Cancer treatment method using anti-CTLA4 antibody

Abstract

The present application provides compositions and methods for treating cancer, including cancers that are resistant or refractory to inhibitors of PD-1 or PD-L1, using anti-CTLA4 antibodies. em ) cells, CD4+T em cells, regulatory T(T reg Biomarkers such as IL-18 (immunoglobulin) and natural killer (NK) cell levels are also provided.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Patent Application No. 63 / 167,111, filed March 28, 2021, the disclosure of which is incorporated by reference in its entirety herein.

[0002] This application is in the field of cancer treatment and relates to compositions and methods for treating cancer using antibodies that bind to human CTLA4. [Background technology]

[0003] Submitting a sequence listing as an ASCII text file The following submission in an ASCII text file, a computer readable form (CRF) of the sequence listing (file name: 695402001340SEQLIST.TXT, date of recording: March 25, 2022, size: 98,612 bytes), is hereby incorporated by reference in its entirety.

[0004] CTLA4 is a member of the immunoglobulin (Ig) superfamily of proteins that act to downregulate T cell activation and maintain 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, as translation from preclinical animal models to human safety is often poor. Therefore, there is a need for anti-CTLA4 antibodies that are cross-reactive between different species, such as humans and experimental animals (e.g., mice, monkeys, rats, etc.), to enable animal model studies while providing suitable human therapeutic candidates. Furthermore, there is a need to develop safer anti-CTLA4 antibodies that are active only in certain contexts, such as the protease-rich tumor microenvironment. Summary of the Invention

[0005] The present application provides methods for treating cancer with anti-CTLA4 antibodies and methods for using biomarkers (e.g., pharmacodynamic biomarkers) to determine patient responsiveness to anti-CTLA4 antibody treatment, adjust the dosage and administration schedule of treatment, and monitor the prognosis of treatment.

[0006] Accordingly, in one aspect, provided herein is a method of treating cancer in a subject, comprising: (a) administering to the subject an effective amount of an anti-CTLA4 antibody that specifically binds to an epitope that includes amino acid residues Y105 and L106, but not including residue I108, of human CTLA4, where the numbering of the amino acid residues is based on SEQ ID NO:108; and (b) then detecting in the subject's sample the level of IL-1β, IL-2, IL-6, IL-10, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, soluble CTLA4 (sCTLA4), soluble PD-L1 (sPD-L1), soluble CD25 (sCD25), CXCL11, FoxP3, Ki67, CD8+ T cells, CD4+ T cells, CD8+ effector memory T cells, CD8+ ... em ) cells, CD4+T em cells, regulatory T(T reg ) cells, CD8+T em Cells and regulatory T(T reg ) cells, CD4+T em Cells and T reg and determining the level of one or more biomarkers selected from the group consisting of: a ratio of CTLA4 cells to NK cells and B cells. In some embodiments, an increase or decrease in the one or more biomarkers after administration of the anti-CTLA4 antibody compared to a baseline level of the one or more biomarkers indicates that the subject is likely to have an effective response to the CTLA4 antibody. In some embodiments, if the sample has an increase or decrease in the one or more biomarkers after administration of the anti-CTLA4 antibody compared to the baseline level of the one or more biomarkers, the method further comprises administering to the subject additional cycles of an effective amount of an anti-CTLA4 antibody.

[0007] Thus, in one aspect, provided herein is a method for providing a prognosis to a subject to which an effective amount of an anti-CTLA4 antibody that specifically binds to an epitope that includes amino acid residues Y105 and L106, but not including residue I108, of human CTLA4, where the numbering of the amino acid residues is based on SEQ ID NO: 108, is administered, comprising detecting in a sample from the subject, IL-1β, IL-2, IL-6, IL-10, IFN-γ, TNF-α, sCTLA4, sPD-L1, sCD25, CXCL11, FoxP3, Ki67, CD8+ T cells, CD4+ T cells, CD8 ... em 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 method includes determining the level of one or more biomarkers selected from the group consisting of: a ratio of CTLA4 cells to NK cells, and B cells, wherein an increase or decrease in the one or more biomarkers after administration of an anti-CTLA4 antibody compared to a baseline level of the one or more biomarkers indicates that the subject is likely to have an effective response to the CTLA4 antibody.

[0008] In some embodiments of any one of the methods described above, the one or more biomarkers are selected from the group consisting of CD8+ T cells, CD4+ T cells, CD8+ T em 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 In some embodiments, the ratio of CD8+ T cells, CD4+ T cells, ... em cells, CD4+T em cells, Treg cells, CD8+T em Cells and T reg Ratio to CD4+T cells em Cells and T regIncreased levels of CD8+ T cells, NK cells, and B cells indicate that the subject is likely to have an effective response to the anti-CTLA4 antibody. em In some embodiments, the one or more biomarkers include CD4+ T cells. em In some embodiments, the one or more biomarkers include NK cells. In some embodiments, the one or more biomarkers include CD8+ T cells. em Cells and T reg In some embodiments, the one or more biomarkers include a ratio of CD4+ T cells. em Cells and T reg In some embodiments, the one or more biomarkers include a ratio of T reg In some embodiments, the T reg T cells after administration of anti-CTLA4 antibody compared to baseline levels reg A decrease in the level of cells indicates that the subject is more likely to have an effective response to the CTLA4 antibody.

[0009] In some embodiments according to any one of the above methods, the sample is a blood sample. In some embodiments, the sample is a tumor biopsy sample.

[0010] 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 conventional therapy 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, e.g., pembrolizumab. In some embodiments, the conventional therapy is an inhibitor of PD-1 ligand (e.g., PD-L1), e.g., an anti-PD-L1 antibody. In some embodiments, the conventional therapy includes both an inhibitor of CTLA4 and an inhibitor of PD-1. In some embodiments, the conventional therapy includes both an inhibitor of CTLA4 and an inhibitor of PD-L1. In some of the above embodiments, the anti-CTLA4 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:87 or an amino acid sequence 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 of the above embodiments, the anti-CTLA4 antibody comprises a human IgG1 Fc region, such as a wild-type IgG1 Fc region or a variant with enhanced ADCC activity. In some embodiments, the anti-CTLA4 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:125 and a light chain comprising the amino acid sequence of SEQ ID NO:127. In some of the above embodiments, the anti-CTLA4 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:126 and a light chain comprising the amino acid sequence of SEQ ID NO:127. In some of the above embodiments, the anti-CTLA4 antibody is TY21580.

[0011] Another aspect of the application provides a method of treating cancer in a subject, comprising administering to the subject an effective amount of an anti-CTLA4 antibody that specifically binds to an epitope that includes amino acid residues Y105 and L106, but not including residue I108, of human CTLA4, as numbered by amino acid residues according to SEQ ID NO: 108, where the cancer is resistant or refractory to conventional treatment, and the conventional treatment is an inhibitor of CTLA4, PD-1, or PD-1 ligand. In some embodiments, the application provides a method of treating cancer in a subject, comprising administering to the subject an effective amount of an anti-CTLA4 antibody that specifically binds to an epitope that includes amino acid residues Y105 and L106, but not including residue I108, of human CTLA4, as numbered by amino acid residues according to SEQ ID NO: 108, where the subject is resistant to or has relapsed from conventional treatment, and the conventional treatment is an inhibitor of CTLA4, PD-1, or PD-1 ligand. In some embodiments, the conventional treatment 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, e.g., pembrolizumab. In some embodiments, the conventional therapy is an inhibitor of a PD-1 ligand (e.g., PD-L1), e.g., an anti-PD-L1 antibody.

[0012] 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 cancer. In some embodiments, the cancer is an advanced stage cancer. In some embodiments, the cancer is a metastatic cancer.

[0013] In some embodiments according to any one of the above methods, the anti-CTLA4 antibody is administered at a dose of about 0.001 mg / kg to about 20 mg / kg. In some embodiments according to any one of the above methods, the anti-CTLA4 antibody is administered at a dose of about 0.001 mg / kg to about 10 mg / kg. In some embodiments, the anti-CTLA4 antibody is administered at a dose of at least about 0.03 mg / kg. In some embodiments, the anti-CTLA4 antibody is administered at a dose of at least about 6 mg / kg. In some embodiments, the anti-CTLA4 antibody is administered at a dose of about 0.03 mg / kg, 0.1 mg / kg, 0.3 mg / kg, 1.0 mg / kg, 3.0 mg / kg, 6.0 mg / kg, 10.0 mg / kg, 15 mg / kg, or 20 mg / kg. In some embodiments, the anti-CTLA4 antibody is administered intravenously. In some embodiments, the anti-CTLA4 antibody is administered subcutaneously. In some embodiments, the anti-CTLA4 antibody is administered about once every three weeks. In some embodiments, the subject receives at least four cycles of treatment with the anti-CTLA4 antibody. In some embodiments, the subject further receives a maintenance treatment comprising administering to the subject an effective amount of the anti-CTLA4 antibody about once every four weeks to about once every twelve weeks (e.g., once every four, six, eight, ten, or twelve weeks).

[0014] Another aspect of the present application provides a method of treating cancer in a subject, comprising administering to the subject an effective amount of an anti-CTLA4 antibody that specifically binds to an epitope including amino acid residues Y105 and L106 but not including residue I108 of human CTLA4, where the amino acid residue numbering is based on SEQ ID NO:108, wherein the anti-CTLA4 antibody is administered at a dose of at least 6 mg / kg.

[0015] In some embodiments according to any one of the above methods, the anti-CTLA4 antibody is administered at a dose of about 6 mg / kg (eg, 6 mg / kg).

[0016] In some embodiments, the anti-CTLA4 antibody is administered at a dose of about 10 mg / kg (eg, 10 mg / kg).

[0017] In some embodiments, the anti-CTLA4 antibody is administered at a dose of about 3 mg / kg (e.g., 3 mg / kg) once every three weeks. In some embodiments, the anti-CTLA4 antibody is administered at a dose of about 6 mg / kg (e.g., 6 mg / kg) once every three weeks. In some embodiments, the anti-CTLA4 antibody is administered at a dose of about 10 mg / kg (e.g., 10 mg / kg) once every three weeks. In some embodiments, the anti-CTLA4 antibody is administered at a dose of about 10 mg / kg (e.g., 10 mg / kg) once every three weeks. In some embodiments, the anti-CTLA4 antibody is administered at a dose of about 15 mg / kg (e.g., 15 mg / kg) once every three weeks. In some embodiments, the anti-CTLA4 antibody is administered at a dose of about 20 mg / kg (e.g., 15 mg / kg) once every three weeks. In some embodiments, the cancer is urothelial carcinoma. In some of the above embodiments, the anti-CTLA4 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:87 or an amino acid sequence 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 of the above embodiments, the anti-CTLA4 antibody comprises a human IgG1 Fc region, such as a wild-type IgG1 Fc region or a variant with enhanced ADCC activity. In some embodiments, the anti-CTLA4 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:125 and a light chain comprising the amino acid sequence of SEQ ID NO:127. In some of the above embodiments, the anti-CTLA4 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:126 and a light chain comprising the amino acid sequence of SEQ ID NO:127. In some of the above embodiments, the anti-CTLA4 antibody is TY21580.

[0018] In some embodiments according to any one of the above methods, the subject is a human.

[0019] In some embodiments according to any one of the above methods, the anti-CTLA4 antibody is cross-reactive with CTLA4 polypeptides from at least one non-human animal selected from the group consisting of cynomolgus monkey, mouse, rat, and dog. In some embodiments, the antibody binds to cynomolgus monkey CTLA4 and mouse CTLA4. In some embodiments, the antibody binds to human CTLA4, cynomolgus monkey CTLA4, mouse CTLA4, rat CTLA4, and / or dog CTLA4 with a dissociation constant (K D In some embodiments, K D is measured by surface plasmon resonance (SPR). In some embodiments, the binding of the antibody to CTLA4 induces antibody-dependent cellular cytotoxicity (ADCC) against CTLA4-expressing cells. In some embodiments, the binding of the antibody to CTLA4 induces ADCC against Treg cells. In some embodiments, the binding of the anti-CTLA4 antibody described herein induces antibody-dependent cellular cytotoxicity (ADCC) against CTLA4-expressing human cells or human Treg cells, and the ADCC activity of the anti-CTLA4 antibody is higher than the ADCC activity of ipilimumab in vitro, provided that both antibodies comprise a wild-type human IgG1 Fc region. In some embodiments, the binding of the anti-CTLA4 antibody described herein induces antibody-dependent cellular cytotoxicity (ADCC) against CTLA4-expressing human cells or human Treg cells, and the ADCC activity of the anti-CTLA4 antibody is more than two-fold higher than the ADCC activity of ipilimumab in vitro, provided that both antibodies comprise a wild-type human IgG1 Fc region. In some embodiments, the EC50 of anti-CTL4 antibody ADCC activity is 50% or less than the EC50 of ipilimumab ADCC activity in vitro. In some embodiments, anti-CTLA4 antibodies selectively deplete Treg cells in the tumor microenvironment (e.g., reduce the percentage of Treg cells among tumor-infiltrating lymphocytes) compared to PBMCs or spleen in mouse cancer models.

[0020] In some embodiments according to any one of the above methods, the antibody specifically binds to an epitope that includes amino acid residues of a ligand binding site of human CTLA4, such as the CD80 and / or CD86 binding site of human CTLA4. In some embodiments, the antibody specifically binds to an epitope that is similar to a ligand binding site of human CTLA4, such as the CD80 and / or CD86 binding site of human CTLA4. In some embodiments, the anti-CTLA4 antibody blocks binding of CD80 and / or CD86 to human CTLA4. In some embodiments, the anti-CTLA4 antibody has an IC50 that is higher than the IC50 of ipilimumab for blocking binding of CD80 and / or CD86 to human CTLA4. In some embodiments, in an assay in which CD86 or CD80 is bound to a plate and CTLA4 is in solution or CTLA4 is displayed on a cell surface, the anti-CTLA4 antibody has an IC50 that is 3.5-fold or more higher (e.g., 3.9-fold or more) than the IC50 of ipilimumab in blocking binding of CD80 and / or CD86 to human CTLA4.

[0021] In some embodiments according to any one of the methods above, the antibody comprises a heavy chain variable region and a light chain variable region, wherein a) the heavy chain variable region comprises HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H1 is a sequence selected from the group consisting of formula (I): X1TFSX2YX3IHWV (SEQ ID NO: 1), where X1 is F or Y, X2 is D or G, and X3 is A, G, or W; formula (II): YSIX1SGX2X3WX4WI (SEQ ID NO: 2), where X1 is S or T, X2 is H or Y, X3 is H or Y, and X4 is A, D, or S; and formula (III): FSLSTGGVAVX1WI (SEQ ID NO: 3) (wherein X1 is G or S), and HVR-H2 comprises an amino acid sequence according to a formula selected from the group consisting of formula (IV): IGX1IX2HSGSTYYSX3SLKSRV (SEQ ID NO: 4) (wherein X1 is D or E, X2 is S or Y, and X3 is P or Q), formula (V): IGX1ISPSX2GX3TX4YAQKFQGRV (SEQ ID NO: 5) (wherein X1 is I or W, X2 is G or S, X3 is G or S, and X4 is K or N), and formula (V): HVR-H3 comprises an amino acid sequence according to a formula selected from the group consisting of: formula (VII): ARX1X2X3X4FDX5 (SEQ ID NO: 7) (wherein X1 is G, R, or S, X2 is A, I, or Y, X3 is D, V, or Y, X4 is A, E, or Y, and X5 is I or Y); formula (VIII): ARX1GX2GY FDX3 (SEQ ID NO: 8) (wherein X1 is D or L, X2 is F or Y, and X3 is V or Y), formula (IX): ARX1X2X3X4AX5X6FDY (SEQ ID NO: 9) (wherein X1 is L or R, X2 is I or P, X3 is A or Y, X4 is S or T, X5 is T or Y, and X6 is A or Y), and formula (X): ARDX1X2X3GSSGYYX4GFDX5 (SEQ ID NO: 10) (wherein X1 is I or V, X2 is A or H, X3 is P or S, and X4 is D or Y,and b) the light chain variable region comprises an amino acid sequence according to a formula selected from the group consisting of HVR-L1, HVR-L2, and HVR-L3, wherein HVR-L1 is selected from the group consisting of formula (XI): RASQX1X2X3SX4LX5 (SEQ ID NO: 11) (wherein X1 is G or S, X2 is I or V, X3 is G or S, X4 is S or Y, and X5 is A or N), formula (XII): RASQX1VX2X3RX4LA (SEQ ID NO: 12) (wherein X X1 is S or T, X2 is F, R, or S, X3 is G or S, and X4 is F or Y; and formula (XIII): RASX1SVDFX2GX3SFLX4 (SEQ ID NO: 13) (wherein X1 is E or Q, X2 is D, F, H, or Y, X3 is F, I, or K, and X4 is A, D, or H); and HVR-L2 comprises an amino acid sequence according to formula (XIV): X1ASX2X3X4X5GX6 (SEQ ID NO: 14) wherein X1 is A or D, X2 is N, S, or T, X3 is L or R, X4 is A, E, or Q, X5 is S or T, and X6 is I or V; and HVR-L3 comprises an amino acid sequence according to formula (XV): YCX1X2X3X4X5X6PX7T (SEQ ID NO: 15), wherein X1 is E, Q, or V, X2 is H or Q, X3 is A, G, H, R, or S, X4 is D, L, S, or Y, and X5 is E, G, P, Q, or or S, X6 is L, T, V, or W, and X7 is F, L, P, W, or Y), formula (XVI): YCQQX1X2X3WPPWT (SEQ ID NO: 16) (wherein X1 is S or Y, X2 is D or Y, and X3 is Q or Y), and formula (XVII): YCQX1YX2SSPPX3YT (SEQ ID NO: 17) (wherein X1 is H or Q, X2 is T or V, and X3 is E or V). In some embodiments, HVR-H1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 29, HVR-H2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 30 to 39, and HVR-H3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 40 to 52,HVR-L1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53 to 65, HVR-L2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 66 to 69, and HVR-L3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 70 to 81. In some embodiments, the antibody is selected from the group consisting of: a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 18, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 30, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 40, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 53, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 66, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 70; b) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 19, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 31, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 41, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 54, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 67, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 71; c) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 32, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 42, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 55, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 66, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 72. d) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 21, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 33, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 43, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 56, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 68, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 73; e) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 22, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 34, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 44. -H3, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 57, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 66, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 74; f) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 23, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 35, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 45, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 58, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 66, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 75;g) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 24, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 32, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 46, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 59, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 66, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 76; h) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 25, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 47, and HVR-L1 comprising the amino acid sequence of SEQ ID NO: 60; HVR-L2 comprising the amino acid sequence of SEQ ID NO: 69, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 77; i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 26, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 37, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 48, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 61, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 66, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 78; j) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 27, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 32, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 49, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 62, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 67, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 79; k) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 28, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 37, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: HVR-H3 comprising the amino acid sequence of SEQ ID NO: 50, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 63, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 67, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 80; l) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 18, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 38, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 51, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 64, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 67, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 81; or m) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 29, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 39, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 52, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 65, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 68, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 77. In some embodiments, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 82-94, and / or the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 95-107. In some embodiments, the antibody comprises: a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 82, 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: 82;and a light chain variable region comprising the amino acid sequence of SEQ ID NO:95 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:95; b) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:83 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:83; and c) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 84 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: 84, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 97 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: 97; d) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:85 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:85, and a light chain variable region comprising an amino acid sequence of SEQ ID NO:98 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:98; e) a light chain variable region comprising an amino acid sequence of SEQ ID NO:86 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:86; 99 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: 99; f) 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; g) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 88 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: 88; h) a light chain variable region comprising the amino acid sequence of SEQ ID NO:89 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:89; and i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:102 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:102. i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:90 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:90, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:103 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:103; j) an amino acid sequence of SEQ ID NO:91 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:91. and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 92 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: 104; k) a light chain variable region comprising an amino acid sequence of SEQ ID NO: 92 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: 92;99% or more) sequence identity to the amino acid sequence of SEQ ID NO: 105; and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 105 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: 105; l) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 93 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: 93; and or m) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 94 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: 94, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 107 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: 107.

[0022] In some embodiments according to any one of the above methods, the anti-CTLA4 antibodies described herein comprise a heavy chain variable region and a light chain variable region, and one, two, three, four, five or six HVRs of the antibody comprise the HVR sequences set forth in Table A. In some embodiments, the anti-CTLA4 antibodies comprise a heavy chain variable region comprising HVR-H1, HVR-H2 and HVR-H3, and HVR-H1 comprises the amino acid sequence of SEQ ID NO:23, or HVR-H2 comprises the amino acid sequence of SEQ ID NO:35, or HVR-H3 comprises the amino acid sequence of SEQ ID NO:45. In some embodiments, the anti-CTLA4 antibodies comprise a light chain variable region comprising HVR-L1, HVR-L2 and HVR-L3, and HVR-L1 comprises the amino acid sequence of SEQ ID NO:58, or HVR-L2 comprises the amino acid sequence of SEQ ID NO:66, or HVR-L3 comprises the amino acid sequence of SEQ ID NO:75. In some embodiments, the anti-CTLA4 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, one, two, three, four, five, or six of the HVRs of the antibody may contain one, two, or three conservative amino acid substitutions in the HVR. In some embodiments, the anti-CTLA4 antibody comprises (b) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:87, or an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO:87, and / or a light chain variable region comprising an amino acid sequence of SEQ ID NO:100, or an amino acid sequence having at least 90% (e.g., 91%, 92%, 93%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO:100.

[0023] In some embodiments according to any one of the above methods, the antibody is a human antibody. In some embodiments, the antibody comprises an IgG1, IgG2, IgG3, or IgG4 Fc region (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region). In some embodiments, the antibody comprises a human IgG1 or a variant with enhanced ADCC activity. In some embodiments, the antibody comprises a human IgG1 with reduced fucosylation (or no fucosylation).

[0024] In some embodiments according to any one of the above methods, the method further comprises administering to the subject an effective amount of at least one additional therapeutic agent. In some embodiments, the at least one additional therapeutic agent is selected from the group consisting of viral gene therapy, immune checkpoint inhibitors, targeted therapy, radiation therapy, vaccine therapy, and chemotherapy. In some embodiments, the method comprises administering to the subject an effective amount of an anti-CTLA4 antibody described herein before or after surgery to remove the subject's tumor.

