A cancer treatment method using an activatable anti-CTLA4 antibody in combination with pembrolizumab.

Abstract

This application provides a composition and method for treating cancer, including advanced metastatic cancer, using an activatable anti-CTLA4 antibody in combination with pembrolizumab.

Description

Cross - reference to related applications

[0001] This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 495,968, filed on April 13, 2023, U.S. Provisional Patent Application 63 / 584,327, filed on September 21, 2023, and U.S. Provisional Patent Application 63 / 594,734, filed on October 31, 2023. The content of each application is hereby incorporated by reference in its entirety. Submission of a Sequence Listing in ASCII text file

[0002] The content of the electronic sequence list (695402002740SEQLIST.xml, size: 93,905 bytes, creation date: April 3, 2024) is hereby incorporated by reference in its entirety.

Technical Field

[0003] This application relates to the field of cancer treatment, and compositions and methods for treating cancer using an antibody that binds to human CTLA4 in combination with the anti - PD - 1 antibody pembrolizumab.

Background Art

[0004] CTLA4 is a member of the immunoglobulin (Ig) superfamily of proteins that downregulates T cell activation and maintains immunogenic homeostasis. In a syngeneic mouse prostate cancer model, in vivo antibody-mediated blockade of CTLA4 has been shown to enhance the anti-cancer immune response (Kwonetal. (1997) ProcNatlAcadSciUSA, 94(15):8099-103). In addition, in tumor-bearing mice, blockade of CTLA4 function has been shown to enhance the anti-tumor T cell response at various stages of tumor growth (Yangetal. (1997) CancerRes 57(18):4036-41, Hurwitzetal. (1998) ProcNatlAcadSciUSA 95(17):10067-7). However, the development of antibody-based therapeutics suitable for human use remains challenging. This is because the transition from preclinical animal models to human safety is often poor. Therefore, there is a need for anti-CTLA4 antibodies that cross-react between humans and different species of experimental animals (e.g., mice, monkeys, rats, etc.) in order to enable animal model research while simultaneously providing appropriate therapeutic candidates for human use. In addition, there is a need for the development of safer anti-CTLA4 antibodies that are active only in specific situations, such as within the protease-rich tumor microenvironment.

[0005] PD-1 is recognized as an important molecule in immunomodulation and maintenance of peripheral tolerance. PD-1 is moderately expressed in naive T cells, B cells, and NKT cells, and is upregulated by T / B cell receptor signaling on lymphocytes, monocytes, and myeloid cells (Sharpe, Arlene H et al., The function of programmed cell death 1 and its ligands in regulating autoimmunity and infection. Nature Immunology (2007); 8:239-245).

[0006] PD-L1 (B7-H1) and PD-L2 (B7-DC), known ligands for PD-1, are expressed in human cancers that occur in various tissues. In large sample sets of ovarian cancer, kidney cancer, colorectal cancer, liver cancer, and melanoma, PD-L1 expression was shown to correlate with poor prognosis and reduce overall survival regardless of subsequent treatment (Dong, Haidong et al., Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion. Nat Med. 2002 Aug;8(8):793-800; Yang, Wanhua et al., PD-1 interaction contributes to the functional suppression of T-cell responses to human uveal melanoma cells in vitro. Invest Ophthalmol Vis Sci. 2008 Jun; 49(6 (2008): 49: 2518-2525; Ghebeh, Hazem et al., The B7-H1 (PD-L1) T lymphocyte-inhibitory molecule is expressed in breast cancer patients with infiltrating ductal carcinoma: correlation with important high-risk prognostic factors. Neoplasia (2006) 8: 190-198; Hamanishi, Junzo et al., Programmed cell death 1 ligand 1 and tumor-infiltrating CD8+ T lymphocytes are prognostic factors of human ovarian cancer. Proc. Natl. Acad. Sci.USA (2007): 104: 3360-3365; Thompson, R Houston, and Eugene D Kwon, Significance of B7-H1 overexpression in kidney cancer. Clinical genitourin Cancer (2006): 5: 206-211; Nomi, Takeo et al., Clinical significance and therapeutic potential of the programmed death-1 ligand / programmed death-1 pathway in human pancreatic cancer. Clinical Cancer Research (2007);13:2151-2157; Ohigashi, Yuichiro et al., Clinical significance of programmed death-1 ligand-1 and programmed death-1 ligand 2 expression in human esophageal cancer. Clin. Cancer Research (2005): 11: 2947-2953; Inman, Brant A et al., PD-L1 (B7-H1) expression by urothelial carcinoma of the bladder and BCG-induced granulomata: associations with localized stage progression. Cancer (2007): 109: 1499-1505; Shimauchi, Takatoshi et al., Augmented expression of programmed death-1 in both neoplasmatic and nonneoplastic CD4+ T-cells in adult T-cell Leukemia / Lymphoma. Int. J. Cancer (2007): 121:2585-2590; Gao, Qiang et al., Overexpression of PD-L1 significantly associates with tumor aggressiveness and postoperative recurrence in human hepatocellular carcinoma. Clinical Cancer Research (2009) 15: 971-979; Nakanishi, Juro et al., Overexpression of B7-H1 (PD-L1) significantly associates with tumor grade and postoperative prognosis in human urothelial cancers. Cancer Immunol Immunother. (2007) 56: 1173-1182; Hino et al., Tumor cell expression of programmed cell death-1 is a prognostic factor for malignant melanoma.Cancer (2010): 00: 1-9)。.

[0007] Several monoclonal antibodies that inhibit the interaction between PD-1 and one or both of its ligands, PD-L1 and PD-L2, have been approved as cancer treatments. Pembrolizumab (KEYTRUDA®, Merck Sharp & Dohme LLC, Rahway, NJ, USA) is a potent humanized immunoglobulin G4 (IgG4) mAb with high binding specificity to the programmed cell death 1 (PD-1) receptor, inhibiting the interaction with programmed cell death ligand 1 (PD-L1) and programmed cell death ligand 2 (PD-L2). Based on preclinical in vitro data, pembrolizumab exhibits high affinity for PD-1 and potent receptor inhibitory activity. Keytruda® (pembrolizumab) has been indicated for patient treatment across numerous directives and is indicated as first-line treatment for patients with unresectable or metastatic CRC that is microsatellite instability or mismatch repair deficiency (MSL-H / dMMR). Pembrolizumab is currently the standard first-line treatment for MSLH / dMMR mCRC. [Overview of the project]

[0008] This application provides a method for treating cancer by combining the activatable anti-CTLA4 antibody of the present invention with the anti-PD-1 antibody pembrolizumab. This application further provides a method for treating cancer with the anti-CTLA4 antibody, pembrolizumab, and at least one additional therapeutic agent.

[0009] In one embodiment, the present invention provides a method for treating cancer in a subject (e.g., a human patient), comprising (a) administering to the subject (e.g., a human patient) an effective amount of an activatable antibody, the activatable antibody comprising a polypeptide comprising a masking moiety (MM), a cleavable moiety (CM), and an anti-CTLA4 antibody as described herein, from the N-terminus to the C-terminus, wherein the MM comprises the amino acid sequence EVGSYPNPSSDCVPYYYACAY (SEQ ID NO: 192), and the cleavable moiety comprises the amino acid sequence SGRSAGGGGTPLGLAGSGGS (SEQ ID NO: 221); and (b) comprising an effective amount of pembrolizumab. The MM and CM comprise the amino acid sequence EVGSYPNPSSDCVPYYYACAYSGRSAGGGGTPLGLAGSGGS (SEQ ID NO: 200) from the N-terminus to the C-terminus. In certain embodiments, the MM and CM are covalently bound to the N-terminus of the light chain of the anti-CTLA4 antibody. In some embodiments, the MM and CM sequences from the N-terminus to the C-terminus include amino acid sequences having at least 90% or at least 95% sequence identity with SEQ ID NO: 200.

[0010] In some embodiments, the activatable anti-CTLA4 antibody includes HVR-H1 containing the amino acid sequence of formula YSISSGYHWSWI (SEQ ID NO: 23), HVR-H2 containing the amino acid sequence of formula LARIDWDDDKYYSTSLKSRL (SEQ ID NO: 35), HVR-H3 containing the amino acid sequence of formula ARSYVYFDY (SEQ ID NO: 45), HVR-L1 containing the amino acid sequence of formula RASQSVRGRFLA (SEQ ID NO: 58), HVR-L2 containing the amino acid sequence of formula DASNRATGI (SEQ ID NO: 66), and HVR-L3 containing the amino acid sequence of formula YCQQSSSWPPT (SEQ ID NO: 75).

[0011] In some embodiments, the activatable anti-CTLA4 antibody, upon cleavage of CM, comprises a) a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 87, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 100.

[0012] In some embodiments, the activatable anti-CTLA4 antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 320 and a light chain containing the amino acid sequence of SEQ ID NO: 322. The activatable antibody having the heavy chain SEQ ID NO: 320 and the light chain SEQ ID NO: 322 is called TY22404. In some embodiments, the activatable antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 320 and a light chain containing 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 with respect to the amino acid sequence of SEQ ID NO: 320. In some embodiments, the activatable anti-CTLA4 antibody includes 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 includes 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 includes 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.

[0013] In any of the embodiments described above, the activatable anti-CTLA4 antibody (e.g., TY22404), when administered in combination with pembrolizumab, can be administered in a dose that results in a steady-state plasma concentration of approximately 50 nM to approximately 100 nM of the cleaved antibody (i.e., the active antibody after cleavage of the masking portion (MM) and the cleavable portion (CM)). In some embodiments, the activatable anti-CTLA4 antibody is administered in a dose that results in a steady-state plasma concentration of approximately 50 nM to approximately 100 nM of the cleaved antibody. In some embodiments, the activatable anti-CTLA4 antibody is administered in a dose that results in a steady-state plasma concentration of approximately 50 nM to approximately 75 nM of the cleaved antibody. In some embodiments, the activatable anti-CTLA4 antibody is administered in a dose that results in a steady-state plasma concentration of approximately 70 nM to approximately 80 nM of the cleaved antibody. In some embodiments, the activatable anti-CTLA4 antibody is administered in a dose that results in a steady-state plasma concentration of approximately 100 nM to approximately 175 nM of the cleaved antibody. In some embodiments, the activatable anti-CTLA4 antibody is administered in a dose that results in a steady-state plasma concentration of approximately 100 nM to approximately 200 nM of the cleaved antibody. In some embodiments, the activatable anti-CTLA4 antibody is administered in a dose that results in a steady-state plasma concentration of approximately 125 nM to approximately 200 nM of the cleaved antibody. In some embodiments, the activatable anti-CTLA4 antibody is administered in a dose that results in a steady-state plasma concentration of approximately 100 nM to approximately 150 nM of the cleaved antibody. In some embodiments, the activatable anti-CTLA4 antibody is administered in a dose that results in a steady-state plasma concentration of approximately 125 nM to approximately 175 nM of the cleaved antibody. In some embodiments, the activatable anti-CTLA4 antibody is administered in a dose that results in a steady-state plasma concentration of approximately 125 nM to approximately 150 nM of the cleaved antibody. In some embodiments, the activatable anti-CTLA4 antibody is administered in a dose that results in a steady-state plasma concentration of approximately 150 nM to approximately 200 nM of the cleaved antibody. In some embodiments, the activatable anti-CTLA4 antibody is administered in a dose such that the steady-state plasma concentration of the cleaved antibody is approximately 200 nM to approximately 600 nM.In some embodiments, the activatable anti-CTLA4 antibody is administered at a dose that results in a steady-state plasma concentration of approximately 200 nM to approximately 400 nM of the cleaved antibody. In some embodiments, the activatable anti-CTLA4 antibody is administered at a dose that results in a steady-state plasma concentration of approximately 300 nM to approximately 500 nM of the cleaved antibody. In some embodiments, the activatable anti-CTLA4 antibody is administered at a dose that results in a steady-state plasma concentration of approximately 400 nM to approximately 600 nM of the cleaved antibody. In some of the embodiments described above, the plasma concentration can be measured at the trough level of the activatable anti-CTLA4 antibody (i.e., the minimum concentration in each administration cycle). For example, the plasma concentration for a particular cycle can be measured immediately before administration in the next cycle.

[0014] In some embodiments, the activatable anti-CTLA4 antibody and pembrolizumab are administered in combination with one or more additional therapeutic agents. In some embodiments, the activatable anti-CTLA4 antibody is administered in a dose that results in a steady-state plasma concentration of approximately 50 nM to approximately 150 nM of the cleaved antibody. In other such embodiments, the activatable anti-CTLA4 antibody is administered in a dose that results in a steady-state plasma concentration of approximately 50 nM to approximately 100 nM of the cleaved antibody. In other such embodiments, the activatable anti-CTLA4 antibody is administered in a dose that results in a steady-state plasma concentration of approximately 50 nM to approximately 75 nM of the cleaved antibody. In other such embodiments, the activatable anti-CTLA4 antibody is administered in a dose that results in a steady-state plasma concentration of approximately 75 nM to approximately 100 nM of the cleaved antibody.

[0015] In any of the embodiments described above, the activatable anti-CTLA4 antibody (e.g., TY22404), when administered in combination with pembrolizumab, can be administered in a dose such that the steady-state plasma concentration ratio of cleaved to uncleaved antibodies is approximately 0.3 to approximately 1.0 at trough levels for a particular dosing cycle. In some embodiments, the activatable anti-CTLA4 antibody can be administered in a dose such that the steady-state plasma concentration ratio of cleaved to uncleaved antibodies is approximately 0.3 to approximately 0.8 at trough levels for a particular dosing cycle. In some embodiments, the activatable anti-CTLA4 antibody can be administered in a dose such that the steady-state plasma concentration ratio of cleaved to uncleaved antibodies is approximately 0.5 to approximately 0.8 at trough levels for a particular dosing cycle. In some embodiments, the activatable anti-CTLA4 antibody can be administered in a dose such that the steady-state plasma concentration ratio of cleaved to uncleaved antibodies is approximately 0.7 to approximately 1.0 at trough levels for a particular dosing cycle.

[0016] In one embodiment, the present disclosure provides a method for treating cancer in a subject (e.g., a human patient) comprising administering an effective dose of the activatable anti-CTLA4 antibody (e.g., TY22404) described above to the subject (e.g., a human patient) in combination with pembrolizumab, wherein the activatable anti-CTLA4 antibody is administered at a dose of approximately 3 mg / kg to approximately 30 mg / kg. In some embodiments, the activatable anti-CTLA4 antibody (e.g., TY22404) is administered at a dose of approximately 10 mg / kg to approximately 20 mg / kg. In some embodiments, the activatable anti-CTLA4 antibody (e.g., TY22404) is administered at a dose of approximately 20 mg / kg to approximately 30 mg / kg. In some embodiments, the activatable anti-CTLA4 antibody (e.g., TY22404) is administered at a dose of approximately 3 mg / kg. In some embodiments, the activatable anti-CTLA4 antibody (e.g., TY22404) is administered at a dose of approximately 5 mg / kg. In some embodiments, the activatable anti-CTLA4 antibody (e.g., TY22404) is administered at a dose of approximately 6 mg / kg. In some embodiments, the activatable anti-CTLA4 antibody (e.g., TY22404) is administered at a dose of approximately 8 mg / kg. In some embodiments, the activatable anti-CTLA4 antibody (e.g., TY22404) is administered at a dose of approximately 10 mg / kg. In some embodiments, the activatable anti-CTLA4 antibody (e.g., TY22404) is administered at a dose of approximately 8 mg / kg. In some embodiments, the activatable anti-CTLA4 antibody (e.g., TY22404) is administered at a dose of approximately 20 mg / kg. In some embodiments, the activatable anti-CTLA4 antibody (e.g., TY22404) is administered at a dose of approximately 25 mg / kg. In some embodiments, the activatable anti-CTLA4 antibody (e.g., TY22404) is administered at a dose of approximately 30 mg / kg. In any of the embodiments described above, the activatable anti-CTLA4 antibody can be administered once every three weeks or once every six weeks. For example, in some embodiments, the activatable anti-CTLA4 antibody can be administered at a dose of 10 mg / kg once every three weeks or once every six weeks.In other embodiments, the activatable anti-CTLA4 antibody may be administered at a dose of 20 mg / kg once every three weeks or once every six weeks. In other embodiments, the activatable anti-CTLA4 antibody may be administered at a dose of 30 mg / kg once every three weeks or once every six weeks.

[0017] In some embodiments, the activatable anti-CTLA4 antibody is administered at a first higher dose (e.g., about 10 mg / kg to about 100 mg / kg) in at least one therapeutic cycle (as defined herein), followed by lower doses (e.g., about 3 mg / kg to about 20 mg / kg) in subsequent cycles. Herein, the higher initial dose is also referred to as the loading dose, and the subsequent doses are also referred to as the maintenance dose. As shown in the examples, at least one loading dose administration more rapidly establishes a steady-state plasma concentration of the activatable anti-CTLA4 antibody. In some embodiments, the loading dose of the activatable anti-CTLA4 antibody is administered in combination with pembrolizumab. In other embodiments, the loading dose of the activatable anti-CTLA4 antibody is not administered in combination with pembrolizumab. In some embodiments, pembrolizumab is administered in combination with a maintenance dose of the activatable anti-CTLA4 antibody, as described herein.

[0018] In one embodiment, the activatable anti-CTLA4 antibody is administered at a dose of approximately 20 mg / kg to approximately 50 mg / kg for at least one treatment cycle (e.g., 1 to 3 treatment cycles), and at a dose of approximately 6 mg / kg to approximately 20 mg / kg for subsequent treatment cycles (e.g., once every 3 weeks or once every 6 weeks). In another embodiment, the activatable anti-CTLA4 antibody is administered at a dose of approximately 20 mg / kg to approximately 50 mg / kg for at least one treatment cycle (e.g., 1 to 3 treatment cycles), and at a dose of approximately 10 mg / kg to approximately 20 mg / kg for subsequent treatment cycles (e.g., once every 3 weeks or once every 6 weeks). In one embodiment, the activatable anti-CTLA4 antibody is administered at a dose of approximately 20 mg / kg to approximately 50 mg / kg for at least one treatment cycle (e.g., 1 to 3 treatment cycles), and at a dose of approximately 6 mg / kg to approximately 10 mg / kg for subsequent treatment cycles (e.g., once every 3 weeks or once every 6 weeks). In one embodiment, the activatable anti-CTLA4 antibody is administered at a dose of approximately 20 mg / kg to approximately 50 mg / kg for at least one treatment cycle (e.g., 1 to 3 treatment cycles), and at a dose of approximately 6 mg / kg for subsequent treatment cycles. In one embodiment, the activatable anti-CTLA4 antibody is administered at a dose of approximately 20 mg / kg to approximately 50 mg / kg for at least one treatment cycle (e.g., 1 to 3 treatment cycles), and at a dose of approximately 10 mg / kg for subsequent treatment cycles (e.g., once every 3 weeks or once every 6 weeks).

[0019] In one embodiment, the activatable anti-CTLA4 antibody is administered at a dose of approximately 20 mg / kg to approximately 40 mg / kg for at least one treatment cycle (e.g., 1 to 3 treatment cycles), and at a dose of approximately 6 mg / kg to approximately 20 mg / kg for subsequent treatment cycles (e.g., once every 3 weeks or once every 6 weeks). In another embodiment, the activatable anti-CTLA4 antibody is administered at a dose of approximately 20 mg / kg to approximately 40 mg / kg for at least one treatment cycle (e.g., 1 to 3 treatment cycles), and at a dose of approximately 10 mg / kg to approximately 20 mg / kg for subsequent treatment cycles (e.g., once every 3 weeks or once every 6 weeks). In one embodiment, the activatable anti-CTLA4 antibody is administered at a dose of approximately 20 mg / kg to approximately 40 mg / kg for at least one treatment cycle (e.g., 1 to 3 treatment cycles), and at a dose of approximately 6 mg / kg to approximately 10 mg / kg for subsequent treatment cycles (e.g., once every 3 weeks or once every 6 weeks). In one embodiment, the activatable anti-CTLA4 antibody is administered at a dose of approximately 20 mg / kg to approximately 40 mg / kg for at least one treatment cycle (e.g., 1 to 3 treatment cycles), and at a dose of approximately 6 mg / kg for subsequent treatment cycles. In one embodiment, the activatable anti-CTLA4 antibody is administered at a dose of approximately 20 mg / kg to approximately 40 mg / kg for at least one treatment cycle (e.g., 1 to 3 treatment cycles), and at a dose of approximately 10 mg / kg for subsequent treatment cycles (e.g., once every 3 weeks or once every 6 weeks).

[0020] In one embodiment, the activatable anti-CTLA4 antibody is administered at a dose of approximately 20 mg / kg for at least one treatment cycle (e.g., 1 to 3 treatment cycles), and then at a dose of approximately 6 mg / kg in subsequent treatment cycles (e.g., once every 3 weeks). In another embodiment, the activatable anti-CTLA4 antibody is administered at a dose of approximately 20 mg / kg for one treatment cycle, and then at a dose of approximately 6 mg / kg in subsequent treatment cycles (e.g., once every 3 weeks). In yet another embodiment, the activatable anti-CTLA4 antibody is administered at a dose of approximately 20 mg / kg for two treatment cycles, and then at a dose of approximately 6 mg / kg in subsequent treatment cycles (e.g., once every 3 weeks). In one embodiment, the activatable anti-CTLA4 antibody is administered as a single dose of 20 mg / kg (loading dose), followed by a dose of 6 mg / kg (maintenance dose) three weeks later. In subsequent cycles, the maintenance dose (6 mg / kg) is administered once every 3 weeks. In another embodiment, the activatable anti-CTLA4 antibody is administered twice at a loading dose of 20 mg / kg every three weeks, followed by a maintenance dose of 6 mg / kg three weeks later. In subsequent cycles, the maintenance dose (6 mg / kg) is administered once every three weeks.

[0021] In one embodiment, the activatable anti-CTLA4 antibody is administered at a dose of approximately 20 mg / kg for at least one treatment cycle (e.g., 1 to 3 treatment cycles), and at a dose of approximately 10 mg / kg in subsequent treatment cycles (e.g., once every 3 weeks). In another embodiment, the activatable anti-CTLA4 antibody is administered at a dose of approximately 20 mg / kg for one treatment cycle, and at a dose of approximately 10 mg / kg in subsequent treatment cycles (e.g., once every 3 weeks). In yet another embodiment, the activatable anti-CTLA4 antibody is administered at a dose of approximately 20 mg / kg for two treatment cycles, and at a dose of approximately 10 mg / kg in subsequent treatment cycles (e.g., once every 3 weeks). In one embodiment, the activatable anti-CTLA4 antibody is administered as a single dose of 20 mg / kg (loading dose). Three weeks later, a maintenance dose of 10 mg / kg is administered. In subsequent cycles, the maintenance dose (10 mg / kg) is administered once every 3 weeks. In another embodiment, the activatable anti-CTLA4 antibody is administered twice at a loading dose of 20 mg / kg every three weeks, followed by a maintenance dose of 10 mg / kg three weeks later. In subsequent cycles, the maintenance dose (10 mg / kg) is administered once every three weeks.

[0022] In one embodiment, the activatable anti-CTLA4 antibody is administered at a dose of approximately 30 mg / kg to approximately 50 mg / kg (e.g., 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, or 50 mg / kg) over at least one treatment cycle (e.g., 1 to 3 treatment cycles), and at a dose of approximately 10 mg / kg in subsequent treatment cycles (e.g., once every 3 weeks). In one embodiment, the activatable anti-CTLA4 antibody is administered at a dose of approximately 30 mg / kg to approximately 50 mg / kg (e.g., 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, or 50 mg / kg) in one treatment cycle, and at a dose of approximately 10 mg / kg in subsequent treatment cycles (e.g., once every 3 weeks). In another embodiment, the activatable anti-CTLA4 antibody is administered in doses of approximately 30 mg / kg to approximately 50 mg / kg (e.g., 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, or 50 mg / kg) for two treatment cycles, and then in doses of approximately 10 mg / kg for subsequent treatment cycles (e.g., once every three weeks). In one embodiment, the activatable anti-CTLA4 antibody is administered as a single dose of approximately 30 mg / kg to approximately 50 mg / kg (e.g., 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, or 50 mg / kg) (loading dose), followed by a dose of 10 mg / kg (maintenance dose) three weeks later. In subsequent cycles, the maintenance dose (10 mg / kg) is administered once every three weeks. In another embodiment, the activatable anti-CTLA4 antibody is administered twice at a loading dose of approximately 30 mg / kg to approximately 50 mg / kg (e.g., 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, or 50 mg / kg) once every three weeks, followed by a maintenance dose of 10 mg / kg three weeks later. In subsequent cycles, the maintenance dose (10 mg / kg) is administered once every three weeks.

[0023] In some embodiments, pembrolizumab is administered once every three weeks in doses of approximately 100 mg to approximately 300 mg. In some such embodiments, both an activatable anti-CTLA4 antibody (e.g., TY22404) and pembrolizumab are administered once every three weeks.

[0024] In some embodiments, an activatable anti-CTLA4 antibody (e.g., TY22404) is administered at a dose of approximately 6 mg / kg to approximately 10 mg / kg once every 3 to 6 weeks, and pembrolizumab is administered at a dose of approximately 200 mg to approximately 600 mg once every 6 weeks. In some such embodiments, both the activatable anti-CTLA4 antibody (e.g., TY22404) and pembrolizumab are administered once every 6 weeks.

