Anti-CTLA-4 antibodies

Abstract

To provide an anti-CTLA-4 antibody and a production method and a use method thereof; nucleic acid encoding the anti-CTLA-4 antibody and a host cell comprising the nucleic acid; a polypeptide comprising a variant Fc region comprising amino acid modifications in a parent Fc region and a production method and a use method of the same.SOLUTION: There is provided herein, an anti-CTLA-4 antibody comprising: (A) a variable region having CTLA-binding activity that is dependent on concentration of an adenosine-containing compound; and (B) a variant Fc region comprising multiple amino acid alterations in a parent Fc region, where the parent Fc region is composed of two polypeptide chains and the variant Fc region comprises amino acid alterations at specific positions.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to anti-CTLA-4 antibodies and methods for using the same. The present invention also relates to polypeptides comprising mutant Fc regions that contain amino acid modifications in the parent Fc region, and methods for producing the polypeptides. [Background technology]

[0002] Mutant cells in the body due to genetic mutations and other factors are monitored and eliminated by the immune surveillance system. However, persistent excessive immune responses can be harmful to the body itself, such as by causing autoimmune damage to normal tissues. Therefore, the immune system is equipped with a negative feedback mechanism (immune checkpoint) to suppress immune responses once activated (see, for example, Non-Patent Document 1). Immune checkpoints are thought to play an important role in maintaining homeostasis in the immune system. On the other hand, it has become clear that some tumors utilize immune checkpoints to evade the immune system. Currently, extensive research is being conducted on the immunosuppressive functions mediated by major immune checkpoint molecules such as cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), programmed cell death 1 (PD-1), and programmed cell death ligand 1 (PD-L1). CTLA-4 is a glycoprotein belonging to the immunoglobulin superfamily whose gene was cloned from a cDNA library of a mouse-derived killer T cell clone in 1987 (see, for example, Non-Patent Document 2). It is known that T cell immune responses are suppressed via CTLA-4. Based on the idea that suppressing CTLA-4 function and promoting T cell activation could lead to cancer regression, it was reported in 1996 that administration of an anti-CTLA-4 antibody to tumor-bearing mice resulted in tumor regression (see, for example, Non-Patent Document 3). Since 2000, evaluation of the efficacy of anti-CTLA-4 antibodies in humans has been ongoing, and in 2011, an anti-human CTLA-4 monoclonal antibody (ipilimumab) was approved by the U.S. Food and Drug Administration (FDA) as the world's first immunostimulatory antibody drug. In addition to ipilimumab, numerous other anti-CTLA-4 monoclonal antibodies have been produced (see, for example, Patent Documents 1, 2, 3, and 4), and attempts are being made to develop them as pharmaceuticals. These drugs, which inhibit immune checkpoints to release the immune suppression mechanism and thereby enhance immune activity, are called immune checkpoint inhibitors. On the other hand, it has long been known that some T cells have immunosuppressive functions. In 1995, these cells were identified as CD25+CD4+ T cells and named regulatory T cells (see, for example, Non-Patent Document 4). In 2003, the Foxp3 gene was identified as a master gene that is specifically expressed in regulatory T cells and controls their development and function. Foxp3 acts as a transcription factor and controls the expression of various immune response-related genes. Foxp3 is particularly involved in the constitutive expression of CTLA-4 in regulatory T cells, which is thought to play an important role in the immunosuppressive function of regulatory T cells (see, for example, Non-Patent Document 5). It is believed that the infiltration of regulatory T cells into tumor tissue results in the weakening or inhibition of immune surveillance against tumors. In fact, it has been revealed that regulatory T cells are increased in many human carcinomas (see, for example, Non-Patent Document 6), and it has been reported that the infiltration of regulatory T cells into the local tumor can be a factor in poor prognosis for cancer patients. Conversely, it is expected that the removal or reduction of regulatory T cells from tumor tissue will lead to the enhancement of anti-tumor immunity. Currently, the development of cancer immunotherapy targeting regulatory T cells is being actively pursued. Administration of the anti-CTLA-4 antibody ipilimumab enhances antitumor immunity, but it has also been reported that its systemic enhancement of immune activity can lead to the development of autoimmune diseases. In one clinical trial, 60% of patients treated with ipilimumab experienced adverse events, most of which were autoimmune diseases related to the skin or gastrointestinal tract. In other clinical trials, approximately half of patients treated with ipilimumab also developed similar autoimmune diseases. To prevent these side effects, patients treated with ipilimumab are sometimes administered immunosuppressants. The development of new drugs that can maintain antitumor immune responses while minimizing the side effects of immune checkpoint inhibitors is desirable. When a therapeutic antibody is administered in vivo, it is desirable that the target antigen be specifically expressed only at the lesion site. However, in many cases, the same antigen is also expressed in non-lesioned normal tissues, which can cause undesirable side effects from a therapeutic perspective. For example, while an antibody against a tumor antigen can exert cytotoxic activity against tumor cells through ADCC or other mechanisms, if the same antigen is also expressed in normal tissues, it may also cytotoxicize normal cells. To solve the above problems, a technology has been developed that focuses on the phenomenon in which specific compounds are present in large amounts in target tissues (e.g., tumor tissues) to create antigen-binding molecules whose antigen-binding activity changes depending on the concentration of such compounds (see, for example, Patent Document 11).