[0025] 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 description of the drawings]

[0026] [Figure 1] 1 shows the change in T cell levels over time relative to baseline in patients treated with the anti-CTLA4 antibody TY21580. The x-axis shows the time of sample collection, the y-axis shows the % change in absolute T cell counts per μL relative to baseline, and each line represents values ​​from a different subject. [Diagram 2]1 shows the change in NK cell levels over time relative to baseline in patients treated with TY21580. The x-axis shows the time of sample collection, the y-axis shows the % change in absolute T cell counts per μL relative to baseline, and each line represents values ​​from a different subject. [Diagram 3] Figure 1 shows the percentage of Treg cells in the total CD4+ T cell population over time in patients treated with TY21580. The x-axis shows the time of sampling, the y-axis shows the % change in Treg levels relative to baseline, and each line represents values ​​from a different subject. [Figure 4] Figure 1 shows the percent change in T cell levels versus baseline in patients treated with TY21580 at different dose cohorts of TY21580. The x-axis shows dose, the y-axis shows % change in absolute T cell counts per μL versus baseline, and each line represents values ​​from a different subject. [Diagram 5] Figure 1 shows the percent change in T cell levels versus baseline in patients treated with TY21580 at different dose cohorts of TY21580. The x-axis shows dose, the y-axis shows % change in absolute T cell counts per μL versus baseline, and each line represents values ​​from a different subject. [Figure 6] Figure 1 shows the percentage of Treg cells in the total CD4+ T cell population in patients treated with TY21580 at different dose cohorts of TY21580. The x-axis shows dose, the y-axis shows the % change in Treg levels relative to baseline, and each line represents values ​​from a different subject. [Figure 7] Figure 1 shows the percentage of effector memory (EM) CD8+ T cells in the total CD8+ T cell population in patients treated with TY21580 across different dose cohorts of TY21580. The x-axis shows dose, the y-axis shows the % change in EM CD8+ T cell levels relative to baseline, and each line represents values ​​from a different subject. [Figure 8]Figure 1 shows the percentage of EM CD4+ T cells in the total CD4+ T cell population in patients treated with TY21580 across different dose cohorts of TY21580. The x-axis shows dose, the y-axis shows the % change in EM CD4+ T cell levels relative to baseline, and each line represents values ​​from a different subject. [Figure 9A] Shown are lymphocyte profiling results for three subjects at 0.1 mg / kg or 0.3 mg / kg doses of TY21580. Shown (from left to right) are the % change in CD8+TEM / Treg ratio, % change in CD8+TEM cells, and % change in Treg cells for subject 6102-003 at the 0.1 mg / kg dose. [Figure 9B] Shown are lymphocyte profiling results for three subjects at 0.1 mg / kg or 0.3 mg / kg doses of TY21580. Shown (from left to right) are the % change in CD8+TEM / Treg ratio, % change in CD8+TEM cells, and % change in Treg cells for subject 6101-004 at the 0.1 mg / kg dose. [Figure 9C] Shown are lymphocyte profiling results for three subjects at 0.1 mg / kg or 0.3 mg / kg doses of TY21580. Shown (from left to right) are the % change in CD8+TEM / Treg ratio, % change in CD8+TEM cells, and % change in Treg cells for subject 6101-005 at the 0.3 mg / kg dose. For each plot, the x-axis shows the time of sample collection and the y-axis shows the % change relative to baseline. [Figure 10] 1 shows the results of lymphocyte profiling for subject 6102-002 at a dose of 0.03 mg / kg of TY21580. The plots show (top left to right) the percentage change in CD8+TEM / Treg ratio, percentage change in CD8+TEM cells, and percentage change in Treg cells, and (bottom left to right) the percentage change in CD4+TEM cells, percentage change in NK cells, and percentage change in B cells. In each plot, the x-axis shows the time of sample collection and the y-axis shows the percentage change relative to baseline. [Figure 11]The results of a population pharmacokinetic model using a two-compartment model for TY21580 in a Phase 1 study are shown. [Figure 12A] 1 shows the results of the population pharmacokinetic model from a Phase 1 study. A goodness-of-fit plot is shown. [Figure 12B] 1 shows the results of a population pharmacokinetic model from a Phase 1 study. Diagnostic plots are shown. [Figure 13] Figure 1 shows the percent change in CD8+TEM / Treg cell ratio in patients treated with TY21580 across different dose cohorts of TY21580. The x-axis shows dose, the y-axis shows percentage, and each line represents values ​​from a different subject. [Figure 14] Figure 1 shows the percent change in CD4+TEM / Treg cell ratio in patients treated with TY21580 across different dose cohorts of TY21580. The x-axis shows dose, the y-axis shows percentage, and each line represents values ​​from a different subject. [Figure 15A] Provide information about ongoing trials. Explain the dosing schedule for the trial. [Figure 15B] Information on subjects in ongoing trials is provided, along with information on the dose and duration of each TY21580 dose. [Figure 15C] 1 shows information about ongoing trials. FIG. 1 shows the increase in CD8+ T cells in subject 23 between pretreatment with C1D1 and C2D1. [Figure 15D] Provides information about ongoing trials. Provides treatment-related adverse events experienced by patients in the trials. [Figure 16] 1 shows the serum pharmacokinetics of TY21580. The x-axis shows days after dosing and the y-axis shows the measured drug concentration in patient serum, with each line representing a different dose. [Figure 17A] 1 shows the kinetics of serum IFN-γ levels in patients treated with TY21580. The percent change in relative abundance of IFN-γ versus baseline in patients treated with TY21580 is shown. The x-axis shows dose cohort, the y-axis shows percent change, and each line represents values ​​from a different subject. [Figure 17B]Figure 1 shows the kinetics of serum IFN-γ levels in patients treated with TY21580. Figure 1 shows the percent change in relative abundance of IFN-γ between baseline and C1D2 in patients treated with TY21580. The x-axis shows dose cohort, the y-axis shows percent change, and each line represents values ​​from a different subject. [Figure 17C] 4 shows the kinetics of serum IFN-γ levels in patients treated with TY21580. A scatter plot of the percent change in IFN-γ from C1D2 versus baseline in patients treated with TY21580 is shown. The x-axis shows dose cohort, the y-axis shows percent change, and each dot represents a value from a different subject. [Figure 18] Figure 1 shows the percent change in TNFα in patients treated with TY21580. (A) shows the percent change in relative abundance of TNFα versus baseline in patients treated with TY21580. The x-axis shows dose cohort, the y-axis shows percent change, and each line represents a value from a different subject. (B) shows a scatter plot of the percent change in TNFα from C1D2 versus baseline in patients treated with TY21580. The x-axis shows dose cohort, the y-axis shows percent change, and each dot represents a value from a different subject. [Figure 19] Figure 1 shows the percent change in IL-6 in patients treated with TY21580. (A) shows the percent change in relative abundance of IL-6 versus baseline in patients treated with TY21580. The x-axis shows dose cohort, the y-axis shows percent change, and each line represents a value from a different subject. (B) shows a scatter plot of the percent change in IL-6 from C1D2 versus baseline in patients treated with TY21580. The x-axis shows dose cohort, the y-axis shows percent change, and each dot represents a value from a different subject. [Figure 20]Figure 1 shows the percent change in IL-10 in patients treated with TY21580. (A) shows the percent change in relative abundance of IL-10 versus baseline in patients treated with TY21580. The x-axis shows dose cohort, the y-axis shows percent change, and each line represents a value from a different subject. (B) shows a scatter plot of the percent change in IL-10 from C1D2 versus baseline in patients treated with TY21580. The x-axis shows dose cohort, the y-axis shows percent change, and each dot represents a value from a different subject. [Figure 21A] Figure 1 shows the percent change in sPD-L1 in patients treated with TY21580. Figure 2 shows the abundance of sPD-L1 in patients treated with TY21580. The x-axis shows dose cohort, the y-axis shows sPD-L1 concentration, and each line represents values ​​from a different subject. [Figure 21B] Figure 1 shows the percent change in sPD-L1 in patients treated with TY21580. Figure 2 shows the percent change in relative abundance of sPD-L1 versus baseline in patients treated with TY21580. The x-axis shows dose cohort, the y-axis shows % change versus baseline, and each line represents values ​​from a different subject. [Figure 21C] Figure 1 shows the percent change in sPD-L1 in patients treated with TY21580. Figure 2 shows a scatter plot of the percent change in sPD-L1 from C1D8 versus baseline in patients treated with TY21580. The x-axis shows dose cohort, the y-axis shows percent change, and each dot represents a value from a different subject. [Figure 22] Figure 1 shows the percent change in sCD25 in patients treated with TY21580. (A) shows the percent change in relative abundance of sCD25 versus baseline in patients treated with TY21580. The x-axis shows dose cohort, the y-axis shows percent change, and each line represents a value from a different subject. (B) shows a scatter plot of the percent change in sCD25 from C1D8 versus baseline in patients treated with TY21580. The x-axis shows dose cohort, the y-axis shows percent change, and each dot represents a value from a different subject. [Figure 23]Figure 1 shows the percent change in CXCL-11 in patients treated with TY21580. (A) shows the percent change in relative abundance of CXCL-11 versus baseline in patients treated with TY21580. The x-axis shows dose cohort, the y-axis shows percent change, and each line represents a value from a different subject. (B) shows a scatter plot of the percent change in CXCL-11 from C1D8 versus baseline in patients treated with TY21580. The x-axis shows dose cohort, the y-axis shows percent change, and each dot represents a value from a different subject. [Figure 24A] Figure 1 shows the percent change in CD4+ T cells in patients treated with TY21580. Figure 2 shows the change in CD4+ T cell levels over time relative to baseline in patients treated with TY21580. The x-axis shows dose cohort and the y-axis shows the percent change in absolute T cell counts per μL relative to baseline, with each line representing values ​​from a different subject. Values ​​from multiple patients are highlighted (#4, #19, #22, #23). [Figure 24B] Figure 1 shows the percent change in CD4+ T cells in patients treated with TY21580. Figure 2 shows the percent change in absolute CD4+ T cell levels at C1D8 versus baseline in patients treated with TY21580. The x-axis shows dose cohort, the y-axis shows percent change, and each dot represents a value from a different subject. [Figure 24C] Figure 1 shows the percent change in CD4+ T cells in patients treated with TY21580. Figure 2 shows the percent change in absolute CD4+ T cell counts from baseline to C1D8 in patients treated with TY21580. The x-axis shows time points, the y-axis shows absolute counts per μL, and each line represents values ​​from a different subject. [Figure 24D] Figure 1 shows the percent change in CD4+ T cells in patients treated with TY21580. Figure 2 shows the percent change in absolute CD4+ T cell levels at C1D15 versus baseline in patients treated with TY21580. The x-axis shows dose cohort, the y-axis shows percent change, and each dot represents a value from a different subject. [Figure 24E]Figure 1 shows the percent change in CD4+ T cells in patients treated with TY21580. Figure 2 shows the percent change in absolute CD4+CD8- T cell counts from baseline to C1D15 in patients treated with TY21580. The x-axis shows time points, the y-axis shows absolute counts per μL, and each line represents values ​​from a different subject. [Figure 25A] Figure 1 shows the percent change in CD8+ T cells in patients treated with TY21580. Figure 2 shows the change in CD8+ T cell levels over time relative to baseline in patients treated with TY21580. The x-axis shows dose cohort and the y-axis shows the percent change in absolute T cell counts per μL relative to baseline, with each line representing values ​​from a different subject. Values ​​from multiple patients are highlighted (#4, #19, #22, #23). [Figure 25B] Figure 1 shows the percent change in CD8+ T cells in patients treated with TY21580. Figure 2 shows the percent change in absolute CD4+ T cell levels at C1D8 versus baseline in patients treated with TY21580. The x-axis shows dose cohort, the y-axis shows percent change, and each dot represents a value from a different subject. [Figure 25C] Figure 1 shows the percent change in CD8+ T cells in patients treated with TY21580. Figure 2 shows the percent change in absolute CD8+ T cell counts from baseline to C1D8 in patients treated with TY21580. The x-axis shows time points, the y-axis shows absolute counts per μL, and each line represents values ​​from a different subject. [Figure 25D] Figure 1 shows the percent change in CD8+ T cells in patients treated with TY21580. Figure 2 shows the percent change in absolute CD8+ T cell levels at C1D15 versus baseline in patients treated with TY21580. The x-axis shows dose cohort, the y-axis shows percent change, and each dot represents a value from a different subject. [Figure 25E] Figure 1 shows the percent change in CD8+ T cells in patients treated with TY21580. Figure 2 shows the percent change in absolute CD8+ T cell counts from baseline to C1D15 in patients treated with TY21580. The x-axis shows time points, the y-axis shows absolute counts per μL, and each line represents values ​​from a different subject. [Figure 26] Figure 1 shows the percent change in NK cell counts in patients treated with TY21580. (A) shows the percent change in absolute NK cell counts at C1D8 versus baseline in patients treated with TY21580. The x-axis shows time points, the y-axis shows absolute counts per μL, and each line represents a value from a different subject. (B) shows the percent change in absolute NK cell counts at C1D15 versus baseline in patients treated with TY21580. The x-axis shows time points, the y-axis shows absolute counts per μL, and each line represents a value from a different subject. [Figure 27] 3 shows the 3D structure of CTLA4 and its interactions with ipilimumab and TY21580. [Figure 28] Shows Treg depletion and CD8 / Treg ratio in tumor infiltrating lymphocytes (TILs) of MC38 tumors treated with a hCTLA4 knock-in background. [Figure 29] 1 shows the plasma Cmax of TY21580 at various dose levels following intravenous administration of TY21580 to human subjects. [Diagram 30] 1 shows the predicted AUC(0-∞) at various dose levels following intravenous administration of TY21580 to human subjects. [Diagram 31] 1 shows a simulated PK study used to predict serum concentration levels of TY21580 at various doses administered to human subjects. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] I. Definition Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meanings commonly understood by those of ordinary skill in the art. Furthermore, unless otherwise required by context, singular terms shall include plural referents and plural terms shall include singular referents. In general, the nomenclature used in connection with and techniques of antibody engineering, immunotherapy, cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry described herein are those well known and commonly used in the art.

[0028] The term "antibody" is used herein in the broadest sense and specifically covers monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies), and antibody fragments (e.g., Fab, Fab', Fab'-SH, F(ab')2, Fv and / or single chain variable fragments or scFv), so long as they exhibit the desired biological activity.

[0029] 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 a 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 the N-terminus. Hと 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 It consists of. V L is V H Aligned with C L is aligned with the first constant domain (CH1) of the heavy chain. Hand V L The pairings of the tetramers together form a single antigen-binding site. IgM antibodies consist of five basic heterotetrameric units plus an additional polypeptide called the J chain and thus 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.

[0030] V H and V L Based on structural and sequence analysis, the regions 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). 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.

[0031] As used herein, the term "CDR" or "complementarity determining region" is intended to mean the non-contiguous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. These specific regions were identified by 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 amino acid residues that comprise the CDRs as defined by each of the above references are set forth below in Table I for comparison.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. [Table 1] 1 Residue numbering follows the nomenclature of Kabat et al. (supra). 2 Residue numbering follows the nomenclature of Chothia et al. (supra). 3 Residue numbering follows the nomenclature of MacCallum et al. (supra). 4 Residue numbering follows the nomenclature of Lefranc et al. (supra). 5 Residue numbering follows the nomenclature of Honegger and Pluckthun (supra).

[0032] Each of the variable regions of the heavy and light chains contains a binding domain that interacts with an antigen. The constant region of the antibody may 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).

[0033] L 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, with heavy chains designated α (alpha), δ (delta), ε (epsilon), γ (gamma), and μ (mu), respectively. The IgG class of antibodies can be further classified into four subclasses, IgG1, IgG2, IgG3, and IgG4, by the gamma heavy chains Y1-Y4, respectively.

[0034] The term "antibody derivative" or "derivative" of an antibody refers to a molecule that can bind to the same antigen (e.g., CTLA4) that the antibody binds and that contains the amino acid sequence of the antibody linked to an additional molecular entity. The amino acid sequence of the antibody contained in the antibody derivative can be the full-length heavy chain, the full-length light chain, any portion(s) of the full-length heavy chain, any portion(s) of the full-length light chain of the antibody, any other fragment(s) of the antibody, or a complete antibody. The additional molecular entity can be a chemical or biological molecule. Examples of additional molecular entities include chemical groups, amino acids, peptides, proteins (enzymes, antibodies, etc.), and chemical compounds. The additional molecular entity can have any utility, such as use as a detection agent, label, marker, pharmaceutical or therapeutic agent. The amino acid sequence of the antibody can be attached or linked to the additional molecular entity by chemical coupling, genetic fusion, non-covalent bonding, etc. The term "antibody derivative" also encompasses chimeric antibodies, humanized antibodies, and molecules derived from modifications of the amino acid sequence of the CTLA4 antibody, such as conservative amino acid substitutions, additions, and insertions.

[0035] The term "antigen-binding fragment" or "antigen-binding portion" of an antibody refers to one or more portions of an antibody that retain the ability to bind to the antigen to which the antibody binds (e.g., CTLA4). Examples of "antigen-binding fragments" of antibodies include: (i) V L , V H , C L , and CH1 (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; and (iii) a V H and C H1 (iv) an Fd fragment consisting of the V domain of a single arm of an antibody; L and V H (v) a dAb fragment consisting of the VH domain (Ward et al., Nature 341:544-546 (1989)); and (vi) an isolated complementarity determining region (CDR).

[0036] 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, the anti-CTLA4 antibodies defined and disclosed herein may also bind to CTLA4 from species other than human. In other cases, the anti-CTLA4 antibodies may be completely specific for human CTLA4 and may not exhibit species or other types of cross-reactivity.

[0037] As defined herein, the term "CTLA4 antibody" refers to an antibody capable of binding to human CTLA4.

[0038] The term "chimeric antibody" refers to an antibody that contains amino acid sequences derived from different animal species, such as those having a variable region derived from a human antibody and a murine immunoglobulin constant region.

[0039] The term "compete for binding" refers to the interaction of two antibodies in binding to a binding target. A first antibody competes with a second antibody for binding if the binding of the first antibody to its cognate epitope is detectably reduced in the presence of the second antibody compared to the binding of the first antibody in the absence of the second antibody. The alternative that the binding of the second antibody to its epitope is also detectably reduced in the presence of the first antibody can be, but need not be, the case. That is, a first antibody can inhibit the binding of a second antibody to its epitope without the second antibody inhibiting the binding of the first antibody to its respective epitope. However, if each antibody detectably inhibits the binding of the other antibody to its cognate epitope, whether to the same extent, more or less, the antibodies are said to "cross-compete" with each other for binding of their respective epitope(s).

[0040] The term "epitope" refers to a portion of an antigen to which an antibody (or an 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, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes can include a variable number of amino acids in unique spatial conformations. Methods for determining the spatial conformation of an epitope include, for example, X-ray crystallography, 2D nuclear magnetic resonance, deuterium and hydrogen exchange combined with mass spectrometry, or site-directed mutagenesis, or any method used in combination with a computational model of the antigen and its complex structure with its bound antibody and its variants (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996)). Once the desired epitope of an antigen is 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. An approach to achieve this is to perform cross-competition studies to find 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 WO03 / 48731.

[0041] The term "glycosylation site" refers to an amino acid residue that is recognized by a eukaryotic cell as a location for the attachment of a sugar residue. The amino acids to which carbohydrates, such as oligosaccharides, are attached are typically asparagine (N-linked), serine (O-linked), and threonine (O-linked) residues. The particular attachment site is typically signaled by a sequence of amino acids, referred to herein as a "glycosylation site sequence." The glycosylation site sequence for N-linked glycosylation is -Asn-X-Ser- or -Asn-X-Thr-, where X can be any conventional amino acid except proline. The terms "N-linked" and "O-linked" refer to chemical groups that serve as attachment sites between a sugar molecule and an amino acid residue. N-linked sugars are attached through an amino group. O-linked sugars are attached through a hydroxyl group. The term "glycan occupancy" refers to the presence of a carbohydrate moiety linked to a glycosylation site (i.e., the glycan site is occupied). When there are at least two potential glycosylation sites on a polypeptide, either none (0-glycan site occupation), one (1-glycan site occupation), or both (2-glycan site occupation) sites can be occupied by a sugar moiety.

[0042] The term "host cell" refers to a cell line that can be engineered to produce a protein, protein fragment, or peptide of interest. Host cells include, but are not limited to, cultured cells, e.g., mammalian cultured cells derived from rodents (rat, mouse, guinea pig, or hamster), such as CHO, BHK, NSO, SP2 / 0, YB2 / 0; human cells (e.g., HEK293F cells, HEK293T cells; or human tissue or hybridoma cells, yeast cells, insect cells (e.g., S2 cells), bacterial cells (e.g., E. coli cells), as well as cells contained within transgenic animals or cultured tissues. This term encompasses not only the particular subject cell, but also the progeny of such cells. Since certain modifications may occur in subsequent generations due to either mutations or environmental influences, such progeny may not be identical to the parent cell, but are still included within the scope of the term "host cell".

[0043] A "human antibody" is one that possesses an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human or human cell, or derived from a non-human source that utilizes the human antibody repertoire, or other human antibody coding sequence. This definition of a human antibody specifically excludes humanized antibodies which contain non-human antigen-binding residues.

[0044] The term "humanized antibody" refers to a chimeric antibody that contains amino acid residues derived from human antibody sequences. A humanized antibody may contain some or all of the CDRs or HVRs from a non-human animal or synthetic antibody, but the framework and constant regions of the antibody contain amino acid residues derived from human antibody sequences.

[0045] The term "exemplary antibody" refers to any one of the antibodies described in this disclosure and named as listed in Tables A and B, as well as any antibody that contains the six HVRs and / or VHs and VLs of the antibodies listed in Tables A and B. These antibodies can be of any class (e.g., IgA, IgD, IgE, IgG, and IgM). Thus, each of the antibodies identified above includes the V L and V H The present invention encompasses antibodies of all five classes that have the same amino acid sequence for the V domain. Furthermore, antibodies of the IgG class can be of any subclass (e.g., IgG1, IgG2, IgG3, and IgG4). Thus, each of the above-identified antibodies of the IgG subclasses includes the V L and V H It includes antibodies of all four subclasses which have the same amino acid sequence for that region. The amino acid sequences of the heavy chain constant regions of human antibodies of the five classes and four IgG subclasses are known in the art.

[0046] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" is a form of cytotoxicity in which secreted immunoglobulins bind to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., NK cells, neutrophils, and macrophages), allowing these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells with cytotoxins. NK cells, the primary cells for mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess the ADCC activity of a molecule of interest, an in vitro ADCC assay may be performed, such as that described in U.S. Pat. No. 5,500,362 or U.S. Pat. No. 5,821,337 or U.S. Pat. No. 6,737,056 (Presta). Useful effector cells for such assays include PBMCs and NK cells. Alternatively or additionally, the ADCC activity of a molecule of interest may be assessed in vivo, for example in an animal model, such as that disclosed in Clynes et al., PNAS (USA) 95:652-656 (1998). Exemplary assays for assessing ADCC activity are provided in the Examples herein.

[0047] "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway begins with the binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass), which are bound to their cognate antigen. To assess complement activation, a CDC assay, for example, as described in Gazzano-Santoro et al., J. Immunol.Methods 202:163 (1996) can be performed. Polypeptide variants with altered Fc region amino acid sequences and increased or decreased C1q binding capacity (polypeptides with variant Fc regions) are described, for example, in U.S. Pat. No. 6,194,551 B1 and WO 1999 / 51642. See also Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0048] An "isolated" antibody is an antibody that has been separated from the components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, for example, as determined 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).

[0049] "K a The term "k" refers to the binding rate constant of a particular antibody-antigen interaction. d The term "dissociation rate constant" refers to the dissociation rate constant of a particular antibody-antigen interaction.

[0050] "K D The term "dissociation constant" refers to the equilibrium dissociation constant of a particular antibody-antigen interaction. This is k d vs. k a (i.e., k d / k a ) and expressed as molar concentration (M). Dis used as a measure of the affinity of binding of an antibody to a binding partner. D The smaller the K, the stronger the antibody binds, or the higher the affinity between the antibody and the antigen. For example, an antibody with a nanomolar (nM) dissociation constant will bind more tightly to a particular antigen than an antibody with a micromolar (μM) dissociation constant. D Values ​​can be determined using methods well established in the art. D One method for determining is by using surface plasmon resonance, typically by using a biosensor system such as the BIACORE® system. For example, an assay procedure using the BIACORE™ system (BIAcore assay) is described in at least Example 3 of the present application.

[0051] The term "mammal" refers to any animal species of the class Mammalia. Examples of mammals include humans; laboratory animals such as rats, mice, hamsters, rabbits, non-human primates, and guinea pigs; farm animals such as cats, dogs, cows, sheep, goats, horses, and pigs; and captive wild animals such as lions, tigers, and elephants.

[0052] As used herein, "sequence identity" between two polypeptide sequences refers to the percentage of amino acids that are identical between sequences.The amino acid sequence identity of polypeptides can be determined conventionally 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 such that the percentage of identity is calculated over the entire length of the reference amino acid sequence, allowing 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 the percentage of identity between polypeptides is applicable to all proteins, fragments, or variants thereof disclosed herein.

[0053] As used herein, the terms "bind", "bind to", "specifically bind 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 longer 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.

[0054] The terms "treat", "treating" or "treatment" refer to a particular disease state in a mammal, and to causing a desired or beneficial effect in a mammal having a disease state. A desired or beneficial effect may include a reduction in the frequency or severity of one or more symptoms of the disease (i.e., tumor growth and / or metastasis, or other effects mediated by 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 desired or beneficial effect may include inhibiting further growth or metastasis of cancer cells, killing cancer cells, inhibiting recurrence of cancer, reducing pain associated with cancer, or improving the survival of the mammal. The effect may be either subjective or objective. For example, if the animal is a human, the human may perceive improved vitality or survival, or reduced pain, as subjective symptoms of improvement or response to treatment. Alternatively, the clinician may perceive a reduction in tumor size or burden based on physical examination, clinical laboratory values, tumor markers, or x-ray 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. In addition, 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.

[0055] The terms "prevent" or "prophylaxis" in reference 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.

[0056] The term "isolated nucleic acid" refers to a nucleic acid molecule of genomic, cDNA, or synthetic origin, or a combination thereof, that is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid. For example, with respect to genomic DNA, the term "isolated" includes a nucleic acid molecule that is separated from the chromosome with which the genomic DNA is naturally associated. Preferably, an "isolated" nucleic acid is free of sequences that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid of interest).

[0057] The term "vector" refers to a nucleic acid molecule capable of transporting a foreign nucleic acid molecule. The foreign nucleic acid molecule is linked to the vector nucleic acid molecule by recombinant techniques such as ligation or recombination. This allows the foreign nucleic acid molecule to be propagated, selected, further manipulated, or expressed in a host cell or organism. The vector can be a plasmid, phage, transposon, cosmid, chromosome, virus, or virion. Some types of vectors can be integrated into the genome of the host cell upon introduction into the host cell, and are thereby replicated along with the host genome (e.g., non-episomal mammalian vectors). Other types of vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors and episomal mammalian vectors having a bacterial origin of replication). Another specific type of vector capable of directing the expression of an expressible foreign nucleic acid to which they are operably linked is usually referred to as an "expression vector". Expression vectors generally have control sequences that drive the expression of an expressible foreign nucleic acid. Simpler vectors, known as "transcription vectors", are capable of only transcription but not translation, and they can replicate but not express in target cells. The term "vector" encompasses all types of vectors, regardless of their function. Vectors capable of directing the expression of an expressible nucleic acid to which they are operatively linked are commonly referred to as "expressible vectors." Other examples of "vectors" can include display vectors (e.g., vectors that direct the expression and display of an encoded polypeptide on the surface of a virus or a cell, such as a bacterial cell, yeast cell, insect cell, and / or mammalian cell).