[0025] In some embodiments, an activatable anti-CTLA4 antibody (e.g., TY22404) is administered at a dose of 6 mg / kg once every 3 to 6 weeks, and pembrolizumab is administered at a dose of approximately 200 mg to approximately 600 mg once every 6 weeks. In some such embodiments, both the activatable anti-CTLA4 antibody (e.g., TY22404) and pembrolizumab are administered once every 6 weeks. In some such embodiments, the above-described dosing regimen is a maintenance dose and may be preceded by one or more loading doses of the activatable anti-CTLA4 antibody as described herein.

[0026] In some embodiments, an activatable anti-CTLA4 antibody (e.g., TY22404) is administered at a dose of 10 mg / kg once every 3 to 6 weeks, and pembrolizumab is administered at a dose of approximately 200 mg to approximately 600 mg once every 6 weeks. In some such embodiments, both the activatable anti-CTLA4 antibody (e.g., TY22404) and pembrolizumab are administered once every 6 weeks. In some such embodiments, the above-described dosing regimen is a maintenance dose and may be preceded by one or more loading doses of the activatable anti-CTLA4 antibody, as described herein.

[0027] In some embodiments, an activatable anti-CTLA4 antibody (e.g., TY22404) is administered at a dose of 20 mg / kg once every 3 to 6 weeks, and pembrolizumab is administered at a dose of about 200 mg to about 600 mg once every 6 weeks. In some such embodiments, both the activatable anti-CTLA4 antibody (e.g., TY22404) and pembrolizumab are administered once every 6 weeks. In some such embodiments, the aforementioned dosing schedule is a maintenance dose, and as described herein, administration of one or more loading doses of the activatable anti-CTLA4 antibody may precede.

[0028] In some embodiments, an activatable anti-CTLA4 antibody (e.g., TY22404) is administered at a dose of 30 mg / kg once every 3 to 6 weeks, and pembrolizumab is administered at a dose of about 200 mg to about 600 mg once every 6 weeks. In some such embodiments, both the activatable anti-CTLA4 antibody (e.g., TY22404) and pembrolizumab are administered once every 6 weeks. In some such embodiments, the aforementioned dosing schedule is a maintenance dose, and as described herein, administration of one or more loading doses of the activatable anti-CTLA4 antibody may precede.

[0029] In some embodiments, an activatable anti-CTLA4 antibody (e.g., TY22404) is administered at a dose of about 6 mg / kg to about 10 mg / kg once every 3 to 6 weeks, and pembrolizumab is administered at a dose of about 100 mg to about 300 mg once every 3 weeks.

[0030] In one embodiment, an activatable anti-CTLA4 antibody (e.g., TY22404) is administered to a subject (e.g., a human patient) at doses of approximately 1 mg / kg to approximately 30 mg / kg, approximately 6 mg / kg to approximately 10 mg / kg, approximately 10 mg / kg to approximately 20 mg / kg, or approximately 10 mg / kg to approximately 20 mg / kg. In some such embodiments, the activatable anti-CTLA4 antibody (e.g., TY22404) is administered to a subject (e.g., a human patient) at a dose of approximately 6 mg / kg. In some embodiments, the activatable anti-CTLA4 antibody (e.g., TY22404) is administered to a subject (e.g., a human patient) at a dose of approximately 10 mg / kg (e.g., 10 mg / kg). In some such embodiments, the activatable anti-CTLA4 antibody (e.g., TY22404) is administered to a subject (e.g., a human patient) once every three weeks. In other such embodiments, an activatable anti-CTLA4 antibody (e.g., TY22404) is administered to the subject (e.g., a human patient) once every six weeks. In some embodiments, an activatable anti-CTLA4 antibody (e.g., TY22404) is administered to the subject (e.g., a human patient) at a dose of approximately 20 mg / kg (e.g., 20 mg / kg). In some such embodiments, an activatable anti-CTLA4 antibody (e.g., TY22404) is administered to the subject (e.g., a human patient) once every three weeks. In other such embodiments, an activatable anti-CTLA4 antibody (e.g., TY22404) is administered to the subject (e.g., a human patient) once every six weeks. In some embodiments, an activatable anti-CTLA4 antibody (e.g., TY22404) is administered to the subject (e.g., a human patient) at a dose of approximately 25 mg / kg (e.g., 25 mg / kg). In some such embodiments, an activatable anti-CTLA4 antibody (e.g., TY22404) is administered to the subject (e.g., a human patient) once every three weeks. In other such embodiments, an activatable anti-CTLA4 antibody (e.g., TY22404) is administered to the subject (e.g., a human patient) once every six weeks. In some embodiments, an activatable anti-CTLA4 antibody (e.g., TY22404) is administered to the subject (e.g., a human patient) at a dose of approximately 30 mg / kg (e.g., 30 mg / kg).In some such embodiments, an activatable anti-CTLA4 antibody (e.g., TY22404) is administered to a subject (e.g., a human patient) once every three weeks. In other such embodiments, an activatable anti-CTLA4 antibody (e.g., TY22404) is administered to a subject (e.g., a human patient) once every six weeks. In any of the foregoing embodiments, pembrolizumab can be administered in combination with the activatable anti-CTLA4 antibody on the same day of a particular dosing schedule or on different days of a particular dosing schedule. In some embodiments, both the activatable anti-CTLA4 antibody and pembrolizumab are administered on the first day of a three-week or six-week dosing schedule. In some such embodiments, the foregoing dosing schedule is a maintenance dose, and as described herein, one or more loading doses of the activatable anti-CTLA4 antibody may precede it.

[0031] In some embodiments, pembrolizumab is administered once every three weeks at a dose of about 100 mg to about 300 mg. In some embodiments, pembrolizumab is administered once every three weeks at a dose of about 200 mg. In some such embodiments, the activatable anti-CTLA4 antibody is administered concurrently with pembrolizumab. For example, the activatable anti-CTLA4 antibody and pembrolizumab can each be administered to a subject (e.g., a human patient) that requires it on the first day of a three-week or six-week dosing schedule.

[0032] In some embodiments, an activatable anti-CTLA4 antibody (e.g., TY22404) and pembrolizumab are administered to a subject (e.g., a human patient) that requires it, where the activatable anti-CTLA4 antibody (e.g., TY22404) is administered at a dose of 6 mg / kg once every three weeks and pembrolizumab is administered at a dose of 200 mg once every three weeks. In some embodiments, the activatable anti-CTLA4 antibody and pembrolizumab are administered concurrently. In some such embodiments, the foregoing dosing schedule is a maintenance dose, and as described herein, one or more loading doses of the activatable anti-CTLA4 antibody may precede it.

[0033] In some embodiments, an activatable anti-CTLA4 antibody (e.g., TY22404) and pembrolizumab are administered to a subject requiring them (e.g., a human patient), where the activatable anti-CTLA4 antibody (e.g., TY22404) is administered at a dose of 10 mg / kg every three weeks, and pembrolizumab is administered at a dose of 200 mg every three weeks. In some embodiments, the activatable anti-CTLA4 antibody and pembrolizumab are administered simultaneously. In some such embodiments, the above-described dosing regimen is a maintenance dose, and may be preceded by one or more loading doses of the activatable anti-CTLA4 antibody, as described herein.

[0034] In some embodiments, an activatable anti-CTLA4 antibody (e.g., TY22404) and pembrolizumab are administered to a subject in need (e.g., a human patient), with the activatable anti-CTLA4 antibody (e.g., TY22404) administered at a dose of 20 mg / kg every three weeks, and pembrolizumab administered at a dose of 200 mg every three weeks. In some embodiments, the activatable anti-CTLA4 antibody and pembrolizumab are administered simultaneously. In some such embodiments, the above-described dosing regimen is a maintenance dose, and may be preceded by one or more loading doses of the activatable anti-CTLA4 antibody, as described herein.

[0035] In some embodiments, an activatable anti-CTLA4 antibody (e.g., TY22404) and pembrolizumab are administered once every six weeks to subjects requiring it (e.g., human patients). In some such embodiments, the activatable anti-CTLA4 antibody is administered in doses of approximately 3 mg / kg to approximately 20 mg / kg (e.g., 3 mg / kg, 6 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, or 30 mg / kg), and pembrolizumab is administered in doses of approximately 200 mg to approximately 400 mg (e.g., approximately 400 mg). In certain embodiments, an activatable anti-CTLA4 antibody and pembrolizumab are administered simultaneously. In some such embodiments, the aforementioned dosing regimen is a maintenance dose, and may be preceded by one or more loading doses of the activatable anti-CTLA4 antibody, as described herein.

[0036] In some embodiments, an activatable anti-CTLA4 antibody (e.g., TY22404) and pembrolizumab are administered to a subject requiring them (e.g., a human patient), where the activatable anti-CTLA4 antibody (e.g., TY22404) is administered at a dose of 6 mg / kg once every 6 weeks, and pembrolizumab is administered at a dose of 200 mg once every 6 weeks. In some embodiments, the activatable anti-CTLA4 antibody and pembrolizumab are administered simultaneously. In some such embodiments, the above-described dosing regimen is a maintenance dose, and may be preceded by one or more loading doses of the activatable anti-CTLA4 antibody, as described herein.

[0037] In some embodiments, an activatable anti-CTLA4 antibody (e.g., TY22404) and pembrolizumab are administered to a subject requiring them (e.g., a human patient), where the activatable anti-CTLA4 antibody (e.g., TY22404) is administered at a dose of 10 mg / kg once every six weeks, and pembrolizumab is administered at a dose of 200 mg once every six weeks. In some embodiments, the activatable anti-CTLA4 antibody and pembrolizumab are administered simultaneously. In some such embodiments, the above-described dosing regimen is a maintenance dose, and may be preceded by one or more loading doses of the activatable anti-CTLA4 antibody, as described herein.

[0038] In some embodiments, an activatable anti-CTLA4 antibody (e.g., TY22404) and pembrolizumab are administered to a subject in need (e.g., a human patient), where the activatable anti-CTLA4 antibody (e.g., TY22404) is administered at a dose of 20 mg / kg once every 6 weeks, and pembrolizumab is administered at a dose of 200 mg once every 6 weeks. In some embodiments, the activatable anti-CTLA4 antibody and pembrolizumab are administered simultaneously. In some such embodiments, the aforementioned administration schedule is a maintenance dose, and one or more loading doses of the activatable anti-CTLA4 antibody may be administered first, as described herein.

[0039] In some embodiments, an activatable anti-CTLA4 antibody (e.g., TY22404) and pembrolizumab are administered to a subject requiring them (e.g., a human patient), where the activatable anti-CTLA4 antibody (e.g., TY22404) is administered at a dose of 30 mg / kg once every six weeks, and pembrolizumab is administered at a dose of 200 mg once every six weeks. In some embodiments, the activatable anti-CTLA4 antibody and pembrolizumab are administered simultaneously. In some such embodiments, the above-described dosing regimen is a maintenance dose, and may be preceded by one or more loading doses of the activatable anti-CTLA4 antibody, as described herein.

[0040] In some embodiments following any one of the methods described above, the cancer is resistant or refractory to conventional therapy, and the conventional therapy is an inhibitor of CTLA4, PD-1, or a PD-1 ligand. In some embodiments, the subject (e.g., a human patient) is resistant to conventional therapy or has relapsed from conventional therapy, and the conventional therapy is an inhibitor of CTLA4, PD-1, or a PD-1 ligand. In some embodiments, the conventional therapy is a CTLA4 inhibitor such as ipilimumab. In some embodiments, the conventional therapy is a PD-1 inhibitor such as an anti-PD-1 antibody. In some embodiments, the conventional therapy is an inhibitor of a PD-1 ligand (e.g., PD-L1), such as an anti-PD-L1 antibody.

[0041] In some embodiments following any one of the methods described above, cancer is liver cancer, gastrointestinal cancer (e.g., colorectal cancer, colon cancer), lung cancer, bone cancer, heart cancer, brain cancer, kidney cancer, bladder cancer, blood cancer (e.g., leukemia), skin cancer, breast cancer, thyroid cancer, pancreatic cancer, head and / or neck cancer, eye-related cancer, male reproductive system cancer (e.g., prostate cancer, testicular cancer), or female reproductive system cancer (e.g., uterine cancer, cervical cancer). In some embodiments, cancer is a solid tumor. In some embodiments, cancer is pancreatic cancer. In some embodiments, cancer is advanced-stage cancer. In some embodiments, cancer is colorectal cancer. In some embodiments, cancer is cervical cancer. In some embodiments, cancer is neuroendocrine cancer. In some embodiments, cancer is endometrial cancer. In some embodiments, cancer is cecal cancer. In some embodiments, cancer is ovarian cancer. In one embodiment, cancers include, but are not limited to, colorectal cancer, gastric cancer, gastroesophageal junction cancer, esophageal cancer, endometrial cancer, or head and neck cancer. In another embodiment, cancers include, but are not limited to, melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCDC), head and neck squamous cell carcinoma (HNSCC), classical Hodgkin lymphoma (cHL), primary mediastinal large B-cell lymphoma (PMBCL), urothelial carcinoma, high microsatellite instability or mismatch repair deficiency cancer, high microsatellite instability or mismatch repair deficiency colorectal cancer, gastric cancer, esophageal cancer, cervical cancer, hepatocellular carcinoma (HCC), Merkel cell carcinoma (MCC), renal cell carcinoma (RCC), endometrial cancer, high tumor mutation burden (TMB-H) cancer, squamous cell carcinoma (cSCC), or triple-negative breast cancer (TNBC). The present invention relates to a method for treating cancer in a subject (e.g., a human patient) that requires such treatment, comprising administering a combination therapy comprising at least two pharmaceutical compositions to the subject (e.g., a human patient).

[0042] In some embodiments, both the activatable anti-CTLA4 antibody and pembrolizumab are administered intravenously. In some embodiments, the activatable anti-CTLA4 antibody is administered subcutaneously. In some embodiments, the activatable anti-CTLA4 antibody and pembrolizumab are administered intravenously or subcutaneously every three weeks. In some embodiments, the activatable anti-CTLA4 antibody and pembrolizumab are administered intravenously or subcutaneously every six weeks. In some embodiments, the subject (e.g., a human patient) receives at least four cycles of treatment with the activatable anti-CTLA4 antibody and pembrolizumab. In some embodiments, the subject (e.g., a human patient) receives further maintenance treatment (e.g., after four or more cycles), which includes administering an effective dose of the activatable anti-CTLA4 antibody to the subject (e.g., a human patient) at intervals ranging from approximately four weeks to approximately twelve weeks (e.g., every four, six, eight, ten, or twelve weeks). In some embodiments, doses of the activatable anti-CTLA4 antibody and pembrolizumab can be administered simultaneously. In other embodiments, the doses of the activatable anti-CTLA4 antibody and pembrolizumab may be administered at different times. For example, pembrolizumab may be administered approximately 0.5 to 5 hours before or after the administration of the activatable anti-CTLA4 antibody on day 1 of the administration schedule (e.g., a 3-week administration schedule).

[0043] In some embodiments following any one of the methods described above, the subject is a human.

[0044] It should be understood that some or all of the embodiments following any one of the methods described above may be combined to form other embodiments of the present application. These and other embodiments of the present application will be apparent to those skilled in the art. These and other embodiments of the present application are further described by the detailed description below. [Brief explanation of the drawing]

[0045] [Figure 1A-1B]This shows the response to the combination of the activatable CTLA4 antibody TY22404 and pembrolizumab. Figure 1A shows a swimmer plot of patients treated with TY22404 + pembrolizumab. At this data cut point, four patients were still receiving treatment. All patients had received at least two cycles of treatment. The longest treatment duration was eight cycles or more. Figure 1B shows a waterfall plot of patients (n=10) treated with TY22404 + pembrolizumab during dose escalation for whom target lesion measurements were available both at baseline and post-baseline. One patient (10 mg / kg, Q3W, PD due to new lesions) had not completed post-treatment target lesion measurements and still does not have them.

[0046] [Figure 2] This shows serum IFN-γ levels during treatment with TY22404 monotherapy or in combination with pembrolizumab. Serum IFN-γ was quantified using the V-Plex Proflammable Panel 1 from Mesoscale Discovery (MSD) Technologies.

[0047] [Figure 3A-3B] This shows a representative minimal physiological pharmacokinetic (mPBPK) model fitted to pharmacokinetic (PK) data (e.g., complete and cleaved plasma) of TY22404 observed at a dose of 10 mg / kg once every three weeks. Figure 3A shows the concentration of complete (uncleaved) antibody across three dosing schedules. Figure 3B shows the concentration of cleaved antibody across three dosing schedules.

[0048] [Figure 4] This shows the circulation of TY22404 after administration to various species.

[0049] [Figure 5] This shows a typical PK simulation (mean value including 95% CI) of TY22404 using mPBPK modeling at 10 mg / kg Q3W.

[0050] [Figure 6] This study shows that, at steady state (SS), the maximum cleavage TY22404 tumor interstitial fluid (ISF) concentration at 10 mg / kg Q3W administration is predicted to be significantly higher on average in the tumor microenvironment (TME) than at 3 mg / kg Q3W*4 or 1 mg / kg Q6W ipilimumab, respectively.

[0051] [Figure 7] This study shows that, at steady state (SS), a plasma or serum concentration of 10 mg / kg Q3W administration of truncated TY22404 is predicted to reduce active drug exposure in normal tissues compared to ipilimumab administration at 3 mg / kg Q3W*4 or 1 mg / kg Q6W.

[0052] [Figure 8] This shows the predicted drug concentrations of cleaved TY22404 in various dosing cycles.

[0053] [Figure 9] This paper presents PK modeling to predict the effects of a single loading dose and subsequent maintenance dose of TY22404. The model predicts that steady-state plasma concentrations can be achieved after a single dosing cycle.

[0054] [Figure 10] This diagram shows a mechanism-based safety model of TY22404 combined with an anti-PD1 antibody and ipilimumab. The vertical line represents the PD marker levels modeled from TY22404 in the specified dosing schedule on day 42 post-administration. TY22404 has been shown to have significantly fewer treatment-related side effects compared to ipilimumab when administered in combination with various anti-PD1 antibodies.

[0055] [Figure 11] TY22404 demonstrates the ability to overcome pembrolizumab resistance in patients with 3L cervical cervix.

[0056] [Figure 12] Compared to TY22404 administered at 10 mg / kg Q3W, the mPBPK model predicts greater variation in tumor cleavage PK and shows a decrease in cleavage AUC / Cmax.

[0057] [Figure 13] The mPBPK model predicts that the steady-state tumor cleavage PK exceeds the upper limit of in vitro EC90 (approximately 90 nM) for cleaved TY22404 (human T cell binding).

[0058] [Figure 14] This study demonstrates that the mPBPK model can adequately characterize the plasma and tumor PK of mice with tumors after a single dose of 10 mg / kg, enabling the estimation of tumor traction parameters for TY22404.

[0059] [Figure 15] This shows the mPBKB model-predicted mean plasma cleavage TY22404 concentrations (nM) over time with different dosing regimens using surrogate loading doses, compared to target concentrations for clinical efficacy based on population exposure response (ER) analysis.

[0060] [Figures 16A-16B] The mPBPK model predicts mean cleavage TY22404 concentrations over time in tumor interstitial fluid (ISF; Figure 16A) or leaky normal tissue (Figure 16B) under different administration regimens using alternative loading doses. [Modes for carrying out the invention]

[0061] I. Definition Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have meanings generally understood by those skilled in the art. Furthermore, unless otherwise required by context, singular terms shall include plural terms, and plural terms shall include singular terms. In general, the nomenclature and techniques used in connection with antibody manipulation, immunotherapy, cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry described herein are well known and commonly used in the art.

[0062] The term "antibody" is used in its broadest sense herein 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-strand variable fragments, i.e., scFv), as long as they exhibit the desired biological activity.

[0063] An "antibody fragment" or "antigen-binding fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Antibody fragments retain the ability to specifically bind to the antigen to which the full-length antibody binds. For example, a fragment may contain one or more CDR regions (e.g., all six CDRs). Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules formed from antibody fragments (e.g., scFv), and multispecific antibodies.

[0064] In some embodiments, the term “antibody” refers to an antigen-binding protein (i.e., immunoglobulin) having a basic four-polypeptide chain structure consisting of two identical heavy (H) chains and two identical light (L) chains. Each L chain is linked to an H chain by one disulfide covalent bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each heavy chain has a variable region (abbreviated herein as VH) at its N-terminus, followed by a constant region. The heavy chain constant region consists of three domains CH1, CH2, and CH3. Each light chain has a variable region (abbreviated herein as VI) at its N-terminus, followed by a constant region at its opposite end. The light chain constant region consists of one domain CL. VL aligns with VH, and CL aligns with the first constant domain (CH1) of the heavy chain. The pairing of VH and VL together forms a single antigen-binding site. IgM antibodies consist of five basic heterotetrameric units and an additional polypeptide called a J chain, and thus contain 10 antigen-binding sites. However, secreted IgA antibodies can polymerize to form a multivalent aggregate containing 2 to 5 basic tetrameric units and a J chain.

[0065] The VH and VL regions can be further subdivided into hypervariable regions called hypervariable regions (HVRs) based on structural and sequence analysis. The HVRs are flanked by more conserved regions called framework regions (FWs) (see, for example, Chenetal. (1999) J. Mol. Biol. (1999) 293, 865-881). Each VH and VL consists of three HVRs and four FWs, arranged in the order FW-1_HVR-1_FW-2_HVR-2_FW-3_HVR-3_FW4 from the amino terminus to the carboxyl terminus. Throughout this application, the three HVRs of the heavy chain are referred to as HVR-H1, HVR-H2, and HVR-H3. Similarly, the three HVRs of the light chain are referred to as HVR-L1, HVR-L2, and HVR-L3.

[0066] As used herein, the terms “CDR” or “CDRs” are intended to mean complementarity-determining regions within the immunoglobulin variable region, or discontinuous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. Unless otherwise specified herein, CDRs are defined using the Kabat numbering system. For example, Kabat et al.,J.Biol.Chem.252:6609-6616(1977);Kabat et al.,USDept.of Health and Human Services,“Sequences of proteins of immunological interest”(1991);Chothia et al.,J.Mol.Biol.196:901-917(1987);Al-Lazikani B.et al., J.Mol.Biol.,273:927-948(1997);MacCallum et al., J.Mol.Biol.262:732-745(1996);Abhinandan and Martin, 45:3832-3839(2008);Lefranc MPet al.,Dev. Comp. Immunol., 27:55-77 (2003); and Honegger and Pluckthun, J. As described in Mol. Biol., 309:657-670 (2001), the definition includes duplication or subsets of amino acid residues when compared to one another. Nevertheless, the application of any definition to refer to the CDR of an antibody or graft antibody or its variant is intended to be within the scope of the terms defined and used herein.For example, the contents of the references cited in this paragraph, in which CDR prediction algorithms and interfaces are known in the art, including Mol.Immunol.,45:3832-3839(2008);Ehrenmann F. et al.,Nucleic Acids Res.,38:D301-D307(2010);andAdolf-Bryfogle J. et al.,Nucleic Acids Res.,43:D432-D438(2015), are incorporated herein by reference in their entirety for use in this application and for the possibility of being included in one or more claims herein.

[0067] Each variable region of the heavy and light chains contains a binding domain that interacts with the antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to various cells of the immune system (e.g., effector cells) and to host tissues or factors, including the first component (C1q) of the classical complement system. In the light and heavy chains, the variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 10 or more amino acids (see, for example, Fundamental Immunology Ch.7 (Paul, W., ed., 2nd ed. Raven Press, NY) (1989)).

[0068] Light chains (L chains) derived from any vertebrate species can be assigned to one of two distinct types, called kappa and lambda, based on the amino acid sequence of their constant domains. Antibodies can be assigned to different classes or isotypes depending on the amino acid sequence of the constant domain of their heavy chain (CH). There are five classes of antibodies: IgA, IgD, IgE, IgG, and IgM, each having heavy chains named α (alpha), δ (delta), ε (epsilon), γ (gamma), and μ (mu), respectively. The IgG class of antibodies can be further classified into four subclasses, IgG1, IgG2, IgG3, and IgG4, based on the gamma heavy chains Y1-Y4.

[0069] The term "CTLA4" is used herein and includes human CTLA4 (e.g., UniProt accession number P16410), as well as its variants, isoforms, and species homologs (e.g., mouse CTLA4 (UniProt accession number P09793), rat CTLA4 (UniProt accession number Q9Z1A7), canine CTLA4 (UniProt accession number Q9XSI1), cynomolgus monkey CTLA4 (UniProt accession number G7PL88), etc.). Therefore, anti-CTLA4 antibodies as defined and disclosed herein may also bind to CTLA4 of non-human species. In other cases, anti-CTLA4 antibodies may be perfectly specific to human CTLA4 and may not exhibit interspecies cross-reactivity or other types of cross-reactivity.

[0070] The term "CTLA4 antibody" refers to an antibody capable of binding to human CTLA4, as defined herein.

