Use of anti-CTLA-4 antibodies

Abstract

The present disclosure provides anti-CTLA-4 antibodies and methods of producing and using the antibodies. The disclosure also provides nucleic acids encoding an anti-CTLA-4 antibody and host cells containing the nucleic acids. In addition, the present disclosure provides polypeptides containing a variant Fc region comprising an amino acid change in a parent Fc region, as well as methods of producing and using the polypeptides.

Description

[0001] This application is a divisional application of the application with an international application date of June 24, 2022, international application number PCT / JP2022 / 025222, entering the Chinese national phase application number 202280044888.1, and invention name “Use of anti-CTLA-4 antibodies”. Technical Field

[0002] The present invention relates to anti-CTLA-4 antibodies and methods of using the same. The present invention also relates to polypeptides comprising variant Fc regions comprising amino acid changes in a parent Fc region and methods of producing the same. Background Art

[0003] The immune surveillance system monitors and eliminates cells in living organisms that have undergone mutations, such as those caused by genetic changes. However, the persistence of an excessive immune response can also be harmful, such as autoimmune damage to normal tissues. Therefore, the immune system has negative feedback mechanisms (immune checkpoints) that, once activated, suppress immune responses (see, for example, NPL 1). Immune checkpoints are believed to play a vital role in maintaining immune homeostasis. On the other hand, it has been revealed that some tumors exploit immune checkpoints for immune evasion. Currently, research on the immunosuppressive functions of the major immune checkpoint molecules—cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), programmed cell death 1 (PD-1), and programmed cell death ligand 1 (PD-L1)—is gaining traction.

[0004] CTLA-4 is a glycoprotein belonging to the immunoglobulin superfamily. Its gene was cloned in 1987 from a cDNA library of a mouse-derived killer T cell clone (see, for example, NPL 2). CTLA-4 is known to suppress T cell immune responses. In 1996, it was reported that tumor regression was observed by administering anti-CTLA-4 antibodies to cancer-bearing mice, based on the idea that inhibiting CTLA-4 function promotes T cell activation and leads to cancer regression (see, for example, NPL 3). Evaluation of the efficacy of anti-CTLA-4 antibodies in humans has been ongoing since 2000, and in 2011, the anti-human CTLA-4 monoclonal antibody (ipilimumab) was approved by the US Food and Drug Administration (FDA) as the world's first immunostimulatory therapeutic antibody. In addition to ipilimumab, a number of anti-CTLA-4 monoclonal antibodies have been developed (see, for example, PTL1, PTL2, PTL3, and PTL4), and their development as pharmaceuticals is ongoing. Drugs that inhibit immune checkpoints to abolish immunosuppressive mechanisms and thus enhance immune responsiveness are called immune checkpoint inhibitors.

[0005] Meanwhile, it was previously known that some T cells possess immunosuppressive functions. These cells were identified in 1995 as CD25- and CD4-positive T cells and designated as regulatory T cells (regulatory T cells) (see, for example, NPL 4). In 2003, the Foxp3 gene was identified as a master gene specifically expressed in regulatory T cells and regulating their development and function. Foxp3 acts as a transcription factor, regulating the expression of various genes involved in immune responses. In particular, Foxp3 is involved in the constitutive expression of CTLA-4 in regulatory T cells and is believed to play a key role in the immunosuppressive function of regulatory T cells (see, for example, NPL 5).

[0006] The infiltration of regulatory T cells into tumor tissues is thought to lead to a weakening or suppression of tumor immune surveillance mechanisms. Indeed, increased levels of regulatory T cells have been shown in many human cancers (see, for example, NPL 6), and localized infiltration of regulatory T cells into tumors has been reported to be a negative prognostic factor for cancer patients. Conversely, if regulatory T cells could be eliminated or reduced from tumor tissues, this could potentially lead to enhanced anti-tumor immunity. Currently, the development of cancer immunotherapies targeting regulatory T cells is booming.

[0007] Administration of the anti-CTLA-4 antibody ipilimumab enhances anti-tumor immunity, but it has been reported to cause autoimmune diseases because it systemically enhances immune reactivity. In a certain clinical trial, adverse events were observed in 60% of patients to whom ipilimumab was administered, and many of them were autoimmune diseases related to the skin or gastrointestinal tract. Similarly, in another clinical trial, it has been reported that about half of the patients to whom ipilimumab was administered developed similar autoimmune diseases. In order to suppress such side effects, in some cases, immunosuppressants are administered to patients to whom ipilimumab has been administered. It is expected that new drugs will be developed that can maintain anti-tumor immune responses while suppressing such side effects of immune checkpoint inhibitors.

[0008] It is desirable that when therapeutic antibodies are administered to a living organism, their target antigens are specifically expressed only at diseased sites. However, in many cases, the same antigens are also expressed in non-lesional sites and normal tissues, potentially causing undesirable therapeutic side effects. For example, while antibodies against tumor antigens can exert cytotoxic activity against tumor cells through ADCC and other methods, when the same antigen is also expressed in normal tissues, the antibodies may also damage normal cells. To address this issue, technologies have been developed that focus on the phenomenon that large amounts of specific compounds are present in target tissues (e.g., tumor tissue) and produce antigen-binding molecules whose antigen-binding activity varies depending on the concentration of the compound (see, for example, PTL 11).

[0009] Antibodies have attracted considerable attention as pharmaceuticals due to their high stability in the blood and minimal side effects (NPL 12 and NPL 13). Currently, the majority of commercially available antibody drugs are human IgG1 subclass antibodies. To date, extensive research has been conducted on the effector functions of IgG antibodies, namely antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Among human IgG antibodies, IgG1 subclass antibodies have been reported to exhibit the highest ADCC and CDC activities (NPL 14). Furthermore, antibody-dependent cell-mediated phagocytosis (ADCP), the engulfment of target cells by IgG antibodies, has also been demonstrated as an antibody effector function (NPL 15 and NPL 16).

[0010] In order for IgG antibodies to exert ADCC, CDC, and ADCP, the antibody Fc region must bind to antibody receptors (hereinafter referred to as FcγRs) and various complement components present on the surfaces of effector cells such as killer cells, natural killer cells, and activated macrophages. In humans, the FcγR protein family is reported to include subtypes: FcγRIa, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb, and the isotypes of each have also been reported (NPL 17).

[0011] Enhancing cytotoxic effector functions such as ADCC, ADCP, and CDC has attracted attention as a promising approach to enhance the anti-tumor effects of antibodies. The importance of FcγR-mediated effector functions for the anti-tumor effects of antibodies has been reported using mouse models (NPL 7 and NPL 8). Furthermore, a correlation has been observed between clinical outcomes in humans and the presence of the high-affinity polymorphic allotype (V158) and the low-affinity polymorphic allotype (F158) of FcγRIIIa (NPL 18). Similarly, clinical efficacy has been shown to differ depending on the FcγRIIa allotype (H131 and R131) (NPL 19). These reports suggest that antibodies with Fc regions optimized for binding to specific FcγRs mediate enhanced effector functions, leading to effective anti-tumor effects.

[0012] The balance of antibody binding activity for activating receptors (FcγRIa, FcγRIIa, FcγRIIIa, and FcγRIIIb) and inhibitory receptors (FcγRIIb) is a crucial factor in optimizing antibody effector functions. By employing an Fc region with enhanced binding activity for activating receptors and reduced binding activity for inhibitory receptors, it is possible to confer optimal effector functions on antibodies (NPL 20). Several amino acid residues within the antibody hinge region and CH2 domain, as well as the sugar chain attached to Asn 297 (according to EU numbering), have been shown to be crucial for binding between the Fc region and FcγRs (NPL 14, NPL 21, and NPL 22). By focusing on this binding site, Fc region variants with various FcγR binding properties have been developed, and Fc region variants with enhanced binding activity for activating FcγRs have been obtained (PTL 5, PTL 6, NPL 9, and NPL 10). For example, Lazar et al. replaced Ser at position 239, Ala at position 330, and Ile at position 332 (EU numbering) of human IgG1 with Asp, Leu, and Glu, respectively, successfully increasing the binding affinity of this antibody to human FcγRIIIa (V158) by approximately 370-fold (NPL 9 and PTL 6). Shinkawa et al. successfully increased the binding affinity of this antibody to FcγRIIIa by approximately 100-fold by deleting fucose from the sugar chain attached to Asn at position 297 (EU numbering) (NPL 23). These methods introduce the same alteration or sugar chain modification into both H chain Fc regions of the antibody. Furthermore, it has been reported that, although the antibody Fc is a homodimer, it binds to FcγRs in a 1:1 ratio, asymmetrically recognizing FcγRs via the lower hinge and CH2 domains (NPL 11). Given that the Fc region interacts asymmetrically with FcγRs, it is possible to more finely optimize IgG-FcγR interactions by introducing different modifications to each H chain. Based on this concept, methods for optimizing antibody-FcγR interactions by asymmetrically modifying the Fc region by modifying each H chain differently have been reported (PTL 7, PTL 8, PTL 9, and PTL 10). In fact, asymmetric modification of the Fc region resulted in variants with higher ADCC activity than existing ADCC-enhancing antibodies, including non-fucosylated antibodies (PTL 9 and PTL 10).

[0013] In addition to ADCC activity, ADCP activity is also an important effector function of antibodies, reportedly contributing to anti-tumor effects (NPL 24). ADCP activity can be enhanced by inhibiting "don't eat me" signals, such as CD47 (NPL 24), or by enhancing FcγRIIa binding (NPL 25). However, the activating FcγRs (FcγRIIa) and inhibitory FcγRIIb share high homology in their extracellular amino acid sequences, making it difficult to selectively enhance FcγRIIa binding (NPL 26). Therefore, when FcγRIIa binding is enhanced, binding activity to the inhibitory receptor FcγRIIb may also be enhanced, thereby weakening effector function. In fact, variants with significantly improved FcγRIIa binding compared to native IgG1 also have stronger FcγRIIb binding (PTL 9 and PTL 10). Therefore, in order to exhibit higher ADCC / ADCP activity, it is preferred to enhance binding to FcγRIIIa and / or FcγRIIa as much as possible without enhancing binding to FcγRIIb; however, there are currently no reports on such variants.

[0014] Citation List

[0015] Patent Literature

[0016] [PTL 1] WO 2000 / 037504

[0017] [PTL 2] WO 2001 / 014424

[0018] [PTL 3] WO 2012 / 120125

[0019] [PTL 4] WO 2016 / 196237

[0020] [PTL 5] WO 2000 / 042072

[0021] [PTL 6] WO 2006 / 019447

[0022] [PTL 7] WO 2012 / 058768

[0023] [PTL 8] WO 2012 / 125850

[0024] [PTL 9] WO 2013 / 002362

[0025] [PTL 10] WO 2014 / 104165

[0026] [PTL 11] WO 2013 / 180200

[0027] Non-Patent Literature

[0028] [NPL 1] Pardoll, Nat Rev Cancer (2012) 12: 252-264

[0029] [NPL 2] Brunet et al., Nature (1987) 328: 267-270

[0030] [NPL 3] Leach et al., Science (1996) 271: 1734-1736

[0031] [NPL 4] Sakaguchi et al., J Immunol (1995) 155: 1151-1164

[0032] [NPL 5] Takahashi et al., J Exp Med (2000) 192: 303-310

[0033] [NPL 6] Nishikawa & Sakaguchi, Int J Cancer (2010) 127: 759-767

[0034] [NPL 7] Clynes et al., Proc Natl Acad Sci U S A (1998) 95: 652-656

[0035] [NPL 8] Clynes et al., Nat Med (2000) 6: 443-446

[0036] [NPL 9] Lazar et al., Proc Natl Acad Sci U S A (2006) 103: 4005-4010

[0037] [NPL 10] Chu et al., Mol Immunol (2008) 45: 3926-3933

[0038] [NPL 11] Radaev et al., J Biol Chem (2001) 276: 16469-16477

[0039] [NPL 12] Reichert et al.,Nat Biotechnol (2005) 23: 1073-1078

[0040] [NPL 13] Pavlou & Belsey,Eur J Pharm Biopharm (2005) 59: 389-396

[0041] [NPL 14] Clark,Chem Immunol (1997) 65: 88-110

[0042] [NPL 15] Horton et al.,Cancer Res (2008) 68: 8049-8057

[0043] [NPL 16] Zalevsky et al.,Blood (2009) 113: 3735-3743

[0044] [NPL 17] Jefferis & Lund,Immunol Lett (2002) 82: 57-65

[0045] [NPL 18] Carton et al.,Blood (2002) 99: 754-758

[0046] [NPL 19] Tamura et al.,Ann Oncol (2011) 22: 1302-1307

[0047] [NPL 20] Nimmerjahn & Ravetch,Science (2005) 310: 1510-1512

[0048] [NPL 21] Greenwood et al.,Eur J Immunol (1993) 23: 1098-1104

[0049] [NPL 22] Morgan et al.,Immunology (1995) 86: 319-324

[0050] [NPL 23] Shinkawa et al.,J Biol Chem (2003) 278: 3466-3473

[0051] [NPL 24] Tsao et al., JCI Insight (2019) 4: e131882

[0052] [NPL 25] Richards et al., Mol Cancer Ther (2008) 7: 2517-2527

[0053] [NPL 26] Mimoto et al., Protein Eng Des Sel (2013) 26: 589-598 Summary of the Invention

[0055] Technical issues

[0056] The present invention provides anti-CTLA-4 antibodies and methods of using the same. The present invention also provides polypeptides comprising variant Fc regions and methods of producing the same.

[0057] Solution to the problem

[0058] More specifically, the present invention provides the following [1] to

[34] .

[0059] [1] An anti-CTLA-4 antibody for treating tumors, wherein the anti-CTLA-4 antibody comprises:

[0060] (A) a variable region having CTLA-binding activity that is dependent on the concentration of an adenosine-containing compound; and

[0061] (B) A variant Fc region comprising multiple amino acid changes in a parent Fc region, wherein the parent Fc region is composed of two polypeptide chains, and wherein the variant Fc region comprises amino acid changes at the following positions:

[0062] (i) positions 234, 235, 236, 239, 268, 270, 298 and 330 according to EU numbering in the first polypeptide of the parent Fc region; and

[0063] (ii) positions 270, 298, 326, 330 and 334 according to EU numbering in the second polypeptide of the parent Fc region.

[0064] [2] An anti-CTLA-4 antibody for use according to [1], wherein the tumor is a solid tumor in which regulatory T (Treg) cells have infiltrated.

[0065] [3] An anti-CTLA-4 antibody for use in damaging cells, wherein the anti-CTLA-4 antibody comprises:

[0066] (A) a variable region having CTLA-binding activity that is dependent on the concentration of an adenosine-containing compound; and

[0067] (B) A variant Fc region comprising multiple amino acid changes in a parent Fc region, wherein the parent Fc region is composed of two polypeptide chains, and wherein the variant Fc region comprises amino acid changes at the following positions:

[0068] (i) positions 234, 235, 236, 239, 268, 270, 298 and 330 according to EU numbering in the first polypeptide of the parent Fc region; and

[0069] (ii) positions 270, 298, 326, 330 and 334 according to EU numbering in the second polypeptide of the parent Fc region.

[0070] [4] An anti-CTLA-4 antibody for use in [3], wherein the cells to be damaged are Treg cells.

[0071] [5] The anti-CTLA-4 antibody for use according to [3], wherein the cells are damaged by ADCC activity, CDC activity or ADCP activity.

[0072] [6] The anti-CTLA-4 antibody for use in [3] or [4], wherein immunity is activated by damaging Treg cells.

[0073] [7] The anti-CTLA-4 antibody for use according to [1] or [2], wherein the tumor is at least one selected from carcinoma, lymphoma, blastoma, sarcoma and leukemia.

[0074] [8] An anti-CTLA-4 antibody for use in accordance with [7], wherein the tumor is selected from lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, colorectal cancer, rectal cancer, colon cancer, breast cancer, liver cancer, gastric cancer, pancreatic cancer, kidney cancer, prostate cancer, ovarian cancer, thyroid cancer, bile duct cancer, peritoneal cancer, mesothelioma, squamous cell carcinoma, cervical cancer, endometrial cancer, bladder cancer, esophageal cancer, head and neck cancer, nasopharyngeal cancer, salivary gland tumor, thymoma, skin cancer, basal cell tumor, melanoma, malignant melanoma, anal cancer, penile cancer, testicular cancer, Wilms' tumor, acute myeloid leukemia (including acute myeloid leukemia, acute myeloid leukemia), leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, and acute monocytic leukemia), chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma (including Burkitt lymphoma, chronic lymphocytic leukemia, mycosis fungoides, mantle cell lymphoma, follicular lymphoma, diffuse large cell lymphoma, marginal zone lymphoma, hairy cell leukemia plasmacytoma, peripheral T-cell lymphoma, and adult T-cell leukemia / lymphoma), Langerhans cell histiocytosis, multiple myeloma, myelodysplastic syndrome, At least one of the following: brain tumors (including gliomas, astrocytomas, glioblastomas, meningiomas, and ependymomas), neuroblastoma, retinoblastoma, osteosarcoma, Kaposi's sarcoma, Ewing's sarcoma, angiosarcoma, and hemangiopericytoma.

[0075] [9] The anti-CTLA-4 antibody for use in [8], wherein the tumor is at least one selected from lung cancer, colorectal cancer, colon cancer, breast cancer, breast cancer, liver cancer, pancreatic cancer, kidney cancer, bladder cancer, head and neck cancer, and melanoma.

[0076]

[10] The anti-CTLA-4 antibody for use according to any one of [1] to [9], wherein the anti-CTLA-4 antibody is an immunoconjugate.

[0077]

[11] The anti-CTLA-4 antibody for use according to any one of [1] to

[10] , wherein the anti-CTLA-4 antibody is mixed with one or more pharmaceutically acceptable carriers and used.

[0078]

[12] The anti-CTLA-4 antibody for use according to any one of [1] to

[11] , wherein the anti-CTLA-4 antibody is used in combination with at least one additional therapeutic agent.

[0079]

[13] An anti-CTLA-4 antibody for use in

[12] , wherein the additional therapeutic agent is at least one selected from an immune checkpoint inhibitor, an EGFR inhibitor, a HER2 inhibitor, and a chemotherapeutic agent.

[0080]

[14] The anti-CTLA-4 antibody for use according to any one of [1] to

[13] , wherein the variable region has at least one feature selected from the following (a) to (i):

[0081] (a) The binding activity in the presence of 100 μM adenosine-containing compounds is two or more times greater than that in the absence of adenosine-containing compounds;

[0082] (b) The KD value in the presence of 100 μM adenosine-containing compounds was 5× 10 -7 M or lower;

[0083] (c) The KD value in the absence of adenosine-containing compounds is 1×10 -6 M or higher;

[0084] (d) forming a ternary complex with adenosine-containing compounds and CTLA-4;

[0085] (e) binds to the region from amino acid 97 to amino acid 106 of human CTLA-4 (extracellular domain, SEQ ID NO: 28);

[0086] (f) competes with ABAM004 (VH, SEQ ID NO: 10; and VL, SEQ ID NO: 11) for binding to CTLA-4;

[0087] (g) binds to the same epitope as that bound by ABAM004 (VH, SEQ ID NO: 10; and VL, SEQ ID NO: 11);

[0088] (h) exhibit cytotoxic activity against cells expressing CTLA-4; and

[0089] (i) Binds to human and mouse derived CTLA-4.

[0090]

[15] The anti-CTLA-4 antibody for use according to any one of [1] or

[14] , wherein the anti-CTLA-4 antibody is a monoclonal antibody.

[0091]

[16] The anti-CTLA-4 antibody for use according to any one of [1] to

[15] , wherein the antibody is a human antibody, a humanized antibody or a chimeric antibody.

[0092]

[17] The anti-CTLA-4 antibody for use of any one of [1] to

[16] , wherein the anti-CTLA-4 antibody comprises: (a) HVR-H1 (SEQ ID NO:223) comprising the amino acid sequence SX1TMN, wherein X1 is H, A, R or K; (b) HVR-H2 (SEQ ID NO:224) comprising the amino acid sequence SISX1X2SX3YIYYAX4SVX5G, wherein X1 is S or T, X2 is R or Q, X3 is G or H, X4 is D, E or R, and X5 is K or R; and (c) HVR-H3 (SEQ ID NO:225) comprising the amino acid sequence YGX1REDMLWVFDY, wherein X1 is K or A.

[0093]

[18] The anti-CTLA-4 antibody for use of

[17] , wherein the anti-CTLA-4 antibody further comprises: (a) HVR-L1 (SEQ ID NO: 226) comprising the amino acid sequence X1GX2STX3VGDYX4X5VX6, wherein X1 is T, D, Q or E, X2 is T or P, X3 is D or G, X4 is N or T, X5 is Y or W, and X6 is S or H; (b) HVR-L2 (SEQ ID NO: 227) comprising the amino acid sequence X1TX2X3KPX4, wherein X1 is E, F or Y, X2 is S or I, X3 is K or S, and X4 is S, E or K; and (c) HVR-L3 (SEQ ID NO: 228) comprising the amino acid sequence X1TYAAPLGPX2, wherein X1 is S or Q, and X2 is M or T.

[0094]

[19] The anti-CTLA-4 antibody for use of

[17] , wherein the anti-CTLA-4 antibody further comprises: a heavy chain variable domain FR1 comprising the amino acid sequence of any one of SEQ ID NOs: 229 to 232; FR2 comprising the amino acid sequence of SEQ ID NO: 233; FR3 comprising the amino acid sequence of SEQ ID NO: 234; and FR4 comprising the amino acid sequence of SEQ ID NO: 235.

[0095]

[20] The anti-CTLA-4 antibody for use of

[18] , wherein the anti-CTLA-4 antibody further comprises: a light chain variable domain FR1 comprising the amino acid sequence of any one of SEQ ID NOs: 236 to 238; FR2 comprising the amino acid sequence of any one of SEQ ID NOs: 240 and 241; FR3 comprising the amino acid sequence of any one of SEQ ID NOs: 242 to 244; and FR4 comprising the amino acid sequence of any one of SEQ ID NOs: 245 and 246.

[0096]

[21] The anti-CTLA-4 antibody for use of any one of [1] to

[16] , wherein the anti-CTLA-4 antibody further comprises: (a) a VH sequence having at least 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 83 to 86, 98, and 135 to 141; (b) a VL sequence having at least 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 88 to 95, 97, 99, 134, and 144 to 149; or (c) a VH sequence having the amino acid sequence of any one of SEQ ID NOs: 83 to 86, 98, and 135 to 141 and a VL sequence having the amino acid sequence of any one of SEQ ID NOs: 88 to 95, 97, 99, 134, and 144 to 149.

[0097]

[22] The anti-CTLA-4 antibody for use of

[21] , wherein the anti-CTLA-4 antibody comprises:

[0098] (1) VH sequence of SEQ ID NO:98 and VL sequence of SEQ ID NO:99;

[0099] (2) the VH sequence of SEQ ID NO:83 and the VL sequence of SEQ ID NO:88;

[0100] (3) VH sequence of SEQ ID NO:83 and VL sequence of SEQ ID NO:89;

[0101] (4) VH sequence of SEQ ID NO:83 and VL sequence of SEQ ID NO:90;

[0102] (5) VH sequence of SEQ ID NO:83 and VL sequence of SEQ ID NO:91;

[0103] (6) VH sequence of SEQ ID NO:83 and VL sequence of SEQ ID NO:92;

[0104] (7) VH sequence of SEQ ID NO:83 and VL sequence of SEQ ID NO:93;

[0105] (8) VH sequence of SEQ ID NO:83 and VL sequence of SEQ ID NO:94;

[0106] (9) VH sequence of SEQ ID NO:83 and VL sequence of SEQ ID NO:97;

[0107] (10) VH sequence of SEQ ID NO:83 and VL sequence of SEQ ID NO:95;

[0108] (11) VH sequence of SEQ ID NO:84 and VL sequence of SEQ ID NO:97;

[0109] (12) VH sequence of SEQ ID NO:85 and VL sequence of SEQ ID NO:97;

[0110] (13) VH sequence of SEQ ID NO:86 and VL sequence of SEQ ID NO:97;

[0111] (14) VH sequence of SEQ ID NO:86 and VL sequence of SEQ ID NO:134;

[0112] (15) VH sequence of SEQ ID NO:136 and VL sequence of SEQ ID NO:97;

[0113] (16) VH sequence of SEQ ID NO:135 and VL sequence of SEQ ID NO:97;

[0114] (17) VH sequence of SEQ ID NO:136 and VL sequence of SEQ ID NO:95;

[0115] (18) VH sequence of SEQ ID NO:137 and VL sequence of SEQ ID NO:97;

[0116] (19) VH sequence of SEQ ID NO:138 and VL sequence of SEQ ID NO:97;

[0117] (20) VH sequence of SEQ ID NO:138 and VL sequence of SEQ ID NO:144;

[0118] (21) VH sequence of SEQ ID NO:138 and VL sequence of SEQ ID NO:145;

[0119] (22) VH sequence of SEQ ID NO:138 and VL sequence of SEQ ID NO:146;

[0120] (23) VH sequence of SEQ ID NO:139 and VL sequence of SEQ ID NO:146;

[0121] (24) VH sequence of SEQ ID NO:140 and VL sequence of SEQ ID NO:146;

[0122] (25) VH sequence of SEQ ID NO:141 and VL sequence of SEQ ID NO:146;

[0123] (26) VH sequence of SEQ ID NO:140 and VL sequence of SEQ ID NO:147;

[0124] (27) VH sequence of SEQ ID NO:141 and VL sequence of SEQ ID NO:147;

[0125] (28) VH sequence of SEQ ID NO:140 and VL sequence of SEQ ID NO:148;

[0126] (29) VH sequence of SEQ ID NO:141 and VL sequence of SEQ ID NO:148;

[0127] (30) VH sequence of SEQ ID NO:136 and VL sequence of SEQ ID NO:149;

[0128] (31) a first variable region comprising a VH sequence of SEQ ID NO: 140 and a VL sequence of SEQ ID NO: 146, and a second variable region comprising a VH sequence of SEQ ID NO: 141 and a VL sequence of SEQ ID NO: 146; or

[0129] (32) a first variable region comprising a VH sequence of SEQ ID NO: 140 and a VL sequence of SEQ ID NO: 147, and a second variable region comprising a VH sequence of SEQ ID NO: 141 and a VL sequence of SEQ ID NO: 147.

[0130]

[23] The anti-CTLA-4 antibody for use according to any one of [1] to

[22] , wherein the anti-CTLA4 antibody is a full-length IgG1 antibody.

[0131]

[24] The anti-CTLA-4 antibody for use of any one of [1] to

[23] , wherein the variant Fc region further comprises an amino acid alteration at position 332 according to EU numbering in the first polypeptide of the parent Fc region.

[0132]

[25] The anti-CTLA-4 antibody for use of any one of [1] to

[24] , wherein the variant Fc region further comprises an amino acid alteration at position 332 according to EU numbering in the second polypeptide of the parent Fc region.

[0133]

[26] The anti-CTLA-4 antibody for use of any one of [1] to

[25] , wherein the variant Fc region further comprises an amino acid alteration at position 236 according to EU numbering in the second polypeptide of the parent Fc region.

[0134]

[27] The anti-CTLA-4 antibody for use of any one of [1] to

[26] , wherein the variant Fc region further comprises amino acid changes at positions 250 and 307 according to EU numbering in the first polypeptide of the parent Fc region.