[0058] As used herein, a "subject," "patient," or "individual" may refer to a human or non-human animal. A "non-human animal" may refer to any animal not classified as a human, such as farm animals, livestock, or zoo animals, sports animals, pet animals (e.g., dogs, horses, cats, cows, etc.), and animals used in research. A research animal may refer to, but is 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.

[0059] "Effective amount" refers to at least an amount effective at the dosage and for the period 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 a monotherapy or combination therapy). Thus, an "effective amount" may be considered in the context of administration of one or more therapeutic agents, and a monotherapy may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desired result can be achieved or is achieved.

[0060] The terms "recurrence," "recurrence," or "recurrence" refer to the recurrence of cancer or disease after a clinical assessment of disease resolution. A diagnosis of distant metastasis or local recurrence may be considered a recurrence.

[0061] The terms "refractory" or "resistant" refer to a cancer or disease that has not responded to treatment.

[0062] 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% decrease 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 starting treatment to qualify for PR nor sufficient increase to qualify for PD based on the smallest SLD.

[0063] 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.

[0064] 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 or partial response, and the amount of time a patient experiences stable disease.

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

[0066] As used herein, "overall survival rate" refers to the proportion of individuals in a group who are likely to be alive after a particular period of time.

[0067] As used herein, the term "biomarker" or "marker" generally refers to a molecule (e.g., pre-mRNA, mRNA, protein, etc.) or a cell population (e.g., effector memory T cells or T em cells, or regulatory T cells, or T regCTLA4 antibody or a CTLA5 antibody that is capable of detecting a subject's responsiveness to a therapeutic regimen (e.g., treatment with an anti-CTLA4 antibody), and in some embodiments, can be used to predict or predict (or help predict) the subject's sensitivity to a therapeutic regimen (e.g., treatment with an anti-CTLA4 antibody), and in some embodiments, to predict (or help predict) the subject's responsiveness to a therapeutic regimen.

[0068] As used herein, the term "sample" refers to a composition obtained or derived from a subject of interest, including cells and / or other molecular entities that are characterized and / or identified based on, for example, physical, biochemical, chemical, and / or physiological characteristics.

[0069] As used herein, a "tissue or cell sample" refers to a collection of similar cells obtained from a subject's or patient's tissue. The source of the tissue or cell sample can be solid tissue from a fresh, frozen, and / or archived organ or tissue sample or biopsy or aspirate; blood or any blood constituent; bodily fluids such as cerebrospinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; cells from any time point in the subject's gestation or development. Also, the tissue sample can be primary or cultured cells. Optionally, the tissue or cell sample is obtained from a diseased tissue or organ. The tissue sample can include compounds that are not naturally mixed with the tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc. As used herein, a "reference value" or "reference level" can be an absolute value; a relative value; a value with upper and / or lower limits; a range of values; an average value; a median value; a mean value; or a value compared to a particular level or baseline level.

[0070] 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.

[0071] 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 (e.g., tissue or cell) of the body 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 cell 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 (e.g., tissue or cell) of the body of an individual other than the subject or individual. In yet another embodiment, the reference sample, reference 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.

[0072] "Correlate" or "correlating" means to compare, in any manner, the performance and / or results of a first analysis or protocol with the performance and / or results of a second analysis or protocol. For example, the results of a first analysis or protocol can be used in performing a second protocol and / or can be used to determine whether a second analysis or protocol should be performed. For biomarker analysis or protocol embodiments, the results of the biomarker level analysis or protocol can be used to determine whether a particular treatment regimen should be performed. For biomarker level analysis or protocol embodiments, the results of the biomarker level analysis or protocol can be used to determine whether a particular treatment regimen should be performed.

[0073] 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.

[0074] A patient who "does not respond effectively" to a treatment is one who does not have any of the following: prolonged survival (including overall survival and progression-free survival), failed to produce an objective response (including a complete or partial response), or failed to improve the signs or symptoms of cancer.

[0075] The methods and techniques of the present 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 can be carried out according to manufacturer's specifications, as commonly performed in the art, or as described herein. The nomenclatures used in connection with analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein, as well as the laboratory procedures and methods thereof, are well known and commonly used in the art. Standard methods are used for chemical synthesis, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0076] 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)).

[0077] 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.

[0078] The term "about" as used herein 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 are directed to that value or parameter in itself.

[0079] It is to be understood that the aspects and embodiments of the present application described herein include "comprising," "consisting," and "consisting essentially of" aspects and embodiments.

[0080] As used herein, reference to a value or parameter "not" generally means and describes the value or parameter "other than." 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.

[0081] As used herein, the term "about X to Y" has the same meaning as "about X to about Y."

[0082] 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" (single), and "B" (single). 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 (single); B (single); and C (single).

[0083] 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 IV, "Anti-CTLA4 Antibodies," including full-length antibodies and antigen-binding fragments thereof, can be used in the methods described herein.

[0084] In some embodiments, a method of treating cancer in a subject is provided, comprising administering to the subject an effective amount of an anti-CTLA4 antibody that specifically binds to an epitope that includes amino acid residues Y105 and L106, but not including residue I108, of human CTLA4, where the numbering of the amino acid residues is based on SEQ ID NO: 108, and the cancer is resistant or refractory to conventional treatments, and the conventional treatments are inhibitors of CTLA4, PD-1, or PD-1 ligand. In some embodiments, the cancer is a solid cancer, such as advanced and / or metastatic cancer. In some embodiments, the cancer is a urothelial carcinoma.

[0085] In some embodiments, a method of treating cancer in a subject is provided, comprising administering to the subject an effective amount of an anti-CTLA4 antibody that specifically binds to an epitope that includes amino acid residues Y105 and L106, but not including residue I108, of human CTLA4, where the numbering of the amino acid residues is based on SEQ ID NO: 108, and the cancer is resistant or refractory to a different anti-CTLA4 antibody, such as ipilimumab. In some embodiments, the cancer is a solid cancer, such as advanced and / or metastatic cancer. In some embodiments, the cancer is a urothelial carcinoma.

[0086] In some embodiments, a method of treating cancer in a subject is provided, comprising administering to the subject an effective amount of an anti-CTLA4 antibody that specifically binds to an epitope that includes amino acid residues Y105 and L106, but not including residue I108, of human CTLA4, where the numbering of the amino acid residues is based on SEQ ID NO: 108, and the cancer is resistant or refractory to an inhibitor of PD-1 or a PD-1 ligand (PD-L1 or PD-L2). In some embodiments, the cancer is a solid cancer, such as an advanced stage cancer and / or a metastatic cancer. In some embodiments, the cancer is a urothelial cancer.

[0087] In some embodiments, a method of treating cancer in a subject is provided, comprising administering to the subject an effective amount of an anti-CTLA4 antibody that specifically binds to an epitope that includes amino acid residues Y105 and L106, but not including residue I108, of human CTLA4, where the numbering of the amino acid residues is based on SEQ ID NO: 108, and the cancer is resistant or refractory to an anti-PD-1 antibody, such as pembrolizumab. In some embodiments, the cancer is a solid cancer, such as advanced and / or metastatic cancer. In some embodiments, the cancer is a urothelial carcinoma.

[0088] In some embodiments, a method is provided for treating a cancer in a subject 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, 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, such as pembrolizumab. 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 advanced stage cancer and / or metastatic cancer. In some embodiments, the cancer is urothelial carcinoma. 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 a human IgG1 Fc region, such as a wild-type IgG1 Fc region or a variant with enhanced ADCC activity. In some embodiments, the antibody is TY21580.

[0089] In some embodiments, a method of treating cancer in a subject is provided comprising administering to a subject an effective amount of an anti-CTLA4 antibody that specifically binds to an epitope including amino acid residues Y105 and L106 but not including residue I108 of human CTLA4, where the numbering of the amino acid residues is based on SEQ ID NO: 108, wherein the anti-CTLA4 antibody is administered at a dose of at least about 6 mg / kg (e.g., 6 mg / kg or 10 mg / kg). In some embodiments, the anti-CTLA4 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 antibody comprises a heavy chain variable region comprising an 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 an 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 a human IgG1 Fc region, such as a wild-type IgG1 Fc region or a variant with enhanced ADCC activity. In some embodiments, the antibody is TY21580. In some embodiments, the anti-CTLA4 antibody is administered about once every three weeks. In some embodiments, the anti-CTLA4 antibody is administered intravenously.

[0090] In some embodiments, a method of treating cancer in a subject is provided, comprising administering to the subject (a) an effective amount of an anti-CTLA4 antibody that specifically binds to an epitope that includes amino acid residues Y105 and L106 but not including residue I108 of human CTLA4, where the numbering of the amino acid residues is based on SEQ ID NO: 108, and (b) an effective amount of an anti-PD-1 antibody. In some embodiments, the anti-PD-L1 antibody is pembrolizumab, a biosimilar thereof, or a derivative thereof. In some embodiments, the anti-PD-L1 antibody is toripalimab, a biosimilar thereof, or a derivative thereof. In some embodiments, the anti-CTLA4 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 antibody comprises a heavy chain variable region comprising an 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 an 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 a human IgG1 Fc region, such as a wild-type IgG1 Fc region or a variant with enhanced ADCC activity. In some embodiments, the antibody is TY21580. In some embodiments, the anti-CTLA4 antibody is administered at a dose of at least about 6 mg / kg (e.g., 6 mg / kg or 10 mg / kg). In some embodiments, the anti-CTLA4 antibody is administered about once every three weeks. In some embodiments, the anti-CTLA4 antibody is administered intravenously. In some embodiments, the cancer is resistant or refractory to an inhibitor of CTLA-4, PD-1, or PD-1 ligand (e.g., PD-L1 or PD-L2). In some embodiments, the cancer is a solid cancer, such as an advanced stage cancer and / or a metastatic cancer, hi some embodiments, the cancer is a urothelial carcinoma.Cancer treatment can be evaluated, for example, by tumor regression, reduction in tumor weight or size, time to progression, survival, progression-free survival, overall response rate, duration of response, quality of life, protein expression, and / or activity. Approaches to determine efficacy of therapy can be used, including, for example, measuring response by radiological imaging.

[0091] 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.

[0092] In some embodiments, the anti-CTLA4 antibody is administered at a dose not exceeding any one of 20 mg / kg, 15 mg / kg, 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.8 mg / kg, 0.6 mg / kg, 0.5 mg / kg, 0.4 mg / kg, 0.3 mg / kg, 0.2 mg / kg, 0.1 mg / kg, 0.08 mg / kg, 0.05 mg / kg, 0.04 mg / kg, 0.03 mg / kg, 0.01 mg / kg, 0.003 mg / kg, or 0.001 mg / kg. In some embodiments, the dose of the anti-CTLA4 antibody is within any one of the following ranges, with an upper limit of any one of 20 mg / kg, 15 mg / kg, 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.8 mg / kg, 0.6 mg / kg, 0.5 mg / kg, 0.4 mg / kg, 0.3 mg / kg, 0.2 mg / kg, 0.1 mg / kg, 0.08 mg / kg, 0.05 mg / kg, 0.04 mg / kg, 0.03 mg / kg, or 0.003 mg / kg, and an upper limit of any one of 15 mg / kg, 10 mg / kg, 20 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 15 mg / kg, 10 mg / kg, 20 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, 60 mg / kg, 5 mg / kg, 7 mg / kg, 8 mg / kg, 5 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.8 mg / kg, 0.6 mg / kg, 0.5 mg / kg, 0.4 mg / kg, 0.3 mg / kg, 0.2 mg / kg, 0.1 mg / kg, 0.08 mg / kg, 0.05 mg / kg, 0.04 mg / kg, 0.03 mg / kg, or 0.003 mg / kg. and an independently selected lower limit of any one of: 0.8 mg / kg, 0.6 mg / kg, 0.5 mg / kg, 0.4 mg / kg, 0.3 mg / kg, 0.2 mg / kg, 0.1 mg / kg, 0.08 mg / kg, 0.05 mg / kg, 0.04 mg / kg, 0.03 mg / kg, 0.01 mg / kg, 0.003 mg / kg or 0.001 mg / kg, wherein the lower limit is lower than the upper limit.In some embodiments, the anti-CTLA4 antibody is administered at a dose of about 0.03 mg / kg to about 20 mg / kg, about 0.1 mg / kg to about 20 mg / kg, about 0.3 mg / kg to about 20 mg / kg, about 1 mg / kg to about 20 mg / kg, about 5 mg / kg to about 20 mg / kg, about 0.03 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 10 mg / kg, about 0.3 mg / kg to about 10 mg / kg, about 1 mg / kg to about 10 mg The therapeutic agent may be administered at a dose of any one of about 0.03 mg / kg to about 0.1 mg / kg, about 0.1 mg / kg to about 0.3 mg / kg, about 0.3 mg / kg to about 1 mg / kg, about 1 mg / kg to about 3 mg / kg, about 3 mg / kg to about 5 mg / kg, about 0.1 mg / kg to about 3 mg / kg, or about 1 mg / kg to about 5 mg / kg. Doses described herein may refer to doses suitable for humans, or equivalent doses in the particular species of subject. In some embodiments, the anti-CTLA4 antibody is administered in an amount equivalent to about 0.03 mg / kg to about 10 mg / kg, or about 0.03 mg / kg to about 20 mg / kg in a human subject, e.g., about 0.03 mg / kg, about 0.1 mg / kg, about 0.3 mg / kg, about 1.0 mg / kg, about 3.0 mg / kg, about 6.0 mg / kg, about 10 mg / kg, about 15 mg / kg, or about 20 mg / kg.

[0093] In embodiments where the anti-CTLA4 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 87 or an amino acid sequence having at least 90% 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, the anti-CTLA4 antibody is administered at a dose of about 0.5 mg / kg to about 10 mg / kg once every three weeks, about 1 mg / kg to about 10 mg / kg once every three weeks, about 3 mg / kg to about 20 mg / kg once every three weeks, about 3 mg / kg to about 15 mg / kg once every three weeks, about 6 mg / kg to about 15 mg / kg once every three weeks, or about 6 mg / kg to about 10 mg / kg once every three weeks. In some such embodiments, the anti-CTLA4 antibody is administered at a dose of about 0.5 mg / kg once every three weeks. In some such embodiments, the anti-CTLA4 antibody is administered at a dose of about 1 mg / kg once every three weeks. In some such embodiments, the anti-CTLA4 antibody is administered at a dose of about 3 mg / kg once every three weeks. In other such embodiments, the anti-CTLA4 antibody is administered at a dose of about 6 mg / kg once every three weeks. In other such embodiments, the anti-CTLA4 antibody is administered at a dose of about 10 mg / kg once every three weeks. In some of the above embodiments, the anti-CTLA4 antibody comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO:23, HVR-H2 comprising the amino acid sequence of SEQ ID NO:35, HVR-H3 comprising the amino acid sequence of SEQ ID NO:45, HVR-L1 comprising the amino acid sequence of SEQ ID NO:58, HVR-L2 comprising the amino acid sequence of SEQ ID NO:66, and HVR-L3 comprising the amino acid sequence of SEQ ID NO:75. In some embodiments, the anti-CTLA4 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:125 and a light chain comprising the amino acid sequence of SEQ ID NO:127. In some embodiments, the anti-CTLA4 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 126 and a light chain comprising the amino acid sequence of SEQ ID NO: 127. In some of the above embodiments, the anti-CTLA4 antibody is TY21580. In some of the above embodiments, the anti-CTLA4 antibody is administered to a patient with melanoma, non-small cell lung cancer, renal cell carcinoma, or hepatocellular carcinoma.In other of the above embodiments, the anti-CTLA4 antibody is administered to patients with MSI-H or dMMR cancer. In other of the above embodiments, the anti-CTLA4 antibody is administered to patients with metastatic cancer. In some of the above embodiments, the anti-CTLA4 antibody is administered to patients who are resistant or refractory to conventional cancer therapies, including other anti-CTLA4 antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, or combinations thereof.

[0094] In those embodiments in which the anti-CTLA4 antibody comprises a heavy chain variable region comprising an 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 an 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, the anti-CTLA4 antibody is administered at a first higher dose (e.g., about 10 mg / kg to about 20 mg / kg) for at least one treatment cycle (as defined herein) followed by a lower dose (e.g., about 0.5 mg / kg to about 10 mg / kg, about 0.5 mg / kg to about 6 mg / kg, about 3 mg / kg to about 10 mg / kg) in subsequent cycles. In some such embodiments, the anti-CTLA4 antibody is administered at a dose of about 10 mg / kg for at least one treatment cycle (e.g., 1 to 3 treatment cycles) followed by a dose of about 6 mg / kg in subsequent treatment cycles. In other such embodiments, the anti-CTLA4 antibody is administered at a dose of about 10 mg / kg for at least one treatment cycle (e.g., 1-3 treatment cycles) and at a dose of about 3 mg / kg for a subsequent treatment cycle. In other such embodiments, the anti-CTLA4 antibody is administered at a dose of about 15 mg / kg for at least one treatment cycle (e.g., 1-3 treatment cycles) and at a dose of about 10 mg / kg for a subsequent treatment cycle. In other such embodiments, the anti-CTLA4 antibody is administered at a dose of about 20 mg / kg for at least one treatment cycle (e.g., 1-3 treatment cycles) and at a dose of about 10 mg / kg for a subsequent treatment cycle. In some of the above embodiments, the anti-CTLA4 antibody comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 23, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 35, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 45, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 58, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 66, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 75. In some embodiments, the anti-CTLA4 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 125 and a light chain comprising the amino acid sequence of SEQ ID NO: 127.In some embodiments, the anti-CTLA4 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 126 and a light chain comprising the amino acid sequence of SEQ ID NO: 127. In some of the above embodiments, the anti-CTLA4 antibody is TY21580. In some of the above embodiments, the anti-CTLA4 antibody is administered to patients with melanoma, non-small cell lung cancer, renal cell carcinoma, or hepatocellular carcinoma. In other of the above embodiments, the anti-CTLA4 antibody is administered to patients with MSI-H or dMMR cancer. In other of the above embodiments, the anti-CTLA4 antibody is administered to patients with metastatic cancer. In some of the above embodiments, the anti-CTLA4 antibody is administered to patients who are resistant or refractory to conventional cancer therapies, including other anti-CTLA4 antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, or combinations thereof.

[0095] In those embodiments where the anti-CTLA4 antibody comprises a heavy chain variable region comprising an 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 an 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, the anti-CTLA4 antibody is administered at a dose that provides a steady state concentration in the systemic circulation of about 50 nM to about 700 nM when measured on the last day of the administration cycle (i.e., serum trough concentration). For example, if the administration cycle is once every three weeks, the serum trough concentration is measured on day 21 of the administration cycle (i.e., 504 hours later). If the administration cycle is once every four weeks, the serum trough concentration is measured on day 28 of the administration cycle (i.e., 672 hours later). In some embodiments, the anti-CTLA4 antibody is administered at a dose that provides a steady state serum trough concentration of about 100 nM to about 500 nM. In other embodiments, the anti-CTLA4 antibody is administered at a dose that provides a steady-state serum trough concentration of about 100 nM to about 400 nM. In other embodiments, the anti-CTLA4 antibody is administered at a dose that provides a steady-state serum trough concentration of about 100 nM to about 200 nM. In other embodiments, the anti-CTLA4 antibody is administered at a dose that provides a steady-state serum trough concentration of about 150 nM to about 400 nM. In other embodiments, the anti-CTLA4 antibody is administered at a dose that provides a steady-state serum trough concentration of about 150 nM to about 450 nM. In some of the above embodiments, the anti-CTLA4 antibody is administered once every three weeks. In some of the above embodiments, the anti-CTLA4 antibody is administered once every four weeks. In some of the above embodiments, the anti-CTLA4 antibody is administered once every two weeks. In some of the above embodiments, the anti-CTLA4 antibody comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 23, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 35, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 45, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 58, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 66, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 75. In some embodiments, the anti-CTLA4 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 125 and a light chain comprising the amino acid sequence of SEQ ID NO: 127.In some embodiments, the anti-CTLA4 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 126 and a light chain comprising the amino acid sequence of SEQ ID NO: 127. In some of the above embodiments, the anti-CTLA4 antibody is TY21580. In some of the above embodiments, the anti-CTLA4 antibody is administered to patients with melanoma, non-small cell lung cancer, renal cell carcinoma, or hepatocellular carcinoma. In other of the above embodiments, the anti-CTLA4 antibody is administered to patients with MSI-H or dMMR cancer. In other of the above embodiments, the anti-CTLA4 antibody is administered to patients with metastatic cancer. In some of the above embodiments, the anti-CTLA4 antibody is administered to patients who are resistant or refractory to conventional cancer therapies, including other anti-CTLA4 antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, or combinations thereof.

[0096] In embodiments where the anti-CTLA4 antibody comprises a heavy chain variable region comprising an 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 an 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, the anti-CTLA4 antibody is administered at a dose that provides a concentration in the systemic circulation of about 100 nM to about 2000 nM when measured one week (i.e., 168 hours) after a dosing cycle. In some embodiments, the anti-CTLA4 antibody is administered at a dose that provides a concentration in the systemic circulation of about 150 nM to about 1500 nM when measured one week (i.e., 168 hours) after a dosing cycle. In some embodiments, the anti-CTLA4 antibody is administered at a dose that provides a concentration in the systemic circulation of about 150 nM to about 1000 nM when measured one week (i.e., 168 hours) after a dosing cycle. In some embodiments, the anti-CTLA4 antibody is administered at a dose that provides a concentration in the systemic circulation of about 1000 nM to about 2000 nM when measured one week (i.e., 168 hours) after the administration cycle. In other embodiments, the anti-CTLA4 antibody is administered at a dose that provides a concentration in the systemic circulation of about 150 nM to about 500 nM when measured one week (i.e., 168 hours) after the administration cycle. In other embodiments, the anti-CTLA4 antibody is administered at a dose that provides a concentration in the systemic circulation of about 150 nM when measured one week (i.e., 168 hours) after the administration cycle. In some of the above embodiments, the anti-CTLA4 antibody is administered once every three weeks. In some of the above embodiments, the anti-CTLA4 antibody is administered once every four weeks. In some of the above embodiments, the anti-CTLA4 antibody is administered once every two weeks. In some of the above embodiments, the anti-CTLA4 antibody comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 23, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 35, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 45, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 58, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 66, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 75.In some embodiments, the anti-CTLA4 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 125 and a light chain comprising the amino acid sequence of SEQ ID NO: 127. In some embodiments, the anti-CTLA4 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 126 and a light chain comprising the amino acid sequence of SEQ ID NO: 127. In some of the above embodiments, the anti-CTLA4 antibody is TY21580. In some of the above embodiments, the anti-CTLA4 antibody is administered to patients with melanoma, non-small cell lung cancer, renal cell carcinoma, or hepatocellular carcinoma. In other of the above embodiments, the anti-CTLA4 antibody is administered to patients with MSI-H or dMMR cancer. In other of the above embodiments, the anti-CTLA4 antibody is administered to patients with metastatic cancer. In some of the above embodiments, the anti-CTLA4 antibody is administered to patients who are resistant or refractory to conventional cancer therapies, including other anti-CTLA4 antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, or combinations thereof.

