[0260]

[0261] As shown in Table 11, a 74-year-old patient with MSS CRC and liver metastases at baseline received TY22404 6 mg / kg every 3 weeks plus toripalimab 240 mg every 3 weeks. His baseline ECOGPS was 1. He had previously received three lines of therapy: XELOX plus bevacizumab; irinotecan plus cetuximab; and irinotecan plus panitumumab. A 21% reduction in the total number of target lesions was observed (treatment was ongoing at the time of data cutoff).

[0262] Table 11. Tumor Assessments in Patients with MSS CRC

[0263]

[0264] As shown in Table 12, a 56-year-old woman with pancreatic ductal adenocarcinoma (PDAC) received TY22404 6 mg / kg every 3 weeks plus toripalimab 240 mg every 3 weeks. Her baseline ECOG PS was 0. She had previously received radical surgery and four lines of therapy: gemcitabine / nab-paclitaxel; FOLFIRINOX; oxaliplatin, raltitrexed, irinotecan, and leucovorin; and gemcitabine / capecitabine. Long-term tumor growth control (stable disease) was observed, with a 5% reduction in the total number of target lesions (treatment ongoing).

[0265] Table 12. Tumor Assessments in Patients with PDAC

[0266]

[0267] Figure 6 A-6B shows the response of TY22404 + toripalimab in selected gastrointestinal "cold tumors" as described above. Figure 6 A shows a lane diagram. Figure 6 B shows a waterfall plot.

[0268] TY22404 monotherapy was well tolerated at up to 20 mg / kg Q3W and showed promising efficacy signals in heavily pretreated patients. Long-term stable disease was observed in 5 patients. Increased Teff / Treg ratios, Treg depletion, and increased CD8+ T cells were observed in paired tumor biopsies. The safety profile of TY22404+toripalimab dose escalation showed the best potential compared to other anti-CTLA-4 molecules combined with anti-PD-1 antibodies at similar doses / schedules. TY22404+toripalimab showed encouraging efficacy, including two confirmed PRs, as well as long-term stable disease and reduced target lesions in "cold" gastrointestinal tumors. In 7 evaluable patients treated with TY22404 10 mg / kg Q3W + toripalimab 240 mgQ3W, ORR=28% and DCR=57%. In two patients with MSS CRC and liver metastases, long-term stable disease and a 58% and 21% reduction in the total number of target lesions compared to baseline were observed; similar findings were observed in patients with PDAC, with a 5% reduction in the total number of target lesions. The good safety profile of continuous dosing for more than 4 cycles enables its use in combination with drugs other than anti-PD-1 therapy: a study of TY22404 + atezolizumab + bevacizumab (NCT04524871) is being planned. The unique mechanism of Treg depletion may unlock the potential of TY22404 to address significant unmet needs in "cold tumors." The indications for the TY22404 10mg / kg + toripalimab dose expansion program include MSS CRC.

[0269]

[0270]

[0271]

[0272]

[0273]

[0274]

[0275]

Claims

1. A method of treating cancer in a subject, comprising administering to the subject: (a) an effective amount of an activatable antibody, wherein the activatable antibody comprises an HVR-H1 comprising an amino acid sequence according to the formula YSISSGYHWSWI (SEQ ID NO: 23), an HVR-H2 comprising an amino acid sequence according to the formula LARIDWDDDKYYSTSLKSRL (SEQ ID NO: 35), an HVR-H3 comprising an amino acid sequence according to the formula ARSYVYFDY (SEQ ID NO: 45), an HVR-L1 comprising an amino acid sequence according to the formula RASQSVRGRFLA (SEQ ID NO: 58), an HVR-L2 comprising an amino acid sequence according to the formula DASNRATGI (SEQ ID NO: 66), and an HVR-H3 comprising an amino acid sequence according to the formula YCQQSSSWPPT (SEQ ID NO: 70). NO:75), and wherein the activatable antibody further comprises: a polypeptide covalently linked to the N-terminus of the anti-CTLA4 antibody light chain, the polypeptide comprising, from N-terminus to C-terminus, a blocking moiety (MM) and a cleavable moiety (CM), wherein the MM comprises the amino acid sequence EVGSYPNPSSDCVPYYYACAY (SEQ ID NO:192), and the cleavable moiety comprises the amino acid sequence SGRSAGGGGTPLGLAGSGGS (SEQ ID NO:221); and (b) an effective amount of toripalimab, wherein the activatable antibody is administered at a dose of about 6 mg / kg to about 10 mg / kg once every three to six weeks, and wherein toripalimab is administered at a dose of about 200 mg to about 400 mg once every three weeks or about 300 mg to about 600 mg once every six weeks.