[0003] Antibodies are attracting attention as pharmaceuticals due to their high stability in the blood and minimal side effects (Non-Patent Documents 12 and 13). Most antibody drugs currently on the market are antibodies of the human IgG1 subclass. Numerous studies have been conducted on the effector functions of IgG class antibodies, antibody-dependent cellular cytotoxicity (hereinafter referred to as ADCC) and complement-dependent cytotoxicity (hereinafter referred to as CDC), and it has been reported that, among human IgG class antibodies, IgG1 subclass antibodies have the highest ADCC and CDC activities (Non-Patent Document 14). Furthermore, antibody-dependent cell-mediated phagocytosis (ADCP), which is the phagocytosis of target cells mediated by IgG class antibodies, has also been shown to be one of the effector functions of antibodies (Non-Patent Documents 15 and 16).

[0004] The expression of ADCC, CDC, and ADCP by IgG antibodies requires the binding of the antibody Fc region to antibody receptors (hereinafter referred to as FcγR) and various complement components present on the surface of effector cells such as killer cells, natural killer cells, and activated macrophages. In humans, the FcγR protein family has been reported to include isoforms FcγRIa, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb, and each allotype has also been reported (Non-Patent Document 17).

[0005] Enhancement of cytotoxic effector functions, such as ADCC, ADCP, and CDC, has attracted attention as a promising means to enhance the antitumor effects of antibodies. The importance of FcγR-mediated effector functions for the antitumor effects of antibodies has been reported using mouse models (Non-Patent Document 7, Non-Patent Document 8). Furthermore, a correlation was observed between the high-affinity polymorphic allotype (V158) of FcγRIIIa and the low-affinity polymorphic allotype (F158) of FcγRIIIa in humans (Non-Patent Document 18). Similarly, it has been shown that clinical effects differ depending on the FcγRIIa allotype (H131 vs. R131) (Non-Patent Document 19). These reports indicate that antibodies with Fc regions optimized for binding to specific FcγRs mediate stronger effector functions and thereby exert effective antitumor effects.

[0006] The balance of antibody binding activity toward activating receptors (FcγRIa, FcγRIIa, FcγRIIIa, and FcγRIIIb) and toward inhibitory receptors (FcγRIIb) is an important factor in optimizing antibody effector function. Using an Fc region with enhanced binding activity toward activating receptors and reduced binding activity toward inhibitory receptors may confer optimal effector function to an antibody (Non-Patent Document 20). It has been shown that several amino acid residues in the antibody hinge region and CH2 domain, as well as the sugar chain attached to Asn at position 297 (EU numbering) attached to the CH2 domain, are important for the binding between the Fc region and FcγR (Non-Patent Document 14, Non-Patent Document 21, Non-Patent Document 22). Focusing on this binding site, various Fc region mutants with FcγR binding properties have been studied, and Fc region mutants with enhanced activating FcγR binding activity have been identified (Patent Document 5, Patent Document 6, Non-Patent Document 9, Non-Patent Document 10). For example, Lazar et al. succeeded in increasing human FcγRIIIa (V158) binding by approximately 370-fold by substituting Ser at position 239 (EU numbering), Ala at position 330, and Ile at position 332 (EU numbering) with Asp, Leu, and Glu, respectively (Non-Patent Document 9, Patent Document 6). Shinkawa et al. succeeded in increasing FcγRIIIa binding by approximately 100-fold by deleting the fucose attached to Asn at position 297 (EU numbering) (Non-Patent Document 23). These methods introduce the same alterations or the same glycosylation modifications into the Fc regions of both antibody H chains. On the other hand, it has been reported that antibody Fc, despite being a homodimer, binds to FcγR in a 1:1 ratio and asymmetrically recognizes FcγR at the lower hinge and CH2 regions (Non-Patent Document 11). Considering that the Fc region interacts asymmetrically with FcγR, it is thought that introducing different alterations into each H chain will enable more precise optimization of the interaction between IgG and FcγR. Based on this concept, methods have been reported in which different alterations are made to the Fc region of each H chain of an antibody to asymmetrically modify the Fc, thereby optimizing the interaction with FcγR (Patent Document 7, Patent Document 8, Patent Document 9, Patent Document 10).In fact, by asymmetrically modifying the Fc region, variants have been obtained that exhibit higher ADCC activity than existing afucosylated antibodies, which are ADCC-enhancing antibodies (Patent Documents 9 and 10).