[0097] In embodiments in which the anti-CTLA4 antibody comprises a heavy chain variable region comprising an 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 an 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, the anti-CTLA4 antibody has a steady-state AUC of about 500,000 day*ng / mL to about 2,500,000 day*ng / mL. (0~∞) In some embodiments, the anti-CTLA4 antibody is administered at a dose that results in a steady-state AUC of about 500,000 day*ng / mL to about 1,500,000 day*ng / mL. (0~∞) In some embodiments, the anti-CTLA4 antibody is administered at a dose that results in a steady-state AUC of about 500,000 day*ng / mL to about 1,000,000 day*ng / mL. (0~∞) In some embodiments, the anti-CTLA4 antibody is administered at a dose that results in a steady-state AUC of about 500,000 day*ng / mL to about 1,000,000 day*ng / mL. (0~∞)In some embodiments, the anti-CTLA4 antibody is administered at a dose that results in a steady-state AUC of about 1,500,000 day*ng / mL to about 2,000,000 day*ng / mL. (0~∞) In some embodiments, the anti-CTLA4 antibody is administered at a dose that results in a steady-state AUC of about 2,000,000 day*ng / mL to about 2,500,000 day*ng / mL. (0~∞) In some embodiments, the steady-state AUC (0~∞) is achieved by administering an anti-CTLA4 antibody once every three weeks. In some embodiments, the steady-state AUC (0~∞) is achieved by administering an anti-CTLA4 antibody once every four weeks. In some embodiments, the steady-state AUC (0~∞) is achieved by administering an anti-CTLA4 antibody once every 5 weeks. In some embodiments, the steady-state AUC (0~∞)is achieved by administering the anti-CTLA4 antibody once every two weeks. In some of the above embodiments, the anti-CTLA4 antibody comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO:23, HVR-H2 comprising the amino acid sequence of SEQ ID NO:35, HVR-H3 comprising the amino acid sequence of SEQ ID NO:45, HVR-L1 comprising the amino acid sequence of SEQ ID NO:58, HVR-L2 comprising the amino acid sequence of SEQ ID NO:66, and HVR-L3 comprising the amino acid sequence of SEQ ID NO:75. In some embodiments, the anti-CTLA4 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:125 and a light chain comprising the amino acid sequence of SEQ ID NO:127. In some embodiments, the anti-CTLA4 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:126 and a light chain comprising the amino acid sequence of SEQ ID NO:127. In some of the above embodiments, the anti-CTLA4 antibody is TY21580. In some of the above embodiments, the anti-CTLA4 antibody is administered to a patient with melanoma, non-small cell lung cancer, renal cell carcinoma, or hepatocellular carcinoma. In other of the above embodiments, the anti-CTLA4 antibody is administered to patients with MSI-H or dMMR cancer. In other of the above embodiments, the anti-CTLA4 antibody is administered to patients with metastatic cancer. In some of the above embodiments, the anti-CTLA4 antibody is administered to patients who are resistant or refractory to conventional cancer therapies, including other anti-CTLA4 antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, or combinations thereof.

[0098] An effective amount of anti-CTLA4 antibody can be administered in one dose or multiple doses. In methods involving administration of multiple doses of anti-CTLA4 antibody, exemplary administration frequencies include, but are not limited to, weekly, weekly without interruption, 2 weeks out of 3 weeks, 3 weeks out of 4 weeks, once every 3 weeks, once every 2 weeks, monthly, every 6 months, annually, etc. In some embodiments, anti-CTLA4 antibody is administered about once a week, once every 2 weeks, once every 3 weeks, once every 6 weeks, or once every 12 weeks. In some embodiments, the interval between each administration is about any of 3 years, 2 years, 12 months, 11 months, 10 months, 9 months, 8 months, 7 months, 6 months, 5 months, 4 months, 3 months, 2 months, 1 month, 4 weeks, 3 weeks, 2 weeks, or less than 1 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.

[0099] 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, 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 about once every three weeks.

[0100] In some embodiments, the anti-CTLA4 antibody is administered for two or more cycles, such as about any one of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles. In some embodiments, the anti-CTLA4 antibody is administered for at least four cycles.

[0101] In some embodiments, the treatment includes 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 the same frequency in the maintenance phase as in the initial phase. In some embodiments, the treatment includes 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, the subject may be administered an anti-CTLA4 antibody and the subject may then be adjusted according to one or more biomarkers, such as CD8+ T cells, the ratio of CD8+ T cells to NK cells, and / or 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.

[0102] Administration of the anti-CTLA4 antibody can be extended for an extended period of time, e.g., from about one week to about one month, from about one month to about one year, from about one year to about several years, etc. In some embodiments, the anti-CTLA4 antibody is administered for at least one week, two weeks, three weeks, four weeks, five weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, one year, two years, three years, four years, or more.

[0103] The methods described herein are useful for treating various cancers. In some embodiments, the cancer is a solid cancer. In some embodiments, the cancer is a liquid cancer. Various cancers in which CTLA4 is involved, whether malignant or benign, and whether primary or secondary, can be treated or prevented by the methods provided by the present disclosure. Examples of cancers include, but are not limited to, 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 kidney cancer, such as renal cell carcinoma, or urothelial cancer.

[0104] In some embodiments, the subject has previously been treated for cancer with conventional therapy. In some embodiments, the subject has previously been treated with 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 recurrent disease 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, three months. In some embodiments, the subject has not previously been treated with conventional therapy.

[0105] 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 recurrent disease following 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, 3 months. In some embodiments, the subject has not previously received standard treatment. In some embodiments, the subject has rejected standard treatment or is ineligible for standard treatment.

[0106] 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 PD-1 ligand (e.g., PD-L1 or PD-L2). In some embodiments, the conventional therapy is an inhibitor of CTLA4, such as an anti-CTLA4 antibody that is different from the anti-CTLA4 antibodies described herein. In some embodiments, the conventional therapy is ipilimumab. In some embodiments, the conventional therapy is an inhibitor of CTLA4 (e.g., an anti-CTLA4 antibody) and an inhibitor of PD-1 (e.g., an anti-PD-1 antibody). In some embodiments, the conventional therapy is an inhibitor of CTLA4 (e.g., an anti-CTLA4 antibody) and an inhibitor of PD-1 ligand (e.g., an anti-PD-L1 antibody or an anti-PD-L2 antibody).

[0107] In some embodiments, the conventional therapy is an inhibitor of PD-1 or PD-1 ligand, including PD-1 binding antagonists, PDL1 binding antagonists, and PDL2 binding antagonists. Alternative names for "PD-1" include CD279 and SLEB2. Alternative names for "PDL1" include B7-H1, B7-4, CD274, and B7-H. Alternative names for "PDL2" include B7-DC, Btdc, and CD273. In some embodiments, the PD-1, PDL1, and PDL2 are human PD-1, PDL1, and PDL2.

[0108] In some embodiments, the inhibitor of PD-1 is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In some embodiments, the inhibitor of PD-1 ligand is an inhibitor of PD-L1 and / or PD-L2. In some embodiments, the inhibitor of PD-L1 is a molecule that inhibits 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.

[0109] In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, 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. 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. Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA®, and SCH-900475, are anti-PD-1 antibodies described in WO2009 / 114335. CT-011, also known as hBAT or hBAT-1, are anti-PD-1 antibodies described in WO2009 / 101611. AMP-224, also known as B7-DCIg, are PDL2-Fc fusion soluble receptors described in WO2010 / 027827 and WO2011 / 066342. In some embodiments, the anti-PD-1 antibody is nivolumab (CAS Registry Number: 946414-94-4). In some embodiments, the anti-PD-1 antibody is pembrolizumab (CAS Registry Number: 1374853-91-4).

[0110] In some embodiments, the PD-L1 inhibitor is an anti-PD-L1 antibody. In some embodiments, the PD-L1 inhibitor is YW243.55.S70, MPDL3280A, MDX-1105, and MEDI4736. MDX-1105, also known as BMS-936559, is an anti-PD-L1 antibody described in WO2007 / 005874. Antibody YW243.55.S70 (heavy and light chain variable region sequences are set forth in SEQ ID NOs:20 and 21, respectively) is an anti-PD-L1 antibody described in WO2010 / 077634A1. MEDI4736 is an anti-PD-L1 antibody described in WO2011 / 066389 and US2013 / 034559. Examples of anti-PD-L1 antibodies useful in the methods of the application, and methods for making them, are described in PCT Patent Application No. WO2010 / 077634 A1 and U.S. Patent No. 8,217,149, which are incorporated herein by reference.

[0111] Conventional treatments (e.g., standard treatments) also include surgery to remove tumors and radiation therapy. Exemplary radiation therapies include, but are not limited to, ionizing (electromagnetic) radiation therapy (e.g., X-rays or gamma rays) and particle beam radiation therapy (e.g., high linear energy radiation). The source of radiation can be external or internal to the subject.

[0112] The methods described herein are useful in various aspects of cancer treatment. In some embodiments, a method of inhibiting cell proliferation (e.g., tumor growth) in an individual is provided, comprising administering to the individual an effective amount of any one of the anti-CTLA4 antibodies described herein. In some embodiments, at least about 10% (e.g., including at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, 95% or more) of cell proliferation is inhibited.

[0113] In some embodiments, methods are provided for inhibiting metastasis of a tumor in an individual, comprising administering to the individual an effective amount of any one of the anti-CTLA4 antibodies described herein, in some embodiments, at least about 10% (including, for example, at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, 95% or more) metastasis is inhibited.

[0114] 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 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).

[0115] In some embodiments, a method is 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.

[0116] In some embodiments, methods are provided for reducing the size of a tumor in an individual, comprising administering to the individual an effective amount of any one of the anti-CTLA4 antibodies described herein. In some embodiments, the method reduces the size of the tumor by at least about 10% (including, for example, at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, 95% or more).

[0117] In some embodiments, a method is provided for extending the time to disease progression of cancer in an individual, comprising administering to the individual an effective amount of any one of the anti-CTLA4 antibodies described herein. 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.

[0118] In some embodiments, methods are provided for extending the survival (e.g., overall survival or progression-free survival) of an individual with cancer, comprising administering to the individual an effective amount of any one of the anti-CTLA4 antibodies described herein. In some embodiments, the method extends the survival of the individual by at least any of 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, or 24 months.

[0119] 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.

[0120] In some embodiments, a method of improving the quality of life of an individual having cancer is provided, comprising administering to the individual an effective amount of any one of the anti-CTLA4 antibodies described herein.

[0121] The anti-CTLA4 antibody may be administered alone as a monotherapy or in combination with one or more additional therapeutic agents or treatments. In some embodiments, the anti-CTLA4 antibody is administered in combination with one or more additional therapeutic agents for separate, sequential, or simultaneous administration. 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, 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 chemotherapeutic agents, immunotherapeutic agents, and / or hormonal therapy agents. 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.

[0122] The term "chemotherapeutic agent" refers to a chemical or biological substance that can cause the death of cancer cells or interfere with the growth, division, repair, and / or function of cancer cells. Examples of chemotherapeutic agents include those disclosed in WO2006 / 129163 and US20060153808, the disclosures of which are incorporated herein by reference. Examples of specific chemotherapeutic agents include: (1) alkylating agents such as chlorambucil (LEUKERAN®), cyclophosphamide (CYTOXAN®), ifosfamide (IFEX®), mechlorethamine hydrochloride (MUSTARGEN®), thiotepa (THIOPLEX®), streptozotocin (ZANOSAR®), carmustine (BICNU, GLIADEL WAFER®), lomustine (CEENU®), and dacarbazine (DTIC-DOME®); (2) doxorubicin (ADRIAMYCIN®), epirubicin (ELLENCE®, PHARMORUBICIN®), daunorubicin (CERUBIDINE®, DAUNOXOME®), nemorubicin, idarubicin (IDAMYCIN®), ribavirin (RIBA ... PFS®, ZAVEDOS®), mitoxantrone (DHAD®, NOVANTRONE®), dactinomycin (Actinomycin D, COSMEGEN®), plicamycin (MITHRACIN®), mitomycin (MUTAMYCIN), and bleomycin (BLENOXANE®), vinorelbine tartrate (NAVELBINE®), vinblastine (VELBAN®), vincristine (O alkaloids or plant vinca alkaloids, including cytotoxic antibiotics such as NCOVIN®, and vindesine (ELDISINE®); (3) capecitabine (XELODA®), cytarabine (CYTOSAR-U®), fludarabine (FLUDARA®), gemcitabine (GEMZAR®), hydroxyurea (HYDRA®), methotrexate (FOLEX®, MEXATE, TREXALL®);(4) antimetabolites, such as 5-fluorouracil (5-FU), capecitabine (XELODA®), raltitrexed (TOMUDEX®), tegafur-uracil (UFTORAL®), and gemcitabine (GEMZAR®); (5) pyrimidine antagonists, such as docetaxel (TAXOTERE®), paclitaxel (PACL®), ribavirin (RIBA ... (6) platinum agents, such as cisplatin (PLATINOL®) and carboplatin (PARAPLATIN®), and oxaliplatin (ELOXATIN®); (7) topoisomers, such as irinotecan (CAMPTOSAR®), topotecan (HYCAMTIN®), etoposide (ETOPOPHOS®, VEPESSID®, TOPOSAR®), and teniposide (VUMON®). (8) epipodophyllotoxins (podophyllotoxin derivatives (registered trademarks)) such as etoposide (ETOPOPHOS (registered trademark), VEPESSID (registered trademark), TOPOSAR (registered trademark)), (9) folic acid derivatives such as leucovorin (WELLCOVORIN (registered trademark)), (10) nitrosoureas such as carmustine (BICNU (registered trademark)) and lomustine (CEENU (registered trademark), (11) gefitinib (IRESSA (registered trademark), erlotinib (TARCEVA (registered trademark), bortezomib (V ELCADE®), imatinib mesylate (GLEEVEC®), genefitinib, lapatinib, sorafenib, thalidomide, sunitinib (SUTENT®), axitinib, rituximab (RITUXAN, MABTHERA®), trastuzumab (HERCEPTIN®), cetuximab (ERBITUX®), bevacizumab (AVASTIN®), and ranibizumab (LUCENTIS®), lym-1 (ONCOLYM®),Inhibitors of receptor tyrosine kinases, including epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), insulin receptor, insulin-like growth factor receptor (IGFR), hepatocyte growth factor receptor (HGFR), and platelet-derived growth factor receptor (PDGFR), such as antibodies against insulin-like growth factor-1 receptor (IGF-1R) disclosed in WO2002 / 053596; (12) angiogenesis inhibitors, such as bevacizumab (AVASTIN®), suramin (GERMANIN®), angiostatin, SU5416, thalidomide, and matrix metalloproteinase inhibitors (such as batimastat and marimastat), and those disclosed in WO2002055106; and (13) proteasome inhibitors, such as bortezomib (VELCADE®).

[0123] The term "immunotherapeutic agent" refers to a chemical or biological substance capable of enhancing the immune response of a mammal. Examples of immunotherapeutic agents include bacillus Calmette-Guerin (BCG); cytokines such as interferons; vaccines such as MyVax personalized immunotherapy, Onyvax-P, Oncophage, GRNVAC1, Favld, Provenge, GVAX, Lovaxin C, BiovaxID, GMXX, and NeuVax; and immunomodulators such as alemtuzumab (CAMPATH®), bevacizumab (AVASTIN®), cetuximab (ERBITUX®), gemtuzumab ozogamicin (MYLOTARG®), ibritumomab tiuxetan (ZEVALIN®), panitumumab (VECTIBIX®), rituximab (RITUXAN®, MABTHERA®), trastuzumab (HERCEPTIN®), and other immunomodulators. and antibodies such as agonist antibodies against the OX40 receptor (e.g., those disclosed in WO2009 / 079335), agonist antibodies against the CD40 receptor (e.g., those disclosed in WO2003 / 040170), and TLR-9 agonists (e.g., those disclosed in WO2003 / 015711, WO2004 / 016805, and WO2009 / 022215).

[0124] The term "hormonal therapy" refers to a chemical or biological substance that inhibits or eliminates the production of hormones or inhibits or interferes with the effect of hormones on the growth and / or survival of cancerous cells. Examples of such agents suitable for the methods herein include those disclosed in US20070117809. Examples of specific hormonal therapy agents include tamoxifen (NOLVADEX®), toremifene (FARESTON), fulvestrant (FASLODEX®), anastrozole (ARIMIDEX®), exemestane (AROMASIN®), letrozole (FEMARA), megestrol acetate (MEGACE®), goserelin (ZOLADEX®), and leuprolide (LUPRON). The anti-CTLA4 antibodies of the present disclosure may also be used in combination with non-drug hormone therapies such as (1) surgical procedures to remove all or part of the organs or glands involved in the production of hormones, such as the ovaries, testes, adrenal glands, and pituitary gland, and (2) radiation therapy, in which a patient's organs or glands are irradiated in an amount sufficient to inhibit or eliminate production of the targeted hormone.

[0125] In some embodiments, the additional therapeutic agent is one or more of pomalyst, Revlimid, lenalidomide, pomalidomide, thalidomide, DNA alkylating platinum-containing derivatives, cisplatin, 5-fluorouracil, cyclophosphamide, anti-CD137 antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-CD20 antibody, anti-CD40 antibody, anti-DR5 antibody, anti-CD1d antibody, anti-TIM3 antibody, SLAMF7 antibody, anti-KIR receptor antibody, anti-OX40 antibody, anti-HER2 antibody, anti-ErbB-2 antibody, anti-EGFR antibody, cetuximab, rituximab, trastuzumab, pembrolizumab, radiation therapy, single radiation, fractionated radiation, focal radiation, whole organ radiation, IL-12, IFNα, GM-CSF, chimeric antigen receptor, adoptive transfer T cells, anti-cancer vaccine, and oncolytic virus. In some embodiments, the additional therapeutic agent is an anti-PD-1 antibody. In some embodiments, the additional therapeutic agent is an anti-PD-1 antibody.

[0126] Combination therapies for treating cancer also include the combination of CTLA4 antibodies with surgery to remove tumors. The anti-CTLA4 antibodies can be administered to the mammal before, during, or after surgery.

[0127] Combination therapies for treating cancer also include the use of anti-CTLA4 antibodies in combination with radiation therapy, such as 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 mammal. The anti-CTLA4 antibodies can be administered to the mammal before, during, or after radiation therapy.

[0128] Also provided are compositions of any one of the anti-CTLA4 antibodies described herein for use in the methods described in this section, and the use of an anti-CTLA4 antibody in the manufacture of a medicament for treating cancer (e.g., a solid cancer, e.g., urothelial carcinoma).

[0129] III. Biomarkers The present application also provides biomarkers that may be used in combination with any one of the therapeutic methods described herein. Suitable biomarkers include IL-1β, IL-2, IL-6, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, soluble CTLA4 (sCTLA4), soluble PD-L1 (sPD-L1), soluble CD25 (sCD25), CXCL11, FoxP3, Ki67, CD8+ T cells, CD4+ T cells, CD8+ effector memory T (T em ) cells, CD4+T em cells, regulatory T(T reg ) cells, CD8+T em Cells and T reg Ratio to CD4+T cells em Cells and T reg The ratio of NK cells to B cells is also included.

[0130] In some embodiments, the present invention relates to a method for detecting IL-1β, IL-2, IL-6, IL-10, IFN-γ, TNF-α, sCTLA4, sPD-L1, sCD25, CXCL11, FoxP3, Ki67, CD8+ T cells, CD4+ T cells, CD8+ T cells, or CD8+ T cells in one or more samples obtained from a subject. em 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 In some embodiments, the one or more biomarkers are selected from the group consisting of CD8+ T cells, CD4+ T cells, ... em 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 In some embodiments, the one or more biomarkers include the ratio of CD8+ T cells, NK cells, and B cells. em In some embodiments, the one or more biomarkers include CD4+ T cells. em In some embodiments, the one or more biomarkers include T reg In some embodiments, the one or more biomarkers include CD8+ T cells. em Cells and T reg In some embodiments, the one or more biomarkers include a ratio of CD4+ T cells. em Cells and T reg In some embodiments, the one or more biomarkers include NK cells.

[0131] In some embodiments, the present invention relates to a method for detecting IL-1β, IL-2, IL-6, IL-10, IFN-γ, TNF-α, sCTLA4, sPD-L1, sCD25, CXCL11, FoxP3, Ki67, CD8+ T cells, CD4+ T cells, CD8+ T cells, or CD8+ T cells in one or more samples obtained from a subject. em 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 Methods are provided for determining whether a subject is likely to respond to a treatment comprising an anti-CTLA4 antibody by determining the level of one or more biomarkers selected from the group consisting of CD8+ T cells, CD4+ T cells, CD8+ T cells, NK cells, and B cells. In some embodiments, methods are provided for treating or slowing the progression of cancer in a subject by administering an effective amount of an anti-CTLA4 antibody after determining that the subject is likely to respond to an anti-CTLA4 antibody. In some embodiments, the one or more biomarkers are CD8+ T cells, CD4+ T cells, CD8 ... em 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 In some embodiments, the one or more biomarkers include the ratio of CD8+ T cells, NK cells, and B cells. em In some embodiments, the one or more biomarkers include CD4+ T cells. em In some embodiments, the one or more biomarkers include T reg In some embodiments, the one or more biomarkers include CD8+ T cells. em Cells and T reg In some embodiments, the one or more biomarkers include a ratio of CD4+ T cells. em Cells and T reg In some embodiments, the one or more biomarkers include NK cells.

[0132] In some embodiments, the present invention relates to a method for detecting IL-1β, IL-2, IL-6, IL-10, IFN-γ, TNF-α, sCTLA4, sPD-L1, sCD25, CXCL11, FoxP3, Ki67, CD8+ T cells, CD4+ T cells, CD8+ T cells, or CD8+ T cells in one or more samples obtained from a subject. em 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 In some embodiments, the one or more biomarkers are selected from the group consisting of CD8+ T cells, CD4+ T cells, ... em 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 In some embodiments, the one or more biomarkers include the ratio of CD8+ T cells, NK cells, and B cells. em In some embodiments, the one or more biomarkers include CD4+ T cells. em In some embodiments, the one or more biomarkers include T reg In some embodiments, the one or more biomarkers include CD8+ T cells. em Cells and T reg In some embodiments, the one or more biomarkers include a ratio of CD4+ T cells. em Cells and T reg In some embodiments, the one or more biomarkers include NK cells.

[0133] In some embodiments, the present invention relates to a method for detecting IL-1β, IL-2, IL-6, IL-10, IFN-γ, TNF-α, sCTLA4, sPD-L1, sCD25, CXCL11, FoxP3, Ki67, CD8+ T cells, CD4+ T cells, CD8+ T cells, or CD8+ T cells in one or more samples obtained from a subject. em 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 Methods are provided for predicting and / or monitoring a treatment and / or predicting responsiveness of a subject to a treatment comprising an anti-CTLA4 antibody by determining the level of one or more biomarkers selected from the group consisting of CD8+ T cells, CD4+ T cells, ... em 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 In some embodiments, the one or more biomarkers include the ratio of CD8+ T cells, NK cells, and B cells. em In some embodiments, the one or more biomarkers include CD4+ T cells. em In some embodiments, the one or more biomarkers include T reg In some embodiments, the one or more biomarkers include CD8+ T cells. em Cells and T reg In some embodiments, the one or more biomarkers include a ratio of CD4+ T cells. em Cells and T reg In some embodiments, the one or more biomarkers include NK cells.

[0134] In some embodiments, the present invention relates to a method for detecting IL-1β, IL-2, IL-6, IL-10, IFN-γ, TNF-α, sCTLA4, sPD-L1, sCD25, CXCL11, FoxP3, Ki67, CD8+ T cells, CD4+ T cells, CD8+ T cells, or CD8+ T cells in one or more samples obtained from a subject. em 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 Methods are provided for positively and / or negatively stratifying patients into specific treatment regimens based on the levels of one or more biomarkers selected from the group consisting of CD8+ T cells, CD4+ T cells, CD8+ T cells, NK cells, and B cells. In some embodiments, the treatment comprises administration of an anti-CTLA4 antibody. In some embodiments, the one or more biomarkers are CD8+ T cells, CD4+ T cells, ... em 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 In some embodiments, the one or more biomarkers include the ratio of CD8+ T cells, NK cells, and B cells. em In some embodiments, the one or more biomarkers include CD4+ T cells. em In some embodiments, the one or more biomarkers include T reg In some embodiments, the one or more biomarkers include CD8+ T cells. em Cells and T reg In some embodiments, the one or more biomarkers include a ratio of CD4+ T cells. em Cells and T reg In some embodiments, the one or more biomarkers include NK cells.

[0135] In some embodiments, the present invention relates to a method for detecting IL-1β, IL-2, IL-6, IL-10, IFN-γ, TNF-α, sCTLA4, sPD-L1, sCD25, CXCL11, FoxP3, Ki67, CD8+ T cells, CD4+ T cells, CD8+ T cells, or CD8+ T cells in one or more samples obtained from a subject. em 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 Assays are provided for determining the level of one or more biomarkers selected from the group consisting of CD8+ T cells, CD4+ T cells, NK cells, and B cells. In some embodiments, the assay is a flow cytometry assay. In some embodiments, the assay is an immunohistochemistry (IHC) assay. In some embodiments, the assay is a multiplex IHC assay capable of detecting two or more biomarkers. In some embodiments, the assay is an immunoassay, such as a mesoscale discovery (MSD) assay. In some embodiments, the one or more biomarkers are CD8+ T cells, CD4+ T cells, ... em 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 In some embodiments, the one or more biomarkers include the ratio of CD8+ T cells, NK cells, and B cells. em In some embodiments, the one or more biomarkers include CD4+ T cells. em In some embodiments, the one or more biomarkers include T reg In some embodiments, the one or more biomarkers include CD8+ T cells. em Cells and T reg In some embodiments, the one or more biomarkers include a ratio of CD4+ T cells. em Cells and T regIn some embodiments, the one or more biomarkers include NK cells.