[0135]

[28] The anti-CTLA-4 antibody for use of any one of [1] to

[27] , wherein the variant Fc region further comprises amino acid changes at positions 250 and 307 according to EU numbering in the second polypeptide of the parent Fc region.

[0136]

[29] The anti-CTLA-4 antibody for use of any one of [1] to

[28] , wherein the variant Fc region further comprises at least one amino acid alteration selected from the group consisting of:

[0137] (i) Phe at position 234, Gln at position 235, Trp at position 236, Met at position 239, Val at position 250, Asp at position 268, Glu at position 270, Ala at position 298, Pro at position 307, Met at position 330, Glu at position 332 in the parent Fc region first polypeptide according to EU numbering; and

[0138] (ii) Ala at position 236, Val at position 250, Glu at position 270, Ala at position 298, Pro at position 307, Asp at position 326, Met at position 330, Glu at position 332, and Glu at position 334 in the parent Fc region second polypeptide according to EU numbering.

[0139]

[30] The anti-CTLA-4 antibody for use of any one of [1] to

[29] , wherein the variant Fc region further comprises any one of the following amino acid changes (a) to (f):

[0140] (a) Lys at position 356 by EU numbering in a first parent Fc-region polypeptide and Glu at position 439 by EU numbering in a second parent Fc-region polypeptide,

[0141] (b) Glu at position 439 by EU numbering in the parent Fc-region first polypeptide and Lys at position 356 by EU numbering in the parent Fc-region second polypeptide,

[0142] (c) Trp at position 366 by EU numbering in the parent Fc-region first polypeptide, and Ser at position 366, Ala at position 368, and Val at position 407 by EU numbering in the parent Fc-region second polypeptide,

[0143] (d) Ser at position 366, Ala at position 368, and Val at position 407 according to EU numbering in a first parent Fc-region polypeptide, and Trp at position 366, according to EU numbering in a second parent Fc-region polypeptide,

[0144] (e) Cys at position 349 and Trp at position 366 according to EU numbering in the first parent Fc-region polypeptide, and Cys at position 356, Ser at position 366, Ala at position 368, and Val at position 407 according to EU numbering in the second parent Fc-region polypeptide, and

[0145] (f) Cys at position 356, Ser at position 366, Ala at position 368 and Val at position 407 by EU numbering in the first parent Fc-region polypeptide, and Cys at position 349 and Trp at position 366 by EU numbering in the second parent Fc-region polypeptide.

[0146]

[31] The anti-CTLA-4 antibody for use according to any one of [1] to

[30] , wherein the variant Fc region further comprises an amino acid change of any one of (a) to (d) in the first polypeptide and / or the second polypeptide of the parent Fc region:

[0147] (a) Ala at position 434 according to EU numbering,

[0148] (b) Ala at position 434, Thr at position 436, Arg at position 438, and Glu at position 440, according to EU numbering,

[0149] (c) Leu at position 428, Ala at position 434, Thr at position 436, Arg at position 438, and Glu at position 440, according to EU numbering, and

[0150] (d) Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440 according to EU numbering.

[0151]

[32] The anti-CTLA-4 antibody for use of any one of [1] to

[31] , comprising a heavy chain constant region comprising a variant Fc region.

[0152]

[33] The anti-CTLA-4 antibody for use of

[32] , wherein the heavy chain constant region comprises:

[0153] (1) a first polypeptide of SEQ ID NO: 350 and a second polypeptide of SEQ ID NO: 351, or

[0154] (2) a first polypeptide of SEQ ID NO: 343 and a second polypeptide of SEQ ID NO: 344.

[0155]

[34] An anti-CTLA-4 antibody for treating tumors, wherein the anti-CTLA-4 antibody comprises:

[0156] (1) the first H chain polypeptide of SEQ ID NO: 352, the second H chain polypeptide of SEQ ID NO: 353, and the L chain polypeptide of SEQ ID NO: 161; or

[0157] (2) The first H chain polypeptide of SEQ ID NO: 335, the second H chain polypeptide of SEQ ID NO: 336, and the L chain polypeptide of SEQ ID NO: 161.

[0158] In one non-limiting embodiment, the present disclosure provides the following:

[0159]

[101] A polypeptide comprising a variant Fc region comprising an amino acid alteration in a parent Fc region, wherein the parent Fc region is composed of two polypeptide chains, and wherein the variant Fc region comprises an amino acid alteration at:

[0160] (i) positions 234, 235, 236, 239, 268, 270 and 298 according to EU numbering in the first polypeptide of the parent Fc region; and

[0161] (ii) positions 270, 298, 326 and 334 according to EU numbering in the second polypeptide of the parent Fc region.

[0162]

[102] The polypeptide of

[101] , wherein the variant Fc region further comprises an amino acid alteration at position 326 according to EU numbering in the first polypeptide of the parent Fc region.

[0163]

[103] The polypeptide of

[101] or

[102] , wherein the variant Fc region further comprises an amino acid alteration at position 236 according to EU numbering in the second polypeptide of the parent Fc region.

[0164]

[104] A polypeptide comprising a variant Fc region comprising an amino acid alteration in a parent Fc region, wherein the parent Fc region is composed of two polypeptide chains, and wherein the variant Fc region comprises an amino acid alteration at:

[0165] (i) positions 234, 235, 236, 239, 268, 270, 298, and 326 according to EU numbering in the first polypeptide of the parent Fc region; and

[0166] (ii) positions 236, 270, 298, 326 and 334 according to EU numbering in the second polypeptide of the parent Fc region.

[0167]

[105] The polypeptide of any one of

[101] to

[104] , wherein the variant Fc region further comprises an amino acid alteration at position 332 according to EU numbering in the first polypeptide of the parent Fc region.

[0168]

[106] The polypeptide of any one of

[101] to

[105] , wherein the variant Fc region further comprises an amino acid alteration at position 330 according to EU numbering in the first polypeptide of the parent Fc region.

[0169]

[107] The polypeptide of any one of

[101] to

[106] , wherein the variant Fc region further comprises an amino acid alteration at position 332 according to EU numbering in the second polypeptide of the parent Fc region.

[0170]

[108] The polypeptide of any one of

[101] to

[107] , wherein the variant Fc region further comprises an amino acid alteration at position 330 according to EU numbering in the second polypeptide of the parent Fc region.

[0171]

[109] A polypeptide comprising a variant Fc region comprising an amino acid alteration in a parent Fc region, wherein the parent Fc region is composed of two polypeptide chains, and wherein the variant Fc region comprises an amino acid alteration at:

[0172] (i) positions 234, 235, 236, 239, 268, 270, 298, 330, and 332 according to EU numbering in the first polypeptide of the parent Fc region; and

[0173] (ii) positions 236, 270, 298, 326, 330, 332 and 334 according to EU numbering in the second polypeptide of the parent Fc region.

[0174]

[110] The polypeptide of any one of

[101] to

[109] , wherein the variant Fc region further comprises amino acid alterations at positions 250 and 307 according to EU numbering in the first polypeptide of the parent Fc region.

[0175]

[111] The polypeptide of any one of

[101] to

[110] , wherein the variant Fc region further comprises amino acid alterations at positions 250 and 307 according to EU numbering in the second polypeptide of the parent Fc region.

[0176]

[112] A polypeptide comprising a variant Fc region comprising an amino acid alteration in a parent Fc region, wherein the parent Fc region is composed of two polypeptide chains, and wherein the variant Fc region comprises an amino acid alteration at:

[0177] (i) positions 234, 235, 236, 239, 250, 268, 270, 298 and 307 according to EU numbering in the first polypeptide of the parent Fc region; and

[0178] (ii) positions 250, 270, 298, 307, 326 and 334 according to EU numbering in the second polypeptide of the parent Fc region.

[0179]

[113] A polypeptide comprising a variant Fc region comprising an amino acid alteration in a parent Fc region, wherein the parent Fc region is composed of two polypeptide chains, and wherein the variant Fc region comprises an amino acid alteration at:

[0180] (i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, and 326 according to EU numbering in the first polypeptide of the parent Fc region; and

[0181] (ii) positions 236, 250, 270, 298, 307, 326 and 334 according to EU numbering in the second polypeptide of the parent Fc region.

[0182]

[114] A polypeptide comprising a variant Fc region comprising an amino acid alteration in a parent Fc region, wherein the parent Fc region is composed of two polypeptide chains, and wherein the variant Fc region comprises an amino acid alteration at:

[0183] (i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 330, and 332 according to EU numbering in the first polypeptide of the parent Fc region; and

[0184] (ii) positions 236, 250, 270, 298, 307, 326, 330, 332 and 334 according to EU numbering in the second polypeptide of the parent Fc region.

[0185]

[115] The polypeptide of any one of

[101] to

[114] , comprising at least one amino acid change selected from the group consisting of:

[0186] (i) Tyr or Phe at position 234, Gln or Try at position 235, Trp at position 236, Met at position 239, Val at position 250, Asp at position 268, Glu at position 270, Ala at position 298, Pro at position 307, Asp at position 326, Met at position 330, Glu at position 332 in the first polypeptide of the parent Fc region according to EU numbering; and

[0187] (ii) Ala at position 236, Val at position 250, Glu at position 270, Ala at position 298, Pro at position 307, Asp at position 326, Met or Lys at position 330, Asp or Glu at position 332, Glu at position 334 according to EU numbering in the second polypeptide of the parent Fc region.

[0188]

[116] The polypeptide of any one of

[101] to

[115] , wherein the variant Fc region further comprises any one of the following amino acid changes (a) to (f):

[0189] (a) Lys at position 356 according to EU numbering in a first parent Fc region polypeptide, and Glu at position 439 according to EU numbering in a second parent Fc region polypeptide;

[0190] (b) Glu at position 439 according to EU numbering in the first parent Fc region polypeptide, and Lys at position 356 according to EU numbering in the second parent Fc region polypeptide;

[0191] (c) Trp at position 366 according to EU numbering in a first parent Fc region polypeptide, and Ser at position 366, Ala at position 368, and Val at position 407 according to EU numbering in a second parent Fc region polypeptide;

[0192] (d) Ser at position 366 according to EU numbering, Ala at position 368, Val at position 407 in the parent Fc region first polypeptide, Trp at position 366 according to EU numbering in the parent Fc region second polypeptide;

[0193] (e) Cys at position 349 according to EU numbering and Trp at position 366 of the first parent Fc region polypeptide, Cys at position 356 according to EU numbering, Ser at position 366, Ala at position 368 and Val at position 407 of the second parent Fc region polypeptide;

[0194] (f) Cys at position 356 according to EU numbering, Ser at position 366, Ala at position 368 and Val at position 407 of the first parent Fc region polypeptide, and Cys at position 349 and Trp at position 366 according to EU numbering of the second parent Fc region polypeptide.

[0195]

[117] The polypeptide of any one of

[101] to

[116] , wherein the variant Fc region further comprises any one of the following amino acid changes (a) to (d) in the first polypeptide and / or the second polypeptide of the parent Fc region:

[0196] (a) Ala at position 434 according to EU numbering;

[0197] (b) Ala at position 434, Thr at position 436, Arg at position 438, and Glu at position 440 according to EU numbering;

[0198] (c) Leu at position 428, Ala at position 434, Thr at position 436, Arg at position 438, and Glu at position 440, according to EU numbering; and

[0199] (d) Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440 according to EU numbering.

[0200]

[118] The polypeptide of any one of

[101] to

[117] , wherein the variant Fc region has enhanced binding activity to at least one Fcγ receptor selected from FcγRIa, FcγRIIa, FcγRIIb and FcγRIIIa compared to the parent Fc region.

[0201]

[119] The polypeptide of

[118] , wherein the variant Fc region has enhanced binding activity to FcγRIIa and FcγRIIIa compared to the parent Fc region.

[0202]

[120] The polypeptide of any one of

[101] to

[119] , wherein the selectivity between activating Fcγ receptors and inhibitory Fcγ receptors in the variant Fc region is improved compared to the parent Fc region.

[0203] [120-2] The polypeptide of any one of

[101] to

[119] , wherein the binding activity to an activating Fcγ receptor is selectively enhanced in the variant Fc region compared to the binding activity to an inhibitory Fcγ receptor compared to the parent Fc region.

[0204] [120-3] The polypeptide of any one of

[101] to

[119] , wherein the ratio of the binding activity to an activating Fcγ receptor and the binding activity to an inhibitory Fcγ receptor (A / I ratio) of the variant Fc region is higher than that of the parent Fc region.

[0205] [120-4] The polypeptide of [120-3], wherein the ratio (A / I ratio) in the polypeptide comprising the variant Fc region is 1.1 times or more, 1.2 times or more, 1.3 times or more, 1.4 times or more, 1.5 times or more, 1.6 times or more, 1.7 times or more, 1.8 times or more, 1.9 times or more, 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times or more, 7 times or more, 8 times or more, 9 times or more, 10 times or more, 20 times or more, 30 times or more, 40 times or more. times or more, 50 times or more, 60 times or more, 70 times or more, 80 times or more, 90 times or more, 100 times or more, 200 times or more, 300 times or more, 400 times or more, 500 times or more, 600 times or more, 700 times or more, 800 times or more, 900 times or more, 1000 times or more, 2000 times or more, 3000 times or more, 4000 times or more, 5000 times or more, 6000 times or more, 7000 times or more, 8000 times or more, 9000 times or more, or 10000 times or more.

[0206] [120-5] The polypeptide of [120-3], wherein the ratio (A / I ratio) of the polypeptide comprising the variant Fc region is 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 200 or more, 300 or more, 400 or more, 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, 1000 or more, 2000 or more, 3000 or more, 4000 or more, 5000 or more, 6000 or more, 7000 or more, 8000 or more, 9000 or more, 10000 or more, 11000 or more, 12000 or more, 13000 or more, 14000 or more, or 15000 or more.

[0207]

[121] The polypeptide of any one of

[120] to [120-5], wherein the activating Fcγ receptor is at least one Fcγ receptor selected from FcγRIa, FcγRIIa and FcγRIIIa, and wherein the inhibitory Fcγ receptor is FcγRIIb.

[0208]

[122] The polypeptide of any one of

[101] to

[121] , wherein the polypeptide comprising the variant Fc region is an antibody.

[0209]

[123] A method of producing a polypeptide comprising a variant Fc region, the method comprising introducing an amino acid change into a parent Fc region, wherein the parent Fc region is composed of two polypeptide chains, and wherein the amino acid change is introduced at:

[0210] (i) positions 234, 235, 236, 239, 268, 270 and 298 according to EU numbering in the first polypeptide of the parent Fc region; and

[0211] (ii) positions 270, 298, 326 and 334 according to EU numbering in the second polypeptide of the parent Fc region.

[0212]

[124] A method of producing a polypeptide comprising a variant Fc region, the method comprising introducing an amino acid change into a parent Fc region, wherein the parent Fc region is composed of two polypeptide chains, and wherein the amino acid change is introduced at:

[0213] (i) positions 234, 235, 236, 239, 268, 270, 298 and 326 according to EU numbering in the first polypeptide of the parent Fc region; and

[0214] (ii) positions 236, 270, 298, 326 and 334 according to EU numbering in the second polypeptide of the parent Fc region.

[0215]

[125] A method of producing a polypeptide comprising a variant Fc region, the method comprising introducing an amino acid change into a parent Fc region, wherein the parent Fc region is composed of two polypeptide chains, and wherein the amino acid change is introduced at:

[0216] (i) positions 234, 235, 236, 239, 268, 270, 298, 330, and 332 according to EU numbering in the first polypeptide of the parent Fc region; and

[0217] (ii) positions 236, 270, 298, 326, 330, 332 and 334 according to EU numbering in the second polypeptide of the parent Fc region.

[0218]

[126] An isolated nucleic acid encoding the polypeptide of any one of

[101] to

[122] .

[0219]

[127] A host cell comprising the nucleic acid of

[126] .

[0220]

[128] A method for producing a polypeptide, comprising culturing the host cell of

[127] so as to produce the polypeptide.

[0221]

[129] The polypeptide of any one of

[101] to

[122] , wherein the polypeptide is used to treat tumors.

[0222]

[130] The polypeptide of any one of

[101] to

[122] , wherein the polypeptide is used to damage cells.

[0223]

[131] The polypeptide of

[130] , wherein the cell is damaged by ADCC activity, CDC activity or ADCP activity.

[0224]

[132] A pharmaceutical composition comprising the polypeptide of any one of

[101] to

[122] and a pharmaceutically acceptable carrier.

[0225]

[133] The pharmaceutical composition of

[132] is a pharmaceutical composition for treating tumors.

[0226]

[134] The pharmaceutical composition of

[132] is a pharmaceutical composition for damaging cells.

[0227]

[135] The pharmaceutical composition of

[134] , wherein the cells are damaged by ADCC activity, CDC activity or ADCP activity.

[0228]

[136] A method for treating tumors, comprising administering the polypeptide of any one of

[101] to

[122] or the pharmaceutical composition of

[132] .

[0229]

[137] A method for damaging cells, comprising administering the polypeptide of any one of

[101] to

[122] or the pharmaceutical composition of

[132] .

[0230]

[138] The method of

[137] , wherein the cells are damaged by ADCC activity, CDC activity or ADCP activity.

[0231]

[139] Use of the polypeptide of any one of

[101] to

[122] in the preparation of an agent for treating tumors.

[0232]

[140] Use of the polypeptide of any one of

[101] to

[122] in the preparation of an agent for damaging cells.

[0233]

[141] The use of

[140] , wherein the cells are damaged by ADCC activity, CDC activity or ADCP activity.

[0234]

[142] A method for modifying the function of a polypeptide comprising an Fc region, comprising introducing an amino acid change into a parent Fc region, wherein the parent Fc region is composed of two polypeptide chains, and the amino acid change is introduced at the following positions:

[0235] (i) positions 234, 235, 236, 239, 268, 270 and 298 according to EU numbering in the first polypeptide of the parent Fc region; and

[0236] (ii) positions 270, 298, 326 and 334 according to EU numbering in the second polypeptide of the parent Fc region.

[0237]

[143] A method for modifying the function of a polypeptide comprising an Fc region, comprising introducing an amino acid change into a parent Fc region, wherein the parent Fc region is composed of two polypeptide chains, and the amino acid is introduced at the following positions:

[0238] (i) positions 234, 235, 236, 239, 268, 270, 298 and 326 according to EU numbering in the first polypeptide of the parent Fc region; and

[0239] (ii) positions 236, 270, 298, 326 and 334 according to EU numbering in the second polypeptide of the parent Fc region.

[0240]

[144] A method for modifying the function of a polypeptide comprising an Fc region, comprising introducing an amino acid change into a parent Fc region, wherein the parent Fc region is composed of two polypeptide chains, and the amino acid is introduced at the following positions:

[0241] (i) positions 234, 235, 236, 239, 268, 270, 298, 330, and 332 according to EU numbering in the first polypeptide of the parent Fc region; and

[0242] (ii) positions 236, 270, 298, 326, 330, 332 and 334 according to EU numbering in the second polypeptide of the parent Fc region.

[0243]

[145] The method of any one of

[142] to

[144] , wherein the functional modification is enhancement of binding activity to FcγRIIa and FcγRIIIa.

[0244]

[146] The method of any one of

[142] to

[144] , wherein the modification of function is an increase in selectivity between activating Fcγ receptors and inhibitory Fcγ receptors.

[0245]

[147] The method of any one of

[142] to

[144] , wherein the functional modification is an increase in the selectivity of the binding activity to an activating Fcγ receptor compared to the binding activity to an inhibitory Fcγ receptor.

[0246]

[148] The method of any one of

[142] to

[144] , wherein the modification of function is an increase in the ratio of binding activity to an activating Fcγ receptor to binding activity to an inhibitory Fcγ receptor (A / I ratio).

[0247]

[149] The method of

[148] , wherein the ratio (A / I ratio) is increased by 1.1-fold or more, 1.2-fold or more, 1.3-fold or more, 1.4-fold or more, 1.5-fold or more, 1.6-fold or more, 1.7-fold or more, 1.8-fold or more, 1.9-fold or more, 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, 6-fold or more, 7-fold or more, 8-fold or more, 9-fold or more, 10-fold or more, 20-fold or more, 30-fold or more, 40-fold or more, 50-fold or more compared to a polypeptide comprising a parent Fc region. , 60 times or more, 70 times or more, 80 times or more, 90 times or more, 100 times or more, 200 times or more, 300 times or more, 400 times or more, 500 times or more, 600 times or more, 700 times or more, 800 times or more, 900 times or more, 1000 times or more, 2000 times or more, 3000 times or more, 4000 times or more, 5000 times or more, 6000 times or more, 7000 times or more, 8000 times or more, 9000 times or more or 10000 times or more.

[0248]

[150] The method of any one of

[142] to

[149] , wherein the activating Fcγ receptor is at least one Fcγ receptor selected from FcγRIa, FcγRIIa and FcγRIIIa, and wherein the inhibitory Fcγ receptor is FcγRIIb.

[0249]

[151] The method of any one of

[142] to

[144] , wherein the modification of function is enhancement of ADCC activity, CDC activity or ADCP activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0250] [ Figure 1 ] Figure 1The anti-tumor effects of the anti-CTLA-4 antibodies mNS-mFa55 (control antibody) and SW1208-mFa55 (switch antibody) in a mouse model transplanted with the CT26 cell line (colon cancer), as described in Example 2-1-5. The control antibody was administered via the tail vein at 1 mg / kg, 2 mg / kg, 4 mg / kg, and 8 mg / kg, while the switch antibody was administered at 30 mg / kg. Each point represents the mean tumor volume for one group (n = 4).

[0251] [ Figure 2 ] Figure 2 The anti-tumor effects of the anti-CTLA-4 antibodies mNS-mFa55 (control antibody) and SW1208-mFa55 (switch antibody) in a mouse model transplanted with the MBT2 cell line (bladder cancer), as described in Example 2-2-4. The control antibody was administered at 1 mg / kg, while the switch antibody was administered at 30 mg / kg via the tail vein. Each point represents the mean tumor volume for one group (n = 5).

[0252] [ Figure 3 ] Figure 3 The anti-tumor effect of the anti-CTLA-4 antibody hNS-mFa55 (control antibody) in a mouse model transplanted with the Colon38 cell line (colon cancer), as described in Example 2-3-5, is shown. The control antibody was administered via the tail vein at 0.3 mg / kg, 1 mg / kg, and 10 mg / kg. Each point represents the mean tumor volume for one group (n = 4).

[0253] [ Figure 4 ] Figure 4 The anti-tumor effect of the anti-CTLA-4 antibody SW1610-mFa55 (switch antibody) in a mouse model transplanted with the Colon38 cell line (colon cancer), as described in Example 2-3-5, is shown. The switch antibody was administered via the tail vein at 0.3 mg / kg, 1 mg / kg, and 10 mg / kg. Each point represents the mean tumor volume for one group (n = 4).

[0254] [ Figure 5 ] Figure 5 The anti-tumor effect of the anti-CTLA-4 antibody SW1612-mFa55 (switch antibody) in a mouse model transplanted with the Colon38 cell line (colon cancer), as described in Example 2-3-5, is shown. The switch antibody was administered via the tail vein at 0.3 mg / kg, 1 mg / kg, and 10 mg / kg. Each point represents the mean tumor volume for one group (n = 4).

[0255] [ Figure 6 ] Figure 6The anti-tumor effects of the anti-CTLA-4 antibodies hNS-mFa55 (control antibody) and SW1610-mFa55 and SW1389-mFa55 (switch antibodies) in a mouse model transplanted with the A755 cell line (breast cancer), as described in Example 2-4-5. The control antibody was administered at 1 mg / kg, while the switch antibodies were each administered at 30 mg / kg via the tail vein. Each point represents the mean tumor volume for one group (n = 4).

[0256] [ Figure 7 ] Figure 7 The anti-tumor effects of the anti-CTLA-4 antibodies hNS-mFa55 (control antibody) and SW1610-mFa55 and SW1389-mFa55 (switch antibodies) in a mouse model transplanted with the B16F1 / OVA cell line (melanoma), as described in Example 2-5-5. The control antibody was administered at 10 mg / kg, while the switch antibodies were each administered at 100 mg / kg via the tail vein. Each point represents the mean tumor volume for one group (n = 4).

[0257] [ Figure 8 ] Figure 8 The Treg depletion activity of ipilimumab, SW1610-ART12, and SW1389-ART5+ACT1 (switch antibody) on PBMCs from colorectal cancer patients is shown, as described in Example 3. Each antibody was used at 10 μg / mL or 50 μg / mL. Each point represents a sample from a different patient, and the horizontal line represents the mean.

[0258] [ Figure 9 ] Figure 9 The Treg depletion activity of ipilimumab, SW1610-ART12, and SW1389-ART5+ACT1 (switch antibody) on PBMCs from patients with head and neck cancer is shown, as described in Example 3. Each antibody was used at 10 μg / mL or 50 μg / mL. Each point represents a sample from a different patient, and the horizontal line represents the mean.

[0259] [ Figure 10 ] Figure 10 The Treg depletion activity of ipilimumab, SW1610-ART12, and SW1389-ART5+ACT1 (switch antibodies) on PBMCs from pancreatic cancer patients is shown, as described in Example 3. Each antibody was used at 10 μg / mL or 50 μg / mL. Each point represents a sample from a different patient, and the horizontal line represents the mean.

[0260] [ Figure 11 ] Figure 11The Treg depletion activity of ipilimumab, SW1610-ART12, and SW1389-ART5+ACT1 (switch antibody) on PBMCs from patients with renal cancer is shown, as described in Example 3. Each antibody was used at 10 μg / mL or 50 μg / mL. Each point represents a sample from a different patient, and the horizontal line represents the mean.

[0261] [ Figure 12 ] Figure 12 Shown are the Treg depletion activities of ipilimumab, SW1610-ART12, and SW1389-ART5+ACT1 (switch antibodies) on PBMCs from lung cancer patients, as described in Example 3. Each antibody was used at 10 μg / mL or 50 μg / mL.

[0262] [ Figure 13 ] Figure 13 The anti-tumor effect of the combination of anti-CTLA-4 antibody SW1610-mFa55 (switch antibody) and anti-PD-L1 antibody in a mouse model transplanted with the Colon38 cell line (colon cancer), as described in Example 5, is shown. The switch antibody was administered via the tail vein at 0.3 mg / kg on day 14 after transplantation, while the anti-PD-L1 antibody was administered at 10 mg / kg on days 14, 17, and 20 after transplantation. Each point represents the mean tumor volume of one group (n=5).

[0263] [ Figure 14 ] Figure 14 The anti-tumor effect of the combination of anti-CTLA-4 antibody SW1389-mFa55 (switch antibody) and anti-PD-L1 antibody in a mouse model transplanted with the Colon38 cell line (colon cancer), as described in Example 5, is shown. The switch antibody was administered at 1 mg / kg on day 14 after transplantation, while the anti-PD-L1 antibody was administered at 10 mg / kg on days 14 and 17 after transplantation via the tail vein. Each point represents the mean tumor volume of one group (n=5).

[0264] [ Figure 15 ] Figure 15 The binding activity of the anti-CTLA-4 antibody ABAM004 to CTLA-4 was shown, and the binding activity depended on the concentration of ATP, ADP, or AMP, as described in Reference Examples 1-9.

[0265] [ Figure 16 ] Figure 16 The binding activity of the anti-CTLA-4 antibody ABAM004 to cells expressing CTLA-4 was shown, and the binding activity was dependent on the concentration of AMP, as described in Reference Examples 1-10.