[0071] As used herein, “monoclonal antibody,” “mAb,” or “Mab” refers to a substantially homogeneous population of antibodies, i.e., the antibody molecules constituting the population have identical amino acid sequences, apart from any potentially trace amounts of spontaneously occurring mutations. In contrast, conventional (polyclonal) antibody preparations typically contain a number of different antibodies with different amino acid sequences in their variable domains, particularly the CDR, and are often specific to different epitopes. The modifier “monoclonal” indicates a characteristic of antibodies that would be obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring antibody production by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be produced by the hybridoma method first described by Kohler et al. (1975) Nature 256: 495, or by the recombinant DNA method (see, for example, U.S. Patent No. 4,816,567). Monoclonal antibodies may also be isolated from phage antibody libraries using techniques described in Clackson et al. (1991) Nature 352: 624-628 and Marks et al. (1991) J. Mol. Biol. 222: 581-597, for example. See also Presta (2005) J. Allergy Clin. Immunol. 116:731.

[0072] A "PD-1 antagonist" means any chemical compound or biological molecule that inhibits the binding of PD-L1, expressed on cancer cells, to PD-1, expressed on immune cells (T cells, B cells, or natural killer T cells). In specific embodiments, it also blocks the binding of PD-L2, expressed on cancer cells, to immune cells that express PD-1. Other names or synonyms for PD-1 and its ligands include PDCD1, PD1, CD279, SLEB2 for PD-1; PDCD1L1, PDL1, B7H1, B7-4, CD274, B7-H for PD-L1; and PDCD1L2, PDL2, B7-DC, Btdc, CD273 for PD-L2. In any of the therapeutic methods, agents, and uses of the present invention for treating a human individual, a PD-1 antagonist inhibits the binding of human PD-L1 to human PD-1, and in specific embodiments, inhibits the binding of both human PD-L1 and PD-L2 to human PD-1. The amino acid sequence for human PD-1 is located at NCBI Locus No.: NP_005009. The amino acid sequences for human PD-L1 and PD-L2 are located at NCBI Locus No.: NP_054862 and NP_079515, respectively.

[0073] "Pembrolizumab" (formerly known as MK-3475, SCH900475, and lambrolizumab), as used herein, is a humanized IgG4 mAb having the structure described in WHO Drug Information, Vol. 27, No. 2, pages 161-162 (2013), and includes the heavy-chain amino acid sequence, light-chain amino acid sequence, and CDR described in Table B. Pembrolizumab is approved by the U.S. FDA as described in the prescribing information of KEYTRUDA® (Merck Sharp & Dohme LLC, Rahway, NJ, USA, first approved in the U.S.: 2014, updated: March 2021).

[0074] In this specification, "pembrolizumab variant" or "its variant" with respect to the pembrolizumab sequence means a monoclonal antibody containing heavy and light chain sequences substantially identical to those of pembrolizumab, except that it has three, two, or one conserved amino acid substitutions located outside the light chain CDR and six, five, four, three, two, or one conserved amino acid substitutions located outside the heavy chain CDR. For example, the mutation site is located in the FR region or constant region, and optionally a C-terminal lysine residue of the heavy chain is deleted. In other words, pembrolizumab and pembrolizumab variants consist of the same CDR sequence, but they differ from each other because they have three or six or fewer conserved amino acid substitutions at other positions within their full-length light and heavy chain sequences. The pembrolizumab variant is substantially identical to pembrolizumab in terms of its binding affinity to PD-1, and its ability to inhibit the binding of PD-L1 and PD-L2 to PD-1, respectively.

[0075] The term "epitope" refers to the portion of an antigen to which an antibody (or its antigen-binding fragment) binds. Epitopes can be formed from both adjacent amino acids or non-adjacent amino acids that are paralleled by the three-dimensional folding of the protein. Epitopes formed from adjacent amino acids are typically retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes can contain a variety of amino acids in a unique spatial conformation. Methods for determining the spatial conformation of an epitope include, for example, X-ray crystallography, two-dimensional nuclear magnetic resonance, deuterium and hydrogen exchange combined with mass spectrometry, or site-directed mutagenesis, or all methods used in combination with computational models of the complex structure of the antigen and its bound antibody and its variants (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GEMorris, Ed. (1996)). Once the desired epitope of an antigen is determined, an antibody against that epitope can be generated, for example, using the techniques described herein. Furthermore, antibody generation and characterization can elucidate information about the desired epitope. From this information, it is possible to competitively screen antibodies for binding to the same epitope. An approach to achieve this is to conduct cross-competition studies to discover antibodies that bind competitively to each other, i.e., antibodies compete for binding to the antigen. A high-throughput process for "binning" antibodies based on cross-competition is described in PCT Publication WO03 / 48731.

[0076] An “isolated” antibody is an antibody that has been separated from its natural environment. In some embodiments, the antibody is purified to a purity of 95% or greater than 99%, as determined, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). For an overview of methods for evaluating antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0077] As used herein, “sequence identity” between two polypeptide sequences refers to the percentage of amino acids that are identical between the sequences. The amino acid sequence identity of polypeptides can conventionally be determined using known computer programs such as Bestfit, FASTA, or BLAST (see, for example, Pearson, Methods Enzymol. 183:63-98 (1990), Pearson, Methods Mol. Biol. 132:185-219 (2000), Altschul et al., J. Mol. Biol. 215:403-410 (1990), and Altschul 25:3389-3402 (1997)). When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for example, 95% identical to a reference amino acid sequence, the parameters are set such that the percentage of identity is calculated over the entire length of the reference amino acid sequence, and a difference in homology of up to 5% of the total number of amino acid residues in the reference sequence is permitted. This aforementioned method for determining the percentage of identity between polypeptides is applicable to all proteins, fragments, or variants thereof disclosed herein.

[0078] As used herein, the terms “binding,” “binding,” “specifically binding to,” or “specific to” refer to measurable and reproducible interactions, such as binding between a target and an antibody, which determines the presence of the target in the presence of a heterogeneous population of molecules, including biological molecules. For example, an antibody that binds to a target (which may be an epitope), or specifically binds to it, is an antibody that binds to this target more easily and / or for a longer period of time, with higher affinity or binding strength, than an antibody that binds to other targets. In one embodiment, the degree of antibody binding to an unrelated target is less than about 10% of the antibody binding to the target, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that specifically binds to a target has a dissociation constant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, or ≤0.1 nM. In certain embodiments, the antibody specifically binds to an epitope on a protein that is conserved between proteins of different species. In another embodiment, specific binding may include, but does not require, exclusive binding. An antibody that "specifically binds" to a particular target protein is an antibody that shows preferential binding to its target compared to other proteins, but this specificity does not require absolute binding specificity. An antibody is considered "specific" to its intended target if its binding is conclusive in determining the presence of the target protein in the sample (for example, if no undesirable results such as false positives occur). In this invention, the antibody or its binding fragment useful binds to the target protein with an affinity at least twice, preferably at least 10 times, more preferably at least 20 times, and most preferably at least 100 times greater than its affinity to the non-target protein. In this specification, an antibody is said to specifically bind to a polypeptide containing a given amino acid sequence, for example, the amino acid sequence of a mature human PD-1 or human PD-L1 molecule. This applies when the antibody binds to a polypeptide containing that sequence but does not bind to a protein lacking that sequence.

[0079] The terms “to treat,” “to treat,” or “treatment” refer to producing a desirable or beneficial effect in a mammal with respect to a particular disease condition. Such desirable or beneficial effects may include a reduction in the frequency or severity of one or more symptoms of the disease (i.e., tumor growth and / or metastasis, or other effects mediated by the number and / or activity of immune cells), or the suppression or inhibition of further manifestation of the disease, condition, or disorder. With respect to treating cancer in a mammal, such desirable or beneficial effects may include inhibition of further growth or spread of cancer cells, death of cancer cells, inhibition of cancer recurrence, reduction of cancer-related pain, or improvement in the survival rate of the mammal. Such effects may be subjective or objective. For example, if the mammal is a human, the human may recognize improved vitality or survivability, or reduced pain, as subjective symptoms of improvement or treatment response. Alternatively, a clinician may recognize a reduction in tumor size or tumor volume based on physical examination, laboratory values, tumor markers, or radiographic findings. Some clinical laboratory indicators that clinicians may observe regarding the response to treatment include the normalization of laboratory results such as white blood cell count, red blood cell count, platelet count, erythrocyte sedimentation rate, and levels of various enzymes. In addition, clinicians may observe a decrease in detectable tumor markers. Alternatively, other tests such as sonograms, magnetic resonance imaging, and positron emission tomography can be used to assess objective improvement.

[0080] The terms "prevent" or "prevent" refer to preventing or delaying the onset of a particular disease in mammals, or preventing the manifestation of its clinical or subclinical symptoms.

[0081] As used herein, “subject,” “patient,” or “individual” may refer to a human or a non-human animal. “Non-human animal” may refer to any animal not classified as human, such as a domesticated animal, livestock or zoo animal, competition animal, pet animal (dog, horse, cat, cow, etc.), and animal used in research. Animals used in research 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 macaques, crab-eating macaques, chimpanzees, etc.). In some embodiments, the subject, patient, or individual is human.

[0082] "Effective dose" refers to the dose and duration required to achieve one or more desired or demonstrated effects, including therapeutic or preventive outcomes, and is at least an effective amount. An effective dose can be delivered in one or more doses. For the purposes of this application, an effective dose of an antibody, drug, compound, or pharmaceutical composition is an amount sufficient to directly or indirectly provide a preventive or therapeutic treatment. As understood in clinical contexts, an effective dose of a drug, compound, or pharmaceutical composition may or may not be achieved in combination with another drug, compound, or pharmaceutical composition (e.g., an effective dose administered as monotherapy or combination therapy). That is, an "effective dose" may be considered in the context of administering one or more therapeutic agents, or a single agent may be considered to be administered in an effective dose if, in combination with one or more other agents, the desired outcome is possible or has been achieved.

[0083] The terms "recurrence," "relapse," or "relapsed" refer to the return of cancer or disease after a clinical assessment of disease clearance. A diagnosis of distant metastasis or local recurrence can be considered a recurrence.

[0084] The terms "refractory" or "resistant" refer to cancer or disease that has not responded to treatment.

[0085] As used herein, “complete response” or “CR” means the disappearance of all target lesions; “partial response” or “PR” means a total reduction of at least 30% in the longest diameter (SLD) of target lesions relative to the baseline SLD; and “stable” or “SD” means that, relative to the smallest SLD since the start of treatment, the shrinkage of target lesions is not sufficient to constitute a PR, or the enlargement is not sufficient to constitute a PD.

[0086] As used herein, “disease progression” or “PD” means that the SLD of the target lesion has increased by at least 20% relative to the lowest SLD recorded since the start of treatment, or that one or more new lesions have been present.

[0087] As used herein, “progression-free survival” (PFS) refers to the length of time during or after treatment during which the treated disease (e.g., cancer) does not worsen. Progression-free survival may include the amount of time the patient experienced complete or partial response, and the amount of time the patient experienced stability.

[0088] As used herein, "overall response rate" (ORR) refers to the sum of the complete response (CR) rate and the partial response (PR) rate.

[0089] As used herein, “overall survival” refers to the percentage of individuals in a group that are likely to survive after a specific period of time.

[0090] As used herein, “baseline level” or “baseline value” refers to the level or value of a subject (e.g., a human patient) before initiating treatment such as therapy with an anti-CTLA4 antibody.

[0091] As used herein, “reference sample,” “reference cell,” “reference tissue,” “control sample,” “control cell,” or “control tissue” refers to a sample, cell, tissue, standard, or level used for comparative purposes. In one embodiment, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy and / or unaffected portion (e.g., tissue or cell) of the body of the same subject or individual. For example, healthy and / or unaffected cells or tissue adjacent to affected cells or tissue (e.g., cells or tissue adjacent to a tumor). In another embodiment, the reference sample is obtained from untreated tissue and / or cells of the body of the same subject or individual. In yet another embodiment, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy and / or unaffected portion (e.g., tissue or cell) of the body of an individual other than the subject or individual. In yet another embodiment, the reference sample, reference cells, reference tissue, control sample, control cells, or control tissue is obtained from untreated tissue and / or cells of the body of an individual that is not the subject or individual.

[0092] A patient’s “effective response” or “responsiveness” to medical treatment, and similar expressions, refer to the clinical or therapeutic benefit received by a patient who is at risk of or has a disease or disorder such as cancer. In one embodiment, such benefit may include one or more of the following: extended survival (including overall survival and progression-free survival), objective response (including complete response or partial response), or improvement of signs or symptoms of cancer.

[0093] A patient who "does not show an effective response" to treatment is a patient who does not show any of the following: an extension of survival (including overall survival and progression-free survival), an objective response (including complete or partial response), or improvement in signs or symptoms of cancer.

[0094] The methods and techniques described herein are generally carried out in accordance with methods well known in the art, unless otherwise indicated, and as described in the various general and more specific references cited and discussed throughout this specification. Such references include, for example, Sambrook and Russell, Molecular Cloning, Alaboratory Approach, Cold Spring Harbor Press, Cold Spring Harbor, NY (2001); Ausubeletal, Current Protocols in Molecular Biology, John Wiley & Sons, NY (2002); and Harlow and Lane Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1990). Enzymatic reactions and purification techniques can be carried out in accordance with the manufacturer's specifications, as is generally done in the art, or as described herein. The nomenclature used in relation to analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein, as well as the experimental procedures and methods thereof, are well known and commonly used in the art. Standard methods are used for chemical synthesis, preparation, formulation, and delivery of pharmaceuticals, as well as for the treatment of patients.

[0095] In this specification, the 20 conventional amino acids and their abbreviations follow conventional usage. See Immunology-ASynthesis (2nd Edition, E.S. Goluband, D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)).

[0096] As used herein, the singular forms preceded by "a," "an," and "the" include multiple references unless the context clearly indicates otherwise. For example, when referring to a "molecule," it may optionally include two or more such molecules combined together.

[0097] As used herein, the term “approximately” refers to the normal margin of error for each value, as would be readily understood by those skilled in the art. Whereever a value or parameter is referred to herein with “approximately,” embodiments relating to that value or parameter itself are included (and described).

[0098] It should be understood that the aspects and embodiments of the Application described herein include "including," "consisting of," and "essentially consisting of."

[0099] The term "approximately X to Y" as used herein has the same meaning as "approximately X to approximately Y".

[0100] As used herein, the term "and / or" is intended to include, in expressions such as "A and / or B," both A and B, A or B, A (alone), and B (alone). Similarly, as used herein, the term "and / or" is intended to include, in expressions such as "A, B and / or C," each of the 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).

[0101] References to “several embodiments,” “embodiment,” “one embodiment,” or “other embodiments” in this specification mean that certain features, structures, or properties described in relation to the embodiments are included in at least some embodiments of the present invention, but not necessarily in all embodiments. II. Treatment Method

[0102] This application provides a method for treating cancer in a subject (e.g., a human patient) using the activatable anti-CTLA4 antibody of the present invention. Any one of the anti-CTLA4 antibodies (including the full-length antibody and the antigen-binding fragment) in Section III, “Anti-CTLA4 Antibodies” can be used in the method described herein.

[0103] In some embodiments, a method is provided for treating cancer in a subject (e.g., a human patient) that is resistant or refractory to inhibitors of CTLA4, PD-1, or a PD-1 ligand (PD-L1 or PD-L2), comprising administering to the subject (e.g., a human patient) an effective amount of an activatable anti-CTLA4 antibody in combination with pembrolizumab, wherein the antibody comprises (a) a heavy chain variable region comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO: 23, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 35, and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 45, and / or a light chain variable region comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO: 58, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 66, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 75. In some embodiments, the cancer is resistant or refractory to an anti-PD-1 antibody. In some embodiments, the cancer is resistant or refractory to a different anti-CTLA4 antibody, such as ipilimumab. In some embodiments, the cancer is resistant or refractory to the anti-PD-L1 antibody. In some embodiments, the cancer is a solid tumor such as advanced-stage cancer and / or metastatic cancer. In some embodiments, the antibody includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 87 or an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 87, and / or a light chain variable region containing the amino acid sequence of SEQ ID NO: 100 or an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 100. In some embodiments, the antibody includes the Fc region of wild-type IgG1 or the Fc region of human IgG1 such as a variant with enhanced ADCC activity.

[0104] In some embodiments, a method is provided for treating cancer in a subject (e.g., a human patient), comprising administering an effective amount of the anti-CTLA4 antibody disclosed herein in combination with pembrolizumab to the subject (e.g., a human patient), wherein the activatable anti-CTLA4 antibody is administered in doses of approximately 3 mg / kg to approximately 20 mg / kg. In some embodiments, the activatable anti-CTLA4 is administered in doses of approximately 3 mg / kg. In some embodiments, the activatable anti-CTLA4 is administered in doses of approximately 5 mg / kg. In some embodiments, the activatable anti-CTLA4 is administered in doses of approximately 6 mg / kg. In some embodiments, the activatable anti-CTLA4 is administered in doses of approximately 8 mg / kg. In some embodiments, the activatable anti-CTLA4 is administered in doses of approximately 10 mg / kg. In some embodiments, the activatable anti-CTLA4 is administered in doses of approximately 20 mg / kg. In some embodiments, the activatable anti-CTLA4 is administered in doses of approximately 25 mg / kg. In some embodiments, the activatable anti-CTLA4 antibody is administered at a dose of approximately 30 mg / kg. In some of the embodiments described above, the activatable anti-CTLA4 antibody and pembrolizumab are administered once every three weeks. In some of the embodiments described above, the activatable anti-CTLA4 antibody and pembrolizumab are administered once every six weeks. In some of the embodiments described above, the activatable anti-CTLA4 antibody and pembrolizumab are administered over at least five treatment cycles (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20).

[0105] In one embodiment, the present disclosure provides a method for treating cancer in a subject (e.g., a human patient), the method comprising administering to the subject (e.g., a human patient) an effective amount of the above-mentioned activatable anti-CTLA4 antibody (e.g., TY22404) in combination with pembrolizumab, wherein the activatable anti-CTLA4 antibody is administered in a dose of about 3 mg / kg to about 30 mg / kg. In some embodiments, the activatable anti-CTLA4 antibody (e.g., TY22404) is administered in a dose of about 5 mg / kg to about 10 mg / kg. In some embodiments, the activatable anti-CTLA4 antibody (e.g., TY22404) is administered in a dose of about 10 mg / kg to about 20 mg / kg. In some embodiments, the activatable anti-CTLA4 antibody (e.g., TY22404) is administered in a dose of about 20 mg / kg to about 30 mg / kg. In some embodiments, the activatable anti-CTLA4 antibody (e.g., TY22404) is administered at a dose of approximately 3 mg / kg. In some embodiments, the activatable anti-CTLA4 antibody (e.g., TY22404) is administered at a dose of approximately 5 mg / kg. In some embodiments, the activatable anti-CTLA4 antibody (e.g., TY22404) is administered at a dose of approximately 6 mg / kg. In some embodiments, the activatable anti-CTLA4 antibody (e.g., TY22404) is administered at a dose of approximately 8 mg / kg. In some embodiments, the activatable anti-CTLA4 antibody (e.g., TY22404) is administered at a dose of approximately 10 mg / kg. In some embodiments, the activatable anti-CTLA4 antibody (e.g., TY22404) is administered at a dose of approximately 8 mg / kg. In some embodiments, the activatable anti-CTLA4 antibody (e.g., TY22404) is administered at a dose of approximately 20 mg / kg. In some embodiments, the activatable anti-CTLA4 antibody (e.g., TY22404) is administered at a dose of approximately 25 mg / kg. In some embodiments, the activatable anti-CTLA4 antibody (e.g., TY22404) is administered at a dose of approximately 30 mg / kg. In any of the embodiments described above, the activatable anti-CTLA4 antibody can be administered once every three weeks or once every six weeks.For example, in some embodiments, the activatable anti-CTLA4 antibody can be administered at a dose of 10 mg / kg once every three weeks or once every six weeks. In other embodiments, the activatable anti-CTLA4 antibody can be administered at a dose of 20 mg / kg once every three weeks or once every six weeks. In yet another embodiment, the activatable anti-CTLA4 antibody can be administered at a dose of 30 mg / kg once every three weeks or once every six weeks.

[0106] In some embodiments, the activatable anti-CTLA4 antibody is administered in a first higher dose (e.g., about 10 mg / kg to about 100 mg / kg) in at least one therapeutic cycle (as defined herein), followed by lower doses (e.g., about 3 mg / kg to about 20 mg / kg) in subsequent cycles. Herein, the higher initial dose is also referred to as the loading dose, and the subsequent doses are also referred to as the maintenance dose. In some embodiments, the loading dose of the activatable anti-CTLA4 antibody is administered in combination with pembrolizumab. In other embodiments, the loading dose of the activatable anti-CTLA4 antibody is not administered in combination with pembrolizumab. In such embodiments, pembrolizumab is administered in combination with the maintenance dose of the activatable anti-CTLA4 antibody, as described herein.

[0107] In one embodiment, the activatable anti-CTLA4 antibody is administered at a dose of approximately 20 mg / kg in at least one treatment cycle (e.g., 1 to 3 treatment cycles), followed by doses of approximately 5 to 10 mg / kg or 6 to 8 mg / kg in subsequent treatment cycles (e.g., once every 3 weeks). In one embodiment, the activatable anti-CTLA4 antibody is administered at a dose of approximately 20 mg / kg in at least one treatment cycle (e.g., 1 to 3 treatment cycles), followed by doses of approximately 6 mg / kg in subsequent treatment cycles (e.g., once every 3 weeks). In one embodiment, the activatable anti-CTLA4 antibody is administered at a dose of approximately 20 mg / kg in at least one treatment cycle (e.g., 1 to 3 treatment cycles), followed by doses of approximately 10 mg / kg in subsequent treatment cycles (e.g., once every 3 weeks).

[0108] In one embodiment, the activatable anti-CTLA4 antibody is administered at a dose of approximately 10 mg / kg in one treatment cycle, followed by a dose of approximately 6 mg / kg in subsequent treatment cycles (e.g., once every 3 weeks or once every 6 weeks). In another embodiment, the activatable anti-CTLA4 antibody is administered at a dose of approximately 10 mg / kg in two treatment cycles, followed by a dose of approximately 6 mg / kg in subsequent treatment cycles (e.g., once every 3 weeks or once every 6 weeks).

[0109] In one embodiment, the activatable anti-CTLA4 antibody is administered at a dose of approximately 20 mg / kg in one treatment cycle, followed by a dose of approximately 6 mg / kg in subsequent treatment cycles (e.g., once every 3 weeks or once every 6 weeks). In another embodiment, the activatable anti-CTLA4 antibody is administered at a dose of approximately 20 mg / kg in one treatment cycle, followed by a dose of approximately 10 mg / kg in subsequent treatment cycles (e.g., once every 3 weeks or once every 6 weeks).

[0110] In another embodiment, the activatable anti-CTLA4 antibody is administered at a dose of approximately 20 mg / kg over two treatment cycles, followed by a dose of approximately 6 mg / kg in subsequent treatment cycles (e.g., every three weeks or every six weeks). In yet another embodiment, the activatable anti-CTLA4 antibody is administered at a dose of approximately 20 mg / kg over two treatment cycles, followed by a dose of approximately 10 mg / kg in subsequent treatment cycles (e.g., every three weeks or every six weeks).

[0111] In one embodiment, an activatable anti-CTLA4 antibody is administered as a single dose of 20 mg / kg (loading dose), followed by a dose of 6 mg / kg (maintenance dose) three weeks later. In subsequent cycles, the maintenance dose (e.g., 6 mg / kg) is administered once every three weeks. In another embodiment, the activatable anti-CTLA4 antibody is administered twice at a loading dose of 20 mg / kg every three weeks, followed by a maintenance dose of 6 mg / kg three weeks later. In subsequent cycles, the maintenance dose (6 mg / kg) is administered once every three weeks.

[0112] In one embodiment, the activatable anti-CTLA4 antibody is administered as a single dose of 20 mg / kg (loading dose), followed by a dose of 10 mg / kg (maintenance dose) three weeks later. In subsequent cycles, the maintenance dose (e.g., 10 mg / kg) is administered once every three weeks. In another embodiment, the activatable anti-CTLA4 antibody is administered twice at doses of 20 mg / kg (loading dose) every three weeks, followed by a dose of 10 mg / kg (maintenance dose) three weeks later. In subsequent cycles, the maintenance dose (10 mg / kg) is administered once every three weeks.

[0113] In some embodiments, the activatable anti-CTLA4 antibody is administered at a dose of approximately 20 mg / kg in one treatment cycle, and then at a dose of approximately 5 mg / kg to approximately 10 mg / kg in subsequent treatment cycles (e.g., every 3 weeks or every 6 weeks). For example, in one embodiment, the activatable anti-CTLA4 antibody is administered at an initial (loading) dose of 20 mg / kg, followed by additional doses (maintenance doses) of approximately 5 mg / kg to approximately 10 mg / kg every 3 weeks. In some embodiments, the activatable anti-CTLA4 antibody is administered at a dose of approximately 20 mg / kg in two treatment cycles, and then at a dose of approximately 5 mg / kg to approximately 10 mg / kg in subsequent treatment cycles (e.g., every 3 weeks). In one embodiment, an activatable anti-CTLA4 antibody is administered at an initial (loading) dose of 20 mg / kg, followed by another loading dose of 20 mg / kg three weeks later, and then additional doses (maintenance doses) of approximately 5 mg / kg to 10 mg / kg every three weeks thereafter.