[0007] In addition to ADCC activity, ADCP activity is also an important effector function of antibodies and has been reported to contribute to antitumor effects (Non-Patent Document 24). ADCP activity can be enhanced by inhibiting "don't eat me" signals, such as those mediated by CD47 (Non-Patent Document 24), or by enhancing FcγRIIa-binding ability (Non-Patent Document 25). However, the amino acid sequences of the activating FcγR FcγRIIa and the inhibitory FcγRIIb share high homology, making it difficult to selectively enhance FcγRIIa-binding ability (Non-Patent Document 26). Therefore, enhancing FcγRIIa-binding ability also enhances FcγRIIb-binding ability, an inhibitory receptor, potentially resulting in attenuation of effector function. In fact, variants with significantly improved FcγRIIa-binding ability also exhibit enhanced FcγRIIb-binding ability compared to native IgG1 (Patent Documents 9 and 10). Therefore, in order to exhibit high ADCC / ADCP activity, it is preferable to enhance the binding to FcγRIIIa and FcγRIIa as much as possible without enhancing the binding ability to FcγRIIb, but such variants have not been reported. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] WO 2000 / 037504 [Patent Document 2] WO 2001 / 014424 [Patent Document 3] WO 2012 / 120125 [Patent Document 4] WO 2016 / 196237 [Patent Document 5] WO 2000 / 042072

Patent document 6

Patent document 7

Patent document 8

Patent Document 9

Patent document 10

Patent document 11

Non-licensed literature

[0009] [Non-licensed document 1] Pardoll, Nat Rev Cancer (2012) 12: 252-264 [Non-licensed document 2] Brunet et al., Nature (1987) 328: 267-270 [Non-licensed document 3] Leach et al., Science (1996) 271: 1734-1736

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Non-licensed Document 6

Non-licensed Document 7

Non-licensed literature 9

Non-licensed literature 10

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Non-licensed Document 14

Non-licensed Document 15

Non-licensed Document 16

Non-licensed Document 17

Non-licensed Document 18

Non-licensed Document 19

Non-licensed Document 20

Non-licensed Document 21

[0010] The present invention provides anti-CTLA-4 antibodies and methods of using them. The present invention also provides polypeptides comprising variant Fc regions and methods of producing them. [Means for solving the problem]

[0011] More specifically, the present invention provides the following [1] to

[26] . [1] (A) A variable region having CTLA-4 binding activity dependent on the concentration of an adenosine-containing compound; and (B) A mutant Fc region containing multiple amino acid alterations in the parent Fc region. an anti-CTLA-4 antibody comprising: wherein the parent Fc region is composed of two polypeptide chains, and the variant Fc region comprises amino acid modifications at the following positions: (i) positions 234, 235, 236, 239, 268, 270, 298, and 330, as indicated by EU numbering, in the first polypeptide of the parent Fc region; and (ii) positions 270, 298, 326, 330, and 334, as expressed in EU numbering, in the second polypeptide of the parent Fc region. [2] The anti-CTLA-4 antibody of [1], wherein the variable region has at least one characteristic selected from the following (a) to (i): (a) the binding activity in the presence of 100 μM of an adenosine-containing compound is at least two-fold higher than the binding activity in the absence of the adenosine-containing compound; (b) KD value of 5 × 10 in the presence of 100 μM adenosine-containing compound. -7 M or less, (c) KD value of 1 × 10 in the absence of adenosine-containing compounds -6 M or more, (d) forming a ternary complex with an adenosine-containing compound and CTLA-4; (e) binds to the region of amino acids 97 to 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 that bound by ABAM004 (VH, SEQ ID NO: 10; and VL, SEQ ID NO: 11); (h) exhibiting cytotoxic activity against CTLA-4 expressing cells; and (i) It binds to human and mouse CTLA-4. [3] The anti-CTLA-4 antibody of [1] or [2], which is a monoclonal antibody. [4] The anti-CTLA-4 antibody of any one of [1] to [3], which is a human antibody, a humanized antibody, or a chimeric antibody. [5] The anti-CTLA-4 antibody of any one of [1] to [4], comprising: (a) an HVR-H1 (SEQ ID NO: 223) comprising the amino acid sequence SX1TMN, where X1 is H, A, R, or K; (b) an HVR-H2 (SEQ ID NO: 224) comprising the amino acid sequence SISX1X2SX3YIYYAX4SVX5G, where 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) an HVR-H3 (SEQ ID NO: 225) comprising the amino acid sequence YGX1REDMLWVFDY, where X1 is K or A. [6] The anti-CTLA-4 antibody of [5], further comprising: (a) an HVR-L1 (SEQ ID NO: 226) comprising the amino acid sequence X1GX2STX3VGDYX4X5VX6, where 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) an HVR-L2 (SEQ ID NO: 227) comprising the amino acid sequence X1TX2X3KPX4, where 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) an HVR-L3 (SEQ ID NO: 228) comprising the amino acid sequence X1TYAAPLGPX2, where X1 is S or Q and X2 is M or T. [7] The anti-CTLA-4 antibody of [5], further comprising 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. [8] The anti-CTLA-4 antibody of [6], further comprising a light chain variable domain FR1 comprising any one of the amino acid sequences of SEQ ID NOs: 236 to 238, FR2 comprising any one of the amino acid sequences of SEQ ID NOs: 240 to 241, FR3 comprising any one of the amino acid sequences of SEQ ID NOs: 242 to 244, and FR4 comprising any one of the amino acid sequences of SEQ ID NOs: 245 to 246. [9] The anti-CTLA-4 antibody of any one of [1] to [4], comprising: (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.