[0266] [ Figure 17 ] Figure 17 ADCC activity of the anti-CTLA-4 antibody ABAM004 against cells expressing CTLA-4 in the presence and absence of AMP is shown, as described in Reference Examples 1-11.

[0267] [ Figure 18 ] Figure 18 The binding pattern of the ABAM004 Fab fragment to AMP is shown, as described in Reference Examples 2-13. In the figure, the heavy chain of the antibody is shown in black, the light chain is shown in gray, and AMP is shown as a ball-and-stick model. The amino acid residues that interact with AMP are shown as stick models. The dashed lines and their values ​​represent the distance (Å) between each amino acid residue and AMP.

[0268] [ Figure 19 ] Figure 19 The binding patterns of the ABAM004 Fab fragment and AMP to human CTLA4 (hCTLA4) are shown, as described in Reference Examples 2-14. In the figure, the heavy chain of the antibody is depicted in black, the light chain in gray, hCTLA4 in white, and the AMP in a ball-and-stick model. Amino acid residues in hCTLA4 containing one or more non-hydrogen atoms within 4.2 Å of any portion of the antibody or AMP are considered epitopes and are depicted in stick models.

[0269] [ Figure 20 ] Figure 20 Mapping of the epitope of the ABAM004 Fab fragment within the hCTLA4 amino acid sequence is shown, as described in Reference Example 2-14. In the figure, the amino acid residues shown in black are those of hCTLA4 that contain one or more non-hydrogen atoms that are located within 4.2 Å of any portion of ABAM004 or AMP in the crystal structure. The amino acid residues shown in gray indicate residues for which the model was not constructed because they are disordered in the crystal structure.

[0270] [ Figure 21 ] Figure 21 This figure shows an overlay of antibody and AMP structures extracted from the crystal structures of the ABAM004 Fab fragment alone, the complex of the ABAM004 Fab fragment and AMP, and the ternary complex of the ABAM004 Fab fragment, AMP, and CTLA4, as described in Reference Examples 2-15. In the figure, the heavy chain of the antibody is shown in black, the light chain in gray, and the AMP is a ball-and-stick model. The structure of the ABAM004 Fab fragment alone is shown with a thin line, the structure of the binary complex with AMP is shown with a medium-bold line, and the structure of the ternary complex is shown with a bold line.

[0271] [ Figure 22 ] Figure 22The CTLA-4 binding activity of the anti-CTLA-4 antibody ABAM004 and its variant 04H0150 / 04L0072 is shown, and the binding activity is dependent on the concentration of ATP, ADP, or AMP, as described in Reference Example 3-2. As annotated in the figure, WT and H150L072 represent ABAM004 and 04H0150 / 04L0072, respectively.

[0272] [ Figure 23 ] Figure 23 The neutralizing activity of the anti-CTLA-4 antibody SW1077 against CTLA-4 was shown to be dependent on the concentration of ATP, as described in Reference Examples 3-6.

[0273] [ Figure 24 ] Figure 24 This figure shows the anti-tumor effect of the anti-CTLA-4 antibody mNS-mFa55 (control antibody) in a mouse model transplanted with the FM3A cell line, as described in Reference Example 3-7-4. The antibody was administered via the tail vein at doses of 0.01 mg / kg, 0.1 mg / kg, 0.25 mg / kg, 1 mg / kg, 10 mg / kg, 30 mg / kg, and 100 mg / kg. Each point represents the mean tumor volume for one group, n = 4.

[0274] [ Figure 25 ] Figure 25 This figure shows the anti-tumor effect of the anti-CTLA-4 antibody SW1208-mFa55 (switch antibody) in a mouse model transplanted with the FM3A cell line, as described in Reference Example 3-7-4. The antibody was administered via the tail vein at doses of 0.1 mg / kg, 1 mg / kg, 10 mg / kg, 100 mg / kg, and 500 mg / kg. Each point represents the mean tumor volume for one group (n = 4).

[0275] [ Figure 26 ] Figure 26 The figure shows changes in the ratio of effector Treg cells in tumors when the anti-CTLA-4 antibody mNS-mFa55 (control antibody) or SW1208-mFa55 (switch antibody) is administered in a mouse model transplanted with the FM3A cell line, as described in Reference Example 3-7-7. mNS-mFa55 was administered via the tail vein at 0.1 mg / kg, 1 mg / kg, 10 mg / kg, and 100 mg / kg, and SW1208-mFa55 was administered via the tail vein at 0.1 mg / kg, 1 mg / kg, 10 mg / kg, 100 mg / kg, and 500 mg / kg. Tumors were harvested six days after administration, and the increase or decrease in effector Tregs was assessed by FACS analysis. The vertical axis represents effector Tregs (CD4 + FoxP3+ KLRG1 + ) and CD45 + The ratio of cells. The average value of n = 3 is shown.

[0276] [ Figure 27 ] Figure 27 The figure shows changes in the ratio of activated helper T cells in the spleen when the anti-CTLA-4 antibody mNS-mFa55 (control antibody) or SW1208-mFa55 (switch antibody) is administered to a mouse model transplanted with the FM3A cell line, as described in Reference Example 3-7-8. mNS-mFa55 was administered via the tail vein at 0.1 mg / kg, 1 mg / kg, 10 mg / kg, and 100 mg / kg, and SW1208-mFa55 was administered via the tail vein at 0.1 mg / kg, 1 mg / kg, 10 mg / kg, 100 mg / kg, and 500 mg / kg. Spleens were collected six days after administration, and the increase or decrease in activated helper T cells was evaluated by FACS analysis. The vertical axis is the number of activated helper T cells (CD4 + Foxp3 - ICOS + ) and CD45 + The ratio of cells is shown. The mean values ​​of n = 3 are shown.

[0277] [ Figure 28 ] Figure 28 This figure shows the anti-tumor effect of the anti-CTLA-4 antibody SW1389-mFa55 (switch antibody) in a mouse model transplanted with the Hepa1-6 / hGPC3 cell line, as described in Reference Example 4-3-5. The antibody was administered via the tail vein at 0.1 mg / kg, 1 mg / kg, 10 mg / kg, and 100 mg / kg. Each point represents the mean tumor volume of a group (n = 4).

[0278] [ Figure 29 ] Figure 29 The anti-tumor effect of the anti-CTLA-4 antibody hNS-mFa55 (control antibody) in a mouse model transplanted with the Hepa1-6 / hGPC3 cell line is shown, as described in Reference Example 4-3-5. The antibody was administered via the tail vein at 0.1 mg / kg, 1 mg / kg, 10 mg / kg, and 30 mg / kg. Each point represents the mean tumor volume of a group, n = 4.

[0279] [ Figure 30 ] Figure 30The figure shows changes in the ratio of effector Treg cells in tumors when the anti-CTLA-4 antibody hNS-mFa55 (control antibody) or SW1389-mFa55 (switch antibody) is administered in a mouse model transplanted with the Hepa1-6 / hGPC3 cell line, as described in Reference Example 4-3-8. hNS-mFa55 was administered at 0.1 mg / kg, 1 mg / kg, 10 mg / kg, and 30 mg / kg, and SW1389-mFa55 was administered at 0.1 mg / kg, 1 mg / kg, 10 mg / kg, 100 mg / kg, and 500 mg / kg via the tail vein. Tumors were harvested six days after administration, and the increase or decrease in effector Tregs was assessed by FACS analysis. The vertical axis represents the effector Tregs (CD4 + FoxP3 + CCR7 low KLRG1 + ) and CD45 + The ratio of cells. The average value of n = 3 is shown.

[0280] [ Figure 31 ] Figure 31 The figure shows changes in the ratio of activated helper T cells in the spleen when the anti-CTLA-4 antibody hNS-mFa55 (control antibody) or SW1389-mFa55 (switch antibody) was administered to a mouse model transplanted with the Hepa1-6 / hGPC3 cell line, as described in Reference Example 4-3-9. hNS-mFa55 was administered via the tail vein at 0.1 mg / kg, 1 mg / kg, 10 mg / kg, and 30 mg / kg, and SW1389-mFa55 was administered at 0.1 mg / kg, 1 mg / kg, 10 mg / kg, 100 mg / kg, and 500 mg / kg. Spleens were harvested six days after administration, and the increase or decrease in activated helper T cells was evaluated by FACS analysis. The vertical axis represents the number of activated helper T cells (CD4 + Foxp3 - ICOS + ) and CD45 + The ratio of cells. The average value of n = 3 is shown.

[0281] [ Figure 32 ] Figure 32 This figure shows the anti-tumor effect of the anti-CTLA-4 antibody SW1610-mFa55 (switch antibody) in a mouse model transplanted with the Hepa1-6 / hGPC3 cell line, as described in Reference Example 5-4-5. The antibody was administered via the tail vein at doses of 0.3 mg / kg, 1 mg / kg, and 3 mg / kg. Each point represents the mean tumor volume for one group (n = 5).

[0282] [ Figure 33 ] Figure 33 This figure shows the anti-tumor effect of the anti-CTLA-4 antibody SW1612-mFa55 (switch antibody) in a mouse model transplanted with the Hepa1-6 / hGPC3 cell line, as described in Reference Example 5-4-5. The antibody was administered via the tail vein at doses of 0.3 mg / kg, 1 mg / kg, and 3 mg / kg. Each point represents the mean tumor volume for one group (n = 5).

[0283] [ Figure 34 ] Figure 34 This figure shows the anti-tumor effect of the anti-CTLA-4 antibody SW1615-mFa55 (switch antibody) in a mouse model transplanted with the Hepa1-6 / hGPC3 cell line, as described in Reference Example 5-4-5. The antibody was administered via the tail vein at doses of 0.3 mg / kg, 1 mg / kg, and 3 mg / kg. Each point represents the mean tumor volume for one group (n = 5).

[0284] [ Figure 35 ] Figure 35 This study shows changes in the ratio of effector Treg cells in tumors following administration of the anti-CTLA-4 antibodies SW1610-mFa55, SW1612-mFa55, or SW1615-mFa55 (all switch antibodies) in a mouse model transplanted with the Hepa1-6 / hGPC3 cell line, as described in Reference Example 5-4-8. SW1610-mFa55 was administered via the tail vein at 50 mg / kg, 100 mg / kg, and 200 mg / kg; SW1612-mFa55 was administered at 50 mg / kg, 100 mg / kg, and 200 mg / kg; SW1615-mFa55 was administered at 50 mg / kg, 100 mg / kg, 200 mg / kg, and 400 mg / kg; and the negative control antibody KLH-mFa55 was administered at 400 mg / kg. Tumors were harvested six days after administration, and the increase or decrease in effector Treg cells was assessed by FACS analysis. The vertical axis is the effector Treg (CD4 + FoxP3 + CCR7 low KLRG1 + ) and CD45 + The ratio of cells. The average value of n = 3 is shown.

[0285] [ Figure 36 ] Figure 36This figure shows changes in the ratio of activated helper T cells in the spleen following administration of the anti-CTLA-4 antibodies SW1610-mFa55, SW1612-mFa55, or SW1615-mFa55 (all switch antibodies) in a mouse model transplanted with the Hepa1-6 / hGPC3 cell line, as described in Reference Example 5-4-9. SW1610-mFa55 was administered via the tail vein at 50 mg / kg, 100 mg / kg, and 200 mg / kg; SW1612-mFa55 was administered at 50 mg / kg, 100 mg / kg, and 200 mg / kg; SW1615-mFa55 was administered at 50 mg / kg, 100 mg / kg, 200 mg / kg, and 400 mg / kg; and the negative control antibody KLH-mFa55 was administered at 400 mg / kg. Spleens were harvested six days after administration, and increases or decreases in activated helper T cells were assessed by FACS analysis. The vertical axis is activated helper T cells (CD4 + Foxp3 - ICOS + ) and CD45 + The ratio of cells. The average value of n = 3 is shown.

[0286] [ Figure 37 ] Figure 37 Comparison of in vitro ADCC activity of antibodies with various altered constant regions, which have enhanced FcγR binding, as described in Reference Example 6-2, is shown. As annotated in the figure, IgG1 represents MDX10D1H-G1m / MDX10D1L-k0MT, GASDALIE represents MDX10D1H-GASDALIE / MDX10D1L-k0MT, ART6 represents MDX10D1H-Kn462 / MDX10D1H-H1445 / MDX10D1L-k0MT, and ART8 represents MDX10D1H-Kn461 / MDX10D1H-H1443 / MDX10D1L-k0MT. Here, IgG1 is an antibody with a control constant region, GASDALIE is an antibody with a constant region described in prior art literature, and ART6 and ART8 are antibodies with altered constant regions generated in Reference Example 6-1.

[0287] [ Figure 38 ] Figure 38Comparison of in vitro ADCP activity of antibodies with various altered constant regions, which have enhanced FcγR binding, as described in Reference Example 6-3, is shown. As annotated in the figure, IgG1 represents MDX10D1H-G1m / MDX10D1L-k0MT, GASDIE represents MDX10D1H-GASDIE / MDX10D1L-k0MT, ART6 represents MDX10D1H-Kn462 / MDX10D1H-H1445 / MDX10D1L-k0MT, and ART8 represents MDX10D1H-Kn461 / MDX10D1H-H1443 / MDX10D1L-k0MT. Here, IgG1 is an antibody with a control constant region, GASDIE is an antibody with a constant region described in prior art literature, and ART6 and ART8 are antibodies with altered constant regions generated in Reference Example 6-1.

[0288] [ Figure 39 ] Figure 39 The in vitro ADCC activity of the anti-CTLA4 switch antibody SW1389-ART6 having an altered constant region and enhanced binding to FcγR was shown, as described in Reference Example 6-4.

[0289] [ Figure 40 ] Figure 40 The in vitro ADCC activity of the anti-CTLA4 switch antibody SW1610-ART6 having an altered constant region and enhanced binding to FcγR was shown, as described in Reference Example 6-4.

[0290] [ Figure 41 ] Figure 41 The in vitro ADCC activity of the anti-CTLA4 switch antibody SW1612-ART6 having an altered constant region and enhanced binding to FcγR was shown, as described in Reference Example 6-4.

[0291] [ Figure 42 ] Figure 42 The neutralizing activity of the anti-CTLA4 switch antibody SW1389 against CTLA4 (the activity of eliminating activated CTLA4 signals that act on effector cells in an inhibitory manner) was shown, as described in Reference Example 6-5.

[0292] [ Figure 43 ] Figure 43 The neutralizing activity of the anti-CTLA4 switch antibody SW1610 against CTLA4 (the activity of eliminating activated CTLA4 signals that act on effector cells in an inhibitory manner) was shown, as described in Reference Example 6-5.

[0293] [ Figure 44 ] Figure 44The neutralizing activity of the anti-CTLA4 switch antibody SW1612 against CTLA4 (the activity of eliminating activated CTLA4 signals that act on effector cells in an inhibitory manner) was shown, as described in Reference Example 6-5.

[0294] [ Figure 45 ] Figure 45 The neutralizing activity of the anti-CTLA4 switch antibody SW1615 on CTLA4 (the activity of eliminating the activated CTLA4 signal that acts on effector cells in an inhibitory manner) was shown, as described in Reference Example 6-5.

[0295] [ Figure 46 ] Figure 46 The in vitro cytotoxic activity of the anti-CTLA4 switch antibody SW1389-ART5+ACT1 against CTLA4-positive regulatory T cells was shown, as described in Reference Example 6-6.

[0296] [ Figure 47 ] Figure 47 The in vitro cytotoxic activity of the anti-CTLA4 switch antibody SW1389-ART6+ACT1 against CTLA4-positive regulatory T cells was shown, as described in Reference Example 6-6.

[0297] [ Figure 48 ] Figure 48 The in vitro cytotoxic activity of the anti-CTLA4 switch antibody SW1610-ART5+ACT1 against CTLA4-positive regulatory T cells was shown, as described in Reference Example 6-6.

[0298] [ Figure 49 ] Figure 49 The in vitro cytotoxic activity of the anti-CTLA4 switch antibody SW1610-ART6+ACT1 against CTLA4-positive regulatory T cells was shown, as described in Reference Example 6-6.

[0299] [ Figure 50 ] Figure 50 The results of an ADCC reporter gene assay are shown, in which Hepa1-6 / hEREG cells were used as target cells and Jurkat cells expressing hFcγRIIIaV were used as effector cells, as described in Reference Example 9-2. Each point represents the average fold induction value, n=2.

[0300] [ Figure 51 ] Figure 51 The results of an ADCC reporter gene assay are shown, in which Hepa1-6 / hEREG cells were used as target cells and hFcγRIIaH-expressing Jurkat cells were used as effector cells, as described in Reference Example 10. Each point represents the average fold induction value, n=3.

[0301] [ Figure 52 ] Figure 52 The anti-tumor effects of EGL-G1d, EGL-afucosylated, and EGL-ART6 in a human FcγR transgenic mouse model transplanted with the Hepa1-6 / hEREG cell line, as described in Reference Example 11-5, are shown. Antibodies were administered via the tail vein at 10 mg / kg. Each point represents the mean tumor volume for one group, n = 5.

[0302] [ Figure 53 ] Figure 53 The hC1q binding activity of each antibody having a modified Fc region is shown, as described in Reference Example 12. Each point represents the average ELISA color development value, n=2.

[0303] [ Figure 54 ] Figure 54 Also shown is the hC1q binding activity of each antibody having a modified Fc region, as described in Reference Example 12. Each point represents the average ELISA color development value, n=2.

[0304] Implementation Plan Description

[0305] The techniques and procedures described or referenced herein are generally well understood by those skilled in the art and are routinely used using conventional methodology, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd ed. (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (F. M. Ausubel, et al., eds. (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M. J. MacPherson, B. D. Hames, and G. R. Taylor, eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (R. I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1998) Academic Press, Inc. Press; Animal Cell Culture (RI Freshney, eds., 1987); Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JB Griffiths, and DG Newell, eds., 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MPCalos, ed., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, ed., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (VT DeVita et al., eds., JB Lippincott Company, 1993).

[0306] I. Definitions

[0307] Unless otherwise defined, technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which the invention pertains. Singleton et al., Dictionary of Microbiology and Molecular Biology, 2nd edition, J. Wiley & Sons (New York, NY 1994), and March, Advanced Organic Chemistry Reactions, Mechanisms and Structure, 4th edition, John Wiley & Sons (New York, NY 1992) provide those skilled in the art with a general guide to many of the terms used in this application. All references cited herein, including patent applications and publications, are incorporated herein by reference in their entirety.

[0308] For the purposes of interpreting this specification, the following definitions apply, and whenever appropriate, terms used in the singular also include the plural, and vice versa. It should be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting. If any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth below shall prevail.

[0309] As used herein, the term "and / or" refers to each item or any combination of items written before and after the term "and / or." For example, the phrase "A, B, and / or C" includes the individual items "A," "B," and "C," as well as any combination selected from "A and B," "A and C," "B and C," and "A and B and C."

[0310] For the purposes of this article, an "acceptor human framework" is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework can comprise the same amino acid sequence thereof, or it can comprise amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0311] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted immunoglobulins that bind to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., NK cells, neutrophils, and macrophages) enable 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 that mediate ADCC, express only FcγRIII, while 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 ADCC activity of a molecule of interest, an in vitro ADCC assay, such as those described in U.S. Pat. Nos. 5,500,362 or 5,821,337 or U.S. Pat. No. 6,737,056 (Presta), can be performed. Useful effector cells for such assays include PBMCs and NK cells. Alternatively or additionally, ADCC activity of the target molecule can be assessed in vivo, for example, in an animal model such as that disclosed in Clynes et al. PNAS (USA) 95: 652-656 (1998).

[0312] "Cytotoxic activity" includes, for example, antibody-dependent cell-mediated cytotoxicity (ADCC) activity as described above, complement-dependent cytotoxicity (CDC) activity as described below, and T cell-mediated cytotoxicity. CDC activity refers to the cytotoxic activity of the complement system. On the other hand, ADCC activity refers to an activity in which an antibody binds to an antigen present on the cell surface of a target cell, and effector cells further bind to the antibody, thereby damaging the target cell. Whether an antibody of interest has ADCC activity and whether an antibody of interest has CDC activity can be determined by known methods (e.g., Current Protocols in Immunology, Chapter 7, Immunologic Studies in Humans, edited by Coligan et al. (1993)).

[0313] "Neutralizing activity" refers to the ability of an antibody to inhibit a biological activity by binding to a molecule involved in the biological activity. In some embodiments, the biological activity is caused by binding between a ligand and a receptor. In certain embodiments, the antibody inhibits the binding between a ligand and a receptor by binding to a ligand or receptor. Antibodies with this neutralizing activity are called neutralizing antibodies. The neutralizing activity of a test substance can be determined by comparing the biological activity of the ligand in the presence and absence of the test substance.

[0314] The term "antibody-dependent cellular phagocytosis" or "ADCP" refers to a process in which all or part of a cell coated with antibodies is incorporated into phagocytic immune cells (eg, macrophages, neutrophils, and dendritic cells) that bind the Fc region of an immunoglobulin.

[0315] The terms "binding activity" and "binding capacity" are used interchangeably herein and refer to the strength of the sum of non-covalent interactions between one or more binding sites (e.g., variable regions or Fc regions) of a molecule (e.g., an antibody or other polypeptide) and its binding partner (e.g., an antigen or an Fcγ receptor). As used herein, "binding activity" is not strictly limited to a 1:1 interaction between members of a binding pair (e.g., an antibody and antigen, or an Fc region and an Fcγ receptor). For example, when the members of a binding pair exhibit a monovalent 1:1 interaction, binding activity refers to intrinsic binding affinity ("avidity"). When the members of a binding pair are capable of both monovalent and multivalent binding, binding activity is the sum of the strengths of each binding. The binding activity of a molecule X for its partner Y can generally be expressed in terms of a dissociation constant (KD) or "amount of analyte bound per unit amount of ligand." Binding activity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding activity are described below.

[0316] An "affinity matured" antibody is one with one or more alterations in one or more hypervariable regions (HVRs) which result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody which does not possess such alterations.

[0317] The terms "anti-CTLA-4 antibody" and "antibody that binds to CTLA-4" refer to an antibody that binds to CTLA-4 with sufficient affinity so that the antibody can be used as a diagnostic and / or therapeutic agent in targeting CTLA-4. In one embodiment, the extent of binding of the anti-CTLA-4 antibody to unrelated, non-CTLA-4 proteins is less than about 10% of the binding of the antibody to CTLA-4 as determined by radioimmunoassay (RIA). In certain embodiments, the antibody that binds to CTLA-4 has a specific affinity of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 -8 M or smaller, for example, from 10 -8 M to 10 -13 M, for example, from 10 -9 M to 10 -13 In certain embodiments, the anti-CTLA-4 antibody binds to an epitope of CTLA-4 that is conserved among CTLA-4 from different species.

[0318] The term "antibody" herein is used in the broadest sense and includes a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0319] An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0320] An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks binding of the reference antibody to its antigen, e.g., by 50% or more, in a competition assay, and / or the reference antibody blocks binding of the antibody to its antigen, e.g., by 50% or more, in a competition assay. Exemplary competition assays are provided herein.

[0321] "Autoimmune disease" refers to a non-malignant disease or condition that is caused by and directed against an individual's own tissues. Autoimmune diseases herein specifically exclude malignant or cancerous diseases or conditions, and especially exclude B-cell lymphomas, acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia, and chronic myeloblastic leukemia. Examples of autoimmune diseases or disorders include, but are not limited to, inflammatory responses, such as inflammatory skin diseases, including psoriasis and dermatitis (e.g., atopic dermatitis); systemic scleroderma and sclerosis; responses associated with inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis); respiratory distress syndrome (including adult respiratory distress syndrome; ARDS); dermatitis; meningitis; encephalitis; uveitis; colitis; glomerulonephritis; allergic diseases, such as eczema and asthma, and other conditions involving T cell infiltration and chronic inflammatory responses; atherosclerosis; leukocyte adhesion defects; rheumatoid arthritis; systemic lupus erythematosus (SLE) (including, but not limited to, lupus nephritis, cutaneous lupus); diabetes (e.g., type 1 diabetes or insulin-dependent diabetes mellitus); multiple sclerosis; Raynaud's syndrome; autoimmune thyroiditis; Hashimoto's thyroiditis; allergic encephalomyelitis; Sjögren's syndrome; juvenile onset diabetes; and autoimmune thyroiditis caused by cytokines and T cells. Immune reactions associated with lymphocyte-mediated acute and delayed hypersensitivity reactions, typically seen in tuberculosis, sarcoidosis, polymyositis, granulomas, and vasculitis; pernicious anemia (Addison's disease); disorders involving leukocytic diapedesis; inflammatory conditions of the central nervous system (CNS); multiple organ injury syndrome; hemolytic anemias (including but not limited to cryoglobulinemia or Coombs-positive anemia); myasthenia gravis; antigen-antibody complex-mediated diseases; anti-glomerular basement membrane disease; antiphospholipid syndrome; allergic neuritis; Graves' disease; Lambert-Eaton myasthenic syndrome; bullous pemphigoid; pemphigus; autoimmune polyendocrinopathy; Rett's disease; stiff-person syndrome; Behçet's disease; giant cell arteritis; immune complex nephritis; IgA nephropathy; IgM polyneuropathy; immune thrombocytopenic purpura (ITP) or autoimmune thrombocytopenia.

[0322] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth / proliferation. Examples of cancer include breast cancer and liver cancer.

[0323] The term "complement-dependent cytotoxicity," or "CDC," refers to a mechanism of cell death in which the Fc effector domain of an antibody bound to its target activates a series of enzymatic reactions that lead to the formation of pores in the target cell's membrane. Typically, the antigen-antibody complex formed on the target cell binds and activates the complement component C1q, which in turn activates the complement cascade and leads to target cell death. Additionally, complement activation may result in the deposition of complement components on the target cell surface, which leads to binding to complement receptors (e.g., CR3) on leukocytes, thereby promoting ADCC.

