Activatable anti-CTLA4 antibodies for treating cancer - Patents.com

Abstract

The present application provides compositions and methods for treating cancer using anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA4) antibodies, such as activatable anti-CTLA4 antibodies, in some embodiments, combination therapies comprising an anti-CTLA4 antibody and a PD-1 inhibitor.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 405,293, filed September 9, 2022, and U.S. Provisional Patent Application No. 63 / 495,965, filed April 13, 2023, the disclosures of which are incorporated herein by reference in their entireties.

[0002] Submitting a sequence listing as an ASCII text file The contents of the electronic sequence listing (6954020024xxSEQLIST.xml, size: 364,433 bytes, creation date: September 5, 2023) are incorporated herein by reference in their entirety.

[0003] The present application is in the field of cancer therapy and relates to antibodies that bind to human cytotoxic T-lymphocyte-associated protein 4 (CTLA4). [Background technology]

[0004] Cytotoxic T-lymphocyte protein 4 (CTLA4) is a member of the immunoglobulin (Ig) superfamily of proteins that downregulates T cell activation and maintains immunogenic homeostasis. Antibody-mediated blockade of CTLA4 in vivo has been shown to enhance anti-cancer immune responses in a syngeneic mouse prostate cancer model (Kwon et al. (1997) Proc Natl Acad Sci USA, 94(15):8099-103). Additionally, blockade of CTLA4 function in tumor-bearing mice has been shown to enhance anti-tumor T cell responses at various stages of tumor development (Yang et al. (1997) Cancer Res 57(18):4036-41; Hurwitz et al. (1998) Proc Natl Acad Sci USA 95(17):10067-7). However, developing antibody-based therapeutics suitable for human use remains challenging because the transition 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 while also providing suitable human therapeutic candidates. In addition, there is a need for the development of safer anti-CTLA4 antibodies that are active only in specific contexts, such as within the protease-rich tumor microenvironment. Summary of the Invention

[0005] The present disclosure provides methods of treating cancer with an anti-CTLA4 antibody and an activatable anti-CTLA4 antibody. The present application further provides methods of treating cancer with an anti-CTLA4 antibody and a second therapeutic agent. The present application further provides methods of treating cancer with an activatable anti-CTLA4 antibody and a second therapeutic agent. The present application further provides methods of treating cancer with an anti-CTLA4 antibody and at least two additional (e.g., two or three) therapeutic agents.

[0006] In one aspect, the invention provides a method of treating cancer in a subject, comprising: (a) administering to the subject an effective amount of an activatable antibody, the activatable antibody comprising 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 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), wherein m is 2-10 and 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 the MM inhibits binding of the activatable antibody to human CTLA4 when the CM is uncleaved; the CM comprises at least a first cleavage site; and 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). a) the TBM comprises a peptide; b) the TBM comprises an antibody heavy chain variable region (VH) and the activatable antibody further comprises a second polypeptide comprising an antibody light chain variable region (VL); c) the TBM comprises, from N-terminus to C-terminus, an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH); or d) the TBM comprises, from N-terminus to C-terminus, an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL), wherein upon cleavage of the CM, the activatable antibody binds to human CTLA4 via its 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.

[0007] In some embodiments, the activatable antibody comprises, from N- to C-terminus, a masking moiety (MM), a cleavable moiety (CM), and an anti-CTLA4 antibody polypeptide described herein, 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). The MM and CM comprise, from N- to C-terminus, the amino acid sequence of EVGSYPNPSSDCVPYYYACAYSGRSAGGGGTPLGLAGSGGS (SEQ ID NO: 200). In certain embodiments, the MM and CM are covalently linked to the N-terminus of the light chain of the anti-CTLA4 antibody. In some embodiments, the MM and CM, from N- to C-terminus, comprise amino acid sequences having at least 90% or at least 95% sequence identity to SEQ ID NO: 200.

[0008] In some embodiments, the activatable anti-CTLA4 antibody can be administered to a patient in need thereof as monotherapy. In another embodiment, the activatable anti-CTLA4 antibody can be administered in combination with one or more additional agents as described herein. In some embodiments, the cancer is ovarian cancer, pancreatic cancer, cholangiocarcinoma, 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-stable colorectal cancer, microsatellite instability-high (MSI-H) colorectal cancer, or mismatch repair-deficient (dMMR+) colorectal cancer. In some embodiments, the melanoma is uveal (UV) melanoma. In some embodiments, the cancer is squamous cell carcinoma (SCC) (e.g., anal, anorectal, penile, or cutaneous). In some embodiments, the cancer is anal squamous cell carcinoma or penile squamous cell carcinoma.

[0009] In some embodiments, the MM of the activatable anti-CTLA4 antibody further comprises an additional amino acid sequence at its N-terminus, hi some embodiments, the additional amino acid sequence comprises the amino acid sequence of SEQ ID NO:148.

[0010] In some embodiments, the first cleavage site of the activatable anti-CTLA4 antibody is a protease cleavage site for 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, 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.

[0011] In some embodiments, the CM further comprises a first linker (L1) C-terminal to the first cleavage site, wherein L1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 156-163.

[0012] In some embodiments, the CM further comprises a second cleavage site, hi some embodiments, the second cleavage site is C-terminal to L1. In some embodiments, the second cleavage site is a protease cleavage site for 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, 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 cleavage site and the second cleavage site are different.

[0013] In some embodiments, the CM further comprises a second linker (L2) C-terminal to the second cleavage site. In some embodiments, the 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) N-terminal to the first cleavage site.

[0014] 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.

[0015] In some embodiments, the activatable anti-CTLA4 antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 165-179.

[0016] In some embodiments, the activatable anti-CTLA4 antibody has a MM of an 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 of an 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 of an amino acid sequence selected from the group consisting of SEQ ID NOs: 141-147. In certain embodiments, the activatable anti-CTLA4 antibody comprises a MM of the amino acid sequence of SEQ ID NO: 200.

[0017] In some embodiments, the activatable anti-CTLA4 antibody comprises a MM / CM combination of an amino acid sequence selected from the group consisting of SEQ ID NOs: 197 to 209. In certain embodiments, the activatable anti-CTLA4 antibody has a MM / CM combination of the amino acid sequence of SEQ ID NO: 192.

[0018] In some embodiments, when the MM is cleaved, the activatable anti-CTLA4 antibody specifically binds to an epitope comprising amino acid residues Y105 and L106 of human CTLA4 but not including residue I108, the amino acid residue numbering being according to SEQ ID NO: 207.

[0019] In some embodiments, upon cleavage of the MM, a) the cleaved anti-CTLA4 antibody binds to human CTLA4, cynomolgus monkey CTLA4, mouse CTLA4, rat CTLA4, and dog CTLA4 with a dissociation constant (K) of about 350 nM or less. D ), b) binding of the anti-CTLA4 antibody induces antibody-dependent cellular cytotoxicity (ADCC) against CTLA4-expressing human cells or human Treg cells, and the ADCC activity of the anti-CTLA4 antibody is higher than the ADCC activity of ipilimumab, and / or c) in an assay where CD80 and / or CD86 are bound to a plate or human CTLA4 is present on the cell surface, the anti-CTLA4 antibody has a higher IC50 for blocking the binding of CD80 and / or CD86 to human CTLA4 than the IC50 of ipilimumab.

[0020] 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.

[0021] In some 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 above embodiments, the anti-CTLA4 antibody comprises a heavy chain variable region comprising a variant thereof 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 a variant thereof 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.

[0022] 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. An activatable antibody having a heavy chain SEQ ID NO: 320 and a light chain SEQ ID NO: 322 is designated 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.

[0023] 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 anti-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, the combination of the activatable anti-CTLA4 antibody and the PD-1 inhibitor (e.g., an anti-PD-1 antibody) exhibits a synergistic effect when administered in combination. In some of the above 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 that has at least 90% sequence identity (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%) 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 that has at least 90% sequence identity (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%) to the amino acid sequence of SEQ ID NO:100. In some of the above embodiments, the anti-CTLA4 antibody is an activatable anti-CTLA4 antibody that, when cleaved, comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 87 or an amino acid sequence that has at least 90% sequence identity (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%) 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 that has at least 90% sequence identity (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%) to the amino acid sequence of SEQ ID NO: 100.

[0024] In some embodiments, the present invention provides a method of treating cancer in a subject, comprising administering to the subject (a) an effective amount of an activatable antibody, the activatable antibody comprising, from N-terminus to C-terminus, a masking moiety (MM), a cleavable moiety (CM), and an anti-CTLA4 antibody described herein, wherein the MM comprises, from N-terminus to C-terminus, 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 (b) an effective amount of toripalimab. The MM and CM comprise, from N-terminus to C-terminus, the amino acid sequence of EVGSYPNPSSDCVPYYYACAYSGRSAGGGGTPLGLAGSGGS (SEQ ID NO: 200). In certain embodiments, the MM and CM are covalently linked to the N-terminus of the light chain of the anti-CTLA4 antibody. In some embodiments, the MM and CM, from N-terminus to C-terminus, comprise amino acid sequences having at least 90% or at least 95% sequence identity to SEQ ID NO: 200.

[0025] In some embodiments of any one of the above methods, the cancer is resistant or refractory to conventional therapy, and the conventional therapy is an inhibitor of CTLA4, PD-1, or PD-1 ligand. In some embodiments, the subject is resistant to conventional therapy or has relapsed after conventional therapy, and the conventional therapy is an inhibitor of CTLA4, PD-1, or PD-1 ligand. In some embodiments, the conventional therapy is a CTLA4 inhibitor such as an anti-CTLA4 antibody, e.g., ipilimumab. In some embodiments, the conventional therapy is a PD-1 inhibitor such as an anti-PD-1 antibody, e.g., nivolumab or toripalimab. In some embodiments, the conventional therapy is a PD-1 ligand (e.g., PD-L1) inhibitor, e.g., an anti-PD-L1 antibody. In some embodiments, the conventional therapy includes both a CTLA4 inhibitor and a PD-1 inhibitor. In some embodiments, the conventional therapy includes both a CTLA4 inhibitor and a PD-L1 inhibitor. In some of the above 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 that has at least 90% sequence identity (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%) 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 that has at least 90% sequence identity (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%) to the amino acid sequence of SEQ ID NO: 100. In some of the above embodiments, the activatable anti-CTLA4 antibody comprises the Fc region of human IgG1, e.g., the Fc region of wild-type IgG1, or a variant with enhanced ADCC activity. In some of the above embodiments, the activatable anti-CTLA4 antibody comprises a human IgG1 Fc region, eg, a wild-type IgG1 Fc region or a variant with enhanced ADCC activity.In some of the above 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 that has at least 90% sequence identity (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%) 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 that has at least 90% sequence identity (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%) to the amino acid sequence of SEQ ID NO: 322.

[0026] In any of the foregoing embodiments, the activatable anti-CTLA4 antibody (e.g., TY22404), when administered as monotherapy or in combination with other agents, can be administered at a dose that provides a steady-state concentration of the cleaved antibody (i.e., active antibody following cleavage of the masking moiety (MM) and cleavable moiety (CM)) that exceeds the EC50 value of the cleaved antibody. In some embodiments, the activatable anti-CTLA4 antibody (e.g., TY22404), when administered as monotherapy or in combination with other agents, can be administered at a dose that provides a steady-state concentration of the cleaved antibody that exceeds the EC50 value of the cleaved antibody. In some of the above embodiments, the concentration of the cleaved antibody is measured at the trough level of the anti-CTLA4 antibody.

[0027] In any of the foregoing embodiments, the activatable anti-CTLA4 antibody (e.g., TY22404), when administered as monotherapy or in combination with other agents, can be administered at a dose that results in a steady-state concentration of the cleaved antibody (i.e., active antibody following cleavage of the masking moiety (MM) and cleavable moiety (CM)) of about 100 nM to about 600 nM. In some embodiments, the activatable anti-CTLA4 antibody is administered at a dose that results in a steady-state concentration of the cleaved antibody of about 100 nM to about 175 nM. In some embodiments, the activatable anti-CTLA4 antibody is administered at a dose that results in a steady-state concentration of the cleaved antibody of about 100 nM to about 200 nM. In some embodiments, the activatable anti-CTLA4 antibody is administered at a dose that results in a steady-state concentration of the cleaved antibody of about 125 nM to about 200 nM. In some embodiments, the activatable anti-CTLA4 antibody is administered at a dose that results in a steady-state concentration of the cleaved antibody of about 100 nM to about 150 nM. In some embodiments, the activatable anti-CTLA4 antibody is administered at a dose that results in a steady-state concentration of the cleaved antibody of about 125 nM to about 175 nM. In some embodiments, the activatable anti-CTLA4 antibody is administered at a dose that results in a steady-state concentration of the cleaved antibody of about 125 nM to about 150 nM. In some embodiments, the activatable anti-CTLA4 antibody is administered at a dose that results in a steady-state concentration of the cleaved antibody of about 150 nM to about 200 nM. In some embodiments, the activatable anti-CTLA4 antibody is administered at a dose that results in a steady-state concentration of the cleaved antibody of about 200 nM to about 600 nM. In some embodiments, the activatable anti-CTLA4 antibody is administered at a dose that results in a steady-state concentration of the cleaved antibody of about 200 nM to about 400 nM. In some embodiments, the activatable anti-CTLA4 antibody is administered at a dose that results in a steady-state concentration of the cleaved antibody of about 300 nM to about 500 nM. In some embodiments, the activatable anti-CTLA4 antibody is administered at a dose that results in a steady-state concentration of cleaved antibody of about 400 nM to about 600 nM. In some of these embodiments, the plasma concentration can be measured at the trough level (i.e., the minimum concentration during each dosing cycle) of the anti-CTLA4 antibody.For example, plasma concentrations for a particular cycle can be measured immediately before administration in the next cycle.

[0028] In some embodiments, the activatable anti-CTLA4 antibody is administered in combination with two or more therapeutic agents. In some embodiments, at least one of the therapeutic agents is an anti-PD1 antibody. In some embodiments, the activatable anti-CTLA4 antibody is administered at a dose that results in 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 results in 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 results in 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 results in a steady-state concentration of the cleaved antibody of about 75 nM to about 100 nM.

[0029] In any of the foregoing embodiments, the activatable anti-CTLA4 antibody (e.g., TY22404), when administered as monotherapy or in combination with other agents, can be administered at a dose that provides a steady-state plasma concentration ratio of cleaved antibody to uncleaved antibody at trough levels for a particular administration cycle of about 0.3 to about 1.0. In some embodiments, the activatable anti-CTLA4 antibody can be administered at a dose that provides a steady-state plasma concentration ratio of cleaved antibody to uncleaved antibody at trough levels for a particular administration cycle of about 0.3 to about 1.0. In some embodiments, the activatable anti-CTLA4 antibody can be administered at a dose that provides a steady-state plasma concentration ratio of cleaved antibody to uncleaved antibody at trough levels for a particular administration cycle of about 0.5 to about 0.8. In some embodiments, the activatable anti-CTLA4 antibody can be administered at a dose that provides a steady-state plasma concentration ratio of cleaved antibody to uncleaved antibody at trough levels for a particular administration cycle of about 0.7 to about 1.0.

[0030] In any of the foregoing embodiments, the activatable anti-CTLA4 antibody can be administered to the subject at a dose of about 3 mg / kg to about 20 mg / kg, e.g., 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 embodiments, the activatable anti-CTLA4 antibody is administered once every three weeks. In some embodiments, the activatable anti-CTLA4 antibody is administered once every six weeks. In some embodiments, the activatable antibody is administered once every three weeks at a dose of about 10 mg / kg. In some embodiments, the activatable antibody is administered once every three weeks at a dose of about 20 mg / kg.