[0114] In some embodiments, the activatable anti-CTLA4 antibody is administered at a dose of approximately 20 mg / kg in one treatment cycle, and then at a dose of approximately 6 mg / kg in subsequent treatment cycles (e.g., once every 3 weeks or once every 6 weeks). For example, in one embodiment, the activatable anti-CTLA4 antibody is administered at an initial (loading) dose of 6 mg / kg, followed by an additional dose (maintenance dose) of 6 mg / kg every 3 weeks. In some embodiments, the activatable anti-CTLA4 antibody is administered at a dose of approximately 20 mg / kg in two treatment cycles, and then at a dose of approximately 6 mg / kg in subsequent treatment cycles. In one embodiment, the activatable anti-CTLA4 antibody is administered at an initial (loading) dose of 20 mg / kg, followed by another loading dose of 20 mg / kg after 3 weeks, followed by an additional dose (maintenance dose) of 6 mg / kg every 3 weeks.

[0115] In some embodiments, the activatable anti-CTLA4 antibody is administered at a dose of approximately 20 mg / kg in one treatment cycle, and then at a dose of approximately 10 mg / kg in subsequent treatment cycles (e.g., every 3 weeks or every 6 weeks). For example, in one embodiment, the activatable anti-CTLA4 antibody is administered at an initial (loading) dose of 20 mg / kg, followed by an additional dose (maintenance dose) of 10 mg / kg every 3 weeks. In some embodiments, the activatable anti-CTLA4 antibody is administered at a dose of approximately 20 mg / kg in two treatment cycles, and then at a dose of approximately 10 mg / kg in subsequent treatment cycles (e.g., every 3 weeks). In one embodiment, the activatable anti-CTLA4 antibody is administered at an initial (loading) dose of 20 mg / kg, followed by another loading dose of 20 mg / kg after 3 weeks, followed by an additional dose (maintenance dose) of 10 mg / kg every 3 weeks.

[0116] In one embodiment, the activatable anti-CTLA4 antibody is administered at a dose of approximately 30 mg / kg to approximately 50 mg / kg (e.g., 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, or 50 mg / kg) over at least one treatment cycle (e.g., 1 to 3 treatment cycles), and then at a dose of approximately 5 mg / kg in subsequent treatment cycles (e.g., once every 3 weeks or once every 6 weeks). In another embodiment, the activatable anti-CTLA4 antibody is administered at a dose of approximately 30 mg / kg to approximately 50 mg / kg (e.g., 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, or 50 mg / kg) over at least one treatment cycle (e.g., 1 to 3 treatment cycles), and then at a dose of approximately 6 mg / kg in subsequent treatment cycles (e.g., once every 3 weeks or once every 6 weeks). In one embodiment, the activatable anti-CTLA4 antibody is administered at a dose of approximately 30 mg / kg to approximately 50 mg / kg (e.g., 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, or 50 mg / kg) over at least one treatment cycle (e.g., 1 to 3 treatment cycles), and then at a dose of approximately 10 mg / kg in subsequent treatment cycles (e.g., once every 3 weeks or once every 6 weeks). In another embodiment, the activatable anti-CTLA4 antibody is administered at a dose of approximately 30 mg / kg to approximately 50 mg / kg (e.g., 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, or 50 mg / kg) for at least one treatment cycle (e.g., 1 to 3 treatment cycles), and then at a dose of approximately 15 mg / kg in subsequent treatment cycles (e.g., once every 3 weeks or once every 6 weeks). In another embodiment, the activatable anti-CTLA4 antibody is administered at a dose of approximately 30 mg / kg to approximately 50 mg / kg (e.g., 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, or 50 mg / kg) for at least one treatment cycle (e.g., 1 to 3 treatment cycles), and at a dose of approximately 20 mg / kg in subsequent treatment cycles (e.g., once every 3 weeks or once every 6 weeks).

[0117] In some embodiments, the activatable anti-CTLA4 antibody is administered in doses ranging from approximately 30 to 50 mg / kg in a single treatment cycle, and then in subsequent treatment cycles (e.g., every three weeks) at a dose of approximately 6 mg / kg. For example, in one embodiment, the activatable anti-CTLA4 antibody is administered as an initial (loading) dose of 30 mg / kg, followed by an additional dose (maintenance dose) of 6 mg / kg every three weeks. In another embodiment, the activatable anti-CTLA4 antibody is administered as an initial (loading) dose of 40 mg / kg, followed by an additional dose (maintenance dose) of 6 mg / kg every three weeks. In yet another embodiment, the activatable anti-CTLA4 antibody is administered as an initial (loading) dose of 50 mg / kg, followed by an additional dose (maintenance dose) of 6 mg / kg every three weeks. The maintenance dose can be initiated at a predetermined time after the administration of the loading dose. For example, in some embodiments, the first maintenance dose can be administered three weeks after the administration of the loading dose.

[0118] In some embodiments, the activatable anti-CTLA4 antibody is administered in doses ranging from approximately 30 to 50 mg / kg in a single treatment cycle, and then in subsequent treatment cycles (e.g., every three weeks) at doses of approximately 10 mg / kg. For example, in one embodiment, the activatable anti-CTLA4 antibody is administered as an initial (loading) dose of 30 mg / kg, followed by an additional dose (maintenance dose) of 10 mg / kg every three weeks. In another embodiment, the activatable anti-CTLA4 antibody is administered as an initial (loading) dose of 40 mg / kg, followed by an additional dose (maintenance dose) of 10 mg / kg every three weeks. In yet another embodiment, the activatable anti-CTLA4 antibody is administered as an initial (loading) dose of 50 mg / kg, followed by an additional dose (maintenance dose) of 10 mg / kg every three weeks. The maintenance dose can be initiated at a predetermined time after the administration of the loading dose. For example, in some embodiments, the first maintenance dose can be administered three weeks after the administration of the loading dose.

[0119] In some embodiments, the activatable anti-CTLA4 antibody is administered at a dose of approximately 40–50 mg / kg per treatment cycle, and at a dose of approximately 30 mg / kg in subsequent treatment cycles (e.g., every three weeks). For example, in one embodiment, the activatable anti-CTLA4 antibody is administered at an initial (loading) dose of 40 mg / kg, followed by an additional dose (maintenance dose) of 30 mg / kg every three weeks. In another embodiment, the activatable anti-CTLA4 antibody is administered at an initial (loading) dose of 40 mg / kg, followed by an additional dose (maintenance dose) of 30 mg / kg every three weeks. In yet another embodiment, the activatable anti-CTLA4 antibody is administered at an initial (loading) dose of 50 mg / kg, followed by an additional dose (maintenance dose) of 30 mg / kg every three weeks. The maintenance dose can be initiated at a predetermined time after the administration of the loading dose. For example, in some embodiments, the first maintenance dose can be administered three weeks after the administration of the loading dose.

[0120] As described above, it has been shown that administering a single loading dose followed by a maintenance dose leads to a faster establishment of steady-state plasma concentrations of cleaved antibodies than when activatable antibodies are administered without a loading dose. As described above, it has been shown that continuing with maintenance dose administration after two loading doses leads to a faster establishment of steady-state plasma concentrations of cleaved antibodies than when activatable antibodies are administered without a loading dose. For example, in some embodiments, steady-state plasma concentrations of cleaved antibodies can be established within 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, or 7 weeks after the initial administration of one or more loading doses.

[0121] In any of the embodiments described above, the activatable anti-CTLA4 antibody (e.g., TY22404), when administered in combination with pembrolizumab, can be administered in a dose that results in a steady-state plasma concentration of approximately 50 nM to approximately 100 nM of the cleaved antibody (i.e., the active antibody after cleavage of the masking portion (MM) and the cleavable portion (CM)). In some embodiments, the activatable anti-CTLA4 antibody is administered in a dose that results in a steady-state plasma concentration of approximately 50 nM to approximately 100 nM of the cleaved antibody. In some embodiments, the activatable anti-CTLA4 antibody is administered in a dose that results in a steady-state plasma concentration of approximately 50 nM to approximately 75 nM of the cleaved antibody. In some embodiments, the activatable anti-CTLA4 antibody is administered in a dose that results in a steady-state plasma concentration of approximately 70 nM to approximately 80 nM of the cleaved antibody. In some embodiments, the activatable anti-CTLA4 antibody is administered so that the steady-state plasma concentration of the cleaved antibody is approximately 100 nM to approximately 175 nM. In some embodiments, the activatable anti-CTLA4 antibody is administered in such a dose that the steady-state plasma concentration of the cleaved antibody is approximately 100 nM to approximately 200 nM. In some embodiments, the activatable anti-CTLA4 antibody is administered in such a dose that the steady-state plasma concentration of the cleaved antibody is approximately 125 nM to approximately 200 nM. In some embodiments, the activatable anti-CTLA4 antibody is administered in a dose that results in a steady-state plasma concentration of the cleaved antibody from approximately 100 nM to approximately 150 nM. In some embodiments, the activatable anti-CTLA4 antibody is administered in a dose that results in a steady-state plasma concentration of the cleaved antibody from approximately 125 nM to approximately 175 nM. In some embodiments, the activatable anti-CTLA4 antibody is administered in a dose that results in a steady-state plasma concentration of the cleaved antibody from approximately 125 nM to approximately 150 nM. In some embodiments, the activatable anti-CTLA4 antibody is administered in a dose that results in a steady-state plasma concentration of the cleaved antibody from approximately 150 nM to approximately 200 nM. In some embodiments, the activatable anti-CTLA4 antibody is administered in a dose such that the steady-state plasma concentration of the cleaved antibody is approximately 200 nM to approximately 600 nM.In some embodiments, the activatable anti-CTLA4 antibody is administered at a dose that results in a steady-state plasma concentration of approximately 200 nM to approximately 400 nM of the cleaved antibody. In some embodiments, the activatable anti-CTLA4 antibody is administered at a dose that results in a steady-state plasma concentration of approximately 300 nM to approximately 500 nM of the cleaved antibody. In some embodiments, the activatable anti-CTLA4 antibody is administered at a dose that results in a steady-state plasma concentration of approximately 400 nM to approximately 600 nM of the cleaved antibody. In some of the embodiments described above, the plasma concentration can be measured at the trough level of the activatable anti-CTLA4 antibody (i.e., the minimum concentration for each administration cycle). For example, the plasma concentration for a particular cycle can be measured immediately before administration in the next cycle.

[0122] In some embodiments, the activatable anti-CTLA4 antibody and pembrolizumab are administered in combination with two or more therapeutic agents. In some such embodiments, the activatable anti-CTLA4 antibody is administered so that the steady-state plasma concentration of the cleaved antibody is about 50 nM to about 150 nM. In other such embodiments, the activatable anti-CTLA4 antibody is administered so that the steady-state plasma concentration of the cleaved antibody is about 50 nM to about 100 nM. In other such embodiments, the activatable anti-CTLA4 antibody is administered so that the steady-state plasma concentration of the cleaved antibody is about 50 nM to about 75 nM. In other such embodiments, the activatable anti-CTLA4 antibody is administered so that the steady-state plasma concentration of the cleaved antibody is about 75 nM to about 100 nM.

[0123] In some embodiments, the cancer is resistant or refractory to inhibitors of CTLA-4, PD-1, or PD-1 ligands (e.g., PD-L1 or PD-L2). In some embodiments, the cancer is a solid tumor, such as advanced-stage cancer and / or metastatic cancer. Cancer treatment can be evaluated, for example, by tumor regression, reduction in tumor weight or size, progression-free survival, overall survival, progression-free survival rate, overall response rate, duration of response, quality of life, and protein expression and / or activity. Approaches can be used to determine the effectiveness of therapy, including, for example, measuring the response by radiographic imaging.

[0124] The activatable anti-CTLA4 antibodies and compositions provided herein can be administered via any suitable enteral or parenteral route. The term “enteral route” refers to administration via any part of the gastrointestinal tract. Examples of enteral routes include oral, mucosal, oral, and rectal routes, or intragastric routes. “Pareral routes” refers to routes of administration other than enteral routes. Examples of parenteral routes include intravenous, intramuscular, intradermal, intraperitoneal, intratumoral, intravesical, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, transorgan, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, intrasternal, subcutaneous, or local administration. The antibodies and compositions of this disclosure can be administered by any suitable method, such as 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 specific antibody used, the desired absorption rate, the specific formulation or dosage form used, the type or severity of the disorder being treated, the specific site of action, and the patient's condition, and can be easily selected by those skilled in the art. In some embodiments, the anti-CTLA4 antibody is administered intravenously.

[0125] The effective dose of the activatable anti-CTLA4 antibody may be administered in a single dose or multiple doses. Examples of administration frequencies for multiple doses of the activatable anti-CTLA4 antibody include, but are not limited to, once a week, once a week for two weeks out of three, once a week for three weeks out of four, once a week, once every three weeks, once every two weeks, once a month, once every six months, or once a year. In some embodiments, the activatable anti-CTLA4 antibody is administered approximately once a week, once every two weeks, once every three weeks, once every six weeks, or once every twelve weeks. In some embodiments, the interval between each dose is shorter than any of the following periods: approximately 3 years, approximately 2 years, approximately 12 months, approximately 11 months, approximately 10 months, approximately 9 months, approximately 8 months, approximately 7 months, approximately 6 months, approximately 5 months, approximately 4 months, approximately 3 months, approximately 2 months, approximately 1 month, approximately 4 weeks, approximately 3 weeks, approximately 2 weeks, or approximately 1 week. In some embodiments, the interval between each dose is longer than any of the following periods: approximately 1 week, approximately 2 weeks, approximately 3 weeks, approximately 4 weeks, approximately 1 month, approximately 2 months, approximately 3 months, approximately 4 months, approximately 5 months, approximately 6 months, approximately 7 months, approximately 8 months, approximately 9 months, approximately 10 months, approximately 11 months, approximately 12 months, approximately 2 years, or approximately 3 years. In some embodiments, there is no interruption in the dosing schedule.

[0126] In some embodiments, the activatable anti-CTLA4 antibody is administered at a low frequency, for example, once a week, once every two weeks, once every three weeks, once a month, once every two months, once every three months, once every four months, once every five months, once every six months, once every seven months, once every eight months, once every nine months, once every ten months, once every eleven months, or once a year or less, or at any one of these frequencies or less. In some embodiments, the activatable anti-CTLA4 antibody is administered in a single dose. In some embodiments, the activatable anti-CTLA4 antibody is administered approximately once every three weeks. In some embodiments, the activatable anti-CTLA4 antibody is administered approximately once every six weeks.

[0127] In some embodiments, the activatable anti-CTLA4 antibody is administered over two or more cycles, for example, approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles. In some embodiments, the activatable anti-CTLA4 antibody is administered over at least four cycles.

[0128] Activatable anti-CTLA4 antibodies can be administered to patients in combination with pembrolizumab at doses that achieve high levels of receptor (CTLA-4) occupancy, and are therefore effective with minimal side effects. Thus, the activatable anti-CTLA4 antibodies of this disclosure exhibit an improved therapeutic index compared to anti-CTLA4 antibodies such as ipilimumab. For example, in one embodiment, the activatable antibody (e.g., TY22404) can be administered as a single dose (in combination with pembrolizumab) to achieve a receptor occupancy of more than 50% within 3 weeks or even 6 weeks after administration. In some embodiments, the activatable anti-CTLA4 antibody (e.g., TY22404) can be administered as a single dose (in combination with pembrolizumab) to achieve a receptor occupancy of more than 60% within 3 weeks after administration. In other such embodiments, the activatable anti-CTLA4 antibody (e.g., TY22404) can be administered as a single dose (in combination with pembrolizumab) to achieve a receptor occupancy of more than 70% within 3 weeks after administration. In other such embodiments, an activatable anti-CTLA4 antibody (e.g., TY22404) can be administered as a single dose (in combination with pembrolizumab) to achieve a receptor occupancy of more than 80% three weeks after administration. In other such embodiments, an activatable anti-CTLA4 antibody (e.g., TY22404) can be administered as a single dose (in combination with pembrolizumab) to achieve a receptor occupancy of about 50% to about 80% three weeks after administration. In other such embodiments, an activatable anti-CTLA4 antibody (e.g., TY22404) can be administered as a single dose (in combination with pembrolizumab) to achieve a receptor occupancy of about 60% to about 75% three weeks after administration. In other such embodiments, an activatable anti-CTLA4 antibody (e.g., TY22404) can be administered as a single dose (in combination with pembrolizumab) to achieve a receptor occupancy of more than 60% six weeks after administration. In other such embodiments, an activatable anti-CTLA4 antibody (e.g., TY22404) can be administered as a single dose (in combination with pembrolizumab) that achieves a receptor occupancy rate of more than 70% within 6 weeks after administration.In other such embodiments, an activatable anti-CTLA4 antibody (e.g., TY22404) can be administered as a single dose (in combination with pembrolizumab) that achieves approximately 50% to approximately 70% receptor occupancy within 6 weeks after administration.

[0129] In some embodiments, the treatment comprises an initial phase and a subsequent maintenance phase. In some embodiments, the activatable anti-CTLA4 antibody (e.g., TY22404) is administered at a lower frequency in the maintenance phase than in the initial phase. In some embodiments, the activatable anti-CTLA4 antibody is administered at the same frequency in the maintenance phase as in the initial phase. In some embodiments, the treatment comprises an initial phase in which the activatable anti-CTLA4 antibody is administered approximately every three weeks for at least four cycles, and a maintenance phase in which the activatable anti-CTLA4 antibody is administered approximately every four weeks to every twelve weeks, for example, every four weeks, every six weeks, every eight weeks, every ten weeks, or every twelve weeks. In some embodiments, the administration frequency in the maintenance phase is adjusted according to one or more biomarkers such as Treg cells, CD8+ Tem cells, CD4+ Tem cells, the ratio of CD8+ Tem cells to Treg cells, the ratio of CD4+ Tem cells to Treg cells, and / or NK cells. For example, if a subject (e.g., a human patient) shows an increase in the ratio of CD8+ Temp cells to Treg cells after administration of an anti-CTLA4 antibody, the subject (e.g., a human patient) can be further administered an activatable anti-CTLA4 antibody approximately once every four weeks.

[0130] The combination therapy of an activatable anti-CTLA4 antibody and pembrolizumab can be extended over long periods, such as approximately one week to one month, one month to one year, or one year to several years. In some embodiments, the activatable anti-CTLA4 antibody is administered for at least one week, two weeks, three weeks, four weeks, five weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, one year, two years, three years, or four years, or longer.

[0131] The methods described herein are useful for treating a variety of cancers. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a liquid tumor. The various cancers in which CTLA4 is involved, whether malignant or benign, and primary or secondary, can be treated or prevented in the manner provided herein. Examples of cancers, but not limited to, include liver cancer, gastrointestinal cancers (e.g., colon cancer, colorectal cancer), lung cancer, bone cancer, heart cancer, brain cancer, kidney cancer, bladder cancer, hematological cancers (e.g., leukemia), skin cancer, breast cancer, thyroid cancer, pancreatic cancer, head and neck cancer, eye-related cancers, male reproductive system cancers (e.g., prostate cancer, testicular cancer), or female reproductive system cancers (e.g., uterine cancer, cervical cancer). In some embodiments, the cancer is a kidney cancer such as renal cell carcinoma, or urothelial carcinoma. In some embodiments, the cancer is a cold tumor. In some embodiments, the cancer is resistant or refractory to one or more conventional treatments, such as immunotherapy, including immune checkpoint inhibitors. In some embodiments, cancer is a tumor in which T cells cannot penetrate because the tumor is not recognized by the immune system or does not trigger an immune response.

[0132] In some embodiments, the activatable anti-CTLA4 antibody of this disclosure (in combination with pembrolizumab) can be used to treat colorectal cancer (CRC). In some embodiments, the colorectal cancer has not metastasized to other organs such as the lungs or liver. In some embodiments, the colorectal cancer has metastasized to other organs such as the lungs or liver. In some embodiments, the colorectal cancer patient has previously been treated with other chemotherapy reagents. Such chemotherapy reagents include, but are not limited to, FOLFOX, FOLFIRI / Avastin, Erbitux, Lonsurf, IO-202, APN401, or IPH5201.

[0133] In some embodiments, the activatable anti-CTLA4 antibody of this disclosure (in combination with pembrolizumab) can be used to treat colorectal cancer (CRC), wherein the CRC is microsatellite-stable (MSS) colorectal cancer (MSS CRC). In some embodiments, the MSS CRC has metastasized to other organs such as the lungs or liver. In some embodiments, the MSS CRC has not metastasized to other organs such as the lungs or liver. In some embodiments, the MSS CRC has not metastasized to the peritoneum. In some embodiments, the MSS CRC has not metastasized to the liver or peritoneum. In some embodiments, the MSS CRC patient has previously been treated with other chemotherapy reagents. Such chemotherapy reagents include, but are not limited to, FOLFOX, FOLFIRI / Avastin, Erbitux, Lonsurf, IO-202, APN401, or IPH5201.

[0134] In some embodiments, the activatable anti-CTLA4 antibody of this disclosure can be used for the treatment of Kaposi's sarcoma.

[0135] In some embodiments, the activatable anti-CTLA4 antibody of this disclosure (in combination with pembrolizumab) can be used to treat head and neck squamous cell carcinoma (HNSCC).

[0136] In some embodiments, the anti-CTLA4 antibody of this disclosure (in combination with pembrolizumab) can be used to treat pancreatic cancer.

[0137] In some embodiments, the anti-CTLA4 antibody of this disclosure (in combination with pembrolizumab) can be used to treat ovarian cancer.

[0138] In some embodiments, the subject (e.g., a human patient) has previously been treated with conventional therapy. In some embodiments, the subject (e.g., a human patient) has previously received one, two, three, or four or more prior treatments. In some embodiments, the subject (e.g., a human patient) has exhausted all other available therapies. In some embodiments, the subject (e.g., a human patient) is unresponsive to or resistant to conventional therapy. In some embodiments, the subject (e.g., a human patient) has experienced a relapse of the disease after conventional therapy. In some embodiments, the subject (e.g., a human patient) is refractory to conventional therapy. In some embodiments, the subject (e.g., a human patient) has experienced failure of conventional therapy within approximately one year, six months, or three months. In some embodiments, the subject (e.g., a human patient) has never received conventional therapy before.

[0139] In some embodiments, the subject (e.g., a human patient) has previously been treated with standard therapy for the cancer. In some embodiments, the subject (e.g., a human patient) is unresponsive to or resistant to standard therapy. In some embodiments, the subject (e.g., a human patient) has experienced a recurrence of the disease after standard therapy. In some embodiments, the subject (e.g., a human patient) is refractory to standard therapy. In some embodiments, the subject (e.g., a human patient) has experienced failure of standard therapy within approximately one year, six months, or three months. In some embodiments, the subject (e.g., a human patient) has never previously received standard therapy. In some embodiments, the subject (e.g., a human patient) has refused or is ineligible for standard therapy.

[0140] In some embodiments, the conventional therapy (e.g., standard therapy) is selected from the group consisting of viral gene therapy, immunotherapy, targeted therapy, radiotherapy, and chemotherapy. In some embodiments, the 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 antibody described herein. In some embodiments, the conventional therapy is ipilimumab.

[0141] In some embodiments, the conventional therapy is an inhibitor of PD-1 or a PD-1 ligand, including PD-1 binding antagonists, PDL1 binding antagonists, and PDL2 binding antagonists. 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.

[0142] In some embodiments, conventional therapy is a PD-1 inhibitor, which is a molecule that inhibits the binding of PD-1 to its ligand-binding partner. In some embodiments, the PD-1 ligand inhibitor is a PD-L1 and / or PD-L2 inhibitor. In some embodiments, the PD-L1 inhibitor is a molecule that inhibits PD-L1 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 PD-1 ligand inhibitor 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, its antigen-binding fragment, an immunoadhesin, a fusion protein, or an oligopeptide.

[0143] In some embodiments, conventional treatments include pembrolizumab, 2E5 (Cstone Pharmaceuticals), tisrelizumab (BGB-A317), BGB-108, STI-A1110, AM0001, BI754091, cintilimab (IBI308), cetrelimab (JNJ-63723283), tripalimab (JS-001), camrelizumab (SHR-1210, INCSHR-1210, HR-301210), MEDI-0680 (AMP-514), and MGA-012 (INCMGA). 0012), Nivolumab (BMS-936558, MDX1106, ONO-4538), Spartalizumab (PDR00l), PF-06801591, Semiprimab (REGN-2810, REGEN2810), Dostallimab (TSR-042, ANB011), Pizilizumab (CT-011), FITC-YT-16 (PD-1 binding peptide), APL-501, CBT-501 or Geptanolimab (GB-226), AB-122, AK105, AMG404, BCD-100, F520, HLX10, HX008, JTX-4014, LZM009, Sym021, PSB205, AMP-224 (PD-1 targeting fusion protein), CX-188 (PD-1 probody), AGEN-2034, GLS-010, buzigalimab (ABBV-181), AK-103, BAT-1306, CS-1003, AM-0001, TILT-123, BH-2922, BH-2941, BH-2950, ​​ENUM-244C8, ENUM-388D4, HAB-21, H EISCOI 11-003, IKT-202, MCLA-134, MT-17000, PEGMP-7, PRS-332, RXI-762, STI-1110, VXM-10, XmAb-2 3104, AK-112, HLX-20, SSI-361, AT-16201, SNA-01, AB122, PD1-PIK, PF-06936308, RG-7769, CAB PD-1 Abs, AK-123, MEDI-3387, MEDI-5771, 4H1128Z-E27, REMD-288, SG-001, BY-24.3. The anti-PD-1 antibody is selected from CB-201, IBI-319, ONCR-177, Max-1, CS-4100, JBI-426, CCC-0701, CCX-4503, their biosimilars, or derivatives thereof. 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 containing an extracellular or PD-1 binding moiety of PDL1 or PDL2 fused to a constant region (e.g., the Fc region of an immunoglobulin sequence)). In some embodiments, the PD-1 inhibitor is AMP-224. In some embodiments, the anti-PD-1 antibody is nivolumab (CAS registry number: 946414-94-4). Nivolumab, also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-1 antibody described in WO2006 / 121168. CT-011, also known as hBAT or hBAT-1, is an anti-PD-1 antibody described in WO2009 / 101611. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in WO2010 / 027827 and WO2011 / 066342.