[10] (1) a VH sequence of SEQ ID NO: 98 and a VL sequence of SEQ ID NO: 99; (2) the VH sequence of SEQ ID NO: 83 and the VL sequence of SEQ ID NO: 88; (3) the VH sequence of SEQ ID NO: 83 and the VL sequence of SEQ ID NO: 89; (4) the VH sequence of SEQ ID NO: 83 and the VL sequence of SEQ ID NO: 90; (5) the VH sequence of SEQ ID NO: 83 and the VL sequence of SEQ ID NO: 91; (6) the VH sequence of SEQ ID NO: 83 and the VL sequence of SEQ ID NO: 92; (7) the VH sequence of SEQ ID NO: 83 and the VL sequence of SEQ ID NO: 93; (8) the VH sequence of SEQ ID NO: 83 and the VL sequence of SEQ ID NO: 94; (9) the VH sequence of SEQ ID NO: 83 and the VL sequence of SEQ ID NO: 97; (10) the VH sequence of SEQ ID NO: 83 and the VL sequence of SEQ ID NO: 95; (11) the VH sequence of SEQ ID NO: 84 and the VL sequence of SEQ ID NO: 97; (12) the VH sequence of SEQ ID NO: 85 and the VL sequence of SEQ ID NO: 97; (13) the VH sequence of SEQ ID NO: 86 and the VL sequence of SEQ ID NO: 97; (14) the VH sequence of SEQ ID NO: 86 and the VL sequence of SEQ ID NO: 134; (15) the VH sequence of SEQ ID NO: 136 and the VL sequence of SEQ ID NO: 97; (16) the VH sequence of SEQ ID NO: 135 and the VL sequence of SEQ ID NO: 97; (17) the VH sequence of SEQ ID NO: 136 and the VL sequence of SEQ ID NO: 95; (18) the VH sequence of SEQ ID NO: 137 and the VL sequence of SEQ ID NO: 97; (19) the VH sequence of SEQ ID NO: 138 and the VL sequence of SEQ ID NO: 97; (20) the VH sequence of SEQ ID NO: 138 and the VL sequence of SEQ ID NO: 144; (21) the VH sequence of SEQ ID NO: 138 and the VL sequence of SEQ ID NO: 145; (22) the VH sequence of SEQ ID NO: 138 and the VL sequence of SEQ ID NO: 146; (23) the VH sequence of SEQ ID NO: 139 and the VL sequence of SEQ ID NO: 146; (24) the VH sequence of SEQ ID NO: 140 and the VL sequence of SEQ ID NO: 146; (25) the VH sequence of SEQ ID NO: 141 and the VL sequence of SEQ ID NO: 146; (26) the VH sequence of SEQ ID NO: 140 and the VL sequence of SEQ ID NO: 147; (27) the VH sequence of SEQ ID NO: 141 and the VL sequence of SEQ ID NO: 147; (28) the VH sequence of SEQ ID NO: 140 and the VL sequence of SEQ ID NO: 148; (29) the VH sequence of SEQ ID NO: 141 and the VL sequence of SEQ ID NO: 148; (30) the VH sequence of SEQ ID NO: 136 and the VL sequence of SEQ ID NO: 149; (31) A first variable region comprising the VH sequence of SEQ ID NO: 140 and the VL sequence of SEQ ID NO: 146, and a second variable region comprising the VH sequence of SEQ ID NO: 141 and the VL sequence of SEQ ID NO: 146; or (32) A first variable region comprising the VH sequence of SEQ ID NO: 140 and the VL sequence of SEQ ID NO: 147, and a second variable region comprising the VH sequence of SEQ ID NO: 141 and the VL sequence of SEQ ID NO: 147; The anti-CTLA-4 antibody of [9], comprising:

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

[10] , which is a full-length IgG1 antibody.

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

[11] , wherein the mutant Fc region further comprises an amino acid modification at position 332 (EU numbering) in the first polypeptide of the parent Fc region.

[13] The anti-CTLA-4 antibody of any one of [1] to

[12] , wherein the mutant Fc region further comprises an amino acid modification at position 332 (EU numbering) in the second polypeptide of the parent Fc region.

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

[13] , wherein the mutant Fc region further comprises an amino acid modification at position 236 (EU numbering) in the second polypeptide of the parent Fc region.

[15] The anti-CTLA-4 antibody of any one of [1] to

[14] , wherein the mutant Fc region further comprises amino acid modifications at positions 250 and 307, as represented by EU numbering, in the first polypeptide of the parent Fc region.

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

[15] , wherein the mutant Fc region further comprises amino acid modifications at positions 250 and 307, as represented by EU numbering, in the second polypeptide of the parent Fc region.

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

[16] , wherein the mutant Fc region comprises at least one amino acid modification selected from the amino acid modifications described below: (i) in a first polypeptide of a parent Fc region, a Phe at position 234, a Gln at position 235, a Trp at position 236, a Met at position 239, a Val at position 250, an Asp at position 268, a Glu at position 270, an Ala at position 298, a Pro at position 307, a Met at position 330, and a Glu at position 332, as indicated by EU numbering; and (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, as indicated by EU numbering, in the second polypeptide of the parent Fc region.