[0136] In some embodiments, a method of treating cancer in a subject includes: (a) administering to the subject an effective amount of an anti-CTLA4 antibody that specifically binds to an epitope that includes amino acid residues Y105 and L106, but not including residue I108, of human CTLA4, where the numbering of the amino acid residues is based on SEQ ID NO: 108; and (b) then detecting CD8+ T cells, CD4+ T cells, CD8+ T cells, or CD8+ T cells in a sample from the subject. em 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 and determining the level of one or more biomarkers selected from the group consisting of CD8+ T cells, CD4+ T cells, ... em cells, CD4+T em cells, Treg cells, CD8+T em Cells and T reg Ratio to CD4+T cells em Cells and T reg An increase in the level of one or more biomarkers selected from the group consisting of T cells, NK cells, and B cells indicates that the subject is likely to have an effective response to the anti-CTLA4 antibody. reg T cells after administration of anti-CTLA4 antibody compared to baseline levels regA decrease in the level of cells indicates that the subject is likely to have an effective response to the CTLA4 antibody. In some embodiments, the cancer is a solid cancer, such as advanced stage cancer and / or metastatic cancer. In some embodiments, the cancer is urothelial carcinoma. In some embodiments, the cancer is resistant or refractory to conventional treatments, where the conventional treatments are inhibitors of CTLA4 (e.g., ipilimumab), PD-1 (e.g., pembrolizumab), or PD-1 ligands. In some embodiments, the one or more biomarkers are CD8+ T em In some embodiments, the one or more biomarkers include CD4+ T cells. em In some embodiments, the one or more biomarkers include T reg In some embodiments, the one or more biomarkers include CD8+ T cells. em Cells and T reg In some embodiments, the one or more biomarkers include a ratio of CD4+ T cells. em Cells and T reg In some embodiments, the one or more biomarkers include NK cells. In some embodiments, the antibody is TY21580.

[0137] In some embodiments, a method of treating cancer in a subject includes: (a) administering to the subject an effective amount of an anti-CTLA4 antibody that specifically binds to an epitope that includes amino acid residues Y105 and L106, but not including residue I108, of human CTLA4, where the numbering of the amino acid residues is based on SEQ ID NO: 108; and (b) then detecting CD8+ T cells, CD4+ T cells, CD8+ T cells, or CD8+ T cells in a sample from the subject. em 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(c) determining the level of one or more biomarkers selected from the group consisting of CD8+ T cells, CD4+ T cells, ... em 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 and / or the sample has an increased level of one or more biomarkers selected from the group consisting of: T cells, NK cells, and B cells. reg T cells after administration of anti-CTLA4 antibody compared to baseline levels reg and administering to the subject an additional cycle of an effective amount of an anti-CTLA4 antibody if the subject has a decreased level of the cells. In some embodiments, the cancer is a solid cancer, such as advanced stage cancer and / or metastatic cancer. In some embodiments, the cancer is urothelial carcinoma. In some embodiments, the cancer is resistant or refractory to conventional treatments, and the conventional treatments are inhibitors of CTLA4 (e.g., ipilimumab), PD-1 (e.g., pembrolizumab), or PD-1 ligands. In some embodiments, the one or more biomarkers are CD8+ T em In some embodiments, the one or more biomarkers include CD4+ T cells. em In some embodiments, the one or more biomarkers include T reg In some embodiments, the one or more biomarkers include CD8+ T cells. em Cells and T reg In some embodiments, the one or more biomarkers include a ratio of CD4+ T cells. em Cells and T reg In some embodiments, the one or more biomarkers include NK cells. In some embodiments, the antibody is TY21580.

[0138] In some embodiments, there is provided a method of providing a prognosis for a subject to which an effective amount of an anti-CTLA4 antibody has been administered, the antibody specifically binds to an epitope that includes amino acid residues Y105 and L106, but not including residue I108, of human CTLA4, where the numbering of the amino acid residues is based on SEQ ID NO: 108, and detects CD8+ T cells, CD4+ T cells, CD8+ T cells, or CD8+ T cells in a sample from the subject. em 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 and (a) determining the level of one or more biomarkers selected from the group consisting of CD8+ T cells, CD4+ T cells, ... em cells, CD4+T em cells, Treg cells, CD8+T em Cells and T reg Ratio to CD4+T cells em Cells and T reg an increase in the level of one or more biomarkers selected from the group consisting of: T cells, NK cells, and B cells, indicates that the subject is likely to have an effective response to the anti-CTLA4 antibody; and / or (b) an increase in the level of one or more biomarkers selected from the group consisting of T cells, NK cells, and B cells, indicates that the subject is likely to have an effective response to the anti-CTLA4 antibody. reg Methods are provided in which a decrease in the level of Treg cells after administration of an anti-CTLA4 antibody compared to the baseline level of Treg cells indicates that the subject is likely to have an effective response to the CTLA4 antibody. In some embodiments, the cancer is a solid cancer, such as advanced stage cancer and / or metastatic cancer. In some embodiments, the cancer is urothelial carcinoma. In some embodiments, the cancer is resistant or refractory to conventional treatments, and the conventional treatments are inhibitors of CTLA4 (e.g., ipilimumab), PD-1 (e.g., pembrolizumab), or PD-1 ligands. In some embodiments, the one or more biomarkers are CD8+ T em In some embodiments, the one or more biomarkers include CD4+ T cells. emIn some embodiments, the one or more biomarkers include T reg In some embodiments, the one or more biomarkers include CD8+ T cells. em Cells and T reg In some embodiments, the one or more biomarkers include a ratio of CD4+ T cells. em Cells and T reg In some embodiments, the one or more biomarkers include NK cells. In some embodiments, the antibody is TY21580.

[0139] In some embodiments, the biomarkers are tumor infiltrating T cells, CD8+ T cells, CD4+ T cells, CD8+ T em cells, CD4+T em cells, NK cells and / or cell populations, or CD8+ T cells em Cells and T reg cell ratio, or CD4+T em Cells and T reg In some embodiments, the ratio is between two cell populations, such as the ratio between T cells and T cells. Suitable methods for determining the level of a cell population in a sample are well known in the art, and include, for example, fluorescence-activated cell sorting (FACS). In some embodiments, the sample is a blood sample. In some embodiments, the sample is a tumor sample. In some embodiments, the T cells are tumor-infiltrating lymphocytes.

[0140] In some embodiments, the method comprises determining whether the T em In some embodiments, determining the level of T em The cells are CD8+T em In some embodiments, the T em The cells are CD4+T em In some embodiments, the T em The cells are CD45RO+CCR7-L-selectin- T cells. In some embodiments, the T em The cells have moderate to high expression of CD44. In some embodiments, the T em Cells (e.g., CD8+ T emCells or CD4+T em The level of T cells) is increased by at least about 10%, 20%, 30%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 2-fold, 3-fold, 4-fold, 5-fold or more in a subject likely to respond to an anti-CTLA4 antibody after administration of the anti-CTLA4 antibody, compared to a baseline level, e.g., before the subject was administered the anti-CTLA4 antibody. In some embodiments, a reference level (e.g., T cells in a healthy subject) is increased by at least about 10%, 20%, 30%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 2-fold, 3-fold, 4-fold, 5-fold or more in a subject likely to respond to the anti-CTLA4 antibody after administration of the anti-CTLA4 antibody, compared to a baseline level, e.g., before the subject was administered the anti-CTLA4 antibody. em cell levels) in subjects before administration of anti-CTLA4 antibody em A low level of cells indicates that the subject is more likely to respond to anti-CTLA4 antibody treatment. In some embodiments, a low level is no more than any one of 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or less than the reference level.

[0141] In some embodiments, the method comprises determining whether the T reg In some embodiments, determining the level of T reg In some embodiments, the T cells are CD4+CD25+FOXP3+ T cells. reg A decrease in the level of T cells indicates that the subject is likely to respond to anti-CTLA4 antibody treatment (e.g., the subject is likely to have stable disease or a partial response). reg After administration of an anti-CTLA4 antibody, the level of cells is reduced by at least about 10%, 20%, 30%, 50%, 60%, 70%, 80%, 90% or more in a subject likely to respond to the anti-CTLA4 antibody compared to before the subject was administered the anti-CTLA4 antibody.

[0142] In some embodiments, the method includes administering an anti-CTLA4 antibody to a subject. em Cell levels and T reg Ratio of CD8+ T cells, e.g. em Cell levels and T reg Ratio to CD8+ T cells and / or em Cell levels and Treg In some embodiments, the ratio of CD8+ T cells after administration of an anti-CTLA4 antibody compared to baseline is determined. em Cells and T reg An increase in the ratio of CD8+ T cells indicates that the subject is more likely to respond to anti-CTLA4 antibody treatment (e.g., the subject is more likely to have stable disease or a partial response). em Cells and T reg The ratio of CD4+ T cells to CD4+ T cells is reduced by at least about 10%, 20%, 30%, 50%, 60%, 70%, 80%, 90% or more in subjects likely to respond to anti-CTLA4 antibodies after administration of the anti-CTLA4 antibody compared to before the subject was administered the anti-CTLA4 antibody. In some embodiments, the ratio of CD4+ T cells to CD4+ T cells after administration of the anti-CTLA4 antibody compared to baseline is em Cells and T reg An increase in the ratio of CD4+ T cells indicates that the subject is more likely to respond to anti-CTLA4 antibody treatment (e.g., the subject is more likely to have stable disease or a partial response). em Cells and T reg After administration of an anti-CTLA4 antibody, the ratio of cells to CTLA4 cells increases by at least about 10%, 20%, 30%, 50%, 60%, 70%, 80%, 90% or more in a subject likely to respond to the anti-CTLA4 antibody compared to before the subject was administered the anti-CTLA4 antibody.

[0143] In some embodiments, the method includes determining the level of NK cells in a sample, such as a blood sample, e.g., a peripheral blood sample. In some embodiments, an increase in the level of NK cells in the subject after administration of an anti-CTLA4 antibody compared to a baseline level of NK cells indicates that the subject is likely to have an effective response to the anti-CTLA4 antibody (e.g., the subject is likely to have stable disease or a partial response). In some embodiments, the level of NK cells increases by at least about 10%, 20%, 30%, 50%, 60%, 70%, 80%, 90% or more in subjects likely to respond to the anti-CTLA4 antibody after administration of the anti-CTLA4 antibody compared to before the subject was administered the anti-CTLA4 antibody. In some embodiments, a lower level of NK cells in a subject before administration of the anti-CTLA4 antibody compared to a reference level (e.g., the level of NK cells in a healthy subject) indicates that the subject is likely to respond to anti-CTLA4 antibody treatment. In some embodiments, a lower level is not more than any one of 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or less than a reference level.

[0144] In some embodiments, the level of a biomarker in a sample is measured by determining the level of RNA transcript expression of the biomarker. Suitable methods for measuring RNA transcript levels in a sample are known in the art and include, for example, Northern blot analysis, nuclease protection assays, in situ hybridization, PCR analysis (e.g., qPCR, RT-PCR, RT-qPCR, etc.), and next-generation sequencing (e.g., RNAseq). In some embodiments, the level of transcript expression of a biomarker is measured by RT-PCR, in situ hybridization, and / or RNAseq.

[0145] In some embodiments, the level of a biomarker in a sample is measured by determining the level of protein expression of the biomarker. Suitable methods for measuring protein expression in a sample are known in the art and include, for example, immunoassays (e.g., Meso Scale Discovery or MSD assays), immunohistochemistry (IHC), PET imaging, Western blotting, enzyme-linked immunosorbent assay (ELISA), flow cytometry, and mass spectrometry. In some embodiments, the level of protein expression of a biomarker is measured by immunoassays, Western blotting, ELISA, IHC, and / or flow cytometry.

[0146] In some embodiments, the level of one or more biomarkers is measured in one or more (e.g., one or more, two or more, three or more, four or more, etc.) samples obtained from a subject. Any suitable sample in the form of tissue and / or bodily fluid known or believed to contain disease cells and / or targets of interest can be used in the methods described herein, including, for example, sputum, pleural fluid, lymphatic fluid, bone marrow, blood, plasma, serum, urine, tissue samples (samples known or suspected to contain cancer cells), tumor samples, tumor biopsies, etc. In some embodiments, the sample is a blood sample. In some embodiments, the sample is a serum sample. In some embodiments, the sample is a tumor sample. In some embodiments, the sample is a tumor biopsy. In some embodiments, the sample contains one or more cancer cells.

[0147] Methods for obtaining appropriate tissue and / or fluid samples (e.g., methods suitable for obtaining a representative sample from a particular type, location, diseased tissue, etc.) are well known to those of skill in the art and include, for example, resection, bone marrow biopsy or aspirate, endoscopic biopsy or aspiration (e.g., cystoscopy, bronchoscopy, colonoscopy, etc.), needle biopsy or aspiration (e.g., fine needle aspiration, core needle biopsy, aspiration biopsy, image-guided biopsy, etc.), skin biopsy (e.g., shave biopsy, punch biopsy, incision biopsy, excision biopsy, etc.), a variety of other surgical tissue (e.g., tumor tissue) biopsy and / or resection strategies, and bodily fluid collection (e.g., urine collection, blood, serum, plasma, sputum, etc.).

[0148] In some embodiments, disease (e.g., cancerous) cells are enriched in one or more samples obtained from a subject. Methods for enriching disease (e.g., cancerous) cells in tissue or body fluid preparations are known in the art, including, for example, by separating disease (e.g., cancerous) cells from normal cells by flow cytometry. In some embodiments, the level of one or more biomarkers is measured in the enriched sample. In some embodiments, the level of one or more biomarkers is measured in a sample that is not enriched or otherwise modified after isolation.

[0149] In some embodiments, one or more samples are fixed (i.e., preserved) by conventional methodologies (see, e.g., “Manual of Histological Staining Method of the Armed Forces Institute of Pathology,” 3 rdSee The Armed Forces Institute of Pathology Advanced Laboratory Methods in Histology and Pathology (1994) Ulreka V. Mikel, Editor, Armed Forces Institute of Pathology, American Registry of Pathology, Washington, DC). The choice of fixative can be determined by one of skill in the art for the purpose for which the sample is to be analyzed. The length of fixation will depend on the size and type of tissue sample and fixative (e.g., neutral buffered formalin, paraformaldehyde, etc.) used, as will be recognized by one of skill in the art. In some embodiments, the level of one or more biomarkers is measured in a sample that has been fixed. In some embodiments, the level of one or more biomarkers is measured in a sample that has not been fixed or otherwise altered after isolation.

[0150] In some embodiments, one or more samples are obtained from the subject prior to administration of the anti-CTLA4 antibody. In some embodiments, one or more samples are obtained from the subject after administration of a first and / or subsequent dose of the anti-CTLA4 antibody. In some embodiments, one or more samples are obtained from the subject after completion of anti-CTLA4 antibody treatment. In some embodiments, one or more samples are obtained from the subject before, during, or after completion of anti-CTLA4 antibody treatment.

[0151] In some embodiments, the method includes comparing the level of the biomarker in a sample obtained from the subject to a reference level of the biomarker. In some embodiments, the reference level is the level of the biomarker in a reference sample (e.g., a reference cell (such as a cell line, including but not limited to Raji (ATCC, CC-86) or Daudi (ATCC, CCL-213) cell line), corresponding samples taken from one or more patients determined to be responsive to anti-CTLA4 antibody treatment, corresponding samples taken from one or more patients determined to be non-responsive to anti-CTLA4 antibody treatment, corresponding adjacent normal tissue, etc.). In some embodiments, the reference level is measured in the reference sample using the same method as used to measure the level of the biomarker in the subject's sample. In some embodiments, the reference level is measured in the reference sample using a different method than used to measure the level of the biomarker in the subject's sample.

[0152] In some embodiments, the reference level is a predetermined level of the biomarker (e.g., the average level of the biomarker in a database of disease samples (e.g., tissue biopsies or serum samples, etc.) isolated from multiple reference patients; the average level of the biomarker in a database of samples (e.g., tissue biopsies or serum samples, etc.) isolated from multiple healthy reference patients; etc.).

[0153] In some embodiments, the reference level is the baseline level of the biomarker before the subject is administered the anti-CTLA4 antibody.

[0154] In some embodiments, the reference level of a biomarker refers to a detectable level of expression, i.e., in some embodiments, the level of a biomarker measured in a sample obtained from a subject is considered to be lower than the reference level when the level of the biomarker in the sample is undetectable, e.g., below the limit of detection.

[0155] In some embodiments, the level of a biomarker measured in a sample obtained from a subject is considered to be lower than the reference level when the level of the biomarker in the sample is at least about 25% lower than the reference level.For example, the expression level of a biomarker measured in a sample obtained from a subject is considered to be lower than the reference level when the expression level of the biomarker in the sample is at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% lower than the reference level.In some embodiments, the level of a biomarker measured in a sample obtained from a subject is considered to be lower than the reference level when the level of the biomarker in the sample is at least about 1-fold lower than the reference level. For example, the level of a biomarker measured in a sample obtained from a subject is considered to be lower than the reference level when the level of the biomarker in the sample is at least about 1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold, at least about 9.5-fold, at least about 10-fold, at least about 100-fold, or at least about 1000-fold lower than the reference level. In some embodiments, the level of a biomarker in a sample obtained from a subject is below the detection limit. In some embodiments, the level of a biomarker measured in a sample obtained from a subject is considered to be lower than the reference level when the level of the biomarker in the sample is below the detection limit while the reference level is above the detection limit, detectable, and / or non-zero.In some embodiments, a level is considered below the limit of detection when, when an assay to measure the level of a biomarker is performed, the level does not result in a discernible signal, a detectable signal, and / or is not significantly different from an appropriate negative control (e.g., below the limit of detection of an assay that measures RNA transcript expression of the biomarker (e.g., RT-PCR, in situ hybridization, and / or next generation sequencing, etc.), below the limit of detection of an assay that measures protein expression of the biomarker (e.g., immunoassay, PET imaging, Western blotting, ELISA, immunohistochemistry, and / or flow cytometry, etc.), etc.).

[0156] In some embodiments, the subject is administered an effective amount of an anti-CTLA4 antibody when the level of the biomarker in the sample obtained from the subject is lower than the reference level. In some embodiments, the subject is determined to be likely to respond to the anti-CTLA4 antibody when the level of the biomarker in the sample obtained from the subject is lower than the reference level. In some embodiments, the subject is administered an effective amount of an anti-CTLA4 antibody after being determined to be likely to respond to the anti-CTLA4 antibody. In some embodiments, the subject with cancer is selected for treatment with anti-CTLA4 when the level of the biomarker in the sample obtained from the subject is lower than the reference level. In some embodiments, the subject is classified as positive for enrollment in anti-CTLA4 antibody treatment when the level of the biomarker in the sample obtained from the subject is lower than the reference level.

[0157] In some embodiments, the level of a biomarker measured in a sample obtained from a subject is considered to be higher than the reference level when the level of the biomarker in the sample is at least about 5% higher than the reference level.For example, the level of a biomarker measured in a sample obtained from a subject is considered to be higher than the reference level when the expression level of the biomarker in the sample is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% higher than the reference level.In some embodiments, the level of a biomarker measured in a sample obtained from a subject is considered to be higher than the reference level when the level of the biomarker in the sample is at least about 1-fold higher than the reference level. For example, the level of a biomarker measured in a sample obtained from a subject is considered to be higher than the reference level when the level of the biomarker in the sample is at least about 1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold, at least about 9.5-fold, at least about 10-fold, at least about 100-fold, or at least about 1000-fold higher than the reference level. In some embodiments, the level of the biomarker in the reference sample is below the detection limit. In some embodiments, the level of a biomarker measured in a sample obtained from a subject is considered to be higher than the reference level when the level of the biomarker in the sample is above the detection limit, detectable, and / or non-zero, while the level of the biomarker in the reference sample is below the detection limit.In some embodiments, a level is considered below the limit of detection when, when an assay to measure the level of a biomarker is performed, the level does not result in a discernible signal, a detectable signal, and / or is not significantly different from an appropriate negative control (e.g., below the limit of detection of an assay that measures RNA transcript expression of the biomarker (e.g., RT-PCR, in situ hybridization, and / or next generation sequencing, etc.), below the limit of detection of an assay that measures protein expression of the biomarker (e.g., immunoassay, PET imaging, Western blotting, ELISA, immunohistochemistry, and / or flow cytometry, etc.), etc.).

[0158] In some embodiments, the subject is administered an effective amount of an anti-CTLA4 antibody when the level of the biomarker in the sample obtained from the subject is higher than the reference level. In some embodiments, the subject is determined to be likely to respond to the anti-CTLA4 antibody when the level of the biomarker in the sample obtained from the subject is higher than the reference level. In some embodiments, the subject is administered an effective amount of an anti-CTLA4 antibody after being determined to be likely to respond to the anti-CTLA4 antibody. In some embodiments, the subject with cancer is selected for treatment with an anti-CTLA4 antibody when the level of expression of the biomarker in the sample obtained from the subject is higher than the reference level. In some embodiments, the subject is classified as positive for enrollment in anti-CTLA4 antibody treatment when the level of the biomarker in the sample obtained from the subject is higher than the reference level.

[0159] In some embodiments, the method includes determining the level of a biomarker at two or more time points during anti-CTLA4 antibody treatment. In some embodiments, the method includes determining the level of a biomarker (e.g., CD8+ T) in a sample obtained from the subject prior to administration of the anti-CTLA4 antibody. em cells, CD4+T em cells, CD8+T em Cells and T reg Ratio to CD4+T cells em Cells and Treg In some embodiments, the method includes determining the level of a biomarker in a sample obtained from the subject after administration of an anti-CTLA4 antibody. In some embodiments, the method includes determining the level of a biomarker in a sample obtained from the subject after each cycle of anti-CTLA4 antibody treatment. In some embodiments, the method includes determining the ratio between the difference between the level of the biomarker after administration of an anti-CTLA4 antibody (C2) and the level of the biomarker before administration of an anti-CTLA4 antibody (C1) and the level of the biomarker before administration of an anti-CTLA4 antibody (C1) (the "induction ratio"): (C2-C1) / C1. In some embodiments, an induction ratio of at least about 50%, any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more indicates a high likelihood of responding to anti-CTLA4 antibody treatment (i.e., having stable disease (SD) or partial response (PR) after treatment).

[0160] IV. Anti-CTLA4 antibody The methods described herein include administration of 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.

[0161] In some embodiments, the anti-CTLA4 antibody is any one of the antibodies described herein, including those described with respect to the 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. In some embodiments, the anti-CTLA4 antibody binds to human CTLA4 and has the following functional properties: (a) a K of 500 nM or less for human, cynomolgus monkey, mouse, rat, and / or dog CTLA4. D(b) has antagonist activity against human CTLA4; (c) does not bind to human PD-1, PD-L1, PD-L2, LAG3, TIM3, B7-H3, CD95, CD120a, OX40, CD40, BTLA, VISTA, ICOS, and / or B7-H4 at concentrations up to 100 nM; (d) is cross-reactive with monkey, mouse, rat, and / or dog CTLA4; (e) induces an ADCC effect (e.g., against Tregs); (f) inhibits the expression of human PB At least one of (e.g., at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or all nine) of the following functions are achieved: activate MCs (e.g., stimulate secretion of IL-2 and / or IFNγ); (g) can inhibit tumor cell proliferation; (h) has a therapeutic effect against cancer; and (i) blocks binding of human CTLA4 to human CD80 and / or human CD86. In some embodiments, the anti-CTLA4 antibodies described herein have a lower activity of blocking binding of CD80 and / or CD86 to human CTLA4 compared to ipilimumab in assays in which human CD80 and / or CD86 are immobilized (or bound to a plate) or human CTLA4 protein is present on the cell surface. In some embodiments, the anti-CTLA4 antibodies described herein selectively deplete Treg cells in the tumor microenvironment compared to Treg depletion in PBMCs or spleen. In some embodiments, the anti-CTLA4 antibodies described herein have higher Treg depletion activity in the tumor microenvironment compared to ipilimumab.Also provided herein is one or more anti-CTLA4 antibodies or antigen-binding fragments that cross-compete with one or more of the antibodies or antigen-binding fragments described herein for binding to human CTLA4.