[0144] Conventional treatments (e.g., standard treatments) include surgical removal of tumors and radiotherapy. Exemplary radiotherapys, but not limited to these, include ionizing (electromagnetic) radiation therapy (e.g., X-rays or gamma rays) and particle beam radiation therapy (e.g., high-linear-energy radiation). The radiation source may be external to or internal to the subject (e.g., a human patient).

[0145] The methods described herein are useful in various aspects of cancer treatment. In some embodiments, a method is provided for inhibiting cell proliferation (such as tumor growth) in an individual, comprising administering to the individual an effective dose of the activatable anti-CTLA4 antibody of this disclosure in combination with pembrolizumab. In some embodiments, cell proliferation is inhibited by at least about 10% (e.g., including at least about 20%, about 30%, about 40%, about 60%, about 70%, about 80%, about 90%, or about 95% or more).

[0146] In some embodiments, administration of pembrolizumab in combination with an activatable antibody of the Disclosure (e.g., TY22404) significantly increases IFN-γ levels compared to monotherapy with the activatable antibody of the Disclosure (e.g., TY22404). For example, in some embodiments, administration of pembrolizumab in combination with an activatable antibody of the Disclosure (e.g., TY22404) at a specific dose (e.g., 6 mg / kg or 10 mg / kg) results in a 2- to 10-fold increase in IFN-γ compared to administration of activatable anti-CTLA4 monotherapy at the same dose.

[0147] In some embodiments, a method is provided for inhibiting tumor metastasis in an individual, comprising administering to the individual an effective amount of one of the anti-CTLA4 antibodies described herein in combination with pembrolizumab. In some embodiments, at least about 10% of metastasis (e.g., including at least about 20%, 30%, 40%, 60%, 70%, 80%, 90%, 95%, or more) is inhibited.

[0148] In some embodiments, a method is provided for reducing (e.g., eliminating) existing tumor metastases in an individual (e.g., metastases to lymph nodes), comprising administering to the individual an effective amount of one of the activatable anti-CTLA4 antibodies described herein in combination with pembrolizumab. In some embodiments, metastases are reduced by at least about 10% (e.g., including at least about 20%, 30%, 40%, 60%, 70%, 80%, 90%, 95%, or more).

[0149] In some embodiments, a method is provided for reducing the occurrence or burden of existing tumor metastases (e.g., metastases to lymph nodes) in an individual, comprising administering to the individual an effective amount of one of the activatable anti-CTLA4 antibodies described herein in combination with pembrolizumab.

[0150] In some embodiments, a method is provided for reducing the size of a tumor in an individual, comprising administering to the individual an effective amount of one of the activatable anti-CTLA4 antibodies described herein in combination with pembrolizumab. In some embodiments, the method reduces the size of the tumor by at least about 10% (including, for example, at least about 20%, 30%, 40%, 60%, 70%, 80%, 90%, 95%, or more).

[0151] In some embodiments, a method is provided for extending the time to disease progression of cancer in an individual, comprising administering to the individual an effective amount of one of the activatable anti-CTLA4 antibodies described herein in combination with pembrolizumab. In some embodiments, the method extends the time to disease progression by at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, sixteen, twenty, twenty, twenty-four, twenty-eight, thirty-two, or thirty-six weeks, or beyond.

[0152] In some embodiments, a method is provided for extending the survival time (e.g., overall survival or progression-free survival) of an individual with cancer, comprising administering to the individual an effective amount of one of the activatable anti-CTLA4 antibodies described herein in combination with pembrolizumab. In some embodiments, the method extends the survival time of the individual by at least 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.

[0153] In some embodiments, a method is provided for alleviating one or more symptoms of a cancer individual, comprising administering to the individual an effective amount of either an activatable anti-CTLA4 antibody described herein in combination with pembrolizumab.

[0154] In some embodiments, a method is provided for improving the quality of life of an individual having cancer, comprising administering to the individual an effective amount of either an activatable anti-CTLA4 antibody described herein in combination with pembrolizumab.

[0155] The activatable anti-CTLA4 antibody and pembrolizumab may be administered in combination with one or more additional therapeutic agents or therapies. In some embodiments, the activatable anti-CTLA4 antibody and pembrolizumab are administered separately, sequentially, or in combination with one or more additional therapeutic agents for administration simultaneously. The term “additional therapeutic agent” refers to any therapeutic agent other than the activatable anti-CTLA4 antibody provided herein. In some embodiments, a combination therapy is provided for treating cancer in a subject (e.g., a human patient), comprising administering an effective amount of the activatable anti-CTLA4 antibody described herein to the subject (e.g., a human patient) in combination with one or more additional therapeutic agents. In some embodiments, the activatable anti-CTLA4 antibody is administered in combination with one or more additional therapeutic agents, including chemotherapeutic agents, immunotherapeutic agents, and / or hormonal therapeutic agents. In some embodiments, one or more therapeutic agents are selected from the group consisting of viral gene therapy, immune checkpoint inhibitors, targeted therapies, radiotherapy, vaccine therapy, and chemotherapy. III. Activatable Anti-CTLA4 Antibody

[0156] This disclosure relates in part to precision / situation-dependent activatable conjugating polypeptides (i.e., activatable antibodies) that conjugate to human CTLA4 in combination with pembrolizumab. Activatable antibodies include any of the anti-CTLA4 antibodies described herein (e.g., anti-CTLA4 antibodies, conjugated fragments of anti-CTLA4 antibodies, and / or anti-CTLA4 antibody derivatives), antigen-conjugated fragments of activatable anti-CTLA4 antibodies, and / or derivatives of activatable anti-CTLA4 antibodies. In some embodiments, the activatable anti-CTLA4 antibodies described herein may have an improved safety profile. For example, the activatable anti-CTLA4 antibodies described herein may have an increased safety margin as assessed by changes in spleen weight. The safety margin of candidate drugs is evaluated by using changes in spleen size as a benchmark with increasing drug doses. The activatable anti-CTLA4 antibodies described herein have an increased safety margin compared to the parent antibody (antibody without a masking moiety). In some embodiments, the activatable antibody is TY22404.

[0157] In some embodiments, the activatable antibody of this disclosure comprises (a) a masking moiety (MM), (b) a cleavable moiety (CM), and (c) a target-binding moiety (e.g., an anti-CTLA4 antibody). In some embodiments, the MM is one of the masking moieties described herein. In some embodiments, the CM is one of the cleavable moieties described herein. In some embodiments, the TBM is one of the target-binding moieties described herein (e.g., a target-binding moiety (TBM) comprising the light chain variable region and / or heavy chain variable region of an anti-CTLA4 antibody, e.g., a VH and / or VL of any of the anti-CTLA4 antibodies described herein).

[0158] In some embodiments, the activatable antibody comprises (a) a polypeptide comprising a masking moiety (MM), a cleavable moiety (CM), and a target-binding moiety (TBM) from the N-terminus to the C-terminus, wherein the MM is one of the masking moieties described herein, the CM is one of the cleavable moieties described herein, and the TBM comprises an antibody light chain variable region (VL); and (b) an anti-CTLA4 antibody heavy chain variable region (VH).

[0159] In some embodiments, the activatable antibody comprises (a) a polypeptide comprising a masking moiety (MM), a cleavable moiety (CM), and a target-binding moiety (TBM) from the N-terminus to the C-terminus, wherein the MM is one of the masking moieties described herein, the CM is one of the cleavable moieties described herein, and the TBM comprises an anti-CTLA4 antibody heavy chain variable region (VH); and (b) an anti-CTLA4 antibody light chain variable region (VL).

[0160] In some embodiments, the activatable antibody comprises a polypeptide comprising a masking moiety (MM), a cleavable moiety (CM), and a target-binding moiety (TBM) from the N-terminus to the C-terminus, wherein the MM is one of the masking moieties described herein, the CM is one 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).

[0161] The terms “activatable binding polypeptide,” “ABP,” or “activatable antibody” include polypeptides comprising a target-binding moiety (TBM), a cleavable moiety (CM), and a masking moiety (MM). In some embodiments, the TBM comprises an amino acid sequence that binds to the target. In some embodiments, the TBM (anti-CTLA4 antibody) comprises an antigen-binding domain (ABD) of the antibody or an antibody fragment (e.g., either the antibody or antigen-binding fragment described herein). Anti-CTLA4 antibody

[0162] The methods described herein involve the administration of an activatable anti-CTLA4 antibody that specifically binds to human CTLA4, comprising a CTLA4 antibody, an antigen-binding fragment of a CTLA4 antibody, and a derivative of a CTLA4 antibody. Exemplary anti-CTLA4 antibodies are described, for example, in International Publication No. WO2019149281A1, which is incorporated herein by reference in its entirety.

[0163] In some embodiments, the antibodies include an activatable anti-CTLA4 antibody, HVR-H1 comprising the amino acid sequence of formula YSISSGYHWSWI (SEQ ID NO: 23), HVR-H2 comprising the amino acid sequence of formula LARIDWDDDKYYSTSLKSRL (SEQ ID NO: 35), HVR-H3 comprising the amino acid sequence of formula ARSYVYFDY (SEQ ID NO: 45), HVR-L1 comprising the amino acid sequence of formula RASQSVRGRFLA (SEQ ID NO: 58), HVR-L2 comprising the amino acid sequence of formula DASNRATGI (SEQ ID NO: 66), and HVR-L3 comprising the amino acid sequence of formula YCQQSSSWPPT (SEQ ID NO: 75).

[0164] In some embodiments, the activatable anti-CTLA4 antibody, upon cleavage of CM, comprises a) a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 87, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 100. In some embodiments, the activatable anti-CTLA4 antibody comprises a heavy chain variable region containing 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 containing 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.

[0165] In some embodiments, the activatable anti-CTLA4 antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 320 and a light chain containing the amino acid sequence of SEQ ID NO: 322. The activatable antibody having the heavy chain SEQ ID NO: 320 and the light chain SEQ ID NO: 322 is called TY22404. In some embodiments, the activatable antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 320 and a light chain containing 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 with respect to the amino acid sequence of SEQ ID NO: 320. In some embodiments, the activatable anti-CTLA4 antibody includes 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 includes 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 includes 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.

[0166] In some embodiments, the activatable antibody comprises a polypeptide having a masking moiety (MM)-cleavable moiety (CM)-VL structure from the N-terminus to the C-terminus, and the activatable antibody further comprises a second polypeptide containing VH (e.g., a Fab fragment). In some embodiments, the activatable antibody comprises a polypeptide having a masking moiety (MM)-cleavable moiety (CM)-VL-VH structure from the N-terminus to the C-terminus (e.g., scFv). In some embodiments, the activatable antibody comprises a polypeptide having a masking moiety (MM)-cleavable moiety (CM)-VH structure from the N-terminus to the C-terminus, and the activatable antibody further comprises a second polypeptide containing VL (e.g., a Fab fragment). In some embodiments, the activatable antibody comprises a polypeptide having a masking moiety (MM)-cleavable moiety (CM)-VH-VL structure from the N-terminus to the C-terminus (e.g., scFv).

[0167] These CMs generally contain cleavable amino acid sequences, such as amino acid sequences that function as substrates for enzymes, and / or cysteine-cysteine ​​pairs capable of forming reducible disulfide bonds. Therefore, when terms such as “cleaved,” “cleavable,” and “cleaved” are used in relation to CMs, these terms include the rupture of the disulfide bond between cysteine-cysteine ​​pairs through enzymatic cleavage, such as cleavage by proteases, and reduction of the disulfide bond, which may result from exposure to reducing agents.

[0168] The MM refers to the amino acid sequence in which the CM of the activatable antibody is in an intact state (e.g., not cleaved by the corresponding enzyme and / or containing an unreduced cysteine-cysteine ​​disulfide bond), and the MM interferes with or inhibits the binding of the TBM to its target. In some embodiments, the MM interferes with or inhibits the binding of the TBM to its target so very efficiently that the binding of the TBM to its target is extremely low and / or below the detection limit (e.g., binding cannot be detected by an ELISA assay or flow cytometry assay). The amino acid sequence of the CM may overlap with or be contained within the MM. For convenience, it should be noted that "ABP" or "activatable antibody" is used herein to refer to both the uncleaved (or "natural") and cleaved states of ABP or activatable antibody. In some embodiments, it will be apparent to those skilled in the art that, for example, CM is cleaved by a protease, causing the cleaved ABP to lack MM, resulting in the release of at least MM (for example, if MM is not covalently linked to ABP by a disulfide bond between cysteine ​​residues). Exemplary ABPs are described in further detail below.

[0169] In some embodiments, the masking moiety (MM) interferes with, blocks, reduces, inhibits, or competes with the target binding moiety (e.g., an "inactive, activatable antibody") in relation to binding to its target. In some embodiments, the masking moiety (MM) interferes with, blocks, reduces, inhibits, or competes with the target binding moiety (e.g., activation by a change in pH, activation by a temperature shift, activation after contact with a second molecule (such as a small molecule or protein ligand)) in relation to binding to its target. In some embodiments, activation induces cleavage of the polypeptide within the cleavage moiety. In some embodiments, activation induces a structural change (e.g., replacement of the masking moiety (MM)) in the polypeptide, making the masking moiety activatable. The masking moiety (MM) will no longer block the binding of an effective antibody to its target. In some embodiments, the masking moiety (MM) will interfere with, block, reduce the ability of, block, inhibit, or compete with the target binding moiety, with respect to binding to its target, only when the cleavable moiety (CM) is not cleaved by one or more proteases that cleave within the CM. In some embodiments, the masking moiety (MM) has a masking efficiency of at least about 2.0 before activation (e.g., at least about 2.0, at least about 3.0, at least about 4.0, at least about 5.0, at least about 6.0, at least about 7.0, at least about 8.0, at least about 9.0, at least about 10, at least about 25, at least about 50, at least about 75, at least about 100, at least about 150, at least about 200, at least about 300, at least about 400, at least about 500, etc.).In some embodiments, masking efficiency is measured as the difference between the affinity of an activatable antibody (before activation) containing the masking moiety (MM) to its target and the affinity of a polypeptide lacking the masking moiety to its target (for example, the difference between the affinity of the activatable antibody (before activation) containing the masking moiety (MM) to the target antigen (e.g., CTLA4) and the affinity of the parent antibody lacking the masking moiety (MM), or the difference between the affinity of the activatable antibody (before activation) containing the masking moiety (MM) to the target antigen (e.g., CTLA4) and the affinity of the activatable antibody (after activation) to the target antigen). In some embodiments, masking efficiency is measured by dividing the binding EC50 of the activatable antibody (before activation) containing the masking moiety (MM) by the EC50 of the parent antibody (for example, by measuring EC50 by ELISA; see, for example, the method in Example 8). In some embodiments, masking efficiency is measured as the difference between the affinity of an activatable antibody containing a masking moiety (MM) to its target before activation and the affinity of an activatable antibody containing a masking moiety (MM) to its target after activation (e.g., the difference between the affinity of the activatable antibody to the target antigen (CTLA4) before activation and the affinity of the activatable antibody after activation). In some embodiments, the masking moiety (MM) binds to a target-binding moiety (TBM), preventing the activatable antibody from binding to its target (e.g., an "inactive" activatable antibody). In some embodiments, the dissociation constant of the masking moiety (MM) for binding to the target-binding moiety (TBM) is greater than the dissociation constant of the target-binding moiety (TBM) for its target.

[0170] In some embodiments, after the activatable antibody is activated (for example, by treatment with one or more proteases that cleave within its cleavable portion (CM), by a change in pH, by a temperature shift, or after contact with a second molecule (such as an enzyme or protein ligand), the masking portion (MM) does not interfere with, block, reduce the ability of the target binding portion (TBM) to bind to its target, inhibit, or compete with the target binding portion (TBM). In some embodiments, after the cleavable portion (CM) is cleaved by one or more proteases that cleave within the CM, the masking portion (MM) does not interfere with, block, reduce the ability of the target binding portion (TBM) to bind to its target, block, inhibit, or compete with the target binding portion (TBM). In some embodiments, after activation, the masking efficiency of the masking portion (MM) is up to about 1.75 (e.g., up to about 1.75, up to about 1.5, up to about 1.4, up to about 1.3, up to about 1.2, up to about 1.1, up to about 1.0, up to about 0.9, up to about 0.8, up to about 0.7, up to about 0.6, or up to about 0.5, etc.) (e.g., relative affinity of the activated activatable antibody compared to the affinity of the parent antibody).

[0171] In some embodiments, the activatable antibodies of this disclosure include a masking moiety (MM) containing a pair of cysteine ​​residues at a specific position, thereby fixing the conformation of the activatable antibody and / or having few or no chemically unstable residues (such as methionine or tryptophan). Beneficially, the inclusion of a pair of cysteine ​​residues at a specific position tends to fix the conformation of the activatable antibody, thereby improving binding affinity and / or specificity. Furthermore, the activatable antibodies of this disclosure include a masking moiety that has few or no residues undesirable in the manufacturing process, such as methionine or tryptophan. Mashing section and cuttable linker

[0172] In certain embodiments, MM includes the amino acid sequence EVGSYPNPSSDCVPYYYACAY (SEQ ID NO: 192), and the cleavable portion includes the amino acid sequence SGRSAGGGGTPLGLAGSGGS (SEQ ID NO: 221). In some embodiments, MM and CM include the amino acid sequence EVGSYPNPSSDCVPYYYACAYSGRSAGGGGTPLGLAGSGGS (SEQ ID NO: 200) from the N-terminus to the C-terminus. In some embodiments, MM and CM are covalently bound to the N-terminus of the light chain of the anti-CTLA4 antibody. In some embodiments, MM and CM from the N-terminus to the C-terminus include an amino acid sequence having at least 90% or at least 95% sequence identity with SEQ ID NO: 200.

[0173] In some embodiments, any of the masking moieties (MMs) described herein may further include one or more additional amino acid sequences (e.g., one or more polypeptide tags). Suitable examples of additional amino acid sequences include, but are not limited to, purification tags (e.g., His tags, FLAG tags, maltose-binding proteins, and glutathione-S-transferase tags), detection tags (e.g., tags detectable by photometry (e.g., red or green fluorescent proteins)), tags having detectable enzymatic activity (e.g., alkaline phosphatase), tags including secretion sequences, leader sequences, and / or stabilization sequences, and protease cleavage sites (e.g., furin cleavage sites, TEV cleavage sites, thrombin cleavage sites). In some embodiments, the one or more additional amino acid sequences are located at the N-terminus of the masking moiety (MM). In some embodiments, the additional amino acid sequences include or consist of the sequence of EVGSY (SEQ ID NO: 148).

[0174] In some embodiments, the masking portion binds to the target-binding portion (TBM) and inhibits the binding of the activatable antibody to its target before activation (e.g., before treatment with one or more proteases that cleave within the cleavable portion (CM), before a (local) change (increase or decrease) in pH, before a (increase or decrease) in temperature, 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 the binding of the activatable antibody to its target after activation (e.g., after treatment with one or more proteases that cleave within the cleavable portion (CM), after a (local) change (increase or decrease) in pH, after a (increase or decrease) in temperature, after contact with a second molecule (such as a small molecule or protein ligand), etc.). In some embodiments, the masking portion (MM) inhibits the binding of the activatable antibody to its target when its CM is not cleaved, but does not inhibit the binding of the activatable antibody to its target once its CM is cleaved. In some embodiments, the masking portion (MM) has a dissociation constant for binding to the TBM that is greater than the dissociation constant of the activatable antibody for its target (when in the active form) (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.). Activatable anti-CTLA4 antibody

[0175] In some embodiments, the activatable anti-CTLA4 antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 320 and a light chain containing the amino acid sequence of SEQ ID NO: 322. In some embodiments, the activatable anti-CTLA4 antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 321 and a light chain containing the amino acid sequence of SEQ ID NO: 322. The activatable antibody having heavy chain SEQ ID NO: 320 and light chain SEQ ID NO: 322 is called TY22404. In some embodiments, the activatable antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 320 and a light chain containing 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 with respect to the amino acid sequence of SEQ ID NO: 320. In some embodiments, the activatable anti-CTLA4 antibody includes 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 includes 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 includes 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.

[0176] The activatable antibodies described herein may be further modified. In some embodiments, the activatable antibody is linked to additional molecules. Examples of additional molecules include pharmaceuticals, peptides or proteins, detection agents or labels, and antibodies.

[0177] In some embodiments, the activatable antibodies of this disclosure are linked to pharmaceutical agents. Examples of pharmaceutical agents include cytotoxic agents or other cancer treatments, and radioisotopes. Specific examples of cytotoxic agents include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracine dione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as their analogues or homologues. Examples of therapeutic agents include antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechloretamine, thiotepa, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiammineplatin(II) (DDP), which is cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mitramycin 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, but are not limited to, iodine-131, indium-111, yttrium-90, and lutetium-177. Methods for linking polypeptides to pharmaceutical agents are known in the art, including the use of various linker technologies. Examples of linker types include hydrazones, thioethers, esters, disulfides, and peptides.For further discussion of linkers and methods for linking therapeutic agents to antibodies, see, for example, Saito et al., Adv. DrugDeliv. Rev. 55:199-215 (2003), Trail, et al., CancerImmunol.Immunother. 52:328-337 (2003), Payne, CancerCell3:207-212 (2003), Allen, Nat. Rev. Cancer2:750-763 (2002), Pastan and Kreitman, Curr. Opin. Investig. Drugs3:1089-1091 (2002), and Senter and Springer (2001) Adv. DrugDeliv. Rev. 53:247-264. V. PD-1 antagonists

[0178] In one embodiment, the PD-1 antagonist useful for the treatment, drug, and use of the present invention includes a monoclonal antibody (mAb) or its antigen-binding fragment that specifically binds to PD-1 or PD-L1, preferably to human PD-1 or human PD-L1. The mAb is a human antibody, a humanized antibody, or a chimeric antibody, and may include a human constant region. In some embodiments, the human constant region is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4 constant regions, and in some embodiments, the human constant region is IgG1 or IgG4 constant region. In some embodiments, the antigen-binding fragment is selected from the group consisting of Fab, Fab'-SH, F(ab')2, scFv, and Fv fragments.

[0179] Examples of mAbs that bind to human PD-1 and are useful for the therapeutic methods, pharmaceuticals, and use of the present invention are described in U.S. Patent Nos. US7488802, US7521051, US8008449, US8354509, and US8168757, and in International Patent 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 therapeutic methods, pharmaceuticals, and uses of the present invention include pembrolizumab (also known as MK-3475), a humanized IgG4 mAb having the structure described in WHO Drug Information, Vol. 27, No. 2, pages 161-162 (2013) and containing the heavy and light chain amino acid sequences shown in Table B; nivolumab (BMS-936558), a human IgG4 mAb having the structure described 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, semiprimab, camrelizumab, scintirimab, tislerizumab, and tripalimab, which are under development by MedImmune. Additional anti-PD-1 antibodies intended for use in the present invention include MEDI0680 (U.S. Patent No. 8609089), 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).

[0180] Examples of mAbs that bind to human PD-1 and are useful in the therapeutic methods, pharmaceuticals, and uses of the present invention are described in US8383796. Specific anti-human PD-L1 mAbs useful as PD-1 antagonists in the therapeutic methods, pharmaceuticals, and uses of the present invention include BMS-936559, MEDI4736, and MSB0010718C.

[0181] In some embodiments, the PD-1 antagonist is pembrolizumab (KEYTRUDA®, Merck Sharp & Dohme LLC, Rahway, NJ, USA), nivolumab (OPDIVO®, Bristol-Myers Squibb Company, Princeton, NJ, USA), atezolizumab (TECENTRIQ®, Genentech, San Francisco, CA, USA), durvalumab (IMFINZI®, AstraZeneca Pharmaceuticals LP, Wilmington, DE), semiprimab (LIBTAYO®, Regeneron Pharmaceuticals, Tarrytown, NY, USA), avelumab (BAVENCIO®, Merck KGaA, Darmstadt, Germany), or dostallimab (JEMPERLI®, GlaxoSmithKline LLC, Philadelphia, USA). (PA). In other embodiments, the PD-1 antagonist is pizilizumab (US Patent No. 7,332,582), AMP-514 (MedImmune LLC, Gaithersburg, MD, USA), PDR001 (US Patent No. 9,683,048), BGB-A317 (US Patent No. 8,735,553), or MGA012 (MacroGenics, Rockville, MD).