[0324] "Chemotherapeutic agent" refers to a compound that can be used to treat cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN (registered trademark)); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and hydroxymethylmelamines including hexamethylmelamine, triethylenemelamine, Triethylenephosphamide, triethylenethiophosphamide, and trimethylmelamine; acetogenins (particularly bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL (registered trademark)); beta-lapachone; lapachol; colchicine; betulinic acid; camptothecins (including the synthetic analogs topotecan (HYCAMTIN (registered trademark)), CPT -11 (irinotecan, CAMPTOSAR (registered trademark)), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); podophyllotoxin podophyllinic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycins (including synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin;Nitrogen mustards, such as chlorambucil, naphthyl mustard, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, nitrogen oxide mustard hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicin, in particular calicheamicin gamma 1I and calicheamicin omega 1I (see, for example, Nicolaou et al., Angew. Chem Intl. Ed. Engl., 33: 183-186 (1994); CDP323, an oral α-4 integrin inhibitor; dynemicins, including dynemicin A; esperamicin;and new carcinostaticin chromophores and related chromoprotein enediyne antibiotic chromophores), aclacinomycins, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, carubicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin bicin), 6-diazo-5-oxo-L-norleucine, doxorubicin (including ADRIAMYCIN (registered trademark)), morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin hydrochloride liposome injection (DOXIL (registered trademark)), liposomal doxorubicin TLC D-99 (MYOCET (registered trademark)), pegylated liposomal doxorubicin (CAELYX (registered trademark) and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid acid), nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin;Antimetabolites, such as methotrexate, gemcitabine (GEMZAR(registered trademark)), tegafur (UFTORAL(registered trademark)), capecitabine (XELODA(registered trademark)), epothilone, and 5-fluorouracil (5-FU); folic acid analogs, such as denopterin, methotrexate, pteropterin, and trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, and thiabendazole; iprine), thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens, such as calusterone and dromostanolone propionate. propionate, epitiostanol, mepitiostane, testolactone; antiadreners such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as folinic acid; aceglucuronolide; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; amustine; bisantrene; edatrexate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate acetate); epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitraerine; pentostatin; phenamet; pirarubicin;losoxantrone; 2-ethylhydrazide; procarbazine; PSK (registered trademark) polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2'-trichlorotriethylamine; trichothecenes (particularly T-2 toxin, verracurin A, and baculozolin A) (roridin A and anguidine); urethane; vindesine (ELDISINE (registered trademark), FILDESIN (registered trademark)); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; cytosine; cytarabine ("Ara-C"); thiotepa; taxoids, such as paclitaxel (TAXOL (registered trademark)), albumin-modified nanoparticle formulations of paclitaxel (ABRAXANE; TM) and docetaxel (TAXOTERE (registered trademark)); chlorambucil; 6-thioguanine; mercaptopurine; methotrexate; platinum agents, such as cisplatin, oxaliplatin (e.g., ELOXATIN (registered trademark)), and carboplatin; vincas, which prevent tubulin from polymerizing to form microtubules, including vinblastine (VELBAN (registered trademark)), vincristine (ONCOVIN (registered trademark)), and oxaliplatin (e.g., ELOXATIN (registered trademark)). (Registered Trademark)), vindesine (ELDISINE (Registered Trademark), FILDESIN (Registered Trademark)), and vinorelbine (NAVELBINE (Registered Trademark)); etoposide (VP-16); ifosfamide; mitoxantrone; folinic acid; novantrone; edatrexate; daunomycin; aminopterin; ibandronate; the topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid, including bexarotene (TARGRETIN (Registered Trademark)); bisphosphonates, such as clodronate (e.g., BONEFOS (Registered Trademark) or OSTAC (Registered Trademark)), etidronate (DIDROCAL (Registered Trademark)), NE-58095, zoledronic acid (Registered Trademark)). acid / zoledronate (ZOMETA(registered trademark)), alendronate (FOSAMAX(registered trademark)), pamidronate (AREDIA(registered trademark)), tiludronate (SKELID(registered trademark)), or risedronate (ACTONEL(registered trademark)); troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit the expression of genes in signaling pathways involved in abnormal cell proliferation, such as PKC-α, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines, such as THERATOPE(registered trademark) vaccine, and gene therapy vaccines, such as ALLOVECTIN(registered trademark) vaccine, LEUVECTIN(registered trademark) vaccine, and VAXID(registered trademark) vaccine; topoisomerase 1 inhibitors (e.g., LURTOTECAN(registered trademark)); rmRH (e.g., ABARELIX(registered trademark));BAY439006 (sorafenib; Bayer); SU-11248 (sunitinib, SUTENT (registered trademark), Pfizer); perifosine, COX-2 inhibitors (e.g., celecoxib or etoricoxib), proteosome inhibitors (e.g., PS341); bortezomib (VELCADE (registered trademark)); CCI-779; tipifarnib (R11577); sorafenib, ABT510; Bcl-2 inhibitors, such as oblimersen sodium (GENASEENSE (registered trademark)); pixantrone; EGFR inhibitors (see definition below); tyrosine kinase inhibitors (see definition below); serine-threonine kinase inhibitors, such as rapamycin (sirolimus, RAPAMUNE (registered trademark)); farnesyltransferase inhibitors, such as lonafarnib (SCH 6636, SARASAR; TM ); and pharmaceutically acceptable salts, acids or derivatives of any of the above substances; and combinations of two or more of the above, such as CHOP (abbreviation for cyclophosphamide, doxorubicin, vincristine and prednisolone combined therapy); and FOLFOX (oxaliplatin (ELOXATIN TM ) is an abbreviation for the combined 5-FU and folinic acid treatment regimen.

[0325] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0326] The "class" of an antibody refers to the type of constant domain, or constant region, possessed by its heavy chain. There are five major antibody classes: IgA, IgD, IgE, IgG, and IgM, and some of these are further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0327] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents the function of cells and / or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., 211 At 131 I. 125 I. 90 Y.186 Re、 188 Re、 153 Sm, 212 Bi, 32 P. 212 radioisotopes of Pb and Lu); chemotherapeutic agents or drugs (e.g., methotrexate, doxorubicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof, such as nucleolytic enzymes; antibiotics; toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof; and a variety of chemotherapeutic agents disclosed above.

[0328] "Effector cells" refer to leukocytes that express one or more FcRs and perform effector functions. In certain embodiments, the cells express at least FcγRIII and perform ADCC effector functions. Examples of leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils. Effector cells can be isolated from natural sources, such as blood. In certain embodiments, the effector cells can be human effector cells.

[0329] "Effector functions" refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); antibody-dependent cell-mediated phagocytosis (ADCP); downregulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

[0330] The term "epitope" includes any determinant capable of being bound by an antibody. An epitope is a region of an antigen that is bound by an antibody that targets the antigen, including specific amino acids that directly contact the antibody. Epitope determinants may include chemically active surface groups of molecules, such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and may have specific three-dimensional structural characteristics and / or specific charge characteristics. Typically, antibodies specific for a particular target antigen preferentially recognize epitopes on the target antigen in a complex mixture of proteins and / or macromolecules.

[0331] The term "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. In some embodiments, an FcR is a native human FcR. In some embodiments, an FcR is a receptor that binds to IgG antibodies (gamma receptors) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See, e.g., Daeron, Annu. Rev. Immunol. 15: 203-234 (1997)). FcRs are generally described, for example, in Ravetch and Kinet, Annu. Rev. Immunol 9: 457-492 (1991); Capel et al., Immunomethods 4: 25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126: 330-341 (1995). The term "FcR" herein encompasses additional FcRs, including those to be identified in the future.

[0332] The term "Fc receptor" or "FcR" also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117: 587 (1976); and Kim et al., J. Immunol. 24: 249 (1994)) and the regulation of immunoglobulin homeostasis. Methods for measuring binding to FcRn are known (see, e.g., Ghetie and Ward., Immunol. Today 18(12): 592-598 (1997); Ghetie et al., Nature Biotechnology, 15(7): 637-640 (1997); Hinton et al., J. Biol. Chem. 279(8): 6213-6216 (2004); WO 2004 / 92219 (Hinton et al.)).

[0333] In vivo binding to human FcRn and the serum half-life of high-affinity human FcRn binding polypeptides can be determined, for example, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates to which polypeptides having variant Fc regions are administered. WO 2000 / 42072 (Presta) describes antibody variants with improved or reduced binding to FcRs. See also, for example, Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).

[0334] The term "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain, which comprises at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) or glycine-lysine (Gly446-Lys447) in the Fc region may or may not be present. Unless otherwise indicated herein, the amino acid residues in the Fc region or constant region are numbered according to the EU numbering system, also referred to as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0335] The term "antibody comprising an Fc region" refers to an antibody comprising an Fc region. The C-terminal lysine (residue 447 according to the EU numbering system) or the C-terminal glycine-lysine (residues 446-447) of the Fc region can be removed, for example, during antibody purification or by recombinant engineering of the nucleic acid encoding the antibody. Thus, a composition comprising an antibody having an Fc region according to the present invention can comprise an antibody having G446-K447, an antibody having G446 and not having K447, an antibody with all of G446-K447 removed, or a mixture of the three types of antibodies.

[0336] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of native antibodies (VH and VL, respectively) generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs) (see, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., p. 91 (2007)). A single VH or VL domain can be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen can be isolated by using VH or VL domains from an antigen-binding antibody to screen libraries of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0337] "Framework" or "FR" refers to variable domain residues excluding the hypervariable region (HVR) residues. The FR of a variable domain is generally composed of four FR domains: FR1, FR2, FR3, and FR4. Thus, in a VH (or VL) sequence, the HVR and FR sequences generally appear in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0338] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that comprise an Fc region as defined herein.

[0339] A "functional Fc region" possesses the "effector functions" of a native sequence Fc region. Exemplary "effector functions" include C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc. Such effector functions generally require the Fc region to be associated with a binding domain (e.g., an antibody variable domain) and can be assessed using, for example, the various assays disclosed in the definitions herein.

[0340] A "human antibody" is an antibody having an amino acid sequence corresponding to an antibody produced by a human or human cell or derived from a non-human source utilizing human antibody libraries or other human antibody encoding sequences. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.

[0341] A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Typically, the sequence subgroup is as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, NIH Publication 91-3242, Bethesda MD (1991), volumes 1-3. In one embodiment, for VL, the subgroup is subgroup κI as described in Kabat et al., supra. In one embodiment, for VH, the subgroup is subgroup III as described in Kabat et al., supra.

[0342] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally comprises at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, such as a non-human antibody, refers to an antibody that has undergone humanization.

[0343] As used herein, the term "hypervariable region" or "HVR" refers to each of the regions of an antibody variable domain that are hypervariable in sequence ("complementarity determining regions" or "CDRs") and / or form structurally defined loops ("hypervariable loops") and / or contain antigen-contacting residues ("antigen contacts"). Generally, antibodies comprise six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Exemplary HVRs herein include:

[0344] (a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987));

[0345] (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991));

[0346] (c) antigenic contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al., J. Mol. Biol. 262: 732-745 (1996)); and

[0347] (d) a combination of (a), (b) and / or (c), including HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3) and 94-102 (H3).

[0348] Unless otherwise indicated, HVR residues and other residues in the variable domain (eg, FR residues) are numbered herein according to Kabat et al., supra.

[0349] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, including but not limited to a cytotoxic agent.

[0350] An "isolated" antibody is one that has been separated from components of its natural environment. In some embodiments, the antibody is purified to a purity greater than 95% or 99%, such as by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr, B 848: 79-87 (2007).

[0351] An "isolated" nucleic acid is one that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0352] "Isolated nucleic acid encoding an antibody" refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such nucleic acid molecules in a single vector or separate vectors, and such nucleic acid molecules are present at one or more locations in a host cell.

[0353] The "first polypeptide" and "second polypeptide" herein refer to polypeptides that comprise the Fc region of an antibody. The terms "first polypeptide" and "second polypeptide" mean that their sequences differ from each other, preferably differing in at least the CH2 domain sequence. Furthermore, the CH3 domain sequences may differ. The polypeptides may be, for example, polypeptides that comprise the Fc region of a naturally occurring (native) IgG, or polypeptides produced by altering a polypeptide that comprises the Fc region of a naturally occurring (native) IgG.

[0354] "Native IgG" refers to a polypeptide belonging to a class of antibodies that are actually encoded by immunoglobulin gamma genes and contain the same amino acid sequence as IgG found in nature. For example, natural human IgG refers to natural human IgG1, natural human IgG2, natural human IgG3, natural human IgG4, etc. Natural IgG also includes mutants that arise spontaneously from these.

[0355] "Polypeptides" in the present invention generally refer to peptides or proteins of approximately 10 amino acids or longer. Furthermore, these are generally polypeptides derived from organisms, but are not particularly limited. For example, they may include artificially designed sequences. Furthermore, they may be naturally occurring polypeptides, synthetic polypeptides, or recombinant polypeptides. Protein molecules in the present invention refer to molecules comprising polypeptides.

[0356] Preferred examples of polypeptides of the present invention include antibodies. More preferred examples include natural IgG and antibodies produced by introducing modifications into natural IgG. Examples of natural IgG particularly include natural human IgG. "Native IgG" refers to a polypeptide belonging to a class of antibodies that is actually encoded by the immunoglobulin gamma gene and has an amino acid sequence identical to IgG found in nature. For example, natural human IgG refers to natural human IgG1, natural human IgG2, natural human IgG3, natural human IgG4, etc. Natural IgG also includes spontaneously generated mutants thereof.

[0357] The term "polypeptide comprising an Fc region" is not particularly limited; any polypeptide comprising an Fc region is eligible. For example, an antibody comprising an Fc region. The C-terminal lysine (residue 447 according to the EU numbering system) or the C-terminal glycine-lysine (residues 446-447) of the Fc region can be removed, for example, during purification of the polypeptide (e.g., antibody) or by recombinant engineering of the nucleic acid encoding the polypeptide. Thus, a composition comprising a polypeptide comprising an Fc region according to the present invention may include a polypeptide comprising an Fc region having G446-K447, a polypeptide comprising an Fc region having G446 but not K447, a polypeptide comprising an Fc region with all of G446-K447 removed, or a mixture of the three.

[0358] An "isolated" polypeptide is one that has been separated from components of its natural environment. In some embodiments, the polypeptide is purified to a purity greater than 95% or 99%, such as by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods for assessing polypeptide purity, see, for example, Flatman et al., J. Chromatogr., B 848: 79-87 (2007).

[0359] An "isolated nucleic acid encoding a polypeptide" refers to one or more nucleic acid molecules encoding a polypeptide (such as an antibody Fc region or antibody heavy and light chains or fragments thereof), including such nucleic acid molecules in a single vector or separate vectors, and such nucleic acid molecules are present at one or more locations in a host cell.

[0360] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors." Vectors can be introduced into host cells via viral or electroporation methods. The introduction of vectors is not limited to in vitro introduction; vectors can also be introduced directly in vivo.

[0361] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably to refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, without regard to the number of passages. Progeny need not be identical in nucleic acid content to the parent cell but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0362] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies comprising the population are identical and / or bind to the same epitope, except for possible variant antibodies, which, for example, contain naturally occurring mutations or arise during the production process of the monoclonal antibody preparation, wherein these variants are generally present in small amounts. Compared to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates the characteristic of an antibody obtained from a substantially homogeneous antibody population and should not be interpreted as requiring the antibody to be produced by any particular method. For example, the monoclonal antibodies used in accordance with the present invention can be prepared by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci. Such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0363] A "naked antibody" is an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or a radiolabel. Naked antibodies can be present in pharmaceutical formulations.

[0364] "Natural antibodies" refer to naturally occurring immunoglobulin molecules with different structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, consisting of two identical light chains and two identical heavy chains bonded by disulfide bonds. From N-terminus to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy chain domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from N-terminus to C-terminus, each light chain has a variable region (VL), also called a variable light chain domain or a light chain variable domain, followed by a constant light chain (CL) domain. Based on the amino acid sequence of its constant domain, the light chain of an antibody can be assigned to one of two types, called kappa (κ) and lambda (λ).

[0365] A "native sequence Fc region" comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc regions include native sequence human IgG1 Fc regions (non-A and A allotypes); native sequence human IgG2 Fc regions; native sequence human IgG3 Fc regions; and native sequence human IgG4 Fc regions, and naturally occurring variants thereof.

[0366] A "variant Fc region" comprises an amino acid sequence that differs from a native sequence Fc region by virtue of at least one amino acid modification (alteration), preferably one or more amino acid substitutions. Preferably, the variant Fc region has at least one amino acid substitution in the native or parent Fc region, for example, about 1 to about 30 amino acid substitutions, preferably about 1 to about 20 amino acid substitutions, more preferably about 1 to about 10 amino acid substitutions, and most preferably about 1 to about 5 amino acid substitutions, compared to the native sequence Fc region or the parent Fc region. The variant Fc region herein preferably has at least about 80% homology to the native sequence Fc region or the parent Fc region, preferably at least about 85% homology thereto, more preferably at least about 90% homology thereto, and most preferably at least about 95% homology thereto.

[0367] "Percent (%) amino acid sequence identity" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing spaces (if necessary) to achieve maximum sequence identity and disregarding any conservative substitutions as part of sequence identity. Alignment for determining percent amino acid sequence identity can be achieved in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software or GENETYX (registered trademark) (Genetyx Co., Ltd.). Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required for achieving maximum alignment over the full length of the compared sequences.

[0368] The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code and user documentation have been filed with the U.S. Copyright Office, Washington, D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from the source code. The ALIGN-2 program should be compiled for UNIX operating systems, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not change.

[0369] In cases where ALIGN-2 is used for amino acid sequence comparison, the percent amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be expressed as a given amino acid sequence A having or comprising a specific amino acid sequence identity with a given amino acid sequence B) is calculated as follows:

[0370] Multiply 100 by the fraction X / Y

[0371] where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in the program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be understood that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values ​​used herein are obtained as described in the preceding paragraph using the ALIGN-2 computer program.

[0372] The terms "pharmaceutical formulation" and "pharmaceutical composition" are used interchangeably to refer to a preparation that is in such form as to permit the biological activity of the active ingredient contained therein to be effective, and that contains no additional components that are unacceptably toxic to a subject to which the formulation would be administered.

[0373] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0374] "Pharmaceutically acceptable carrier" refers to a component of a pharmaceutical preparation or pharmaceutical composition other than the active ingredient that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0375] An "effective amount" of an agent (eg, a pharmaceutical formulation) refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0376] The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and / or warnings concerning the use of such therapeutic products.

[0377] Unless otherwise indicated, the term "CTLA-4" as used herein refers to any native CTLA-4 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term includes "full-length," unprocessed CTLA-4 as well as any form of CTLA-4 produced by processing in cells. The term also includes naturally occurring CTLA-4 variants, such as splice variants or allelic variants. The amino acid sequence of an exemplary human CTLA-4 is shown in SEQ ID NO: 214, the amino acid sequence of mouse CTLA-4 is shown in SEQ ID NO: 247, the amino acid sequence of monkey CTLA-4 is shown in SEQ ID NO: 248, and the amino acid sequence of the extracellular domain of human CTLA-4 is shown in SEQ ID NO: 28. CTLA-4 may also be described herein as CTLA4.

[0378] The term "regulatory T (Treg) cells" refers to a subset of T cells that regulate the immune system, maintain tolerance to self-antigens, and suppress autoimmune diseases. These cells typically inhibit or downregulate the induction and proliferation of effector T cells. The best-understood Treg cells are those that express CD4, CD25, and Foxp3 (CD4 + CD25 + Treg cells are cells that are not T cells. These Treg cells are different from helper T cells. Several different methods are used to identify and monitor Treg cells. + CD25 + Treg cells constitute mature CD4 + T cell subsets are about 5% to about 10%, while Tregs can be detected in whole blood at about 1% to about 2%. + CD25 + Foxp3 + Treg cells can be identified and monitored using the methods described in the following examples. Furthermore, the absence or low expression of CD127 can be used in combination with the presence of CD4 and CD25 as another marker. Treg cells also express high levels of CTLA-4 and GITR. Tregs can also be identified using the methods described in the following examples.

[0379] As used herein, the terms "substantially similar," "substantially equal," or "substantially identical" refer to a sufficiently high degree of similarity between two values ​​(e.g., one associated with an antibody of the invention and the other associated with a reference / comparator antibody) such that one of skill in the art would consider the difference between the two values ​​to have little or no biological and / or statistical significance in the context of the biological characteristic being measured by the value (e.g., KD value).

[0380] As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to clinical intervention to attempt to alter the natural course of the individual being treated, and can be performed prophylactically or during clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviation of symptoms, reduction in any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of disease progression, amelioration or alleviation of the disease state, and alleviation or improvement of prognosis. In some embodiments, the antibodies of the invention are used to delay the development of a disease or slow the progression of a disease. In some embodiments, polypeptides comprising a variant Fc region of the invention are used to delay the development of a disease or slow the progression of a disease.

[0381] The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive herein.

[0382] The term "tumor tissue" refers to tissue containing at least one tumor cell. Tumor tissue typically consists of a cluster of tumor cells that form the main mass (parenchyma) of the tumor, and connective tissue and blood vessels ("stroma") that exist between these cells and support the tumor. In some cases, the two are clearly distinct, while in others, they are intermingled. Tumor tissue may be infiltrated by immune cells and other tissues. On the other hand, "non-tumor tissue" refers to tissue other than tumor tissue within a living organism. Healthy / normal tissue that is not in a diseased state is a typical example of non-tumor tissue.

[0383] II. Compositions and Methods

[0384] In one aspect, the present invention is based in part on anti-CTLA-4 antibodies and their uses. In certain embodiments, antibodies that bind to CTLA-4 are provided. The antibodies of the present invention can be used, for example, in the diagnosis or treatment of cancer.

[0385] A. Exemplary Anti-CTLA-4 Antibodies

[0386] In one aspect, the invention provides isolated antibodies that bind to CTLA-4. In certain embodiments, the anti-CTLA-4 antibodies of the invention have CTLA-4 binding activity that is dependent on the concentration of an adenosine-containing compound. In some embodiments, the binding activity to CTLA-4 is higher in the presence of an adenosine-containing compound than in the absence of an adenosine-containing compound. In another embodiment, the binding activity to CTLA-4 is higher in the presence of a high concentration of an adenosine-containing compound than in the presence of a low concentration of an adenosine-containing compound. In further embodiments, the difference in binding activity to CTLA-4 is, for example, 2-fold or higher, 3-fold or higher, 5-fold or higher, 10-fold or higher, 20-fold or higher, 30-fold or higher, 50-fold or higher, 100-fold or higher, 200-fold or higher, 300-fold or higher, 500-fold or higher, 1×10 3 times or higher, 2×10 3 times or higher, 3×10 3 times or higher, 5×10 3 times or higher, 1×10 4 or higher, 2×10 4 or higher, 3×10 4 times or higher, 5×10 4 times or higher or 1×10 5 times or higher.

[0387] In some embodiments, the binding activity of an anti-CTLA-4 antibody can be represented by a KD (dissociation constant) value. In further embodiments, the KD value of the anti-CTLA-4 antibody in the presence of an adenosine-containing compound is less than the KD value in the absence of the adenosine-containing compound. Alternatively, in another embodiment, the KD value of the anti-CTLA-4 antibody in the presence of a high concentration of an adenosine-containing compound is less than the KD value in the presence of a low concentration of an adenosine-containing compound. In further embodiments, the difference in KD value of the anti-CTLA-4 antibody is, for example, 2-fold or more, 3-fold or more, 5-fold or more, 10-fold or more, 20-fold or more, 30-fold or more, 50-fold or more, 100-fold or more, 200-fold or more, 300-fold or more, 500-fold or more, 1×10 3 times or higher, 2×10 3 times or higher, 3×10 3 times or higher, 5×10 3 times or higher, 1×10 4 or higher, 2×10 4 or higher, 3×10 4 times or higher, 5×10 4 times or higher, or 1×10 5The KD value of the anti-CTLA-4 antibody in the presence of adenosine-containing compounds or in the presence of high concentrations of adenosine-containing compounds can be, for example, 9 × 10 -7 M or less, 8×10 -7 M or less, 7×10 -7 M or less, 6×10 -7 M or less, 5 × 10 -7 M or less, 4×10 -7 M or less, 3×10 -7 M or less, 2×10 -7 M or less, 1 × 10 -7 M or less, 9×10 -8 M or less, 8×10 -8 M or less, 7×10 -8 M or less, 6 × 10 -8 M or less, 5×10 -8 M or less, 4×10 -8 M or less, 3×10 -8 M or less, 2 × 10 -8 M or less, 1×10 -8 M or less, 9×10 -9 M or less, 8×10 -9 M or less, 7 × 10 -9 M or less, 6×10 -9 M or less, 5×10 -9 M or less, 4×10 -9 M or less, 3 × 10 -9 M or less, 2×10 -9 M or less, 1×10 -9 M or less, 9 × 10 -10 M or less, 8×10 -10 M or less, 7×10 -10 M or less, 6×10 -10 M or less, 5×10 -10 M or less, 4×10 -10 M or less, 3×10 -10 M or less, 2×10 -10 M or less, or 1×10 -10 M or less. The KD value of the anti-CTLA-4 antibody in the absence of an adenosine-containing compound or in the presence of a low concentration of an adenosine-containing compound may be, for example, 1×10 -8 M or more, 2×10 -8 M or more, 3×10 -8 M or more, 4×10-8 M or more, 5×10 -8 M or more, 6×10 -8 M or more, 7×10 -8 M or more, 8×10 -8 M or more, 9×10 -8 M or more, 1×10 -7 M or more, 2×10 -7 M or more, 3×10 -7 M or more, 4×10 -7 M or more, 5×10 -7 M or more, 6×10 -7 M or more, 7×10 -7 M or more, 8×10 -7 M or more, 9×10 -7 M or more, 1×10 -6 M or more, 2×10 -6 M or more, 3×10 -6 M or more, 4×10 -6 M or more, 5×10 -6 M or more, 6×10 -6 M or more, 7×10 -6 M or more, 8×10 -6 M or more, 9×10 -6 M or more.

[0388] In another embodiment, the binding activity of an anti-CTLA-4 antibody can be expressed using a kd (dissociation rate constant) value instead of a KD value.

[0389] In another embodiment, the binding activity of an anti-CTLA-4 antibody can be expressed by the amount of CTLA-4 bound per unit amount of antibody. For example, in a surface plasmon resonance assay, the amount of antibody bound to a sensor chip and the amount of antigen bound thereto are each measured as resonance units (RU). The value obtained by dividing the amount of antigen bound by the amount of antibody bound can be defined as the amount of antigen bound per unit amount of antibody. Specific methods for determining and calculating such binding amounts are described in the examples below. In some embodiments, the amount of CTLA-4 bound in the presence of an adenosine-containing compound is greater than the amount bound in the absence of the adenosine-containing compound. Alternatively, in another embodiment, the amount of CTLA-4 bound in the presence of a high concentration of an adenosine-containing compound is greater than the amount bound in the presence of a low concentration of an adenosine-containing compound. In further embodiments, the difference in the amount of CTLA-4 bound is, for example, 2-fold or more, 3-fold or more, 5-fold or more, 10-fold or more, 20-fold or more, 30-fold or more, 50-fold or more, 100-fold or more, 200-fold or more, 300-fold or more, 500-fold or more, 1×10 3 times or higher, 2× 10 3 times or higher, 3× 10 3 times or higher, 5× 10 3 times or higher, 1×10 4 or higher, 2×10 4 or higher, 3×10 4 times or higher, 5×10 4 times or higher or 1×10 5The value of the amount of CTLA-4 bound in the presence of an adenosine-containing compound or in the presence of a high concentration of an adenosine-containing compound can be, for example, 0.01 or more, 0.02 or more, 0.03 or more, 0.04 or more, 0.05 or more, 0.06 or more, 0.07 or more, 0.08 or more, 0.09 or more, 0.1 or more, 0.2 or more, 0.3 or more, 0.4 or more, 0.5 or more, 0.6 or more, 0.7 or more, 0.8 or more, 0.9 or more, or 1 or more. The value of the amount of CTLA-4 bound in the absence of an adenosine-containing compound or in the presence of a low concentration of an adenosine-containing compound can be, for example, 0.5 or less, 0.4 or less, 0.3 or less, 0.2 or less, 0.1 or less, 0.09 or less, 0.08 or less, 0.07 or less, 0.06 or less, 0.05 or less, 0.04 or less, 0.03 or less, 0.02 or less, 0.01 or less, 0.009 or less, 0.008 or less, 0.007 or less, 0.006 or less, 0.005 or less, 0.004 or less, 0.003 or less, 0.002 or less, or 0.001 or less.

[0390] In some embodiments, KD values, kd values, binding values, etc. described herein are determined or calculated by surface plasmon resonance assay at 25°C or 37°C (see, eg, Reference Example 3 herein).

