Cancer treatment method combining anti-CTLA4 antibody with pembrolizumab

Abstract

The present application provides compositions and methods for treating cancer, including advanced metastatic cancer, using anti-CTLA4 antibodies in combination with pembrolizumab.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 422,947, filed November 5, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Reference to Electronic Sequence Listing The contents of the electronic sequence listing (695402002640seqlist.xml, size: 50,377 bytes, creation date: November 1, 2023) are incorporated herein by reference in their entirety.

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

[0004] CTLA4 is a member of the immunoglobulin (Ig) superfamily of proteins that downregulates T cell activation and maintains immunogenic homeostasis. In vivo antibody-mediated blockade of CTLA4 has been shown to enhance anti-cancer immune responses in a syngeneic mouse prostate cancer model (Kwon et al. (1997) Proc Natl Acad Sci USA, 94(15):8099-103). Furthermore, blockade of CTLA4 function has been shown to enhance anti-tumor T cell responses at various stages of tumor growth in tumor-bearing mice (Yang et al. (1997) Cancer Res 57(18):4036-41; Hurwitz et al. (1998) Proc Natl Acad Sci USA 95(17):10067-7). However, the development of antibody-based therapeutics suitable for human use remains challenging because translation from preclinical animal models to human safety is often insufficient. Therefore, there is a need for anti-CTLA4 antibodies that are cross-reactive between different species, such as humans and experimental animals (e.g., mice, monkeys, rats, etc.), to enable animal model studies while also providing suitable human therapeutic candidates. Furthermore, there is a need for the development of safer anti-CTLA4 antibodies that are active only in specific contexts, such as the protease-rich tumor microenvironment.

[0005] PD-1 is recognized as an important molecule in immune regulation and the maintenance of peripheral tolerance. PD-1 is moderately expressed on 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] The known ligands of PD-1, PD-L1 (B7-H1) and PD-L2 (B7-DC), are expressed in human cancers arising in a variety of tissues. In large sample sets of ovarian, renal, colorectal, liver, and melanoma, PD-L1 expression has been 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). Similarly, PD-1 expression on tumor-infiltrating lymphocytes has been found to characterize dysfunctional T cells in breast cancer and melanoma (Ghebeh, Hazem et al., Foxp3+ Tregs and B7-H1+ / PD-1+ T lymphocytes co-infiltrate the tumor tissues of high-risk breast cancer patients: Implication for immunotherapy. BMC Cancer. 2008 Feb 23;8:57; Ahmadzadeh, Mojgan et al., Tumor antigen-specific CD8 T cells infiltrating the tumor express high levels of PD-1 and are functionally impaired. Blood (2009) 114: 1537-1544) and correlates with poor prognosis in kidney cancer (Thompson, R Houston et al.PD-1 is expressed by tumor-infiltrating cells and is associated with poor outcome for patients with renal carcinoma. Clinical Cancer Research (2007) 15: 1757-1761). Therefore, it is thought that PD-L1-expressing tumor cells may interact with PD-1-expressing T cells to attenuate T cell activation and evasion of immune surveillance, thereby contributing to impaired immune responses against tumors.

[0007] Several monoclonal antibodies that inhibit the interaction of PD-1 with one or both of its ligands, PD-L1 and PD-L2, have been approved for the treatment of cancer. 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 its 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) is indicated for the treatment of patients across multiple indications, including as first-line treatment for patients with unresectable or metastatic CRC that is microsatellite instability- or mismatch repair-deficient (MSLH / dMMR). Pembrolizumab is currently the standard of care for first-line treatment of MSLH / dMMR mCRC. Summary of the Invention

[0008] The present application provides methods for treating cancer by combining an anti-CTLA4 antibody of the present invention with the anti-PD-1 antibody pembrolizumab. The anti-CTLA4 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HVR-H1, HVR-H2, and HVR-H3, and the light chain variable region comprises HVR-L1, HVR-L2, and HVR-L3, wherein HVR-H1 comprises an amino acid sequence according to the formula YSISSGYHWSWI (SEQ ID NO: 23), HVR-H2 comprises an amino acid sequence according to the formula LARIDWDDDKYYSTSLKSRL (SEQ ID NO: 35), HVR-H3 comprises an amino acid sequence according to the formula ARSYVYFDY (SEQ ID NO: 45), HVR-L1 comprises an amino acid sequence according to the formula RASQSVRGRFLA (SEQ ID NO: 58), HVR-L2 comprises an amino acid sequence according to the formula DASNRATGI (SEQ ID NO: 66), and HVR-L3 comprises an amino acid sequence according to the formula YCQQSSSWPPT (SEQ ID NO: 75).

[0009] In some embodiments, the anti-CTLA4 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 87, or a variant thereof having at least about 90% (e.g., at least about 92%, 95%, 98%, 99% or more) sequence identity to the amino acid sequence of SEQ ID NO: 87, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 100, or a variant thereof having at least about 90% (e.g., at least about 92%, 95%, 98%, 99% or more) sequence identity to the amino acid sequence of SEQ ID NO: 100. In some embodiments, the anti-CTLA4 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 87, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 100.

[0010] In some embodiments, the anti-CTLA4 antibody is EVQLVESGGGLVQPGGSLRLSCAASGYSISSGYHWSWIRQAPGKGLEWLARIDWDDDKYYSTSLKSRLTISRDNSKNTLYLQLNSLRAEDTAVYYCARSYVYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF a heavy chain region comprising the amino acid sequence of SCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 126) or a variant thereof having at least about 90% (e.g., at least about 92%, 95%, 98%, 99% or more) sequence identity to the amino acid sequence of SEQ ID NO: 126, and and a light chain region comprising the amino acid sequence of PTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 127), or a variant thereof having at least about 90% (e.g., at least about 92%, 95%, 98%, 99% or more) sequence identity to the amino acid sequence of SEQ ID NO: 127. In some embodiments, the anti-CTLA4 antibody is TY21580, which comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO: 126, and a light chain region comprising the amino acid sequence of SEQ ID NO: 127.

[0011] In one aspect, the present disclosure provides a method of treating cancer in a subject, the method comprising administering to the subject an effective amount of the anti-CTLA4 antibody (e.g., TY21580) in combination with pembrolizumab, wherein the anti-CTLA4 antibody is administered at a dose of about 3 mg / kg to about 10 mg / kg. In some embodiments, the anti-CTLA4 antibody (e.g., TY21580) is administered at a dose of about 3 mg / kg. In some embodiments, the anti-CTLA4 antibody (e.g., TY21580) is administered at a dose of about 5 mg / kg. In some embodiments, the anti-CTLA4 antibody (e.g., TY21580) is administered at a dose of about 6 mg / kg. In some embodiments, the anti-CTLA4 antibody (e.g., TY21580) is administered at a dose of about 8 mg / kg. In some embodiments, the anti-CTLA4 antibody (e.g., TY21580) is administered at a dose of about 10 mg / kg.

[0012] In one embodiment, the anti-CTLA4 antibody (e.g., TY21580) is administered to the subject at a dose of about 1 mg / kg to about 10 mg / kg, or about 2 mg / kg to about 5 mg / kg. In some embodiments, the anti-CTLA4 antibody (e.g., TY21580) is administered to the subject at a dose of about 3 mg / kg. In some embodiments, the anti-CTLA4 antibody (e.g., TY21580) is administered to the subject once every three weeks. In other embodiments, the anti-CTLA4 antibody (e.g., TY21580) is administered to the subject once every six weeks. In certain embodiments, the anti-CTLA4 antibody (e.g., TY21580) is administered to the patient at a dose of about 3 mg / kg once every three weeks or once every six weeks. In any of the foregoing embodiments, pembrolizumab can be administered in combination with the anti-CTLA4 antibody on the same day of a particular dosing regimen or on different days of a particular dosing regimen. In some embodiments, both the anti-CTLA4 antibody and pembrolizumab are administered on the first day of a three-week or six-week dosing regimen.

[0013] In some embodiments, pembrolizumab is administered at a dose of about 100 mg to about 300 mg once every three weeks. In some embodiments, pembrolizumab is administered at a dose of about 200 mg once every three weeks. In some embodiments, the anti-CTLA4 antibody is administered simultaneously with pembrolizumab. For example, the anti-CTLA4 antibody and pembrolizumab can be administered to a patient in need thereof on day 1 of a three-week or six-week administration schedule, respectively.

[0014] In some embodiments, the anti-CTLA4 antibody and pembrolizumab are administered to a patient in need thereof once every three weeks. In some embodiments, the anti-CTLA4 antibody is administered at a dose of about 2 mg / kg to about 5 mg / kg (e.g., 3 mg / kg), and the pembrolizumab is administered at a dose of about 100 mg / kg to about 300 mg / kg (e.g., about 200 mg). In certain embodiments, the anti-CTLA4 antibody and pembrolizumab are administered simultaneously.

[0015] In some embodiments, pembrolizumab is administered at a dose of about 200 mg to about 400 mg once every six weeks. In some embodiments, pembrolizumab is administered at a dose of about 400 mg once every six weeks. In some embodiments, the anti-CTLA4 antibody is administered simultaneously with pembrolizumab. For example, the anti-CTLA4 antibody and pembrolizumab can be administered to a patient in need thereof on day 1 of a 3-week or 6-week administration schedule, respectively.

[0016] In some embodiments, the anti-CTLA4 antibody and pembrolizumab are administered to a patient in need thereof once every six weeks. In some embodiments, the anti-CTLA4 antibody is administered at a dose of about 2 mg / kg to about 5 mg / kg (e.g., 3 mg / kg), and the pembrolizumab is administered at a dose of about 200 mg to about 400 mg (e.g., about 400 mg). In certain embodiments, the anti-CTLA4 antibody and pembrolizumab are administered simultaneously.

[0017] In some embodiments according to any one of the above methods, the cancer is resistant or refractory to conventional therapy, and the conventional therapy is an inhibitor of CTLA4, PD-1, or PD-1 ligand. In some embodiments, the subject is resistant to or has relapsed from conventional therapy, and the conventional therapy is an inhibitor of CTLA4, PD-1, or PD-1 ligand. In some embodiments, the conventional therapy is an inhibitor of CTLA4, such as ipilimumab. In some embodiments, the conventional therapy is an inhibitor of PD-1, e.g., an anti-PD-1 antibody. In some embodiments, the conventional therapy is an inhibitor of PD-1 ligand (e.g., PD-L1), e.g., an anti-PD-L1 antibody.

[0018] In some embodiments according to any one of the above methods, the cancer is liver cancer, cancer of the digestive system (e.g., colon cancer, colorectal 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 neck cancer, eye-related cancer, cancer of the male reproductive system (e.g., prostate cancer, testicular cancer), or cancer of the female reproductive system (e.g., uterine cancer, cervical cancer). In some embodiments, the cancer is a solid cancer. In some embodiments, the cancer is urothelial carcinoma. In some embodiments, the cancer is renal cell carcinoma. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is advanced stage cancer. In some embodiments, the cancer is metastatic cancer. In some embodiments, the cancer is Kaposi's sarcoma. In one embodiment, the cancer includes, but is not limited to, colorectal cancer, gastric cancer, gastroesophageal junction cancer, esophageal cancer, endometrial cancer, or head and neck cancer. In another embodiment, the cancer includes, but is not limited to, melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), head and neck squamous cell carcinoma (HNSCC), classical Hodgkin lymphoma (cHL), primary mediastinal large B-cell lymphoma (PMBCL), urothelial carcinoma, microsatellite instability-high or mismatch repair deficient cancer, microsatellite instability-high or mismatch repair deficient 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, cutaneous squamous cell carcinoma (cSCC), or triple-negative breast cancer (TNBC). The present invention relates to a method of treating cancer in a subject in need thereof, comprising administering to the subject a combination therapy comprising at least two pharmaceutical compositions.