[0162] In some embodiments, the antibody or antigen-binding fragment has a K for human, cynomolgus monkey, mouse, rat, and / or dog CTLA4 of about 500 nM or less (e.g., about 500 nM or less, about 450 nM or less, about 400 nM or less, about 350 nM or less, about 300 nM or less, about 250 nM or less, about 200 nM or less, about 150 nM or less, about 100 nM or less, about 90 nM or less, about 80 nM or less, about 70 nM or less, about 60 nM or less, about 50 nM or less, about 40 nM or less, about 30 nM or less, about 25 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less, about 0.1 nM or less, etc.). D In some embodiments, the antibody or antigen-binding fragment binds to human, cynomolgus monkey, mouse, rat, and / or dog CTLA4 with a K of about 350 nM or less. D In some embodiments, the antibody or antigen-binding fragment binds with a K of about 100 nM or less. D In some embodiments, the antibody or antigen-binding fragment binds to human CTLA4 with a K of about 50 nM or less. D In some embodiments, the antibody or antigen-binding fragment binds to human CTLA4 with a K of about 10 nM or less. D The K of the antibody or antigen-binding fragment binds to human CTLA4. D Methods for measuring K can be performed using any method known in the art, including, for example, by surface plasmon resonance, ELISA, isothermal titration calorimetry, filter binding assays, EMSA, etc. In some embodiments, K D is measured by surface plasmon resonance or ELISA (see, for example, Example 3 below).

[0163] 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 CLA4 blockade reporter gene assay) when a cell expressing human CTLA4 (e.g., a human cell) is contacted by the antibody or antigen-binding fragment.

[0164] 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, and the like. In some embodiments, cross-reactivity is measured by ELISA.

[0165] In some embodiments, the antibody induces an ADCC effect on CTLA4 expressing cells (e.g., on CTLA4 expressing human cells such as Tregs) after the antibody binds to the cell expressing 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 more than about 10% compared to a control (e.g., an isotype control or ipilimumab) (e.g., induces ADCC of more than about 10%, more than about 15%, more than about 20%, more than about 25%, more than about 30%, more than about 35%, more than about 40%, etc.).

[0166] 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.

[0167] In some embodiments, the antibody or antigen-binding fragment has a therapeutic effect against cancer. In some embodiments, the antibody or antigen-binding fragment relieves one or more signs or symptoms of cancer. In some embodiments, a subject suffering from cancer undergoes partial or complete remission when administered the antibody or antigen-binding fragment.

[0168] In another aspect, the present disclosure provides an isolated antibody that competes or cross-competes with any of the exemplary antibodies of the present disclosure, e.g., TY21585, TY21586, TY21587, TY21588, TY21589, TY21580, TY21591, TY21686, TY21687, TY21689, TY21680, TY21691, and / or TY21692, for binding to human CTLA4. In certain embodiments, the present application provides an isolated antibody that competes or cross-competes with any of the exemplary antibodies of the present disclosure for binding to the same epitope on human CTLA4. The ability of an antibody to compete or cross-compete for binding with another antibody can be determined using standard binding assays known in the art, such as BIAcore analysis, ELISA assays, or flow cytometry. For example, the exemplary antibodies of the present disclosure can be allowed to bind to human CTLA4 under saturating conditions, and then the ability of the test antibody to bind to CTLA4 can be measured. If the test antibody can bind to CTLA4 at the same time as the exemplary antibody, then the test antibody binds to a different epitope than the exemplary antibody. However, if the test antibody cannot bind to CTLA4 at the same time, then the test antibody binds to the same epitope, an overlapping epitope, or an epitope that is very close to the epitope bound by the exemplary antibody. This experiment can be performed using a variety of methods, such as ELISA, RIA, FACS, or surface plasmon resonance.

[0169] 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 inhibitory concentration (IC50) 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. In some embodiments, the antibody or antigen-binding fragment has a half inhibitory concentration (IC50) 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 binding of human CTLA4 to CD80 and / or CD86 when provided at a concentration of about 100 nM or more (e.g., about 100 nM or more, about 500 nM or more, about 1 μM or more, about 10 μM or more, 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 assays, and flow cytometry. In some embodiments, the anti-CTLA4 antibodies described herein have less activity in blocking ligand binding than ipilimumab.

[0170] In some embodiments, the anti-CTLA4 antibody has a K of 1000 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.

[0171] In some embodiments, the anti-CTLA4 antibody specifically binds to an epitope similar to the ligand binding site of human CTLA4. In some embodiments, the antibody specifically binds to an epitope similar to the CD80 binding site of human CTLA4. In some embodiments, the antibody specifically binds to an epitope similar to the CD86 binding site of human CTLA4. In some embodiments, the antibody specifically binds to an epitope that includes one or more amino acid residues in the ligand binding site (e.g., the CD80 and / or CD86 binding site) of human CTLA4. In some embodiments, the antibody specifically binds to an epitope on human CTLA4 that is distinct from the epitope of ipilimumab. In some embodiments, the epitope does not include amino acid residues in the CC' loop motif of human CTLA4. In some embodiments, the epitope does not include amino acid residues L106 or I108 of human CTLA4. In some embodiments, the antibody specifically binds to an epitope that includes amino acid residues Y105 and L106, but not I108, of human CTLA4, where the numbering of the amino acid residues is based on the following sequence: KAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPE (SEQ ID NO: 108).

[0172] In some embodiments, the anti-CTLA4 antibody comprises a heavy chain variable region and a light chain variable region, a) the heavy chain variable region comprises HVR-H1, HVR-H2, and HVR-H3, wherein HVR-H1 is a variable region selected from the group consisting of formula (I): X1TFSX2YX3IHWV (SEQ ID NO: 1), where X1 is F or Y, X2 is D or G, and X3 is A, G, or W; formula (II): YSIX1SGX2X3WX4WI (SEQ ID NO: 2), where X1 is S or T, X2 is H or Y, X3 is H or Y, and X4 is A, D, or S; and formula (I II): FSLSTGGVAVX1WI (SEQ ID NO: 3) (wherein X1 is G or S), and HVR-H2 comprises an amino acid sequence according to a formula selected from formula (IV): IGX1IX2HSGSTYYSX3SLKSRV (SEQ ID NO: 4) (wherein X1 is D or E, X2 is S or Y, and X3 is P or Q), formula (V): IGX1ISPSX2GX3TX4YAQKFQGRV (SEQ ID NO: 5) (wherein X1 is I or W, X2 is G or S, X3 is G or S, and X4 is K or N), and formula (VI): V SX1ISGX2GX3X4TYYADSVKGRF (SEQ ID NO: 6) (wherein X1 is A, G, or S, X2 is S or Y, X3 is G or S, and X4 is S or T); HVR-H3 comprises an amino acid sequence according to a formula selected from formula (VII): ARX1X2X3X4FDX5 (SEQ ID NO: 7) (wherein X1 is G, R, or S, X2 is A, I, or Y, X3 is D, V, or Y, X4 is A, E, or Y, and X5 is I or Y); Column No. 8) (wherein X1 is D or L, X2 is F or Y, and X3 is V or Y), formula (IX): ARX1X2X3X4AX5X6FDY (SEQ ID NO: 9) (wherein X1 is L or R, X2 is I or P, X3 is A or Y, X4 is S or T, X5 is T or Y, and X6 is A or Y), and formula (X): ARDX1X2X3GSSGYYX4GFDX5 (SEQ ID NO: 10) (wherein X1 is I or V, X2 is A or H, X3 is P or S, and X4 is D or Y,and / or b) the light chain variable region comprises an amino acid sequence according to a formula selected from HVR-L1, HVR-L2, and HVR-L3, wherein HVR-L1 is selected from the group consisting of formula (XI): RASQX1X2X3SX4LX5 (SEQ ID NO: 11) (wherein X1 is G or S, X2 is I or V, X3 is G or S, X4 is S or Y, and X5 is A or N), formula (XII): RASQX1VX2X3RX4LA (SEQ ID NO: 12) (wherein X1 is G or S, X2 is I or V, X3 is G or S, X4 is S or Y, and X5 is A or N). wherein X1 is S or T, X2 is F, R, or S, X3 is G or S, and X4 is F or Y; and formula (XIII): RASX1SVDFX2GX3SFLX4 (SEQ ID NO: 13) (wherein X1 is E or Q, X2 is D, F, H, or Y, X3 is F, I, or K, and X4 is A, D, or H); and HVR-L2 comprises an amino acid sequence according to a formula selected from formula (XIV): X1ASX2X3X4X5GX6 (SEQ ID NO: No. 14) (wherein X1 is A or D, X2 is N, S, or T, X3 is L or R, X4 is A, E, or Q, X5 is S or T, and X6 is I or V); and HVR-L3 comprises an amino acid sequence according to the formula (XV): YCX1X2X3X4X5X6PX7T (SEQ ID NO: 15) (wherein X1 is E, Q, or V, X2 is H or Q, X3 is A, G, H, R, or S, X4 is D, L, S, or Y, and X5 is E , G, P, Q, or S, X6 is L, T, V, or W, and X7 is F, L, P, W, or Y), formula (XVI): YCQQX1X2X3WPPWT (SEQ ID NO: 16) (wherein X1 is S or Y, X2 is D or Y, and X3 is Q or Y), and formula (XVII): YCQX1YX2SSPPX3YT (SEQ ID NO: 17) (wherein X1 is H or Q, X2 is T or V, and X3 is E or V).

[0173] In some embodiments, the antibody comprises (a) HVR-H1 comprising an amino acid sequence selected from SEQ ID NOs: 18-29, HVR-H2 comprising an amino acid sequence selected from SEQ ID NOs: 30-39, and HVR-H3 comprising an amino acid sequence selected from SEQ ID NOs: 40-52, and / or (b) HVR-L1 comprising an amino acid sequence selected from SEQ ID NOs: 53-65, HVR-L2 comprising an amino acid sequence selected from SEQ ID NOs: 66-69, and HVR-L3 comprising an amino acid sequence selected from SEQ ID NOs: 70-81. In some embodiments, the antibody comprises one, two, three, four, five, or all six of the HVRs set forth for any of the exemplary antibodies described in Table A below. [Table 2-1] [Table 2-2]

[0174] In some embodiments, the antibody comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 18, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 30, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 40, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 53, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 66, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 70. In some embodiments, the antibody comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 19, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 31, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 41, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 54, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 67, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 71. In some embodiments, the antibody comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 20, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 32, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 42, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 55, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 66, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 72. In some embodiments, the antibody comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 21, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 33, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 43, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 56, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 68, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 73. In some embodiments, the antibody comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 22, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 34, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 44, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 57, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 66, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 74. In some embodiments, the antibody comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 23, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 35, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 45, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 58, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 66, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 75.In some embodiments, the antibody comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 24, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 32, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 46, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 59, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 66, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 76. In some embodiments, the antibody comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 25, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 36, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 47, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 60, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 69, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 77. In some embodiments, the antibody comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO:26, HVR-H2 comprising the amino acid sequence of SEQ ID NO:37, HVR-H3 comprising the amino acid sequence of SEQ ID NO:48, HVR-L1 comprising the amino acid sequence of SEQ ID NO:61, HVR-L2 comprising the amino acid sequence of SEQ ID NO:66, and HVR-L3 comprising the amino acid sequence of SEQ ID NO:78. In some embodiments, the antibody comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO:27, HVR-H2 comprising the amino acid sequence of SEQ ID NO:32, HVR-H3 comprising the amino acid sequence of SEQ ID NO:49, HVR-L1 comprising the amino acid sequence of SEQ ID NO:62, HVR-L2 comprising the amino acid sequence of SEQ ID NO:67, and HVR-L3 comprising the amino acid sequence of SEQ ID NO:79. In some embodiments, the antibody comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO:28, HVR-H2 comprising the amino acid sequence of SEQ ID NO:37, HVR-H3 comprising the amino acid sequence of SEQ ID NO:50, HVR-L1 comprising the amino acid sequence of SEQ ID NO:63, HVR-L2 comprising the amino acid sequence of SEQ ID NO:67, and HVR-L3 comprising the amino acid sequence of SEQ ID NO:80. In some embodiments, the antibody comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO:18, HVR-H2 comprising the amino acid sequence of SEQ ID NO:38, HVR-H3 comprising the amino acid sequence of SEQ ID NO:51, HVR-L1 comprising the amino acid sequence of SEQ ID NO:64, HVR-L2 comprising the amino acid sequence of SEQ ID NO:67, and HVR-L3 comprising the amino acid sequence of SEQ ID NO:81.In some embodiments, the antibody comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 29, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 39, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 52, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 65, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 68, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 77.

[0175] In some embodiments, the antibody comprises a) a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 82-94, and / or b) a light chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 95-107. 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 a sequence selected from SEQ ID NOs: 82-94, 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 NOs: 95-107. In some embodiments, the antibody comprises the heavy chain variable region and the light chain variable region of any of the exemplary antibodies set forth in Table B below. In some embodiments, the antibody comprises one, two, or all three HVRs of the heavy chain variable region, and / or one, two, or all three HVRs of the light chain variable region, as set forth for any of the exemplary antibodies set forth in Table B below. [Table 3-1] [Table 3-2] [Table 3-3]

[0176] In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:82, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:95. In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:83, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:96. In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:84, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:97. In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:85, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:98. In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:86, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:99. In some embodiments, 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. In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:88, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:101. In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:89, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:102. In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:90, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:103. In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:91, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:104. In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:92, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:105. In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:93, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:106. In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:94, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:107.

[0177] In some embodiments, an antibody of the present application cross-competes for binding to human CTLA4 with an antibody comprising (a) an HVR-H1 comprising an amino acid sequence selected from SEQ ID NOs: 18-29, an HVR-H2 comprising an amino acid sequence selected from SEQ ID NOs: 30-39, and an HVR-H3 comprising an amino acid sequence selected from SEQ ID NOs: 40-52, and / or (b) an HVR-L1 comprising an amino acid sequence selected from SEQ ID NOs: 53-65, an HVR-L2 comprising an amino acid sequence selected from SEQ ID NOs: 66-69, and an HVR-L3 comprising an amino acid sequence selected from SEQ ID NOs: 70-81. In some embodiments, an antibody of the present application cross-competes for binding to human CTLA4 with an antibody comprising one, two, three, four, five, or all six of the HVRs set forth for any of the exemplary antibodies described in Table A. In some embodiments, an antibody of the present application cross-competes for binding to human CTLA4 with an antibody comprising a) a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 82-94, and / or b) a light chain variable region comprising an amino acid sequence selected from SEQ ID NOs: 95-107. In some embodiments, an antibody of the present application cross-competes for binding to human CTLA4 with an antibody comprising a VH and / or VL as shown for any of the exemplary antibodies described in Table B.

[0178] 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 the IgG class, such as 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 an antibody of a desired class or subclass is to isolate nucleic acid encoding a heavy chain of a CTLA4 antibody and nucleic acid encoding a light chain of a CTLA4 antibody, and convert the nucleic acid to a V-type antibody. H The coding sequence for the region was isolated and HThe method includes the steps of linking the sequence to a sequence encoding a heavy chain constant region of the desired class or subclass, expressing the light chain genes and heavy chain constructs in a cell, and harvesting 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 can include one or more Fc mutations (e.g., that modulate (increase or decrease) ADCC or CDC activity). Any suitable Fc mutation known in the art can be used in the CTLA4 antibodies of the present application.

[0179] 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 antibody refers 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.

[0180] In some embodiments, the anti-CTLA4 antibody has the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYAIHWVRQAPGKGLEWIGIISPSGGSTKYAQKFQGRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARLGYGYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA and a light chain comprising the amino acid sequence: DIQLTQSPSSLSASVGDRVTITCRASQSVDFYGISFLHWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCVQALQLPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 130).In some embodiments, the anti-CTLA4 antibody has the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYAIHWVRQAPGKGLEWIGIISPSGGSTKYAQKFQGRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARLGYGYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA and a light chain comprising the amino acid sequence: DIQLTQSPSSLSASVGDRVTITCRASQSVDFYGISFLHWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCVQALQLPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 130). In some embodiments, anti-CTLA4 antibody refers to a mixture of antibody species, each antibody species comprising a light chain comprising the amino acid sequence of SEQ ID NO: 130 and a heavy chain comprising the amino acid sequence of either SEQ ID NO: 128 or 129.

[0181] In some embodiments, the anti-CTLA4 antibody has the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGYSITSGYYWAWIRQAPGKGLEWVSSISGSGSTTYYADSVKGRFTISRDNSKNTLYLQLNSLRAEDTAVYYCARDGFGYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI and a light chain comprising the amino acid sequence DIQLTQSPSSLSASVGDRVTITCSASSSVSYVYWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCVQGLQTPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 133).In some embodiments, the anti-CTLA4 antibody has the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGYSITSGYYWAWIRQAPGKGLEWVSSISGSGSTTYYADSVKGRFTISRDNSKNTLYLQLNSLRAEDTAVYYCARDGFGYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI and a light chain comprising the amino acid sequence DIQLTQSPSSLSASVGDRVTITCSASSSVSYVYWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCVQGLQTPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 133). In some embodiments, anti-CTLA4 antibody refers to a mixture of antibody species, each antibody species comprising a light chain comprising the amino acid sequence of SEQ ID NO: 133 and a heavy chain comprising the amino acid sequence of either SEQ ID NO: 131 or 132.

[0182] In some embodiments, the anti-CTLA4 antibody has the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFSGYAIHWVRQAPGKGLEWIGIISPSGGGTKYAQKFQGRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARHPFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG and a light chain comprising the amino acid sequence: DIQLTQSPSSLSASVGDRVTITCRASQSVDFYGISFLDWYQQKPGKAPKLLIYDASNRATGIPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYVSSPPEYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 136).In some embodiments, the anti-CTLA4 antibody has the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFSGYAIHWVRQAPGKGLEWIGIISPSGGGTKYAQKFQGRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARHPFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ and a light chain comprising the amino acid sequence DIQLTQSPSSLSASVGDRVTITCRASQSVDFYGISFLDWYQQKPGKAPKLLIYDASNRATGIPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYVSSPPEYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 136). In some embodiments, anti-CTLA4 antibody refers to a mixture of antibody species, each antibody species comprising a light chain comprising the amino acid sequence of SEQ ID NO: 136 and a heavy chain comprising the amino acid sequence of either SEQ ID NO: 134 or 135.

[0183] In some embodiments, the anti-CTLA4 antibody has the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGYTFSGYAIHWVRQAPGKGLEWIGIISPSGGGTKYAQKFQGRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARLYDVAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK and a light chain comprising the amino acid sequence DIQLTQSPSSLSASVGDRVTITCRASQSVDFHGKSFLHWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCEQSLEVPFTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 139).In some embodiments, the anti-CTLA4 antibody has the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGYTFSGYAIHWVRQAPGKGLEWIGIISPSGGGTKYAQKFQGRVTISRDNSKNTLYLQLNSLRAEDTAVYYCARLYDVAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK and a light chain comprising the amino acid sequence DIQLTQSPSSLSASVGDRVTITCRASQSVDFHGKSFLHWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCEQSLEVPFTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 139). In some embodiments, anti-CTLA4 antibody refers to a mixture of antibody species, each antibody species comprising a light chain comprising the amino acid sequence of SEQ ID NO: 139 and a heavy chain comprising the amino acid sequence of either SEQ ID NO: 137 or 138.

[0184] In some embodiments, the anti-CTLA4 antibody is an antigen-binding fragment of an anti-CTLA4 antibody. The antigen-binding fragment of a 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 V of a single arm of an antibody; L and V H (v) an Fv fragment consisting of a V H (vi) isolated CDRs, and (vii) antibody V domains (Ward et al., (1989) Nature 341:544-546). H V of antibody linked to domain L and single chain antibodies (scFv), which are polypeptides comprising 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).

[0185] 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 the modification of the amino acid sequence of an exemplary antibody of the present application (e.g., a "parent antibody"), 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. The types of modifications include substitution, insertion, deletion, or combinations thereof of one or more amino acids of the parent antibody.

[0186] In some embodiments, the antibody derivative has a V sequence that is at least 65%, at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence set forth in any of SEQ ID NOs: 82-107. L or V HIn some embodiments, the antibody derivative comprises an HVR-H1 amino acid sequence region that is at least 65%, at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in any of SEQ ID NOs: 18-29. In some embodiments, the antibody derivative comprises an HVR-H2 amino acid sequence region that is at least 65%, at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in any of SEQ ID NOs: 30-39. In some embodiments, the antibody derivative comprises an HVR-H3 amino acid sequence region that is at least 65%, at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in any of SEQ ID NOs: 40-52. In some embodiments, the antibody derivative comprises an HVR-L1 amino acid sequence region that is at least 65%, at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in any of SEQ ID NOs: 53-65. In some embodiments, the antibody derivative comprises an HVR-L2 amino acid sequence region that is at least 65%, at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in any of SEQ ID NOs:66-69.In some embodiments, the antibody derivative comprises an HVR-L3 amino acid sequence region that is at least 65%, at least 75%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in any of SEQ ID NOs:70-81.

[0187] 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 amino acid sequence set forth in any of SEQ ID NOs: 18-107.

[0188] Amino acid substitution includes both conservative and non-conservative substitution. The term "conservative amino acid substitution" refers to the replacement of one amino acid with another amino acid when the two amino acids have similarities in certain physicochemical properties such as polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity of the related residues. For example, typically, substitutions can be made within each of the following groups: (a) non-polar (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.

[0189] 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 VVRs of exemplary antibodies of the present disclosure. 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 of 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, USDepartment 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 sequences identical to those found in the germline immunoglobulin gene from which the framework sequences are derived, or the HVR sequences can be grafted into framework regions that contain one or more mutations compared to the germline sequences.

[0190] 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.

[0191] Another type of modification is the V H and / or V L The preferred method is to mutate amino acid residues in the HVR regions of the heavy chain. Site-directed mutagenesis or PCR-mediated mutagenesis can be used to introduce the mutation(s) and the effect on antibody binding or other functional properties of interest can be evaluated in 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 1, 2, 3, 4, or 5 residues in the HVR regions are altered. In some embodiments, the antibody derivative comprises 1, 2, 3, or 4 amino acid substitutions in the heavy chain HVR and / or light chain HVR. In another embodiment, the amino acid substitution is to change 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 1, 2, 3, or 4 conservative amino acid substitutions in the heavy chain HVR regions compared to the amino acid sequence of the exemplary antibody.

[0192] Also, V H and / or V LModifications can also be made to framework residues within the region. Typically, such framework variants are made to reduce the immunogenicity of the antibody. One approach is to "back-mutate" one or more framework residues to the corresponding germline sequence. An antibody that has undergone somatic mutations 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, the somatic mutations can be "back-mutated" to the germline sequence, for example, by site-directed mutagenesis or PCR-mediated mutagenesis.

[0193] In addition, modifications can also be made within the Fc region of an exemplary antibody, typically altering one or more functional properties of the antibody, such as serum half-life, complement binding, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity. In one example, the hinge region of CH1 is modified to change, e.g., increase or decrease, the number of cysteine ​​residues in the hinge region. This approach is further described in U.S. Pat. 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.

[0194] Additionally, the antibodies of the present application may 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 pattern of glycosylation in the variable region. Such antibody derivatives may have increased affinity and / or altered specificity for binding antigen. The mutations may add a new glycosylation site to the V region, change the location of one or more V region glycosylation site(s), or remove an existing V region glycosylation site. In one embodiment, the present application provides derivatives of CTLA4 antibodies with a potential N-linked glycosylation site at an asparagine in the heavy chain variable region, whereby a potential N-linked glycosylation site in one heavy chain variable region is removed. In another embodiment, the present application provides derivatives of CTLA4 antibodies with a potential N-linked glycosylation site at an asparagine in the heavy chain variable region, whereby a potential N-linked glycosylation site in both heavy chain variable regions is removed. Methods for altering the glycosylation pattern of antibodies are known in the art and are described, for example, in U.S. Pat. No. 6,933,368, the disclosure of which is incorporated herein by reference.

[0195] Manufacturing method The antibodies of the present application can be produced by techniques known in the art, including conventional monoclonal antibody methodology, e.g., standard somatic cell hybridization techniques (see, e.g., Kohler and Milstein, Nature 256:495 (1975)), viral or oncogenic transformation of B lymphocytes, or recombinant antibody techniques as described in detail herein.