[0031] In some embodiments, the activatable anti-CTLA4 antibody is administered at a first higher dose (e.g., about 10 mg / kg to about 20 mg) for at least one treatment cycle (as defined herein), followed by lower doses (e.g., about 3 mg / kg to about 10 mg / kg) in subsequent cycles. 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., 1 to 3 treatment cycles) and at a dose of about 10 mg / kg for subsequent treatment cycles (e.g., once every 3 weeks). In one embodiment, 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 for subsequent treatment cycles (e.g., once every 3 weeks).

[0032] Based on noncompartmental analysis (NCA) and population PK modeling, intact drugs (i.e., uncleaved drugs) show a dose-dependent increase in plasma concentrations. Surprisingly, however, body weight was not modeled as a significant covariate (e.g., exponent values ​​greater than 0.8 in allometric scaling, which are considered significant) for key PK parameters such as clearance (CL) and volume of distribution (V). Virtual patient simulations, as described below, demonstrate that flat dosing regimens are readily feasible for anti-CTLA4 antibodies, whether used as monotherapy or as part of a combination therapy. Notably, in some embodiments, a fixed dose of an activatable anti-CTLA4 antibody (e.g., TY22404) can be approximately 500 mg to approximately 1000 mg once every three weeks. In other embodiments, a fixed dosing schedule for an activatable anti-CTLA4 antibody (e.g., TY22404) can be approximately 700 mg to approximately 1000 mg once every three weeks. In another embodiment, the routine administration schedule for an activatable anti-CTLA4 antibody (e.g., TY22404) can be about 500 mg to about 750 mg once every three weeks. In another embodiment, the routine administration schedule for an activatable anti-CTLA4 antibody (e.g., TY22404) can be about 750 mg to about 1,000 mg once every three weeks. In another embodiment, the routine administration schedule for an activatable anti-CTLA4 antibody (e.g., TY22404) can be about 500 mg to about 1,200 mg once every six weeks. In another embodiment, the routine administration schedule for an activatable anti-CTLA4 antibody (e.g., TY22404) can be about 500 mg to about 1,000 mg once every six weeks. In another embodiment, the routine administration schedule for an activatable anti-CTLA4 antibody (e.g., TY22404) can be about 500 mg to about 750 mg once every six weeks. In other embodiments, the routine administration schedule for an activatable anti-CTLA4 antibody (e.g., TY22404) can be about 750 mg to about 1,000 mg once every six weeks. In other embodiments, the routine administration schedule for an activatable anti-CTLA4 antibody (e.g., TY22404) can be about 500 mg once every three weeks or once every six weeks.In other embodiments, the regular dosing schedule for an activatable anti-CTLA4 antibody (e.g., TY22404) can be about 700 mg every three weeks or every six weeks. In other embodiments, the dosing schedule for a flat-dose activatable anti-CTLA4 antibody (e.g., TY22404) can be about 1,000 mg every three weeks or every six weeks.

[0033] It should be understood that one, some, or all of the features of the various embodiments described above and herein may be combined to form additional embodiments of the present application. These and other aspects of the present application will be apparent to those skilled in the art. These and other embodiments of the present application are further described in the detailed description below. [Brief explanation of the drawings]

[0034] [Figure 1] demonstrated a CA125 response in a patient with ovarian serous carcinoma, with a 90% reduction in CA125 from 303 to 31 U / ml (normal <35 U / ml) at the end of cycle 16. [Figure 2] Clinical activity evaluation of TY22404 monotherapy. (A) Maximum tumor burden reduction in patients across dose levels (0.1-20 mg / kg). (B) Duration of patient treatment across dose levels (0.1-20 mg / kg). [Figure 3] Plasma PK of total and cleaved TY22404 over the first four cycles in (A) 20 patients with PK data and (B) an ovarian serous carcinoma patient (case study) is shown. Total and tactic TY22404 were measured by LC-MS using the signature peptide. Tactic TY22404 is calculated by subtracting tactic TY22404 from total TY22404. C = treatment cycles [Figures 4A-4C]We demonstrate the efficacy of TY22404 monotherapy in a 39-year-old male patient with stage IIIB hepatocellular carcinoma. Figure 4A shows that an increase in the Teff / Treg ratio was observed in the patient's paired tumor biopsy. Figure 4B shows that a decrease in Tregs was observed in the patient's paired tumor biopsy. Figure 4C shows that an increase in CD8+ T cells was observed in the patient's paired tumor biopsy. [Figure 5A-5B] Response to TY22404 in combination with toripalimab is shown in 18 evaluable patients* across three dose-escalation cohorts who received TY22404 (6 mg / kg Q3W and 10 mg / kg Q3W or Q6W) plus toripalimab (240 mg Q3W). Figure 5A shows the swimmer plot. Figure 5B shows the waterfall plot. *Evaluable patients with at least one valid post-treatment tumor assessment. For the swimmer plot, the bar ends at the end-of-treatment (EOT) study date, the date of last dose, or the date of last tumor assessment, whichever is most recent. [Figures 6A-6B] Figure 6A shows the response of TY22404 plus toripalimab in selected gastrointestinal "cold tumors," including MSS-CRC and PDAC with liver metastases at baseline. Figure 6B shows the waterfall plot. DETAILED DESCRIPTION OF THE INVENTION

[0035] I. Definition Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meanings commonly understood by those of ordinary skill in the art. Furthermore, unless otherwise required by context, singular terms shall include the plural terms and plural terms shall include the singular terms. Generally, the nomenclature used in connection with, and techniques of, antibody engineering, immunotherapy, cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry described herein are those well known and commonly used in the art.

[0036] The term "antibody" is used herein in the broadest sense and specifically encompasses 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, i.e., scFv), so long as they exhibit the desired biological activity.

[0037] V H Area and V L Based on structural and sequence analysis, the V region can be further subdivided into regions of hypervariability called hypervariable regions (HVRs). HVRs are flanked by more conserved regions called framework regions (FWs) (see, e.g., Chenetal. (1999) J. Mol. Biol. (1999) 293, 865-881). Each V H and V L consists of three HVRs and four FWs, arranged from amino to carboxy terminus in the following order: FW-1_HVR-1_FW-2_HVR-2_FW-3_HVR-3_FW4. Throughout this application, the three HVRs in the heavy chain are referred to as HVR-H1, HVR-H2, and HVR-H3. Similarly, the three HVRs in the light chain are referred to as HVR-L1, HVR-L2, and HVR-L3.

[0038] As used herein, the term "CDR" or "complementarity determining region" is intended to mean the discontinuous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. These specific regions are described in Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., US Department 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); MacCallume tal., J. Mol. Biol. 262:732-745 (1996), Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008), Lefranc M. P. et al., Dev. Comp. Immunol., 27:55-77 (2003), and Honegger and Pluckthun, J. Mol. Biol., 309:657-670 (2001), and the definition includes overlapping amino acid residues or subsets of amino acid residues when compared with each other.

[0039] 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 (e.g., CTLA4).

[0040] As used herein, the term "CTLA4" 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), canine 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 of species other than human. In other cases, anti-CTLA4 antibodies may be completely specific for human CTLA4 and may not exhibit species or other types of cross-reactivity.

[0041] The term "CTLA4 antibody," as defined herein, refers to an antibody capable of binding to human CTLA4 (eg, an activatable anti-CTLA4 antibody).

[0042] The term "epitope" refers to the portion of an antigen to which an antibody (or antigen-binding fragment thereof) binds. Epitopes can be formed both from contiguous amino acids or from non-contiguous amino acids juxtaposed by folding of a protein into a tertiary structure.

[0043] A "human antibody" is an antibody having an amino acid sequence that corresponds to that of an antibody produced by a human or a human cell, or an antibody derived from the human antibody repertoire or other non-human source that utilizes human antibody-encoding sequences.

[0044] The term "humanized antibody" refers to a chimeric antibody containing amino acid residues derived from human antibody sequences.

[0045] The term "exemplary antibody" refers to any one of the antibodies described in this disclosure and defined as those listed in Tables A and B, as well as any antibody comprising the six HVRs and / or VH and VL of the antibodies listed in Tables A and B. These antibodies may be of any class (e.g., IgA, IgD, IgE, IgG, and IgM).

[0046] The term "mammal" refers to any animal species of the class Mammalia.

[0047] As used herein, "sequence identity" between two polypeptide sequences indicates the percentage of amino acids that are identical between the sequences.

[0048] The terms "treat," "treating," or "treatment," with respect to a particular disease state in a mammal, refer to producing a desired or beneficial effect in a mammal having the disease state. The desired or beneficial effect may include reducing the frequency or severity of one or more symptoms of the disease (i.e., tumor growth and / or metastasis, or other effects mediated by immune cell number and / or activity, etc.), or arresting or inhibiting further development of the disease, condition, or disorder. With respect to treating cancer in a mammal, the desired or beneficial effect may include inhibiting further growth or spread of cancer cells, killing cancer cells, inhibiting cancer recurrence, reducing pain associated with cancer, or improving survival of the mammal. The effect can be either subjective or objective. For example, if the mammal is a human, the human may perceive improved vitality or viability or reduced pain as subjective symptoms of improvement or therapeutic response. Alternatively, a clinician may perceive a decrease in tumor size or tumor burden based on physical examination, clinical laboratory values, tumor markers, or x-ray findings. Some laboratory signs that a clinician may observe for response to treatment include normalization of test results such as white blood cell count, red blood cell count, platelet count, erythrocyte sedimentation rate, and various enzyme levels. In addition, a clinician may observe a decrease in detectable tumor markers. Alternatively, other tests such as sonograms, nuclear magnetic resonance imaging, and positron emission tomography can be used to assess objective improvement.

[0049] The term "prevent" or "preventing" refers to preventing or delaying the onset of disease or preventing the manifestation of clinical or subclinical symptoms of a particular condition in a mammal.

[0050] As used herein, a "subject," "patient," or "individual" may refer to a human or a non-human animal. A "non-human animal" may refer to any animal not classified as a human, such as farm animals, livestock or zoo animals, sport animals, pet animals (dogs, horses, cats, cows, etc.), and animals used in research. Research animals may refer to, but are not limited to, 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 human.

[0051] An "effective amount" refers to at least an amount effective, at dosages and for periods of time necessary, to achieve one or more desired or indicated effects, including therapeutic or prophylactic results. An effective amount can be provided in one or more administrations. For purposes of this application, an effective amount of an antibody, drug, compound, or pharmaceutical composition is an amount sufficient to effect prophylactic or therapeutic treatment, either directly or indirectly. As understood in a clinical context, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition (e.g., an effective amount administered as monotherapy or combination therapy). That is, an "effective amount" may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be administered in an effective amount if, in conjunction with one or more other agents, a desired result is likely or is being achieved.

[0052] The terms "recurrence," "relapse," or "relapsed" refer to the recurrence of cancer or disease after a clinical assessment that the disease had disappeared. A diagnosis of distant metastasis or local recurrence can be considered a recurrence.

[0053] The terms "refractory" or "resistant" refer to a cancer or disease that has not responded to treatment.

[0054] As used herein, "complete response" or "CR" refers to the disappearance of all target lesions, "partial response" or "PR" refers to at least a 30% reduction in the sum of the longest diameter (SLD) of the target lesions, based on the baseline SLD, and "stable disease" or "SD" refers to target lesions that have not shrunk enough to warrant PR or have not increased enough to warrant PD, based on the smallest SLD since treatment began.

[0055] As used herein, "progression" or "PD" refers to an increase in the SLD of a target lesion by at least 20% based on the smallest recorded SLD since treatment began, or the presence of one or more new lesions.

[0056] As used herein, "progression-free survival" (PFS) refers to the length of time during or after treatment during which the disease being treated (e.g., cancer) does not worsen. Progression-free survival can include the amount of time a patient experiences a complete or partial response, as well as the amount of time a patient experiences stable disease.

[0057] As used herein, "overall response rate" (ORR) refers to the sum of the complete response (CR) rate and the partial response (PR) rate.

[0058] As used herein, "overall survival rate" refers to the percentage of individuals in a group who are likely to be alive after a particular period of time.

[0059] As used herein, "baseline level" or "baseline value" refers to a subject's level or value before the subject begins treatment, such as treatment with an anti-CTLA4 antibody.

[0060] A "reference sample," "reference cell," "reference tissue," "control sample," "control cell," or "control tissue," as used herein, refers to a sample, cell, tissue, standard, or level used for comparison purposes.

[0061] "Correlate" or "correlating" means comparing, in any manner, the outcomes and / or results of a first analysis or protocol with the outcomes and / or results of a second analysis or protocol. For example, the results of the first analysis or protocol may be used in performing the second protocol and / or to determine whether the second analysis or protocol should be performed.

[0062] A patient "effective response" or patient "responsiveness," and similar phrases, to pharmaceutical treatment refers to a clinical or therapeutic benefit experienced by a patient at risk for or suffering from a disease or disorder, such as cancer. In one embodiment, such benefit includes any one or more of: increased survival (including overall survival and progression-free survival), achieving an objective response (including a complete or partial response), or amelioration of signs or symptoms of cancer.

[0063] A patient who "does not respond effectively" to treatment is one who does not demonstrate any of the following: an increase in survival (including overall survival and progression-free survival), an objective response (including a complete or partial response), or an improvement in the signs or symptoms of cancer.

[0064] As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology - A Synthesis (2nd Edition, E.S. Goluband and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)).

[0065] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise. Thus, for example, reference to a "molecule" includes any combination of two or more such molecules, and the like.

[0066] As used herein, the term "about" refers to a normal error range for the respective value, which is readily understood by one of ordinary skill in the art. When a value or parameter to which "about" is attached is referred herein, embodiments directed to the value or parameter itself are included (and described).

[0067] It is to be understood that the aspects and embodiments of the present application described herein include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0068] As used herein, reference to "not being" a value or parameter generally means and describes "other than" a value or parameter. For example, not using the method to treat type X of cancer means using the method to treat types of cancer other than X.

[0069] As used herein, the term "about X to Y" has the same meaning as "about X to about Y."

[0070] As used herein, the term "and / or" is intended to include both A and B, A or B, A alone, and B alone in phrases such as "A and / or B." Similarly, as used herein, the term "and / or" is intended to include 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 in phrases such as "A, B, and / or C."

[0071] II. Treatment Methods The present application provides methods of treating cancer in a subject using an activatable anti-CTLA4 antibody that specifically binds to human CTLA4, which may be administered alone as monotherapy or in combination with one or more additional therapeutic agents or therapies.

[0072] The methods described herein are useful for treating various cancers. In some embodiments, the cancer is a solid cancer. In some embodiments, the cancer is a liquid cancer. Regardless of whether the cancer is malignant or benign, and regardless of whether it is a primary cancer or a secondary cancer, various cancers in which CTLA4 is involved can be treated or prevented by the methods provided by the present disclosure. Exemplary cancers include, but are not limited to, liver cancer, cancer of the digestive system (e.g., colon cancer, colorectal cancer (CRC), gastrointestinal stromal tumor (GIST), cecal adenocarcinoma), lung cancer, bone cancer, heart cancer, brain cancer, kidney cancer, bladder cancer, blood cancer (e.g., leukemia), skin cancer, breast cancer, thyroid cancer, neuroendocrine cancer, pancreatic cancer (e.g., pancreatic ductal adenocarcinoma (PDAC)), head and neck cancer, eye-related cancer, cancer of the male reproductive system (e.g., prostate cancer, testicular cancer), or cancer of the female reproductive system (e.g., uterine cancer, cervical cancer (e.g., cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), endometrial carcinoma, uterine carcinoma). In some embodiments, the cancer is squamous cell carcinoma (SCC) (e.g., anal, anorectal, penile, or skin). In some embodiments, the cancer is anal squamous cell carcinoma or penile squamous cell carcinoma. In some embodiments, the cancer is an adenocarcinoma. In some embodiments, the cancer is a 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, cholangiocarcinoma, 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 carcinoma, microsatellite stable (MSS) colorectal carcinoma). 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 carcinoma, microsatellite stable (MSS) colorectal carcinoma).