[0019] In some embodiments, the anti-CTLA4 antibody and pembrolizumab are both administered intravenously. In some embodiments, the anti-CTLA4 antibody is administered subcutaneously. In some embodiments, the anti-CTLA4 antibody and pembrolizumab are administered intravenously or subcutaneously once every three weeks. In some embodiments, the anti-CTLA4 antibody and pembrolizumab are administered intravenously or subcutaneously once every six weeks. In some embodiments, the subject receives at least four cycles of treatment with the anti-CTLA4 antibody and pembrolizumab. In some embodiments, the subject further receives maintenance treatment comprising administering an effective amount of the anti-CTLA4 antibody to the subject about once every four weeks to about once every 12 weeks (e.g., once every 4 weeks, 6 weeks, 8 weeks, 10 weeks, or 12 weeks). In some embodiments, the doses of the anti-CTLA4 antibody and pembrolizumab may be administered simultaneously. In other embodiments, the doses of the anti-CTLA4 antibody and pembrolizumab may be administered simultaneously. For example, pembrolizumab can be administered about 0.5 hours to about 5 hours before or after administration of the anti-CTLA4 antibody on day 1 of the administration schedule (eg, a 3-week administration schedule).

[0020] In some embodiments according to any one of the above methods, the subject is a human.

[0021] It should be understood that one, some, or all of the features of the various embodiments described above and herein may be combined to form other embodiments of the present application. These and other aspects of the present application will be apparent to those skilled in the art. These and other embodiments of the present application are further described in the detailed description that follows. [Brief explanation of the drawings]

[0022] [Figure 1] We demonstrate a reduction in serum carcinoembryonic antigen (CEA) in MSS CRC patients with lung metastases after combination treatment with TY21580 and pembrolizumab. [Figure 2]We demonstrate a reduction in serum oncofetal CEA in MSS CRC patients with liver and lung metastases after combination treatment with TY21580 and pembrolizumab. [Figure 3] Figure 1 shows modulation of immune biomarkers by combination treatment with TY21580 and pembrolizumab. [Figure 4] Serum PK of TY21580 when administered at 3 mg / kg Q3W in combination with pembrolizumab. DETAILED DESCRIPTION OF THE INVENTION

[0023] I. Definition Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meanings commonly understood by those of ordinary skill in the art. Furthermore, unless the context otherwise requires, singular terms shall include plural referents and plural terms shall include the singular referent. Generally, the nomenclature used in connection with and the techniques of antibody engineering, immunotherapy, cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry described herein are those well known and commonly used in the art.

[0024] The term "antibody" is used herein in the broadest sense and specifically encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies), and antibody fragments (e.g., Fab, Fab', Fab'-SH, F(ab')2, Fv and / or single-chain variable fragments or scFv), so long as they exhibit the desired biological activity.

[0025] 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 bound by the intact antibody. An antibody fragment retains the ability to specifically bind to the antigen bound by the full-length antibody. For example, fragments that retain one or more CDR regions (e.g., all six CDRs) are included. 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.

[0026] In some embodiments, the term "antibody" refers to an antigen-binding protein (i.e., an immunoglobulin) with 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 covalent disulfide 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 contains a variable region (referred to herein as V) at its N-terminus. H The heavy chain constant region is divided into three domains, C H1 , C H2 , and C H3 Each light chain consists of a variable region (referred to herein as V I The light chain constant region consists of one domain, C L V L is V H Align with C L is aligned with the first constant domain (CH1) of the heavy chain. H and V L The pairings of these together form a single antigen-binding site. IgM antibodies consist of five basic heterotetrameric units plus an additional polypeptide called the J chain and therefore contain 10 antigen-binding sites, whereas secreted IgA antibodies can polymerize to form multivalent assemblies containing two to five basic four-chain units plus the J chain.

[0027] VH and V L Based on structural and sequence analysis, the V region can be further subdivided into regions of hypervariability called hypervariable regions (HVRs). HVRs are interspersed with more conserved regions called framework regions (FWs) (see, e.g., Chen et al. (1999) J. Mol. Biol. (1999) 293, 865-881). Each V H and V L is composed of three HVRs and four FWs, arranged from amino to carboxy terminus in the following order: FW-1_HVR-1_FW-2_HVR-2_FW-3_HVR-3_FW4. Throughout this application, the three HVRs 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.

[0028] As used herein, the term "CDR" or "CDRs" is intended to mean a complementarity-determining region within an immunoglobulin variable region, or the noncontiguous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. Unless otherwise specified, CDRs are defined herein 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. Mol. Biol., 309:657-670 (2001), and the definitions include overlapping or subsets of amino acid residues when compared against each other. Nevertheless, application of either definition to refer to the CDRs of an antibody or grafted antibody or variants thereof is intended to be within the scope of the term as defined and used herein.The contents of the references cited in this paragraph in which CDR prediction algorithms and interfaces are known in the art, including, for example, Mol. Immunol., 45:3832-3839 (2008); Ehrenmann F. et al., Nucleic Acids Res., 38:D301-D307 (2010); and Adolf-Bryfogle J. et al., Nucleic Acids Res., 43:D432-D438 (2015), are hereby incorporated by reference in their entirety for use in this application and for possible inclusion in one or more claims herein.

[0029] Each variable region of the heavy and light chains contains a binding domain that interacts with an antigen. The constant regions of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Within the light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also containing a "D" region of about 10 or more amino acids (see, e.g., Fundamental Immunology Ch. 7 (Paul, W., ed., 2002)). nd ed. Raven Press, NY (1989).

[0030] Light chains from any vertebrate species can be assigned to one of two clearly 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 chains (CH). There are five classes of antibodies: IgA, IgD, IgE, IgG, and IgM, each with heavy chains designated α (alpha), δ (delta), ε (epsilon), γ (gamma), and μ (mu). The IgG class of antibodies can be further classified into four subclasses, IgG1, IgG2, IgG3, and IgG4, respectively, based on the gamma heavy chains Y1-Y4.

[0031] The term "CTLA4" is used in this application to include human CTLA4 (e.g., UniProt Accession No. P16410), as well as variants, isoforms, and species homologs thereof (e.g., mouse CTLA4 (UniProt Accession No. P09793), rat CTLA4 (UniProt Accession No. Q9Z1A7), canine CTLA4 (UniProt Accession No. Q9XSI1), cynomolgus monkey CTLA4 (UniProt Accession No. G7PL88), etc.). Thus, anti-CTLA4 antibodies as defined and disclosed herein may also bind to CTLA4 from species other than human. In other cases, anti-CTLA4 antibodies may be completely specific for human CTLA4 and may not exhibit species or other types of cross-reactivity.

[0032] As defined herein, the term "CTLA4 antibody" refers to an antibody capable of binding to human CTLA4.

[0033] As used herein, "monoclonal antibody" or "mAb" or "Mab" refers to a population of substantially homogeneous antibodies, i.e., the antibody molecules comprising the population are identical in amino acid sequence except for possible minor naturally occurring mutations. In contrast, conventional (polyclonal) antibody preparations typically contain a large number of different antibodies with different amino acid sequences in the variable domains, particularly the CDRs, and are often specific for different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody 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 may be produced by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). "Monoclonal antibodies" may also be isolated from phage antibody libraries using the 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.

[0034] The term "PD-1 antagonist" refers to 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), and in specific embodiments, blocks the binding of PD-L2 expressed on cancer cells to immune cells that also express PD-1. Alternative or synonymous terms for PD-1 and its ligands include PDCD1, PD1, CD279, and SLEB2 for PD-1; PDCD1L1, PDL1, B7H1, B7-4, CD274, and B7-H for PD-L1; and PDCD1L2, PDL2, B7-DC, Btdc, and CD273 for PD-L2. In any of the therapeutic methods, medicaments, and uses of the present invention for treating a human individual, the 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 of human PD-1 is available at NCBI Locus No.: NP_005009. The amino acid sequences of human PD-L1 and PD-L2 are available at NCBI Locus No.: NP_054862 and NP_079515, respectively.

[0035] "Pembrolizumab" (formerly known as MK-3475, SCH900475, and lambrolizumab), alternatively referred to herein as "pembro," is a humanized IgG4 mAb having the structure set forth in WHO Drug Information, Vol. 27, No. 2, pages 161-162 (2013), and containing the heavy and light chain amino acid sequences and CDRs set forth in Table B. Pembrolizumab is approved by the U.S. FDA as set forth in the prescribing information for KEYTRUDA® (MerckSharp & Dohme LLC, Rahway, NJ, USA, first approved in the U.S. in 2014, updated March 2021).

[0036] As used herein, "pembrolizumab variant" or "variant thereof" with respect to a pembrolizumab sequence refers to a monoclonal antibody comprising heavy and light chain sequences substantially identical to those of pembrolizumab, except that it has three, two, or one conservative amino acid substitution at a position located outside the light chain CDR and six, five, four, three, two, or one conservative amino acid substitution at a position located outside the heavy chain CDR (e.g., the mutation positions are located in the FR or constant region, and optionally, the C-terminal lysine residue of the heavy chain is deleted). In other words, pembrolizumab and a pembrolizumab variant are composed of identical CDR sequences but differ from each other by having conservative amino acid substitutions at no more than three or six other positions within the full-length light and heavy chain sequences, respectively. The pembrolizumab variants are substantially identical to pembrolizumab with respect to their binding affinity to PD-1 and their ability to inhibit the binding of PD-1 to PD-L1 and PD-L2, respectively.

[0037] The term "epitope" refers to the portion of an antigen to which an antibody (or antigen-binding fragment thereof) binds. Epitopes can be formed from both contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous 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 comprise a variable number of amino acids in unique spatial conformations. Methods for determining the spatial conformation of epitopes include, for example, X-ray crystallography, two-dimensional nuclear magnetic resonance, deuterium and hydrogen exchange coupled with mass spectrometry, or site-directed mutagenesis, all methods used in conjunction with computational models of the structure of the antigen and its complex with its binding antibody and its variants (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996)). Once a desired epitope of an antigen has been determined, antibodies against that epitope can be generated, for example, using the techniques described herein. Antibody generation and characterization can also elucidate information about the desired epitope. From this information, it is possible to competitively screen antibodies for binding to the same epitope. One approach to accomplish this is to perform cross-competition studies to discover antibodies that competitively bind to each other, i.e., the antibodies compete for binding to the antigen. A high-throughput process for "binning" antibodies based on mutual competition is described in PCT Publication WO 03 / 48731.

[0038] An "isolated" antibody is one that has been separated from a component of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, 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 a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0039] As used herein, "sequence identity" between two polypeptide sequences refers to the percentage of identical amino acids between the sequences.The amino acid sequence identity of polypeptides can be conventionally 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); 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, parameters are set so that the percent identity is calculated over the entire length of the reference amino acid sequence and allows for a difference in homology of up to 5% of the total number of amino acid residues in the reference sequence. This aforementioned method of determining percent identity between polypeptides is applicable to all proteins, fragments, or variants thereof disclosed herein.

[0040] As used herein, the terms "bind," "binds to," "specifically binds to," or "specific for" refer to a measurable and reproducible interaction, such as binding between a target and an antibody, that 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 or specifically binds to a target (which may be an epitope) is an antibody that binds to this target with higher affinity, avidity, more readily, and / or for a longer period of time than it binds to other targets. In one embodiment, the extent of binding of an antibody to an unrelated target is less than about 10% of the binding of the antibody 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 or less, 100 nM or less, 10 nM or less, 1 nM or less, or 0.1 nM or less. In certain embodiments, an antibody specifically binds to an epitope on a protein that is conserved among proteins from different species. In another embodiment, specific binding can include, but does not require, exclusive binding. An antibody that "specifically binds" to a particular target protein is one that exhibits preferential binding to that target relative to other proteins, although this specificity does not require absolute binding specificity. An antibody is considered "specific" for its intended target if its binding is conclusive about the presence of the target protein in a sample (e.g., without producing undesirable results such as false positives). Antibodies or binding fragments thereof useful in the present invention bind to the target protein with an affinity that is at least 2-fold, preferably at least 10-fold, more preferably at least 20-fold, and most preferably at least 100-fold greater than its affinity for non-target proteins. As used herein, an antibody is said to specifically bind to a polypeptide containing a given amino acid sequence, e.g., the amino acid sequence of a mature human PD-1 or human PD-L1 molecule. This is true when the antibody binds to a polypeptide containing that sequence but not to proteins lacking that sequence.