[0391] Any concentration of adenosine-containing compound can be selected so long as a difference in binding activity of the anti-CTLA-4 antibody is detected. In certain embodiments, a high concentration can include, for example, 1 nM or higher, 3 nM or higher, 10 nM or higher, 30 nM or higher, 100 nM or higher, 300 nM or higher, 1 μM or higher, 3 μM or higher, 10 μM or higher, 30 μM or higher, 100 μM or higher, 300 μM or higher, 100 μM or higher, 300 μM or higher, 100 μM or higher, 300 μM or higher, 1 mM or higher, 1 mM or higher, 3 mM or higher, 10 mM or higher, 30 mM or higher, 100 mM or higher, 300 mM or higher, and 1 M or higher. Alternatively, the high concentration herein can be an amount sufficient for each anti-CTLA-4 antibody to exhibit maximum binding activity. In one embodiment, 1 μM, 10 μM, 100 μM, 1 mM, or an amount sufficient for each anti-CTLA-4 antibody to exhibit maximum binding activity can be selected as the high concentration herein. In certain embodiments, low concentrations can include, for example, 1 mM or less, 300 μM or less, 100 μM or less, 30 μM or less, 10 μM or less, 3 μM or less, 1 μM or less, 300 nM or less, 100 nM or less, 30 nM or less, 10 nM or less, 3 nM or less, 1 nM or less, 300 pM or less, 100 pM or less, 30 pM or less, 10 pM or less, 3 pM or less, and 1 pM or less. Alternatively, the low concentration herein can be the concentration at which various anti-CTLA-4 antibodies exhibit minimal binding activity. Alternatively, the case where the substantial concentration is zero (absence of an adenosine-containing compound) can be selected as the embodiment of the low concentration. In one embodiment, the concentrations at which various anti-CTLA-4 antibodies show minimal binding activity are 1 mM, 100 μM, 10 μM, 1 μM, or absence of an adenosine compound. In another embodiment, the following values ​​can be selected as the ratio of the high concentration to the low concentration: for example, 3 times or more, 10 times or more, 30 times or more, 100 times or more, 300 times or more, 1×10 3 times or higher, 3×10 3 times or higher, 1×104 times or higher, 3×10 4 times or higher, 1×10 5 times or higher, 3×10 5 times or higher, 1×10 6 times or higher, 3×10 6 times or higher, 1×10 7 times or higher, 3×10 7 times or higher, 1×10 8 times or higher, 3×10 8 times or higher, 1×10 9 times or higher, 3×10 9 times or higher, 1×10 10 times or higher, 3×10 10 times or higher, 1×10 11 times or higher, 3×10 11 times or higher, or 1×10 12 times or higher.

[0392] In another embodiment, the anti-CTLA-4 antibodies of the present invention also have binding activity with adenosine-containing compounds. The amount of adenosine-containing compound bound per unit amount of the anti-CTLA-4 antibody can be calculated using the above-described method and used as the binding activity of the antibody with the adenosine-containing compound. Specific methods for determining and calculating such binding amount are described in the following examples. The value of the amount of adenosine-containing compound bound per unit amount of the anti-CTLA-4 antibody of the present invention can be, for example, 0.0001 or more, 0.0002 or more, 0.0003 or more, 0.0004 or more, 0.0005 or more, 0.0006 or more, 0.0007 or more, 0.0008 or more, 0.0009 or more, 0.001 or more, 0.002 or more, 0.003 or more, 0.004 or more, 0.005 or more, 0.006 or more, 0.007 or more, 0.008 or more, 0.009 or more, or 0.01 or more.

[0393] In another embodiment, an anti-CTLA-4 antibody of the invention forms a ternary complex with an adenosine-containing compound and CTLA-4. In one embodiment, the anti-CTLA-4 antibody binds to the adenosine-containing compound via the heavy chain CDR1, CDR2, and CDR3. In one embodiment, the anti-CTLA-4 antibody has a binding motif for an adenosine-containing compound. The binding motif for an adenosine-containing compound can consist of, for example, at least one amino acid present at positions 33, 52, 52a, 53, 56, 58, 95, 96, 100a, 100b, and 100c according to Kabat numbering. In a further embodiment, the anti-CTLA-4 antibody binds to the adenosine-containing compound, for example, via at least one amino acid selected from positions 33, 52, 52a, 53, 56, 58, 95, 96, 100a, 100b, and 100c according to Kabat numbering. In certain embodiments, the anti-CTLA-4 antibody has at least one amino acid selected from the group consisting of Thr at position 33, Ser at position 52, Ser at position 52a, Arg at position 53, Tyr at position 56, Tyr at position 58, Tyr at position 95, Gly at position 96, Met at position 100a, Leu at position 100b, and Trp at position 100c, according to Kabat numbering. CTLA-4 can further bind to a complex formed by the combination of the anti-CTLA-4 antibody and the adenosine-containing compound. Furthermore, the adenosine-containing compound can be present at the interface where the anti-CTLA-4 antibody and CTLA-4 interact and can bind to both. The formation of a ternary complex between the anti-CTLA-4 antibody, the adenosine-containing compound, and CTLA-4 can be confirmed by techniques such as crystal structure analysis described below (see Examples).

[0394] In another embodiment, an anti-CTLA-4 antibody of the present invention binds to at least one amino acid selected from amino acids 3 (Met), 33 (Glu), 35 (Arg), 53 (Thr), 97 (Glu), 99 (Met), 100 (Tyr), 101 (Pro), 102 (Pro), 103 (Pro), 104 (Tyr), 105 (Tyr), and 106 (Leu) of human CTLA-4 (extracellular domain; SEQ ID NO: 28). These amino acids may constitute an epitope of the anti-CTLA-4 antibody of the present invention. In another embodiment, the anti-CTLA-4 antibody of the present invention binds to a region from amino acids 97 (Glu) to amino acids 106 (Leu) of human CTLA-4 (extracellular domain; SEQ ID NO: 28). In another embodiment, the anti-CTLA-4 antibodies of the invention bind to the region from amino acid 99 (Met) to amino acid 106 (Leu) of human CTLA-4 (extracellular domain; SEQ ID NO: 28).

[0395] In another embodiment, the anti-CTLA-4 antibodies of the invention compete for CTLA-4 binding with ABAM004 (VH, SEQ ID NO: 10; VL, SEQ ID NO: 11; HVR-H1, SEQ ID NO: 100; HVR-H2, SEQ ID NO: 101; HVR-H3, SEQ ID NO: 102; HVR-L1, SEQ ID NO: 113; HVR-L2, SEQ ID NO: 114; HVR-L3, SEQ ID NO: 115). In another embodiment, the anti-CTLA-4 antibodies of the invention bind to the same epitope as ABAM004. When an excess of anti-CTLA-4 antibody is present, the binding of ABAM004 to CTLA-4 can be reduced, for example, by 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more. Exemplary competition assays are provided herein.

[0396] In another embodiment, the anti-CTLA-4 antibodies of the present invention exhibit cytotoxic activity against cells expressing CTLA-4. When CTLA-4 is expressed on the surface of target cells and the anti-CTLA-4 antibody binds to them, the cells are damaged. The cell damage may be caused by effector cells bound to the antibody, such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP), or by complement bound to the antibody, such as complement-dependent cytotoxicity (CDC). Alternatively, the damage may be caused by a cytotoxic agent (e.g., a radioisotope or chemotherapeutic agent) conjugated to the antibody, such as an immunoconjugate. Cytotoxicity in this context may include inducing cell death, inhibiting cell proliferation, and impairing cell function. When an anti-CTLA-4 antibody is present in sufficient amounts, it can cause damage to, for example, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more of the cells expressing CTLA-4. Such cytotoxic activity can be measured by comparing it to an assay in the absence of the antibody or in the presence of a negative control antibody. Exemplary cytotoxicity assays are provided herein.

[0397] In another embodiment, the anti-CTLA-4 antibodies of the present invention exhibit neutralizing activity against CTLA-4. CTLA-4 is known to exert its effects by interacting with its ligands CD80 (B7-1) or CD86 (B7-2). In certain embodiments, the anti-CTLA-4 antibodies inhibit the interaction of CTLA-4 with CD80 (B7-1) or CD86 (B7-2). When the anti-CTLA-4 antibody is present in a sufficient amount, it can inhibit the interaction of CTLA-4 with CD80 (B7-1) or CD86 (B7-2), for example, by 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more. This determination of inhibitory activity can be compared to that in the absence of the antibody or in the presence of a negative control antibody. Specific methods for determining neutralizing activity are provided herein.

[0398] In another embodiment, the anti-CTLA-4 antibodies of the present invention bind to CTLA-4 derived from a variety of animal species. Exemplary animal species can include mammals, such as humans, monkeys, mice, rats, hamsters, guinea pigs, rabbits, pigs, cattle, goats, horses, sheep, camels, dogs and cats. In certain embodiments, anti-CTLA-4 antibodies bind to CTLA-4 derived from humans and non-humans (e.g., monkeys, mice and rats). The amino acid sequence of human CTLA-4 is shown in SEQ ID NO: 214, the amino acid sequence of ape CTLA-4 is shown in SEQ ID NO: 247, and the amino acid sequence of mouse CTLA-4 is shown in SEQ ID NO: 248. The amino acid sequence of CTLA-4 derived from other animal species can also be appropriately determined by methods known to those skilled in the art.

[0399] In certain embodiments, adenosine-containing compounds of the present invention may include, for example, adenosine (ADO), adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), cyclic adenosine monophosphate (cAMP), deoxyadenosine (dADO), deoxyadenosine triphosphate (dATP), deoxyadenosine diphosphate (dADP), deoxyadenosine monophosphate (dAMP), and adenosine (γ-thio) triphosphate (ATPγS).

[0400] In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 223; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 224; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 225. In one embodiment, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 223; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 224; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 225.

[0401] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from the group consisting of: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 226; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 227; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 228. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 226; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 227; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 228.

[0402] In another aspect, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two or all three VH HVR sequences selected from the group consisting of: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 223, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 224, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 225; and (b) a VL domain comprising at least one, at least two or all three VL HVR sequences selected from the group consisting of: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 226, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 227, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 228.

[0403] In another aspect, the present invention provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 223; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 224; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 225; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 226; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 227; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 228.

[0404] In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 100; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 101; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102. In one embodiment, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 100; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 101; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102.

[0405] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from the group consisting of: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 113; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 114; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 115. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 113; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 114; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 115.

[0406] In another aspect, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two or all three VH HVR sequences selected from the group consisting of: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 100, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 101, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102; and (b) a VL domain comprising at least one, at least two or all three VL HVR sequences selected from the group consisting of: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 113, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 114, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 115.

[0407] In another aspect, the present invention provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 100; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 101; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 113; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 114; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 115.

[0408] In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 100; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 104; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102. In one embodiment, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 100; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 104; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102.

[0409] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from the group consisting of: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 116; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 115. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 116; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 115.

[0410] In another aspect, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two or all three VH HVR sequences selected from the group consisting of: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 100, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 104, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102; and (b) a VL domain comprising at least one, at least two or all three VL HVR sequences selected from the group consisting of: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 116, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 115.

[0411] In another aspect, the present invention provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 100; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 104; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 116; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 115.

[0412] In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 105; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 106; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102. In one embodiment, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 105; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 106; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102.

[0413] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from the group consisting of: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133.

[0414] In another aspect, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two or all three VH HVR sequences selected from the group consisting of: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 105, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 106, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102; and (b) a VL domain comprising at least one, at least two or all three VL HVR sequences selected from the group consisting of: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133.

[0415] In another aspect, the present invention provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 105; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 106; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133.

[0416] In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 108; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102. In one embodiment, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 108; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102.

[0417] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from the group consisting of: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 121; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 123; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 153. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 121; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 123; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 153.

[0418] In another aspect, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two or all three VH HVR sequences selected from the group consisting of: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 108, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102; and (b) a VL domain comprising at least one, at least two or all three VL HVR sequences selected from the group consisting of: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 121, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 123, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 153.

[0419] In another aspect, the present invention provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 108; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 121; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 123; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 153.

[0420] In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 110; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102. In one embodiment, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 110; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102.

[0421] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from the group consisting of: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133.

[0422] In another aspect, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two or all three VH HVR sequences selected from the group consisting of: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 110, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102; and (b) a VL domain comprising at least one, at least two or all three VL HVR sequences selected from the group consisting of: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133.

[0423] In another aspect, the present invention provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 110; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133.

[0424] In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 112; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102. In one embodiment, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 112; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102.

[0425] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from the group consisting of: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 128; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 128; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133.

[0426] In another aspect, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two or all three VH HVR sequences selected from the group consisting of: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 112, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102; and (b) a VL domain comprising at least one, at least two or all three VL HVR sequences selected from the group consisting of: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 128, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133.

[0427] In another aspect, the present invention provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 112; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 128; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133.

[0428] In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 111; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 152. In one embodiment, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 111; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 152.

[0429] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from the group consisting of: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 128; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 128; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133.

[0430] In another aspect, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two or all three VH HVR sequences selected from the group consisting of: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 111, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 152; and (b) a VL domain comprising at least one, at least two or all three VL HVR sequences selected from the group consisting of: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 128, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133.

[0431] In another aspect, the present invention provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 111; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 152; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 128; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133.

[0432] In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 112; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102. In one embodiment, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 112; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102.

[0433] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from the group consisting of: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 129; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 129; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133.

[0434] In another aspect, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two or all three VH HVR sequences selected from the group consisting of: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 112, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102; and (b) a VL domain comprising at least one, at least two or all three VL HVR sequences selected from the group consisting of: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 129, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133.

[0435] In another aspect, the present invention provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 112; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 129; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133.

[0436] In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 111; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 152. In one embodiment, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 111; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 152.

[0437] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from the group consisting of: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 129; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 129; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133.

[0438] In another aspect, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two or all three VH HVR sequences selected from the group consisting of: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 111, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 152; and (b) a VL domain comprising at least one, at least two or all three VL HVR sequences selected from the group consisting of: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 129, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133.

[0439] In another aspect, the present invention provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 111; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 152; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 129; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133.

[0440] In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 109; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102. In one embodiment, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 109; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102.

[0441] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from the group consisting of: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 130; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 130; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133.

[0442] In another aspect, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two or all three VH HVR sequences selected from the group consisting of: (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 109, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102; and (b) a VL domain comprising at least one, at least two or all three VL HVR sequences selected from the group consisting of: (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 130, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117, and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133.

[0443] In another aspect, the present invention provides an antibody comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 109; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 130; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133.

[0444] In certain embodiments, any one or more amino acids of an anti-CTLA-4 antibody provided above are substituted at the following HVR positions:

[0445] - In HVR-H1 (SEQ ID NO: 223): position 2

[0446] - In HVR-H2 (SEQ ID NO: 224): positions 4, 5, 7, 13 and 16

[0447] - In HVR-H3 (SEQ ID NO: 225): position 3

[0448] - in HVR-L1 (SEQ ID NO: 226): positions 1, 3, 6, 11, 12 and 14

[0449] - In HVR-L2 (SEQ ID NO: 227): positions 1, 3, 4 and 7

[0450] - In HVR-L3 (SEQ ID NO: 228): positions 1 and 10

[0451] In certain embodiments, the substitution is a conservative substitution as provided herein. In certain embodiments, any one or more of the following substitutions can be made in any combination:

[0452] - In HVR-H1 (SEQ ID NO: 100): H2A, R or K

[0453] - In HVR-H2 (SEQ ID NO: 101): S4T; R5Q; G7H; D13E or R; K16R

[0454] - In HVR-H3 (SEQ ID NO: 102): K3A

[0455] - In HVR-L1 (SEQ ID NO: 113): T1D, Q, or E; T3P; D6G; N11T; Y12W; S14H

[0456] - In HVR-L2 (SEQ ID NO: 114): E1F or Y; S3I; K4S; S7E or K

[0457] - In HVR-L3 (SEQ ID NO: 115): S1Q; M10T

[0458] For HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2 and HVR-L3, all possible combinations of the above substitutions are contained in the consensus sequences of SEQ ID NOs: 223, 224, 225, 226, 227 and 228, respectively.

[0459] In any of the above embodiments, the anti-CTLA-4 antibody is humanized. In one embodiment, the anti-CTLA-4 antibody comprises HVRs as in any of the above embodiments and further comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework. In another embodiment, the anti-CTLA-4 antibody comprises HVRs as in any of the above embodiments and further comprises a VH or VL comprising a FR sequence. In a further embodiment, the anti-CTLA-4 antibody comprises the following heavy and / or light chain variable domain FR sequences: for the heavy chain variable domain, FR1 comprises the amino acid sequence of any one of SEQ ID NOs: 229 to 232, FR2 comprises the amino acid sequence of SEQ ID NO: 233, FR3 comprises the amino acid sequence of SEQ ID NO: 234, and FR4 comprises the amino acid sequence of SEQ ID NO: 235; for the light chain variable domain, FR1 comprises the amino acid sequence of any one of SEQ ID NOs: 236 to 238, FR2 comprises the amino acid sequence of any one of SEQ ID NOs: 240 to 241, FR3 comprises the amino acid sequence of any one of SEQ ID NOs: 242 to 244, and FR4 comprises the amino acid sequence of any one of SEQ ID NOs: 245 to 246.

[0460] In another aspect, the anti-CTLA-4 antibody comprises a heavy chain variable domain (VH) sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 10. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-CTLA-4 antibody comprising the sequence retains the ability to bind to CTLA-4. In certain embodiments, a total of 1 to 10, 11, 12, 13, 14, or 15 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 10. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-CTLA-4 antibody comprises the VH sequence of SEQ ID NO: 10, including post-translational modifications of that sequence. In specific embodiments, the VH comprises one, two, or three HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 100; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 101; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102. Post-translational modifications include, but are not limited to, modification of the N-terminal glutamine or glutamic acid of the heavy or light chain to pyroglutamic acid by pyroglutamylation.

[0461] In another aspect, an anti-CTLA-4 antibody is provided, wherein the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 11. In certain embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity contains substitutions (e.g., conservative substitutions), insertions or deletions relative to the reference sequence, but the anti-CTLA-4 antibody comprising the sequence retains the ability to bind to CTLA-4. In certain embodiments, a total of 1 to 10, 11, 12, 13, 14 or 15 amino acids are substituted, inserted and / or deleted in SEQ ID NO: 11. In certain embodiments, the substitutions, insertions or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-CTLA-4 antibody comprises the VL sequence of SEQ ID NO: 11, including post-translational modifications of that sequence. In specific embodiments, the VL comprises one, two, or three HVRs selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 113, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 114, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 115. Post-translational modifications include, but are not limited to, modification of the N-terminal glutamine or glutamic acid of the heavy or light chain to pyroglutamic acid by pyroglutamylation.

[0462] In another aspect, an anti-CTLA-4 antibody is provided, wherein the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 149. In certain embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity contains substitutions (e.g., conservative substitutions), insertions or deletions relative to the reference sequence, but the anti-CTLA-4 antibody comprising the sequence retains the ability to bind to CTLA-4. In certain embodiments, a total of 1 to 10, 11, 12, 13, 14 or 15 amino acids are substituted, inserted and / or deleted in SEQ ID NO: 149. In certain embodiments, the substitutions, insertions or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-CTLA-1 antibody comprises the VL sequence of SEQ ID NO: 149, including post-translational modifications thereof. In specific embodiments, the VL comprises one, two, or three HVRs selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 130, (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133. Post-translational modifications include, but are not limited to, modification of the N-terminal glutamine or glutamic acid of the heavy or light chain to pyroglutamic acid by pyroglutamylation.

[0463] In another aspect, an anti-CTLA-4 antibody is provided, wherein the antibody comprises a VH as in any embodiment provided above and a VL as in any embodiment provided above. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 10 and SEQ ID NO: 11, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 98 and SEQ ID NO: 99, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 83 and SEQ ID NO: 88, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 83 and SEQ ID NO: 89, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 83 and SEQ ID NO: 90, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 83 and SEQ ID NO: 91, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 83 and SEQ ID NO: 92, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 83 and SEQ ID NO: 93, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 83 and SEQ ID NO: 94, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 83 and SEQ ID NO: 97, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 83 and SEQ ID NO: 95, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 84 and SEQ ID NO: 97, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 85 and SEQ ID NO: 97, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 86 and SEQ ID NO: 97, respectively, including post-translational modifications of those sequences.In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 86 and SEQ ID NO: 134, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 136 and SEQ ID NO: 97, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 135 and SEQ ID NO: 97, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 136 and SEQ ID NO: 95, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 137 and SEQ ID NO: 97, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 138 and SEQ ID NO: 97, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 138 and SEQ ID NO: 144, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 138 and SEQ ID NO: 145, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 138 and SEQ ID NO: 146, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 139 and SEQ ID NO: 146, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 140 and SEQ ID NO: 146, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 141 and SEQ ID NO: 146, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 140 and SEQ ID NO: 147, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 141 and SEQ ID NO: 147, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 140 and SEQ ID NO: 148, respectively, including post-translational modifications of those sequences.In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 141 and SEQ ID NO: 148, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 136 and SEQ ID NO: 149, respectively, including post-translational modifications of those sequences. In another aspect, heteromeric anti-CTLA-4 antibodies are provided, wherein the antibody comprises at least two different variable regions selected from the variable regions comprising the VH and VL sequences provided above. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 140 and SEQ ID NO: 146, respectively, and the VH and VL sequences in SEQ ID NO: 141 and SEQ ID NO: 146, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 140 and SEQ ID NO: 147, respectively, and the VH and VL sequences in SEQ ID NO: 141 and SEQ ID NO: 147, respectively, including post-translational modifications of those sequences. Post-translational modifications include, but are not limited to, modification of the N-terminal glutamine or glutamic acid of the heavy or light chain to pyroglutamic acid by pyroglutamylation.

[0464] When the N-terminal amino acid of the heavy or light chain of the anti-CTLA-4 antibodies provided herein is glutamine, the amino acid can be substituted with glutamic acid. When the N-terminal amino acid of the heavy or light chain of the anti-CTLA-4 antibodies provided herein is glutamic acid, the amino acid can be substituted with glutamine.

[0465] In another aspect, the present invention provides antibodies that bind to the same epitope as the anti-CTLA-4 antibodies provided herein. For example, in certain embodiments, antibodies that bind to the same epitope as any one of the antibodies listed in Table 17, Table 22, Table 27, and Table 32 are provided. In certain embodiments, antibodies are provided that bind to an epitope within a CTLA-4 fragment comprising at least one amino acid selected from the group consisting of amino acids at position 3 (Met), position 33 (Glu), position 35 (Arg), position 53 (Thr), position 97 (Glu), position 99 (Met), position 100 (Tyr), position 101 (Pro), position 102 (Pro), position 103 (Pro), position 104 (Tyr), position 105 (Tyr), and position 106 (Leu) of SEQ ID NO: 28. In certain embodiments, antibodies are provided that bind to an epitope within a CTLA-4 fragment consisting of amino acids at positions 97 (Glu) to 106 (Leu) of SEQ ID NO: 28. In certain embodiments, antibodies are provided that bind to an epitope within a CTLA-4 fragment consisting of amino acids from position 99 (Met) to position 106 (Leu) of SEQ ID NO: 28.

[0466] In other aspects of the invention, the anti-CTLA-4 antibody according to any of the above embodiments is a monoclonal antibody, including a chimeric antibody, a humanized antibody, or a human antibody. In one embodiment, the anti-CTLA-4 antibody is an antibody fragment, such as an Fv, Fab, Fab', scFv, a diabody, or a F(ab')2 fragment. In another embodiment, the antibody is a full-length antibody, such as an intact IgG1 antibody, an intact IgG4 antibody, or other antibody classes or isotypes as defined herein.

[0467] In other aspects, the anti-CTLA-4 antibodies of the present invention comprise an Fc region. In other aspects, the anti-CTLA-4 antibodies of the present invention comprise a constant region. The constant region can be a heavy chain constant region (including the Fc region), a light chain constant region, or both. In some embodiments, the Fc region is a native sequence Fc region. Exemplary heavy chain constant regions derived from natural antibodies can include, for example, heavy chain constant regions such as human IgG1 (SEQ ID NO: 249), human IgG2 (SEQ ID NO: 250), human IgG3 (SEQ ID NO: 251), and human IgG4 (SEQ ID NO: 252). In addition, other exemplary heavy chain constant regions can include the heavy chain constant regions of SEQ ID NOs: 82, 158, and 334. Exemplary light chain constant regions derived from natural antibodies can include, for example, light chain constant regions such as human kappa chain (SEQ ID NOs: 33, 63, and 159) and human lambda chain (SEQ ID NOs: 53 and 87).

[0468] In another embodiment, the Fc region is a variant Fc region generated by adding amino acid changes to the native sequence Fc region. In certain embodiments, the variant Fc region has enhanced binding activity to at least one Fcγ receptor selected from FcγRIa, FcγRIIa, FcγRIIb and FcγRIIIa compared to the native sequence Fc region. In other embodiments, the variant Fc region has enhanced binding activity to FcγRIIa and FcγRIIIa compared to the native sequence Fc region. Examples of heavy chain constant regions comprising such variant Fc regions include, for example, the heavy chain constant regions listed in Tables 39 to 43 and the heavy chain constant regions of SEQ ID NOs: 31, 32, 41 to 46, 65, 66, 81, 207, 239, 253 to 271, 276, 277, 278, 308, 309, 311 to 333, 343 to 348, 350 and 351.

[0469] A native sequence Fc region typically consists of a homodimer composed of two identical polypeptide chains. In certain embodiments, a variant Fc region may be a homodimer composed of polypeptide chains having the same sequence, or a heterodimer composed of polypeptide chains having different sequences. Similarly, the heavy chain constant region comprising the Fc region may be a homodimer composed of polypeptide chains having the same sequence, or a heterodimer composed of polypeptide chains having different sequences.Examples of heteromeric heavy chain constant regions include, for example, a heavy chain constant region comprising a polypeptide chain of SEQ ID NOs: 31 and 32; a heavy chain constant region comprising a polypeptide chain of SEQ ID NOs: 43 and 44; a heavy chain constant region comprising a polypeptide chain of SEQ ID NOs: 45 and 46; a heavy chain constant region comprising a polypeptide chain of SEQ ID NOs: 254 and 256; a heavy chain constant region comprising a polypeptide chain of SEQ ID NOs: 257 and 258; a heavy chain constant region comprising a polypeptide chain of SEQ ID NOs: 259 and 260; a heavy chain constant region comprising a polypeptide chain of SEQ ID NOs: 261 and 263; a heavy chain constant region comprising a polypeptide chain of SEQ ID NOs: 262 and 264; a heavy chain constant region comprising a polypeptide chain of SEQ ID NOs: 265 and 267; a heavy chain constant region comprising a polypeptide chain of SEQ ID NOs: 266 and 268; a heavy chain constant region comprising a polypeptide chain of SEQ ID NOs: the heavy chain constant region of a polypeptide chain comprising SEQ ID NOs: 269 and 270; the heavy chain constant region of a polypeptide chain comprising SEQ ID NOs: 271 and 81; the heavy chain constant region of a polypeptide chain comprising SEQ ID NOs: 65 and 66; the heavy chain constant region of a polypeptide chain comprising SEQ ID NOs: 239 and 207; the heavy chain constant region of a polypeptide chain comprising SEQ ID NOs: 259 and 276; the heavy chain constant region of a polypeptide chain comprising SEQ ID NOs: 65 and 278; the heavy chain constant region of a polypeptide chain comprising SEQ ID NOs: 308 and 309; the heavy chain constant region of a polypeptide chain comprising SEQ ID NOs: 311 and 312; the heavy chain constant region of a polypeptide chain comprising SEQ ID NOs: 313 and 314; the heavy chain constant region of a polypeptide chain comprising SEQ ID NOs: 315 and 316; the heavy chain constant region of a polypeptide chain comprising SEQ ID NOs: 317 and 318; the heavy chain constant region of a polypeptide chain comprising SEQ ID NOs: the heavy chain constant region of a polypeptide chain comprising SEQ ID NOs: 319 and 320; the heavy chain constant region of a polypeptide chain comprising SEQ ID NOs: 321 and 322; the heavy chain constant region of a polypeptide chain comprising SEQ ID NOs: 323 and 324; the heavy chain constant region of a polypeptide chain comprising SEQ ID NOs: 325 and 326; the heavy chain constant region of a polypeptide chain comprising SEQ ID NOs: 327 and 328; the heavy chain constant region of a polypeptide chain comprising SEQ ID NOs: 330 and 331; the heavy chain constant region of a polypeptide chain comprising SEQ ID NOs: 332 and 333; the heavy chain constant region of a polypeptide chain comprising SEQ ID NOs: 343 and 344; the heavy chain constant region of a polypeptide chain comprising SEQ ID NOs: 345 and 347; the heavy chain constant region of a polypeptide chain comprising SEQ ID NOs: 346 and 348; and the heavy chain constant region of a polypeptide chain comprising SEQ ID NOs: 350 and 351.