[0073] In some embodiments, the activatable anti-CTLA4 is administered to a cancer patient as monotherapy. In some embodiments, the activatable anti-CTLA4 is administered to a cancer patient as combination therapy. In some embodiments, the cancer is squamous cell carcinoma (e.g., anal, anorectal, penile, 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, cholangiocarcinoma, 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 MSS CRC. In some embodiments, the cancer is anal squamous cell carcinoma or penile squamous cell carcinoma.

[0074] Cancer treatment can be evaluated, for example, by tumor regression, reduction in tumor weight or size, time to progression, survival, progression-free survival, overall response rate, duration of response, quality of life, protein expression and / or activity. Approaches to determining efficacy of therapy can be used, including measuring response, for example, by radiological imaging.

[0075] The activatable anti-CTLA4 antibodies, one or more additional therapeutic agents, and compositions provided by the present disclosure can be administered via any suitable enteral or parenteral route of administration. The term "enteral administration route" refers to administration via any portion of the gastrointestinal tract. Examples of enteral routes include oral, mucosal, buccal, and rectal, or intragastric routes. A "parenteral administration route" refers to a route of administration other than enteral. Examples of parenteral administration routes include intravenous, intramuscular, intradermal, intraperitoneal, intratumoral, intravesical, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, transvisceral, intraarticular, subcapsular, intrathecal, intraspinal, epidural, intrasternal, subcutaneous, or topical administration. The antibodies and compositions of the present disclosure can be administered using any suitable method, including oral ingestion, nasogastric tube, gastrostomy tube, injection, infusion, implantable infusion pump, and osmotic pump. The appropriate route and method of administration can vary depending on many factors, such as the particular antibody used, the desired rate of absorption, the particular formulation or dosage form used, the type or severity of the disorder being treated, the particular site of action, and the condition of the patient, and can be readily selected by one of skill in the art. In some embodiments, the anti-CTLA4 antibody is administered intravenously.

[0076] An effective amount of the activatable anti-CTLA4 antibody and / or one or more additional therapeutic agents may be administered in a single dose or multiple doses. For methods involving administration of the anti-CTLA4 antibody in multiple doses, exemplary dosing frequencies include, but are not limited to, once per week, once per week without interruption, two weeks out of three weeks, once per week, three weeks out of four weeks, once per week, once per three weeks, once per two weeks, once per month, once per six months, once per year, etc. In some embodiments, the anti-CTLA4 antibody is administered about once per week, once per two weeks, once per three weeks, once per six weeks, or once per 12 weeks. In some embodiments, the interval between each administration is less than about 3 years, about 2 years, about 12 months, about 11 months, about 10 months, about 9 months, about 8 months, about 7 months, about 6 months, about 5 months, about 4 months, about 3 months, about 2 months, about 1 month, about 4 weeks, about 3 weeks, about 2 weeks, or about 1 week. In some embodiments, the interval between each administration is greater than about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 2 years, or about 3 years. In some embodiments, there is no break in the administration schedule.

[0077] In some embodiments, the activatable anti-CTLA4 antibody and / or one or more additional therapeutic agents are administered infrequently, for example, once per week, once per two weeks, once per three weeks, once per month, once per two months, once per three months, once per four months, once per five months, once per six months, once per seven months, once per eight months, once per nine months, once per ten months, once per eleven months, once per 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 about once every three weeks. In some embodiments, the activatable anti-CTLA4 antibody and / or one or more additional therapeutic agents are administered about once every six weeks.

[0078] In some embodiments, the activatable anti-CTLA4 antibody and / or one or more additional therapeutic agents are administered for two or more cycles, e.g., any one of about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, or about 12 or more cycles. In some embodiments, the anti-CTLA4 antibody is administered for at least four cycles.

[0079] In some embodiments, the treatment comprises an initial phase followed by a maintenance phase. In some embodiments, the activatable anti-CTLA4 antibody and / or one or more additional therapeutic agents are administered less frequently during the maintenance phase than during the initial phase. In some embodiments, the anti-CTLA4 antibody and / or one or more additional therapeutic agents are administered at the same frequency during the maintenance phase as during the initial phase. In some embodiments, the treatment comprises an initial phase in which 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 in which the anti-CTLA4 antibody and / or one or more additional therapeutic agents are administered approximately once every four weeks to once every twelve weeks, e.g., once every four weeks, once every six weeks, once every eight weeks, once every ten weeks, or once every twelve weeks. In some embodiments, the frequency of administration during the maintenance phase is determined by the level of one or more biomarkers, e.g., T reg cells, CD8+T em cells, CD4+T em CD8+ T cells, Treg cells em Ratio of CD4+ T cells to Treg cells em For example, if the subject has a high percentage of T cells, CD25+ T cells, and / or NK cells after administration of an anti-CTLA4 antibody, reg CD8+ T cells em If an increase in the proportion of cells is observed, the subject may receive further administration of anti-CTLA4 antibody approximately once every four weeks.

[0080] Administration of the anti-CTLA4 antibody and / or one or more additional therapeutic agents can be extended over an extended period of time, e.g., from about one week to about one month, from about one month to about one year, or 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 one week, about two weeks, about three weeks, about four weeks, about five weeks, about one month, about two months, about three months, about four months, about five months, about six months, about seven months, about eight months, about nine months, about ten months, about 11 months, about 12 months, about one year, about two years, about three years, or about four years or more.

[0081] Upon cleavage of the MM, the activatable 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% sequence identity (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%) to the amino acid sequence of SEQ ID NO:87, and / or the amino acid sequence of SEQ ID NO:100 or an amino acid sequence having at least 90% sequence identity (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%) to the amino acid sequence of SEQ ID NO:100. In some embodiments, the activatable anti-CTLA4 antibody comprises a light chain variable region comprising an amino acid sequence that is 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% ribonucleotides (RIs) of the activatable anti-CTLA4 antibody. In some such embodiments, the activatable anti-CTLA4 antibody is administered at a dose of about 3 mg / kg to about 20 mg / kg once every three weeks, about 3 mg / kg to about 15 mg / kg once every three weeks, about 6 mg / kg to about 15 mg / kg once every three weeks, or about 6 mg / kg to about 10 mg / kg once every three weeks. In some such embodiments, the activatable anti-CTLA4 antibody is administered at a dose of about 3 mg / kg once every three weeks. In other such embodiments, the activatable anti-CTLA4 antibody is administered at a dose of about 6 mg / kg once every three weeks. In other such embodiments, the activatable anti-CTLA4 antibody is administered at a dose of about 10 mg / kg once every three weeks. In some of the above 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 above embodiments, 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 above embodiments, the activatable anti-CTLA4 antibody comprises a MM of an amino acid sequence selected from the group consisting of SEQ ID NOs: 189-196.In some of the above embodiments, the activatable anti-CTLA4 antibody comprises a MM of an amino acid sequence selected from the group consisting of SEQ ID NOs: 213-216. In some of the above embodiments, the activatable anti-CTLA4 antibody comprises a MM of an amino acid sequence selected from the group consisting of SEQ ID NOs: 141-147. In some of the above embodiments, the activatable anti-CTLA4 antibody comprises a MM of the amino acid sequence of SEQ ID NO: 200. In some of the above 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% sequence identity (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%) 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% sequence identity (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%) to the amino acid sequence of SEQ ID NO: 322. In some of the above 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 of the above embodiments, the activatable anti-CTLA4 antibody is administered to a patient with MSI-H or dMMR cancer. In other of the above embodiments, the activatable anti-CTLA4 antibody is administered to a patient with metastatic cancer. In some of the above embodiments, the activatable anti-CTLA4 antibody is administered to a patient who is resistant or refractory to conventional cancer therapies, including other anti-CTLA4 antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, or combinations thereof.

[0082] In some embodiments, when MM is cleaved, the activatable anti-CTLA4 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 87, or an amino acid sequence that has at least 90% sequence identity (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%) 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 that has at least 90% sequence identity (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%) to the amino acid sequence of SEQ ID NO: 100, the activatable anti-CTLA4 antibody is administered at a first higher dose (e.g., about 10 mg / kg to about 20 mg) for at least one treatment cycle (as defined herein), followed by lower doses (e.g., about 3 mg / kg to about 10 mg / kg) in subsequent cycles. 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., 1-3 treatment cycles), followed by 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., 1-3 treatment cycles), followed by 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., 1-3 treatment cycles), followed by 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., 1-3 treatment cycles), followed by a dose of about 10 mg / kg in subsequent treatment cycles.In some of the above 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 above 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% sequence identity (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%) 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% sequence identity (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%) to the amino acid sequence of SEQ ID NO: 322. In some of the above 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 of the above embodiments, the activatable anti-CTLA4 antibody is administered to a patient with MSI-H or dMMR cancer. In other of the above embodiments, the activatable anti-CTLA4 antibody is administered to a patient with metastatic cancer. In some of the above embodiments, the activatable anti-CTLA4 antibody is administered to a patient who is resistant or refractory to conventional cancer therapies, including other anti-CTLA4 antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, or combinations thereof.

[0083] In some embodiments, the activatable anti-CTLA4 antibody is administered at a dose of about 20 mg / kg for one treatment cycle, followed by a dose of about 10 mg / kg for subsequent treatment cycles (e.g., once every three weeks). For example, in one embodiment, the anti-CTLA4 antibody is administered at 20 mg / kg as an initial (loading) dose, followed by additional doses (maintenance doses) of 10 mg / kg every three weeks. The maintenance doses can be initiated at a predetermined time after administration of the loading dose. For example, in some embodiments, the first maintenance dose can be administered three weeks after administration of the loading dose.

[0084] As described above, it has been found that administering a single loading dose followed by a maintenance dose establishes steady-state concentrations of the cleaved antibody more quickly than administering an activated antibody 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 administering the initial loading dose.

[0085] In any of the foregoing cases, the activatable anti-CTLA4 antibody is administered at a dose that results in a steady-state concentration of the cleaved antibody of about 100 nM to about 200 nM. In some embodiments, the activatable anti-CTLA4 antibody is administered at a dose that results in a steady-state concentration of the cleaved antibody of about 125 nM to about 200 nM. In some embodiments, the activatable anti-CTLA4 antibody is administered at a dose that results in a steady-state concentration of the cleaved antibody of about 100 nM to about 175 nM. In some embodiments, the activatable anti-CTLA4 antibody is administered at a dose that results in a steady-state concentration of the cleaved antibody of about 125 nM to about 175 nM. In some embodiments, the activatable anti-CTLA4 antibody is administered at a dose that results in a steady-state concentration of the cleaved antibody of about 100 nM to about 150 nM. In some embodiments, the activatable anti-CTLA4 antibody is administered at a dose that results in a steady-state concentration of the cleaved antibody of about 125 nM to about 150 nM. In some embodiments, the activatable anti-CTLA4 antibody is administered at a dose that results in a steady-state concentration of the cleaved antibody of about 150 nM to about 200 nM. In some embodiments, the activatable anti-CTLA4 antibody is administered at a dose that results in a steady-state concentration of the cleaved antibody of about 200 nM to about 600 nM. In some embodiments, the activatable anti-CTLA4 antibody is administered at a dose that results in a steady-state concentration of the cleaved antibody of about 200 nM to about 400 nM. In some embodiments, the activatable anti-CTLA4 antibody is administered at a dose that results in a steady-state concentration of the cleaved antibody of about 300 nM to about 500 nM. In some embodiments, the activatable anti-CTLA4 antibody is administered at a dose that results in a steady-state concentration of the cleaved antibody of about 400 nM to about 600 nM. In some of the above embodiments, the plasma concentration can be measured at the trough level of the anti-CTLA4 antibody (i.e., the minimum concentration of each administration cycle). For example, plasma concentrations for a particular cycle can be measured immediately before administration in the next cycle.

[0086] In some embodiments, the activatable anti-CTLA4 antibody is administered in combination with two or more therapeutic agents. In some embodiments, at least one of the therapeutic agents is an anti-PD1 antibody. In some embodiments, the activatable anti-CTLA4 antibody is administered at a dose that results in 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 results in 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 results in 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 results in a steady-state concentration of the cleaved antibody of about 75 nM to about 100 nM.

[0087] In either of the embodiments of the previous two paragraphs, the anti-CTLA4 antibody can be administered as monotherapy or in combination with one or more anti-cancer agents as 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.

[0088] In some embodiments, treatment with an anti-CTLA4 antibody and / or one or more additional therapeutic agents depletes Treg cells in tumors, hi some embodiments, treatment with an anti-CTLA4 antibody and / or one or more additional therapeutic agents does not deplete Treg cells in peripheral tissues.

[0089] In some embodiments, the subject has previously been treated with conventional therapy. In some embodiments, the subject has previously received any one of one, two, three, or more prior therapies. In some embodiments, the subject has exhausted all other available therapies. In some embodiments, the subject is unresponsive or resistant to conventional therapy. In some embodiments, the subject has experienced a disease relapse after conventional therapy. In some embodiments, the subject is refractory to conventional therapy. In some embodiments, the subject has failed conventional therapy within about 1 year, about 6 months, or about 3 months. In some embodiments, the subject has not previously received conventional therapy.

[0090] In some embodiments, the subject has previously been treated with standard therapy for the cancer. In some embodiments, the subject does not respond to or is resistant to standard therapy. In some embodiments, the subject has a recurrence of disease after standard therapy. In some embodiments, the subject is refractory to standard therapy. In some embodiments, the subject has failed standard therapy within about 1 year, about 6 months, or about 3 months. In some embodiments, the subject has not previously received standard therapy. In some embodiments, the subject has refused standard therapy or is ineligible for standard therapy.

[0091] In some embodiments, the conventional therapy (e.g., standard therapy) is selected from the group consisting of viral gene therapy, immunotherapy, targeted therapy, radiation therapy, and chemotherapy. In some embodiments, the conventional therapy is an immune checkpoint inhibitor. In some embodiments, the conventional therapy is an inhibitor of CTLA4, PD-1, or a PD-1 ligand (e.g., PD-L1 or PD-L2). In some embodiments, the conventional therapy is a CTLA4 inhibitor, such as an anti-CTLA4 antibody different from the anti-CTLA4 antibodies described herein. In some embodiments, the conventional therapy is ipilimumab. In some embodiments, the conventional therapy is ipilimumab and / or nivolumab.

[0092] In some embodiments, the conventional therapy is an inhibitor of PD-1 or a PD-1 ligand, including a PD-1 binding antagonist, a PDL1 binding antagonist, and a PDL2 binding antagonist. Alternative names for "PD-1" include CD279 and SLEB2. Alternative names for "PDL1" include B7-H1, B7-4, CD274, and B7-H. Alternative names for "PDL2" include B7-DC, Btdc, and CD273. In some embodiments, PD-1, PDL1, and PDL2 are human PD-1, human PDL1, and human PDL2.

[0093] In some embodiments, the inhibitor of PD-1 is a molecule that inhibits PD-1 from binding to its ligand binding partner. In some embodiments, the inhibitor of PD-1 ligand is an inhibitor of PD-L1 and / or PD-L2. In some embodiments, the inhibitor of PD-L1 is a molecule that inhibits PDL1 from binding to its binding partner. In some embodiments, the binding partner of PD-L2 is PD-1 and / or B7-1. In some embodiments, the inhibitor of PD-1 ligand is a molecule that inhibits PD-L2 from binding to its binding partner. In some embodiments, the binding partner of PD-L2 is PD-1. The inhibitor may be an antibody, antigen-binding fragment thereof, immunoadhesin, fusion protein, or oligopeptide.

[0094] 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 portion 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 OPDIVO®, is an anti-PD-1 antibody described in WO 2006 / 121168. CT-011, also known as hBAT or hBAT-1, is an anti-PD-1 antibody described in WO 2009 / 101611. AMP-224, also known as B7-DCIg, is a soluble receptor that is a PDL2-Fc fusion, described in WO 2010 / 027827 and WO 2011 / 066342. In some embodiments, the anti-PD-1 antibody is nivolumab (CAS Registry Number: 946414-94-4).