2. The method of claim 1, wherein the activatable antibody is administered at a dose of about 6 mg / kg once every 3 to 6 weeks.

3. The method of claim 1, wherein the activatable antibody is administered at a dose of about 10 mg / kg once every 3 to 6 weeks.

4. The method of claim 1, wherein the toripalimab is administered at a dose of about 240 mg once every three weeks.

5. The method of claim 1, wherein the toripalimab is administered at a dose of about 480 mg once every six weeks.

6. A method of treating cancer in a subject, comprising administering to the subject an effective amount of an activatable antibody, wherein the activatable antibody comprises an HVR-H1 comprising an amino acid sequence according to the formula YSISSGYHWSWI (SEQ ID NO: 23), an HVR-H2 comprising an amino acid sequence according to the formula LARIDWDDDKYYSTSLKSRL (SEQ ID NO: 35), an HVR-H3 comprising an amino acid sequence according to the formula ARSYVYFDY (SEQ ID NO: 45), an HVR-L1 comprising an amino acid sequence according to the formula RASQSVRGRFLA (SEQ ID NO: 58), an HVR-L2 comprising an amino acid sequence according to the formula DASNRATGI (SEQ ID NO: 66), and an HVR-H4 comprising an amino acid sequence according to the formula YCQQSSSWPPT (SEQ ID NO: 70). NO:75), and wherein the activatable antibody further comprises: a polypeptide covalently linked to the N-terminus of the anti-CTLA4 antibody light chain, the polypeptide comprising, from N-terminus to C-terminus, a blocking moiety (MM) and a cleavable moiety (CM), wherein the MM comprises the amino acid sequence of EVGSYPNPSSDCVPYYYACAY (SEQ ID NO:192), and the cleavable moiety comprises the amino acid sequence of SGRSAGGGGTPLGLAGSGGS (SEQ ID NO:221); and wherein the activatable antibody is administered at a dose of about 6 mg / kg to about 20 mg / kg once every three to six weeks.

7. The method of any one of claims 1 to 6, wherein the cancer is resistant or refractory to prior therapy, wherein the prior therapy is a CTLA4, PD-1, or PD-1 ligand inhibitor.

8. The method of claim 7, wherein the prior therapy is ipilimumab.

9. The method of any one of claims 1 to 8, wherein the cancer is colorectal cancer (CRC).

10. The method of claim 9, wherein the CRC is a microsatellite stable (MSS) CRC.

11. The method of any one of claims 1 to 8, wherein the cancer is squamous cell carcinoma.

12. The method of any one of claims 1 to 8, wherein the cancer is anal squamous cell carcinoma or penile squamous cell carcinoma.

13. The method of any one of claims 1 to 8, wherein the cancer is pancreatic cancer.

14. The method of any one of claim 13, wherein the cancer is pancreatic ductal adenocarcinoma (PDAC).

15. The method of any one of claims 1 to 8, wherein the cancer is ovarian cancer.

16. The method of any one of claims 1 to 8, wherein the cancer is NSCLC.

17. The method of any one of claims 1 to 8, wherein the cancer is hepatocellular carcinoma.

18. The method of any one of claims 1 to 17, wherein the cancer is an advanced metastatic cancer.

19. The method of claim 18, wherein the cancer has metastasized to the lungs or liver.

20. The method of any one of claims 1 to 18, wherein the activatable anti-CTLA4 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 87, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 87, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 100, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:

100.

21. The method of claim 20, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 87 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:

100.

22. The method of claim 21, wherein the activatable antibody comprises the full-length heavy chain region of SEQ ID NO: 320 or SEQ ID NO:

321.

23. The method of claim 22, wherein the activatable antibody comprises the full-length light chain region of SEQ ID NO: 322 or SEQ ID NO:

323.

24. The method of any one of claims 1 to 23, wherein the subject is a human.

25. The method of any one of claims 1 to 24, wherein the activatable anti-CTLA4 antibody and toripalimab are both administered on day 1 of a dosing schedule of weeks 3 to 6.