[18] The anti-CTLA-4 antibody of any one of [1] to

[17] , wherein the mutant Fc region further comprises any one of the following amino acid modifications (a) to (f): (a) a Lys at position 356 (EU numbering) in a first polypeptide of a parent Fc region and a Glu at position 439 (EU numbering) in a second polypeptide of a parent Fc region; (b) Glu at position 439 (EU numbering) in the first polypeptide of the parent Fc region, and Lys at position 356 (EU numbering) in the second polypeptide of the parent Fc region. (c) a Trp at position 366, as indicated by EU numbering, in a first polypeptide of a parent Fc region, and a Ser at position 366, an Ala at position 368, and a Val at position 407, as indicated by EU numbering, in a second polypeptide of a parent Fc region; (d) Ser at position 366, Ala at position 368, and Val at position 407, as indicated by EU numbering, in a first polypeptide of the parent Fc region, and Trp at position 366, as indicated by EU numbering, in a second polypeptide of the parent Fc region; (e) a Cys at position 349 and a Trp at position 366, as indicated by EU numbering, in a first polypeptide of a parent Fc region, and a Cys at position 356, a Ser at position 366, an Ala at position 368, and a Val at position 407, as indicated by EU numbering, in a second polypeptide of a parent Fc region; (f) Cys at position 356, Ser at position 366, Ala at position 368, and Val at position 407, as indicated by EU numbering, in a first polypeptide of the parent Fc region, and Cys at position 349 and Trp at position 366, as indicated by EU numbering, in a second polypeptide of the parent Fc region.

[19] The anti-CTLA-4 antibody of any one of [1] to

[18] , wherein the variant Fc region further comprises any of the following amino acid modifications (a) to (d) in the first polypeptide and / or second polypeptide of the parent Fc region: (a) Ala at position 434, as expressed in EU numbering; (b) Ala at position 434, Thr at position 436, Arg at position 438, and Glu at position 440, as expressed in EU numbering; (c) Leu at position 428, Ala at position 434, Thr at position 436, Arg at position 438, and Glu at position 440, as indicated by EU numbering; (d) Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440, as expressed in EU numbering.

[20] The anti-CTLA-4 antibody of any one of [1] to

[19] , comprising a heavy chain constant region comprising a mutated Fc region.

[21] The heavy chain constant region is (1) a first polypeptide of SEQ ID NO: 358 and a second polypeptide of SEQ ID NO: 359, or (2) a first polypeptide of SEQ ID NO: 360, and a second polypeptide of SEQ ID NO: 361; The anti-CTLA-4 antibody of

[20] , comprising: 〔twenty two〕 (1) a first heavy chain polypeptide of SEQ ID NO: 335, a second heavy chain polypeptide of SEQ ID NO: 336, and a light chain polypeptide of SEQ ID NO: 161, or (2) a first H chain polypeptide of SEQ ID NO: 337, a second H chain polypeptide of SEQ ID NO: 338, and a L chain polypeptide of SEQ ID NO: 161; an anti-CTLA-4 antibody, comprising

[23] An isolated nucleic acid encoding the anti-CTLA-4 antibody of any one of [1] to

[22] .

[24] A host cell comprising the nucleic acid according to

[23] .

[25] A method for producing an anti-CTLA-4 antibody, comprising culturing the host cell of

[24] so that the anti-CTLA-4 antibody is produced.

[26] A pharmaceutical formulation comprising the anti-CTLA-4 antibody of any one of [1] to

[22] and a pharmaceutically acceptable carrier.

[0012] In one non-limiting embodiment, the present disclosure provides the following:

[101] A polypeptide comprising a mutant Fc region containing amino acid modifications in a parent Fc region, wherein the parent Fc region is composed of two polypeptide chains, and the mutant Fc region contains amino acid modifications at the following positions: (i) positions 234, 235, 236, 239, 268, 270, and 298, as represented by EU numbering, in the first polypeptide of the parent Fc region; and (ii) positions 270, 298, 326, and 334, as expressed in EU numbering, in the second polypeptide of the parent Fc region.

[102] The polypeptide described in

[101] , wherein the mutant Fc region further comprises an amino acid modification at position 326 (EU numbering) in the first polypeptide of the parent Fc region.

[103] The polypeptide described in

[101] or

[102] , wherein the mutant Fc region further comprises an amino acid modification at position 236 (EU numbering) in a second polypeptide of the parent Fc region.

[104] A polypeptide comprising a mutant Fc region containing amino acid modifications in a parent Fc region, wherein the parent Fc region is composed of two polypeptide chains, and the mutant Fc region contains amino acid modifications at the following positions: (i) positions 234, 235, 236, 239, 268, 270, 298, and 326, as represented by EU numbering, in the first polypeptide of the parent Fc region; and (ii) positions 236, 270, 298, 326, and 334, as expressed in EU numbering, in the second polypeptide of the parent Fc region.

[105] A polypeptide described in any of

[101] to

[104] , wherein the mutant Fc region further comprises an amino acid modification at position 332 (EU numbering) in the first polypeptide of the parent Fc region.

[106] A polypeptide described in any of

[101] to

[105] , wherein the mutant Fc region further comprises an amino acid modification at position 330 (EU numbering) in the first polypeptide of the parent Fc region.