[0095] Prior treatment (e.g., standard therapy) also includes surgery to remove the tumor and radiation therapy. Exemplary radiation therapies include, but are not limited to, ionizing (electromagnetic) radiation therapy (e.g., X-rays or gamma rays) and particle radiation therapy (e.g., high-energy radiation therapy). The source of radiation can be external or internal to the subject's body.

[0096] The methods described herein are useful for various aspects of cancer treatment. In some embodiments, provided are methods of inhibiting cell proliferation (such as tumor growth) in an individual, comprising administering to the individual an effective amount of any one of the anti-CTLA4 antibodies described herein. In some embodiments, cell proliferation 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%, or about 95% or more).

[0097] In some embodiments, methods are provided for inhibiting tumor metastasis in an individual, comprising administering to the individual an effective amount of any one of the anti-CTLA4 antibodies described herein, in some embodiments, inhibiting metastasis by at least about 10% (including, for example, at least about 20%, about 30%, about 40%, about 60%, about 70%, about 80%, about 90%, or about 95% or more).

[0098] In some embodiments, methods are provided for reducing (e.g., eradicating) metastasis (e.g., metastasis to lymph nodes) of an existing tumor in an individual, comprising administering to the individual an effective amount of any one 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%, or about 95% or more).

[0099] In some embodiments, a method is provided for reducing the incidence or amount of metastasis (such as metastasis to lymph nodes) of an existing tumor in an individual, comprising administering to the individual an effective amount of any one of the anti-CTLA4 antibodies described herein.

[0100] In some embodiments, methods are provided for reducing tumor size in an individual, comprising administering to the individual an effective amount of any one 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%, or about 95% or more).

[0101] In some embodiments, provided are methods of increasing the time to disease progression of cancer in an individual, comprising administering to the individual an effective amount of any one of the anti-CTLA4 antibodies described herein, hi some embodiments, the method increases the time to disease progression by at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 16 weeks, 20 weeks, 24 weeks, 28 weeks, 32 weeks, or 36 weeks or more.

[0102] In some embodiments, methods are provided for extending survival (e.g., overall survival or progression-free survival) of an individual with cancer, comprising administering to the individual an effective amount of any one of the anti-CTLA4 antibodies described herein. In some embodiments, the method extends survival of the individual by at least any of 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, or 24 months.

[0103] In some embodiments, there is provided a method of alleviating one or more symptoms in an individual with cancer, comprising administering to the individual an effective amount of any one 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.

[0104] In some embodiments, provided are methods of improving quality of life in an individual with cancer, comprising administering to the individual an effective amount of any one of the anti-CTLA4 antibodies described herein.

[0105] III. Activatable Binding Polypeptides Targeting CTLA4 The present disclosure also relates, in part, to precision / context-dependent activatable binding polypeptides (i.e., activatable antibodies) that bind to human CTLA4, including any of the anti-CTLA4 antibodies described herein (e.g., anti-CTLA4 antibodies, binding fragments of anti-CTLA4 antibodies, and / or anti-CTLA4 antibody derivatives), antigen-binding fragments of the activatable anti-CTLA4 antibodies, and / or derivatives of the 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 an increased margin of safety as assessed by change in spleen weight. The change in spleen size with increasing drug dose is used as a benchmark to evaluate the safety margin of a candidate drug. The activatable anti-CTLA4 antibodies described herein have an increased margin of safety relative to the parent antibody (antibody without a masking moiety). In some embodiments, the activatable antibody is TY22404.

[0106] In some embodiments, an activatable antibody of the present disclosure comprises (a) a masking moiety (MM), (b) a cleavable moiety (CM), and (c) a target binding moiety (TBM). In some embodiments, the MM is any of the masking moieties described herein. In some embodiments, the CM is any of the cleavable moieties described herein. In some embodiments, the TBM is any of the target binding moieties described herein (e.g., a target binding moiety (TBM) comprising an antibody light chain variable region and / or an antibody heavy chain variable region, e.g., the VH and / or VL of any of the anti-CTLA4 antibodies described herein).

[0107] 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 of the masking moieties described herein, the CM is any of the cleavable moieties described herein, and the TBM comprises an antibody light chain variable region (VL); and (b) an antibody heavy chain variable region (VH).

[0108] 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 of the masking moieties described herein, the CM is any of the cleavable moieties described herein, and the TBM comprises an antibody heavy chain variable region (VH); and (b) an antibody light chain variable region (VL).

[0109] In some embodiments, the activatable antibody comprises, from N-terminus to C-terminus, a masking moiety (MM), a cleavable moiety (CM), and a target binding moiety (TBM) polypeptide, wherein the MM is any of the masking moieties described herein, the CM is any of the cleavable moieties described herein, and the TBM comprises a polypeptide comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL).

[0110] The term "activatable binding polypeptide," "ABP," or "activatable antibody" includes a polypeptide 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 the antigen-binding domain (ABD) of an antibody or antibody fragment thereof (e.g., any of the antibodies or antigen-binding fragments 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 region HVRs described herein, and a light chain variable region comprising one, two, or three of the light chain variable region HVRs 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, including all six HVRs of any of the exemplary antibodies as shown in Table A). In some embodiments, the antigen-binding domain comprises a heavy chain variable region comprising any of the heavy chain variable region sequences described herein, and a light chain variable region comprising any of the light chain variable region sequences 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., a TBM comprising an ABD) comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), wherein the VH and VL form a binding domain that binds to a target in the absence of MM. In some embodiments, the VH and VL are covalently linked, for example, in an scFv. In some embodiments, the VH and VL are not covalently linked. In some embodiments, the VH and VL form a Fab fragment. In some embodiments, the VH is linked to an antibody heavy chain constant region, and the VL is linked to an antibody light chain constant region. TIFF2025530196000001.tif255162TIFF2025530196000002.tif21170TIFF2025530196000003.tif252170TIFF2025530196000004.tif106170

[0111] In some embodiments, the activatable antibody comprises a polypeptide comprising, from N- to C-terminus, the structure masking moiety (MM)-cleavable moiety (CM)-VL, and the activatable antibody further comprises a second polypeptide comprising a VH (e.g., a Fab fragment). In some embodiments, the activatable antibody comprises a polypeptide comprising, from N- to C-terminus, the structure masking moiety (MM)-cleavable moiety (CM)-VL-VH (e.g., an scFv). In some embodiments, the activatable antibody comprises a polypeptide comprising, from N- to C-terminus, the structure masking moiety (MM)-cleavable moiety (CM)-VH, and the activatable antibody further comprises a second polypeptide comprising a VL (e.g., a Fab fragment). In some embodiments, the activatable antibody comprises a polypeptide comprising, from N- to C-terminus, the structure masking moiety (MM)-cleavable moiety (CM)-VH-VL (e.g., an scFv).

[0112] The CM generally comprises an amino acid sequence that is cleavable, e.g., that serves as a substrate for an enzyme, and / or a cysteine-cysteine ​​pair that can form a reducible disulfide bond. Thus, the terms "cleavage," "cleavable," "cleaved," and the like, when used in connection with a CM, include disruption of the disulfide bond between the cysteine-cysteine ​​pair via enzymatic cleavage, e.g., cleavage by a protease, and reduction of the disulfide bond, which may result from exposure to a reducing agent.

[0113] The MM refers to the amino acid sequence of the CM of the activatable antibody in its intact state (e.g., uncleaved by the corresponding enzyme and / or containing an unreduced cysteine-cysteine ​​disulfide bond), which interferes with or inhibits TBM binding to its target. In some embodiments, the MM interferes with or inhibits TBM binding to its target so efficiently that binding of TBM to its target is extremely low and / or below the limit of detection (e.g., undetectable binding in an ELISA or flow cytometry assay). The amino acid sequence of the CM may overlap with or be contained within the MM. Note that for convenience, "ABP" or "activatable antibody" is used herein to refer to an ABP or activatable antibody in both its uncleaved (or "native") and cleaved states. It will be apparent to one of skill in the art that in some embodiments, cleavage of the CM, e.g., by a protease, can cause the cleaved ABP to lose the MM, resulting in release of at least the MM (e.g., if the MM is not linked to the ABP by a covalent bond (e.g., a disulfide bond between cysteine ​​residues)). Exemplary ABPs are described in further detail below.

[0114] In some embodiments, the masking moiety (MM) interferes with, blocks, reduces the ability of, blocks, inhibits, 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, blocks, reduces, blocks, inhibits, or competes with the target binding moiety for binding to its target only when the polypeptide has not been activated (e.g., activated by a change (increase or decrease) in pH, activated by a temperature shift (increase or decrease), activated after contact with a second molecule (e.g., 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., displacement of the masking moiety (MM)) such that the masking moiety becomes an activatable antibody. In some embodiments, the masking moiety (MM) interferes with, blocks, reduces the ability of, 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, the masking moiety (MM) has a masking efficiency 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.) prior to activation.In some embodiments, the masking efficiency is measured as the difference between the affinity with which an activatable antibody (before activation) comprising the masking moiety (MM) binds to its target and the affinity with which a polypeptide lacking the masking moiety binds to its target (e.g., the difference between the affinity of an activatable antibody (before activation) comprising the masking moiety (MM) for a target antigen (such as CTLA4) and that of a parent antibody lacking the masking moiety (MM), or the difference between the affinity of an activatable antibody (before activation) comprising the masking moiety (MM) for a target antigen (such as CTLA4) and that of the activatable antibody after activation). In some embodiments, the masking efficiency is the EC of binding of an activatable antibody (before activation) comprising the masking moiety (MM). 50 EC of the parent antibody 50 by dividing by (e.g., by ELISA, EC 50 (by measuring the masking efficiency). In some embodiments, masking efficiency is measured as the difference between the affinity with which an activatable antibody comprising a masking moiety (MM) binds to its target before activation and the affinity with which an activatable antibody comprising a masking moiety (MM) binds to its target after activation (e.g., the difference between the affinity of the activatable antibody for its target antigen (CTLA4) before activation and the affinity of the activatable antibody after activation). In some embodiments, the masking 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 masking 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.

[0115] In some embodiments, after the activatable antibody is activated (e.g., by treatment with one or more proteases that cleave within the cleavable moiety (CM), by a change (increase or decrease) in pH, by a temperature shift (increase or decrease), or after contact with a second molecule (such as an enzyme or protein ligand), the masking moiety (MM) does not interfere with, block, reduce the ability of, prevent, inhibit, or compete with the target binding moiety (TBM) for binding to its target. In some embodiments, after the cleavable portion (CM) is cleaved by one or more proteases that cleave within the cleavable portion (CM), the masking moiety (MM) does not interfere with, block, reduce the ability of, prevent, inhibit, or compete with the target binding moiety (TBM) for binding to its target. In some embodiments, the masking moiety (MM) has a masking efficiency 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.) after activation (e.g., the relative affinity of the activatable antibody after activation compared to the affinity of the parent antibody).

[0116] In some embodiments, activatable antibodies of the present disclosure include a masking moiety (MM) comprising a pair of cysteine ​​residues at specific positions, resulting in the activatable antibody having few or no conformationally locked and / or chemically labile residues (e.g., methionine or tryptophan). Advantageously, the inclusion of a pair of cysteine ​​residues at specific positions has been shown to impart a locked conformation to the activatable antibody, tending to improve binding affinity and / or specificity. Additionally, activatable antibodies of the present disclosure include masking moieties that have few or no residues, such as methionine or tryptophan, that are undesirable in manufacturing processes.

[0117] In some embodiments, activatable antibodies of the present disclosure are context-dependent (e.g., they are active (can only bind to their target) in certain contexts, such as in a protease-rich tumor microenvironment). In some embodiments, activatable antibodies of the present disclosure have improved safety characteristics over more conventional non-activatable antibodies (e.g., exhibit reduced toxicity, do not induce significant changes in multiple organ weights, do not alter liver histopathology, hematology, and / or blood biochemistry). In some embodiments, activatable antibodies of the present disclosure have improved pharmacokinetic properties (e.g., have longer in vivo half-lives) over more conventional non-activatable antibodies.

[0118] Activity of activatable anti-CTLA4 antibodies In some embodiments, the disclosure relates to activatable antibodies that, when in their activated form, bind to human CTLA4 (e.g., the activatable antibody is active after cleavage at the cleavable moiety (e.g., with one or more proteases), but is inactive before cleavage at the cleavable moiety (e.g., with one or more proteases). In some embodiments, the activatable antibodies, when in their activated form, (a) bind to human CTLA4, cynomolgus CTLA4, mouse CTLA4, rat CTLA4, and / or dog CTLA4 with a K of 500 nM or less, e.g., about 10 nM or less. D (b) having antagonist activity against human CTLA4; (c) not binding to human PD-1, human PD-L1, human PD-L2, human LAG3, human TIM3, human B7-H3, human CD95, human CD120a, human OX40, human CD40, human BTLA, human VISTA, human ICOS, and / or human B7-H4 at concentrations up to 100 nM; (d) having cross-reactivity with monkey CTLA4, mouse CTLA4, rat CTLA4, and / or canine CTLA4; and (e) against (e.g., Tregs). (f) induce ADCC via activation of human PBMCs (e.g., stimulate secretion of IL-2 and / or IFNγ), (g) be capable of inhibiting tumor cell growth, (h) have a therapeutic effect against cancer, and (i) inhibit the binding of human CTLA4 to human CD80 and / or human CD86 (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 these functional properties. The present invention also provides one or more activatable antibodies that compete or cross-compete with one or more of the activatable CTLA4-targeting antibodies and / or anti-CTLA4 antibodies described herein for binding to human CTLA4.

[0119] In some embodiments, the activatable antibody, when in an inactive form, has a K of about 500 nM or greater for human CTLA4, cynomolgus monkey CTLA4, mouse CTLA4, rat CTLA4, and / or dog CTLA4.D In some embodiments, the activatable antibody, when in its active form, binds to human CTLA4, cynomolgus monkey CTLA4, mouse CTLA4, rat CTLA4, and / or dog CTLA4 with 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 In some embodiments, the activatable antibody, when in its activated form, binds to human CTLA4, cynomolgus monkey CTLA4, mouse CTLA4, rat CTLA4, and / or dog CTLA4 with a K of about 350 nM or less. D In some embodiments, the activatable antibody, when in its activated form, binds to human CTLA4 with a K of about 100 nM or less. D In some embodiments, the activatable antibody, when in its activated form, binds to human CTLA4 with a K of about 50 nM or less. D In some embodiments, the activatable antibody, when in its activated form, binds to human CTLA4 with a K of about 10 nM or less. D The K of the activatable antibody D Measurement of K may 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 is measured by ELISA (see, for example, the Examples below).

[0120] In some embodiments, the activatable antibody, when in an inactive form, has no antagonist activity against human CTLA4. In some embodiments, the activatable antibody, when in an active form, has antagonist activity against human CTLA4 (e.g., inducing ADCC (e.g., against Tregs), activating PBMCs (e.g., by activating, inducing, and / or stimulating the secretion of IL-2 and / or IFNγ), blocking the binding of human CTLA4 to human CD80 and / or human CD86, etc.). In some embodiments, the activatable antibody, when in an active form, inhibits one or more activities of human CTLA4 (e.g., inhibits one or more activities of human CTLA4 when the activatable antibody is contacted with a cell (e.g., a human cell) that expresses human CTLA4).