[0041] The terms "treat," "treating," or "treatment," with respect to a particular disease state in a mammal, refer to causing a desirable or beneficial effect in a mammal having the disease state. A desirable or beneficial effect can include reducing the frequency or severity of one or more symptoms of the disease (i.e., tumor growth and / or metastasis, or other effects mediated by immune cell numbers and / or activity, etc.), or preventing or inhibiting further progression of the disease, condition, or disorder. In the context of treating cancer in a mammal, a desirable or beneficial effect can include inhibiting further growth or metastasis of cancer cells, killing cancer cells, inhibiting cancer recurrence, reducing cancer-associated pain, or improving the mammal's survival. The effect can be either subjective or objective. For example, if the animal is a human, the human may note increased vitality or survival, or reduced pain, as subjective symptoms of improvement or therapeutic response. Alternatively, a clinician may note a decrease in tumor size or burden based on physical examination, clinical laboratory values, tumor markers, or radiological findings. Some clinical signs that a clinician may observe regarding a therapeutic response include normalization of laboratory values ​​such as white blood cell count, red blood cell count, platelet count, erythrocyte sedimentation rate, and various enzyme levels. Additionally, a clinician may observe a decrease in detectable tumor markers. Alternatively, other tests, such as ultrasound imaging, nuclear magnetic resonance imaging, and positron emission tomography, may be used to assess objective improvement.

[0042] The terms "preventing" or "prevention" with respect to a particular disease state in a mammal refer to preventing or delaying the onset of the disease or preventing the manifestation of its clinical or subclinical symptoms.

[0043] As used herein, a "subject," "patient," or "individual" may refer to a human or a non-human animal. A "non-human animal" may refer to any animal not classified as a human, such as farm, livestock, or zoo animals, sport animals, pet animals (e.g., dogs, horses, cats, cows, etc.), and animals used in research. Research animals may refer to, but are not limited to, nematodes, arthropods, vertebrates, mammals, frogs, rodents (e.g., mice or rats), fish (e.g., zebrafish or pufferfish), birds (e.g., chickens), dogs, cats, and non-human primates (e.g., rhesus monkeys, cynomolgus monkeys, chimpanzees, etc.). In some embodiments, the subject, patient, or individual is a human.

[0044] An "effective amount" refers to at least an amount effective, at dosages and for periods of time necessary, to achieve one or more desired or indicated effects, including therapeutic or prophylactic results. An effective amount can be given in one or more administrations. For purposes of this application, an effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to directly or indirectly achieve prophylactic or therapeutic treatment. As understood in a clinical context, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition (e.g., an effective amount when administered as monotherapy or combination therapy). Thus, an "effective amount" may be considered in the context of administration of one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desired result can be or is achieved.

[0045] The terms "recurrence," "recurrence," or "recurrence" refer to the recurrence of cancer or disease after clinical assessment of disease resolution. A diagnosis of distant metastasis or local recurrence may be considered a recurrence.

[0046] The terms "refractory" or "resistant" refer to a cancer or disease that has not responded to treatment.

[0047] As used herein, "complete response" or "CR" refers to the disappearance of all target lesions, "partial response" or "PR" refers to at least a 30% reduction in the sum of the longest diameters (SLD) of the target lesions, based on baseline SLD, and "stable disease" or "SD" refers to neither sufficient shrinkage of target lesions since initiating treatment to qualify for PR nor sufficient increase to qualify for PD, based on the smallest SLD,

[0048] As used herein, "disease progression" or "PD" refers to at least a 20% increase in the SLD of a target lesion or the presence of one or more new lesions, based on the smallest SLD recorded since the start of treatment.

[0049] As used herein, "progression-free survival" (PFS) refers to the length of time during and after treatment during which the disease being treated (e.g., cancer) does not worsen. Progression-free survival can include the amount of time a patient experiences a complete response or a partial response, as well as the amount of time a patient experiences stable disease.

[0050] As used herein, "overall response rate" (ORR) refers to the sum of the complete response (CR) rate and the partial response (PR) rate.

[0051] As used herein, "overall survival rate" refers to the proportion of individuals in a group who are likely to be alive after a specified period of time.

[0052] As used herein, "baseline level" or "baseline value" refers to a subject's level or value before the subject begins treatment, such as anti-CTLA4 antibody treatment.

[0053] As used herein, a "reference sample," "reference cell," "reference tissue," "control sample," "control cell," or "control tissue" refers to a sample, cell, tissue, standard, or level used for comparison purposes. In one embodiment, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy and / or non-diseased part of the body (e.g., tissue or cells) of the same subject or individual. For example, a healthy and / or non-diseased cell or tissue adjacent to a diseased cell or tissue (e.g., a cell or tissue adjacent to a tumor). In another embodiment, the reference sample is obtained from an 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 non-diseased part of the body (e.g., tissue or cells) of an individual other than the subject or individual. In yet another embodiment, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from untreated tissues and / or cells from the body of an individual other than the subject or individual.

[0054] An "effective response" and similar expressions of a patient or a patient's "responsiveness" to treatment with an agent refer to a clinical or therapeutic benefit conferred on a patient at risk for or suffering from a disease or disorder, such as cancer. In one embodiment, such benefit includes any one or more of extending survival (including overall survival and progression-free survival), producing an objective response (including a complete or partial response), or ameliorating the signs or symptoms of cancer.

[0055] A patient who "does not respond effectively" to treatment is one who does not have any of the following: an extension of survival (including overall survival and progression-free survival), an objective response (including a complete response or a partial response), or an improvement in the signs or symptoms of cancer.

[0056] The methods and techniques of this application are generally carried out according to methods well known in the art and as described in various general and more specific references cited and discussed throughout the specification, unless otherwise indicated. Such references include, for example, Sambrook and Russell, Molecular Cloning, A Laboratory Approach, Cold Spring Harbor Press, Cold Spring Harbor, NY (2001); Ausubel et al., 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 may be performed according to manufacturer's specifications, as commonly practiced in the art, or as described herein. The nomenclatures used in connection with, and the laboratory procedures and methods of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard methods are used for chemical synthesis, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0057] As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology—A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)).

[0058] As used herein, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to a "molecule" includes any combination of two or more such molecules, and so forth.

[0059] As used herein, the term "about" refers to a customary error range for each value, which is readily understood by one of ordinary skill in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments that refer to that value or parameter in itself.

[0060] It is to be understood that the aspects and embodiments of the present application described herein include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0061] As used herein, reference to a value or parameter "not" generally means and describes "other than" the value or parameter. For example, a method is not used to treat cancer type X means that the method is used to treat cancers other than type X.

[0062] As used herein, the term "about X to Y" has the same meaning as "about X to about Y."

[0063] The term "and / or," when used herein in expressions such as "A and / or B," is intended to include both "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, as used herein, phrases such as the term "and / or," "A, B and / or C," are intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0064] References herein to "some embodiments," "an embodiment," "one embodiment," or "other embodiments" mean that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least some embodiments of the invention, but not necessarily in all embodiments.

[0065] II. Treatment method The present application provides methods of treating cancer in a subject using anti-CTLA4 antibodies that specifically bind to human CTLA4. Any one of the anti-CTLA4 antibodies in Section III, "Anti-CTLA4 Antibodies," including full-length antibodies and antigen-binding fragments thereof, can be used in the methods described herein.

[0066] In some embodiments, methods are provided for treating a subject's cancer that is resistant or refractory to an inhibitor of CTLA4, PD-1, or PD-1 ligand (PD-L1 or PD-L2), comprising administering to the subject an effective amount of an 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 an anti-PD-L1 antibody. In some embodiments, the cancer is a solid cancer, such as an advanced stage cancer and / or a metastatic cancer. In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 87 or an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 87, and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 100 or an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 100. In some embodiments, the antibody comprises the Fc region of a human IgG1, such as a wild-type IgG1 Fc region or a variant with enhanced ADCC activity. In some embodiments, the antibody is TY21580.

[0067] In some embodiments, methods are provided for treating cancer in a subject, comprising administering to the subject an effective amount of an anti-CTLA4 antibody disclosed herein in combination with pembrolizumab, wherein the anti-CTLA4 antibody is administered at a dose of about 3 mg / kg to about 10 mg / kg. In some embodiments, the anti-CTLA4 antibody is administered at a dose of about 3 mg / kg. In some embodiments, the anti-CTLA4 antibody is administered at a dose of about 5 mg / kg. In some embodiments, the anti-CTLA4 antibody is administered at a dose of about 6 mg / kg. In some embodiments, the anti-CTLA4 antibody is administered at a dose of about 8 mg / kg. In some embodiments, the anti-CTLA4 antibody is administered at a dose of about 10 mg / kg.

[0068] In some embodiments, the cancer is resistant or refractory to inhibitors of CTLA-4, PD-1, or a PD-1 ligand (e.g., PD-L1 or PD-L2). In some embodiments, the cancer is a solid cancer, such as an advanced and / or metastatic cancer. In some embodiments, the cancer is urothelial carcinoma. Cancer treatment can be evaluated by, for example, tumor regression, reduction in tumor weight or size, time to progression, survival, progression-free survival, overall response rate, duration of response, quality of life, protein expression, and / or activity. Approaches to determining the efficacy of therapy can be used, including, for example, measuring response by radiological imaging.

[0069] The anti-CTLA4 antibodies and compositions provided by the present disclosure can be administered via any suitable enteral or parenteral route of administration. The term "enteral route" of administration refers to administration via any part of the gastrointestinal tract. Examples of enteral routes include oral, mucosal, buccal, and rectal routes, or intragastric routes. A "parenteral route" of administration refers to a route of administration other than the enteral route. Examples of parenteral routes of administration include intravenous, intramuscular, intradermal, intraperitoneal, intratumoral, intravesical, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal, subcutaneous, or topical administration. The antibodies and compositions of the present disclosure can be administered using any suitable method, such as oral ingestion, nasogastric tube, gastrostomy tube, injection, infusion, implantable infusion pump, and osmotic pump. Suitable routes and methods of administration can vary depending on many factors, such as the particular antibody used, the desired rate of absorption, the particular formulation or dosage form used, the type or severity of the disorder being treated, the particular site of action, and the condition of the patient, and can be readily selected by one of skill in the art. In some embodiments, the anti-CTLA4 antibody is administered intravenously.

[0070] An effective amount of an anti-CTLA4 antibody can be administered in a single dose or multiple doses. For methods involving administration of multiple doses of an anti-CTLA4 antibody, exemplary administration frequencies include, but are not limited to, weekly, weekly without interruption, two weeks out of three weeks, three weeks out of four weeks, once every three weeks, once every two weeks, monthly, every six months, and annually. In some embodiments, the anti-CTLA4 antibody is administered approximately once a week, once every two weeks, once every three weeks, once every six weeks, or once every 12 weeks. In some embodiments, the interval between each administration is approximately 3 years, 2 years, 12 months, 11 months, 10 months, 9 months, 8 months, 7 months, 6 months, 5 months, 4 months, 3 months, 2 months, 1 month, 4 weeks, 3 weeks, 2 weeks, or less than one week. In some embodiments, the interval between each administration is about 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, or more than 3 years. In some embodiments, there is no break in the administration schedule.