[107] A polypeptide described in any of

[101] to

[106] , wherein the mutant Fc region further comprises an amino acid modification at position 332 (EU numbering) in a second polypeptide of the parent Fc region.

[108] A polypeptide described in any of

[101] to

[107] , wherein the mutant Fc region further comprises an amino acid modification at position 330 (EU numbering) in a second polypeptide of the parent Fc region.

[109] A polypeptide comprising a mutant Fc region containing amino acid modifications in a parent Fc region, wherein the parent Fc region is composed of two polypeptide chains, and the mutant Fc region contains amino acid modifications at the following positions: (i) positions 234, 235, 236, 239, 268, 270, 298, 330, and 332 (EU numbering) in the first polypeptide of the parent Fc region; and (ii) positions 236, 270, 298, 326, 330, 332, and 334, as expressed in EU numbering, in the second polypeptide of the parent Fc region.

[110] A polypeptide described in any of

[101] to

[109] , wherein the mutant Fc region further comprises amino acid modifications at positions 250 and 307, as represented by EU numbering, in the first polypeptide of the parent Fc region.

[111] A polypeptide described in any of

[101] to

[110] , wherein the mutant Fc region further comprises amino acid modifications at positions 250 and 307, as represented by EU numbering, in a second polypeptide of the parent Fc region.

[112] A polypeptide comprising a mutant Fc region containing amino acid modifications in a parent Fc region, wherein the parent Fc region is composed of two polypeptide chains, and the mutant Fc region contains amino acid modifications at the following positions: (i) positions 234, 235, 236, 239, 250, 268, 270, 298, and 307, as represented by EU numbering, in the first polypeptide of the parent Fc region; and (ii) positions 250, 270, 298, 307, 326, and 334, as expressed in EU numbering, in the second polypeptide of the parent Fc region.

[113] A polypeptide comprising a mutant Fc region containing amino acid modifications in a parent Fc region, wherein the parent Fc region is composed of two polypeptide chains, and the mutant Fc region contains amino acid modifications at the following positions: (i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, and 326 (EU numbering) in the first polypeptide of the parent Fc region; and (ii) positions 236, 250, 270, 298, 307, 326, and 334, as expressed in EU numbering, in the second polypeptide of the parent Fc region.

[114] A polypeptide comprising a mutant Fc region containing amino acid modifications in a parent Fc region, wherein the parent Fc region is composed of two polypeptide chains, and the mutant Fc region contains amino acid modifications at the following positions: (i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 330, and 332 (EU numbering) in the first polypeptide of the parent Fc region; and (ii) positions 236, 250, 270, 298, 307, 326, 330, 332, and 334, as expressed in EU numbering, in the second polypeptide of the parent Fc region.

[115] The polypeptide of any one of

[101] to

[114] , comprising at least one amino acid modification selected from the amino acid modifications described below: (i) in a first polypeptide of a parent Fc region, Tyr or Phe at position 234, Gln or Tyr 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, and Glu at position 332, as indicated by EU numbering; and (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, and Glu at position 334 in the second polypeptide of the parent Fc region, as indicated by EU numbering.

[116] The polypeptide of any one of

[101] to

[115] , wherein the mutant Fc region further comprises any one of the following amino acid modifications (a) to (f): (a) a Lys at position 356 (EU numbering) in a first polypeptide of a parent Fc region and a Glu at position 439 (EU numbering) in a second polypeptide of a parent Fc region; (b) Glu at position 439 (EU numbering) in the first polypeptide of the parent Fc region, and Lys at position 356 (EU numbering) in the second polypeptide of the parent Fc region. (c) a Trp at position 366, as indicated by EU numbering, in a first polypeptide of a parent Fc region, and a Ser at position 366, an Ala at position 368, and a Val at position 407, as indicated by EU numbering, in a second polypeptide of a parent Fc region; (d) Ser at position 366, Ala at position 368, and Val at position 407, as indicated by EU numbering, in a first polypeptide of the parent Fc region, and Trp at position 366, as indicated by EU numbering, in a second polypeptide of the parent Fc region; (e) a Cys at position 349 and a Trp at position 366, as indicated by EU numbering, in a first polypeptide of a parent Fc region, and a Cys at position 356, a Ser at position 366, an Ala at position 368, and a Val at position 407, as indicated by EU numbering, in a second polypeptide of a parent Fc region; (f) Cys at position 356, Ser at position 366, Ala at position 368, and Val at position 407, as indicated by EU numbering, in a first polypeptide of the parent Fc region, and Cys at position 349 and Trp at position 366, as indicated by EU numbering, in a second polypeptide of the parent Fc region.

[117] The polypeptide of any one of

[101] to

[116] , wherein the mutant Fc region further comprises any of the following amino acid modifications (a) to (d) in the first polypeptide and / or second polypeptide of the parent Fc region: (a) Ala at position 434, as expressed in EU numbering; (b) Ala at position 434, Thr at position 436, Arg at position 438, and Glu at position 440, as expressed in EU numbering; (c) Leu at position 428, Ala at position 434, Thr at position 436, Arg at position 438, and Glu at position 440, as indicated by EU numbering; (d) Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440, as expressed in EU numbering.