[0470] In other aspects, the anti-CTLA-4 antibodies according to any of the above embodiments can incorporate any of the features, alone or in combination, as described in Sections 1-7 below:

[0471] 1. Antibody Binding Activity

[0472] In certain embodiments, the binding activity of an antibody provided herein is 10 μM or less, 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 -8 M or less, for example, 10 -8 M to 10 -13 M, for example, 10 -9 M to 10 -13 The dissociation constant (KD) of the

[0473] In one embodiment, the binding activity of the antibody is determined by a radiolabeled antigen binding assay (RIA). In one embodiment, the RIA is performed using a Fab version of the antibody of interest and its antigen. For example, the solution binding affinity of the Fab for the antigen is determined by titrating the antibody with a minimal concentration of ( 125 I) labeled antigen equilibrated Fab, and then the bound antigen was captured using an anti-Fab antibody coated plate for measurement (see, for example, Chen et al., J. Mol. Biol. 293: 865-881 (1999)). To establish the assay conditions, MICROTITER (registered trademark) multiwell plates (Thermo Scientific) were coated overnight with 5 μg / ml capture anti-Fab antibody (CappelLabs) in 50 mM sodium carbonate (pH 9.6) and then blocked with 2% (w / v) bovine serum albumin in PBS for 2 to 5 hours at room temperature (about 23°C). In non-adsorbent plates (Nunc #269620), 100 pM or 26 pM [ 125The antigen is mixed with a serial dilution of the Fab of interest (e.g., consistent with the evaluation of the anti-VEGF antibody Fab-12 in Presta et al., Cancer Res. 57: 4593-4599 (1997)). The Fab of interest is then incubated overnight; however, the incubation can be continued for a longer period (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixture is transferred to a capture plate and incubated at room temperature (e.g., one hour). The solution is then removed and the plate is washed eight times with 0.1% polysorbate 20 (TWEEN-20 (registered trademark)) in PBS. Once the plate is dry, 150 μL / well of scintillant (MICROSCINT-20 TM ; Packard), and in TOPCOUNT TM Plates were counted for 10 minutes on a gamma counter (Packard).Concentrations of each Fab that gave less than or equal to 20% of maximal binding were selected for competitive binding assays.

[0474] In one embodiment, the binding activity of the antibody is measured using a ligand capture assay using surface plasmon resonance as the assay principle, for example, using a BIACORE (registered trademark) T200 or BIACORE (registered trademark) 4000 (GE Healthcare, Uppsala, Sweden). BIACORE (registered trademark) control software is used for instrument operation. In one embodiment, an amine coupling kit (GE Healthcare, Uppsala, Sweden) is used according to the supplier's instructions, and a sensor chip coated with carboxymethyl dextran (GE Healthcare, Uppsala, Sweden) is immobilized using a ligand capture molecule, such as an anti-tag antibody, an anti-IgG antibody, and protein A. The ligand capture molecule is diluted with 10 mM sodium acetate solution at an appropriate pH and injected at an appropriate flow rate and injection time. The binding activity assay is performed using a buffer containing 0.05% polysorbate 20 (also known as TWEEN (registered trademark)-20) as the assay buffer, at a flow rate of 10-30 μL / min, and at an assay temperature preferably of 25°C or 37°C. When the assay is performed by allowing the antibody serving as the ligand to be captured by a molecule for ligand capture, serial dilutions of the antigen or Fc receptor prepared in the assay buffer (analyte) are injected after the target amount of the antibody is captured by the antibody injection. When the assay is performed by allowing the antigen or Fc receptor serving as the ligand to be captured by a molecule for ligand capture, serial dilutions of the antibody prepared in the assay buffer (analyte) are injected after the target amount of the antigen or Fc receptor is captured by the antigen or Fc receptor injection.

[0475] In one embodiment, the assay results are analyzed using BIACORE (registered trademark) evaluation software. Kinetic parameters are calculated by simultaneously fitting the association and dissociation sensorgrams using a 1:1 binding model, and the association rate (k or ka), dissociation rate (k or k), and equilibrium dissociation constant (KD) can be calculated. When the binding activity is weak, especially when dissociation is rapid and kinetic parameters are difficult to calculate, the equilibrium dissociation constant (KD) can be calculated using a steady-state model. As another parameter of binding activity, the "analyte binding amount per unit amount of ligand" can be calculated by dividing the amount of analyte bound (RU) at a specific concentration by the amount of captured ligand (RU).

[0476] 2. Antibody fragments

[0477] In certain embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv and scFv fragments, as well as other fragments described below. For a review of certain antibody fragments, see Hudson et al., Nat. Med. 9: 129-134 (2003). For a review of scFv fragments, see, for example, Pluckthün, The Pharmacology of Monoclonal Antibodies, Volume 113, Rosenburg and Moore, eds., (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab')2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Patent No. 5,869,046.

[0478] Diabodies are antibody fragments with two antigen-binding sites that can be bivalent or bispecific. See, for example, EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med. 9: 129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9: 129-134 (2003).

[0479] Single-domain antibodies are antibody fragments that comprise all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, single-domain antibodies are human single-domain antibodies (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 B1).

[0480] As described herein, antibody fragments can be prepared by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (eg, E. coli or phage).

[0481] 3. Chimeric and humanized antibodies

[0482] In certain embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In other examples, a chimeric antibody is a "class-switched" antibody, in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0483] In certain embodiments, chimeric antibodies are humanized antibodies. Typically, non-human antibodies are humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Typically, a humanized antibody comprises one or more variable domains, wherein HVR, such as CDR, (or a portion thereof) is derived from a non-human antibody, and FR (or a portion thereof) is derived from a human antibody sequence. The humanized antibody optionally also comprises at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., an antibody from which HVR residues are derived), for example, to restore or improve antibody specificity or affinity.

[0484] Humanized antibodies and methods for their preparation are reviewed, for example, by Almagro and Franson, Front. Biosci. 13:1619-1633 (2008), and are further described, for example, in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Natl Acad. Sci. USA, 86:10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods. 36:25-34 (2005) (describing specificity-determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing “surface reconstruction”); Dall'Acqua et al., Methods. 36:43-60 (2005) (describing “FR shuffling”); and Osbourn et al., Methods. 36:61-68 (2005) and Klimka et al., Br. J. Cancer. 83:252-260 (2000) (describing the “guided selection” method for FR shuffling).

[0485] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best fit" method (see, e.g., Sims et al., J. Immunol. 151: 2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al., Proc. Natl. Acad. Sci. USA, 89: 4285 (1992); and Presta et al., J. Immunol., 151: 2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272: 10678-10684 (1997) and Rosok et al., J. Immunol., 151: 2623 (1993)). et al., J. Biol. Chem. 271: 22611-22618 (1996)).

[0486] 4. Human Antibodies

[0487] In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be produced using a variety of techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-374 (2001) and Lonberg, Curr. Opin. Immunol. 20: 450-459 (2008).

[0488] Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce complete human antibodies or complete antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or part of the human immunoglobulin loci that replace the endogenous immunoglobulin loci, or are present extrachromosomally or randomly integrated into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin loci are typically inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23: 1117-1125 (2005). (See also, for example, the description of the XENOMOUSE TM The human variable regions of intact antibodies produced by such animals can be further modified, for example, by combining with different human constant regions.

[0489] Human antibodies can also be prepared by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for producing human monoclonal antibodies have been described (see, e.g., Kozbor, J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991)). Human antibodies produced by human B cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103: 3557-3562 (2006). Additional methods include, for example, those described in U.S. Patent No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-191 (2005).

[0490] Human antibodies can also be produced by isolating the Fv clone variable domain sequence selected from the phage display library derived from people. Such variable domain sequence can then be combined with the desired human constant domain. The technology of selecting human antibodies from the antibody library is described below.

[0491] 5. Library-derived antibodies

[0492] Antibodies of the invention can be isolated by screening combinatorial libraries for antibodies with the desired activity.For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies with the desired binding properties. Such methods are summarized in, e.g., Hoogenboom et al., Methods in Molecular Biology 178: 1-37 (O'Brien et al., eds., Human Press, Totowa, NJ, 2001) and further described in, e.g., McCafferty et al., Nature 348: 552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, Methods in Molecular Biology 248: 161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004).

[0493] In some phage display methods, repertoires of VH and VL genes are cloned separately by polymerase chain reaction (PCR) and randomly recombined in a phage library, which can then be screened for antigen-binding phage, as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Phage typically display antibody fragments as single-chain Fv (scFv) fragments or Fab fragments. Libraries from immune sources provide high-affinity antibodies against immunogens without the need to construct hybridomas. Alternatively, as described in Griffiths et al., EMBO J, 12: 725-734 (1993), naive repertoires can be cloned (e.g., from humans) to provide antibodies against a wide range of non-self and self antigens from a single source without the need for any immunization. Finally, as described in Hoogenboom and Winter, J.Mol.Biol., 227:381-388 (1992), also by cloning V gene segments that are not rearranged from stem cells, and using PCR primers containing random sequences to synthesize and prepare immature libraries, to encode highly variable CDR3 and to achieve external rearrangement. Patent publications describing human antibody phage libraries include, for example: U.S. Patent number 5,750,373, and U.S. Patent Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936 and 2009 / 0002360.

[0494] Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.

[0495] 6. Multispecific Antibodies

[0496] In certain embodiments, the antibodies provided herein are multispecific antibodies, such as bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. In certain embodiments, one binding specificity is for CTLA-4, and the other is for any other antigen. In certain embodiments, bispecific antibodies can bind to two different epitopes of CTLA-4. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing CTLA-4. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0497] Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs of different specificities (see Milstein and Cuello, Nature, 305: 537 (1983)), WO 93 / 08829, and Trauneckerd et al., EMBO J. 10: 3655 (1991)), and "knob-in-hole" engineering (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can also be prepared by engineering electrostatic steering effects for preparing antibody Fc-heterodimer molecules (WO 2009 / 089004A1); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980 and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to generate bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5): 1547-1553 (1992)); using "diabody" technology to prepare bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993)); and using single-chain Fv (scFv) dimers (see, e.g., Gruber et al., J. Immunol., 152: 5368-5376). (1994)); and the preparation of trispecific antibodies as described, for example, in Tutt et al., J. Immunol. 147:60 (1991).

[0498] Also included herein are engineered antibodies having three or more functional antigen-binding sites, including "octopus antibodies" (see, eg, US 2006 / 0025576A1).

[0499] The antibodies or fragments herein also include "dual-acting Fabs" or "DAFs," which comprise an antigen binding site that binds CTLA-4 as well as another, different antigen (see, eg, US 2008 / 0069820).

[0500] 7. Antibody variants

[0501] In certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be necessary to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues in the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be performed to obtain the final construct, provided that the final construct has desired characteristics, such as antigen binding.

[0502] a) Substitution, insertion, and deletion variants

[0503] In certain embodiments, antibody variants with one or more amino acid substitutions are provided. The target sites for substitution mutations include HVRs and FRs. Conservative substitutions are shown in Table 1 under the heading "Preferred Substitutions." More substantial changes are provided in Table 1 under the heading "Exemplary Substitutions," and are further described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest, and the product can be screened for desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0504] [Table 1]

[0505]

[0506] Amino acids can be divided into several groups based on the common properties of their side chains:

[0507] (1) Hydrophobicity: norleucine, methionine (Met), alanine (Ala), valine (Val), leucine (Leu) and isoleucine (Ile);

[0508] (2) Neutral and hydrophilic: cysteine ​​(Cys), serine (Ser), threonine (Thr), asparagine (Asn) and glutamine (Gln);

[0509] (3) Acidic: aspartic acid (Asp) and glutamic acid (Glu);

[0510] (4) Basic: histidine (His), lysine (Lys) and arginine (Arg);

[0511] (5) residues that affect chain orientation: glycine (Gly) and proline (Pro); and

[0512] (6) Aromatic: tryptophan (Trp), tyrosine (Tyr) and phenylalanine (Phe).

[0513] Non-conservative substitutions involve exchanging a member of one of these classes for a member of another class.

[0514] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Typically, the resulting variant selected for further study will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and / or will substantially retain certain biological properties of the parent antibody. Exemplary substitutional variants are affinity-matured antibodies, which can be conveniently produced, for example, using phage display-based affinity maturation techniques, such as those described herein. Briefly, one or more HVR residues are mutated, and the variant antibodies are displayed on phage and screened for a specific biological activity (e.g., binding affinity).

[0515] Changes (e.g., substitutions) can be made in the HVRs, for example, to improve antibody affinity. Such changes can be made in HVR "hotspots," i.e., residues encoded by codons that undergo high-frequency mutation during somatic maturation (see, e.g., Chowdhury, Methods Mol. Bol. 207: 179-196 (2008)), and / or residues that contact the antigen, with the resulting variant VH or VL tested for binding affinity. Affinity maturation by construction and reselection from secondary libraries has been described, for example, in Hoogenboom et al., Methods in Molecular Biology 178: 1-37 (O'Brien et al., eds., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another approach to introducing diversity involves HVR-directed approaches, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. For example, alanine scanning mutagenesis or modeling can be used to specifically identify HVR residues involved in antigen binding. In particular, CDR-H3 and CDR-L3 are frequently targeted.

[0516] In certain embodiments, substitutions, insertions or deletions may occur within one or more HVRs, as long as such changes do not substantially reduce the ability of the antibody to bind to antigens. For example, conservative changes that do not significantly reduce binding affinity (e.g., conservative substitutions provided herein) may be made in HVRs. For example, such changes may be outside the antigen contact residues in the HVRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR is either unchanged or contains no more than one, two or three amino acid substitutions.

[0517] As described in Cunningham and Wells (1989) Science, 244: 1081-1085, a useful method for identifying residues or regions of antibodies that can be targeted for mutagenesis is referred to as "alanine scanning mutagenesis." In this method, an identification residue or a group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) is replaced with a neutral or negatively charged amino acid (e.g., alanine or phenylalanine) to determine whether the interaction of the antibody with the antigen is affected. Further replacements can be introduced at amino acid positions that demonstrate functional sensitivity to the initial replacement. Alternatively or in addition, the crystal structure of the antigen-antibody complex can be analyzed to identify contact points between the antibody and the antigen. This type of contact residue and adjacent residue can be targeted or eliminated as a candidate for replacement. Variants can be screened to determine whether they contain desired characteristics.

[0518] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion of an enzyme (e.g., for ADEPT) or a polypeptide that increases the plasma half-life of the antibody to the N- or C-terminus of the antibody.

[0519] b) Glycosylation variants

[0520] In certain embodiments, the antibodies provided herein are altered to increase or decrease the extent to which the antibodies are glycosylated. Addition or deletion of glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0521] In the case where the antibody comprises an Fc region, the carbohydrates attached thereto can be changed. Natural antibodies produced by mammalian cells typically comprise branched biantennary oligosaccharides, which are typically attached to Asn297 of the CH2 domain of the Fc region via an N-linked bond. See, for example, Wright et al., TIBTECH 15: 26-32 (1997). Oligosaccharides can include a variety of carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in the antibodies of the present invention can be modified to produce antibody variants with certain improved properties.

[0522] In one embodiment, antibody variants are provided that have carbohydrate structures that lack fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the carbohydrate chain at Asn297 relative to the sum of all carbohydrate structures (e.g., complex, hybrid, and high-mannose structures) attached to Asn297 as determined by MALDI-TOF mass spectrometry, for example, as described in WO 2008 / 077546. Asn297 refers to the asparagine residue located at approximately position 297 in the Fc region (EU numbering of Fc region residues); however, due to minor sequence variation in antibodies, Asn297 may also be located approximately + / - 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylated variants may have improved ADCC function. See, for example, U.S. Patent Publication Nos. US 2003 / 0157108 (Presta, L.); US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include: US 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US 2003 / 0115614; US 2002 / 0164328; US 2004 / 0093621; US ​​2004 / 0132140; US 2004 / 0110704; US 2004 / 0110282; US 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO2005 / 053742; WO2002 / 031140; Okazaki et al., J. Mol. Biol. 336: 1239-1249 (2004); Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614 (2004).Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells deficient in protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249: 533-545 (1986); U.S. Patent Application No. US 2003 / 0157108 A1, Presta, L; and WO 2004 / 056312 A1, Adams et al., particularly in Example 11), and knockout cell lines, such as α1,6-fucosyltransferase gene FUT8 knockout CHO cells (see, e.g., Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4): 680-688 (2006); and WO 2003 / 085107).

[0523] Antibody variants are also provided with bisected oligosaccharides, for example, wherein a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US 2005 / 0123546 (Umana et al.). Antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0524] c) Fc region variants

[0525] In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0526] In certain embodiments, the present invention contemplates antibody variants that possess some, but not all, effector functions, making them ideal candidates for applications where the in vivo half-life of the antibody is important but certain effector functions (such as complement and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduction / depletion of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and therefore likely lacks ADCC activity) but retains FcRn binding ability. NK cells, the primary cells that mediate ADCC, express only FcγRIII, while 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: 457-492 (1991). Non-limiting examples of in vitro assays for evaluating ADCC activity of a molecule of interest are described in U.S. Pat. Nos. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83: 7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat-7063 (1986) 82: 1499-1502 (1985); 5,821,337 (see, e.g., Bruggemann, M. et al., J. Exp. Med. 166: 1351-1361 (1987)). Alternatively, non-radioactive assays can be used (see, e.g., ACT1 for flow cytometry). TMNon-radioactive cytotoxicity assays (CellTechnology, Inc. Mountain View, CA; and CytoTox 96 (registered trademark) non-radioactive cytotoxicity assays (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the molecule of interest can be assessed in vivo, for example, in the animal models disclosed in Clynes et al., Proc. Nat'l Acad. Sci. USA 95: 652-656 (1998). C1q binding assays can also be performed to confirm that the antibody cannot bind to C1q and, therefore, lacks CDC activity. See, e.g., C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202: 163). (1996); Cragg, MS et al., Blood 101: 1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103: 2738-2743 (2004). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, for example, Petkova, SB et al., Int'l. Immunol. 18 (12): 1759-1769 (2006)).

[0527] Antibodies with reduced effector function include those in which one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 are substituted ( U.S. Pat. No. 6,737,056 ). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant in which residues 265 and 297 are substituted with alanine ( U.S. Pat. No. 7,332,581 ).

[0528] Certain antibody variants with increased or decreased binding to FcRs have been described (see, e.g., U.S. Patent No. 6,737,056; WO 2004 / 056312 and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001)).

[0529] In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, eg, substitutions at positions 298, 333, and / or 334 of the Fc region (EU numbering of residues).

[0530] In some embodiments, alterations are made in the Fc region that result in altered (i.e., increased or decreased) Clq binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol. 164: 4178-4184 (2000).

[0531] Antibodies with increased half-life and increased binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117: 587 (1976); and Kim et al., J. Immunol. 24: 249 (1994)), are described in US 2005 / 0014934A1 (Hinton et al.). These antibodies comprise an Fc region having one or more substitutions therein that increase binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424 or 434, for example, substitution of Fc region residue 434 ( U.S. Pat. No. 7,371,826 ).

[0532] For further examples of Fc region variants, see Duncan & Winter, Nature 322: 738-740 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351.

[0533] d) Cysteine-engineered antibody variants

[0534] In certain embodiments, it is desirable to produce cysteine ​​engineered antibodies, such as "thioMAbs," in which one or more residues of an antibody are substituted with cysteine ​​residues. In specific embodiments, the substituted residues occur at accessible sites of the antibody. By replacing those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to produce immunoconjugates, as further described herein. In certain embodiments, any one or more of the following residues can be substituted with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; S400 (EU numbering) of the heavy chain Fc region. Cysteine ​​engineered antibodies can be produced as described, for example, in U.S. Patent No. 7,521,541.

[0535] e) Antibody derivatives

[0536] In certain embodiments, the antibodies provided herein can be further modified to contain additional non-proteinaceous moieties that are known in the art and readily available. Suitable moieties for derivatizing antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol / propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone) polyethylene glycol, polypropylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer can have any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody may vary; if more than one polymer is attached, they may be the same or different molecules. In general, the amount and / or type of polymer used for derivatization can be determined based on considerations including, but not limited to, the specific property or function of the antibody to be improved, whether the antibody derivative will be used therapeutically under defined conditions, etc.

[0537] In another embodiment, a conjugate of an antibody and a non-protein moiety is provided that can be selectively heated by exposure to radiation. In one embodiment, the non-protein moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation can be of any wavelength and includes, but is not limited to, wavelengths that do not harm normal cells but heat the non-protein moiety to a temperature close to that at which the antibody-non-protein moiety cell is killed.

[0538] The anti-CTLA-4 antibodies herein can be combined with various existing technologies. A non-limiting embodiment of the combination with the technology is, for example, the use of anti-CTLA-4 antibodies to produce cells expressing chimeric antigen receptors (CARs). Examples of cells herein include T cells, γδ T cells, NK cells, NKT cells, cytokine-induced killer (CIK) cells, and macrophages (Int J Mol Sci. (2019) 20(11), 2839, Nat Rev Drug Discov. (2020) 19(5), 308). A non-limiting example of a method for producing CAR-expressing T cells (CAR-T) includes a method of introducing CAR into effector cells such as T cells by genetic modification technology, wherein the CAR comprises an antigen binding domain (e.g., scFv) of an anti-CTLA-4 antibody, a transmembrane domain of a TCR, and a signaling domain of a co-stimulatory molecule such as CD28 to enhance T cell activation.

[0539] Non-limiting examples of technologies that can be combined with the anti-CTLA-4 antibodies herein include the use of anti-CTLA-4 antibodies to produce T cell redirecting antibodies (Nature (1985) 314 (6012), 628-31, Int J Cancer (1988) 41 (4), 609-15, Proc Natl Acad Sci USA (1986) 83 (5), 1453-7). A non-limiting embodiment of the T cell redirecting antibody is a bispecific antibody comprising: a binding domain for any subunit of the T cell receptor (TCR) complex on T cells, particularly a binding domain for the CD3 epsilon chain of CD3; and an antigen binding domain of an anti-CTLA-4 antibody.

[0540] B. Recombinant Methods and Compositions

[0541] Antibodies can be produced using recombinant methods and compositions, for example, as described in U.S. Patent No. 4,816,567. In one embodiment, isolated nucleic acids encoding the anti-CTLA-4 antibodies described herein are provided. Such nucleic acids may encode an amino acid sequence comprising the VL of the antibody and / or an amino acid sequence comprising the VH (e.g., the light chain and / or heavy chain of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In a further embodiment, host cells comprising such nucleic acids are provided. In one such embodiment, the host cell comprises (e.g., has been used for): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody, and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., a Y0, NS0, Sp2 / 0 cell). In one embodiment, a method of making an anti-CTLA-4 antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding an antibody provided above under conditions suitable for expression of the anti-CTLA-4 antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0542] For recombinant production of anti-CTLA-4 antibodies, nucleic acid encoding the antibody is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell, e.g., as described above. Such nucleic acid can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the antibody heavy and light chains).

[0543] Suitable host cells for cloning or expressing vectors encoding antibodies include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523 (see also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, describing expression of antibody fragments in E. coli). After expression, the antibodies can be isolated from the bacterial cell pellet as a soluble fraction and can be further purified.

[0544] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody encoding vectors, including fungi and yeast strains whose glycosylation pathways have been "humanized" to produce antibodies with partially or fully human glycosylation patterns. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004), and Li et al., Nat. Biotech. 24: 210-215 (2006).

[0545] Suitable host cells for expressing glycosylated antibodies also originate from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. A number of baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0546] Plant cell cultures can also be used as hosts. See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing plant cell cultures for producing antibodies in transgenic plants). TM technology).

[0547] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for growth in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 cell line transformed by SV40 (COS-7); human embryonic kidney line (e.g., 293 or 293 cells as described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells as described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human hepatocytes (HepG2); mouse mammary tumor (MMT 060562); TRI cells as described in Mather et al., Annals NY Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR -CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines, such as Y0, NS0, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0548] Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. Bifunctional or derivatizing agents such as maleimidobenzoylsulfosuccinimide ester (conjugated through cysteine ​​residues), N-hydroxysuccinimide (conjugated through lysine residues), glutaraldehyde, succinic anhydride, SOCl2 or R 1 N=C=NR (where R and R 1 It may be useful to conjugate the relevant antigen to a protein that is immunogenic in the species to be immunized, such as keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor.

[0549] An animal (typically a non-human mammal) is immunized with the antigen, immunogenic conjugate, or derivative by combining, for example, 100 μg or 5 μg of the protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later, the animal is boosted with 1 / 5 to 1 / 10 the initial amount of the peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. After 7 to 14 days, blood is drawn from the animal and the serum is assayed for antibody titers. The animal is boosted until the titer plateaus. Preferably, the animal is boosted with the same antigen, but conjugated to a different protein and / or via a different cross-linking agent. Conjugates can also be prepared as protein fusions in recombinant cell culture. In addition, aggregating agents such as alum are also suitable for enhancing the immune response.

[0550] A monoclonal antibody is obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in minor amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies.

[0551] For example, monoclonal antibodies can be prepared using the hybridoma method first described by Kohler et al., Nature 256(5517): 495-497 (1975). In the hybridoma method, a mouse or other suitable host animal, such as a hamster, is immunized as described above to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to the protein used for immunization. Alternatively, lymphocytes can be immunized in vitro.

[0552] Immunizing agents typically include antigenic proteins or fusion variants thereof. Typically, if cells of human origin are required, peripheral blood lymphocytes (PBLs) are used, and if non-human mammalian origin is required, spleen cells or lymph node cells are used. Lymphocytes are then fused with immortalized cell lines using a suitable fusing agent such as polyethylene glycol to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986), pp. 59-103).

[0553] Immortalized cell lines are typically transformed mammalian cells, particularly myeloma cells of rodent, cattle, and human origin. Typically, rat or mouse myeloma cell lines are used. The hybridoma cells thus prepared are inoculated and cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium used for hybridomas will typically contain hypoxanthine, aminopterin, and thymidine (HAT culture medium), which is a substance that prevents the growth of HGPRT-deficient cells.