[0071] In some embodiments, the anti-CTLA4 antibody is administered infrequently, 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 frequently. In some embodiments, the anti-CTLA4 antibody is administered in a single dose. In some embodiments, the anti-CTLA4 antibody is administered approximately once every three weeks.

[0072] In some embodiments, the anti-CTLA4 antibody is administered for two or more cycles, for example, any one of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles, hi some embodiments, the anti-CTLA4 antibody is administered for at least four cycles.

[0073] 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 while having minimal side effects. Accordingly, the anti-CTLA4 antibodies of the present disclosure exhibit an improved therapeutic index compared to anti-CTLA4 antibodies, such as ipilimumab. For example, in one embodiment, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 87 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 100. The anti-CTLA4 antibody can be administered as a single dose (in combination with pembrolizumab) that achieves greater than 50% receptor occupancy three or even six weeks after administration. In some embodiments, the anti-CTLA4 antibody can be administered as a single dose (in combination with pembrolizumab) that achieves greater than 60% receptor occupancy three weeks after administration. In other such embodiments, the anti-CTLA4 antibody can be administered as a single dose (in combination with pembrolizumab) that achieves greater than 70% receptor occupancy three weeks after administration. In other such embodiments, the anti-CTLA4 antibody can be administered in a single dose (in combination with pembrolizumab) that achieves greater than 80% receptor occupancy three weeks after administration. In other such embodiments, the anti-CTLA4 antibody can be administered in a single dose (in combination with pembrolizumab) that achieves about 50% to about 80% receptor occupancy three weeks after administration. In other such embodiments, the anti-CTLA4 antibody can be administered in a single dose (in combination with pembrolizumab) that achieves about 60% to about 75% receptor occupancy three weeks after administration. In other such embodiments, the anti-CTLA4 antibody can be administered in a single dose (in combination with pembrolizumab) that achieves greater than 60% receptor occupancy six weeks after administration. In other such embodiments, the anti-CTLA4 antibody can be administered in a single dose (in combination with pembrolizumab) that achieves greater than 70% receptor occupancy six weeks after administration. In other such embodiments, the anti-CTLA4 antibody can be administered as a single dose (in combination with pembrolizumab) to achieve about 50% to about 70% receptor occupancy 6 weeks after administration.

[0074] In some embodiments, the treatment comprises an initial phase followed by a maintenance phase. In some embodiments, the anti-CTLA4 antibody is administered less frequently in the maintenance phase than in the initial phase. In some embodiments, the 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 anti-CTLA4 antibody is administered about once every 3 weeks for at least 4 cycles, and a maintenance phase in which the anti-CTLA4 antibody is administered about once every 4 weeks to once every 12 weeks, for example, once every 4 weeks, once every 6 weeks, once every 8 weeks, once every 10 weeks, or once every 12 weeks. In some embodiments, the administration frequency in the maintenance phase is T reg cells, CD8+T em cells, CD4+T em cells, CD8+T em Cells and T reg Ratio to CD4+T cells em Cells and T reg For example, if a subject is administered an anti-CTLA4 antibody and the subject is subsequently treated with an anti-CTLA4 antibody, the subject may be adjusted according to one or more biomarkers, such as the ratio of CD8+ T cells to NK cells, and / or the ratio of NK cells to NK cells. em Cells and T reg If the subject shows an increased ratio of CTLA4 cells, the subject can be further administered an anti-CTLA4 antibody about once every four weeks.

[0075] The combined administration of an anti-CTLA4 antibody and pembrolizumab can be extended for an extended period of time, for example, from about 1 week to about 1 month, from about 1 month to about 1 year, from about 1 year to about several years, etc. In some embodiments, the anti-CTLA4 antibody is administered for at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, or more.

[0076] The methods described herein are useful for treating a variety of cancers. In some embodiments, the cancer is a solid cancer. In some embodiments, the cancer is a liquid cancer. Various cancers in which CTLA4 is involved, whether malignant or benign, and whether primary or secondary, can be treated or prevented using the methods provided by the present disclosure. Examples of cancers include, but are not limited to, liver cancer, cancers of the digestive system (e.g., colon cancer, colorectal 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 neck cancer, eye-related cancer, cancers of the male reproductive system (e.g., prostate cancer, testicular cancer), or cancers of the female reproductive system (e.g., uterine cancer, cervical cancer). In some embodiments, the cancer is 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, the cancer is a tumor that cannot be penetrated by T cells because the tumor is not recognized by the immune system or does not elicit an immune response.

[0077] In some embodiments, the anti-CTLA4 antibodies of the present disclosure can be used to treat colorectal cancer (CRC). In some embodiments, the colorectal cancer is microsatellite stable (MSS) colorectal cancer. In some embodiments, the colorectal cancer has metastasized to other organs, such as the lung or liver. In some embodiments, the colorectal cancer patient has previously been treated with other chemotherapy agents. Such chemotherapy agents include, but are not limited to, FOLFOX, FOLFIRI / Avastin, Erbitux, Lonsurf, IO-202, APN401, or IPH5201.

[0078] In some embodiments, the anti-CTLA4 antibodies of the present disclosure can be used to treat Kaposi's sarcoma.

[0079] In some embodiments, the anti-CTLA4 antibodies of the present disclosure can be used to treat head and neck squamous cell carcinoma (HNSCC).

[0080] In some embodiments, the anti-CTLA4 antibodies of the present disclosure can be used to treat pancreatic cancer.

[0081] In some embodiments, the anti-CTLA4 antibodies of the present disclosure can be used to treat ovarian cancer.

[0082] In some embodiments, the subject has previously been treated for cancer with conventional therapy. In some embodiments, the subject has previously received one, two, three, four, or more conventional therapies. In some embodiments, the subject has previously exhausted all other available therapies. In some embodiments, the subject is unresponsive or resistant to conventional therapy. In some embodiments, the subject has relapsed after conventional therapy. In some embodiments, the cancer is refractory to conventional therapy. In some embodiments, the subject has failed conventional therapy within about one year, six months, or three months. In some embodiments, the subject has not previously received conventional therapy.

[0083] In some embodiments, the subject has previously received standard treatment for cancer. In some embodiments, the subject is unresponsive or resistant to standard treatment. In some embodiments, the subject has disease recurrence after standard treatment. In some embodiments, the cancer is refractory to standard treatment. In some embodiments, the subject has failed standard treatment within about 1 year, 6 months, or 3 months. In some embodiments, the subject has not previously received standard treatment. In some embodiments, the subject has refused standard treatment or is ineligible for standard treatment.

[0084] In some embodiments, the conventional therapy (e.g., standard of care) is selected from the group consisting of viral gene therapy, immunotherapy, targeted therapy, radiation therapy, and chemotherapy. In some embodiments, the conventional therapy is an immune checkpoint inhibitor. In some embodiments, the conventional therapy is an inhibitor of CTLA4, PD-1, or a PD-1 ligand (e.g., PD-L1 or PD-L2). In some embodiments, the conventional therapy is an inhibitor of CTLA4, e.g., an anti-CTLA4 antibody different from the anti-CTLA4 antibodies described herein. In some embodiments, the conventional therapy is ipilimumab.

[0085] In some embodiments, the conventional therapy is an inhibitor of PD-1 or PD-1 ligand, including a PD-1 binding antagonist, a PDL1 binding antagonist, and a PDL2 binding antagonist. Other names for "PD-1" include CD279 and SLEB2. Other names for "PDL1" include B7-H1, B7-4, CD274, and B7-H. Other names for "PDL2" include B7-DC, Btdc, and CD273. In some embodiments, the PD-1, PDL1, and PDL2 are human PD-1, PDL1, and PDL2.

[0086] In some embodiments, the conventional treatment is a PD-1 inhibitor, which is a molecule that inhibits the binding of PD-1 to its ligand binding partner. The PD-1 inhibitor is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In some embodiments, the PD-1 ligand inhibitor is an inhibitor of PD-L1 and / or PD-L2. In some embodiments, the PD-L1 inhibitor is a molecule that inhibits the binding of PDL1 to its binding partner. In some embodiments, the PD-L2 binding partner is PD-1 and / or B7-1. In some embodiments, the PD-1 ligand is a molecule that inhibits the binding of PD-L2 to its binding partner. In some embodiments, the PD-L2 binding partner is PD-1. The inhibitor can be an antibody, antigen-binding fragment thereof, immunoadhesin, fusion protein, or oligopeptide.

[0087] In some embodiments, the conventional therapy is selected from the group consisting of pembrolizumab, 2E5 (Cstone Pharmaceuticals), tislelizumab (BGB-A317), BGB-108, STI-A1110, AM0001, BI754091, sintilimab (IBI308), cetrelimab (JNJ-63723283), toripalimab (JS-001), camrelizumab (SHR-1210, INCSHR-1210, HR-301210), MEDI-0680 (AMP-514), MGA-012 (INCMGA 0012), nivolumab (BMS-936558, MDX1106, ONO-4538), spartalizumab (PDR00l), PF-06801591, cemiplimab (REGN-2810, REGEN2810), dostallimab (TSR-042, ANB011), pidilizumab (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 (fusion protein targeting PD-1), CX-188 (PD-1 probody), AGEN-2034, GLS-010, budigalimab (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.In some embodiments, the anti-PD-1 antibody is selected from the group consisting of NIH-3, CB-201, IBI-319, ONCR-177, Max-1, CS-4100, JBI-426, CCC-0701, CCX-4503, biosimilars thereof, 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 comprising an extracellular or PD-1-binding portion of PDL1 or PDL2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence)). In some embodiments, the PD-1 inhibitor is AMP-224. 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 WO 2006 / 121168. CT-011, also known as hBAT or hBAT-1, is an anti-PD-1 antibody described in WO 2009 / 101611. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in WO 2010 / 027827 and WO 2011 / 066342.

[0088] Conventional treatments (e.g., standard treatments) also include surgery to remove tumors and radiation therapy. Exemplary radiation treatments include, but are not limited to, ionizing (electromagnetic) radiation therapy (e.g., X-rays or gamma rays) and particle radiation therapy (e.g., high linear energy radiation). The source of radiation can be external or internal to the subject.

[0089] The methods described herein are useful in various aspects of cancer treatment. In some embodiments, provided are methods of inhibiting cell proliferation (e.g., tumor growth) in an individual, comprising administering to the individual an effective amount of any one of the anti-CTLA4 antibodies described herein in combination with pembrolizumab. In some embodiments, cell proliferation is inhibited by at least about 10% (including, for example, at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, 95% or more).

[0090] In some embodiments, methods are provided for inhibiting tumor metastasis in an individual, comprising administering to the individual an effective amount of any one of the anti-CTLA4 antibodies described herein in combination with pembrolizumab, in which at least about 10% (including, for example, at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, 95% or more) of metastasis is inhibited.

[0091] In some embodiments, methods are provided for reducing (e.g., eliminating) existing tumor metastasis (e.g., lymph node metastasis) in an individual, comprising administering to the individual an effective amount of any one of the anti-CTLA4 antibodies described herein in combination with pembrolizumab. In some embodiments, metastasis is reduced by at least about 10% (including, for example, at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, 95% or more).

[0092] In some embodiments, methods are provided for reducing the incidence or burden of existing tumor metastases (e.g., lymph node metastases) in an individual, comprising administering to the individual an effective amount of any one of the anti-CTLA4 antibodies described herein in combination with pembrolizumab.

[0093] In some embodiments, methods are provided for reducing tumor size in an individual, comprising administering to the individual an effective amount of any one of the anti-CTLA4 antibodies described herein in combination with pembrolizumab. In some embodiments, the method reduces tumor size by at least about 10% (including, for example, at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, 95% or more).