[0196] Hybridoma production is a very well-established procedure. A common animal system for preparing hybridomas is the murine system. Immunization protocols and techniques for isolating immunized splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known. One well-known method that can be used to generate human CTLA4 antibodies provided by the present application includes the use of the XenoMouse™ animal system. XenoMouse™ mice are an engineered mouse strain that contains large fragments of human immunoglobulin heavy and light chain loci and are deficient in mouse antibody production (see, e.g., Green et al., (1994) Nature Genetics 7:13-21, WO2003 / 040170). The animal is immunized with a CTLA4 antigen. The CTLA4 antigen is isolated and / or purified CTLA4. It can be a fragment of CTLA4, such as the extracellular domain of CTLA4. Immunization of animals can be performed by any method known in the art (see, for example, Harlow and Lane, Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Press, 1990). Methods for immunizing non-human animals, such as mice, rats, sheep, goats, pigs, cattle and horses, are well known in the art (see, for example, Harlow and Lane, supra, and U.S. Pat. No. 5,994,619). CTLA4 antigen can be administered with an adjuvant to stimulate the immune response. Exemplary adjuvants include complete or incomplete Freund's adjuvant, RIBI (muramyl dipeptide), or ISCOM (immunostimulating complex). After immunization of an animal with CTLA4 antigen, an immortalized cell line producing antibodies is prepared from cells isolated from the immunized animal. After immunization, the animal is sacrificed and B cells in the lymph nodes and / or spleen are immortalized.Methods for immortalizing cells include, but are not limited to, introducing oncogenes into the cells, curing the cells with oncogenic viruses, culturing the cells under conditions that select for immortalized cells, subjecting the cells to oncogenic or mutating compounds, fusing the cells with immortalized cells, e.g., myeloma cells, and inactivating tumor suppressor genes (see, e.g., Harlow and Lane, supra). When fusion with myeloma cells is used, the myeloma cells preferably do not secrete immunoglobulin polypeptides (non-secretory cell lines). Immortalized cells are screened using CTLA4, a portion thereof, or cells expressing CTLA4. As discussed further below, CTLA4 antibody-producing cells, e.g., hybridomas, are selected, cloned, and further screened for desirable properties, including robust growth, high antibody production, and desirable antibody properties. Hybridomas can be grown in vivo in syngeneic animals, animals lacking an immune system, e.g., nude mice, or in vitro in cell culture. Methods for selecting, cloning, and growing hybridomas are well known to those skilled in the art.

[0197] The antibodies of the present application can also be prepared using phage display or yeast display methods. Such display methods for isolating human antibodies are established in the art (see, for example, Knappik, et al. (2000) J. Mol. Biol. 296, 57-86; Feldhaus et al. (2003) Nat Biotechnol 21:163-170).

[0198] In some embodiments, the anti-CTLA4 antibody is prepared by expressing one or more nucleic acids encoding the anti-CTLA4 antibody or its polypeptide chains in a host cell. In some embodiments, the one or more nucleic acids are DNA or RNA and may or may not contain intron sequences. Typically, the nucleic acid is a cDNA molecule.

[0199] The nucleic acids of the present disclosure can be obtained using any suitable molecular biology technique. In the case of antibodies expressed by hybridomas, cDNAs encoding the light and heavy chains of the antibodies produced by the hybridomas can be obtained by PCR amplification or cDNA cloning techniques. In the case of antibodies obtained from an immunoglobulin gene library (e.g., using phage display technology), nucleic acids encoding the antibodies can be recovered from the library.

[0200] The isolated DNA encoding the VH region can be converted into a full-length heavy chain gene by operably linking the VH-encoding DNA to another DNA molecule encoding a heavy chain constant region (CH1, CH2, and CH3). The sequences of human heavy chain constant region genes are known in the art (see, for example, Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but is most preferably an IgG4 or IgG2 with no ADCC activity. The IgG4 constant region sequence can be any of the various alleles or allotypes known to occur among different individuals. These allotypes are representative of naturally occurring amino acid substitutions in the IgG4 constant region. For a Fab fragment heavy chain gene, the VH-encoding DNA can be operably linked to another DNA molecule encoding only the heavy chain CH1 constant region.

[0201] The isolated DNA encoding the VL region can be converted into a full-length light chain gene (as well as a Fab light chain gene) by operably linking the DNA encoding the VL to another DNA molecule encoding the light chain constant region CL. The sequences of human light chain constant region genes are known in the art (see, for example, Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region.

[0202] To generate an scFv gene, a DNA fragment encoding the VH and VL can be operably linked to another fragment encoding a flexible linker (e.g., encoding the amino acid sequence (Gly4-Ser)3) such that the VH and VL sequences can be expressed as a contiguous single-chain protein having VL and VH regions joined by a flexible linker (see, e.g., Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and McCafferty et al., Nature 348:552-554 (1990)).

[0203] In some embodiments, further provided is a vector comprising a nucleic acid molecule described herein. In some embodiments, the vector is an expression vector or a display vector (e.g., a viral display vector, a bacterial display vector, a yeast display vector, an insect display vector, a mammalian display vector, etc.). The nucleic acid molecule may encode a portion of a light or heavy chain (e.g., a CDR or HVR, a light or heavy chain variable region), a full-length light or heavy chain, a polypeptide comprising a portion or the full-length of a heavy or light chain, or an amino acid sequence of an antibody derivative or antigen-binding fragment. In some embodiments, the vector is an expression vector useful for expressing an anti-CTLA4 antibody. In some embodiments, provided herein is a vector in which a first vector comprises a polynucleotide sequence encoding a heavy chain variable region described herein and a second vector comprises a polynucleotide sequence encoding a light chain variable region described herein. In some embodiments, a single vector comprises a polynucleotide encoding a heavy chain variable region described herein and a light chain variable region described herein.

[0204] To express the anti-CTLA4 antibodies of the present disclosure, DNA encoding partial or full-length light and heavy chains is inserted into an expression vector such that the DNA molecule is operably linked to transcriptional and translational control sequences. In this context, the term "operably linked" means that the antibody gene is ligated into a vector such that the transcriptional and translational control sequences in the vector perform their intended function of regulating the transcription and translation of the DNA molecule. The expression vector and expression control sequences are selected to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors, or both genes can be inserted into the same expression vector. The antibody gene is inserted into the expression vector by any suitable method (e.g., ligation of complementary restriction sites on the antibody gene fragment and the vector, or homologous recombination-based DNA ligation). The light and heavy chain variable regions of the antibodies described herein are used to ligate the V and VD chains into an expression vector already encoding heavy and light chain constant regions of the desired isotype and subclass.H The segment is C in the vector H operatively coupled to the segment(s), L The segment is C in the vector L By inserting them operably linked to the segment, full-length antibody genes of any antibody isotype and subclass can be generated. Additionally or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0205] In addition to the antibody sequence, the expression vector of the present disclosure typically has a regulatory sequence that controls the expression of the antibody sequence in a host cell. The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of antibody chain genes. Such regulatory sequences are described, for example, in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). It will be understood by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences, may depend on factors such as the choice of the host cell to be transformed, the expression level of the desired protein, and the like. Examples of regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus (e.g., adenovirus major late promoter (AdMLP)), and polyoma. Alternatively, non-viral regulatory sequences such as the ubiquitin promoter or the β-globin promoter can be used. Furthermore, regulatory elements are composed of sequences from various sources, such as the SR promoter system containing sequences from the SV40 early promoter and the long terminal repeat of human T-cell leukemia virus type 1 (Takebe, Y. et al. (1988) Mol. Cell. Biol. 8: 466-472).

[0206] In addition to the antibody chain genes and regulatory sequences, expression vectors may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665, and 5,179,017, all by Axel et al.). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin, or methotrexate, on a host cell into which the vector has been introduced. Selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr host cells with methotrexate selection / amplification) and the neo gene (for G418 selection).

[0207] For expression of the light and heavy chains, the expression vector(s) encoding the heavy and light chains are transfected into a host cell by any suitable technique. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, etc. Although the antibodies of the present disclosure can be expressed in either prokaryotic or eukaryotic host cells, expression of the antibodies in eukaryotic cells, and typically mammalian host cells, is most typical.

[0208] The present application further provides host cell uses containing the nucleic acid molecules provided by the present application. The host cell can be virtually any cell for which an expression vector is available. It can be, for example, a higher eukaryotic host cell, such as a mammalian cell, a lower eukaryotic host cell, such as a yeast cell, or a prokaryotic cell, such as a bacterial cell. Methods for introducing recombinant nucleic acid into a host cell are known in the art, and include, for example, calcium phosphate transfection, DEAE, dextran-mediated transfection, electroporation, or phage infection.

[0209] Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium, and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus.

[0210] Suitable eukaryotic hosts for transformation include yeast, insect (e.g., S2 cells), and mammalian cells. Mammalian host cells for expressing the anti-CTLA4 antibodies of the present disclosure include, for example, Chinese hamster ovary (CHO) cells (including dhfr-CHO cells described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216-4220 (1980); Sharp, J. Mol. Biol. 159:601-621 (1982)), NS0 myeloma cells, COS cells, HEK293F cells, HEK293T cells, and Sp2 cells. Another expression system, particularly for use with NS0 myeloma or CHO cells, is the GS (glutamine synthetase) gene expression system disclosed in WO87 / 04462, WO89 / 01036, and EP338,841. In some embodiments, the antibodies of the application are produced in CHO cells. In some embodiments, the antibodies of the application are modified and do not contain a C-terminal lysine residue (e.g., the C-terminal lysine residue of the antibody heavy chain described herein is removed (e.g., before or during antibody production)). When an expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient to allow expression of the antibody in the host cell or secretion of the antibody into the culture medium in which the host cell is growing. The antibody can be recovered from the culture medium using any suitable protein purification method known in the art (e.g., protein A chromatography and / or ion exchange chromatography).

[0211] V. PHARMACEUTICAL COMPOSITIONS, KITS AND ARTICLES OF MANUFACTURE In another aspect, the present application provides a composition comprising any one of the anti-CTLA4 antibodies described herein. In some embodiments, the composition is a pharmaceutical composition comprising an anti-CTLA4 antibody and a pharma- ceutically acceptable carrier. The composition can be prepared by conventional methods known in the art.

[0212] The term "pharmaceutical acceptable carrier" refers to any inert material suitable for use in a formulation for delivery of an active agent (e.g., an anti-CTLA4 antibody). The carrier may be an anti-adherent, binder, coating, disintegrant, filler or diluent, preservative (such as antioxidant, antibacterial, or antifungal), sweetener, absorption retardant, wetting agent, emulsifier, buffer, etc. Examples of suitable pharmaceutical acceptable carriers include water, ethanol, polyol (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), dextrose, vegetable oil (e.g., olive oil), saline, buffer, buffered saline, and isotonic agents such as sugars, polyalcohols, sorbitol, and sodium chloride. The composition may be in any suitable dosage form, such as liquid, semi-solid, and solid dosage forms. Examples of liquid dosage forms include solutions (e.g., injectable and infusible solutions), microemulsions, liposomes, dispersions, or suspensions. Examples of solid dosage forms include tablets, pills, capsules, microcapsules, and powders. The specific form of the composition suitable for delivering anti-CTLA4 antibody is a sterile liquid, such as a solution, suspension, or dispersion for injection or infusion. Sterile solutions can be prepared by incorporating the antibody in the required amount in a suitable carrier, followed by sterilization microfiltration. In general, dispersions are prepared by adding the antibody to a sterile solvent containing a basic dispersion medium and other carriers. In the case of sterile powders for preparing sterile liquids, the preparation method includes vacuum drying and freeze-drying (lyophilization) to obtain a powder containing the active ingredient plus 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.

[0213] The relative amount of anti-CTLA4 antibody included in the composition will vary depending on many factors, such as the particular anti-CTLA4 antibody and carrier used, the dosage form, and the desired release and pharmacodynamic properties. The amount of anti-CTLA4 antibody in a single dosage form will generally be that amount that provides a therapeutic effect, but may be a lesser amount. Generally, this amount will range from about 0.01 percent to about 99 percent, from about 0.1 percent to about 70 percent, or from about 1 percent to about 30 percent, based on the total weight of the dosage form.

[0214] In addition to anti-CTLA4 antibody, one or more additional therapeutic agents can be included in the composition. Examples of additional therapeutic agents are described in the "Methods of Treatment" section of this specification. The suitable amount of additional therapeutic agent to be included in the composition can be easily selected by those skilled in the art and will vary depending on many factors, such as the specific drug and carrier used, dosage form, and desired release and pharmacodynamic properties. The amount of additional therapeutic agent included in a single dosage form is generally the amount of drug that produces a therapeutic effect, but may be a smaller amount.

[0215] In some embodiments, an article of manufacture is provided that includes a substance useful for the treatment of cancer. 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 the treatment of cancer as described herein and can have a sterile access port (e.g., the container is an intravenous solution bag or vial with a stopper pierceable by a hypodermic needle). Package insert refers to instructions typically included in the commercial package of a therapeutic product that include information regarding 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 the treatment of cancer. The label or package insert can further include instructions for administering the composition to a patient.

[0216] The article of manufacture may also further comprise a second container containing a pharma- ceutically 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.

[0217] Kits are also provided that are 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 according to 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 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.

[0218] 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 pharma- ceutically 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 one or more biomarkers described herein (e.g., CD8+ T cells, CD4+ T cells, CD8+ T em 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, B cells, and the ratio of NK cells to B cells).

[0219] 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.

[0220] The containers may be unit doses, bulk packages (e.g., multi-dose packages), or sub-unit doses. The kits may also include a plurality of unit doses of the pharmaceutical composition and instructions for use, and may be packaged in an amount sufficient for storage and use in a pharmacy (e.g., a hospital pharmacy and a compounding pharmacy).

[0221] Working Example 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.

[0222] Example 1: A Phase 1, Open-Label, Dose-Escalation Study of TY21580 in Patients with Advanced / Metastatic Solid Tumors The following example describes an ongoing Phase 1 clinical trial to evaluate the safety and tolerability of TY21580, an anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA4) fully human immunoglobulin G (IgG)1 monoclonal antibody (see ClinicalTrials.gov Identifier: NCT04501276).

[0223] TY21580.TY21580 is a fully human ligand-blocking anti-CTLA-4 mAb that targets a conserved epitope with broad species cross-reactivity for translational fidelity. Without being bound by any theory or hypothesis, TY21580 has been observed to have weaker CTLA-4 ligand blocking and stronger ADCC to deplete regulatory T cells than ipilimumab. In head-to-head in vivo efficacy studies, TY21580 was observed to have at least 5-fold greater preclinical antitumor activity compared to ipilimumab. In preclinical studies, TY21580 was well tolerated in rats and cynomolgus monkeys in a 4-week repeat-dose GLP toxicity study at doses up to 30 mg / kg and was shown to be effective in multiple immunocompetent mouse tumor models as a single agent (approximately 100 mg / kg in the H22 hepatoma model). 3 TY21580 demonstrated significant antitumor responses in a dose-dependent manner both in combination with other therapeutic agents (initial response was observed at 0.02 mg / kg and complete response was observed at 0.1 mg / kg against tumors of 100 mm diameter) and in combination with other therapeutic agents. Without being bound by any theory or hypothesis, TY21580 achieves a balance between safety and efficacy through its novel mechanism of action. That is, TY21580 maintains its inherent physiological function by partially blocking CTLA-4 ligand binding, while inhibiting T cells in the tumor microenvironment through strong antibody-dependent cellular cytotoxicity (ADCC). reg Deplete.

[0224] Objectives The primary objectives of this study are to evaluate the safety and tolerability of TY21580 administered intravenously (IV) at escalating dose levels in adults with advanced / metastatic solid tumors, to determine the maximum tolerated dose (MTD) of TY21580, and to determine the recommended phase 2 dose (RP2D). Secondary objectives of the study are to evaluate the pharmacokinetic (PK) profile of TY21580 and to assess key PK parameters (AUC, C max), evaluate the immunogenicity of TY21580 and characterize the relationship between immunogenicity (anti-drug antibody (ADA) positivity) and PK, safety and efficacy parameters, and evaluate preliminary antitumor activity of TY21580. Study objectives include, but are not limited to, pharmacodynamic biomarkers (cytokines (IL-1β, IL-2, IL-6, IL-10, interferon (IFN)-γ and tumor necrosis factor (TNF)-α), plasma proteins (sCTLA4, sPD-L1, sCD25, CXCL11), immune cells (tumor infiltrating immune (T reg ), CD8+T EM Tissue biomarkers for TY21580 may be evaluated, including immunohistochemistry (IgA, Ki67) (FoxP3, IFNγ, PD-L1).

[0225] Methods: This is a Phase 1, open-label, dose-escalation study in patients with advanced / metastatic solid tumors. The expected number of patients in Phase 1 is approximately 60 maximum and is not based on statistical considerations. The actual total number of patients will be determined by the components of this protocol, the dose escalation and dose level expansion criteria, any observed dose-limiting toxicities (DLTs) (if any), any objective evidence of antitumor activity, and other collectively collected clinical safety data. Safety review will be conducted by a Safety Review Committee (SRC) consisting of the Principal Investigator (PI), medical monitor, and sponsor.

[0226] All potential study candidates will provide informed consent and undergo screening procedures prior to study entry. After a screening period of up to 28 days, eligible patients will be enrolled to receive the assigned dose regimen of TY21580. TY21580 will be administered intravenously (IV) over 60-90 minutes at the scheduled dose every 3 weeks (Q3W) for the first 4 cycles. If no unacceptable toxicity occurs during the first 4 consecutive treatment cycles, TY21580 administration may be continued every 12 weeks (Q12W) for a total period of up to 2 years.

[0227] A treatment cycle is 21 days with a single intravenous dose of TY21580 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 be treated on study with TY21580 until documented disease progression by RECIST v1.1 and / or iRECIST, until the onset of significant toxicity, withdrawal of consent or other reason for discontinuation / withdrawal, whichever occurs first. During the study, patients will be evaluated for safety and toxicity, PK, immunogenicity, objective response rate, DOR, PFS, OS and pre-specified biomarkers per protocol.

[0228] The study will use an accelerated titration design (ATD) at lower dose levels (DL1 and DL2), followed by a conventional 3+3 dose escalation design at higher dose levels until the RP2D is determined. The starting dose will be DL1. The study will be conducted with nine potential dose levels administered by intravenous infusion, as shown in Table 1 below. [Table 4] *The proposed dose, schedule, and PK time points may be revisited and modified during the study based on safety data and observed systemic exposure and as determined by the SRC. DL-1 is a lower titration dose used if a DLT is observed or if a clinically significant grade ≥2 toxic AE is observed at DL1. **If one patient experiences a DLT or two grade ≥2 drug-related toxicities during the 21-day DLT evaluation period, additional patients will be enrolled and traditional 3+3 dose escalation criteria will apply to that dose level, in which case all subsequent dose levels will follow traditional 3+3 dose escalation criteria.

[0229] In the ATD phase, one patient will be treated per dose level. If a patient experiences DLT or two drug-related toxicities of grade 2 or higher, dose levels will be escalated according to a 3+3 design. Dose escalation will follow a traditional 3+3 design from DL3 (0.03 mg / kg) onwards, with three or six patients treated at each dose level depending on the incidence of DLT. Initially, three patients will be enrolled at that dose level, with the sentinel patient treated at least 24 hours before any subsequent patients.

[0230] Dose escalation will follow a traditional 3+3 design from DL3 (0.03mg / kg) onwards, with 3 or 6 patients treated at each dose level depending on the incidence of DLT. Initially, 3 patients will be enrolled at that dose level, with the sentinel patient treated at least 24 hours before the subsequent patients. The decision to escalate will be made by the SRC. The dose escalation rules for the traditional 3+3 design are as shown in Table 2 below. [Table 5]

[0231] The highest potential dose level in this study will not exceed 10 mg / kg as the predefined maximum administered dose (MAD). Once either the MTD or MAD is reached, the RP2D is determined, including the selection and treatment of intermediate dose levels between predefined dose levels. The RP2D is determined primarily based on the observation of the MTD, or based on the MAD if the MTD is not observed. Options for consideration of the RP2D also include dose levels below the MTD or MAD, as well as intermediate doses between predefined dose levels based on an overall evaluation of all available PK and pharmacodynamic data as well as all safety data, and the observation of reported objective response rates at dose escalation. Additional cohorts can be added as expansion cohorts to further evaluate the RP2D.

[0232] For patients who have completed at least two cycles of treatment at the dose level initially enrolled, intrapatient dose escalation will be permitted up to the dose level permitted by the SRC.

[0233] Treatment may continue beyond progressive disease by RECIST v1.1 if there is evidence of clinical benefit without worsening of tumor-related symptoms and / or unacceptable toxicity. Any progressive disease should be confirmed according to iRECIST, and repeat scans are required, preferably after 4 weeks and no later than 6 weeks. If disease progression is confirmed according to iRECIST, ongoing treatment is not permitted. Otherwise, study treatment may continue for a full period of up to 2 years or until disease progression, unacceptable toxicity, or withdrawal of consent, whichever occurs first.

[0234] All patients, except those who withdraw consent, will be followed for safety follow-up assessments at 30 and 90 days from the date of last dose, and for survival every 12 weeks from the date of last dose. Patients who discontinue treatment due to intolerable adverse events related to TY21580 will be followed until the adverse events revert to grade 0 or 1, are stable, or the patient is administered new non-protocol treatment. Survival follow-up will continue until administration of another anti-tumor therapy, or until the patient is lost to follow-up or dies, whichever occurs first. The sponsor may also decide to stop the study at any time.

[0235] Eligibility Criteria. Patients must meet all of the following inclusion criteria to be eligible to participate in this study: 1) Be 18 years of age or older at the time of informed consent. 2) Eastern Cooperative Oncology Group (Ecog) performance status of 0 or 1 and has not deteriorated during the past 2 weeks. 3) Patients with advanced or metastatic solid tumors confirmed by histopathology who have progressed after all standard therapies or for whom no further standard treatment exists. Patients who refuse or cannot receive standard treatment may be enrolled, but the reason for not receiving treatment must be reported. 4) Patients who have resistance or relapsed to conventional anti-CTLA4 checkpoint inhibitors or anti-programmed cell death protein 1 (PD-1) will also be recruited if they meet all eligibility criteria. 5) Have adequate hematological function as defined below. Absolute neutrophil count (ANC) ≥ 1.5 x 10 9 / L (no use of granulocyte colony-stimulating factors such as filgrastim within 2 weeks prior to study treatment). b. Platelet count ≧100×10 9 / L (no blood transfusion within 2 weeks (≦14 days) prior to study treatment). Patients with hepatocellular carcinoma and platelet count ≧75×10 9 / L patients. c. Hemoglobin ≥ 9g / dL (no blood transfusion or erythropoietin administration within 2 weeks (≤ 14 days) prior to study treatment). 6) Aspartate transaminase (AST) and alanine aminotransferase (ALT) ≤ 3 x upper limit of normal (ULN) and total bilirubin ≤ 1.5 x ULN. Exception: Patients with elevated serum bilirubin due to documented underlying Gilbert's syndrome or familial benign unconjugated hyperbilirubinemia. Patients with hepatocellular carcinoma and liver metastases may have AST and ALT ≤ 5 x ULN. 7) Adequate renal function defined as either creatinine clearance ≥ 60 mL / min (by Cockcroft-Gault formula) or serum creatinine (SCr) < 1.5 × ULN 8) Coagulation studies as defined below: Activated partial thromboplastin time (aPTT) ≤ 1.5 x ULN. b. International normalized ratio (INR) ≤ 1.5 x ULN. Exception: INR 2 to ≤ 3 x ULN is acceptable for patients receiving warfarin anticoagulants. 9) Left ventricular ejection fraction (LVEF) ≥ 50% as measured by multi-gate gate acquisition (MUGA) or echocardiogram (ECHO). 10) Prior antitumor therapy (including endocrine therapy, chemoradiotherapy / radiotherapy, targeted therapy or immunotherapy) must have been completed at least 4 weeks prior to administration of TY21580. Patients who have failed a prior anti-CTLA4 checkpoint inhibitor may be considered eligible if they meet all eligibility criteria. 11) Previous adverse events have improved to baseline 1NCI CTCAE v5.0 (excluding patients with alopecia) or ≤ Grade 1.