[118] A polypeptide described in any of

[101] to

[117] , in which the binding activity of the mutant Fc region to at least one Fcγ receptor selected from the group consisting of FcγRIa, FcγRIIa, FcγRIIb, and FcγRIIIa is enhanced compared to the parent Fc region.

[119] The polypeptide described in

[118] , wherein the binding activity to FcγRIIa and FcγRIIIa is enhanced in the mutant Fc region compared to the parent Fc region.

[120] A polypeptide described in any of

[101] to

[119] , in which the selectivity between activating Fcγ receptors and inhibitory Fcγ receptors is improved in the mutant Fc region compared to the parent Fc region. [120-2] A polypeptide described in any of

[101] to

[119] , in which the binding activity to activating Fcγ receptors in the mutant Fc region is selectively enhanced compared to the binding activity to inhibitory Fcγ receptors in the parent Fc region. [120-3] A polypeptide described in any of

[101] to

[119] , in which the ratio of binding activity to activating Fcγ receptors to binding activity to inhibitory Fcγ receptors (A / I ratio) is higher in the mutant Fc region than in the parent Fc region. [120-4] The ratio (A / I ratio) of the polypeptide comprising the mutant Fc region is 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, 60-fold or more, or The polypeptide described in [120-3] above is 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 larger. [120-5] The polypeptide described in [120-3], wherein the value of the ratio (A / I ratio) in the polypeptide comprising a mutant 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.

[121] The polypeptide of any of

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

[122] A polypeptide described in any one of

[101] to

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

[123] A method for producing a polypeptide containing a mutant Fc region, comprising the step of introducing amino acid modifications into a parent Fc region, wherein the parent Fc region is composed of two polypeptide chains, and amino acid modifications are introduced at the following positions: (i) positions 234, 235, 236, 239, 268, 270, and 298, as represented by EU numbering, in the first polypeptide of the parent Fc region; and (ii) positions 270, 298, 326, and 334, as expressed in EU numbering, in the second polypeptide of the parent Fc region.

[124] A method for producing a polypeptide containing a mutant Fc region, comprising the step of introducing amino acid modifications into a parent Fc region, wherein the parent Fc region is composed of two polypeptide chains, and amino acid modifications are introduced at the following positions: (i) positions 234, 235, 236, 239, 268, 270, 298, and 326, as represented by EU numbering, in the first polypeptide of the parent Fc region; and (ii) positions 236, 270, 298, 326, and 334, as expressed in EU numbering, in the second polypeptide of the parent Fc region.

[125] A method for producing a polypeptide containing a mutant Fc region, comprising the step of introducing amino acid modifications into a parent Fc region, wherein the parent Fc region is composed of two polypeptide chains, and amino acid modifications are introduced at the following positions: (i) positions 234, 235, 236, 239, 268, 270, 298, 330, and 332 (EU numbering) in the first polypeptide of the parent Fc region; and (ii) positions 236, 270, 298, 326, 330, 332, and 334, as expressed in EU numbering, in the second polypeptide of the parent Fc region.

[0013]

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

[101] to

[122] .

[127] A host cell comprising the nucleic acid described in

[126] .

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

[127] so that the polypeptide is produced.

[129] The polypeptide of any one of

[101] to

[122] for use in treating a tumor.

[130] A polypeptide according to any one of

[101] to

[122] for use in damaging cells.

[131] The polypeptide of

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

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

[101] to

[122] and a pharmaceutically acceptable carrier.

[133] The pharmaceutical composition according to

[132] , which is a pharmaceutical composition for treating tumors.

[134] The pharmaceutical composition according to

[132] , which is a pharmaceutical composition for cytotoxicity.

[135] The pharmaceutical composition of

[134] , wherein the cell damage is caused by ADCC activity, CDC activity, or ADCP activity.

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

[101] to

[122] or the pharmaceutical composition of

[132] .

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

[101] to

[122] or the pharmaceutical composition of

[132] .

[138] The method according to

[137] , wherein the cell is injured by ADCC activity, CDC activity, or ADCP activity.

[139] Use of the polypeptide of any one of

[101] to

[122] in the manufacture of a tumor therapeutic agent.

[140] Use of a polypeptide according to any one of

[101] to

[122] in the production of a cytotoxic agent.

[141] The use according to

[140] , wherein the cell damage is caused by ADCC activity, CDC activity, or ADCP activity.

[142] A method for modifying the function of a polypeptide comprising an Fc region, comprising introducing amino acid modifications into a parent Fc region, wherein the parent Fc region is composed of two polypeptide chains, and the amino acid modifications are introduced at the following positions: (i) positions 234, 235, 236, 239, 268, 270, and 298, as represented by EU numbering, in the first polypeptide of the parent Fc region; and (ii) positions 270, 298, 326, and 334, as expressed in EU numbering, in the second polypeptide of the parent Fc region.

[143] A method for modifying the function of a polypeptide comprising an Fc region, comprising introducing amino acid modifications into a parent Fc region, wherein the parent Fc region is composed of two polypeptide chains, and the amino acid modifications are introduced at the following positions: (i) positions 234, 235, 236, 239, 268, 270, 298, and 326, as represented by EU numbering, in the first polypeptide of the parent Fc region; and (ii) positions 236, 270, 298, 326, and 334, as expressed in EU numbering, in the second polypeptide of the parent Fc region.