[0554] Preferred immortalized myeloma cells are those cells that effectively fuse, support the selected antibody producing cells to stably produce antibodies at high levels and are sensitive to culture medium such as HAT culture medium. Among them, preferred are mouse myeloma lines, such as the MOPC-21 and MPC-11 mouse tumors derived from the Salk Institute Cell Distribution Center, San Diego, California USA, and the SP-2 cells (and derivatives thereof, e.g., X63-Ag8-653) available from the American Type Culture Collection, Manassas, Virginia USA. Human myeloma and mouse-human heteromyeloma cell lines have also been described for producing human monoclonal antibodies (Kozbor et al., J. Immunol. 133 (6): 3001-3005 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, 51-63 pages (1987)).

[0555] The production of monoclonal antibodies against the antigen in the culture medium in which the hybridoma cells are grown is measured. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art. For example, binding affinity can be determined by Scatchard analysis of Munson, Anal. Biochem. 107(1): 220-239 (1980).

[0556] After identifying hybridoma cells that produce antibodies with the desired specificity, affinity, and / or activity, the clones can be subcloned by limiting dilution procedures and cultured by standard methods (Goding, supra). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, hybridoma cells can be grown as tumors in mammals.

[0557] The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0558] Antibodies can be produced by immunizing an appropriate host animal with an antigen. In one embodiment, the antigen is a polypeptide comprising full-length CTLA-4. In one embodiment, the antigen is a polypeptide comprising soluble CTLA-4. In one embodiment, the antigen is a polypeptide comprising a region corresponding to amino acids from position 97 (Glu) to position 106 (Leu) of human CTLA-4 (extracellular domain, SEQ ID NO: 28). In one embodiment, the antigen is a polypeptide comprising a region corresponding to amino acids from position 99 (Met) to position 106 (Leu) of human CTLA-4 (extracellular domain, SEQ ID NO: 28). Also included in the present invention are antibodies produced by immunizing an animal with an antigen. As described above in "Exemplary anti-CTLA-4 antibodies," the antibodies may bind to any of the features alone or in combination.

[0559] C. Determination

[0560] The anti-CTLA-4 antibodies provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activities by various assays known in the art.

[0561] 1. Binding assays and other assays

[0562] In one aspect, the antigen binding activity of the antibodies of the invention is tested, for example, by known methods such as ELISA, Western blot, surface plasmon resonance assay, and the like.

[0563] In another aspect, competition assays can be used to identify antibodies that compete with the anti-CTLA-4 antibodies described herein (e.g., the anti-CTLA-4 antibodies described in Table 17, Table 22, Table 27, and Table 32) for binding to CTLA-4. In certain embodiments, if such competing antibodies are present in excess, binding of the reference antibody to CTLA-4 is prevented (e.g., reduced) by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more. In some embodiments, binding is prevented by at least 80%, 85%, 90%, 95%, or more. In certain embodiments, such competing antibodies bind to the same epitope (e.g., a linear or conformational epitope) as the epitope bound by the anti-CTLA-4 antibodies described herein (e.g., the anti-CTLA-4 antibodies described in Table 17, Table 22, Table 27, and Table 32). Detailed exemplary methods for mapping epitopes to which antibodies bind are provided in Morris (1996) "Epitope Mapping Protocols," Methods in Molecular Biology vol 66 (Humana Press, Totowa, NJ).

[0564] In an exemplary competition assay, immobilized CTLA-4 is incubated in a solution containing a first labeled antibody that binds to CTLA-4 and a second unlabeled antibody that is being tested for its ability to compete with the first antibody for binding to CTLA-4. The second antibody can be present in the hybridoma supernatant. As a control, immobilized CTLA-4 is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. Following incubation under conditions permissive for binding of the first antibody to CTLA-4, excess unbound antibody is removed, and the amount of label bound to the immobilized CTLA-4 is determined. If the amount of label bound to the immobilized CTLA-4 in the test sample is significantly reduced compared to the control sample, this indicates that the second antibody is competing with the first antibody for binding to CTLA-4. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0565] 2. Activity Assay

[0566] In one aspect, assays are provided for identifying anti-CTLA-4 antibodies that have biological activity. Biological activity can include, for example, cell proliferation inhibition activity, cytotoxic activity (e.g., ADCC / CDC activity and ADCP activity), immunostimulatory activity, and CTLA-4 inhibitory activity. Antibodies having such biological activity in vivo and / or in vitro are also provided.

[0567] In certain embodiments, the antibodies of the invention are tested for such biological activities.

[0568] In certain embodiments, the antibodies of the invention are tested for their ability to inhibit cell growth or proliferation in vitro. Assays for inhibiting cell growth or proliferation are well known in the art. Certain cell proliferation assays, such as the "cell killing" assays described herein, measure cell viability. One such assay is the CellTiter-Glo TMLuminescent Cell Viability Assay, which is commercially available from Promega (Madison, WI). This assay determines the number of viable cells in the culture based on the quantification of the ATP present, which is an indication of metabolically active cells. See Crouch et al. (1993) J. Immunol. Meth. 160: 81-88, US Pat. No. 6602677. The assay can be performed in 96-well or 384-well formats, making it suitable for automated high-throughput screening (HTS). See Cree et al. (1995) AntiCancer Drugs 6: 398-404. The assay procedure involves adding a single reagent (CellTiter-Glo (registered trademark) reagent) directly to the cultured cells. This results in cell lysis and the generation of a luminescent signal generated by the luciferase reaction. The luminescent signal is proportional to the amount of ATP present, which is proportional to the number of viable cells present in the culture. The data can be recorded by a luminometer or CCD camera imaging device. The luminescent output is expressed as relative light units (RLU).

[0569] Another assay for cell proliferation is the "MTT" assay, which is a colorimetric assay that measures the oxidation of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to formazan by mitochondrial reductases. TM As with the CAR T cell assay, this assay indicates the number of metabolically active cells present in a cell culture. See, for example, Mosmann (1983) J. Immunol. Meth. 65: 55-63, and Zhang et al. (2005) Cancer Res. 65: 3877-3882.

[0570] Cells used in any of the above in vitro assays include cells or cell lines that naturally express CTLA-4 or have been engineered to express CTLA-4. Such cells also include cell lines that express CTLA-4 and cell lines that do not normally express CTLA-4 but have been transfected with nucleic acid encoding CTLA-4.

[0571] In one aspect, the ability of anti-CTLA-4 antibodies to inhibit cell growth or proliferation in vivo is tested. In certain embodiments, the ability of anti-CTLA-4 antibodies to inhibit tumor growth in vivo is tested. In vivo model systems, such as xenograft models, can be used for such testing. In an exemplary xenograft system, human tumor cells are introduced into a suitably immunocompromised non-human animal, such as an athymic "nude" mouse. The antibodies of the invention are administered to the animal. The ability of the antibody to inhibit or reduce tumor growth is determined. In certain embodiments of the above-described xenograft systems, the human tumor cells are tumor cells from a human patient. Such xenograft models are commercially available from Oncotest GmbH (Frieberg, Germany). In certain embodiments, human tumor cells are introduced into a suitably immunocompromised non-human animal by subcutaneous injection or by transplantation into a suitable site, such as the mammary fat pad.

[0572] It will be understood that any of the above assays can be performed using the immunoconjugates of the invention in place of or in addition to anti-CTLA-4 antibodies.

[0573] Typical assays used to measure ADCC activity of therapeutic antibodies are based on 51 Cr release assay, and includes the following steps: [ 51 Target cells are labeled with [Cr]Na2CrO4; target cells expressing an antigen on their cell surface are opsonized with an antibody; the opsonized radiolabeled target cells are combined with effector cells at an appropriate ratio in a microtiter plate in the presence or absence of the test antibody; the cell mixture is incubated at 37°C for 16 to 18 hours; the supernatant is collected; and the radioactivity in the supernatant sample is analyzed. The cytotoxicity of the test antibody is then determined, for example, using the following equation: Specific cytotoxicity (%) = (radioactivity in the presence of antibody - radioactivity in the absence of antibody) / (maximum radioactivity - radioactivity in the absence of antibody) × 100. Graphs can be generated by varying the ratio of target cells to effector cells or the antibody concentration.

[0574] To assess complement activation, a complement-dependent cytotoxicity (CDC) assay can be performed, as described, for example, in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996). Briefly, various concentrations of polypeptide variants and human complement are diluted with buffer. Cells expressing an antigen that binds to the polypeptide variant are diluted to approximately 1×10 6The density of cells / ml was 200 μL. A mixture of peptide variants, diluted human complement, and antigen-expressing cells was added to a flat-bottomed 96-well tissue culture plate and incubated at 37°C and 5% CO2 for 2 hours to promote complement-mediated cell lysis. Then, 50 μL of AlamarBlue (Accumed International) was added to each well and incubated overnight at 37°C. Absorbance was measured using a 96-well fluorimeter with excitation at 530 nm and emission at 590 nm. The results are expressed in relative fluorescence units (RFU). Sample concentrations can be calculated from the standard curve, and the percentage of activity compared to the non-variant peptide is reported for the peptide variant of interest.

[0575] An exemplary assay for ADCP activity can include the following: coating target bioparticles, such as E. coli labeled with FITC (Molecular Probes) or Staphylococcus aureus-FITC, with the test antibody; forming opsonized particles; adding the opsonized particles to THP-1 effector cells (a monocytic cell line available from ATCC) at a ratio of 1:1, 10:1, 30:1, 60:1, 75:1, or 100:1 to induce FcγR-mediated phagocytosis; preferably incubating the cells and E. coli-FITC / antibody at 37°C for 1.5 hours; following the incubation, adding trypan blue to the cells (preferably at room temperature for two to three minutes) to quench the fluorescence of bacteria that have not yet incorporated into the cells and are attached to the exterior of the cell surface; transferring the cells to FACS buffer (e.g., 0.1% BSA and 0.1% sodium azide in PBS) for analysis using a FACS (e.g., BD FACS). Fluorescence of THP-1 cells is measured using a Calibur. To determine the extent of ADCP, a gate is preferably set on the THP-1 cells and the median fluorescence intensity is determined. In the most preferred embodiment, ADCP assays are performed using E. coli-FITC in culture medium (control); E. coli-FITC and THP-1 cells (for FcγR-independent ADCP activity); and E. coli-FITC, THP-1 cells, and a test antibody (for FcγR-dependent ADCP activity).

[0576] The cytotoxic activity of an antibody generally involves binding of the antibody to the cell surface. Whether the antigen is expressed on the surface of the target cell can be appropriately confirmed by methods known to those skilled in the art, such as FACS.

[0577] The activation of immunity can be detected by using cellular or humoral immune responses as indicators. The activation of immunity specifically includes increased expression levels of cytokines (e.g., IL-6, G-CSF, IL-12, TNF1, and IFN1) or their receptors, promoting the proliferation of immune cells (e.g., B cells, T cells, NK cells, macrophages, and monocytes), increased activation state, increased function, and enhanced cytotoxic activity. In particular, T cell activation can be detected by measuring the increased expression of activation markers such as CD25, CD69, and ICOS. For example, it is known that patients who have been administered the anti-CTLA-4 antibody ipilimumab have increased ICOS in their peripheral blood after administration. + CD4 + T cells, which is considered to be the effect of systemic immune status activation by administration of anti-CTLA-4 antibodies (Cancer Immunol. Res. (2013) 1(4): 229-234).

[0578] T cell activation requires not only stimulation via the antigen receptor (TCR) but also auxiliary stimulation via CD28. When CD28 on the T cell surface binds to B7-1 (CD80) or B7-2 (CD86) on the surface of antigen-presenting cells, the auxiliary signal is transmitted to the T cell, leading to T cell activation. On the other hand, CTLA-4 is expressed on the surface of activated T cells. Because CTLA-4 binds to CD80 and CD86 with a stronger affinity than CD28, it interacts with CD80 and CD86 in preference to CD28, resulting in inhibition of T cell activation.

[0579] Based on this mechanism of action, inhibitory activity against CTLA-4 can be measured as the activity of inhibiting CTLA-4 binding to CD80 or CD86. In one embodiment, an assay for measuring inhibitory activity against CTLA-4 comprises the following steps: binding purified CTLA-4 protein to a support such as a microtiter plate or magnetic beads; adding a test antibody and labeled soluble CD80 or CD86; washing away unbound components; and quantifying bound labeled CD80 or CD86. Whether the test antibody cross-reacts with CD28 can be confirmed by using a similar assay in which CD28 replaces CTLA-4. In another embodiment, a functional assay for detecting T cell activation as described above can also be used to measure inhibitory activity against CTLA-4. For example, when a test antibody with CTLA-4 inhibitory activity is added to a system in which T cell activation is measured by stimulating a T cell population with cells expressing CD80 or CD86, T cell activation is further enhanced.

[0580] D. Immunoconjugates

[0581] The invention also provides immunoconjugates comprising an anti-CTLA-4 antibody herein conjugated to one or more cytotoxic agents, e.g., chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes.

[0582] In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC) in which the antibody is conjugated to one or more drugs, including but not limited to maytansine (see U.S. Pat. Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 B1); an auristatin, such as the monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483 and 5,780,588 and 7,498,298); dolastatin; calicheamicin or a derivative thereof (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001 and 5,877,296; Hinman et al., Cancer Res. 53: 3336-3342 (1993); and Lode et al., Cancer Res. 58: 2925-2928 (1998)); anthracyclines such as daunomycin or doxorubicin (see Kratz et al., Current Med. Chem. 13: 477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16: 358-362 (2006); Torgov et al., Bioconj. Chem. 16: 717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97: 829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12: 1529-1532 (2002); King et al., J. Med. Chem. 45:4336-4343 (2002); and U.S. Patent No. 6,630,579); methotrexate; vindesine; taxanes, such as docetaxel, paclitaxel, larotaxel, tecitaxel, and otatataxel; trichothecenes; and CC1065.

[0583] In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria A chain, a nonbinding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α-sarcin, Aleurites fordii proteins, dianthin, Phytolacca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, saponaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the trichothecenes.

[0584] In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes can be used to produce radioconjugates. Examples include 211 At 131 I. 125 I. 90 Y. 186 Re、 188 Re、 153 Sm, 212 Bi, 32 P. 212 When the radioconjugate is used for detection, it may contain a radioactive atom such as Tc-99m or 123 I, or spin labels for nuclear magnetic resonance (NMR) imaging (also called magnetic resonance imaging, MRI), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

[0585] Conjugates of antibodies and cytotoxic agents can be prepared using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimide HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionuclides to antibodies. See WO 94 / 11026. The linker can be a "cleavable linker" that promotes release of the cytotoxic drug. For example, an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker can be used (Chari et al., Cancer Res. 52: 127-131 (1992); U.S. Patent No. 5,208,020).

[0586] The immunoconjugates or ADCs herein specifically contemplate, but are not limited to, such conjugates prepared with cross-linking reagents including, but not limited to, commercially available BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB and SVSB (succinimidyl-(4-vinylsulfone)benzoate) (e.g., from Pierce Biotechnology, Inc., Rockford, IL., USA).

[0587] E. Methods and Compositions for Diagnosis and Detection

[0588] In certain embodiments, any of the anti-CTLA-4 antibodies provided herein can be used to detect the presence of CTLA-4 in a biological sample. As used herein, the term "detection" includes quantitative or qualitative detection. In certain embodiments, biological samples include cells or tissues, such as serum, whole blood, plasma, biopsy samples, tissue samples, cell suspensions, saliva, sputum, oral fluid, cerebral fluid, amniotic fluid, ascites, milk, colostrum, mammary secretions, lymph, urine, sweat, tears, gastric juice, synovial fluid, ascites, ocular fluid, and mucus.

[0589] In one embodiment, an anti-CTLA-4 antibody for use in a diagnostic or detection method is provided. In other aspects, a method for detecting the presence of CTLA-4 in a biological sample is provided. In certain embodiments, the method comprises contacting the biological sample with an anti-CTLA-4 antibody as described herein under conditions that allow the anti-CTLA-4 antibody to bind to CTLA-4, and detecting whether a complex is formed between the anti-CTLA-4 antibody and CTLA-4. This method can be an in vitro or in vivo method. In one embodiment, the anti-CTLA-4 antibody is used to select a subject suitable for treatment with the anti-CTLA-4 antibody, for example, where CTLA-4 is a biomarker for selecting a patient.

[0590] The antibodies of the present invention can be used, for example, to examine the state of immune response and diagnose immune system dysfunction.

[0591] In certain embodiments, labeled anti-CTLA-4 antibodies are provided. Labels include, but are not limited to, directly detectable labels or moieties (e.g., fluorescent, chromophore, electron-dense, chemiluminescent, and radiolabeled), as well as indirect detection, for example, moieties detected by enzymatic reactions or molecular interactions, such as enzymes or ligands. Exemplary labels include, but are not limited to, radioisotopes. 32 P. 14 C. 125 I. 3 H and 131 I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases such as firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, sugar oxidases such as glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, those coupled to enzymes that use hydrogen peroxide to oxidize dye precursors (such as HRP, lactoperoxidase or microperoxidase), biotin / avidin, spin labels, phage labels, stable free radicals, and the like.

[0592] F. Pharmaceutical Preparations

[0593] Pharmaceutical formulations of the anti-CTLA-4 antibodies described herein are prepared by mixing such antibodies of the desired purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. ed. (1980)) in the form of a lyophilized formulation or an aqueous solution. Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., zinc-protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersants such as soluble neutral active hyaluronidase glycoprotein (sHASEGP). Certain exemplary sHASEGPs and methods of use are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinase.

[0594] Exemplary lyophilized antibody formulations are described in US Patent No. 6,267,958. Aqueous antibody formulations include those described in US Patent No. 6,171,586 and WO 2006 / 044908, the latter formulations including a histidine-acetate buffer.

[0595] As necessary for the particular indication being treated, the formulations herein may also contain more than one active ingredient, preferably those with complementary activities that do not adversely affect each other. Such active ingredients are suitably present in combination in amounts that are effective for the intended purpose.

[0596] The active ingredient can be embedded in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, such as hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions, respectively. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. ed. (1980).

[0597] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, eg, films, or microcapsules.

[0598] Formulations for in vivo administration are generally sterile. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes.

[0599] G. Methods of Treatment and Compositions

[0600] Any of the anti-CTLA-4 antibodies provided herein can be used in therapeutic methods.

[0601] In one aspect, anti-CTLA-4 antibodies for use as a medicament are provided. In other aspects, anti-CTLA-4 antibodies for use in treating tumors are provided. In certain embodiments, anti-CTLA-4 antibodies for use in methods of treatment are provided. In certain embodiments, the present invention provides anti-CTLA-4 antibodies for use in methods of treating an individual with a tumor, the method comprising administering to the individual an effective amount of an anti-CTLA-4 antibody. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below. In other embodiments, the present invention provides anti-CTLA-4 antibodies for use in damaging cells. In certain embodiments, the present invention provides anti-CTLA-4 antibodies for use in methods of damaging cells in an individual, the method comprising administering to the individual an effective amount of an anti-CTLA-4 antibody to damage the cells. In other embodiments, the present invention provides anti-CTLA-4 antibodies for use in immune activation. In certain embodiments, the present invention provides anti-CTLA-4 antibodies for use in methods of activating immunity in an individual, the method comprising administering to the individual an effective amount of an anti-CTLA-4 antibody to activate immunity. The "individual" according to any of the above embodiments is preferably a human.

[0602] In some embodiments, the tumor is a solid tumor. In solid tumors, tumor cells usually proliferate to form colonies, and tumor tissue is mainly formed by these cells. In addition, tumor tissue in living organisms is often infiltrated by immune cells such as lymphocytes, which also constitute a part of tumor tissue. In some embodiments, tumor tissue is infiltrated by immune cells, particularly regulatory T (Treg) cells. In one embodiment, damage to cells is caused by ADCC activity, CDC activity or ADCP activity. In one embodiment, cells expressing CTLA-4 on their cell surface are damaged. In other embodiments, cells to be damaged are Treg cells. In certain embodiments, Treg cells that have infiltrated into tumor tissue are damaged. In one embodiment, immunity is activated by damage to Treg cells (immunosuppression of Treg cells is cancelled). In other embodiments, immunity (particularly anti-tumor immunity) in tumor tissue is activated. In some embodiments, immune activation is T cell activation.

[0603] In other aspects, the extent of the effect produced by the anti-CTLA-4 antibodies of the present invention as drugs varies depending on the tissue of the individual. In certain embodiments, the extent of the effect varies depending on the concentration of adenosine-containing compounds in the tissue. In further embodiments, the effect is increased in tissues with high concentrations of adenosine-containing compounds compared to tissues with low concentrations of adenosine-containing compounds. Tissues with high concentrations of adenosine-containing compounds include, for example, tumor tissue. Tissues with low concentrations of adenosine-containing compounds include, for example, non-tumor tissues such as normal tissue. In some embodiments, immunity is more strongly activated in tumor tissue than in non-tumor tissue. This difference in response need not be observed for all doses of the anti-CTLA-4 antibody, but only for a specific dose range. In another embodiment, immunity is activated in tumor tissue at a lower dose than in non-tumor tissue. Furthermore, in another embodiment, the therapeutic effect is observed at a dose below that at which side effects are observed. In certain embodiments, the therapeutic effect is the expression of an anti-tumor effect (e.g., tumor regression and induction of cell death or inhibition of tumor cell growth) and the side effect is the development of an autoimmune disease (including damage to normal tissue due to an excessive immune response).

[0604] On the other hand, the extent of the drug effect produced by the anti-CTLA-4 antibody of the present invention varies depending on whether it has an adenosine-containing compound-dependent binding activity to CTLA-4 (i.e., varies depending on the concentration of the adenosine-containing compound). In some embodiments, the anti-CTLA-4 antibody of the present invention is an antibody whose binding activity to CTLA-4 increases with increasing concentration of the adenosine-containing compound. In some embodiments, the control anti-CTLA-4 antibody is an antibody that does not have CTLA-4 binding activity that is dependent on the concentration of the adenosine-containing compound. In certain embodiments, an antibody that does not have CTLA-4 binding activity that is dependent on the concentration of the adenosine-containing compound is an antibody in which the difference in CTLA-4 binding activity in the presence and absence of the compound is, for example, less than 2-fold, less than 1.8-fold, less than 1.5-fold, less than 1.3-fold, less than 1.2-fold, or less than 1.1-fold. It is desirable that the anti-CTLA-4 antibody of the present invention and the control anti-CTLA-4 antibody have approximately the same CTLA-4 binding activity as each other in the presence of a sufficient amount of the adenosine-containing compound.

[0605] In certain aspects, the anti-CTLA-4 antibodies of the present invention and a control anti-CTLA-4 antibody differ in the effects produced by each antibody as a drug. In certain embodiments, they differ in their effects as drugs in tissues with low concentrations of adenosine-containing compounds. Tissues with low concentrations of adenosine-containing compounds include, for example, non-tumor tissues such as normal tissue. The anti-CTLA-4 antibodies can also be provided as pharmaceutical formulations comprising the antibodies. In some embodiments, in tissues with low concentrations of adenosine-containing compounds, the anti-CTLA-4 antibodies of the present invention exhibit lower levels of immune activation compared to a control anti-CTLA-4 antibody. In some embodiments, in tissues with low concentrations of adenosine-containing compounds, the dose of the anti-CTLA-4 antibody of the present invention required for immune activation is higher than that of the control anti-CTLA-4 antibody. In some embodiments, in tissues with low concentrations of adenosine-containing compounds, the anti-CTLA-4 antibodies of the present invention have lower levels of side effects compared to a control anti-CTLA-4 antibody. In some embodiments, in tissues with low concentrations of adenosine-containing compounds, the dose of the anti-CTLA-4 antibody of the present invention required for observed side effects is higher than that of the control anti-CTLA-4 antibody. This difference in response need not be observed in all tissues (e.g., all tissues with low concentrations of adenosine-containing compounds), but only in some tissues. In certain embodiments, the side effect is an autoimmune disease (including damage to normal tissues due to an excessive immune response).

[0606] In certain aspects, the anti-CTLA-4 antibodies of the invention and the control anti-CTLA-4 antibodies each produce substantially the same effect as a drug. In certain embodiments, they produce substantially the same effect as a drug in tissues with high concentrations of adenosine-containing compounds. Tissues with high concentrations of adenosine-containing compounds include, for example, tumor tissue. The anti-CTLA-4 antibodies can also be provided as pharmaceutical formulations comprising the antibodies. In some embodiments, in tissues with high concentrations of adenosine-containing compounds, the anti-CTLA-4 antibodies of the invention and the control anti-CTLA-4 antibodies show substantially equivalent levels of immune activation. In some embodiments, in tissues with high concentrations of adenosine-containing compounds, the anti-CTLA-4 antibodies of the invention and the control anti-CTLA-4 antibodies are substantially equivalent in the doses required to activate immunity. In some embodiments, in tissues with high concentrations of adenosine-containing compounds, the anti-CTLA-4 antibodies of the invention and the control anti-CTLA-4 antibodies have substantially the same level of therapeutic effect. In some embodiments, in tissues with high concentrations of adenosine-containing compounds, the anti-CTLA-4 antibodies of the invention and the control anti-CTLA-4 antibodies are substantially equivalent in the doses at which a therapeutic effect is observed. In certain embodiments, the therapeutic effect is the expression of an anti-tumor effect (eg, tumor regression and induction of cell death or growth inhibition of tumor cells).

[0607] In some embodiments, the tumor can be arbitrarily selected from the group consisting of carcinoma, lymphoma, blastoma, sarcoma and leukemia. In further embodiments, the tumor can be selected from lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, colorectal cancer, rectal cancer, colon cancer, breast cancer, liver cancer, stomach cancer, pancreatic cancer, kidney cancer, prostate cancer, ovarian cancer, thyroid cancer, bile duct cancer, peritoneal cancer, mesothelioma, squamous cell carcinoma, cervical cancer, endometrial cancer, bladder cancer, esophageal cancer, head and neck cancer, nasopharyngeal cancer, salivary gland tumor, thymoma, skin cancer, basal cell tumor, melanoma, malignant melanoma, anal cancer, penile cancer, testicular cancer, Wilms' tumor, acute myeloid leukemia (including acute myeloid leukemia, acute myeloid leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia and acute monocytic leukemia), chronic myeloid leukemia, ... The patient may be selected from the group consisting of acute lymphocytic leukemia, chronic lymphocytic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma (including Burkitt's lymphoma, chronic lymphocytic leukemia, mycosis fungoides, mantle cell lymphoma, follicular lymphoma, diffuse large cell lymphoma, marginal zone lymphoma, hairy cell leukemia plasmacytoma, peripheral T-cell lymphoma and adult T-cell leukemia / lymphoma), Langerhans cell histiocytosis, multiple myeloma, myelodysplastic syndrome, brain tumor (including glioma, astroglioma, glioblastoma, meningioma and ependymoma), neuroblastoma, retinoblastoma, osteosarcoma, Kaposi's sarcoma, Ewing's sarcoma, angiosarcoma and hemangiopericytoma. In certain embodiments, the tumor can be selected from the group consisting of lung cancer, colorectal cancer, colon cancer, breast cancer, liver cancer, pancreatic cancer, kidney cancer, bladder cancer, head and neck cancer, and melanoma.