[0094] In some embodiments, methods are provided for extending the time to disease progression of cancer in an individual, comprising administering to the individual an effective amount of any one of the anti-CTLA4 antibodies described herein in combination with pembrolizumab. In some embodiments, the method extends the time to disease progression by at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 16 weeks, 20 weeks, 24 weeks, 28 weeks, 32 weeks, 36 weeks, or more.

[0095] In some embodiments, methods are provided for extending survival (e.g., overall survival or progression-free survival) of an individual with cancer, comprising administering to the individual an effective amount of any one of the anti-CTLA4 antibodies described herein in combination with pembrolizumab. In some embodiments, the method extends the individual's survival 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.

[0096] In some embodiments, methods are provided for alleviating one or more symptoms in an individual, comprising administering to the individual an effective amount of any one of the anti-CTLA4 antibodies described herein in combination with pembrolizumab.

[0097] In some embodiments, methods are provided for improving the quality of life of an individual with cancer, comprising administering to the individual an effective amount of any one of the anti-CTLA4 antibodies described herein in combination with pembrolizumab.

[0098] The anti-CTLA4 antibody and pembrolizumab may be administered in combination with one or more additional therapeutic agents or treatments. In some embodiments, the anti-CTLA4 antibody and pembrolizumab are administered in combination with one or more additional therapeutic agents, either separately, sequentially, or simultaneously. The term "additional therapeutic agent" refers to any therapeutic agent other than the anti-CTLA4 antibody provided by the present disclosure. In some embodiments, a combination therapy for treating cancer in a subject is provided, the combination therapy comprising administering to the subject an effective amount of an anti-CTLA4 antibody described herein in combination with one or more additional therapeutic agents. In some embodiments, the anti-CTLA4 antibody is administered in combination with one or more additional therapeutic agents, including a chemotherapeutic agent, an immunotherapeutic agent, and / or a hormonal therapy agent. In some embodiments, the one or more therapeutic agents are selected from the group consisting of viral gene therapy, immune checkpoint inhibitors, targeted therapy, radiation therapy, vaccine therapy, and chemotherapy.

[0099] III. Anti-CTLA4 antibody The methods described herein include administering anti-CTLA4 antibodies that specifically bind to human CTLA4, including CTLA4 antibodies, antigen-binding fragments of CTLA4 antibodies, and derivatives of CTLA4 antibodies. Exemplary anti-CTLA4 antibodies are described, for example, in International Publication No. WO2019149281A1, which is incorporated herein by reference in its entirety.

[0100] In some embodiments, the anti-CTLA4 antibody is any one of the antibodies described herein, including those described in terms of HVRs, variable regions (VL, VH), and specific amino acid sequences of the light and heavy chains (e.g., IgG1, IgG2, IgG4). In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is a humanized antibody and / or a chimeric antibody.

[0101] In some embodiments, the antibodies or antigen-binding fragments described herein have antagonist activity against human CTLA4. In some embodiments, the antibodies or antigen-binding fragments inhibit one or more activities of human CTLA4 (e.g., CTLA4 blockade as measured by an increase in reporter gene signal using a CTLA4 blockade reporter gene assay) when cells expressing human CTLA4 (e.g., human cells) are contacted by the antibody or antigen-binding fragment.

[0102] In some embodiments, the antibody or antigen-binding fragment is cross-reactive with monkey (e.g., cynomolgus), mouse, rat, and / or dog CTLA4. In some embodiments, the antibody or antigen-binding fragment is cross-reactive with monkey CTLA4. In some embodiments, the antibody or antigen-binding fragment is cross-reactive with mouse CTLA4. In some embodiments, the antibody or antigen-binding fragment is cross-reactive with rat CTLA4. In some embodiments, the antibody or antigen-binding fragment is cross-reactive with dog CTLA4. In some embodiments, the antibody or antigen-binding fragment is cross-reactive with monkey and mouse CTLA4; monkey and rat CTLA4; monkey and dog CTLA4; mouse and rat CTLA4; mouse and dog CTLA4; rat and dog CTLA4; monkey, mouse, and rat CTLA4; monkey, rat, and dog CTLA4; mouse, rat, and dog CTLA4; or monkey, mouse, rat, and dog CTLA4. In some embodiments, an antibody or antigen-binding fragment is cross-reactive if the antibody or antigen-binding fragment binds to a non-human CTLA4 molecule with a KD of less than about 500 nM (e.g., less than about 1 nM, less than about 10 nM, less than about 25 nM, less than about 50 nM, less than about 75 nM, less than about 100 nM, less than about 150 nM, less than about 200 nM, less than about 250 nM, less than about 300 nM, less than about 350 nM, etc.). Methods for measuring antibody cross-reactivity are known in the art and include, but are not limited to, surface plasmon resonance, ELISA, isothermal titration calorimetry, filter binding assays, EMSA, etc. In some embodiments, cross-reactivity is measured by ELISA.

[0103] In some embodiments, the antibody induces an ADCC effect against CTLA4-expressing cells (e.g., against CTLA4-expressing human cells, such as Tregs) after the antibody binds to the cell-expressed CTLA4. Methods (e.g., in vitro methods) for measuring ADCC effect are well known in the art. In some embodiments, the antibody induces an ADCC effect of greater than about 10% compared to a control (e.g., an isotype control or ipilimumab) (e.g., induces ADCC by greater than about 10%, greater than about 15%, greater than about 20%, greater than about 25%, greater than about 30%, greater than about 35%, greater than about 40%, etc.).

[0104] In some embodiments, the antibody or antigen-binding fragment can inhibit tumor cell growth and / or proliferation. In some embodiments, tumor cell growth and / or proliferation is inhibited by at least about 5% (e.g., at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 99%) when contacted with the antibody or antigen-binding fragment, compared to corresponding tumor cells not contacted with the antibody or antigen-binding fragment (or compared to corresponding tumor cells contacted with an isotype control antibody). In some embodiments, the antibody or antigen-binding fragment can reduce tumor volume in a subject when the antibody or antigen-binding fragment is administered to the subject. In some embodiments, the antibody or antigen-binding fragment can reduce tumor volume in a subject by at least about 5% (e.g., at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 99%) compared to the initial tumor volume in the subject (e.g., compared to a corresponding tumor in a subject administered an isotype control antibody prior to administration of the antibody or antigen-binding fragment). Methods for measuring tumor cell growth / proliferation, tumor volume, and / or tumor inhibition are known in the art.

[0105] In some embodiments, the antibody or antigen-binding fragment has a therapeutic effect against cancer. In some embodiments, the antibody or antigen-binding fragment reduces one or more signs or symptoms of cancer. In some embodiments, a subject with cancer experiences a partial or complete remission when administered the antibody or antigen-binding fragment.

[0106] In some embodiments, the antibody or antigen-binding fragment blocks the binding between CTLA4 and one or more of its binding partners (e.g., human CTLA4 and human CD80, human CTLA4 and human CD86). In some embodiments, the antibody or antigen-binding fragment blocks the binding between CTLA4 and its ligand in vitro. In some embodiments, the antibody or antigen-binding fragment has a half maximal inhibitory concentration (IC) of about 500 nM or less (e.g., about 500 nM or less, about 400 nM or less, about 300 nM or less, about 200 nM or less, about 100 nM or less, about 50 nM or less, about 25 nM or less, about 10 nM or less, about 1 nM or less, etc.) for blocking CTLA4 binding to CD80 and / or CD86. 50 In some embodiments, the antibody or antigen-binding fragment has a half maximal inhibitory concentration (IC) of about 100 nM or less for blocking CTLA4 binding to CD80 and / or CD86. 50 ). In some embodiments, the antibody or antigen-binding fragment completely blocks the binding of human CTLA4 to CD80 and / or CD86 when provided at a concentration of about 100 nM or greater (e.g., about 100 nM or greater, about 500 nM or greater, about 1 μM or greater, about 10 μM or greater, etc.). As used herein, the term "complete blocking" or "completely blocks" refers to the ability of an antibody or antigen-binding fragment to reduce binding between a first protein and a second protein by at least about 80% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, etc.). Methods for measuring the ability of an antibody or antigen-binding fragment to block binding of a first protein (e.g., human CTLA4) and a second protein (e.g., human CD80 or human CD86) are known in the art and include, but are not limited to, by BIAcore analysis, ELISA assay, and flow cytometry. In some embodiments, the anti-CTLA4 antibodies described herein have less activity in blocking ligand binding than ipilimumab.

[0107] In some embodiments, the anti-CTLA4 antibody has a K of 100 nM or less (e.g., 50 nM or less, 10 nM or less) as measured by surface plasmon resonance. D In some embodiments, the antibody is cross-reactive with at least one non-human species selected from cynomolgus monkey, mouse, rat, and dog.

[0108] In some embodiments, the anti-CTLA4 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HVR-H1, HVR-H2, and HVR-H3, and the light chain variable region comprises HVR-L1, HVR-L2, and HVR-L3, wherein HVR-H1 comprises an amino acid sequence according to the formula YSISSGYHWSWI (SEQ ID NO: 23), HVR-H2 comprises an amino acid sequence according to the formula LARIDWDDDKYYSTSLKSRL (SEQ ID NO: 35), HVR-H3 comprises an amino acid sequence according to the formula ARSYVYFDY (SEQ ID NO: 45), HVR-L1 comprises an amino acid sequence according to the formula RASQSVRGRFLA (SEQ ID NO: 58), HVR-L2 comprises an amino acid sequence according to the formula DASNRATGI (SEQ ID NO: 66), and HVR-L3 comprises an amino acid sequence according to the formula YCQQSSSWPPT (SEQ ID NO: 75).

[0109] In some embodiments, the antibody comprises a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 87, and b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 100 (Table A). In some embodiments, the antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the sequence of SEQ ID NO: 87, and / or a light chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to a sequence selected from SEQ ID NO: 100. TIFF2025539236000001.tif37170

[0110] The CTLA4 antibodies described herein can be of any class, such as IgG, IgM, IgE, IgA, or IgD. In some embodiments, the CTLA4 antibody is of an IgG class, such as an IgG1, IgG2, IgG3, or IgG4 subclass. CTLA4 antibodies can be converted from one class or subclass to another using methods known in the art. An exemplary method for producing antibodies of a desired class or subclass includes isolating nucleic acids encoding the heavy chain and the light chain of a CTLA4 antibody, isolating sequences encoding the VH regions, linking the VH sequences to sequences encoding the heavy chain constant regions of the desired class or subclass, expressing the light chain genes and heavy chain constructs in cells, and collecting the CTLA4 antibody. The antibodies of the present application can be monoclonal or polyclonal. The antibodies of the present application can be monospecific or multispecific (e.g., bispecific, trispecific, etc.) antibodies. In some embodiments, the CTLA4 antibodies described herein may contain one or more Fc mutations (e.g., that modulate (increase or decrease) ADCC or CDC activity). Any suitable Fc mutation known in the art may be used in the CTLA4 antibodies of the present application.

[0111] In some embodiments, the anti-CTLA4 antibody has the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGYSISSGYHWSWIRQAPGKGLEWLARIDWDDDKYYSTSLKSRLTISRDNSKNTLYLQLNSLRAEDTAVYYCARSYVYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK and a light chain comprising the amino acid sequence DIQLTQSPSSLSASVGDRVTITCRASQSVRGRFLAWYQQKPGKAPKLLIYDASNRATGIPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSSSWPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 127).In some embodiments, the anti-CTLA4 antibody has the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGYSISSGYHWSWIRQAPGKGLEWLARIDWDDDKYYSTSLKSRLTISRDNSKNTLYLQLNSLRAEDTAVYYCARSYVYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK and a light chain comprising the amino acid sequence DIQLTQSPSSLSASVGDRVTITCRASQSVRGRFLAWYQQKPGKAPKLLIYDASNRATGIPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSSSWPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 127). In some embodiments, anti-CTLA4 antibodies refer to a mixture of antibody species, each antibody species comprising a light chain comprising the amino acid sequence of SEQ ID NO: 127 and a heavy chain comprising the amino acid sequence of either SEQ ID NO: 125 or 126.