26. A method of treating cancer in a subject, comprising administering to the subject an effective amount of an activatable antibody, wherein the activatable antibody comprises an HVR-H1 comprising an amino acid sequence according to the formula YSISSGYHWSWI (SEQ ID NO: 23), an HVR-H2 comprising an amino acid sequence according to the formula LARIDWDDDKYYSTSLKSRL (SEQ ID NO: 35), an HVR-H3 comprising an amino acid sequence according to the formula ARSYVYFDY (SEQ ID NO: 45), an HVR-L1 comprising an amino acid sequence according to the formula RASQSVRGRFLA (SEQ ID NO: 58), an HVR-L2 comprising an amino acid sequence according to the formula DASNRATGI (SEQ ID NO: 66), and an HVR-H4 comprising an amino acid sequence according to the formula YCQQSSSWPPT (SEQ ID NO: 70). NO:75), and wherein the activatable antibody further comprises: a polypeptide covalently linked to the N-terminus of the anti-CTLA4 antibody light chain, the polypeptide comprising, from N-terminus to C-terminus, a blocking moiety (MM) and a cleavable moiety (CM), wherein the MM comprises the amino acid sequence of EVGSYPNPSSDCVPYYYACAY (SEQ ID NO:192), and the cleavable moiety comprises the amino acid sequence of SGRSAGGGGTPLGLAGSGGS (SEQ ID NO:221), wherein the activatable antibody is administered at a single loading dose of about 20 mg / kg followed by a maintenance dose of 10 mg / kg once every three weeks or once every six weeks.

27. The method of claim 26, wherein the first maintenance dose is administered three weeks after the loading dose.

28. The method of claim 26 or claim 27, wherein the maintenance dose is administered once every three weeks.

29. The method of any one of claims 26 to 28, further comprising administering to the subject an effective amount of an anti-PD-1 antibody.

30. The method of claim 29, wherein the anti-PD-1 antibody is toripalimab.

31. The method of claim 30, wherein the toripalimab is administered at a dose of about 200 mg to about 400 mg once every three weeks or about 300 mg to about 600 mg once every six weeks.

32. The method of any one of claims 26 to 31, wherein the cancer is resistant or refractory to prior therapy, wherein the prior therapy is a CTLA4, PD-1, or PD-1 ligand inhibitor.

33. The method of claim 32, wherein the prior therapy is ipilimumab.

34. The method of any one of claims 26 to 33, wherein the cancer is colorectal cancer (CRC).

35. The method of claim 34, wherein the CRC is a microsatellite stable (MSS) CRC.

36. The method of any one of claims 26 to 33, wherein the cancer is squamous cell carcinoma.

37. The method of any one of claims 26 to 33, wherein the cancer is anal squamous cell carcinoma or penile squamous cell carcinoma.

38. The method of any one of claims 26 to 33, wherein the cancer is pancreatic cancer.

39. The method of claim 38, wherein the cancer is pancreatic ductal adenocarcinoma (PDAC).

40. The method of any one of claims 26 to 33, wherein the cancer is ovarian cancer.

41. The method of any one of claims 26 to 33, wherein the cancer is NSCLC.

42. The method of any one of claims 26 to 33, wherein the cancer is hepatocellular carcinoma.

43. The method of any one of claims 26 to 42, wherein the cancer is an advanced metastatic cancer.

44. The method of claim 43, wherein the cancer has metastasized to the lungs or liver.

45. The method of any one of claims 26 to 44, wherein the activatable antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 87, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 87, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 100, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:

100.

46. ​​The method of claim 45, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 87 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:

100.

47. The method of claim 46, wherein the activatable antibody comprises the full-length heavy chain region of SEQ ID NO: 320 or SEQ ID NO:

321.

48. The method of claim 47, wherein the activatable antibody comprises the full-length heavy chain region of SEQ ID NO: 322 or SEQ ID NO:

323.

49. The method of any one of claims 26 to 48, wherein the subject is a human.

50. A method of treating cancer in a subject, comprising administering to the subject an effective amount of an activatable antibody, wherein the activatable antibody comprises an HVR-H1 comprising an amino acid sequence according to the formula YSISSGYHWSWI (SEQ ID NO: 23), an HVR-H2 comprising an amino acid sequence according to the formula LARIDWDDDKYYSTSLKSRL (SEQ ID NO: 35), an HVR-H3 comprising an amino acid sequence according to the formula ARSYVYFDY (SEQ ID NO: 45), an HVR-L1 comprising an amino acid sequence according to the formula RASQSVRGRFLA (SEQ ID NO: 58), an HVR-L2 comprising an amino acid sequence according to the formula DASNRATGI (SEQ ID NO: 66), and an HVR-H4 comprising an amino acid sequence according to the formula YCQQSSSWPPT (SEQ ID NO: 70). NO:75), and wherein the activatable antibody further comprises: a polypeptide covalently linked to the N-terminus of the anti-CTLA4 antibody light chain, the polypeptide comprising, from N-terminus to C-terminus, a blocking moiety (MM) and a cleavable moiety (CM), wherein the MM comprises the amino acid sequence of EVGSYPNPSSDCVPYYYACAY (SEQ ID NO:192), and the cleavable moiety comprises the amino acid sequence of SGRSAGGGGTPLGLAGSGGS (SEQ ID NO:221), wherein the activatable antibody is administered at a dose that provides a steady-state concentration of cleaved antibody that is above the EC50 for cleavage.