[0121] In some embodiments, the activatable antibody, when in an inactive form, has no cross-reactivity with monkey (e.g., cynomolgus) CTLA4, mouse CTLA4, rat CTLA4, and / or dog CTLA4. In some embodiments, the activatable antibody, when in an activated form, has cross-reactivity with monkey (e.g., cynomolgus) CTLA4, mouse CTLA4, rat CTLA4, and / or dog CTLA4. In some embodiments, the activatable antibody, when in an activated form, has cross-reactivity with monkey CTLA4. In some embodiments, the activatable antibody, when in an activated form, has cross-reactivity with mouse CTLA4. In some embodiments, the activatable antibody, when in an activated form, has cross-reactivity with rat CTLA4. In some embodiments, the activatable antibody, when in an activated form, has cross-reactivity with dog CTLA4. In some embodiments, the activatable antibody, when in its activated form, is cross-reactive with monkey CTLA4 and mouse CTLA4, monkey CTLA4 and rat CTLA4, monkey CTLA4 and dog CTLA4, mouse CTLA4 and rat CTLA4, mouse CTLA4 and dog CTLA4, rat CTLA4 and dog CTLA4, monkey CTLA4, mouse CTLA4 and rat CTLA4, monkey CTLA4, mouse CTLA4 and dog CTLA4, monkey CTLA4, rat CTLA4 and dog CTLA4, mouse CTLA4, rat CTLA4 and dog CTLA4, or monkey CTLA4, mouse CTLA4, rat CTLA4 and dog CTLA4. In some embodiments, the activatable binding polypeptide, when in an active form, has a cross-reactivity of about 350 nM (e.g., about 1 nM, about 10 nM, about 25 nM, about 50 nM, about 75 nM, about 100 nM, about 150 nM, about 200 nM, about 250 nM, about 300 nM, 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.

[0122] In some embodiments, the activatable antibody, when in an inactive form, does not induce ADCC (e.g., against human cells expressing CTLA4, such as Tregs). In some embodiments, the activatable antibody, when in an inactive form, has reduced ADCC (e.g., against human cells expressing CTLA4, such as Tregs) compared to a control binding polypeptide (e.g., a parent antibody). In some embodiments, the activatable antibody, when in an activated form, induces ADCC (e.g., against human cells expressing CTLA4, such as Tregs). Methods (e.g., in vitro methods) for measuring ADCC are known in the art and include, but are not limited to, those described in the Examples below. In some embodiments, the activatable antibody, when in an inactive form, induces less than about 10% of the ADCC effect relative to a control (e.g., a parent antibody) (e.g., induces less than about 10%, less than about 5%, less than about 1%, etc.). In some embodiments, the activatable antibody, when in an activated form, induces more than about 10% of the ADCC activity relative to a control (e.g., an isotype control) (e.g., more than about 10%, more than about 15%, more than about 20%, more than about 25%, more than about 30%, more than about 35%, more than about 40%, etc.).

[0123] In some embodiments, the activatable antibody is capable of inhibiting tumor cell growth and / or proliferation. In some embodiments, upon contact 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%) relative to corresponding tumor cells not contacted with the activatable antibody (or relative to corresponding tumor cells contacted with an isotype control antibody). In some embodiments, upon administration of the activatable antibody to a subject, the activatable antibody is capable of reducing tumor volume in the subject. In some embodiments, the activatable antibody can reduce tumor volume in a 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., compared to a corresponding tumor in a subject administered an isotype control antibody prior to administration of the activatable antibody). Methods for monitoring tumor cell growth / proliferation, tumor volume, and / or tumor inhibition are known in the art, and include, for example, those methods described in the Examples below.

[0124] In some embodiments, the activatable antibody has a therapeutic effect against cancer. In some embodiments, the activatable antibody alleviates one or more signs or symptoms of cancer. In some embodiments, a subject suffering from cancer experiences a partial or complete remission when administered the activatable antibody.

[0125] In some embodiments, the disclosure provides activatable isolated antibodies that, when in an activated form, compete or cross-compete for binding to human CTLA4 with an antibody comprising: a) 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, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 45, and / or b) 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 embodiments, the disclosure provides activatable isolated antibodies that, when in an activated 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 for binding 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., an antibody as described above) can be bound to human CTLA4 under saturating conditions, and the ability of the activatable test antibody to bind to CTLA4 (when in an activated form) can be measured. If the activatable test antibody can bind to CTLA4 simultaneously with the antibody, the activatable test antibody binds to a different epitope than the antibody. However, if the activatable test antibody cannot bind to CTLA4 simultaneously, the activatable test antibody binds to the same epitope as, an epitope overlapping with, or an epitope adjacent to the epitope bound by the antibody. This experiment can be performed using a variety of methods, such as ELISA, RIA, FACS, or surface plasmon resonance.

[0126] In some embodiments, the activatable antibody (when in an inactive form) does not inhibit the binding of CTLA4 to one or more of its binding partners (e.g., human CTLA4 to human CD80, human CTLA4 to human CD86). In some embodiments, the activatable antibody (when in an activated form) inhibits the binding of CTLA4 to one or more of its binding partners (e.g., human CTLA4 to human CD80, human CTLA4 to human CD86). In some embodiments, the activatable antibody inhibits the binding of CTLA4 to its ligand in vitro. In some embodiments, the activatable antibody exhibits a half-maximal inhibitory concentration (IC) that inhibits the binding of CTLA4 to CD80 and / or CD86. 50 ) is 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.). In some embodiments, the activatable antibody has a half-maximal inhibitory concentration (IC) that inhibits the binding of CTLA4 to CD80 and / or CD86. 50 ) is about 100 nM or less. In some embodiments, the activatable antibody completely inhibits human CTLA4 binding to CD80 and / or CD86 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.). As used herein, the term "completely inhibiting" or "completely inhibiting" refers to the ability of the activatable antibody to reduce binding between a first protein and a 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 binding between a first protein (e.g., human CTLA4) and a second protein (e.g., human CD80 or human CD86) are known in the art and include, but are not limited to, by BIAcore analysis, ELISA assays, and flow cytometry.

[0127] Masking part (MM) In some embodiments, the present disclosure relates to an activatable antibody comprising a masking moiety (MM). In some embodiments, the masking moiety (MM) is represented by formula (XIX):Z m CZ n CZ o (SEQ ID NO: 135), wherein m is 2-10, n is 3-10, and o is 1-10, 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 6-8. 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 6-8. 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 1-2. In some embodiments, o is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, o is 2.

[0128] 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 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.

[0129] In some embodiments, the masking moiety (MM) comprises an amino acid sequence according to formula (XXII): Z6CX8CZ2 (SEQ ID NO: 138), wherein 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 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.

[0130] 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.

[0131] 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 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), wherein m is 2 to 10 and 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 of 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 of EDCVPYYYACAY (SEQ ID NO: 213), EVGSSDCVPYYYACAY (SEQ ID NO: 214), EDCDAFYPYCDS (SEQ ID NO: 215), or EVGHSDCDAFYPYCDS (SEQ ID NO: 216).

[0132] 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), PNPSSDCVPYYYACAY (SEQ ID NO: 144), YSAYPACDSHYPYCQS (SEQ ID NO: 145), PQPSSDCVPYYYACAY (SEQ ID NO: 146) and PNPASDCVPYYYACAY (SEQ ID NO: 147).

[0133] In some embodiments, any of the masking moieties (MMs) 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, a purification tag (such as a His tag, a FLAG tag, a maltose-binding protein, and a glutathione-S-transferase tag), a detection tag (such as a tag that can be detected photometrically (e.g., red or green fluorescent protein)), a tag with detectable enzymatic activity (such as alkaline phosphatase), a secretion sequence, a tag comprising a leader sequence and / or a stabilizing sequence, a protease cleavage site (e.g., a furin cleavage site, a TEV cleavage site, a thrombin cleavage site), 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 of EVGSY (SEQ ID NO: 148).

[0134] In some embodiments, the masking moiety binds to the target binding moiety (TBM) and inhibits binding of the activatable antibody to its target before activation (e.g., before treatment with one or more proteases that cleave within the cleavable moiety (CM), before a (local) change (increase or decrease) in pH, before a temperature shift (increase or decrease), before contact with a second molecule (such as a small molecule or protein ligand), etc.), but does not bind to the TBM and / or inhibit binding of the activatable antibody to its target after activation (e.g., after treatment with one or more proteases that cleave within the cleavable moiety (CM), after a (local) change (increase or decrease) in pH, after a temperature shift (increase or decrease), after contact with a second molecule (such as a small molecule or protein ligand), etc.). In some embodiments, the masking moiety (MM) inhibits binding of the activatable antibody to its target when the CM is not cleaved, but does not inhibit 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 (when in activated form) for its target (e.g., at least about 1.5 times greater, at least about 2 times greater, at least about 2.5 times greater, at least about 3 times greater, at least about 3.5 times greater, at least about 4 times greater, at least about 4.5 times greater, at least about 5 times greater, at least about 10 times greater, at least about 100 times greater, at least about 500 times greater, etc.).

[0135] Cuttable length (CM) In some embodiments, the present disclosure relates to activatable antibodies that comprise a cleavable moiety (CM), which can be cleaved and / or destroyed, such as 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 (small molecule or protein ligand).

[0136] 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 known in the art that is recognized and / or cleaved by any protease (e.g., a protease known to co-localize with the target of the activatable antibody that comprises the CM) may be used, including, for example, 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-18, 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-60, MMP-61, M MMP-12, MMP-13, MMP-14, MMP-15, MMP-16, MMP-17, MMP-19, MMP-20, MMP-23, 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, ADAMDE C1, ADAMTS1, ADAMTS4 and / or or ADAMTS5), caspases (e.g., 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, and / or caspase-14), aspartic acid proteases (e.g., RACE and / or renin), aspartic acid cathepsins (e.g., cathepsin D and / or cathepsin E), cysteine ​​catecholamines (e.g., cysteine ​​catecholamines), and / or cysteine ​​proteases (e.g., RACE and / or renin). cathepsins (e.g., cathepsin B, cathepsin C, cathepsin K, cathepsin L, cathepsin S, cathepsin V / L2 and / or cathepsin X / Z / P), cysteine ​​proteinases (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),These include protease cleavage sites recognized and / or cleaved by serine proteases (e.g., activated protein C, cathepsin A, cathepsin G, chymase and / or coagulation factor proteases (FVIIa, FIXa, FXa, FXIa, FXIIa, etc.)), elastase, granzyme B, guanidinobenzoatase, HtrA1, human neutrophil elastase, lactoferrin, marapsin, NS3 / 4A, PACE4, tPA, tryptase, type II transmembrane serine proteases (TTSPs) (e.g., DESC1, DPP-4, FAP, hepsin, matriptase-2, MT-SP1 / matriptase, TMPRSS2, TMPRSS3, and / or TMPRSS4), and the like. In some embodiments, the first protease cleavage site is a cleavage site for a protease selected from 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 for a protease selected from 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).

[0137] Any suitable linker (e.g., a flexible linker) known in the art may be used, including, for example, glycine polymers (G)n, where n is an integer of at least 1 (e.g., at least 1, 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.), glycine-serine polymers (GS)n, where n is an integer of at least 1 (e.g., at least 1, 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), glycine-alanine polymers, alanine-serine polymers, and the like. The linker sequence can be 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 glycine-serine polymer), from about 4 amino acids to about 10 amino acids, from about 5 amino acids to about 9 amino acids, or from about 6 amino acids to about 8 amino acids. In some embodiments, the linker is about 1 amino acid, about 2 amino acids, about 3 amino acids, about 4 amino acids, about 5 amino acids, about 6 amino acids, about 7 amino acids, about 8 amino acids, about 9 amino acids, about 10 amino acids, about 11 amino acids, about 12 amino acids, about 13 amino acids, about 14 amino acids, about 15 amino acids, about 16 amino acids, about 17 amino acids, about 18 amino acids, about 19 amino acids, or about 20 amino acids in length. In some embodiments, the linker comprises an amino acid sequence selected from SEQ ID NOs: 159-163. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 156 or 157.

[0138] In some embodiments, the cleavable portion (CM) further comprises at least a second cleavage site (e.g., at least a second cleavage site, at least a third cleavage site, at least a fourth cleavage site, at least a fifth cleavage site, etc.). In some embodiments, the cleavable portion (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 may be any suitable protease cleavage site recognized and / or cleaved by any of the proteases described above. In some embodiments, the first cleavage site (CS1) and the second cleavage site (CS2) are protease cleavage sites recognized and / or cleaved by the same protease. In some embodiments, the first cleavage site (CS1) and the second cleavage site (CS2) 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 and the second protease cleavage site is recognized and / or cleaved by MMP-2, or the first protease cleavage site is recognized and / or cleaved by uPA and the second protease cleavage site is recognized and / or cleaved by MMP-9). In some embodiments, at least the second cleavage site (CS2) is C-terminal to the first linker (L1). In some embodiments, the cleavable portion (CM) comprises the structure, from N- to C-terminus, (CS1)-L1-(CS2).

[0139] In some embodiments, the cleavable moiety (CM) further comprises at least a second linker (e.g., at least a second linker, at least a third linker, at least a fourth linker, at least a fifth linker, etc.). In some embodiments, the cleavable moiety (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: 156-163. In some embodiments, the first linker (L1) and the second linker (L2) are the same (e.g., both linkers comprise the sequence of SEQ ID NO: 156 or 157). In some embodiments, the first linker (L1) and the second linker (L2) 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. In some embodiments, the at least second linker (L2) is C-terminal to the second cleavage site (CS2). In some embodiments, the cleavable portion (CM) comprises the structure, from N- to C-terminus, (CS1)-L1-(CS2)-L2.

[0140] Exemplary MM-CM Sequences In some embodiments, an activatable antibody of the disclosure comprises the structure, from N-terminus to C-terminus, (FP)-(PCS1)-L1-(PCS2)-L2. In some embodiments, an activatable antibody of the disclosure comprises the structure EVGSYNFVADSCPDHPYPCSASGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 168), EVGSYIVHHSDCDAFYPYCDSSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 170), EVGSYYSAYPACDSHYPYCNSSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 172), EVGSYPNPSSDCVPYYYACAYSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 173), SEQ ID NO: 174), EVGSYYSAYPACDSHYPYCQSSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 176), EVGSYYSAYPACDSHYPYCNSAGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 177), EVGSYPQPSSDCVPYYYACAYSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 178), and / or EVGSYPNPASDCVPYYYACAYSGRSAGGGGSPLGLAGSGGS (SEQ ID NO: 179). In some embodiments, a polypeptide of the disclosure comprises the following structure from N- to C-terminus: (FP)-(PCS1)-L1-(PCS2)-L2-(TBM).

[0141] In some embodiments, the activatable antibody comprises the amino acid sequence of SGRSAGGGGTENLYFQGSGGS (SEQ ID NO: 220), SGRSAGGGGTPLGLAGSGGS (SEQ ID NO: 221), or SGRSAPLGLA (SEQ ID NO: 222). In some embodiments, the activatable antibody comprises the amino acid sequence of EV(Z n)C(X)C(Z)SGRSA (SEQ ID NO: 217), EDC(Z)C(Z)SGRSA (SEQ ID NO: 218), or EDC(Z)C(Z)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; n is 1 to 11; 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.

[0142] In certain embodiments, the MM comprises the amino acid sequence of EVGSYPNPSSDCVPYYYACAY (SEQ ID NO: 192), and the cleavable portion comprises the amino acid sequence of SGRSAGGGGTPLGLAGSGGS (SEQ ID NO: 221). In some embodiments, the MM and CM comprise, from N- to C-terminus, the amino acid sequence of EVGSYPNPSSDCVPYYYACAYSGRSAGGGGTPLGLAGSGGS (SEQ ID NO: 200). In some embodiments, the MM and CM are covalently linked to the N-terminus of the light chain of the anti-CTLA4 antibody. In some embodiments, the MM and CM, from N- to C-terminus, comprise amino acid sequences having at least 90% or at least 95% sequence identity to SEQ ID NO: 200.