[0112] In some embodiments, the anti-CTLA4 antibody is an antigen-binding fragment of an anti-CTLA4 antibody. The antigen-binding fragment of an anti-CTLA4 antibody comprises: (i) V L , V H , C L , and C H(ii) a Fab fragment, which is a monovalent fragment consisting of one domain; (iii) a F(ab')2 fragment, which is a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; and (iv) a V H and C H (iv) a single-arm V of an antibody; L and V H Fv fragment consisting of domains, (v) V H (vi) isolated CDRs, and (vii) antibody V domains. H V of antibody linked to domain L and single-chain antibodies (scFv), which are polypeptides containing a region (see, e.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883).

[0113] In some embodiments, the anti-CTLA4 antibody is a derivative of any one of the anti-CTLA4 antibodies described herein. In some embodiments, the antibody derivative is derived from an exemplary antibody of the present application (e.g., a "parent antibody") by modifying the amino acid sequence while preserving the overall molecular structure of the amino acid sequence of the parent antibody. The amino acid sequence of any region of the parent antibody chain, such as the framework region, HVR region, or constant region, may be modified. Types of modifications include substitution, insertion, deletion, or a combination thereof of one or more amino acids of the parent antibody.

[0114] In some particular embodiments, the derivative comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 conservative or non-conservative substitutions and / or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 additions and / or deletions to the above amino acid sequence.

[0115] Amino acid substitutions include both conservative and non-conservative substitutions. The term "conservative amino acid substitution" refers to the replacement of one amino acid with another when the two amino acids have similarities in certain physicochemical properties, such as polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathicity of related residues. For example, substitutions can typically be made within the following groups: (a) nonpolar (hydrophobic) amino acids such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; (b) polar neutral amino acids such as glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; (c) positively charged (basic) amino acids such as arginine, lysine, and histidine; and (d) negatively charged (acidic) amino acids such as aspartic acid and glutamic acid. Those skilled in the art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th Ed.)). Furthermore, substitution of structurally or functionally similar amino acids is unlikely to inhibit biological activity. Exemplary conservative substitutions are shown in Table 1 below: TIFF2025539236000002.tif122170

[0116] As used herein, "framework region" or "FR" means an immunoglobulin variable region excluding the CDR regions.

[0117] Modifications may be made anywhere in the amino acid sequence of the antibody, including in the HVRs, framework regions, or constant regions. In one embodiment, the present application is directed to the V H and V LAntibody derivatives are provided that contain HVR sequences but different framework sequences from those of the exemplary antibodies. Such framework sequences can be obtained from public DNA databases or published references that contain germline antibody gene sequences. For example, germline DNA sequences for human heavy and light chain variable region genes can be found in the Genbank database or the "VBase" human germline sequence database (Kaba et al., Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242 (1991); Tomlinson et al., J. Mol. Biol. 227:776-798 (1992); and Cox et al., Eur. J. Immunol. 24:827-836 (1994)). Framework sequences that can be used in the construction of antibody derivatives include those that are structurally similar to the framework sequences used by the exemplary antibodies of the present disclosure. For example, the HVR-H1, HVR-H2, and HVR-H3 sequences, and the HVR-L1, HVR-L2, and HVR-L3 sequences of an exemplary antibody can be grafted into framework regions having the same sequence as found in the germline immunoglobulin gene from which the framework sequences are derived, or the HVR sequences can be grafted into framework regions containing one or more mutations compared to the germline sequences.

[0118] In some embodiments, the antibody derivative is a chimeric antibody comprising the amino acid sequence of an exemplary antibody of the present disclosure. In one example, one or more HVRs from one or more exemplary antibodies are combined with HVRs from an antibody from a non-human animal, such as a mouse or rat. In another example, all HVRs of the chimeric antibody are derived from one or more exemplary antibodies. In some specific embodiments, the chimeric antibody comprises one, two, or three HVRs from the heavy chain variable region and / or one, two, or three HVRs from the light chain variable region of an exemplary antibody. Chimeric antibodies can be generated using conventional methods known in the art.

[0119] Another type of modification is the V H and / or V L The preferred method involves mutating amino acid residues within the HVR regions of the heavy and / or light chains. Site-directed mutagenesis or PCR-mediated mutagenesis can be used to introduce the mutation(s), and the effect on antibody binding or other desired functional properties can be assessed using in vitro or in vivo assays known in the art. Typically, conservative substitutions are introduced. The mutations can be amino acid additions and / or deletions. Furthermore, typically, no more than one, two, three, four, or five residues within the HVR regions are altered. In some embodiments, the antibody derivative contains one, two, three, or four amino acid substitutions in the heavy and / or light chain HVRs. In another embodiment, the amino acid substitutions involve changing one or more cysteines in the antibody to another residue, such as, but not limited to, alanine or serine. The cysteines can be standard or non-standard cysteines. In one embodiment, the antibody derivative has one, two, three, or four conservative amino acid substitutions in the heavy chain HVR regions compared to the amino acid sequence of an exemplary antibody.

[0120] Also, V H and / or V LModifications can also be made to framework residues within the region. Typically, such framework variants are created to reduce the immunogenicity of antibodies. One approach is to "backmutate" one or more framework residues to the corresponding germline sequence. An antibody that has undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequence with the germline sequence from which the antibody is derived. To return the framework region sequences to their germline configuration, somatic mutations can be "backmutated" to the germline sequence, for example, by site-directed mutagenesis or PCR-mediated mutagenesis.

[0121] Furthermore, modifications can also be made within the Fc region of exemplary antibodies, typically altering one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity. In one example, the hinge region of CH1 is modified to alter, e.g., increase or decrease, the number of cysteine ​​residues in the hinge region. This approach is further described in U.S. Patent No. 5,677,425. The number of cysteine ​​residues in the hinge region of CH1 is altered, for example, to facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody. In another case, the Fc hinge region of an antibody is mutated to decrease the biological half-life of the antibody.

[0122] Furthermore, the antibodies of the present application can be modified to alter their potential glycosylation sites or patterns, according to routine experimentation known in the art. In another aspect, the present application provides derivatives of CTLA4 antibodies containing at least one mutation in the variable region of the light or heavy chain that alters the glycosylation pattern in the variable region. Such antibody derivatives may have increased affinity and / or altered specificity for antigen binding. The mutations may add a new glycosylation site to the V region, change the position of one or more V region glycosylation site(s), or remove an existing V region glycosylation site. In one embodiment, the present application provides a derivative of a CTLA4 antibody with a potential N-linked glycosylation site at an asparagine in the heavy chain variable region, thereby eliminating a potential N-linked glycosylation site in one heavy chain variable region. In another embodiment, the present application provides a derivative of a CTLA4 antibody with a potential N-linked glycosylation site at an asparagine in the heavy chain variable region, thereby eliminating a potential N-linked glycosylation site in both heavy chain variable regions. Methods for altering the glycosylation pattern of antibodies are known in the art and are described, for example, in US Pat. No. 6,933,368, the disclosure of which is incorporated herein by reference.

[0123] IV. PD-1 Antagonists In one embodiment, PD-1 antagonists useful in the treatments, medicaments, and uses of the present invention include a monoclonal antibody (mAb) or antigen-binding fragment thereof that specifically binds to PD-1 or PD-L1, preferably human PD-1 or human PD-L1. The mAb is a human antibody, humanized antibody, or chimeric antibody and may comprise a human constant region. In some embodiments, the human constant region is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4 constant regions, and in some embodiments, the human constant region is an IgG1 or IgG4 constant region. In some embodiments, the antigen-binding fragment is selected from the group consisting of Fab, Fab'-SH, F(ab')2, scFv, and Fv fragments.

[0124] Examples of mAbs that bind to human PD-1 and are useful in the therapeutic methods, medicaments, and uses of the invention are described in U.S. Patent Nos. US7488802, US7521051, US8008449, US8354509, and US8168757, and International Application Publication Nos. WO2004 / 004771, WO2004 / 072286, WO2004 / 056875, US2011 / 0271358, and WO2008 / 156712. Specific anti-human PD-1 mAbs useful as PD-1 antagonists in the methods of treatment, medicaments, and uses of the invention include pembrolizumab (also known as MK-3475), a humanized IgG4 mAb having the structure set forth in WHO Drug Information, Vol. 27, No. 2, pages 161-162 (2013) and comprising the heavy and light chain amino acid sequences set forth in Table B; nivolumab (BMS-936558), a human IgG4 mAb having the structure set forth in WHO Drug Information, Vol. 27, No. 1, pages 68-69 (2013); humanized antibodies h409A11, h409A16, and h409A17, described in WO2008 / 156712; and AMP-514, cemiplimab, camrelizumab, sintilimab, tislelizumab, and toripalimab, which are under development by MedImmune. Additional anti-PD-1 antibodies contemplated for use in the present invention include MEDI0680 (U.S. Patent No. 8,609,089), BGB-A317 (U.S. Patent Publication No. 2015 / 0079109), INCSHR1210 (SHR-1210) (PCT International Application Publication No. WO2015 / 085847), REGN-2810 (PCT International Application Publication No. WO2015 / 112800), PDR001 (PCT International Application Publication No. WO2015 / 112900), TSR-042 (ANB011) (PCT International Application Publication No. WO2014 / 179664), and STI-1110 (PCT International Application Publication No. WO2014 / 194302).

[0125] Examples of mAbs that bind to human PD-L1 and are useful in the therapeutic methods, medicaments, and uses of the present invention are described in US 8383796. Specific anti-human PD-L1 mAbs useful as PD-1 antagonists in the therapeutic methods, medicaments, and uses of the present invention include BMS-936559, MEDI4736, and MSB0010718C.

[0126] In some embodiments, the PD-1 antagonist is selected from the group consisting of 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), cemiplimab (LIBTAYO™, Regeneron Pharmaceuticals, Tarrytown, NY, USA), avelumab (BAVENCIO™, Merck KGaA, Darmstadt, Germany), or dostarlimab (JEMPERLI™, GlaxoSmithKline LLC, Philadelphia, PA, USA). In other embodiments, the PD-1 antagonist is pidilizumab (U.S. Pat. No. 7,332,582), AMP-514 (MedImmune LLC, Gaithersburg, MD, USA), PDR001 (U.S. Pat. No. 9,683,048), BGB-A317 (U.S. Pat. No. 8,735,553), or MGA012 (MacroGenics, Rockville, MD).

[0127] In one embodiment, the PD-1 antagonist useful in the methods of the invention is an anti-PD-1 antibody that inhibits binding of PD-1 to PD-L1 and PD-L2. In some embodiments of the therapeutic methods, medicaments, and uses of the invention, the PD-1 antagonist is a monoclonal antibody or antigen-binding fragment thereof comprising: (a) a light chain variable region comprising the light chain CDR1, CDR2, and CDR3 of SEQ ID NOs: 10, 11, and 12, respectively; and (b) a heavy chain variable region comprising the heavy chain CDR1, CDR2, and CDR3 of SEQ ID NOs: 15, 16, and 17, respectively.

[0128] In other embodiments of the therapeutic methods, medicaments, and uses of the present invention, the PD-1 antagonist is a monoclonal antibody or antigen-binding fragment thereof that specifically binds to human PD-1 and comprises (a) a heavy chain variable region comprising SEQ ID NO: 18 or a variant thereof, and (b) a light chain variable region comprising SEQ ID NO: 13 or a variant thereof. The variant heavy chain variable region sequence is identical to the reference sequence except for up to six conservative amino acid substitutions in the framework regions (i.e., outside the CDRs). The variant light chain variable region sequence is identical to the reference sequence except for up to three conservative amino acid substitutions in the framework regions (i.e., outside the CDRs).