[0182] In one embodiment, a PD-1 antagonist useful in the method of the present invention is an anti-PD-1 antibody that inhibits the binding of PD-1 to PD-L1 and PD-L2. In some embodiments of the therapeutic method, agent and use of the present invention, the PD-1 antagonist is a monoclonal antibody or its antigen-binding fragment comprising (a) a light chain variable region comprising light chain CDR1, CDR2 and CDR3 of SEQ ID NOs. 10, 11 and 12, respectively, and (b) a heavy chain variable region comprising heavy chain CDR1, CDR2 and CDR3 of SEQ ID NOs. 15, 16 and 17, respectively.

[0183] In other embodiments of the therapeutic methods, agents, and uses of the present invention, the PD-1 antagonist is a monoclonal antibody or its antigen-binding fragment that specifically binds to human PD-1 and comprises (a) a heavy chain variable region or a variant thereof containing SEQ ID NO: 18, and (b) a light chain variable region or a variant thereof containing SEQ ID NO: 13. The variant of the heavy chain variable region sequence is identical to the reference sequence except that it has up to six conserved amino acid substitutions in the framework region (i.e., outside the CDR). The variant of the light chain variable region sequence is identical to the reference sequence except that it has up to three conserved amino acid substitutions in the framework region (i.e., outside the CDR).

[0184] In another embodiment of the therapeutic method, agent, and use of the present invention, the PD-1 antagonist is a monoclonal antibody that specifically binds to human PD-1 and comprises (a) a heavy chain comprising SEQ ID NO: 19 and (b) a light chain comprising SEQ ID NO: 14. In one embodiment, the PD-1 antagonist is an anti-PD-1 antibody comprising two heavy chains and two light chains, the heavy chain and the light chain each comprising the amino acid sequences of SEQ ID NO: 19 and SEQ ID NO: 14, respectively.

[0185] In all of the above therapeutic methods, drugs, and uses, the PD-1 antagonist inhibits the binding of PD-L1 to PD-1, and in certain embodiments, also inhibits the binding of PD-L2 to PD-1. In some embodiments of the above therapeutic methods, drugs, and uses, the PD-1 antagonist is a monoclonal antibody or its antigen-binding fragment that specifically binds to PD-1 or PD-L1 and blocks the binding of PD-L1 to PD-1.

[0186] Table B below lists the amino acid sequences of exemplary anti-PD-1 mAbs for use in the therapeutics, pharmaceuticals, and applications of the present invention.

[0187] Table B. Exemplary PD-1 antibody sequences TIFF2026514050000001.tif168170

[0188] Table C. Additional PD-1 antibodies and antigen-binding fragments useful for the formulation, method and use of the present invention. TIFF2026514050000002.tif101170

[0189] In one embodiment, the anti-PD-1 antibody or its antigen-binding fragment includes a heavy chain constant region, such as a human constant region, e.g., the g1, g2, g3, or g4 human heavy chain constant region or a variant thereof. In another embodiment, the anti-PD-1 antibody or its antigen-binding fragment includes a light chain constant region, such as a human light chain constant region, e.g., the lambda or kappa human light chain region, or a variant thereof. For example, but not limited to, the human heavy chain constant region may be g4 and the human light chain constant region may be kappa. In another embodiment, the Fc region of the antibody is g4 with the Ser228Pro mutation (Schuurman, J et.al., Mol. Immunol. 38: 1-8, 2001). In some embodiments, different constant domains may be added to the humanized VL and VH regions derived from the CDR provided herein. For example, if a particular intended use of the antibody (or fragment) of the present invention requires modified effector functionality, a heavy chain constant domain other than human IgG1 may be used, or hybrid IgG1 / IgG4 may be utilized. Human IgG1 antibodies have a long half-life and possess effector functions such as complement activation and antibody-dependent cell-mediated cytotoxicity; however, such activities are not always desirable for all antibody applications. In such cases, for example, a human IgG4 constant domain can be used. The present invention involves the use of an anti-PD-1 antibody or its antigen-binding fragment containing an IgG4 constant domain. In one embodiment, the IgG4 constant domain differs from the natural human IgG4 constant domain (Swiss-Prot accession number P01861.1) in that natural Ser108 is replaced with Pro at positions corresponding to EU system position 228 and KABAT system position 241, thereby preventing potential interchain disulfide bonding between Cysl06 and Cysl09 (corresponding to EU system positions Cys226 and Cys229, and KABAT system positions Cys239 and Cys242), which could interfere with the formation of appropriate intrachain disulfide bonds. See Angal et al. (1993) Mol. Imunol. 30: 105. In other cases, modified IgG1 constant domains can be used that have been altered to extend the half-life or reduce the effector function.

[0190] In another embodiment, the PD-1 antagonist is an antibody or antigen-binding protein having a variable light domain and / or variable heavy domain having at least 95%, 90%, 85%, 80%, 75%, or 50% sequence identity with one of the variable light domains or variable heavy domains described above, and exhibiting specific binding to PD-1. In another embodiment of the therapeutic method of the present invention, the PD-1 antagonist is an antibody or antigen-binding protein comprising a variable light domain and a variable heavy domain having 1, 2, 3, 4, or 5 or more amino acid substitutions, and exhibiting specific binding to PD-1.

[0191] In some embodiments, pembrolizumab is administered at a dose of approximately 400 mg every 6 weeks.

[0192] In some embodiments, pembrolizumab is administered at a dose of approximately 2 mg / kg. In some embodiments, pembrolizumab is administered at a dose of approximately 2 mg / kg every three weeks. In certain embodiments, the patient is a pediatric patient.

[0193] In some embodiments, pembrolizumab is administered as an intravenous infusion over 30 minutes (-5 min / +10 min). In one embodiment, a selected dose of pembrolizumab is administered by IV infusion over 25–40 minutes, or approximately 30 minutes.

[0194] In one embodiment, pembrolizumab is contained in a pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent, which may also include additional pharmaceutically acceptable excipients. VI. Pharmaceutical compositions, kits and products

[0195] The activatable anti-CTLA4 antibody and pembrolizumab described herein can be administered as a pharmaceutical composition containing a pharmaceutically acceptable carrier. The activatable anti-CTLA4 antibody and pembrolizumab can be administered as separate pharmaceutical compositions or as a single pharmaceutical composition. The compositions can be prepared by conventional methods known in the art.

[0196] The term "pharmaceutically acceptable carrier" refers to any inactive substance suitable for use in a formulation to deliver an activator (e.g., an activatable anti-CTLA4 antibody or pembrolizumab). The carrier may be an antifouling agent, binder, coating agent, disintegrant, filler or diluent, preservative (e.g., antioxidant, antibacterial or antifungal agent), sweetener, absorption retarder, wetting agent, emulsifier, buffer, etc. Examples of suitable pharmaceutically acceptable carriers include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol), dextrose, vegetable oil (e.g., olive oil), saline, buffers, buffered saline, and isotonic 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 injectable solutions), microemulsions, liposomes, dispersions, or suspensions. Examples of solid dosage forms include tablets, pills, capsules, microcapsules, and powders. Specific forms of compositions suitable for delivering activatable anti-CTLA4 antibodies are sterile solutions such as solutions, suspensions, or dispersions for injection or infusion. Sterile solutions can be prepared by incorporating the required amount of the antibody into a suitable carrier, followed by sterile 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, preparation methods include vacuum drying and freeze-drying (lyophilization) to obtain the powders from pre-sterilized filtered solutions of the active ingredient and any additional desired ingredients. Compositions in various dosage forms can be prepared by conventional techniques known in the art.

[0197] In some embodiments, a product is provided that contains a material useful for the treatment of cancer. The product may include a container and a label or accompanying information on or attached to the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container may be formed from a variety of materials such as glass or plastic. Generally, the container holds the composition described herein, which is effective for the treatment of cancer, and may have a sterile access port (for example, the container may be a bag or vial for intravenous infusion with a stopper that can be pierced with a subcutaneous needle). An accompanying information document is a document that is typically included in the commercial packaging of a pharmaceutical product and contains information about the indications, use, dosage, administration, contraindications, and / or warnings regarding the use of such a pharmaceutical product. In some embodiments, the accompanying information document indicates that the composition is for use in the treatment of cancer. The label or accompanying information document may further include instructions on how to administer the composition to a patient.

[0198] In addition, the product may further include a second container containing pharmaceutically acceptable buffers, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. Furthermore, it may include other substances desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0199] We also provide kits that can be optionally combined with the product for various purposes, for example, for the treatment of cancer as described herein. The kits of the present application include one or more containers containing any one of the compositions (or unit dosage forms and / or products) described herein. In some embodiments, the kit further includes other agents (e.g., one or more additional therapeutic agents) and / or instructions for use according to any of the methods described herein. The kits may further include instructions for selecting individuals suitable for treatment. The instructions supplied with the kits of the present application are typically instructional documents on a label or accompanying document (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions written on a magnetic or optical storage disk) are also acceptable.

[0200] For example, in some embodiments, a pharmaceutical composition comprising either an activatable anti-CTLA4 antibody and a pharmaceutically acceptable carrier as described herein, pembrolizumab and a pharmaceutically acceptable carrier, and a kit comprising instructions for administering the pharmaceutical composition to a subject with cancer (e.g., a human patient). In some embodiments, the kit further comprises the pharmaceutical composition comprising an additional therapeutic agent, such as a chemotherapeutic agent. In some embodiments, the kit comprises one or more assays or reagents for determining the level of one or more biomarkers described herein (e.g., CD8+ T cells, CD4+ T cells, CD8+ Tem cells, CD4+ Tem cells, Treg cells, the ratio of CD8+ Tem cells to Treg cells, the ratio of CD4+ Tem cells to Treg cells, NK cells, B cells).

[0201] The kit of this application is contained in appropriate packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, and flexible packaging (e.g., sealed Mylar or plastic bags). The kit may optionally provide additional components, such as cushioning and interpretive information. That is, this application also provides products including vials (such as sealed vials), bottles, jars, flexible packaging, etc.

[0202] The container may be a unit dose, a bulk package (e.g., a multi-dose package), or a divided unit dose. The kit may also include multiple unit doses of the pharmaceutical composition and instructions for use and packaging in quantities sufficient for storage and use in a pharmacy, such as a hospital pharmacy and a compounding pharmacy. [Examples]

[0203] The present invention can be further understood by referring to the following embodiments, which are provided as examples and are not intended to limit the scope of the invention. Example 1. Phase 1b / 2, open-label, dose-escalation and expansion study of combination therapy with TY22404 and pembrolizumab (anti-PD-1 antibody) in patients with advanced / metastatic solid tumors.

[0204] In a Phase 1b / 2 trial, TY22404 monotherapy demonstrated an unprecedented safety profile (no Grade 3 or higher TRAEs) up to 20 mg / kg Q3W in terms of clinical activity in patients with repeated dosing and severe treatment history. The following examples describe preliminary results obtained from dose escalation of TY22404 in combination with pembrolizumab in patients with advanced / metastatic solid tumors such as cervical cancer, colorectal cancer, endometrial cancer, neuroendocrine cancer, ovarian cancer, and pancreatic cancer (NCT05405595). TY22404 was administered intravenously over 60-90 minutes. Pembrolizumab (KEYTRUDA®, Merck Sharp & Dohme LLC, Rahway, NJ, USA) was administered intravenously over 30 minutes. In TY22404-pembrolizumab combination therapy, the starting dose of TY22404 was administered 30-60 minutes after the completion of pembrolizumab infusion.

[0205] Objectives. The primary objectives of this study are to evaluate the safety and tolerability of TY22404 in combination with pembrolizumab at escalating doses in adult patients with advanced / metastatic solid tumors, to determine the maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D) of TY22404 in combination with pembrolizumab, and to evaluate the preliminary antitumor activity of TY22404-pembrolizumab combination therapy at dose escalation. Secondary objectives of this study are to evaluate the pharmacokinetic (PK) profiles of TY22404 and pembrolizumab, to assess the dose-proportionality of key PK parameters (area under the time-concentration curve [AUC], maximum concentration [Cmax]) to evaluate the immunogenicity of TY22404 and pembrolizumab, to characterize the relationship between immunogenicity (anti-drug antibody [ADA] positive), PK, safety, and efficacy parameters, and to evaluate the preliminary antitumor activity of TY22404-pembrolizumab combination therapy at dose escalation. This study will evaluate the safety and tolerability of combination therapy with TY22404 and pembrolizumab in adult patients with advanced / metastatic solid tumors. We will assess the PK profiles of TY22404 and pembrolizumab, as well as the immunogenicity of TY22404 and pembrolizumab at dose escalation. Exploratory objectives include evaluating pharmacodynamic and predictive biomarkers, including but not limited to serum proteins such as cytokines, profiling of peripheral immune cell subsets, tumor-infiltrating lymphocytes, pharmacogenomic markers, and TY22404 cleavage in tumor tissue after treatment (where available).

[0206] Study Design: This is a Phase 1b / 2, open-label, multicenter, dose-escalation and dose-expansion study to evaluate the safety, tolerability, pharmacokinetics, and preliminary efficacy of the combination therapy of TY22404 and pembrolizumab in patients with advanced / metastatic solid tumors. After a screening period of up to 28 days, eligible patients were enrolled and to receive an assigned dosing regimen of the combination therapy of TY22404 and pembrolizumab. TY22404 and pembrolizumab were administered at Q3W / Q6W or Q3W, respectively, until disease progression (PD), unacceptable toxicity, withdrawal of consent, or up to 35 cycles (Q3W). IV infusion of TY22404 was administered over 60–90 minutes, 30–60 minutes after pembrolizumab administration.

[0207] Each treatment cycle consists of 21 days. Patients receive treatment with TY22404 and pembrolizumab on day 1 of each treatment cycle in the study, until disease progression (PD) is documented according to RECIST v1.1 and / or iRECIST, or until a major toxicity occurs, consent is withdrawn, or other grounds for discontinuation / withdrawal occur, or up to 35 cycles, whichever comes first. TY22404 was administered intravenously (IV) over 60–90 minutes, 30–60 minutes after the completion of the pembrolizumab infusion. Pembrolizumab was administered according to the approved prescribing information.

[0208] Patients who discontinued treatment due to unacceptable adverse events (AEs) associated with the combination therapy of TY22404 and pembrolizumab were followed until the AEs returned to grade 0 or 1 or stabilized, or until the patient received a new non-protocol treatment. During the study, patient safety and toxicity, PK, immunogenicity, objective response, DCR, DOR, PFS, OS, and biomarkers were evaluated. Dose escalation phase

[0209] With a modified toxicity probability interval (mTPI) design with a target DLT rate of approximately 20% and an equivalence interval (EI) of [0.15, 0.23] for all doses, TY22404 was considered a candidate for the true MTD and was submitted for dose escalation and confirmation to determine the RP2D of TY22404 in combination with pembrolizumab. The dose levels are shown in Table 1. In combination therapy with TY22404 and pembrolizumab, both drugs were administered to TY22404 at Q3W and / or Q6W, and pembrolizumab was maintained at a constant 200 mg Q3W for up to 35 cycles at both dose levels (DL1 and DL2) of TY22404. Table 1: Dose levels of TY22404-pembrolizumab TIFF2026514050000003.tif28170DL=dose level;mTPI=modified toxicity probability interval;Q3W=every 3 weeks;Q6W=every 6 weeks.

[0210] Dose escalation was initiated at 6 mg / kg Q3W according to the mTPI design (DL1). If well tolerated, the dose was increased to 10 mg / kg Q3W. Based on SRC review, if 6 mg / kg Q3W was not tolerated due to early or late toxicity, the dose was increased to 6 mg / kg Q6W. Based on SRC review of early and late toxicity, if 6 mg / kg Q6W was tolerable, the dose was increased to 10 mg / kg Q6W. Similarly, based on SRC review of early and late toxicity, if 6 mg / kg Q3W was tolerable but 10 mg / kg Q3W was not, the dose was increased to 10 mg / kg Q6W.

[0211] DLTs were assessed using the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE) v5.0. Safety and tolerability for each TY22404 dose level were assessed by the SRC after all patients enrolled in the dose level were followed for at least 21 days after the first dose of TY22404-pembrolizumab combination therapy (DLT observation period). The dose and frequency of pembrolizumab administration remained unchanged.

[0212] The effective dose (or range) was defined based on safety, PK / pharmacodynamic data, modeling to predict antitumor response, and early signs of efficacy during dose escalation, and further evaluated at dose expansion. After completion of dose expansion, the RP2D was determined based on a comprehensive evaluation of all safety data, all available PK and pharmacodynamic data, and objective response observations in both dose escalation and dose expansion, including the MTD or MAD, dose levels below the MTD or MAD, and intermediate doses between pre-specified dose levels. The RP2D has been confirmed to be the pharmacologically effective optimal dose. Furthermore, the RP2D may include a maintenance dose at a reduced dose level and / or reduced dosing frequency (or frequency) after the loading dose until data from the DL1 and DL2 combination becomes available. Dosage expansion

[0213] After the effective dose (or range) of TY22404-pembrolizumab combination therapy was determined by dose escalation, a total of 60 patients—up to 20 each—were treated at the effective dose (or dose range) to further evaluate the antitumor activity of the combination therapy. These patients were classified as having microsatellite stable colorectal cancer (MSS CRC), head and neck squamous cell carcinoma (HNSCC) that had not previously received immunotherapy, and HNSCC that had previously received anti-programmed death protein 1 (anti-PD-1) therapy. For each of the three tumor types, the first 10 patients were treated with the first dose / schedule (e.g., TY22404 6 mg / kg every 3 or 6 weeks), and the next 10 patients were treated with the second dose / schedule. This schedule involves administering a higher continuous repeated dose (e.g., TY22404 10 mg / kg every 3 or 6 weeks) or a higher loading dose (e.g., TY22404 10 mg / kg) followed by a lower maintenance dose every 3 or 6 weeks. The higher dose was determined primarily based on pharmacokinetic and safety data in the continuous combination dose escalation phase. For each indicator, the Simon two-stage design outlined in Table 2 was used, with patients having two different dose / scheduling combinations, and the final decision was made by the SRC based on the sum of safety and efficacy information. Note: If the combination dose escalation phase did not identify an active dose or active dose with an acceptable Q3W interval, all 20 patients per tumor type were treated at Q6W. Table 2: Simon two-stage design for dose expansion TIFF2026514050000004.tif60170HNSCC=Head and neck squamous cell carcinoma; IO=Immuno-oncology; MSS CRC=Microsatellite stable colorectal cancer. Stage 1 and the overall study are considered successful if at least the corresponding number of responders are observed.

[0214] If treatment progresses beyond the initial radiological progression (i.e., radiological progression unconfirmed) according to RECIST v1.1, the principal investigator may discontinue treatment if he or she determines it is in the patient's best interest, the patient gives written or verbal consent to continue treatment, and the following criteria are met: no clinical symptoms or signs of clinically significant disease progression; no decline in performance status; no rapid disease progression or threat to vital organs or important anatomical sites requiring urgent alternative medical intervention (e.g., central nervous system [CNS] metastases, respiratory failure due to tumor compression, spinal cord compression); no significant unacceptable or irreversible toxicity associated with the investigational treatment; and no other criteria for discontinuation of treatment are met.

[0215] If disease progression (PD) was confirmed based on iRECIST, a rescan was required, preferably within 4 weeks, or at the latest within 8 weeks. If PD was confirmed according to iRECIST, continued treatment was not permitted. Otherwise, study treatment was continued for a total of up to 35 cycles, or until any of the following occurred: PD, unacceptable toxicity, or withdrawal of consent. Patients who discontinued treatment due to an unacceptable adverse event (AE) associated with TY22404-pembrolizumab combination therapy were followed until the AE resolved to grade 0 or 1 or stabilized, or until the patient received a new non-protocol treatment. Inclusion Criteria

[0216] Patients who meet all of the following inclusion criteria to be eligible to participate in this study: 1. The person must be 18 years of age or older at the time of informed consent. 2. The East Cooperative Oncology Group (ECOG) performance status is 0 or 1 and has not deteriorated over the past two weeks. 3. Dose escalation phase only: Patients with histologically or pathologically confirmed advanced or metastatic solid tumors whose disease has progressed after all standard treatments, or for whom no further standard treatments are available. 4. Dose escalation phase only: Patients must meet one of the following tumor types, including the corresponding criteria: This describes advanced CRC (Chronic Reproductive Cortex) that is not surgically curable and has been diagnosed as having MSS (Medical Stress Syndrome) status by a regional or central laboratory. Previously received at least two and three systemic treatment regimens Liver metastases not present No history of immunotherapy HNSCC with a history of immunotherapy Advanced HNSCC that is not treatable by surgical intervention or radiation therapy I have never previously received treatment with anticytotoxic T lymphocyte antigen (anti-CTLA4) therapy. You must have previously received one or two treatments, one of which must have included PD-1 therapy. For regimens containing PD-1, relapse or secondary resistance must be demonstrated, but primary resistance has not been demonstrated. Secondary resistance to anti-PD-1 / L1 therapy is defined by meeting all of the following criteria: a. At least two doses of an approved anti-PD-1 monoclonal antibody (mAb) have been administered. b. No PD has been recorded within 3 months of the initiation of previous anti-PD-1 treatment. HNSCC patients who developed PD within the first 3 cycles of PD1 / L1 therapy (regardless of whether progression is later confirmed) are considered to have primary resistance and are not eligible for this protocol. c. Post-PD-1 PD as defined in RECIST v1.1 has been demonstrated. Early evidence of PD should be confirmed by a second assessment at least four weeks after the date of the first recorded PD according to iRECIST, in the absence of rapid clinical progression. This decision is made by the investigator. If PD is confirmed, the date of the first recording of PD is considered the date of PD. Patients who have received anticytotoxic T lymphocyte antigen (anti-CTLA4) therapy are not eligible. HNSCC that has not previously received immunotherapy Advanced HNSCC for which surgical treatment or radiation therapy for curative purposes is not possible. Previous immunotherapy must have been administered, and only one line of systemic chemotherapy prior to this treatment is acceptable. A composite PD-L1 positive score (CPS) for tumors based on fresh or archived tumors must be 1 or greater using the PD-L1 IHC 22C3 test. Patients must have at least one measurable lesion at baseline, according to the RECIST v1.1 definition. Lesions located in previously irradiated areas are considered measurable if progression has been demonstrated in such lesions. 6. Appropriate blood function (defined as follows): a. The absolute neutrophil count (ANC) must be 1.5 × 10⁹ / L or higher within two weeks prior to the use of granulocyte colony-stimulating factor (G-CSF) such as filgrastim. b. The platelet count must be 75 × 10⁹ / L or higher without blood transfusion within two weeks (14 days) prior to the experimental treatment. c. Hemoglobin levels must be 9 g / dL or less within two weeks (14 days) prior to the study treatment, without blood transfusion or erythropoietin administration. 7. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels are 2.5 times or less above the upper limit of normal (ULN), and total bilirubin levels are 1.5 times or less above the upper limit of normal. Exception: Patients with Gilbert's syndrome or familial benign unconjugated hyperbilirubinemia as an underlying condition and with elevated serum bilirubin levels are eligible for enrollment. 8. Appropriate renal function is defined as a creatinine clearance of 45 mL / min or higher (according to the Cockcroft-Gault formula). 9. Coagulation tests are defined as follows: a. Activated partial thromboplastin time (aPTT) ≤ 1.5 × ULN. b. International normalized ratio (INR) ≤ 1.5 × ULN. Exception: For patients receiving warfarin anticoagulation therapy, INR ≤ 3 × ULN is acceptable. 10. Washout period from previous antitumor therapy: a. Small molecule inhibitors / chemotherapeutic agents: At least two weeks or five times the half-life, whichever is longer, before the first dose of the investigational drug (six weeks in the case of nitrosourea or mitomycin). b. Macromolecules such as mAbs, bispecific antibodies, antibody-drug conjugates, and fusion proteins: at least 4 weeks before the first dose of the research drug. c. Autologous stem cell transplantation (ASCT) or chimeric antigen receptor T cell (CAR-T) or chimeric antigen receptor natural killer (CAR-NK) cell therapy: at least 3 months prior to the first dose of the investigational drug. d. Radiotherapy for bone metastases or other non-target lesions: at least two weeks prior to the first dose of the study drug. Participants must have recovered from all radiation-related toxicity, not require corticosteroids, and not have radiation pneumonitis. The exceptions are as follows: Hormone therapy for prostate cancer using gonadotropin-releasing hormone agonists or antagonists. b. Hormone replacement therapy or oral contraceptives c. Current or past administration of denosumab (Xgeva), IV bisphosphonate, or oral bisphosphonate to prevent complications related to bone metastases. 11. Previous adverse events (AEs) have improved to baseline or grade ≤1 NCI CTCAE v5.0 (excluding patients with alopecia). Participants with grade ≤2 neuropathy may be eligible. Participants with endocrine-related AEs grade ≤2 may require treatment or hormone replacement therapy.

[0217] Safety assessment. Safety assessments were performed during specified periodic PE, vital signs, ECOG performance status, laboratory variables (e.g., liver tests / monitoring, hematology, coagulation tests, serological chemistry, urine tests, and pregnancy tests), ECG, and AE. AEs were classified according to NCI CTCAE v5.0.