[0236] Exclusion criteria included any one of the following: 1) Pregnant or breastfeeding women 2) Women of childbearing potential and men whose partners are of childbearing potential who do not agree to use two highly effective methods of contraception during treatment and for 120 days after the last dose of study drug. 3) Treatment with any investigational drug within 4 weeks prior to the first dose of study drug. 4) Grade ≥3 immune-related AE (irAE) or irAE that led to discontinuation of previous immunotherapy. 5) Untreated or uncontrolled central nervous system (CNS) tumors or metastases, with the following exceptions: a. Clinically stable magnetic resonance imaging (MRI) scans (at least two consecutive scans within six months) and no progressive or uncontrolled neurological symptoms or signs (e.g., seizures, headache, central nausea / vomiting, progressive neurological deficits) for at least four weeks. b. Any previously untreated asymptomatic brain metastases not requiring immediate local or systemic therapy. 6)Has a history of life-threatening hypersensitivity or known allergy to protein drugs or recombinant proteins or any components contained in the TY21580 formulation. 7) Patients with active autoimmune disease or a history of autoimmune disease or symptoms requiring systemic use of pharmacologic doses of corticosteroids and / or immunosuppressants. Exceptions are patients with vitiligo, resolved childhood asthma / atopy, and type I diabetes or hypothyroidism manageable with replacement therapy. 8) Patients who require systemic treatment with corticosteroids (>15 mg / day prednisone or equivalent) or other immunosuppressants within 21 days prior to the first dose of the proposed study drug. Inhaled or topical steroids at doses equivalent to a daily dose of 15 mg or less of prednisone and adrenal replacement steroids are permitted in the absence of active autoimmune disease. Intraocular, intranasal, inhaled, and intra-articular injections of steroids are permitted. 9) Peripheral neuropathy grade 2 or greater. 10) Patients who received granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), erythropoietin, or blood transfusions (red blood cells [RBCs] or platelets) within 14 days prior to the first dose of study drug. 11) Evidence of underlying liver dysfunction due to other causes; significant alcohol abuse, alcoholic or drug-induced hepatitis, or a reported history of nonalcoholic steatohepatitis 12) Patients with active viral hepatitis (any aetiology) will be excluded. Hepatitis B virus (HBV) carriers will be ineligible. Cured hepatitis C (HCV) patients (HCV ribonucleic acid [RNA] test negative) may be enrolled after consultation with the medical monitor. 13) Any uncontrolled active infection requiring systemic antibiotic treatment (viral, bacterial, etc.) or uncontrolled or poorly controlled diabetes mellitus as indicated by screening (baseline) HgbA-1c ≥ 7.5, asthma, chronic obstructive pulmonary disease (COPD), or other conditions that pose a risk to study participants. 14) Known positive human immunodeficiency virus (HIV) status. 15) Patients with any type of primary immunodeficiency or autoimmune disease requiring treatment. 16) Major surgery within 4 weeks prior to first study drug administration. 17) Conventional organ allotransplantation or allogeneic peripheral blood stem cell (PBSC) / bone marrow (BM) transplantation. 18) Clinically significant cardiac disease (including myocardial infarction within the past 6 months, uncontrolled angina, viral myocarditis, pericarditis, cerebrovascular accident, or other acute uncontrolled cardiac disease within 3 months of first dose of study drug, LVEF <50%, New York Heart Association (NYHA) class III or IV congestive heart failure, or uncontrolled hypertension). 19) Pulmonary embolism or deep vein thrombosis within 3 weeks prior to the first dose of study drug. 20) Live virus vaccine treatment within 4 weeks prior to the first dose of study drug. 21) Known, reported, or suspected history of illegal drug abuse. 22) Other illnesses or clinically significant abnormalities in laboratory parameters, including serious medical or psychiatric illnesses / conditions, that are determined by the investigator to be likely to compromise the patient's safety or the integrity of the study or to prevent the patient's participation in the study or to compromise the objectives of the study.

[0237] Safety Assessments: Safety assessments will be performed during certain periodic physical examination findings, vital signs, ECOG performance status, laboratory variables (e.g., liver function tests / monitoring, hematology, coagulation tests, serum chemistry, urinalysis, and pregnancy test), ECGs, and AEs. AEs will be graded according to NCI CTCAE v5.0. Investigator and site personnel are responsible for appropriate documentation and reporting of AEs / SAEs. Prior to dose escalation, the SRC will review safety data from the current level after all patients have completed the first 21-day cycle and determine whether escalation to the next dose level (or stop escalation) should continue.

[0238] Efficacy Assessments Tumor assessments for response / progression will be performed at baseline and every 6 weeks (± 1 week) for the first 4 cycles. If treatment continues beyond 4 cycles, then every 9 weeks (± 1 week) for the remainder of the treatment period 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 / exit of the study, whichever occurs first. Exploratory efficacy assessments will be based on tumor assessment by the investigator according to RECIST v1.1 and / or iRECIST.

[0239] Pharmacokinetic and immunogenicity evaluations Blood samples will be collected from all patients during the first cycle to measure serum concentrations of TY21580. Pharmacokinetic (PK) parameters will be monitored more intensively during the first treatment cycle. Beginning with the second dose, plasma concentrations of TY21580 will be monitored within 30 minutes (C トラフ ) and at the end of the infusion. PK sampling time points can be adjusted based on cumulative data. Noncompartmental analysis will be performed using WinNonlin 6.4 or higher. PK parameters include, but are not limited to, AUC 0~21d , AUC 最終 , C max , T max , t 1 / 2 , MRT, CL, V d AUC and C max Dose proportionality will also be evaluated.

[0240] Blood samples for ADA will be collected at cycles 1, 2, 3, 4, and every two cycles thereafter if treatment continues beyond four cycles. In addition, ADA samples will be collected at the end of treatment and at follow-up visits 30 and 90 days after the last dose. If ADA is positive, neutralizing activity will be assessed.

[0241] result Interim results of the study are shown in the figures and discussed below.

[0242] General Observations To date, TY21580 has been well tolerated in over 10 patients at dose escalation up to 0.3 mg / kg, with no dose-limiting toxicities (DLTs) or unexpected safety signals observed so far, including treatment-related serious adverse events (SAEs, i.e., drug-related G3 and G4 toxicities), colitis or hepatitis. The terminal half-life of TY21580 was within the normal range for IgG1-based antibodies, approximately 14 days under good exposure conditions.

[0243] Interim pharmacodynamic (PD) results are shown in Figures 1 to 9C and Figures 13 to 14. Specifically, TY21580 induced immune cell responses against T cells at dose levels of 0.03 to 0.3 mg / kg (Figures 1 and 4). Effective CD8 and CD4 T cell increases were observed first, followed by a dose-dependent decrease from doses of 0.03, 0.1, and 0.3 mg / kg, similar to treatment with anti-PD1 and anti-CD137 antibodies, etc. In certain patients, T reg The percentage of CD8+ effector memory T cells (Figure 6), and the percentage of CD8+ effector memory T cells (Figure 7) and CD4+ effector memory T cells (Figure 8) were significantly increased at the 0.03, 0.1, and 0.3 mg / kg doses. Furthermore, in these patients, the ratio of CD8+ effector memory T cells / regulatory T cells (T EM / T reg ) (Figure 13) and the ratio of CD4+ effector memory T cells to regulatory T cells (T EM / T reg ) (Figure 14) showed a significant increase. In certain patients, the levels of NK cells and B cells were increased (Figures 2, 5).

[0244] Figures 4-6 and 13-14 show the relationship between TY21580 dose and immune cell response. CD8, an important pharmacodynamic (PD) biomarker indicative of immune activation, + T EM / T reg Dose-dependent changes in the ratio were observed in patients receiving 0.003 to 0.3 mg / kg TY21580 (Figure 13). Similarly, as shown in Figure 14, the CD4 + TEM / T reg The ratios also showed dose-dependent changes. The significant trends in dose-dependent CD8+TEM / Treg and CD4+TEM / Treg ratios when treated with TY21580 at doses of 0.003-0.3 mg / kg indicate that TY21580 can effectively stimulate immune cells at a dose level as low as 0.03 mg / kg. EM / T reg This dose-dependent change in ratio is a strong clinical proof of mechanism of action (POM) for TY21580. These results are consistent with preclinical observations that TY21580 is potent in highly sensitive tumor models such as H22, where it induces tumor responses at doses as low as 0.02 mg / kg and achieves a complete response (CR) at 0.1 mg / kg. In particular, CD8 + T EM / T reg A steady increase in immune cells, including rheumatoid arthritis, was observed in a patient (subject 6103-002) who received 3 cycles of TY21580 at 0.03 mg / kg after becoming refractory and resistant to >25 cycles of prior pembrolizumab treatment. The patient developed an irAE, pruritus. In addition, subject 6103-003 also developed an irAE, grade 1 rash, at the end of treatment on day 22. This grade 1 treatment-related immune-mediated pruritus was consistent with the mechanism of action of TY21580 treatment.

[0245] Monitoring continues to confirm the dose-dependent response of various biomarkers to TY21580 treatment.

[0246] Significant increases in absolute T and N cell abundances upon treatment with TY21580, as well as T regA reduction in the percentage of cells correlates with favorable clinical outcomes. Phase 1 dose escalation data demonstrates clinical proof of TY21580's mechanism of action in targeting CTLA-4, consistent with preclinical observations of TY21580's efficacy starting at 0.03 mg / kg and up to 0.3 mg / kg. TY21580 has the potential to overcome the limitations of existing anti-CTLA-4 checkpoint inhibitors on the market and is predicted to extend market potential beyond current anti-CTLA-4 inhibitors in both monotherapy and combination therapy settings. The anti-CTLA-4 therapy described herein can improve clinical benefit by expanding clonal diversity, infiltrating cold tumors, and treating patients who are resistant / refractory to current immunotherapies.

[0247] Subject 6102-002 Subject 6102-002 was enrolled in this study. Subject 6102-002 is a Caucasian male, 60 years old, with urothelial carcinoma (kidney) and an ECOG performance status scale of 0. Subject 6102-002 previously received 25 cycles of pembrolizumab + epacadostat from April 15, 2018 to September 19, 2019. The subject initially demonstrated a partial response, then became refractory and resistant to pembrolizumab therapy for a period exceeding 25 cycles. Subject 6102-002 was administered three 0.03 mg / kg doses (2.7+ mg) of TY21580, which was increased to 0.1 mg / kg on February 24, 2021. Subject 6102-002 will be increased to a higher dose when this dose is removed. Subject 6102-002 experienced an immune-related adverse event (irAE) of pruritus. This Grade 1 drug-related immunotoxicity at 0.03 mg / kg suggests target modulation at 0.03 mg / kg with an estimated 60% target binding, consistent with the mechanism of action of TY21580 treatment. A summary of treatment-emergent adverse events that occurred in subject 6102-002 is shown in Table 3 below. [Table 6]

[0248] Figure 10 shows lymphocyte profiling results over time for subject 6102-002. As shown in Figure 10, subject 6102-002 showed a sustained increase in effective CD8 T cells at 0.03 mg / kg, while CD4 T cells increased initially and then decreased. reg The number of cells decreased. + T EM / T reg The ratio of CD4+ T cells to CD8+ B cells was significantly increased. Furthermore, NK cells and B cells were increased. Analysis of immune cell subpopulations by flow cytometry revealed a stimulation of proliferation of T cells, NK cells and B cells, especially CD8+ T cells, during TY21580 treatment. + T EM / T reg A large increase in the ratio of T reg Patients receiving 0.1 mg / kg and 0.3 mg / kg also appear to follow this trend (see above). The PD results for subject 6102-002 are also shown in Figures 1 to 8.

[0249] Population Pharmacokinetic (PK) Modeling Preliminary PK population modeling was performed for this study. The two-compartment linear PK model generally represented the current PK data well across the doses tested (Figure 11). All PK parameters except V2 (marginal volume of distribution) were estimated with high accuracy. The population terminal half-life estimated by the model was approximately 14 days. The population central volume of distribution (V1) estimated by the model was similar to the plasma volume (e.g., 40 mL / kg). Goodness-of-fit plots were generated (Figures 12A-B). The model fit was generally good based on the diagnostic plots. Additional emerging PK data will be used to refine the parameter estimates in real time.

[0250] The second interim analysis of the ongoing study is shown in the figure and described below.

[0251] Clinical overview To date, 25 patients with advanced / metastatic solid tumors have been treated with TY21580 in 9 dose escalation cohorts (i.e., 0.003 mg / kg, 0.01 mg / kg, 0.03 mg / kg, 0.3 mg / kg, 1.0 mg / kg, 3.0 mg / kg, 6.0 mg / kg, and 10 mg / kg, n=24 patients) and 6 mg / kg expansion (n=1). These patients have progressive metastatic disease and have been treated across 15 different tumor types, of which 68% are IO-insensitive tumors (Figure 15B). TY21580 has been well tolerated up to 10 mg / kg, once every 3 weeks (Q3W), with no DLTs or dose-dependent toxicities identified (Figure 15D). Patients are currently entering the study at the next higher dose level, 20 mg / kg, to continue dose escalation according to the modified protocol and 6.0 mg / kg dose expansion.

[0252] Pharmacokinetics TY21580 exhibits a dose-proportional increase in drug exposure with a half-life of approximately 10 days (estimated to be approximately 10±5 days up to 10 mg / kg). Figure 16 shows the serum pharmacokinetics of TY21580 following intravenous infusion, assessed by noncompartmental analysis and population PK modeling approaches.

[0253] Pharmacodynamic results The results of the pharmacodynamic analysis are shown in Figures 17-26. To monitor the dynamic changes of cytokines and immune-related soluble proteins, patients' blood samples were collected at a series of visit points to perform the pharmacodynamic study. Blood was collected at six visits: pre-dose, cycle 1 day 1, 4 hours on cycle 1 day 1, cycle 1 day 2, cycle 1 day 8, cycle 1 day 15, and cycle 2 day 1. Serum was prepared according to standard protocols. An MSD panel was designed to examine whether there were dose-dependent changes in the abundance of five cytokines in peripheral blood during treatment with TY21580: IFN-α, IL-2, IL-6, IL-10, and TNF-γ. In addition, serum concentrations of four soluble proteins, sCTLA-4, sPD-L1, sCD25, and CXCL11, were measured.

[0254] Figure 17A shows dose-dependent changes in serum IFN-γ concentrations in patients treated with TY21580, with most patients showing significant increases in IFN-γ levels over the first 2 days after dosing. These kinetics reflect activation of the immune system and may correlate with antibody concentrations in the patient's blood. Comparison of the absolute concentration changes of each analyte with baseline showed a clear dose-dependent response of IFN-γ to TY21580 treatment at doses up to 6 mg / kg (Figure 17B). Furthermore, quantification of the change in serum concentration of IFN-γ from baseline levels (before C1D1 administration) at C1D2 within each treatment group showed that the increase in IFN-γ abundance was dose-dependent at 0.1 mg / kg and above (Figure 17C), indicating that TY21580 treatment activates the immune system. These data are consistent with one proposed mechanism of action of TY21580, in which TY21580 activates the immune system by alleviating CTLA-4-mediated immunosuppression.

[0255] TNFα concentrations were also measured in patients treated with TY2180 and the percent change relative to baseline levels was plotted. Figure 18 shows a dose-dependent increase in TNFα levels following treatment with TY2180.

[0256] Additionally, IL-6 (Figure 19), IL-10 (Figure 20), and IL-2 (data not shown) were measured in patients treated with TY21580. Figure 19 shows no observable dose-dependent changes in IL-6 concentrations in patients treated with TY21580. Serum concentrations of IL-2 were below the assay detection limit in most patients.

[0257] Serum concentrations of soluble PD-L1 were also measured in TY21580-treated patients at a series of visits (Figure 21A), and the percent change in sPD-L1 abundance relative to baseline in response to treatment is shown in Figure 21B. The percent change in sPD-L1 abundance in each treatment group is shown in Figure 21C. Levels of sPD-L1 were significantly increased in patients treated with 3 mg / kg or greater, which may indicate a clinical response.

[0258] FIG. 22 shows a dose-dependent increase in soluble CD25 in C1D8 compared to baseline levels.

[0259] Serum concentrations of soluble CXCL11 were also measured in TY21580-treated patients at a series of visits, and what appears to be a dose-dependent increase in response following TY21580 treatment is shown in Figure 23. These data indicate that CXCL11 abundance is significantly increased in patients receiving doses of 0.3 mg / kg and above.

[0260] Additionally, whole blood samples from patients were used to assess the numbers of T cells and NK cells in patients treated with TY21580.

[0261] CD4+CD8- T cells were quantified in whole blood from patients treated with TY21580 at C1D1 (pre-dose), C1D8, C1D15, and C2D1. A dose-dependent increase in CD4+ cells was measured by absolute counts, particularly at doses of 3 mg / kg and 6 mg / kg (Figure 24A). Figures 24B-24E compare the percent change in CD4+ T cells between baseline and C1D8 (Figures 24B and 24C) and between baseline and C1D8 (Figures 24D and 24E), with Figures 24B and 24D showing the relative percent change compared to baseline and Figures 24D and 24E showing the percent change in absolute counts per μL. A dose-dependent increase in the absolute number of CD8+ T cells was also measured, as shown in Figure 25. These dose-dependent increases in absolute CD4+ and CD8+ T cell numbers indicate that TY21580 treatment activates the immune system. The data show that TY21580 demonstrated consistent antitumor efficacy even after 2 weeks of treatment (C1D15).

[0262] NK cell counts were also measured in patients treated with TY21580, and Figure 26 shows the percent change in absolute cell counts per μL for C1D8 and C1D15.

[0263] Interim Efficacy Data Subject #23 was a 74-year-old male with renal cell carcinoma who had relapsed on nivolumab and was enrolled in the 10 mg / kg cohort. In this subject, CD8+ T cells increased after the first cycle of treatment (Figure 3), indicating that TY21580 was highly active in inducing T cell activation.

[0264] Subject #22 is a 77 year old male with pancreatic cancer in the 10 mg / kg cohort who has received three prior lines of therapy. This subject had two target lesions, one in the pancreas and one in the liver, with baseline measurements of 35 mm for the pancreatic lesion and 15 mm for the liver lesion. After two cycles of TY21580 treatment, an initial tumor evaluation was performed on this patient. This evaluation showed that the pancreatic lesion had shrunk to 29 mm and the liver lesion had shrunk to 10 mm, reflecting a 22% reduction in the size of the target lesions (Table 4). [Table 7]

[0265] Long-term stable disease was also observed in 17% (4 / 24) of patients, all of whom had received multiple prior therapies and had "cold tumors" (i.e., tumors that T cells cannot penetrate because they are not recognized by the immune system or have not elicited an immune response). Notably, all four patients had increased CD8 and / or CD4 T cells, with subjects #4 and #19 showing a significant increase in CD8 T cells (Figures 24A and 25A).

[0266] Example 2 Dose Selection Criteria for TY21580 Serum pharmacokinetics (PK) following intravenous infusion of TY21580 monotherapy was evaluated using noncompartmental analysis (NCA with Phoenix WinNonlin version 8.3) and population PK modeling (with NONMEM® 7.5) approaches.

[0267] A dose-dependent increase in serum exposure was observed. Maximum serum concentrations (C max、サイクル1) has an approximately linear relationship with dose (e.g., 0.003-10 mg / kg, R2=0.89, Figure 29). Predicted drug exposure AUC based on PK in cycle 1 0~無限大 (AUC INF_pred ) has an almost linear relationship with the dose (e.g., 0.01 to 10 mg / kg, R2 = 0.90, Figure 30).

[0268] The terminal half-life (mean ± SD) of TY21580 in serum during cycle 1 was estimated to be approximately 10 ± 3 days at doses of 0.01 to 10 mg / kg. No clear dose-dependence was observed. max This set of PK analyses, including , AUC and half-life, showed that no significant target-mediated drug elimination (TMDD) was observed for TY21580 at 0.01-10 mg / kg. A two-compartment linear population PK model was developed and can generally represent the PK data well. Body weight (BW) was highly correlated with the model-estimated central volume of distribution (Vc) and is considered a significant covariate (e.g., exponent value >0.8). For patients with PK data from multiple treatment cycles (Q3W, once every 3 weeks), pre-dose PK (C トラフ、反復投与 ) and C max、反復投与 Limited accumulation (e.g., less than 2-fold) was observed in the Q3W dosing group, consistent with the half-life estimated by the PK model and the predicted mean accumulation ratio (e.g., approximately 1.3-fold) with Q3W dosing.

[0269] Potentially effective human doses based on observed PK versus in vitro data were explored. EC 90 is consistent with functional readouts, typically >3 μg / mL to 5 μg / mL, and is consistent with the pre-dose concentration at steady state in minimal tumor tissue (i.e., C トラフ、ss These ECs can be targeted as 90 correlates with a theoretical receptor occupancy (RO) of approximately 90%. As an example, a representative integrated EC2 expression level based on available key in vitro data (e.g., functional readouts of binding, T cell activation, and ADCC) was 90The EC value was chosen to be approximately 5 μg / mL (approximately 34.5 nM). 90 corresponds to 172-345 nM in the systemic circulation, considering an assumed 10-20% tumor tissue drug distribution in targets without significant TMDD. At the 10 mg / kg dose of TY21580, the mean serum trough concentration of TY21580 observed on day 21 of the first cycle is still near the lowest target systemic concentration (e.g., 172 nM assuming 20% ​​tumor drug distribution). These PK data are based on the simulated steady-state PK (Figure 31, C of a 504-hour dosing cycle at 10 mg / kg, e.g., 212 nM). トラフ This, together with the monotherapy clinical PD biomarker data and safety / efficacy data, supports 10 mg / kg intravenously once every three weeks (Q3W) as a potentially efficacious / saturating monotherapy dose.

[0270] Considering the predicted mean accumulation ratio of TY21580 at 6 mg / kg or 3 mg / kg Q3W (e.g., 1.3-fold), the predicted mean serum concentration of TY21580 at day 14 or day 7 would be near or slightly above the lowest target systemic concentration (e.g., 172 nM), respectively (Figure 31). Thus, at steady state with 3 mg / kg Q3W, the predicted time above the lowest target concentration (e.g., 168 hours, Figure 31) is about 33% of the total cycle time, which would be sufficient to achieve an optimal therapeutic index. Alternatively, at steady state with 6 mg / kg Q3W, the predicted time above the lowest target concentration (e.g., 370 hours, Figure 31) is more than 70% of the total cycle time.

Claims

1. A pharmaceutical for treating cancer in a subject, comprising an effective amount of an anti-CTLA4 antibody, the subject is refractory to prior treatment or has relapsed from prior treatment; the conventional treatment is an inhibitor of CTLA4, PD-1, PD-1 ligand, or a PD-L1 antibody; The pharmaceutical, wherein the anti-CTLA4 antibody comprises HVR-H1 comprising the amino acid sequence of SEQ ID NO: 23, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 35, HVR-H3 comprising the amino acid sequence of SEQ ID NO: 45, HVR-L1 comprising the amino acid sequence of SEQ ID NO: 58, HVR-L2 comprising the amino acid sequence of SEQ ID NO: 66, and HVR-L3 comprising the amino acid sequence of SEQ ID NO:

75.

2. A pharmaceutical for treating cancer in a subject, comprising an effective amount of an anti-CTLA4 antibody, A pharmaceutical composition comprising an anti-CTLA4 antibody administered at a dose of 3 mg / kg to 20 mg / kg, an anti-CTLA4 antibody administered approximately once every three weeks, and an HVR-H1 comprising the amino acid sequence of SEQ ID NO:23, an HVR-H2 comprising the amino acid sequence of SEQ ID NO:35, an HVR-H3 comprising the amino acid sequence of SEQ ID NO:45, an HVR-L1 comprising the amino acid sequence of SEQ ID NO:58, an HVR-L2 comprising the amino acid sequence of SEQ ID NO:66, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:

75.

3. The pharmaceutical described in claim 2, wherein the cancer is resistant or refractory to conventional treatment, and the conventional treatment is an inhibitor of CTLA4, PD-1, PD-1 ligand, or an anti-PD-1 antibody.

4. The pharmaceutical described in claim 1 or 3, wherein the anti-PD-1 antibody is pembrolizumab or nivolumab.

5. The pharmaceutical composition of any one of claims 1 to 4, wherein the anti-CTLA4 antibody is administered at a dose of 6 mg / kg to 15 mg / kg.

6. The pharmaceutical composition of any one of claims 1 to 5, wherein the anti-CTLA4 antibody is administered at a dose of 6 mg / kg.

7. The pharmaceutical composition of any one of claims 1 to 5, wherein the anti-CTLA4 antibody is administered at a dose of 10 mg / kg.

8. The pharmaceutical composition of any one of claims 1 to 5, wherein the anti-CTLA4 antibody is administered at a dose of 15 mg / kg.

9. The pharmaceutical composition of any one of claims 1 to 5, wherein the anti-CTLA4 antibody is administered at a dose of 20 mg / kg.

10. The pharmaceutical according to any one of claims 1 to 9, wherein 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.

11. The pharmaceutical agent according to any one of claims 1 to 10, wherein the anti-CTLA4 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 125 or SEQ ID NO: 126, and a light chain comprising the amino acid sequence of SEQ ID NO:

127.

12. The pharmaceutical agent of any one of claims 2 to 11, wherein the anti-CTLA4 antibody is administered at a dose that provides a steady-state serum trough concentration of 50 nM to 700 nM in the systemic circulation when measured on the last day of an administration cycle.

13. The pharmaceutical agent of any one of claims 2 to 12, wherein the anti-CTLA4 antibody is administered at a dose that provides a steady-state serum trough concentration of 100 nM to 500 nM in the systemic circulation when measured on the last day of an administration cycle.

14. The pharmaceutical agent of any one of claims 2 to 13, wherein the anti-CTLA4 antibody is administered at a dose that provides a steady-state serum trough concentration of 100 nM to 400 nM in the systemic circulation when measured on the last day of an administration cycle.

15. The pharmaceutical composition of any one of claims 2 to 14, wherein the subject receives at least four cycles of anti-CTLA4 antibody treatment.

16. The pharmaceutical composition of any one of claims 1 to 15, wherein the cancer is a solid cancer, urothelial carcinoma, renal cell carcinoma, pancreatic cancer, advanced cancer, or metastatic cancer.

Images