[0129] In another embodiment of the methods, medicaments, and uses 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, wherein the heavy and light chains comprise the amino acid sequences of SEQ ID NO: 19 and SEQ ID NO: 14, respectively.

[0130] In all of the above methods of treatment, medicaments, and uses, the PD-1 antagonist inhibits binding of PD-L1 to PD-1, and in certain embodiments, also inhibits binding of PD-L2 to PD-1. In some embodiments of the above methods of treatment, medicaments, and uses, the PD-1 antagonist is a monoclonal antibody or antigen-binding fragment thereof that specifically binds to PD-1 or PD-L1 and blocks binding of PD-L1 to PD-1.

[0131] Table B below provides a list of amino acid sequences of exemplary anti-PD-1 mAbs for use in the therapies, medicaments and applications of the present invention. TIFF2025539236000003.tif188170TIFF2025539236000004.tif104170

[0132] In one embodiment, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain constant region, e.g., a human constant region such as a g1, g2, g3, or g4 human heavy chain constant region or a variant thereof. In another embodiment, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a light chain constant region, e.g., a human light chain constant region, such as a lambda or kappa human light chain region, or a variant thereof. By way of example and not limitation, the human heavy chain constant region can be g4 and the human light chain constant region can be kappa. In another embodiment, the Fc region of the antibody is g4 with a Ser228Pro mutation (Schuurman, J et al., Mol. Immunol. 38: 1-8, 2001). In some embodiments, different constant domains can be added to the humanized VL and VH regions derived from the CDRs provided herein. For example, if a particular intended use of the antibody (or fragment) of the present invention requires altered effector function, a heavy chain constant domain other than human IgG1 can be used, or a hybrid IgG1 / IgG4 can be utilized. While human IgG1 antibodies have long half-lives and effector functions such as complement activation and antibody-dependent cellular cytotoxicity, these activities may not be desirable for all antibody applications. In such cases, for example, a human IgG4 constant domain can be used. The present invention encompasses the use of an anti-PD-1 antibody or antigen-binding fragment thereof comprising an IgG4 constant domain. In one embodiment, the IgG4 constant domain differs from the native human IgG4 constant domain (Swiss-Prot Accession No. P01861.1) at positions corresponding to position 228 in the EU system and position 241 in the KABAT system, with native Ser108 replaced with Pro to prevent a potential interchain disulfide bond between CyslO6 and CyslO9 (corresponding to positions Cys226 and Cys229 in the EU system and positions Cys239 and Cys242 in the KABAT system) that could prevent proper intrachain disulfide bond formation. See Angal et al. (1993) Mol. Immunol. 30:105. In other cases, modified IgG1 constant domains modified to increase half-life or reduce effector function can be used.

[0133] In another embodiment, the PD-1 antagonist is an antibody or antigen binding protein having a variable light domain and / or a variable heavy domain that has at least 95%, 90%, 85%, 80%, 75%, or 50% sequence identity to one of the variable light domains or variable heavy domains described above, and that exhibits specific binding to PD-1. In another embodiment of the therapeutic methods of the invention, the PD-1 antagonist is an antibody or antigen binding protein that comprises a variable light domain and a variable heavy domain with one, two, three, four, five, or more amino acid substitutions, and that exhibits specific binding to PD-1.

[0134] In some embodiments, pembrolizumab is administered at a dose of about 400 mg every six weeks.

[0135] In some embodiments, pembrolizumab is administered at a dose of about 2 mg / kg. In some embodiments, pembrolizumab is administered at a dose of about 2 mg / kg every 3 weeks. In certain embodiments, the patient is a pediatric patient.

[0136] In some embodiments, pembrolizumab is administered as a 30-minute (-5 min / +10 min) intravenous infusion. In one embodiment, the selected dose of pembrolizumab is administered by IV infusion over 25 to 40 minutes, or about 30 minutes.

[0137] In one aspect, pembrolizumab is included in a pharmaceutical composition that includes a pharmaceutically acceptable carrier or diluent, and may include additional pharmaceutically acceptable excipients.

[0138] V. Pharmaceutical Compositions, Kits and Articles of Manufacture The anti-CTLA4 antibody and pembrolizumab described herein can be administered as a pharmaceutical composition containing a pharmaceutically acceptable carrier.The anti-CTLA4 antibody and pembrolizumab can be administered in separate pharmaceutical compositions or in a single pharmaceutical composition.The composition can be prepared by conventional methods known in the art.

[0139] The term "pharmaceutically acceptable carrier" refers to any inert substance suitable for use in a formulation for delivery of an active agent (e.g., an anti-CTLA4 antibody or pembrolizumab). Carriers can be antiadherents, binders, coatings, disintegrants, fillers or diluents, preservatives (such as antioxidants, antibacterial agents, or antifungal agents), sweeteners, absorption delaying agents, wetting agents, emulsifiers, buffers, etc. Examples of suitable pharmaceutically acceptable carriers include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), dextrose, vegetable oils (e.g., olive oil), saline, buffers, buffered saline, and isotonic agents, such as sugars, polyalcohols, sorbitol, and sodium chloride. The composition can be in any suitable dosage form, such as liquid, semisolid, and solid dosage forms. Examples of liquid dosage forms include solutions (e.g., injectable and infusible solutions), microemulsions, liposomes, dispersions, or suspensions. Examples of solid dosage forms include tablets, pills, capsules, microcapsules, and powders. A specific form of the composition suitable for delivering anti-CTLA4 antibodies is a sterile liquid, such as a solution, suspension, or dispersion for injection or infusion. Sterile solutions can be prepared by incorporating the required amount of antibody into a suitable carrier, followed by sterile microfiltration. Generally, dispersions are prepared by adding antibody to a sterile solvent containing a basic dispersion medium and other carriers. For sterile powders for preparing sterile liquids, preparation methods include vacuum drying and freeze-drying (lyophilization) to obtain a powder containing the active ingredient and any additional desired ingredients from its previously sterile-filtered solution. Various dosage forms of the composition can be prepared by conventional techniques known in the art.

[0140] In some embodiments, an article of manufacture containing a substance useful for treating cancer is provided. The article of manufacture can include a container and a label or package insert on or attached to the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The container can be formed from a variety of materials, such as glass or plastic. Generally, the container holds a composition effective for treating cancer as described herein and can have a sterile access port (e.g., the container is an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle). Package insert refers to information typically included in commercial packaging for therapeutic products, including directions, usage, dosage, administration, contraindications, and / or warnings regarding the use of the therapeutic product. In some embodiments, the package insert indicates that the composition is used for treating cancer. The label or package insert can further include instructions for administering the composition to a patient.

[0141] The article of manufacture may further comprise a second container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. The article of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0142] Also provided are kits useful for various purposes, such as, for example, treating cancer as described herein, optionally in combination with an article of manufacture. The kits of the present application include one or more containers containing any one of the compositions (or unit dosage forms and / or articles of manufacture) described herein. In some embodiments, the kits further include other agents (e.g., one or more additional therapeutic agents) and / or instructions for use in accordance with any of the methods described herein. The kits may further include instructions for individual selection of appropriate treatments. The instructions provided in the kits of the present application are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), although machine-readable instructions (e.g., instructions transferred to a magnetic or optical storage disk) are also acceptable.

[0143] For example, in some embodiments, a kit is provided that includes a pharmaceutical composition comprising any one of the anti-CTLA4 antibodies described herein and a pharmaceutically acceptable carrier, pembrolizumab, a pharmaceutically acceptable carrier, and instructions for administering the pharmaceutical composition to a subject with cancer. In some embodiments, the kit includes a pharmaceutical composition that includes an additional therapeutic agent, such as a chemotherapeutic agent. In some embodiments, the kit includes a kit that includes one or more biomarkers described herein (e.g., CD8+ T cells, CD4+ T cells, CD8+ Tem cells, CD4+ T em cell, T reg cells, CD8+T em Cells and T reg Ratio to CD4+T cells em Cells and T reg The present invention includes assays and reagents for determining levels of NK cells (NK cells, NK cells, and B cells) and their ratios.

[0144] The kits of the present application are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. The kits may optionally provide additional components, such as buffers and instructional information. Thus, the present application also provides articles of manufacture that include vials (such as sealed vials), bottles, jars, flexible packaging, and the like.

[0145] The containers may be unit doses, bulk packages (e.g., multi-dose packages), or sub-unit doses. The kits may also include multiple unit doses of the pharmaceutical composition and instructions for use, and may be packaged in quantities sufficient for storage and use in pharmacies (e.g., hospital pharmacies and compounding pharmacies). [Example]

[0146] The present invention may be further understood by reference to the following examples, which are provided by way of illustration and are not intended to be limiting.

[0147] Example 1. A Phase 1b, Open-Label, Dose-Escalation Study of TY21580 in Combination with Pembrolizumab (Anti-PD-1 Antibody) in Patients with Advanced / Metastatic Solid Tumors

[0148] The following example describes a phase 1b clinical trial evaluating the safety, tolerability, PK, and preliminary efficacy of TY21580 in combination with pembrolizumab in patients with advanced / metastatic solid tumors. TY21580 is an anti-CTLA4 fully human IgG1 monoclonal antibody. TY21580 is administered intravenously over 60 to 90 minutes. Pembrolizumab (KEYTRUDA®, Merck Sharp & Dohme LLC, Rahway, NJ, USA) is a PD-1 receptor inhibitor antibody (humanized IgG4 monoclonal antibody) indicated for the treatment of patients with various cancers. Pembrolizumab is administered intravenously over 30 minutes. In the TY21580-pembrolizumab combination, TY21580 is administered 30 to 60 minutes after the end of the pembrolizumab infusion.

[0149] Objectives: The primary objectives of this study are to evaluate the safety and tolerability of TY21580 at escalating dose levels and in combination with pembrolizumab in adult patients with advanced / metastatic solid tumors, to determine the maximum tolerated dose (MTD), and to evaluate the preliminary antitumor activity of the TY21580 and pembrolizumab combination. Secondary objectives of this study are to evaluate the pharmacokinetic (PK) profiles of TY21580 and pembrolizumab, assess dose proportionality of key PK parameters (e.g., area under the time-concentration curve [AUC], maximum [peak] serum concentration [Cmax]), evaluate the immunogenicity of TY21580 and pembrolizumab, characterize the relationship between immunogenicity (anti-drug antibody [ADA] positivity) and PK, safety, and efficacy parameters, evaluate the preliminary antitumor activity of TY21580-pembrolizumab therapy, evaluate the safety and tolerability of the combination of TY21580 and pembrolizumab in adults with advanced / metastatic solid tumors, evaluate the PK profiles of TY21580 and pembrolizumab, and evaluate the immunogenicity of TY21580 and pembrolizumab. Exploratory objectives of this study are to evaluate pharmacodynamic or potentially predictive biomarkers of TY21580. Biomarkers to be evaluated include, but are not limited to, cytokines (e.g., IL-10, IL-2, IL-6, interferon [IFN]-γ, tumor necrosis factor [TNF]-α), serum proteins (e.g., sCTLA4, sPD-Ll, sCD25, CXCL11), tumor-infiltrating lymphocytes (TILs), regulatory T cells (Tregs; e.g., CD4+ Tern, CD8+ Tern, Ki67), and other tissue biomarkers (e.g., MSI, TMB, PD-L1).

[0150] Study Design: This is a Phase 1b, open-label, multicenter, dose-escalation and dose-expansion study to evaluate the safety, tolerability, PK, and preliminary efficacy of TY21580 in combination with pembrolizumab in patients with advanced / metastatic solid tumors.