[0218] Efficacy evaluation. Tumor response / progression was assessed at baseline and every 6 weeks (±1 week) for the first 4 cycles. If treatment continued beyond 4 cycles, assessments were performed every 9 weeks (±1 week) for the remainder of the treatment period until one of the following occurred: PD or death, discontinuation of treatment / study due to treatment toxicity, loss of follow-up, withdrawal of consent, initiation of a new cancer treatment, or completion / termination of the study, whichever occurred first. In this study, response and progression were assessed using the international criteria proposed by the RECIST v1.1 guidelines and / or iRECIST.

[0219] Pharmacokinetic and immunogenicity evaluation. Blood samples were collected from all patients to measure serum concentrations of TY22404 and pembrolizumab. PK parameters for TY22404 were monitored more intensively during the first treatment cycle. PK sampling was reduced. Pembrolizumab sampling is performed. Non-compartmental analysis is performed using Phoenix WinNonlin version 8.3 or later. PK parameters include, but are not limited to, AUC0-21d, AUClast, AUCinf, Cmax, Tmax, t1 / 2, MRT, CL, and Vss. Dose proportionality is also evaluated for AUC and Cmax. ADA blood samples for TY22404 are collected before administration in cycles 1-4, and thereafter every 4 cycles if treatment continues beyond 4 cycles. For pembrolizumab, ADA sampling is reduced. Furthermore, ADA samples were collected at the end of treatment (EOT) and, if possible, 30 days after the last dose.

[0220] Pharmacodynamic evaluation. Pharmacodynamic biomarkers for TY22404 are listed and summarized by time point and treatment as specified in the protocol and include, but are not limited to, serum proteins such as cytokines, peripheral immune cell subset profiling, tumor-infiltrating lymphocytes, and pharmacological genomic markers in tumor tissue (if available).

[0221] Tumor Assessment. Tumor PD-L1 IHC 22C3 testing for CPS assessment was performed on all patients in the expanded cohort. Only patients with head and neck squamous cell carcinoma who had not previously received immunotherapy and had a CPS ≥1 were enrolled in the dose-expansion phase trial. The combination dose-expansion cohort required tumor resection / biopsy formalin-fixed paraffin-embedded (FFPE) samples (block or 10 unstained FFPE slides) taken within 2 years from C1D1. If no archived tumor samples were available, tumor biopsies were collected at screening. Indicator lesions / target lesions or irradiated lesions were not used for biopsy. For CRC patients without existing MSI status reports, tumor samples (archived or fresh) were collected locally or at the central laboratory at the time of MSI screening, and only MSS CRC patients were enrolled in the expanded cohort. Patients with biopsyable tumors could also optionally undergo pre- and post-treatment tumor biopsies at baseline and at week 3 of cycle 2 and / or at EOT. Cutting of TY22404 in fresh post-treatment biopsies (if available) will be investigated in addition to other relevant biomarkers. Patients were given individual, specific written consent to provide baseline, intra-treatment, and / or EOT biopsies. Interim results

[0222] Eleven patients were receiving dose-escalation therapy with TY22404 (6 mg / kg Q3W and 10 mg / kg Q3W or Q6W) plus pembrolizumab (200 mg Q3W). Patients generally had sufficient prior treatment (Table 1). The types of tumors include ovarian cancer, colorectal cancer, pancreatic adenocarcinoma, and endometrial cancer, and most of them (82%) are generally considered "cold" tumors. Table 3. Patient baseline characteristics TIFF2026514050000005.tif85170 Clinical Safety Evaluation

[0223] As shown in Tables 4 and 5, no dose-limiting toxicities were observed with dose-escalation of TY22404 (6 mg / kg Q3W and 10 mg / kg Q3W or Q6W) + pembrolizumab (200 mg Q3W). The most frequently occurring TRAEs were fatigue (3 patients), diarrhea (2 patients), nausea (2 patients), and vomiting (2 patients). Most TRAEs were Grade (G) G1 and G2. Two patients experienced G3 TRAEs, one with G3 diarrhea as a delayed toxicity (C8 in the 6 mg / kg Q3W cohort) and the other with G3 adrenal insufficiency after DLT (C3 in the 10 mg / kg Q6W cohort). No G4 / 5 events were observed, and the initial safety profile was comparable to that of pembrolizumab monotherapy (Tables 4 and 5). Table 4. Frequency of TRAEs of different grades Table 5. TRAEs and frequencies during dose escalation (N=11). (TIFF2026514050000006.tif17170) TIFF2026514050000007.tif98170 Clinical Activity Evaluation

[0224] As shown in Figure 1A, a partial response (PR) was observed in a patient treated with TY22404 10 mg / kg Q3W + pembrolizumab 200 mg Q3W (see Case Study #1). Among 11 patients with tumor-responsiveness after baseline who received TY22404 (6 mg / kg Q3W, 10 mg / kg Q3W, or Q6W) + pembrolizumab (200 mg Q3W), the objective response rate (ORR) was 9% and the disease control rate (DCR) was 36%. Among 6 patients who received TY22404 10 mg / kg Q3W / 6W + pembrolizumab 200 mg Q3W, the ORR was 17% and the DCR was 50%. As shown in Figure 1B, one patient (10 mg / kg, Q3W, PD due to new lesions) did not complete and still does not have complete post-treatment target lesion measurements. Clinical case studies

[0225] Case Study 1: As shown in Table 6, in a patient with advanced endometrial adenocarcinoma (MSI-H) with lung metastases, a definite partial response (PR) with 33% and 37% target lesion reduction was observed at the end of C2 and C4 cycles after administration of TY22404 10 mg / kg Q3W + pembrolizumab 200 mg Q3W. This patient had previously received 6 cycles of carboplatin + paclitaxel and subsequently received anastrozole maintenance therapy until new lung metastatic lesions appeared. Table 6. Tumor evaluation in patients with metastatic endometrial cancer. TIFF2026514050000008.tif34170

[0226] Case Study 2: As shown in Table 7, in a patient with advanced cervical cancer (stage IV squamous cell carcinoma) with mediastinal lymph node metastasis, administration of TY22404 10 mg / kg Q3W + pembrolizumab 200 mg Q3W resulted in a 13% reduction in target lesions and confirmed stable disease (SD) at the end of C6. This patient had a PD-L1 CPS score of 1 and a TMB High of 24 Muts / Mb. This patient had previously received two types of treatment: carboplatin / paclitaxel / bevacizumab x 6 cycles and pembrolizumab monotherapy x 9 cycles. Supported by PK modeling, TY22404 10 mg / kg Q3W + pembrolizumab demonstrates the ability to overcome pembrolizumab resistance in patients with 3L cervical cancer (Figure 11). Table 7. Tumor evaluation in patients with advanced cervical cancer (stage IV squamous cell carcinoma). TIFF2026514050000009.tif49170

[0227] As shown in Table 8, two partial responses (PRs) (2 / 11) were observed in patients treated with TY22404 (6 mg / kg Q3W, 10 mg / kg Q3W, or Q6W) + pembrolizumab (200 mg Q3W) during the dose escalation phase, one of whom was a cervical cancer patient whose disease progressed during pembrolizumab treatment. Two initial PRs were observed in the MSS CRC expanded cohort, and one initial PR was observed in the HNSCC expanded cohort in the dose escalation phase treated with TY22404 (10 mg / kg Q3W or Q6W) + pembrolizumab (200 mg Q3W). Table 8: Tumor evaluation in patients treated with TY22404 / pembrolizumab TIFF2026514050000010.tif76170

[0228] Case Study 3: A 58-year-old patient with advanced rectal adenocarcinoma with brain, lung, and lymph node metastases was treated with TY22404 10 mg / kg Q6W + pembrolizumab 200 mg Q3W. This patient had a PD-L1 CPS score of 0 and a TMB from ctDNA of 7 Muts / Mb. This patient had previously received two types of treatment: FOLFOXIRI + Bev and 5-FU + XRT. As shown in Table 9, the target lesion was reduced by 67% at the end of C4, and confirmed PD due to new lesions (initial PR with a 56% reduction at the end of cycle 2) was observed. In the mPBPK model, greater variability in tumor resection PK and a decrease in resection AUC / Cmax were predicted compared to 10 mg / kg Q3W administration (see Figure 12). Table 9: Tumor evaluation in patients with advanced rectal adenocarcinoma TIFF2026514050000011.tif64170

[0229] Case Study 4: A 66-year-old patient with advanced colorectal adenocarcinoma with lung metastases received TY22404 10 mg / kg Q3W + pembrolizumab 200 mg Q3W. MSS status was good, and TMB from ctDNA was 11 Muts / Mb. This patient had previously received three other treatments: adjuvant FOLFOX, FOLFIRI + Vectibix, and riboceranib (VEGF-R2) + TAS-102. As shown in Table 10, target lesions decreased by 67% at the end of C4, and confirmed PD due to new lesions was observed (initial PR with a 56% decrease at the end of cycle 2). Table 10: Tumor evaluation in patients with advanced colorectal adenocarcinoma TIFF2026514050000012.tif71170

[0230] Case Study 5: A 66-year-old patient with HNSCC squamous cell carcinoma of the head and neck (HNSCC), HNSCC IO-free (stage IVA), and lung metastases, received TY22404 10 mg / kg Q6W + pembrolizumab 200 mg Q3W. This patient had a PD-L1 CPS score of 5. This patient had previously received adjuvant therapy with cisplatin and one line of palliative care, docetaxel / cisplatin. As shown in Table 11, the patient showed a partial response, with a 100% reduction in target lesions at the end of C4 and confirmation at C7. Table 11 Tumor Assessment in HNSCC Patients TIFF2026514050000013.tif57170

[0231] Case Study 6: A 55-year-old patient with advanced adenocarcinoma of the colon (MSS) and para-aortic lymph node metastasis (stage IV) received TY22404 10 mg / kg Q3W + pembrolizumab 200 mg Q3W. This patient had previously received two types of palliative therapy: FOLFOX + bevacizumab and FOLFIRI + aflibercept. As shown in Table 12, the patient showed a partial response (PR), with the target lesion (lymph node) decreasing from 20 mm to 8 mm (normal lymph node size) by the end of C2, and continuing to decrease to 5 mm by the end of C4. TY22404 10 mg / kg Q3W plus pembrolizumab 200 mg Q3W showed a confirmed partial response in MSS CRC, which also demonstrated that model-notified PK and efficacy case studies support the TY22404 dose selection of 10 mg / kg Q3W in microsatellite stability (MSS)-colorectal cancer (CRC) (Figure 13). Table 12: Tumor evaluation in patients with advanced colorectal adenocarcinoma TIFF2026514050000014.tif33170 Biomarker modulation, surrounding area

[0232] In the Phase I clinical trial of TY22404, patients were enrolled in dose-escalation cohorts with monotherapy at 0.1 mg / kg, 0.3 mg / kg, 1.0 mg / kg, 3.0 mg / kg, 10 mg / kg, and 20 mg / kg, and were also enrolled in combination therapy with TY22404 at 6 mg / kg and 10 mg / kg in combination with 200 mg pembrolizumab. Serum samples were prepared from peripheral blood collected at a series of consultation times (before administration on day 1 of cycle 1, day 8 of cycle 1, day 15 of cycle 1, before administration on day 1 of cycle 2, before administration on day 1 of cycle 3, and before administration on day 1 of cycle 4) according to a standard protocol. Serum concentrations of a panel of inflammatory cytokines, including IFN-γ, TNF-α, IL-2, and IL-6, which are early responses to immune activation, were quantified using the V-Plex Proflammable Panel 1 assay (catalog number K151A9H) from Mesoscale Discovery (MSD) Technologies, following manufacturer's instructions. As shown in Figure 2, IFN-γ peak levels in various patients are shown as a multiplicative change compared to the corresponding baseline level (cycle 1, day 1, pre-administration). Each dot represents the change in IFN-γ in one patient. The results indicate that TY22404 monotherapy induces low levels of peripheral immune activation, manifesting as a dose-dependent but limited increase in IFN-γ. However, the magnitude of the IFN-increase or immune activation is more pronounced when TY22404 is combined with pembrolizumab. Example 2: Optimal dose selection of TY22404 in combination with an anti-PD-1 antibody using QSP modeling, which significantly broadened the therapeutic index compared to ipilimumab.

[0233] The developed mPBPK model can be fitted to observed pharmacokinetic (PK) data across dose levels of 10 mg / kg every three weeks (Q3W). As shown in Figures 3A and 3B, PK is used as a representative dose group, showing predicted (e.g., dashed line) and measured (i.e., observed) plasma concentrations of complete and cleaved antibodies, respectively. Despite the accumulation of cleaved TY22404 in plasma over each dosing cycle (Figure 3B), the simulated maximum steady-state exposure (Cmax,ss) of cleaved TY22404 at 10 mg / kg Q3W or Q6W (data not shown) doses is approximately one-sixth or one-twelfth of the mean Cmax,ss of parental Ab TY22404 administered at 3 mg / kg Q3W, respectively (Figure 3B). These results are consistent with the reduction of circulating PD biomarkers and reflect the reduced systemic immune activation and the superior clinical safety profile of TY22404. Therefore, TY22404 can be safely administered as monotherapy or in combination with other therapies (e.g., pembrolizumab). Example 3: Modeling of the physiological pharmacokinetics (mPBPK) of TY22404 across species

[0234] To model the whole, intact, and cleaved forms of TY22404 after administration to different species (mice, rays, cynomolgus monkeys, and humans), minimal physiological pharmacokinetic (mPBPK) models were developed. Known molecular transformations and mass balances of the whole, intact, and cleaved forms of TY22404 were integrated for all compartments. The antibody circulates primarily from the plasma compartment through the ISF_leaky (i.e., leaking normal tissue interstitial fluid) and ISF_tight (i.e., tight normal tissue interstitial fluid) compartments to the lymphatic compartment, and then returns to the plasma (see Figure 4). Additional exchange occurs between the plasma compartment and the tumor_VS (i.e., tumor vascular space) compartment, with a portion continuing to circulate from tumor_VS to tumor_IS (i.e., tumor stromal space) and then flowing into the lymph. Clearance occurs only from the plasma compartment. The mPBPK model successfully characterized the plasma and tumor PK of tumor-bearing mice after a single 10 mg / kg dose, enabling the estimation of tumor cleavage parameters for TY22404 (see Figure 14). For tumor-related parameters, PK data measured in tumor-bearing mice (tumor PK, plasma PK, etc.) were further modeled to estimate the tumor cleavage rate constant in mice, and the same value was maintained in the human model.

[0235] Using PK parameters from a population model fitted to observed PK data, we performed simulations of a virtual patient (VP) to generate mean PK and variability (e.g., 95% confidence interval) at a 10 mg / kg Q3W dose. The simulated PK of cleaved drugs in tumor interstitial fluid (ISF) was higher than the upper limit of EC90 for human T cell binding of MMP9-cleaved TY22404 in vitro (see Figure 5), supporting the effectiveness of 10 mg / kg Q3W as a dose, which has been validated by new clinical efficacy data. The simulated PK of cleaved drugs in normal interstitial fluid (ISF), including leaky and tightly packed tissues, supported the excellent safety profile observed in clinical trials of TY22404. Example 4: Comparison of predictive tumor PK efficacy between TY22404 and ipilimumab

[0236] The predicted tumor ISF concentration of ipilimumab (Ipi) at 1 mg / kg Q6W or 3 mg / kg Q3W cannot cover its EC90, human T cell binding, during each specific dosing interval (see Figure 6). Even at 3 mg / kg Q3W, the predicted tumor Cmax obtained using a 10% tumor tissue compartment is approximately half of the in vitro EC90, human T cell binding, compared to plasma (i.e., systemic concentration).

[0237] In contrast, as shown in Figure 6, at steady state (SS), the maximum cleavage TY22404 tumor interstitial fluid (ISF) concentration at 10 mg / kg Q3W administration is predicted to be significantly higher on average in the tumor microenvironment (TME) than ipilimumab at 3 mg / kg Q3W*4 or 1 mg / kg Q6W, respectively. The simulated PK of cleaved TY22404 in tumor ISF is higher than the upper limit of EC90 for human T cell binding of MMP9-cleaved TY22404 in vitro (e.g., dashed line). TY22404 is predicted to achieve a higher target occupancy rate (RO>90%) throughout the entire steady-state (SS) administration cycle at 10 mg / kg Q3W in the TME compared to ipilimumab at 3 mg / kg Q3W or 1 mg / kg Q6W. TY22404 (10 mg / kg Q3W) is predicted to reduce active drug exposure in normal tissues compared to ipilimumab (3 mg / kg Q3W), which is reflected in the reduced systemic pharmacokinetics (see Figure 7). In summary, PK modeling showed that the therapeutic index (TI) of TY22404, when combined with an anti-PD-1 agent, is improved compared to ipilimumab. Example 5: PK Modeling of Various TY22404 Dosage Schedules

[0238] Model predictions based on preliminary TY22404 clinical PK data suggest that a 10 mg / kg Q3W dosing regimen is likely to cover the upper limit of the in vitro EC90 for cleaved TY22404 in TME (see Figure 8). Furthermore, as shown in Figure 8, a 20 mg / kg loading dose + 10 mg / kg Q3W dosing schedule can achieve tumor cleavage drug concentrations equivalent to those of steady-state 10 mg / kg Q3W dosing in cycle 1, potentially enhancing efficacy while maintaining comparable safety to 10 mg / kg Q3W. Example 6: When a maintenance dose is administered after a single loading dose, the plasma concentration of cleaved TY22404 rapidly reaches a steady-state plasma concentration.

[0239] As shown in Figure 9, we applied a virtual patient simulation of the developed mPBPK model to further investigate the role of the maintenance dose after a single loading dose. The simulation predicts that a loading dose of 20 mg / kg and a maintenance dose of 10 mg / kg Q3W will result in the target steady-state plasma concentration in the tumor ISF in cycle 1 (see tumor_IS.cleft in Figure 9). Despite the accumulation of cleaved TY22404 in plasma, the maximum steady-state exposure of cleaved TY22404 simulated with a loading dose of 20 mg / kg and a Q3W dose of 10 mg / kg (see plasma.cleft under tumor_IS.cleft) is approximately one-sixth of the mean Cmax,ss of the parent antibody TY22404 at a 3 mg / kg Q3W dose, indicating that the safety when combined with anti-PD-1 mAbs (such as pembrolizumab) is manageable. Even considering the 95% upper limit of population PK variation, the mean Cmax,ss of the parent antibody TY22404 administered at 3 mg / kg Q3W was approximately three times smaller, further supporting the idea that this regimen provides manageable safety similar to that of 10 mg / kg Q3W administration, while simultaneously potentially improving efficacy in some patients. Example 7: Mechanism-based safety modeling of TY22404 combined with an anti-PD1 antibody

[0240] A novel mechanism-based model integrating pharmacokinetic (PK), pharmacodynamic (PD), and safety data was constructed using publicly available clinical and in vitro data for ipilimumab (Ipi), pembrolizumab (Pembro), Ipi+Pembro, Ipi+nivolumab (Nivo), and tremelimumab (Treme). As shown in Figure 10, the model predicts that TY22404 at 10 mg / kg Q3W and 20 mg / kg Q3W will show a clear advantage in treatment-related adverse events (TrAEs) compared to Ipi 3 mg / kg Q3W*4 doses in combination settings. Furthermore, repeated administration of TY22404 at 20 mg / kg Q3W resulted in a slight increase in ≥G3 TrAEs compared to repeated administration of 10 mg / kg Q3W (e.g., an increase of less than 10% of the absolute mean). New clinical safety data for TY22404 are consistent with the predicted TrAE range (e.g., with repeated dosing of 10 mg / kg Q3W, the ≥G3 TrAE rate was less than 20% compared to the initial safety data). Example 8: Dose escalation phase (clinical trial design) - Combination therapy with TY22404 and pembrolizumab

[0241] A mTPI design with a target dose-limiting toxicity (DLT) rate of approximately 20% and an equivalence interval (EI) of [0.15, 0.23] (in this design, all doses are considered candidates for the true maximum tolerated dose (MTD)) was applied to dose escalation and confirmation to determine the RP2D in combination with TY22404 and pembrolizumab. The dose levels are shown in Table 13. Table 13: TY22404 - Pembrolizumab Dose Levels TIFF2026514050000015.tif28170DL=dose level; mTPI=modified toxicity probability interval; Q3W=every 3 weeks; Q6W=every 6 weeks. DL1: The dosing regimen will be further determined based on clinical data from the initial repeated dosing at 6 mg / kg. If dosing every 3 weeks is not well tolerated, a reduced dosing frequency schedule (e.g., every 6 weeks) will also be an alternative. DL2 and DL3: These doses and administration regimens are determined sequentially based on the clinical data for DL1 and DL2, respectively. A loading dose (e.g., ≤20 mg / kg) is followed by a reduced dose (e.g., including ≤10 mg / kg, 3 mg / kg, or 6 mg / kg every 3 weeks) and / or a reduced administration schedule (e.g., every 6 weeks).

[0242] Dose escalation will begin with 6 mg / kg Q3W according to the mTPI design (DL1). Based on SRC review, if this dose is tolerable based on early or late toxicity, administration will proceed to 10 mg / kg Q3W. Based on SRC review, if 6 mg / kg Q3W is not tolerable based on early or late toxicity, administration will proceed to 6 mg / kg Q6W. Based on SRC review, if 6 mg / kg Q6W is tolerable based on early and late toxicity, administration will proceed to 10 mg / kg Q6W. Similarly, based on SRC early and late toxicity review, if 6 mg / kg Q3W is tolerable but 10 mg / kg Q3W is not, administration may proceed to 10 mg / kg Q6W. Furthermore, based on SRC early and late toxicity review, if 10 mg / kg Q3W is tolerable, administration may proceed to 20 mg / kg Q3W. The 20 mg / kg dose escalation cohort will include only MSS CRC (less than 50% liver metastases) and NSCLC patients positive for 2L anti-PD-1 / L1 antibodies. The SRC will determine the dose / schedule for the dose expansion phase based on the overall data.

[0243] DLT was evaluated using the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE) v5.0. The safety and tolerability of each dose level of TY22404 were evaluated by SRC after all patients enrolled in that dose level were followed for at least 21 days (DLT observation period) after the first dose of the TY22404-pembrolizumab combination regimen. The dose and frequency of pembrolizumab administration remained unchanged.

[0244] Dose-dependent toxicity of anti-CTLA-4 therapies significantly limits their efficacy and therapeutic index (TI). Ipilimumab, the first anti-CTLA-4 therapy approved by the FDA both as monotherapy and in combination with anti-PD-1 therapy, is limited by safety concerns related to dose levels, frequency, and cycles that may not maximize antitumor efficacy. Tremelimumab, the second anti-CTLA-4 antibody approved by the FDA, faces similar challenges in combination therapy, despite being effective in frontline settings with limited dosing frequency. Next-generation anti-CTLA-4 therapies need to achieve higher efficacy through improved TIs that allow for repeated dosing and sufficiently effective dose levels. TY22404, a masked anti-CTLA-4 SAFEbody, is designed to enable repeated dosing at active dose levels due to its improved TI by targeting a specific and highly conserved epitope of CTLA-4 on Treg cells in the tumor microenvironment (TME), which is preferentially enriched and activated to enable CTLA-4-mediated depletion of Tregs in the TME via epitope-dependent effector functions such as ADCC. Optimal dose selection of TY22404 in combination with anti-PD-1 antibodies requires quantitative evaluation of various dosing regimens, including PK / PD modeling of the effects of masked and cleaved drug concentrations in plasma / tumor on efficacy and safety. Fully activated TY22404 or ADG116 allows for a quantitative approach to TI evaluation by seamlessly integrating preclinical and clinical data, predicting cleaved TY22404 in the TME using the same molecule in patient and in vivo animal models, and features a unified set of physiologically relevant parameters for population PK modeling of more than 50 patients across the entire study.

[0245] A quantitative systems pharmacology (QSP) model was developed by incorporating drug-specific doses across the entire melanoma trial into a publicly available model evaluating ipilimumab and pembrolizumab (Kumar R, Thiagarajan K, Jagannathan L, et al. CPT Pharmacomet Syst Pharmacol. 2021; 10(7): 684-695). Data for ipilimumab and nivolumab were also used. The properties of TY22404 were integrated by mPBPK modeling (Park J, Ariyapperuma M, Richardson G, et al. Journal of Clinical Oncology 2023, 41, no. 16_suppl). Furthermore, a new safety model was developed incorporating data for ipilimumab, tremelimumab, pembrolizumab, and nivolumab. In hot tumors, TY22404 10 mg / kg Q3W is predicted to yield an objective tumor response rate equivalent to ipilimumab 3 mg / kg Q3W*4 using an anti-PD-1 antibody, but with significantly improved safety. In colder, higher tumor-bearing scenarios, administration of TY22404 at 10 mg / kg or higher every three weeks showed superior predictive efficacy compared to administration of ipilimumab 3 mg / kg every three weeks*4. Furthermore, in safety models, a more than twofold reduction in concomitant TRAEs of grade 3 or higher was predicted when comparing TY22404 10 mg / kg Q3W administration with ipilimumab 3 mg / kg Q3W administration*4 (Jedd D. Wolchok, et al. N Engl J Med 2017.377(14): p. 1345-1356.), which was confirmed by the clinical findings of TY22404.