[0143] Target binding moiety (TBM) In some embodiments, the present disclosure relates to an activatable antibody comprising a target binding moiety (TBM). In some embodiments, the target binding moiety (TBM) comprises an antibody light chain variable region and / or an antibody heavy chain variable region. In some embodiments, the target binding moiety (TBM) comprises an antibody light chain variable region. In some embodiments, the target binding moiety (TBM) comprises an antibody heavy chain variable region. In some embodiments, the target binding moiety (TBM) comprises an antibody light chain variable region and an antibody heavy chain variable region.

[0144] 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 κ light chain or a λ light chain. The antibody heavy chain may be of any class, such as IgG, IgM, IgE, IgA, or IgD. In some embodiments, the antibody heavy chain is of the IgG class, for example, the IgG1 subclass, IgG2 subclass, IgG3 subclass, or IgG4 subclass. The antibody heavy chains described herein may be converted from one class or subclass to another using methods known in the art.

[0145] Any one or more of the target binding moieties (TBMs) described herein may incorporate any of the HVR sequences 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 of the heavy chain variable region sequences and / or light chain variable region sequences described herein (e.g., the heavy chain variable region sequences and / or light chain variable region sequences as shown in Table B above), and / or any of the antibodies described herein.

[0146] In some embodiments, the target binding moiety (TBM) comprises the sequence of one or more of the anti-CTLA4 antibodies described herein, including antibodies (e.g., IgG1, IgG2, IgG4) described with respect to the specific amino acid sequences of the HVRs, variable regions (VL, VH), and / or light and heavy chains. In some embodiments, the target binding moiety (TBM) comprises an antibody light chain variable region comprising an HVR-L1 comprising the amino acid sequence of RASQSVRGRFLA (SEQ ID NO:58), an HVR-L2 comprising the amino acid sequence of DASNRATGI (SEQ ID NO:66), and / or an HVR-L3 comprising the amino acid sequence of 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 that has 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 an HVR-H1 comprising the amino acid sequence of YSISSGYHWSWI (SEQ ID NO: 23), an HVR-H2 comprising the amino acid sequence of LARIDWDDDKYYSTSLKSRL (SEQ ID NO: 35), and / or an 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 that has 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.

[0147] Properties of activatable binding polypeptides In some embodiments, an activatable binding polypeptide (i.e., an activatable antibody) of the disclosure comprises (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 binding of the activatable binding moiety to CTLA4 (e.g., human CTLA4) compared to binding of a corresponding binding polypeptide lacking the masking moiety and / or compared to binding of a parent antibody to CTLA4 (e.g., human CTLA4).

[0148] In some embodiments, an "activatable" binding polypeptide refers to a binding polypeptide that exhibits a first level of binding to CTLA4 when inhibited, masked, and / or uncleaved, and a second level of binding to CTLA4 when uninhibited, unmasked, and / or cleaved, where the second level of binding to CTLA4 is greater than the first level of binding to CTLA4. In some embodiments, the accessibility of the activatable binding polypeptide to CTLA4 is increased after cleavage (e.g., by one or more proteases) within the cleavable portion.

[0149] 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. An activatable antibody having a heavy chain SEQ ID NO: 320 and a light chain SEQ ID NO: 322 is designated 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.

[0150] In some embodiments, activatable antibodies of the present disclosure generally have a binding affinity for CTLA4 (e.g., human CTLA4) that is at least about 2-fold (e.g., at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13-fold, at least about 14-fold, at least about 15-fold, at least about 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, at least about 20-fold, at least about 21-fold, at least about 22-fold, at least about 23-fold, at least about 24-fold, at least about 25-fold, at least about 26-fold, at least about 27-fold, at least about 28-fold, at least about 29-fold, at least about 30-fold, at least about 31-fold, at least about 32-fold, at least about 33-fold, at least about 34-fold, at least about 35-fold, at least about 36-fold, at least about 37-fold, at least about 38-fold, at least about 39-fold, at least about 40-fold, at least about 41-fold, at least about 42-fold, at least about 43-fold, at least about 44-fold, at least about 45-fold, at least about 46-fold, at least about 47-fold, at least about 48-fold, at least about 49-fold, at least about 49-fold, at least about 49-fold, at least about 49-fold, A binding polypeptide is considered to be "activatable" when it increases the binding activity of the polypeptide by 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. In some embodiments, an activatable antibody of the disclosure is generally considered to be "activatable" if, following "activation," the EC50 of the activatable antibody is reduced by at least about 2-fold (e.g., at least about 2-fold, 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 more).In some embodiments, an activatable antibody of the disclosure is generally considered to be "activatable" if the EC50 of the polypeptide is reduced by at least about two-fold after treatment with a protease that cleaves within its cleavable moiety (CM) (e.g., as measured by an ELISA assay or a FACS assay; see the Examples below).

[0151] In some embodiments, the masking moiety (MM) when attached to the target binding moiety (TBM) of the activatable antibody reduces the K of the activatable antibody to CTLA4. D is expressed when the masking moiety (MM) is not bound to the target binding moiety (TBM) (e.g., after "activation" of the activatable antibody (e.g., after treatment with a protease to cleave within the cleavable moiety (CM))) and / or the K D The affinity is about 2-fold (e.g., about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 5.5-fold, about 6-fold, about 6.5-fold, about 7-fold, about 7.5-fold, about 8-fold, about 8.5-fold, about 9-fold, about 9.5-fold, about 10-fold, about 25-fold, about 50-fold, about 75-fold, about 100-fold, about 250-fold, about 500-fold, about 750-fold, or about 1000-fold or more) greater than the affinity. Methods for measuring affinity are known in the art, and include, for example, the methods described in the Examples below.

[0152] In some embodiments, the masking moiety, when attached to the target binding portion of the activatable antibody, inhibits the K of the activatable antibody to CTLA4. D is the K of the parent antibody against CTLA4 when the masking moiety is not bound to the target binding moiety (e.g., after "activation" of the activatable antibody (e.g., after treatment with a protease to cleave within the cleavable moiety (CM))). DThe affinity is reduced 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%) relative to the target protein. Methods for measuring affinity are known in the art, and include, for example, the methods described in the Examples below.

[0153] In some embodiments, the masking moiety sterically interferes with binding of the activatable antibody to CTLA4 and / or allosterically interferes with binding of the activatable antibody to CTLA4, hi some embodiments, the masking moiety does not comprise the amino acid sequence of the natural binding partner of the activatable antibody and / or parent antibody.

[0154] In some embodiments, the dissociation constant of the masking moiety for the target binding moiety is greater than the dissociation constant of the activatable antibody (upon activation) for CTLA4. In some embodiments, the dissociation constant of the masking moiety for the target binding moiety is about 2-fold (e.g., about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 5.5-fold, about 6-fold, about 6.5-fold, about 7-fold, about 7.5-fold, about 8-fold, about 8.5-fold, about 9-fold, about 9.5-fold, about 10-fold, about 25-fold, about 50-fold, about 75-fold, about 100-fold, about 250-fold, about 500-fold, about 750-fold, or about 1000-fold or more) greater than the dissociation constant of the activatable antibody (upon activation) for CTLA4. In some embodiments, the dissociation constant of the masking moiety for the target binding moiety is approximately equal to the dissociation constant of the activatable antibody (upon activation) for CTLA4.

[0155] The activatable antibodies described herein may 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.

[0156] In some embodiments, the activatable antibodies of the present disclosure are linked to a pharmaceutical agent. Examples of pharmaceutical agents include cytotoxic agents or other cancer therapeutic agents and radioisotopes. Specific examples of cytotoxic agents include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as analogs or homologs thereof. Therapeutic agents also include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thiotepa, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiammineplatinum(II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and antimitotic agents (e.g., vincristine and vinblastine). Examples of radioisotopes that can be conjugated to antibodies for diagnostic or therapeutic use include iodine 131 ,indium 111 ,yttrium 90 and lutetium 177Methods of linking polypeptides to pharmaceutical agents are known in the art, including, but not limited to, the use of various linker technologies. Exemplary linker types include hydrazones, thioethers, esters, disulfides, and peptide-containing linkers. For further discussion of linkers and methods of linking 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.

[0157] IV. Combination therapy with anti-PD-1 antibody In some embodiments, the activatable antibodies of the present disclosure are administered in combination with a PD-1 antagonist. In one embodiment, PD-1 antagonists useful in the treatments, 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, preferably human PD-1 or human PD-L1. The mAb may be a human antibody, a humanized antibody, or a chimeric antibody and may comprise 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 an 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.

[0158] Examples of mAbs that bind to human PD-1 and are useful in the therapeutic methods, medicaments, and uses of the invention are described in U.S. Patent Nos. US7488802, US7521051, US8008449, US8354509, and US8168757, and International Application Publication Nos. WO2004 / 004771, WO2004 / 072286, WO2004 / 056875, US2011 / 0271358, and WO2008 / 156712. Specific anti-human PD-1 mAbs useful as PD-1 antagonists in the methods of treatment, medicaments, and uses of the present invention include a humanized IgG4 mAb having the structure set forth in WHO Drug Information, Vol. 27, No. 2, pages 161-162 (2013) and comprising the heavy and light chain amino acid sequences set forth in Table B; nivolumab (BMS-936558), a human IgG4 mAb having the structure set forth in WHO Drug Information, Vol. 27, No. 1, pages 68-69 (2013); humanized antibodies h409A11, h409A16, and h409A17, described in WO2008 / 156712; and AMP-514, cemiplimab, camrelizumab, sintilimab, tislelizumab, and toripalimab, which are under development by MedImmune. Additional anti-PD-1 antibodies contemplated for use in the present invention 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).

[0159] In certain embodiments, the activatable antibody of the present disclosure is administered in combination with toripalimab. In some embodiments, the present invention provides a method of treating cancer in a subject, comprising administering to the subject (a) an effective amount of an activatable antibody of the present disclosure, the activatable antibody comprising a polypeptide comprising, from N-terminus to 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 of EVGSYPNPSSDCVPYYYACAY (SEQ ID NO: 192), and the cleavable moiety comprises the amino acid sequence of SGRSAGGGGTPLGLAGSGGS (SEQ ID NO: 221), and (b) an effective amount of toripalimab. The MM and CM comprise, from N-terminus to C-terminus, the amino acid sequence of EVGSYPNPSSDCVPYYYACAYSGRSAGGGGTPLGLAGSGGS (SEQ ID NO: 200). In certain embodiments, the MM and CM are covalently linked to the N-terminus of the light chain of the anti-CTLA4 antibody. In some embodiments, the N- to C-terminal MM and CM comprise an amino acid sequence having at least 90% or at least 95% sequence identity to SEQ ID NO: 200. In some embodiments, the TY2404 is administered in combination with toripalimab.

[0160] In some embodiments, an 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 once every 3 to 6 weeks (e.g., once every 3 weeks, once every 4 weeks, once every 5 weeks, or once every 6 weeks). In some embodiments, toripalimab is administered at a dose of about 200 mg to about 400 mg once every 3 weeks. In another embodiment, toripalimab is administered at a dose of about 240 mg once every 3 weeks. In another embodiment, toripalimab is administered at a dose of about 300 mg to about 600 mg once every 6 weeks. In another embodiment, toripalimab is administered at a dose of about 480 mg once every 5 weeks.

[0161] In one embodiment, the present disclosure provides a method of treating cancer in a subject, the method comprising administering to the subject an effective amount of an activatable anti-CTLA4 antibody (e.g., TY22404) described above in combination with toripalimab, 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 may 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 specific embodiments, both 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 the activatable anti-CTLA4 antibody on the same day of a particular dosing regimen or on different days of a particular dosing regimen. In some embodiments, both the activatable anti-CTLA4 antibody and toripalimab are administered on the first day of a three-week or six-week dosing regimen.

[0162] In some embodiments, an activatable anti-CTLA4 antibody (e.g., TY22404) and toripalimab are administered to a patient in need thereof, where 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.

[0163] In some embodiments, an activatable anti-CTLA4 antibody (e.g., TY22404) and toripalimab are administered to a patient in need thereof, where 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.

[0164] In some embodiments, an activatable anti-CTLA4 antibody (e.g., TY22404) and toripalimab are administered to a patient in need thereof, where 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 200 mg / kg once every three weeks. In some embodiments, the activatable anti-CTLA4 antibody and toripalimab are administered simultaneously.

[0165] In some embodiments, an activatable anti-CTLA4 antibody (e.g., TY22404) and toripalimab are administered to a patient in need thereof, where 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.

[0166] In some embodiments, an activatable anti-CTLA4 antibody (e.g., TY22404) and toripalimab are administered to a patient in need thereof, where 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.

[0167] In some embodiments, an activatable anti-CTLA4 antibody (e.g., TY22404) and toripalimab are administered to a patient in need thereof, where 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.

[0168] 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 four cycles of treatment with the activatable anti-CTLA4 antibody and toripalimab. In some embodiments, the subject further receives maintenance therapy comprising administering to the subject an effective amount of an activatable anti-CTLA4 antibody about once every four weeks to about once every 12 weeks (e.g., once every 4, 6, 8, 10, or 12 weeks). In some embodiments, the administration of the activatable anti-CTLA4 antibody and toripalimab can be administered simultaneously. In other embodiments, doses of the activatable anti-CTLA4 antibody and toripalimab can 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 four cycles of treatment with the activatable anti-CTLA4 antibody and toripalimab. In some embodiments, the subject further receives maintenance therapy comprising administering to the subject an effective amount of an activatable anti-CTLA4 antibody about once every four weeks to about once every 12 weeks (e.g., once every 4, 6, 8, 10, or 12 weeks). In some embodiments, the administration of the activatable anti-CTLA4 antibody and toripalimab can be simultaneous. In other embodiments, doses of the 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 administration of an activatable anti-CTLA4 antibody on day 1 of a dosing schedule (e.g., a 3-week dosing schedule). Toripalimab can be administered about 0.5 hours to about 5 hours before or after administration of an activatable anti-CTLA4 antibody on day 1 of a dosing schedule (e.g., a 3-week dosing schedule), 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 both administered intravenously. In some embodiments, the activatable anti-CTLA4 antibody and toripalimab are 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 four cycles of treatment with an activatable anti-CTLA4 antibody and toripalimab. In some embodiments, the subject further receives maintenance therapy comprising administering to the subject an effective amount of an activatable anti-CTLA4 antibody about once every four weeks to about once every twelve weeks (e.g., once every four, six, eight, ten, or twelve weeks). In some embodiments, the activatable anti-CTLA4 antibody and toripalimab can be administered simultaneously. In other embodiments, doses of the 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 administration of the activatable anti-CTLA4 antibody on day 1 of a dosing schedule (e.g., a three-week dosing schedule). 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 four cycles of treatment with an activatable anti-CTLA4 antibody and toripalimab.In some embodiments, the subject further receives maintenance therapy comprising administering to the subject an effective amount of an activatable anti-CTLA4 antibody about once every 4 weeks to about once every 12 weeks (e.g., once every 4, 6, 8, 10, or 12 weeks). In some embodiments, the activatable anti-CTLA4 antibody and toripalimab may be administered simultaneously. In other embodiments, doses of 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 administration of the activatable anti-CTLA4 antibody on day 1 of a dosing schedule (e.g., a 3-week dosing schedule).

[0169] V. Pharmaceutical Compositions, Kits and Articles of Manufacture In another aspect, the present application provides a composition comprising any one of the anti-CTLA4 antibodies (e.g., activatable antibodies) described herein. In some embodiments, the composition is a pharmaceutical composition comprising the anti-CTLA4 antibody (e.g., activatable antibody) and a pharmaceutically acceptable carrier. In some embodiments, the present invention provides a composition 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.