[0608] In other aspects, the present invention provides the use of an anti-CTLA-4 antibody in the manufacture or preparation of a medicament. In one embodiment, the medicament is used to treat a tumor. In a further embodiment, the medicament is used in a method for treating a tumor, the method comprising administering an effective amount of the medicament to an individual suffering from a tumor. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, such as described below. In a further embodiment, the medicament is used to damage cells. In a further embodiment, the medicament is used in a method for damaging cells in an individual, comprising administering to the individual an effective amount of the medicament to damage the cells. In a further embodiment, the medicament is used to activate immunity. In a further embodiment, the medicament is used in a method for activating immunity in an individual, comprising administering to the individual an effective amount of the medicament to activate immunity. The "individual" according to any of the above embodiments may be a human.

[0609] In other aspects, the present invention provides methods for treating tumors. In one embodiment, the method comprises administering an effective amount of an anti-CTLA-4 antibody to an individual suffering from such a tumor. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, as described below. The "individual" according to any of the above embodiments can be a human.

[0610] In other aspects, the present invention provides a method of damaging cells in an individual. In one embodiment, the method comprises administering to the individual an effective amount of an anti-CTLA-4 antibody to damage the cells. In other aspects, the present invention provides a method of activating immunity in an individual. In one embodiment, the method comprises administering to the individual an effective amount of an anti-CTLA-4 antibody to activate immunity. In one embodiment, the "individual" is a human.

[0611] In other aspects, the present invention provides pharmaceutical formulations (pharmaceutical compositions) comprising any of the anti-CTLA-4 antibodies provided herein, e.g., for use in any of the above-described treatment methods. In one embodiment, the pharmaceutical formulation (pharmaceutical composition) comprises any of the anti-CTLA-4 antibodies provided herein and a pharmaceutically acceptable carrier. In one embodiment, the present invention provides pharmaceutical formulations (pharmaceutical compositions) for treating tumors. In one embodiment, the present invention provides pharmaceutical formulations (pharmaceutical compositions) for damaging cells. In one embodiment, the present invention provides pharmaceutical formulations (pharmaceutical compositions) for activating immunity. In another embodiment, the pharmaceutical formulation (pharmaceutical composition) comprises any of the anti-CTLA-4 antibodies provided herein and at least one additional therapeutic agent, e.g., as described below.

[0612] In other aspects, the present invention provides a method for preparing a medicament or pharmaceutical formulation (e.g., for use in any of the above-described treatment methods), comprising mixing any of the anti-CTLA-4 antibodies provided herein with a pharmaceutically acceptable carrier. In one embodiment, the method for preparing a medicament or pharmaceutical formulation further comprises adding at least one additional therapeutic agent to the medicament or pharmaceutical formulation.

[0613] The antibodies of the present invention can be used alone or in combination with other agents in treatment. For example, the antibodies of the present invention can be co-administered with at least one additional therapeutic agent. In certain embodiments, the additional therapeutic agent is an immune checkpoint inhibitor, an EGFR inhibitor, a HER2 inhibitor, or a chemotherapeutic agent. Immune checkpoint inhibitors can include, for example, anti-CTLA-4 inhibitors, anti-PD-1 inhibitors, anti-PD-L1 inhibitors, anti-PD-L2 inhibitors, anti-TIM-3 inhibitors, anti-LAG-3 inhibitors, anti-TIGIT inhibitors, anti-BTLA inhibitors, and anti-VISTA inhibitors. Anti-CTLA-4 inhibitors can include, for example, ipilimumab and tremelimumab. Anti-PD-1 inhibitors can include, for example, nivolumab and pembrolizumab. Anti-PD-L1 inhibitors can include, for example, atezolizumab, durvalumab, and avelumab. Anti-PD-L2 inhibitors may include, for example, anti-PD-L2 inhibitory antibodies. Anti-TIM-3 inhibitors may include, for example, anti-TIM-3 inhibitory antibodies. Anti-LAG-3 inhibitors may include, for example, anti-LAG-3 inhibitory antibodies. Anti-TIGIT inhibitors may include, for example, anti-TIGIT inhibitory antibodies. Anti-BTLA inhibitors may include, for example, anti-BTLA inhibitory antibodies. Anti-VISTA inhibitors may include, for example, anti-VISTA inhibitory antibodies. EGFR inhibitors may include, for example, cetuximab, panitumumab, nimotuzumab, necitumumab, and zalutumumab. HER2 inhibitors may include, for example, trastuzumab, trastuzumab emtansine, and pertuzumab.

[0614] Such combination therapies include combined administration (where two or more therapeutic agents are contained in the same or separate formulations) and separate administration, in which case administration of the antibody of the invention can occur before, simultaneously with, and / or after administration of the additional therapeutic agent or agents. In one embodiment, administration of the anti-CTLA-4 antibody and administration of the additional therapeutic agent each occur within about one month, or within about one, two, or three weeks, or within about one, two, three, four, five, or six days. The antibodies of the invention can also be used in combination with radiation therapy.

[0615] The antibodies of the present invention (and any additional therapeutic agents) can be administered by any suitable means, including parenteral, intrapulmonary and intranasal, and if necessary for local treatment, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration can be by any suitable route, for example by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-lived or long-term. Various dosing regimens are contemplated herein, including but not limited to single or multiple administrations, bolus administration and pulse infusions at multiple time points.

[0616] The antibodies of the present invention will be formulated, dosed, and applied in a manner consistent with good medical practice. Factors to consider in this case include the specific condition being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the condition, the delivery site of the reagent, the method of administration, the administration time arrangement, and other factors known to the doctor. The antibody need not but is optionally formulated together with one or more reagents currently used to prevent or treat the condition in question. The effective amount of such other reagents depends on the amount of antibody present in the formulation, the type of condition or treatment, and other factors discussed above. These are typically used in the same dosage and route of administration as described herein, or in about 1% to 99% of the dosage described herein, or in any dosage and any route determined to be suitable by experience / clinical practice.

[0617] For the prevention or treatment of disease, the appropriate dosage of the antibodies of the present invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is used for preventive or therapeutic purposes, previous treatments, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The antibody is suitable for administration to the patient at one time or in a series of treatments. Depending on the type and severity of the disease, an antibody of about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg-10 mg / kg) can be an initial candidate dose for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. Depending on the above factors, a typical daily dose may vary in the range of about 1 μg / kg to 100 mg / kg or more. For repeated administration over several days or longer, depending on the condition, treatment will generally continue until the desired suppression of disease symptoms occurs. An exemplary dosage of the antibody will be in the range of about 0.05 mg / kg to about 10 mg / kg. Thus, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg or 10 mg / kg (or any combination thereof) can be administered to the patient. Such doses can be administered intermittently, for example weekly or every three weeks (e.g., so that the patient receives about 2 to about 20, or for example, about 6 doses of the antibody). An initial higher loading dose can be administered, followed by one or more lower doses. The progress of this treatment is easily monitored by conventional techniques and assays.

[0618] It will be understood that any of the above-described formulations or treatment methods can be performed using the immunoconjugates of the invention in place of or in addition to anti-CTLA-4 antibodies.

[0619] The anti-CTLA-4 antibodies herein may be administered in vivo using a vector or the like, or by integrating a nucleic acid encoding the anti-CTLA-4 antibody and directly expressing the anti-CTLA-4 antibody in vivo, or may be administered without using a vector. Examples of vectors include viral vectors and plasmid vectors, and further include adenoviral vectors. Nucleic acid encoding the anti-CTLA-4 antibody may be administered directly in vivo, or cells into which nucleic acid encoding the anti-CTLA-4 antibody has been introduced may be administered in vivo. For example, the anti-CTLA-4 antibody may be expressed in vivo by directly administering mRNA encoding the anti-CTLA-4 antibody (which has been chemically modified to enhance the stability of the mRNA in vivo) into the human body (see EP2101823B and WO2013 / 120629). B cells into which nucleic acid encoding the anti-CTLA-4 antibody has been introduced may be administered (Sci immunol. (2019) 4(35), eaax0644). Bacteria introduced with nucleic acid encoding anti-CTLA-4 antibodies can be administered (Nature Reviews Cancer (2018) 18, 727-743).

[0620] Non-limiting examples of technologies that can be combined with the anti-CTLA-4 antibodies herein include the use of anti-CTLA-4 antibodies to produce T cells that secrete T cell redirecting antibodies (Trends Immunol. (2019) 40(3) 243-257). One non-limiting production method is to introduce a nucleic acid encoding a bispecific antibody into an effector cell (such as a T cell) through genetic modification technology. The antibody comprises a binding domain for any subunit of the T cell receptor (TCR) complex on the T cell, in particular a binding domain for the CD3ε chain in CD3, and an antigen-binding domain of an anti-CTLA-4 antibody.

[0621] H. Products

[0622] In another aspect of the present invention, an article of manufacture containing materials for treating, preventing, and / or diagnosing the above-mentioned conditions is provided. The article of manufacture includes a container and a label on the container or a package insert associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV (intravenous) solution bags, etc. The container can be formed from a variety of materials, such as glass or plastic. The container holds a composition, which is effective alone or in combination with another composition for treating, preventing, and / or diagnosing the condition, and the container can have a sterile access port (for example, the container can be an IV solution bag or a vial with a stopper pierceable by a hypodermic needle). At least one active ingredient in the composition is an antibody of the present invention. The label or package insert indicates that the composition is used to treat a selected condition. In addition, the article of manufacture may include (a) a first container containing a composition comprising an antibody of the present invention; and (b) a second container containing a composition comprising another cytotoxic agent or other therapeutic agent. The article of manufacture in this embodiment of the present invention may also include a package insert indicating that the composition is used to treat a specific condition. Alternatively or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may also include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0623] It will be understood that any of the above-described articles of manufacture may include an immunoconjugate of the invention in place of or in addition to an anti-CTLA-4 antibody.

[0624] In one aspect, the present invention provides isolated polypeptides comprising variant Fc regions. In some aspects, the polypeptides are antibodies. In some aspects, the polypeptides are Fc fusion proteins. In certain embodiments, the variant Fc region comprises at least one amino acid residue alteration (e.g., substitution) compared to the corresponding sequence in a native sequence or reference variant sequence Fc region (collectively referred to herein as the "parent" Fc region). A native sequence Fc region typically consists of a homodimer composed of two identical polypeptide chains. The amino acid alterations in the variant Fc regions of the present invention can be introduced into either or both of the two polypeptide chains of the parent Fc region.

[0625] In some aspects, the present invention provides variant Fc regions whose function has been modified compared to a parent Fc region. In certain aspects, the variant Fc regions of the present invention have enhanced binding activity for Fcγ receptors compared to the parent Fc region. In certain embodiments, the variant Fc regions of the present invention have enhanced binding activity for at least one Fcγ receptor selected from FcγRIa, FcγRIIa, FcγRIIb, and FcγRIIIa compared to the parent Fc region. In some embodiments, the variant Fc regions of the present invention have enhanced binding activity for FcγRIIa. In some embodiments, the variant Fc regions of the present invention have enhanced binding activity for FcγRIIIa. In further embodiments, the variant Fc regions of the present invention have enhanced binding activity for both FcγRIIa and FcγRIIIa. In another aspect, the variant Fc regions of the present invention have enhanced ADCC activity, CDC activity, or ADCP activity compared to the parent Fc region.

[0626] In some embodiments, the variant Fc region of the present invention comprises at least one amino acid alteration at at least one position selected from positions 234, 235, 236, 298, 330, 332, and 334 according to EU numbering. Alternatively, amino acid alterations such as those described in WO 2013 / 002362 and WO 2014 / 104165 can similarly be used in the present invention.

[0627] In certain embodiments, the binding activity of the parent Fc region and the variant Fc region can be expressed as a KD (dissociation constant) value. In one embodiment, the ratio of [KD value of the parent Fc region for FcγRIIa] / [KD value of the variant Fc region for FcγRIIa] is, for example, 1.5 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 40 or more, or 50 or more. In further embodiments, FcγRIIa can be FcγRIIa R or FcγRIIa H, or both. Thus, the KD value of the Fc region of FcγRIIa can be the KD value of the Fc region of FcγRIIa R, the KD value of the Fc region of FcγRIIa H, or the sum or average of the two. In one embodiment, the ratio of [binding activity of the parent Fc region to FcγRIIIa] / [binding activity of the variant Fc region to FcγRIIIa] has a value of, for example, 2 or more, 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, 50 or more, 100 or more, 200 or more, 300 or more, 500 or more, 1 x 10 3 or higher, 2 x 10 3or higher, 3 x 10 3 or higher, or 5 x 10 3 or higher. In further embodiments, FcγRIIIa may be FcγRIIIa F or FcγRIIIa V, or both. Thus, the KD value of the Fc region for FcγRIIIa may be the KD value of the Fc region for FcγRIIIa F, the KD value of the Fc region for FcγRIIIa V, or the sum or average of the two.

[0628] In one embodiment, the variant Fc region has a KD value for FcγRIIa of, for example, 1.0 x 10 -6 M or lower, 5.0x 10 -7 M or lower, 3.0 x 10 -7 M or lower, 2.0 x 10 -7 M or lower, 1.0 x 10 -7 M or smaller, 5.0 x10 -8 M or lower, 3.0 x 10 -8 M or lower, 2.0 x 10 -8 M or lower, 1.0 x 10 -8 M or smaller, 5.0 x 10 -9 M or lower, 3.0 x 10 -9 M or lower, 2.0 x 10 -9 M or lower, or 1.0 x 10 -9 M or less. In other embodiments, FcγRIIa can be FcγRIIa R or FcγRIIa H, or both. In one embodiment, the variant Fc region has a KD value for FcγRIIIa of, for example, 1.0 x 10 -6 M or smaller, 5.0 x 10 -7 M or lower, 3.0 x 10 -7 M or lower, 2.0 x 10 -7 M or lower, 1.0 x 10 -7 M or smaller, 5.0 x 10 -8 M or lower, 3.0 x 10 -8 M or lower, 2.0 x 10 -8 M or lower, 1.0 x10 -8 M or smaller, 5.0 x 10 -9 M or lower, 3.0 x 10 -9 M or lower, 2.0 x 10 -9 M or lower, 1.0 x 10-9 M or smaller, 5.0 x 10 -10 M or lower, 3.0 x 10 -10 M or lower, 2.0 x 10 -10 M or lower, or 1.0 x 10 -10 M or less. In other embodiments, the FcγRIIIa may be FcγRIIIa F or FcγRIIIa V, or both.

[0629] In another embodiment, the binding activity of the parent and variant Fc regions can be expressed using kd (dissociation rate constant) values ​​instead of KD values.

[0630] In another embodiment, the binding activity of the parent and variant Fc regions can be expressed by the amount of binding of the Fc region to the Fcγ receptor per unit amount. For example, in a surface plasmon resonance assay, the amount of binding of the Fc region immobilized on the sensor chip and the amount of binding of the Fcγ receptor further bound thereto are each measured as resonance units (RU). The amount of binding of the Fcγ receptor divided by the amount of binding of the Fc region can be defined as the amount of binding of the Fc region to the Fcγ receptor per unit amount. Specific methods for determining and calculating such binding amounts are described in the examples below. In some embodiments, the ratio of [amount of binding of the variant Fc region to FcγRIIa] / [amount of binding of the parent Fc region to FcγRIIa] is, for example, 1.5 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, or 40 or more, or 50 or more. In some embodiments, the ratio of [the amount of binding of the variant Fc region to FcγRIIIa] / [the amount of binding of the parent Fc region to FcγRIIIa] has a value of, for example, 2 or more, 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, 50 or more, 100 or more, 200 or more, 300 or more, 500 or more, 1 x 10 3 or higher, 2 x 10 3 or higher, 3 x 10 3 or higher, or 5 x 10 3 or higher.

[0631] In certain embodiments, KD values, kd values, binding magnitude values, etc. described herein are determined or calculated by surface plasmon resonance measurements at 25°C or 37°C (see, eg, Reference Example 8 herein).

[0632] In certain aspects, the variant Fc regions of the present invention have improved selectivity between activating and inhibitory Fcγ receptors compared to the parent Fc region. In other words, the variant Fc regions of the present invention have significantly improved binding activity for activating Fcγ receptors compared to inhibitory Fcγ receptors compared to the parent Fc region. In certain embodiments, the activating Fcγ receptor is at least one Fcγ receptor selected from FcγRIa, FcγRIIa R, FcγRIIa H, FcγRIIIa F, and FcγRIIIa V, and the inhibitory Fcγ receptor is FcγRIIb. In some embodiments, the variant Fc regions of the present invention have improved selectivity between FcγRIIa and FcγRIIb. In some embodiments, the variant Fc regions of the present invention have improved selectivity between FcγRIIIa and FcγRIIb. In other embodiments, the variant Fc regions of the invention have improved selectivity between FcγRIIa and FcγRIIb, and between FcγRIIIa and FcγRIIb.

[0633] In some embodiments, the variant Fc region of the present invention comprises at least one amino acid alteration at at least one position selected from positions 236, 239, 268, 270, and 326 according to EU numbering. Alternatively, the amino acid alterations described in WO 2013 / 002362 and WO 2014 / 104165 can be similarly used in the present invention.

[0634] In certain embodiments, the binding activity of the parent and variant Fc regions can be represented by KD (dissociation constant) values. The embodiments of binding activity to FcγRIIa and FcγRIIIa are described above. In one embodiment, the ratio of [KD value of the parent Fc region to FcγRIIb] / [KD value of the variant Fc region to FcγRIIb] is, for example, 10 or less, 5 or less, 3 or less, 2 or less, 1 or less, 0.5 or less, 0.3 or less, 0.2 or less, or 0.1 or less. In another embodiment, the binding activity of the parent and variant Fc regions can be represented by kd (dissociation rate constant) values ​​instead of KD values.

[0635] In another embodiment, the binding activity of the parent and variant Fc regions can be represented by the above-mentioned binding amount of the Fc region to the Fcγ receptor per unit amount. In some embodiments, the value of the ratio of [the amount of binding of the variant Fc region to FcγRIIb] / [the amount of binding of the parent Fc region to FcγRIIb] is, for example, 10 or less, 5 or less, 3 or less, 2 or less, 1 or less, 0.5 or less, 0.3 or less, 0.2 or less, or 0.1 or less. In some embodiments, the amount of binding of the variant Fc region to FcγRIIb is, for example, 0.5 or less, 0.3 or less, 0.2 or less, 0.1 or less, 0.05 or less, 0.03 or less, 0.02 or less, 0.01 or less, 0.005 or less, 0.003 or less, 0.002 or less, or 0.001 or less.

[0636] In certain embodiments, improved selectivity between activating and inhibitory Fcγ receptors refers to enhanced selectivity for binding to activating Fcγ receptors compared to binding to inhibitory Fcγ receptors, or in other words, an increase in the ratio of activating Fcγ receptor binding activity to inhibitory Fcγ receptor binding activity (A / I ratio). This ratio (A / I ratio) is an indicator of excellent effector function. Polypeptides with high A / I ratios can be evaluated as having good effector function. The binding activity of parental and variant Fc regions to Fcγ receptors can be expressed as a KD value, a kd value, or the amount of Fc region binding to an Fcγ receptor per unit amount. The A / I ratio can be expressed as a KD value, kd value, or amount of binding as follows: [KD value of inhibitory Fcγ receptor] / [KD value of activating Fcγ receptor], [kd value of inhibitory Fcγ receptor] / [kd value of activating Fcγ receptor], or [amount of binding to activating Fcγ receptor] / [amount of binding to inhibitory Fcγ receptor].

[0637] In one embodiment, the A / I ratio of a variant Fc region of the invention is increased by 1.1-fold or more, 1.2-fold or more, 1.3-fold or more, 1.4-fold or more, 1.5-fold or more, 1.6-fold or more, 1.7-fold or more, 1.8-fold or more, 1.9-fold or more, 2.0-fold or more, 3.5-fold or more, 4.0-fold or more, 5.0-fold or more, 6.0-fold or more, 7.0-fold or more, 8.0-fold or more, 9.0-fold or more, 10.0-fold or more, 11.0-fold or more, 12.0-fold or more, 13.0-fold or more, 14.0-fold or more, 15.0-fold or more, 16.0-fold or more, 17.0-fold or more, times or more, 1.9 times or more, 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times or more, 7 times or more, 8 times or more, 9 times or more, 10 times or more, 20 times or more, 30 times or more, 40 times or more, 50 times or more, 60 times or more, 70 times or more, 80 times or more, 90 times or more, 100 times or more, 200 times or more, 300 times or more, 400 times or more, 500 time...

Claims

1. Use of an anti-CTLA-4 antibody having CTLA-4 binding activity that depends on the concentration of an adenosine-containing compound in the preparation of a pharmaceutical composition for treating a tumor, wherein the anti-CTLA-4 antibody has at least one feature selected from the following (a) to (i): (a) The binding activity in the presence of 100 μM adenosine-containing compound is two times or more than that in the absence of the adenosine-containing compound; (b) The KD value in the presence of 100 μM adenosine-containing compounds was 5×10 -7 M or lower; (c) The KD value in the absence of adenosine-containing compounds is 1×10 -6 M or higher; (d) forming a ternary complex with adenosine-containing compounds and CTLA-4; (e) a region that binds to amino acid 97 to amino acid 106 of human CTLA-4 (extracellular domain, SEQ ID NO: 28); (f) competes with ABAM004 (VH, SEQ ID NO: 10; and VL, SEQ ID NO: 11) for binding to CTLA-4; (g) binds to the same epitope as ABAM004 (VH, SEQ ID NO: 10; and VL, SEQ ID NO: 11); (h) exhibit cytotoxic activity against cells expressing CTLA-4; and (i) Binds to human and mouse CTLA-4.

2. The use according to claim 1, wherein the anti-CTLA-4 antibody comprises: (a) HVR-H1 (SEQ ID NO:223), comprising the amino acid sequence of SX1TMN, wherein X1 is K, H, R or A; (b) HVR-H2 (SEQ ID NO:224), comprising the amino acid sequence of SISX1X2SX3YIYYAX4SVX5G, wherein X1 is T or S, X2 is Q or R, X3 is H or G, X4 is R, D or E, and X5 is R or K; and (c) HVR-H3 (SEQ ID NO:225), comprising the amino acid sequence of YGX1REDMLWVFDY, wherein X1 is K or A.

3. The use according to claim 2, wherein the anti-CTLA-4 antibody further comprises: (a) HVR-L1 (SEQ ID NO: 226), comprising the amino acid sequence X1GX2STX3VGDYX4X5VX6, wherein X1 is E, T, D or Q, X2 is P or T, X3 is G or D, X4 is N or T, X5 is W or Y, and X6 is H or S; (b) HVR-L2 (SEQ ID NO: 227), comprising the amino acid sequence X1TX2X3KPX4, wherein X1 is F, E or Y, X2 is S or I, X3 is K or S, and X4 is S, E or K; and (c) HVR-L3 (SEQ ID NO: 228), comprising the amino acid sequence X1TYAAPLGPX2, wherein X1 is Q or S, and X2 is M or T.

4. The use according to claim 3, wherein the anti-CTLA-4 antibody comprises: (1) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 112; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 128; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 133; (2) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 112; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 129; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 133; (3) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 100; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 101; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 113; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 114; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 115; (4) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 100; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 104; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 116; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 115; (5) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 105; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 106; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 133; (6) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 108; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 121; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 123; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 153; (7) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 110; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 102; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 122; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 133; (8) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 111; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 152; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 128; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 133; (9) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 111; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 152; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 129; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 117; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 133; or (10) (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:107; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:109; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:102; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:130; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:117; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO:

133.

5. The use according to claim 1, wherein the anti-CTLA-4 antibody comprises: (a) a VH sequence having at least 95% sequence identity to an amino acid sequence of any one of SEQ ID NOs: 140, 141, 83 to 86, 98, and 135 to 139; (b) a VL sequence having at least 95% sequence identity to an amino acid sequence of any one of SEQ ID NOs: 146, 147, 88 to 95, 97, 99, 134 and 144, 145, 148, and 149; or (c) a VH sequence having an amino acid sequence of any one of SEQ ID NOs: 140, 141, 83 to 86, 98, and 135 to 139 and a VL sequence having an amino acid sequence of any one of SEQ ID NOs: 146, 147, 88 to 95, 97, 99, 134 and 144, 145, 148, and 149.

6. Use of an anti-CTLA-4 antibody in the preparation of a pharmaceutical composition for treating a tumor, wherein the anti-CTLA-4 antibody comprises: (1) a first variable region comprising a VH sequence of SEQ ID NO: 140 and a VL sequence of SEQ ID NO: 146, and a second variable region comprising a VH sequence of SEQ ID NO: 141 and a VL sequence of SEQ ID NO: 146; (2) a first variable region comprising a VH sequence of SEQ ID NO: 140 and a VL sequence of SEQ ID NO: 147, and a second variable region comprising a VH sequence of SEQ ID NO: 141 and a VL sequence of SEQ ID NO: 147; (3) the VH sequence of SEQ ID NO: 140 and the VL sequence of SEQ ID NO: 146; (4) the VH sequence of SEQ ID NO: 141 and the VL sequence of SEQ ID NO: 146; (5) the VH sequence of SEQ ID NO: 140 and the VL sequence of SEQ ID NO: 147; (6) the VH sequence of SEQ ID NO: 141 and the VL sequence of SEQ ID NO: 147; (7) the VH sequence of SEQ ID NO:98 and the VL sequence of SEQ ID NO:99; (8) the VH sequence of SEQ ID NO: 83 and the VL sequence of SEQ ID NO: 97; (9) the VH sequence of SEQ ID NO:86 and the VL sequence of SEQ ID NO:134; (10) the VH sequence of SEQ ID NO: 136 and the VL sequence of SEQ ID NO: 95; or (11) The VH sequence of SEQ ID NO:136 and the VL sequence of SEQ ID NO:

149.

7. The method of claim 1 , wherein the tumor is at least one selected from the group consisting of lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, colorectal cancer, rectal cancer, colon cancer, breast cancer, liver cancer, stomach cancer, pancreatic cancer, kidney cancer, prostate cancer, ovarian cancer, thyroid cancer, bile duct cancer, peritoneal cancer, mesothelioma, squamous cell carcinoma, cervical cancer, endometrial cancer, bladder cancer, esophageal cancer, head and neck cancer, nasopharyngeal carcinoma, salivary gland tumors, thymoma, skin cancer, basal cell tumor, melanoma, malignant melanoma, anal cancer, penile cancer, testicular cancer, Wilms' tumor, brain tumor, neuroblastoma, retinoblastoma, osteosarcoma, Kaposi's sarcoma, Ewing's sarcoma, angiosarcoma and hemangiopericytoma.

8. A polypeptide comprising a variant Fc region comprising an amino acid change in a parent Fc region, wherein the parent Fc region comprises two polypeptide chains, and wherein the variant Fc region comprises an amino acid change at: (i) positions 234, 235, 236, 239, 268, 270 and 298 according to EU numbering in the first polypeptide of the parent Fc region; and (ii) positions 270, 298, 326 and 334 according to EU numbering in the second polypeptide of the parent Fc region. 9 . The polypeptide according to claim 8 , wherein the binding activity to at least one Fcγ receptor selected from the group consisting of FcγRIa, FcγRIIa, FcγRIIb and FcγRIIIa in the variant Fc region is enhanced compared to that of the parent Fc region.

10. The polypeptide of claim 8 or 9, wherein the selectivity between activating Fcγ receptors and inhibitory Fcγ receptors in the variant Fc region is improved compared to the parent Fc region.

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