[0246] The unique molecular design and properties of the masked SAFEbody TY22404 enable meaningful mPBPK and QSP modeling evaluations in translational and clinical studies. These models predict increased TI of TY22404 compared to ipi, whether used as monotherapy or in combination with anti-PD-1. The expanded TI of TY22404 allows for repeated administration of TY22404 10 mg / kg Q3W with anti-PD-1 antibodies, resulting in a significantly increased involvement of CTLA-4 by activated TY22404 compared to circulating blood in steady state within tumors. Early clinical data support the finding that TY22404, when used in combination with anti-PD-1, offers greater clinical benefit and demonstrates clinical response in MSS CRCs, etc., with better target engagement in TMEs, while maintaining a favorable safety profile. Example 9: Loading a dose selection of TY22404 with predicted increased therapeutic benefit. Using exposure response (ER) analysis of available safety and efficacy data and virtual patient simulations of previously developed mPBPK models, the efficacy of the following additional loading dose regimens was further investigated: (1) two loading doses of 20 mg / kg Q3W followed by a maintenance dose of 10 mg / kg Q3W; and (2) one loading dose of TY22404 at 30-50 mg / kg Q3W followed by a maintenance dose of 10 mg / kg Q3W.

[0247] For analysis, a maintenance dose of 10 mg / kg Q3W TY22404 in combination with pembrolizumab was selected. This is because this regimen appears to improve clinical efficacy without worsening the safety profile compared to pembrolizumab monotherapy or TY22404 / pembrolizumab combination therapy with a maintenance dose of 6 mg / kg Q3W or 10 mg / kg Q6W Ty22404 over 14 cycles. Based on mPBKB modeling of TY22404 pharmacokinetic data, mg / kg Q3W TY22404 is expected to best cover the target effective exposure at steady state. Furthermore, the TY22404 / pembrolizumab combination therapy showed a similar incidence of Grade 3 treatment-related adverse events (TrAEs) as pembrolizumab monotherapy, with a Grade 3 TrAE incidence of 20% with TY22404 6 mg / kg Q3W and 12.5% ​​with 10 mg / kg Q3W. No Grade 4 or Grade 5 TrAEs were observed.

[0248] As shown in Figure 15, virtual patient simulations indicate that in Cycle 1, a single loading dose of 30 mg / kg or higher is more likely to achieve the target effective plasma concentration of approximately 70 nM for TY22404, based on current population ER analysis, compared to a 10 mg / kg Q3W without an initial high-concentration negative loading dose. Comparing the predicted plasma cleavage pharmacokinetics in Cycle 1 between a 20 mg / kg loading dose and a 30 mg / kg loading dose, the predicted plasma concentrations differ by 80 nM and 120 nM, respectively. Therefore, clinical investigations are needed to confirm whether achieving the target concentration earlier in Cycle 1 using higher loading doses, such as 30 mg / kg or higher, improves the overall clinical response. Taken together, these data suggest that additional loading dose regimens may enhance the therapeutic effect.

[0249] Furthermore, Figure 15 also shows that the model-predicted plasma concentrations of the cleavage drug in Cycles 2-4 are similar between the use of a single loading dose of 30 mg / kg or more and the use of two doses of a 20 mg / kg Q3W loading dose. This result supports the use of the safety information obtained. Starting from two loading doses of 20 mg / kg Q3W, it is possible to introduce a single loading dose of 30 mg / kg or more, followed by a maintenance dose of 10 mg / kg Q3W. Furthermore, as shown in Figure 16A, through modeling, the maximum cleavage TY222404 concentration in interstitial fluid (ISF) at 30 mg / kg in Cycle 1 is estimated to exceed twice the upper limit of the in vitro EC90 (e.g., 90 nM) of the human T cell binding assay described in Example 3, thereby allowing for consideration of translation and modeling uncertainties such as tumor cleavage in patients and PK variability at the population level. Furthermore, as shown in Figure 16B, the model estimated that the maximum cleavage TY22404 concentration in the interstitial fluid of leaky normal tissues in Cycles 1 and 2 using a single loading dose of 30 mg / kg would be approximately 1.5 to 2 times lower than the upper limit of the in vitro EC90 (e.g., 90 nM). The simulation also predicted that there would be no difference in steady-state exposure when comparing any of the proposed loading dose regimens. Taken together, these data indicate that the proposed single loading dose regimen of 30-50 mg / kg may have good tolerability and an acceptable safety profile when combined with pembrolizumab.

[0250] In summary, a single loading dose of 30-50 mg / kg, followed by a maintenance dose of 10 mg / kg Q3W, is predicted to approach the target plasma cleavage drug concentration at which two loading doses of 20 mg / kg Q3W reach Cycle 2 one cycle earlier, thus increasing clinical efficacy and rational dose escalation, while also maintaining the safety of the 10 mg / kg Q3W maintenance dose based on the predicted steady-state level of cleaved TY22404, which is lower than the maximum predicted leakage tissue exposure. Therefore, the proposed loading dose regimen may enhance the clinical effect while maintaining the safety profile. Exemplary sequences Array number 23 Anti-CTLA4 capable of activating HVR-H1 YSISSGYHWSWI Array number 35 Anti-CTLA4 capable of activating HVR-H2 LARIDWDDDKYYSTSLKSRL Array number 45 Anti-CTLA4 capable of activating HVR-H3 ARSYVYFDY Array number 58 Anti-CTLA4 capable of activating HVR-L1 RASQSVRGRFLA Array number 66 Anti-CTLA4 capable of activating HVR-L2 DASNRATGI Array number 75 Anti-CTLA4 capable of activating HVR-L3 YCQQSSSWPPT Array number 87 EVQLVESGGGLVQPGGSLRLSCAASGYSISSGYHWSWIRQAPGKGLEWLARIDWDDDKYYSTSLKSRLTISRDNSKNTLYLQLNSLRAEDTAVYYCARSYVYFDYWGQGTLVTVSS Array number 100: RFLAWYQQKPGKAPKLLIYDASNRATGIPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSSSWPPTFGQGTKVEIKR Array number 192 Masking part (MM) EVGSYPNPSSDCVPYYYACAY [[ID=4⑥]]Array number 221 Cleavable part (CM) SGRSAGGGGTPLGLAGSGGS Array number 200 Masking part (MM) + Cleavable part (CM) EVGSYPNPSSDCVPYYYACAYSGRSAGGGGTPLGLAGSGGS Sequence ID 320: Activatable anti-CTLA4 2-unit full heavy chain (excluding C-terminal lysine) EVQLVESGGGLVQPGGSLRLSCAASGYSISSGYHWSWIRQAPGKGLEWLARIDWDDDKYYSTSLKSRLTISRDNSKNTLYLQLNSLRAEDTAVYYCARSYVYFDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Sequence ID 321: Activatable anti-CTLA4 2-unit full heavy chain (including C-terminal lysine) EVQLVESGGGLVQPGGSLRLSCAASGYSISSGYHWSWIRQAPGKGLEWLARIDWDDDKYYSTSLKSRLTISRDNSKNTLYLQLNSLRAEDTAVYYCARSYVYFDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence ID 322: Activatable anti-CTLA4 2 complete light chain (including N-terminal masking moiety and linker) EVGSYPNPSSDCVPYYYACAYSGRSAGGGGTPLGLAGSGGSDIQLTQSPSSLSASVGDRVTITCRASQSVRGRFLAWYQQKPGKAPKLLIYDASNRATGIPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQSSSWPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Sequence ID 323: Activatable anti-CTLA4 1 full heavy chain (excluding C-terminal lysine) EVQLVESGGGLVQPGGSLRLSCAASGYSISSGYHWSWIRQAPGKGLEWLARIDDDKYYSTLKSRLTISRDNSKNTLYLQLNSLRAEDTAVYYCARSYVYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

Claims

1. A method for treating cancer in a subject, comprising (a) administering an effective amount of an activatable antibody to the subject, wherein the activatable antibody comprises HVR-H1 containing the amino acid sequence of formula YSISSGYHWSWI (SEQ ID NO: 23), HVR-H2 containing the amino acid sequence of formula LARIDWDDDKYYSTSLKSRL (SEQ ID NO: 35), HVR-H3 containing the amino acid sequence of formula ARSYVYFDY (SEQ ID NO: 45), HVR-L1 containing the amino acid sequence of formula RASQSVRGRFLA (SEQ ID NO: 58), HVR-L2 containing the amino acid sequence of formula DASNRATGI (SEQ ID NO: 66), and HVR-L3 containing the amino acid sequence of formula YCQQSSSWPPT (SEQ ID NO: 75), and the activatable antibody. The possible antibody further comprises a polypeptide covalently bonded to the N-terminus of the light chain of an anti-CTLA4 antibody, comprising a polypeptide having a masking portion (MM) and a cleavable portion (CM) from the N-terminus to the C-terminus, wherein the MM comprises the amino acid sequence EVGSYPNPSSDCVPYYYACAY (SEQ ID NO: 192), and the cleavable portion comprises the amino acid sequence SGRSAGGGGTPLGLAGSGGS (SEQ ID NO: 221); and (b) an effective amount of pembrolizumab, wherein the activatable antibody is administered once every 3 to 6 weeks at a dose of approximately 6 mg / kg to approximately 30 mg / kg, and the pembrolizumab is administered once every 3 weeks at a dose of approximately 100 mg to approximately 300 mg, or once every 6 weeks at a dose of approximately 200 mg to approximately 600 mg.

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

3. The method according to claim 1, wherein the activatable antibody is administered once every 3 to 6 weeks at a dose of approximately 10 mg / kg to approximately 20 mg / kg.

4. The method according to claim 1, wherein the activatable antibody is administered once every 3 to 6 weeks at a dose of approximately 20 mg / kg to approximately 30 mg / kg.

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

6. The method according to claim 1, wherein the activatable antibody is administered once every three weeks at a dose of 20 mg / kg.

7. The method according to claim 1, wherein the activatable antibody is administered once every six weeks at a dose of 20 mg / kg.

8. The method according to claim 1, wherein the activatable antibody is administered once every six weeks at a dose of 30 mg / kg.

9. The method according to claim 1, wherein the activatable anti-CTLA4 antibody is administered at a dose of approximately 6 mg / kg once every 3 to 6 weeks.

10. The method according to any one of claims 1 to 6, wherein the pembrolizumab is administered once every three weeks in a dose of approximately 200 mg.

11. The method according to claim 1, 7, or 8, wherein the pembrolizumab is administered once every six weeks in a dose of approximately 400 mg.

12. The method according to any one of claims 1 to 11, wherein the cancer is resistant to or refractory to conventional therapy, and the conventional therapy is an inhibitor of CTLA4, PD-1, or a PD-1 ligand.

13. The method according to claim 12, wherein the conventional therapy is ipilimumab.

14. The method according to any one of claims 1 to 13, wherein the cancer is colorectal cancer.

15. The method according to claim 14, wherein the CRC is microsatellite-stable (MSS) CRC.

16. The method according to claim 15, wherein the MSS CRC has not metastasized to the liver.

17. The method according to claim 15, wherein the MSS CRC has not metastasized to the peritoneum.

18. The method according to claim 15, wherein the MSS CRC has not metastasized to the liver or peritoneum.

19. The method according to any one of claims 1 to 13, wherein the cancer is endometrial cancer.

20. The method according to any one of claims 1 to 13, wherein the cancer is neuroendocrine carcinoma, cecal adenocarcinoma, pancreatic cancer, or ovarian cancer.

21. The method according to any one of claims 1 to 15, 19, or 20, wherein the cancer is advanced metastatic cancer.

22. The method according to claim 21, wherein the cancer has metastasized to the lungs or liver.

23. The activatable anti-CTLA4 antibody comprises a heavy chain variable region including the amino acid sequence of SEQ ID NO: 87 or a variant thereof having at least about 90% sequence identity with respect to the amino acid sequence of SEQ ID NO: 87, and a light chain variable region including the amino acid sequence of SEQ ID NO: 100 or a variant thereof having at least about 90% sequence identity with respect to the amino acid sequence of SEQ ID NO: 100, the method according to any one of claims 1 to 22.

24. The method according to claim 23, 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.

25. The method according to claim 24, wherein the activatable antibody comprises the full heavy chain region of SEQ ID NO: 320 or SEQ ID NO:

321.

26. The method according to claim 25, wherein the activatable anti-CTLA4 antibody comprises the complete light chain region of SEQ ID NO: 322 or SEQ ID NO:

323.

27. The method according to any one of claims 1 to 26, wherein the subject is a human.

28. The method according to any one of claims 1 to 27, wherein both the activatable anti-CTLA4 antibody and pembrolizumab are administered on day 1 of a 3 to 6-week administration schedule.

29. A method for treating cancer in a subject, wherein an effective amount of an activatable antibody is combined with pembrolizumab in the subject, and the activatable antibody is HVR-H1 containing the amino acid sequence of formula YSISSGYHWSWI (SEQ ID NO: 23), HVR-H2 containing the amino acid sequence of formula LARIDWDDDKYYSTSLKSRL (SEQ ID NO: 35), HVR-H3 containing the amino acid sequence of formula ARSYVYFDY (SEQ ID NO: 45), HVR-L1 containing the amino acid sequence of formula RASQSVRGRFLA (SEQ ID NO: 58), HVR-L2 containing the amino acid sequence of formula DASNRATGI (SEQ ID NO: 66), and HVR-L2 containing the amino acid sequence of formula YCQQSSSWPPT (SEQ ID NO: 75). The activatable antibody, comprising HVR-L3, further comprises a polypeptide covalently bonded to the N-terminus of the light chain of the anti-CTLA4 antibody, comprising a polypeptide having a masking moiety (MM) and a cleavable moiety (CM) from the N-terminus to the C-terminus, wherein the MM comprises the amino acid sequence EVGSYPNPSSDCVPYYYACAY (SEQ ID NO: 192), and the cleavable moiety comprises the amino acid sequence SGRSAGGGGTPLGLAGSGGS (SEQ ID NO: 221). The activatable antibody is administered as one to three loading doses of approximately 20 mg / kg to approximately 50 mg / kg, and thereafter as a maintenance dose of approximately 5 mg / kg to approximately 20 mg / kg once every three weeks or once every six weeks.

30. The method according to claim 29, wherein the activatable antibody is administered as one to three loading doses of about 20 mg / kg to about 40 mg / kg, followed by a maintenance dose of about 6 mg / kg to about 20 mg / kg once every three weeks or once every six weeks.

31. The method according to claim 29, wherein the activatable antibody is administered as a loading dose of approximately 20 mg / kg to approximately 40 mg / kg in one to three doses, followed by a maintenance dose of approximately 6 mg / kg to approximately 10 mg / kg once every three weeks or once every six weeks.

32. The method according to claim 29, wherein the activatable antibody is administered as a loading dose of approximately 20 mg / kg to approximately 40 mg / kg in one to three doses, followed by a maintenance dose of approximately 10 mg / kg to approximately 20 mg / kg once every three weeks or once every six weeks.

33. The method according to claim 29, wherein the activatable antibody is administered as a single loading dose of about 20 mg / kg to about 40 mg / kg, followed by a maintenance dose of about 6 mg / kg to about 10 mg / kg once every three weeks or once every six weeks.

34. The method according to any one of claims 29 to 33, wherein the loading dose is 20 mg / kg.

35. The method according to any one of claims 29 to 33, wherein the loading dose is 30 mg / kg.

36. The method according to any one of claims 29 to 33, wherein the loading dose is 40 mg / kg.

37. The method according to any one of claims 29 to 36, wherein a single loading dose is administered to the subject before the administration of the maintenance dose.

38. The method according to any one of claims 29 to 37, wherein two loading doses are administered to the subject before the administration of the maintenance dose.

39. The method according to any one of claims 29 to 37, wherein three loading doses are administered to the subject before the administration of the maintenance dose.

40. The method according to any one of claims 29 to 39, wherein the maintenance dose is administered at 10 mg / kg.

41. The method according to claim 40, wherein the maintenance dose is administered once every three weeks.

42. The method according to claim 40, wherein the maintenance dose is administered once every six weeks.

43. The method according to any one of claims 29 to 39, wherein the maintenance dose is administered at 20 mg / kg.

44. The method according to claim 43, wherein the maintenance dose is administered once every three weeks.

45. The method according to claim 43, wherein the maintenance dose is administered once every six weeks.

46. The method according to any one of claims 29 to 45, wherein the first maintenance dose is administered three weeks after the last loading dose.

47. The method according to any one of claims 29 to 46, wherein the pembrolizumab is administered in a dose of approximately 100 mg to approximately 300 mg once every three weeks, or approximately 200 mg to approximately 600 mg once every six weeks.

48. The method according to any one of claims 29 to 47, wherein the cancer is resistant to or refractory to conventional therapy, and the conventional therapy is an inhibitor of CTLA4, PD-1, or a PD-1 ligand.

49. The method according to claim 48, wherein the conventional therapy is ipilimumab.

50. The method according to any one of claims 29 to 47, wherein the cancer is colorectal cancer (CRC).

51. The method according to claim 50, wherein the CRC is microsatellite-stable (MSS) CRC.

52. The method according to any one of claims 29 to 47, wherein the cancer is squamous cell carcinoma.

53. The method according to any one of claims 29 to 47, wherein the cancer is anal squamous cell carcinoma or penile squamous cell carcinoma.

54. The method according to any one of claims 29 to 47, wherein the cancer is pancreatic cancer.

55. The method according to claim 54, wherein the cancer is pancreatic ductal adenocarcinoma (PDAC).

56. The method according to any one of claims 29 to 47, wherein the cancer is ovarian cancer.

57. The method according to any one of claims 29 to 47, wherein the cancer is NSCLC.

58. The method according to any one of claims 29 to 47, wherein the cancer is hepatocellular carcinoma.

59. The method according to any one of claims 29 to 58, wherein the cancer is advanced metastatic cancer.

60. The method according to claim 59, wherein the cancer has metastasized to the lungs or liver.

61. The method according to any one of claims 29 to 60, wherein the activatable antibody comprises a heavy chain variable region having at least about 90% sequence identity with respect to the amino acid sequence of SEQ ID NO: 87 or a variant thereof, and a light chain variable region having at least about 90% sequence identity with respect to the amino acid sequence of SEQ ID NO: 100 or a variant thereof, the method according to any one of claims 29 to 60.

62. The method according to claim 61, 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.

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

321.

64. The method according to claim 62, wherein the activatable antibody comprises the complete light chain region of SEQ ID NO: 322 or SEQ ID NO:

323.

65. The method according to any one of claims 29 to 64, wherein the subject is a human.

66. A method for treating cancer in a subject, wherein an effective amount of an activatable antibody is combined with pembrolizumab in the subject, and the activatable antibody is HVR-H1 containing the amino acid sequence of formula YSISSGYHWSWI (SEQ ID NO: 23), HVR-H2 containing the amino acid sequence of formula LARIDWDDDKYYSTSLKSRL (SEQ ID NO: 35), HVR-H3 containing the amino acid sequence of formula ARSYVYFDY (SEQ ID NO: 45), HVR-L1 containing the amino acid sequence of formula RASQSVRGRFLA (SEQ ID NO: 58), HVR-L2 containing the amino acid sequence of formula DASNRATGI (SEQ ID NO: 66), and formula YCQQSSSWPP The activatable antibody comprises an HVR-L3 having the amino acid sequence T (SEQ ID NO: 75), further comprising a polypeptide covalently bonded to the N-terminus of the light chain of the anti-CTLA4 antibody, the polypeptide comprising a masking moiety (MM) and a cleavable moiety (CM) from the N-terminus to the C-terminus, wherein the MM comprises the amino acid sequence EVGSYPNPSSDCVPYYYACAY (SEQ ID NO: 192), and the cleavable moiety comprises the amino acid sequence SGRSAGGGGTPLGLAGSGGS (SEQ ID NO: 221), and the activatable antibody is administered in a dose that provides a steady-state plasma concentration of the cleaved antibody with an EC50 greater than that of the cleaved antibody.

67. A method for treating cancer in a subject, wherein an effective amount of an activatable antibody is combined with pembrolizumab in the subject, and the activatable antibody is HVR-H1 containing the amino acid sequence of formula YSISSGYHWSWI (SEQ ID NO: 23), HVR-H2 containing the amino acid sequence of formula LARIDWDDDKYYSTSLKSRL (SEQ ID NO: 35), HVR-H3 containing the amino acid sequence of formula ARSYVYFDY (SEQ ID NO: 45), HVR-L1 containing the amino acid sequence of formula RASQSVRGRFLA (SEQ ID NO: 58), HVR-L2 containing the amino acid sequence of formula DASNRATGI (SEQ ID NO: 66), and formula YCQQSSSWPPT ( The activatable antibody comprises an HVR-L3 having the amino acid sequence of SEQ ID NO: 75, further comprising a polypeptide covalently bonded to the N-terminus of the light chain of the anti-CTLA4 antibody, the polypeptide comprising a masking moiety (MM) and a cleavable moiety (CM) from the N-terminus to the C-terminus, wherein the MM comprises the amino acid sequence EVGSYPNPSSDCVPYYYACAY (SEQ ID NO: 192), and the cleavable moiety comprises the amino acid sequence SGRSAGGGGTPLGLAGSGGS (SEQ ID NO: 221), and the activatable antibody is administered in a dose that provides a steady-state plasma concentration of the cleaved antibody with an EC of 90 or higher.

68. A method for treating cancer in a subject, wherein an effective amount of an activatable antibody is combined with pembrolizumab in the subject, the activatable antibody being HVR-H1 containing the amino acid sequence of formula YSISSGYHWSWI (SEQ ID NO: 23), HVR-H2 containing the amino acid sequence of formula LARIDWDDDKYYSTSLKSRL (SEQ ID NO: 35), HVR-H3 containing the amino acid sequence of formula ARSYVYFDY (SEQ ID NO: 45), HVR-L1 containing the amino acid sequence of formula RASQSVRGRFLA (SEQ ID NO: 58), HVR-L2 containing the amino acid sequence of formula DASNRATGI (SEQ ID NO: 66), and YCQQSSSW The activatable antibody comprises an HVR-L3 containing the amino acid sequence of PPT (SEQ ID NO: 75), further comprising a polypeptide covalently bonded to the N-terminus of the light chain of the anti-CTLA4 antibody, the polypeptide comprising a masking moiety (MM) and a cleavable moiety (CM) from the N-terminus to the C-terminus, wherein the MM contains the amino acid sequence EVGSYPNPSSDCVPYYYACAY (SEQ ID NO: 192), and the cleavable moiety contains the amino acid sequence SGRSAGGGGTPLGLAGSGGS (SEQ ID NO: 221), and the activatable antibody is administered in a dose such that the steady-state plasma concentration of the cleaved antibody is approximately 100 nM to approximately 200 nM.

69. The method according to claim 68, wherein the activatable antibody is administered in a dose such that the steady-state plasma concentration of the cleaved antibody is about 100 nM to about 175 nM.

70. The method according to claim 68, wherein the activatable antibody is administered in a dose such that the steady-state plasma concentration of the cleaved antibody is about 100 nM to about 150 nM.

71. The method according to claim 68, wherein the activatable antibody is administered in a dose such that the steady-state plasma concentration of the cleaved antibody is about 150 nM to about 200 nM.

72. The method according to any one of claims 66 to 71, wherein the steady-state plasma concentration of the cleaved antibody is measured at the trough level of the anti-CTLA4 antibody.

73. The method according to any one of claims 66 to 72, wherein the activatable antibody is administered as a single loading dose or two loading doses, followed by a maintenance dose, wherein the amount of the loading dose is greater than the amount of the maintenance dose.

74. The method according to claim 73, wherein a single loading dose of the activatable antibody is administered to the subject before administration of a maintenance dose.

75. The method according to claim 74, wherein the loading dose is approximately 20 mg / kg.

76. The method according to claim 74, wherein the loading dose is approximately 30 mg / kg.

77. The method according to claim 74, wherein the loading dose is approximately 40 mg / kg.

78. The method according to claim 74, wherein the loading dose is approximately 50 mg / kg.

79. The method according to claim 73, wherein two loading doses of the activatable antibody are administered to the subject before administration of a maintenance dose.

80. The method according to claim 79, wherein the loading dose is approximately 20 mg / kg.

81. The method according to claim 79, wherein the loading dose is approximately 30 mg / kg.

82. The method according to claim 79, wherein the loading dose is approximately 40 mg / kg.

83. The method according to claim 79, wherein the loading dose is approximately 50 mg / kg.

84. The method according to any one of claims 73 to 83, wherein the maintenance dose is approximately 10 mg / kg.

85. The method according to any one of claims 73 to 84, wherein the activatable antibody comprises a heavy chain variable region having at least about 90% sequence identity with respect to the amino acid sequence of SEQ ID NO: 87 or a variant thereof, and a light chain variable region having at least about 90% sequence identity with respect to the amino acid sequence of SEQ ID NO: 100 or a variant thereof,

86. The method according to claim 85, 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.

87. The method according to claim 85, wherein the activatable antibody comprises the full heavy chain region of SEQ ID NO: 320 or SEQ ID NO:

321.

88. The method according to claim 86, wherein the activatable antibody comprises the complete light chain region of SEQ ID NO: 322 or SEQ ID NO:

323.

89. The method according to any one of claims 73 to 88, wherein the subject is a human.

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