[0170] The term "pharmaceutically acceptable carrier" refers to any inert material suitable for use in a formulation to deliver an active agent (e.g., an anti-CTLA4 antibody). Carriers may be antiadherents, binders, coating agents, disintegrants, fillers or diluents, preservatives (e.g., antioxidants, antibacterial or antifungal agents), sweeteners, absorption delaying agents, wetting agents, emulsifiers, buffers, etc. Examples of suitable pharmaceutically acceptable carriers include water, ethanol, polyols (glycerol, propylene glycol, polyethylene glycol, etc.), dextrose, vegetable oils (e.g., olive oil), saline, buffers, buffered saline, and isotonicity agents, such as sugars, polyalcohols, sorbitol, and sodium chloride. The composition may be in any suitable form, e.g., liquid, semi-solid, 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 a composition suitable for delivering an anti-CTLA4 antibody is a sterile liquid, such as a solution, suspension, or dispersion for injection or infusion. Sterile solutions can be prepared by incorporating the antibody in the required amount into a suitable carrier and then sterilizing it through microfiltration. Generally, dispersions are prepared by incorporating the antibody into a sterile vehicle containing a basic dispersion medium and other carriers. In the case of sterile powders for preparing sterile solutions, methods include vacuum drying and freeze-drying (lyophilization) to obtain powders from a previously sterile-filtered solution of the active ingredient and any additional desired ingredients. Compositions in various dosage forms can be prepared by conventional techniques known in the art.

[0171] The relative amount of anti-CTLA4 antibody in the composition will vary depending on many factors, such as the particular anti-CTLA4 antibody and carrier used, the dosage form, and the desired release and pharmacokinetic characteristics. The amount of anti-CTLA4 antibody in a single dosage form will generally be an amount that exerts a therapeutic effect, although lesser amounts may also be used. Generally, this amount will range from about 0.01% to about 99%, from about 0.1% to about 70%, or from about 1% to about 30% of the total weight of the dosage form.

[0172] 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 in the "Methods of Treatment" section herein. The appropriate amount of additional therapeutic agent to be included in the composition can be easily 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 pharmacokinetic characteristics. The amount of additional therapeutic agent included in a single dosage form will generally be the amount of agent that exerts a therapeutic effect, but may be a lesser amount.

[0173] In some embodiments, an article of manufacture containing materials useful for treating cancer is provided. The article of manufacture can include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The container can be formed from a variety of materials, such as glass or plastic. Generally, the container holds a composition described herein that is effective for treating cancer and can have a sterile access port (e.g., the container can be an intravenous infusion bag or vial with a stopper pierceable by a hypodermic needle). Package insert refers to instructions typically included in commercial packaging of pharmaceutical products that include information about the indications, uses, dosage, administration, contraindications, and / or warnings regarding the use of such pharmaceutical products. In some embodiments, the package insert indicates that the composition is for use in treating cancer. The label or package insert can further include instructions for administering the composition to a patient.

[0174] In addition, the article of manufacture may further comprise a second container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0175] Also provided are kits, optionally in combination with the product, that are useful for various purposes, for example, treating cancer as described herein. The kits include one or more containers containing any one of the compositions (or unit dosage forms and / or products) described herein. In some embodiments, the kits further include other agents (e.g., one or more additional therapeutic agents) and / or instructions for use in accordance with any of the methods described herein. The kits may further include instructions for selecting individuals suitable for treatment. The instructions provided with the kits are typically written instructions on a label or package insert (e.g., a paper sheet included with the kit), although machine-readable instructions (e.g., instructions written on a magnetic or optical storage disk) are also acceptable.

[0176] For example, in some embodiments, a kit is provided that includes 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 a subject with cancer. In some embodiments, the kit further includes a pharmaceutical composition comprising an additional therapeutic agent, such as a chemotherapeutic agent. In some embodiments, the kit further includes a pharmaceutical composition comprising an anti-PD-1 antibody. In some embodiments, the kit includes a kit that includes one or more biomarkers described herein (e.g., CD8+ T cells, CD4+ T cells, CD8+ T cells). em cells, CD4+T em cell, T reg cell, T reg CD8+ T cells em Cell ratio, T reg CD4+ T cells emThe assay includes one or more assays or reagents for determining the levels of IgG1-associated ...

[0177] 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 Mylar or plastic bags), etc. The kits may optionally provide additional components, such as buffers and interpretive information. That is, the present application also provides articles of manufacture that include vials (e.g., sealed vials), bottles, jars, flexible packaging, etc.

[0178] The containers may be unit doses, bulk packages (e.g., multi-dose packages), or sub-unit doses. The kits may also include multiple unit doses of the pharmaceutical composition and instructions for use and packaging in sufficient quantities for storage and use in pharmacies, e.g., hospital pharmacies and compounding pharmacies.

[0179] The foregoing description is deemed sufficient to enable one skilled in the art to practice the present disclosure. The following examples are offered for illustrative purposes only and are not intended to limit the scope of the 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 from the foregoing description, and these modifications will fall within the scope of the appended claims. [Example]

[0180] The present invention may be further understood by reference to the following examples, which are offered by way of illustration and not intended to be limiting.

[0181] Example 1. A First-in-Human (FIH), Open-Label, Phase 1, Dose-Escalation Study of TY22404 in Combination with an Anti-PD-1 Antibody in Patients with Advanced / Metastatic Solid Tumors

[0182] method This is a first-in-human, Phase 1, open-label, multicenter, sequential dose-escalation and dose-expansion study to evaluate the safety, tolerability, PK, and preliminary efficacy of TY22404 monotherapy and TY22404-toripalimab combination therapy in patients with advanced / metastatic solid tumors. The study consists of Part 1: TY22404 monotherapy dose escalation followed by biopsy cohorts and expansion (concurrently); and Part 2: TY22404 in combination with toripalimab dose escalation and expansion. The objective of this study is to evaluate the safety and tolerability of escalating dose levels of TY22404 monotherapy and in combination with toripalimab in adult patients with advanced / metastatic solid tumors who have exhausted treatment options.

[0183] Dose Escalation. Dose escalation for TY22404 monotherapy will be a traditional 3+3 cohort design. Three to six patients will be enrolled at up to six dose escalation levels: 0.1, ≤0.3, ≤1, ≤3, ≤10, and ≤20 mg / kg every 3 weeks (Q3W). Dose levels or dosing intervals may be adjusted during the study based on observed safety data, but will not exceed the pre-determined MAD of 20 mg / kg.

[0184] Combination Dose Escalation. The combination will be initiated at a dose lower than that approved in the monotherapy dose-escalation arm and approved by the SRC. Based on nonclinical data, the projected RP2D is 20 mg / kg every 3 weeks. To maximize patient safety, the starting dose in the combination dose-escalation arm will be lower than the RP2D (e.g., 10 mg / kg Q3W). Enrollment in the combination dose-escalation arm (and concurrent enrollment is permitted) may occur before defining the RP2D / MAD in the monotherapy dose-escalation arm. To determine the RP2D for the TY22404 and toripalimab combination, a modified toxicity probability interval (mTPI) design with a target DLT rate of approximately 30% will be applied to dose escalation and confirmation. Dose escalation will include the combination dose escalation and the following three dose escalation levels for each combination: TIFF2025530196000005.tif28170DL=dose level, mTPI=modified toxicity probability interval, SRC=safety review committee, Q3W=every 3 weeks a DL3 indicates dose level 3. The numerical dose level will be determined based on data generated during dose escalation of the combination therapy.

[0185] Key inclusion criteria: Patients with advanced / metastatic solid tumors with ECOG ≤ 1 and at least one measurable lesion per RECIST 1.1. Imaging was performed every 6 weeks for the first 4 cycles, then every 9 weeks thereafter. Tumor response was assessed by the investigator using RECIST 1.1 and iRECIST. Prior treatment with anti-PD-1 or anti-CTLA-4 therapy was permitted.

[0186] Endpoints: The primary endpoints were safety and tolerability, which determined the maximum administered dose (MAD), maximum tolerated dose (MTD), and recommended phase 2 dose (RP2D). Secondary endpoints included PK per RECIST 1.1, antidrug antibodies (ADA), ORR, DCR, DOR, PFS, and OS. Evaluation: Imaging was performed every 6 weeks for the first four cycles, then every 9 weeks thereafter.

[0187] Early interim results of TY22404 monotherapy Twenty-six patients were treated with TY22404 monotherapy. Patients were heavily pretreated. Tumor types included breast cancer, cholangiocarcinoma, colorectal cancer, epithelial ovarian cancer, glioblastoma, hepatocellular carcinoma, melanoma, non-small cell lung cancer, pancreatic adenocarcinoma, renal cell carcinoma, and uveal melanoma.

[0188] TY22404 monotherapy (N=26) was well tolerated, with no dose-limiting toxicities observed up to 20 mg / kg. The most frequent treatment-related adverse events (TRAEs) (>10%) were fatigue (12%), pruritus (12%), rash (12%), and diarrhea (12%) (Table 2). TY22404 demonstrated single-agent antitumor activity with a disease control rate of 39% among 23 evaluable patients (Figure 1A-B).

[0189] A patient who underwent curative salpingo-oophorectomy and received five prior systemic therapies demonstrated a sustained response after administration of TY22404 at 1 mg / kg. CA125 decreased by 90% from 303 to 31 U / ml (normal range: <35 U / ml) (Figure 2), and target lesions decreased by 22% at the end of the 16th cycle (Table 3). Treatment is currently ongoing through the 18th cycle.

[0190] Plasma pharmacokinetics (PK) of the total and activated drugs, measured by LC-MS using their respective signature peptides, was approximately linear with dose, and calculated cleaved (i.e., activated) TY22404 accumulated approximately threefold (e.g., cycle 4 vs. cycle 1) during repeated dosing (Figure 3A-B). Noncompartmental PK analysis (NCA) suggested that the terminal half-life of total TY22404 in circulation increased approximately 1.5-fold on average compared to the parent Ab TY21580. PK of the circulating cleaved drug reflects the prolonged exposure of activated TY22404 in the tumor microenvironment. PK modeling further predicted a target saturation level of approximately 6-10 mg / kg at steady state. TIFF2025530196000006.tif77170TIFF2025530196000007.tif116170TIFF2025530196000008.tif71170

[0191] TY22404 Updated Interim Results 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 were extensively pretreated. Tumor types included breast cancer, cholangiocarcinoma, colorectal cancer, epithelial ovarian cancer, glioblastoma, hepatocellular carcinoma, melanoma, non-small cell lung cancer, pancreatic adenocarcinoma, renal cell carcinoma, uveal melanoma, prostate adenocarcinoma, endometrial carcinoma, gastrointestinal stromal tumor (GIST), squamous cell carcinoma (anal, anorectal, penile, or cutaneous), and adenocarcinoma. These tumor types, including "cold tumors" and immune-experienced "hot tumors," were included in the study. TIFF2025530196000009.tif113170

[0192] Safety - TY22404 monotherapy As shown in Table 5, TY22404 monotherapy was well tolerated at doses up to 20 mg / kg in repeated doses with no dose-limiting toxicities. No treatment-related adverse events (TRAEs) greater than grade 2 or treatment-related serious adverse events (SAEs) were reported. No patients discontinued study treatment due to TRAEs. TIFF2025530196000010.tif41170

[0193] Safety - TY22404 + toripalimab dose escalation Three dose-escalation cohorts were studied: seven patients received TY22404 6 mg / kg Q3W plus toripalimab 240 mg Q3W, seven patients received TY22404 10 mg / kg Q6W plus toripalimab 240 mg Q3W, and nine patients received TY22404 10 mg / kg Q3W plus toripalimab 240 mg Q3W (Table 6). No significant differences in safety were observed among these three dose-escalation cohorts. No DLTs or TRAEs greater than grade 3 were reported. Five patients (21%) experienced G3 TRAEs. Most G3 TRAEs occurred after cycle 4. Three patients experienced treatment-related SAEs, including one G3 hepatitis, one G3 sepsis, and one G3 myocarditis, which led to study discontinuation. TIFF2025530196000011.tif34170

[0194] Efficacy - TY22404 monotherapy Across all dose levels, the disease control rate (DCR) was 37% among 27 evaluable patients who underwent at least one valid post-baseline tumor assessment. Five patients experienced long-term stable disease, including one with NSCLC at 14 cycles and one with ovarian cancer at 22 cycles. This NSCLC patient, whose disease had progressed on pembrolizumab and docetaxel, experienced a 12% reduction in the total number of target lesions. This NSCLC patient completed 14 cycles of TY22404 20 mg / kg Q3W with no TRAEs. A patient with ovarian cancer received TY22404 1 mg / kg Q3W and experienced stable disease with a 90% reduction in CA125 and a 22% reduction in the total number of target lesions.

[0195] A 39-year-old male patient with stage IIIB hepatocellular carcinoma (HCC) was diagnosed with ECOG PS 0 at baseline. He previously received the anti-PD-L1 therapy atezolizumab plus bevacizumab combination therapy as first-line treatment (July 2021–February 2022, PD), followed by lenvatinib as second-line treatment (March–September 2022, PD). The patient maintained stable tumor growth and was receiving TY22404 10 mg / kg Q3W at C6. As shown in Figure 4, paired tumor biopsies demonstrated an increased Teff / Treg ratio (Figure 4A), a decrease in Tregs (Figure 4B), and an increase in CD8+ T cells (Figure 4C).

[0196] Efficacy - TY22404 + toripalimab dose escalation As shown in Table 7 and Figure 5, responses were calculated for 18 evaluable patients in three dose-escalation cohorts receiving TY22404 (6 mg / kg Q3W and 10 mg / kg Q3W or Q6W) plus toripalimab (240 mg Q3W). Among the 18 evaluable patients, the overall ORR was 11% and the DCR was 56%. Among the seven evaluable patients receiving TY22404 10 mg / kg Q3W plus TORI 240 mg Q3W, an ORR of 28% and a DCR of 57% were reported. Confirmed partial responses (PRs) were observed in two patients with penile squamous cell carcinoma (SCC) and anal SCC who had previously been treated with chemotherapy. In PDAC patients, a 5% reduction in total target lesions and prolonged stable disease were observed. In two patients with microsatellite stable colorectal cancer (MSS CRC) who had liver metastases at baseline, a 58% and 21% reduction in total target lesions, respectively, was observed. TIFF2025530196000012.tif47170

[0197] Case study: Confirmation of PR in two patients with anal SCC and penile SCC As shown in Table 8, a 51-year-old female patient with metastatic anal squamous cell carcinoma (SCC) received TY22404 10 mg / kg Q3W plus toripalimab 240 mg Q3W and achieved a PR with a 36% reduction in total target lesions (treatment was ongoing at cycle 5 at the time of this data cut). Her baseline ECOG PS was 0. She had previously received fluorouracil (5FU) / mitomycin C plus radiation therapy. TIFF2025530196000013.tif49170

[0198] As shown in Table 9, a 64-year-old male patient with metastatic penile SCC was administered TY22404 10 mg / kg Q3W plus toripalimab 240 mg Q3W and achieved a confirmed PR with a 72% reduction in total target lesions (treatment was ongoing at cycle 6 at the time of this data cut). The baseline ECOG PS was 1. The patient had previously undergone partial penectomy, bilateral lymph node dissection, chemotherapy (paclitaxel, ifosfamide, cisplatin), and palliative radiation therapy. TIFF2025530196000014.tif47170

[0199] Case study: Promising potential of TY22404 plus toripalimab in 'cold' gastrointestinal tumors As shown in Table 10, a 53-year-old man with MSS rectosigmoid adenocarcinoma with liver metastases at baseline received TY22404 10 mg / kg Q6W plus toripalimab 240 mg Q3W (treatment was ongoing at the time of this data cut). His baseline ECOG PS was 0. He had previously undergone curative and palliative surgery for liver metastases and had received three prior therapies: leucovorin + 5FU + irinotecan + oxaliplatin, bevacizumab + TAS 102, and regorafenib. At week 17, a mixed response was observed, with a 58% reduction in total target lesions. TIFF2025530196000015.tif89170

[0200] As shown in Table 11, a 74-year-old male MSS CRC patient with liver metastases at baseline was administered TY22404 6 mg / kg Q3W and toripalimab 240 mg Q3W. His baseline ECOG PS was 1. He had previously received three prior therapies: XELOX + bevacizumab, irinotecan + cetuximab, and irinotecan + panitumumab. A 21% reduction in total target lesions was observed (treatment was ongoing at the time of this data cut). TIFF2025530196000016.tif61170

[0201] As shown in Table 12, a 56-year-old female patient with pancreatic ductal adenocarcinoma (PDAC) received TY22404 6 mg / kg Q3W and toripalimab 240 mg Q3W. Her baseline ECOG PS was 0. She had previously undergone curative surgery and four prior therapies (gemcitabine / nab-paclitaxel, FOLFIRINOX, oxaliplatin, raltitrexed, irinotecan, leucovorin, and gemcitabine / capecitabine). Prolonged control of tumor growth (stable disease) with a 5% reduction in total target lesions was observed (treatment is ongoing). TIFF2025530196000017.tif56170

[0202] Figures 6A-6B show the response to TY22404 plus toripalimab in selected gastrointestinal "cold tumors," as described above. Figure 6A shows the swimmer plot. Figure 6B shows the waterfall plot.