51. A method of treating cancer in a subject, comprising administering to the subject an effective amount of an activatable antibody, wherein the activatable antibody comprises an HVR-H1 comprising an amino acid sequence according to the formula YSISSGYHWSWI (SEQ ID NO: 23), an HVR-H2 comprising an amino acid sequence according to the formula LARIDWDDDKYYSTSLKSRL (SEQ ID NO: 35), an HVR-H3 comprising an amino acid sequence according to the formula ARSYVYFDY (SEQ ID NO: 45), an HVR-L1 comprising an amino acid sequence according to the formula RASQSVRGRFLA (SEQ ID NO: 58), an HVR-L2 comprising an amino acid sequence according to the formula DASNRATGI (SEQ ID NO: 66), and an HVR-H4 comprising an amino acid sequence according to the formula YCQQSSSWPPT (SEQ ID NO: 70). NO:75), and wherein the activatable antibody further comprises: a polypeptide covalently linked to the N-terminus of the anti-CTLA4 antibody light chain, the polypeptide comprising, from N-terminus to C-terminus, a blocking moiety (MM) and a cleavable moiety (CM), wherein the MM comprises the amino acid sequence of EVGSYPNPSSDCVPYYYACAY (SEQ ID NO:192), and the cleavable moiety comprises the amino acid sequence of SGRSAGGGGTPLGLAGSGGS (SEQ ID NO:221), wherein the activatable antibody is administered at a dose that provides a steady-state concentration of cleaved antibody that is greater than the EC90 for cleavage.

52. A method of treating cancer in a subject, comprising administering to the subject an effective amount of an activatable antibody, wherein the activatable antibody comprises an HVR-H1 comprising an amino acid sequence according to the formula YSISSGYHWSWI (SEQ ID NO: 23), an HVR-H2 comprising an amino acid sequence according to the formula LARIDWDDDKYYSTSLKSRL (SEQ ID NO: 35), an HVR-H3 comprising an amino acid sequence according to the formula ARSYVYFDY (SEQ ID NO: 45), an HVR-L1 comprising an amino acid sequence according to the formula RASQSVRGRFLA (SEQ ID NO: 58), an HVR-L2 comprising an amino acid sequence according to the formula DASNRATGI (SEQ ID NO: 66), and an HVR-H4 comprising an amino acid sequence according to the formula YCQQSSSWPPT (SEQ ID NO: 70). NO:75), and wherein the activatable antibody further comprises: a polypeptide covalently linked to the N-terminus of the anti-CTLA4 antibody light chain, the polypeptide comprising, from N-terminus to C-terminus, a blocking moiety (MM) and a cleavable moiety (CM), wherein the MM comprises the amino acid sequence of EVGSYPNPSSDCVPYYYACAY (SEQ ID NO:192), and the cleavable moiety comprises the amino acid sequence of SGRSAGGGGTPLGLAGSGGS (SEQ ID NO:221), wherein the activatable antibody is administered at a dose that provides a cleaved antibody steady-state concentration of about 100 nM to about 200 nM.

53. The method of claim 52, wherein the activatable antibody is administered at a dose that provides a steady-state concentration of lytic antibody of about 100 nM to about 175 nM.

54. The method of claim 52, wherein the activatable antibody is administered at a dose that provides a steady-state concentration of lytic antibody of about 100 nM to about 150 nM.

55. The method of claim 52, wherein the activatable antibody is administered at a dose that provides a steady-state concentration of lytic antibody of about 150 nM to about 200 nM.

56. The method of any one of claims 50 to 55, wherein the lytic antibody steady-state concentration is measured at a trough level of anti-CTLA4 antibody.

57. The method of any one of claims 50 to 56, wherein the activatable antibody is administered as a single loading dose followed by a maintenance dose, wherein the amount of the loading dose is higher than the amount of the maintenance dose.

58. The method of claim 57, wherein the loading dose is about 20 mg / kg.

59. The method of claim 57 or claim 58, wherein the maintenance dose is about 10 mg / kg.

60. The method of any one of claims 50 to 59, further comprising administering to the subject an effective amount of an anti-PD-1 antibody.

61. The method of claim 60, wherein the anti-PD-1 antibody is toripalimab.

62. The method of any one of claims 50 to 61, wherein the activatable antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 87, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 87, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 100, or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:

100.

63. The method of claim 62, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 87 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:

100.

64. The method of claim 62, wherein the activatable antibody comprises the full-length heavy chain region of SEQ ID NO: 320 or SEQ ID NO:

321.

65. The method of claim 64, wherein the activatable antibody comprises the full-length light chain region of SEQ ID NO: 322 or SEQ ID NO:

323.

66. The method of any one of claims 50 to 65, wherein the subject is a human.

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