[0151] A modified toxicity probability interval (mTPI) design with a target DLT rate of approximately 30% will be applied for dose escalation and confirmation to determine the RP2D. TY21580 in combination with pembrolizumab. Dose escalation will include four dose-escalation cohorts, as shown in Table 2 below. TIFF2025539236000005.tif54170

[0152] Dose escalation of TY21580 in combination with fixed-dose pembrolizumab will begin at one level below the dose at which TY21580 monotherapy was cleared and continue at the next dose level until the RP2D of the combination is determined. If the starting dose of TY21580 is deemed intolerable, reduced doses of TY21580 will be available. All dose escalation and deescalation decisions will be based on the occurrence of DLTs at a specific dose during the first 21 days (Cycle 1) and will be made by a Safety Review Committee (SRC) comprised of the principal investigator (PI), medical monitor, and sponsor.

[0153] For the combination treatment of TY21580 with pembrolizumab, both drugs will be administered Q3W for up to 35 cycles at 200 mg for each dose level of TY21580 in each cohort, with pembrolizumab remaining constant. The dose and frequency of pembrolizumab will remain unchanged.

[0154] The safety and tolerability of each dose level will be evaluated by the SRC after all patients enrolled at that dose level have been followed for at least 21 days (DLT observation period) after their first dose of TY21580-pembrolizumab combination therapy.

[0155] Once either the MTD or maximum administered dose (MAD) is reached, the RP2D is defined. Any changes require submission of a revised protocol to the Independent Ethics Committee (IEC) / Institutional Review Board (IRB) and appropriate regulatory authorities. The RP2D is determined based on observations at the MTD, or, if there is no MTD, based on the MAD. Options for consideration of the RP2D include dose levels below the MTD or MAD, and intermediate doses between pre-specified dose levels (e.g., 3–20 mg / kg) are determined based on a comprehensive evaluation of all safety data, all available PK and pharmacodynamic data, and documented objective response observations during dose escalation.

[0156] Treatment cycles are 21 days long, with a single IV administration of TY21580 in combination with pembrolizumab on day 1. DLTs will be assessed during the first 21 days. Toxicity will be assessed using the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE) v5.0. Patients will receive the TY21580-pembrolizumab Q3W dosing regimen until disease progression according to RECIST v1.1 and / or iRECIST, significant toxicity, withdrawal of consent, or other reason for discontinuation / withdrawal, or for a maximum of 35 cycles (Q3W), whichever occurs first. Patient safety and toxicity, PK, immunogenicity, objective response rate, duration of response (DOR), progression-free survival (PFS), overall survival (OS), and biomarkers will be evaluated during the study.

[0157] Safety Assessment. Safety assessments will be conducted based on specified periodic physical examination findings, vital signs, ECOG performance status, clinical laboratory values ​​(e.g., liver function tests / monitoring, hematology, coagulation tests, serum chemistry, urinalysis, pregnancy test), electrocardiogram, and adverse events. Adverse events will be graded according to the NCI CTCAE v5.0. Investigators and site personnel are responsible for appropriately recording and reporting AEs / SAEs. The Safety Review Committee (SRC) will be composed of investigators and representatives from the sponsor. The SRC will review available safety, clinical activity, PK, and pharmacodynamic data, identify DLTs upon completion of each dose-level cohort, and make recommendations regarding dosing and dose escalation. The SRC may recommend intermediate dose levels to be evaluated within the protocol-specified dose levels. These decisions will be documented.

[0158] Efficacy Assessments. Tumor response / progression assessments will be performed at baseline and every 6 weeks (± 1 week) for the first 4 cycles. If treatment continues beyond 4 cycles, assessments will be performed every 9 weeks (± 1 week) thereafter for the remainder of treatment until disease progression or death, treatment / study discontinuation due to treatment toxicity, loss to follow-up, withdrawal of consent, initiation of new cancer treatment, or completion / termination of study, whichever occurs first. In this study, response and progression will be assessed using the international criteria proposed in the revised RECIST vl.l guidelines and / or iRECIST.

[0159] Pharmacokinetic and immunogenicity assessment. Blood samples will be collected from all patients during the first cycle to measure serum concentrations of TY21580 and pembrolizumab. PK parameters of TY21580 will be monitored more intensively during the first treatment cycle. Reduced PK sampling will be performed for pembrolizumab. Noncompartmental analysis of TY21580 will be performed using WinNonlin 8.3 or later. PK parameters will include AU Co-21d , AUC last , Cmax , T max , t 1 / 2 , MRT, CL, V d These include, but are not limited to, dose proportionality, as measured by AUC and C max ADA blood samples for TY21580 will be collected pre-dose for cycles 1-4, and then every four cycles if treatment continues beyond four cycles. ADA sampling will be scaled down for pembrolizumab. Additionally, ADA samples will be collected at the end of treatment and, if possible, 30 days after the last dose.

[0160] Pharmacodynamic Evaluation: Pharmacodynamic biomarkers for TY21580 are listed and summarized by protocol-specified time points and treatments and include, but are not limited to, soluble proteins (sCTLA-4, MMPs), peripheral immune cell subset profiling, tumor-infiltrating lymphocytes, and pharmacogenomic markers in both peripheral blood and tumor tissue (when available).

[0161] Optional Biopsy Assessment. Patient biopsies before, during, and at the end of treatment are optional but highly recommended for biomarker evaluation. Patients may be able to obtain sufficient and adequate formalin-fixed tumor tissue samples (e.g., 15 FFPE slides) from a tumor lesion biopsy, preferably from a previously unirradiated site, at or after diagnosis of advanced cancer. Alternatively, patients may be biopsied prior to study entry to provide sufficient tissue. Index lesions should not be used for optional biopsies. Patients with biopsy-available tumors may also undergo optional post-treatment tumor biopsies at Cycle 3 and end of treatment. Patients will be given separate, specific written consent to provide baseline, during-treatment, and / or end-of-treatment biopsies. The Cycle 3 biopsy should be collected after the tumor radiation scans scheduled for that cycle have been completed.

[0162] Interim Results Six patients were treated with the combination of TY21580 (3 mg / kg Q3W) and pembrolizumab (200 mg Q3W). Patients were generally well-pretreated (Table 3). Tumor types included breast, colorectal, and pancreatic cancers, which are generally considered "cold" tumors. TIFF2025539236000006.tif103170

[0163] Clinical Safety Evaluation TY21580 demonstrated a manageable safety and tolerability profile in combination with pembrolizumab at a dose of 3 mg / kg, with no dose-limiting toxicities observed. The most frequently observed TRAEs were fatigue (4 / 6, 67%), pruritus (3 / 6, 50%), and nausea (3 / 6, 50%). Two patients experienced grade 3 TRAEs: one grade 3 dehydration (Cycle 3) and one grade 3 rash (Cycle 1) (Tables 4 and 5). TIFF2025539236000007.tif19170TIFF2025539236000008.tif91170

[0164] Clinical Activity Evaluation Two MSS CRC patients with lung or liver metastases demonstrated stable disease (SD) and a decrease in CEA levels after combination therapy. Case 1 was a 47-year-old female patient who had received five prior systemic therapies and had lung nodule target lesions measuring 19 mm and 33 mm at baseline. She demonstrated SD with a 4% decrease in total target lesions and a 27% decrease in CEA levels from baseline at the end of the second cycle (Figure 1). Case 2 was a 47-year-old male patient who had received five prior systemic therapies and had baseline target lesions measuring 74, 22, and 20 mm in the liver, lymph nodes, and lungs, respectively. The patient demonstrated stable disease with a 3% increase in total target lesions and a 27% decrease in CEA levels from baseline at the end of the second cycle (Figure 2).

[0165] Peripheral pharmacodynamic analysis Peripheral immune activation was observed, and treatment with TY21580 plus pembrolizumab increased peripheral CD4+ and CD8+ T cell proliferation (Ki-67+) as demonstrated by flow cytometry analysis, as well as serum levels of pro-inflammatory cytokines, including IFN-γ and TNF-α (Figure 3). All data are compared to pre-treatment baseline.

[0166] Clinical Pharmacokinetics As shown in Figure 4, combination therapy with pembrolizumab did not alter TY21580 serum PK compared to TY21580 monotherapy. The mean terminal half-life of TY21580 was estimated to be approximately 10 days in cycle 1 PK, consistent with minimal accumulation after Q3W repeated dosing in this study. Combination therapy did not have a significant adverse effect on TY21580 PK.

Claims

1. 1. A method of treating cancer in a subject, comprising administering to the subject an effective amount of an anti-CTLA4 antibody and an effective amount of pembrolizumab, wherein the anti-CTLA4 antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region of the antibody comprises HVR-H1, HVR-H2, and HVR-H3, and the light chain variable region of the antibody comprises HVR-L1, HVR-L2, and HVR-L3, wherein HVR-H1 comprises an amino acid sequence according to the formula YSISSGYHWSWI (SEQ ID NO: 23), HVR-H2 comprises an amino acid sequence according to the formula LARIDWDDDKYYSTSLKSRL (SEQ ID NO: 35), and HVR-H3 comprises HVR-L1 comprises an amino acid sequence according to the formula ARSYVYFDY (SEQ ID NO: 45), HVR-L1 comprises an amino acid sequence according to the formula RASQSVRGRFLA (SEQ ID NO: 58), HVR-L2 comprises an amino acid sequence according to the formula DASNRATGI (SEQ ID NO: 66), and HVR-L3 comprises an amino acid sequence according to the formula YCQQSSSWPPT (SEQ ID NO: 75), wherein the anti-CTLA4 antibody is administered at a dose of about 3 mg / kg to about 10 mg / kg once every 3 to 6 weeks, and the pembrolizumab is administered at a dose of about 100 mg to about 300 mg once every 3 weeks or at a dose of about 200 mg to about 600 mg once every 6 weeks.

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

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

4. 10. The method of claim 1, wherein the pembrolizumab is administered at a dose of about 200 mg once every three weeks.

5. 10. The method of claim 1, wherein the pembrolizumab is administered at a dose of about 400 mg once every six weeks.

6. 6. The method of any one of claims 1 to 5, wherein the cancer is resistant or refractory to conventional treatment, and the conventional treatment is an inhibitor of CTLA4, PD-1, or PD-1 ligand.

7. 7. The method of claim 6, wherein the conventional treatment is ipilimumab.

8. The method of any one of claims 1 to 7, wherein the cancer is colorectal cancer.

9. 9. The method of claim 8, wherein the CRC is a microsatellite stable (MSS) CRC.

10. The method of any one of claims 1 to 7, wherein the cancer is Kaposi's sarcoma.

11. The method of any one of claims 1 to 7, wherein the cancer is head and neck squamous cell carcinoma (HNSCC) or angiosarcoma.

12. The method according to any one of claims 1 to 7, wherein the cancer is pancreatic cancer.

13. The method according to any one of claims 1 to 7, wherein the cancer is ovarian cancer.

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

15. 15. The method of claim 14, wherein the cancer has metastasized to the lung or liver.

16. The method of any one of claims 1 to 15, wherein the anti-CTLA4 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 87 or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 87, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 100 or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:

100.

17. 17. The method of claim 16, 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.

18. 18. The method of claim 17, wherein the anti-CTLA4 antibody comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO: 126 and a light chain region comprising the amino acid sequence of SEQ ID NO:

127.

19. 18. The method of claim 17, wherein the anti-CTLA4 antibody comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO: 125 and a light chain region comprising the amino acid sequence of SEQ ID NO:

127.

20. The method of any one of claims 1 to 19, wherein the subject is a human.

21. 21. The method of any one of claims 1-20, wherein both the anti-CTLA4 antibody and pembrolizumab are administered on day 1 of a 3-6 week dosing schedule.

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