[0203] TY22404 monotherapy was well tolerated up to 20 mg / kg Q3W and demonstrated promising efficacy signals in heavily pretreated patients. Prolonged stable disease was observed in five patients. Treg depletion and increased CD8+ T cells, along with an increased Teff / Treg ratio, were observed in paired tumor biopsies. The safety profile of TY22404 + toripalimab dose escalation demonstrates best-in-class potential compared with other anti-CTLA-4 molecules combined with anti-PD-1 antibodies at similar doses / schedules. TY22404 + toripalimab demonstrated promising efficacy, including two confirmed partial response (PRs), prolonged stable disease, and target lesion shrinkage in "cold" gastrointestinal tumors. The ORR was 28% and DCR was 57% in seven evaluable patients receiving TY22404 10 mg / kg Q3W + toripalimab 240 mg Q3W. Two patients with MSS CRC who had liver metastases at baseline experienced prolonged stable disease and a 58% and 21% reduction in the total number of target lesions. Similar results were observed in one patient with PDAC, with a 5% reduction in the total number of target lesions. Sequential dosing beyond four cycles, enabled by a favorable safety profile, may allow for combinations beyond anti-PD-1 therapy: a study of TY22404 + atezolizumab + bevacizumab (NCT04524871) is planned. Due to its unique mechanism of Treg depletion, TY22404 may have the potential to address significant unmet needs in "cold tumor" settings. Dose expansion of TY22404 10 mg / kg + toripalimab is planned for indications including MSS CRC. Exemplary Sequences SEQ ID NO: 23 HVR-H1 activatable anti-CTLA4 YSISSGYHWSWI SEQ ID NO: 35 HVR-H2 activatable anti-CTLA4 LARIDWDDDKYYSTSLKSRL SEQ ID NO: 45 HVR-H3 activatable anti-CTLA4 ARSYVYFDY SEQ ID NO: 58 HVR-L1 activatable anti-CTLA4 RASQSVRGRFLA SEQ ID NO: 66 HVR-L2 activatable anti-CTLA4 DASNRATGI SEQ ID NO: 75 HVR-L3 activatable anti-CTLA4 YCQQSSSWPPT SEQ ID NO: 192 masking portion (MM) EVGSYPNPSSDCVPYYYACAY SEQ ID NO: 221 Cleavable moiety (CM) SGRSAGGGGTPLGLAGSGGS SEQ ID NO: 200 masking moiety (MM) and cleavable moiety (CM) EVGSYPNPSSDCVPYYYACAYSGRSAGGGGTPLGLAGSGGS SEQ ID NO: 312 Toripalimab (anti-PD1) HVR-H1 DYEMH SEQ ID NO: 313 Toripalimab HVR-H2 VIESETGGTAYNQKFKG SEQ ID NO: 314 Toripalimab HVR-H3 EGITTVATTYYWYFDV SEQ ID NO: 315 Toripalimab HVR-L1 RSSQSIVHSNGNTYLE SEQ ID NO: 316 Toripalimab HVR-L2 KVSNRFS SEQ ID NO: 317 Toripalimab HVR-L3 FQGSHVPLT SEQ ID NO: 318 Toripalimab VH QGQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPIHGLEWIGVIESETGGTAYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCAREGITTVATTYYWYFDVWGQGTTVTVSS SEQ ID NO: 319 Toripalimab VL DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPLTFGQGTKLEIK SEQ ID NO: 320 activatable anti-CTLA42 complete heavy chain (excluding C-terminal lysine) EVQLVESGGGLVQPGGSLRLSCAASGYSISSGYHWSWIRQAPGKGLEWLARIDWDDDKYYSTSLKSRLTISRDNSKNTLYLQLNSLRAEDTAVYYCARSYVYFDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 321 activatable anti-CTLA42 complete heavy chain (including C-terminal lysine) EVQLVESGGGLVQPGGSLRLSCAASGYSISSGYHWSWIRQAPGKGLEWLARIDWDDDKYYSTSLKSRLTISRDNSKNTLYLQLNSLRAEDTAVYYCARSYVYFDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 322 Activatable anti-CTLA42 complete light chain with N-terminal masking moiety and linker EVGSYPNPSSDCVPYYYACAYSGRSAGGGGTPLGLAGSGGSDIQLTQSPSSLSASVGDRVTITCRASQSVRGRFLAWYQQKPGKAPKLLIYDASNRATGIPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQSSSWPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 323 activatable anti-CTLA4 1 complete heavy chain (excluding C-terminal lysine) EVQLVESGGGLVQPGGSLRLSCAASGYSISSGYHWSWIRQAPGKGLEWLARIDWDDDKYYSTSLKSRLTISRDNSKNTLYLQLNSLRAEDTAVYYCARSYVYFDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 324 activatable anti-CTLA4 1 complete heavy chain (including C-terminal lysine) EVQLVESGGGLVQPGGSLRLSCAASGYSISSGYHWSWIRQAPGKGLEWLARIDWDDDKYYSTSLKSRLTISRDNSKNTLYLQLNSLRAEDTAVYYCARSYVYFDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 325 Activatable anti-CTLA4 1 complete light chain with N-terminal masking moiety and linker EVGSYNFVADSCPDHPYPCSASGRSAGGGGSPLGLAGSGGSDIQLTQSPSSLSASVGDRVTITCRASQSVRGRFLAWYQQKPGKAPKLLIYDASNRATGIPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQSSSWPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 326 activatable anti-CTLA43 complete heavy chain (excluding C-terminal lysine) EVQLVESGGGLVQPGGSLRLSCAASGYSISSGYHWSWIRQAPGKGLEWLARIDWDDDKYYSTSLKSRLTISRDNSKNTLYLQLNSLRAEDTAVYYCARSYVYFDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 327 activatable anti-CTLA43 complete heavy chain (including C-terminal lysine) EVQLVESGGGLVQPGGSLRLSCAASGYSISSGYHWSWIRQAPGKGLEWLARIDWDDDKYYSTSLKSRLTISRDNSKNTLYLQLNSLRAEDTAVYYCARSYVYFDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 328 Activatable anti-CTLA4 3 complete light chain with N-terminal masking moiety and linker EVGSYIVHHSDCDAFYPYCDSSGRSAGGGGSPLGLAGSGGSDIQLTQSPSSLSASVGDRVTITCRASQSVRGRFLAWYQQKPGKAPKLLIYDASNRATGIPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQSSSWPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Claims

1. 1. A method of treating cancer in a subject, comprising administering to the subject (a) an effective amount of an activatable antibody, the activatable antibody comprising: HVR-H1 comprising an amino acid sequence of the formula YSISSGYHWSWI (SEQ ID NO: 23); HVR-H2 comprising an amino acid sequence of the formula LARIDWDDDKYYSTSLKSRL (SEQ ID NO: 35); HVR-H3 comprising an amino acid sequence of the formula ARSYVYFDY (SEQ ID NO: 45); HVR-L1 comprising an amino acid sequence of the formula RASQSVRGRFLA (SEQ ID NO: 58); HVR-L2 comprising an amino acid sequence of the formula DASNRATGI (SEQ ID NO: 66); and HVR-L3 comprising an amino acid sequence of the formula YCQQSSSWPPT (SEQ ID NO: 75). the antibody further comprises a polypeptide covalently linked to the N-terminus of the light chain of the anti-CTLA4 antibody, the polypeptide comprising, from the N-terminus to the C-terminus, a masking 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 (b) an effective amount of toripalimab, the activatable antibody being administered once every 3 to 6 weeks at a dose of about 6 mg / kg to about 10 mg / kg, the toripalimab being administered once every 3 weeks at a dose of about 200 mg to about 400 mg, or once every 6 weeks at a dose of about 300 mg to about 600 mg.

2. 10. 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. 10. 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. 10. The method of claim 1, wherein the toripalimab is administered at a dose of about 240 mg once every three weeks.

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

6. 1. A method of treating cancer in a subject, comprising administering to the subject an effective amount of an activatable antibody, the activatable antibody comprising: HVR-H1 comprising an amino acid sequence of the formula YSISSGYHWSWI (SEQ ID NO:23); HVR-H2 comprising an amino acid sequence of the formula LARIDWDDDKYYSTSLKSRL (SEQ ID NO:35); HVR-H3 comprising an amino acid sequence of the formula ARSYVYFDY (SEQ ID NO:45); HVR-L1 comprising an amino acid sequence of the formula RASQSVRGRFLA (SEQ ID NO:58); HVR-L2 comprising an amino acid sequence of the formula DASNRATGI (SEQ ID NO:66); and HVR-L3 comprising an amino acid sequence of the formula YCQQSSSWPPT and HVR-L3 comprising the amino acid sequence of (SEQ ID NO: 75), wherein the activatable antibody further comprises a polypeptide covalently linked to the N-terminus of the light chain of the anti-CTLA4 antibody, the polypeptide comprising, from the N-terminus to the C-terminus, a masking 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 of about 6 mg / kg to about 20 mg / kg once every 3 to 6 weeks.

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

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

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

10. 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 according to any one of claims 1 to 8, wherein the cancer is pancreatic cancer.

14. 14. The method of any one of claims 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 according to 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 advanced metastatic cancer.

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

20. 19. 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. 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. 22. The method of claim 21, wherein the activatable antibody comprises the complete heavy chain region of SEQ ID NO: 320 or SEQ ID NO:

321.

23. 23. The method of claim 22, wherein the activatable antibody comprises the complete 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. 25. The method of any one of claims 1-24, wherein both the activatable anti-CTLA4 antibody and toripalimab are administered on day 1 of a 3-6 week dosing schedule.

26. 1. A method of treating cancer in a subject, comprising administering to the subject an effective amount of an activatable antibody, the activatable antibody comprising: HVR-H1 comprising an amino acid sequence of the formula YSISSGYHWSWI (SEQ ID NO:23); HVR-H2 comprising an amino acid sequence of the formula LARIDWDDDKYYSTSLKSRL (SEQ ID NO:35); HVR-H3 comprising an amino acid sequence of the formula ARSYVYFDY (SEQ ID NO:45); HVR-L1 comprising an amino acid sequence of the formula RASQSVRGRFLA (SEQ ID NO:58); HVR-L2 comprising an amino acid sequence of the formula DASNRATGI (SEQ ID NO:66); and HVR-L3 comprising an amino acid sequence of the formula YCQQSSSWPPT (SEQ ID NO:75). and HVR-L3 comprising the amino acid sequence

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

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

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

31. 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 at a dose of about 300 mg to about 600 mg once every six weeks.

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

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

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

35. 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. 34. 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. 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. 44. The method of claim 43, wherein the cancer has metastasized to the lung or liver.

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

321.

48. 48. The method of claim 47, wherein the activatable antibody comprises the complete light 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. 1. A method of treating cancer in a subject, comprising administering to the subject an effective amount of an activatable antibody, the activatable antibody comprising: HVR-H1 comprising an amino acid sequence of the formula YSISSGYHWSWI (SEQ ID NO:23); HVR-H2 comprising an amino acid sequence of the formula LARIDWDDDKYYSTSLKSRL (SEQ ID NO:35); HVR-H3 comprising an amino acid sequence of the formula ARSYVYFDY (SEQ ID NO:45); HVR-L1 comprising an amino acid sequence of the formula RASQSVRGRFLA (SEQ ID NO:58); HVR-L2 comprising an amino acid sequence of the formula DASNRATGI (SEQ ID NO:66); and HVR-L3 comprising an amino acid sequence of the formula YCQQSSSWPPT (SEQ ID NO:67). and HVR-L3 comprising the amino acid sequence of (I) SEQ ID NO: 75), wherein the activatable antibody further comprises a polypeptide covalently linked to the N-terminus of the light chain of the anti-CTLA4 antibody, the polypeptide comprising, from the N-terminus to the C-terminus, a masking 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 the cleaved antibody that exceeds the EC50 of the cleaved antibody.

51. 1. A method of treating cancer in a subject, comprising administering to the subject an effective amount of an activatable antibody, the activatable antibody comprising: HVR-H1 comprising an amino acid sequence of the formula YSISSGYHWSWI (SEQ ID NO: 23); HVR-H2 comprising an amino acid sequence of the formula LARIDWDDDKYYSTSLKSRL (SEQ ID NO: 35); HVR-H3 comprising an amino acid sequence of the formula ARSYVYFDY (SEQ ID NO: 45); HVR-L1 comprising an amino acid sequence of the formula RASQSVRGRFLA (SEQ ID NO: 58); HVR-L2 comprising an amino acid sequence of the formula DASNRATGI (SEQ ID NO: 66); and HVR-L3 comprising an amino acid sequence of the formula YCQQSSSWPPT (SEQ ID NO: 7 5), wherein the activatable antibody further comprises a polypeptide covalently linked to the N-terminus of the light chain of the anti-CTLA4 antibody, the polypeptide comprising, from the N-terminus to the C-terminus, a masking 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 the activatable antibody is administered at a dose that provides a steady-state concentration of the cleaved antibody that exceeds the EC90 of the cleaved antibody.

52. 1. A method of treating cancer in a subject, comprising administering to the subject an effective amount of an activatable antibody, the activatable antibody comprising: HVR-H1 comprising an amino acid sequence of the formula YSISSGYHWSWI (SEQ ID NO:23); HVR-H2 comprising an amino acid sequence of the formula LARIDWDDDKYYSTSLKSRL (SEQ ID NO:35); HVR-H3 comprising an amino acid sequence of the formula ARSYVYFDY (SEQ ID NO:45); HVR-L1 comprising an amino acid sequence of the formula RASQSVRGRFLA (SEQ ID NO:58); HVR-L2 comprising an amino acid sequence of the formula DASNRATGI (SEQ ID NO:66); and HVR-L3 comprising an amino acid sequence of the formula YCQQSSSWPPT (SEQ ID NO:67). and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 75), wherein the activatable antibody further comprises a polypeptide covalently linked to the N-terminus of the light chain of an anti-CTLA4 antibody, the polypeptide comprising, from the N-terminus to the C-terminus, a masking 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 results in a steady-state concentration of cleaved antibody of about 100 nM to about 200 nM.

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

54. 53. The method of claim 52, wherein the activatable antibody is administered at a dose that results in a steady state concentration of cleaved antibody of about 100 nM to about 150 nM.

55. 53. The method of claim 52, wherein the activatable antibody is administered at a dose that results in a steady state concentration of cleaved antibody of about 150 nM to about 200 nM.

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

57. 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, the amount of the loading dose being higher than the amount of the maintenance dose.

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

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

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

62. 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. 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. 63. The method of claim 62, wherein the activatable antibody comprises the complete heavy chain region of SEQ ID NO: 320 or SEQ ID NO:

321.

65. 65. The method of claim 64, wherein the activatable antibody comprises the complete 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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