[144] A method for modifying the function of a polypeptide comprising an Fc region, comprising introducing amino acid modifications into a parent Fc region, wherein the parent Fc region is composed of two polypeptide chains, and the amino acid modifications are introduced at the following positions: (i) positions 234, 235, 236, 239, 268, 270, 298, 330, and 332 (EU numbering) in the first polypeptide of the parent Fc region; and (ii) positions 236, 270, 298, 326, 330, 332, and 334, as expressed in EU numbering, in the second polypeptide of the parent Fc region.

[145] The method according to any one of

[142] to

[144] , wherein the altered function is an enhancement of binding activity to FcγRIIa and FcγRIIIa.

[146] The method of any one of

[142] to

[144] , wherein the functional alteration is an improvement in selectivity between an activating Fcγ receptor and an inhibitory Fcγ receptor.

[147] The method of any of

[142] to

[144] , wherein the functional alteration is a selective enhancement of binding activity to an activating Fcγ receptor compared to binding activity to an inhibitory Fcγ receptor.

[148] The method of any of

[142] to

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

[149] The ratio (A / I ratio) is 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, 60-fold or more, or 70-fold or more, compared to a polypeptide comprising a parent Fc region. , 80-fold or more, 90-fold or more, 100-fold or more, 200-fold or more, 300-fold or more, 400-fold or more, 500-fold or more, 600-fold or more, 700-fold or more, 800-fold or more, 900-fold or more, 1000-fold or more, 2000-fold or more, 3000-fold or more, 4000-fold or more, 5000-fold or more, 6000-fold or more, 7000-fold or more, 8000-fold or more, 9000-fold or more, or 10000-fold or more.

[150] The method of any of

[142] to

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

[151] The method according to any one of

[142] to

[144] , wherein the altered function is an enhancement of ADCC activity, CDC activity, or ADCP activity. [Brief explanation of the drawings]

[0014] [Figure 1] Figure 1 shows the results of measuring the in vitro antibody-dependent cellular cytotoxicity (ADCC) activity of the anti-CTLA4 switch antibody SW1610-ART5+ACT1 against CD4-positive T cells in which CTLA4 expression was induced, in the presence and absence of ATP, as described in Example 2-1. [Figure 2]Figure 2 shows the results of measuring the in vitro antibody-dependent cellular cytotoxicity (ADCC) activity of the anti-CTLA4 switch antibody SW1610-ART12 against CD4-positive T cells in which CTLA4 expression was induced, in the presence and absence of ATP, as described in Example 2-1. [Figure 3] Figure 3 shows the results of measuring the in vitro antibody-dependent cellular cytotoxicity (ADCC) activity of the anti-CTLA4 switch antibody SW1610-ART4 against CD4-positive T cells in which CTLA4 expression was induced, in the presence and absence of ATP, as described in Example 2-1. [Figure 4] Figure 4 shows the proportion of CD4-positive regulatory T (Treg) cells present in human peripheral blood mononuclear cells (PBMCs) when anti-CTLA4 switch antibody SW1610-ART12 was added in the presence and absence of ATP, as described in Example 2-2.

[0015] [Figure 5] FIG. 5 shows the CTLA-4 binding activity of the anti-CTLA-4 antibody ABAM004 depending on the ATP, ADP, or AMP concentration, as described in Reference Examples 1-9. [Figure 6] FIG. 6 shows the AMP concentration-dependent binding activity of the anti-CTLA-4 antibody ABAM004 to CTLA-4 expressing cells, as described in Reference Examples 1-10. [Figure 7] FIG. 7 shows the ADCC activity of the anti-CTLA-4 antibody ABAM004 against CTLA-4 expressing cells in the presence and absence of AMP, as described in Reference Example 1-11. [Figure 8] Figure 8 shows the binding mode between the ABAM004 Fab fragment and AMP, as described in Reference Example 2-13. In the figure, the heavy chain of the antibody is shown in black, the light chain in gray, and AMP as a ball-and-stick model. Amino acid residues that interact with AMP are shown as stick models. The dashed lines and their associated numbers indicate the distance (Å) between each amino acid residue and AMP. [Figure 9] Figure 9 shows the binding mode of the ABAM004 Fab fragment to AMP and human CTLA4 (hCTLA4), as described in Reference Example 2-14. In the figure, the heavy chain of the antibody is shown in black, the light chain in gray, hCTLA4 in white, and AMP in a ball-and-stick model. The epitope is defined as an amino acid residue of hCTLA4 containing one or more non-hydrogen atoms located within 4.2 Å of either the antibody or AMP, and is shown in a stick model. [Figure 10] Figure 10 shows the mapping of the epitope of the ABAM004 Fab fragment to the amino acid sequence of hCTLA4, as described in Reference Example 2-14. In the figure, black amino acid residues indicate hCTLA4 amino acid residues that contain one or more non-hydrogen atoms located within 4.2 Å of either ABAM004 or AMP in the crystal structure. Gray amino acid residues indicate residues that were disordered in the crystal structure and therefore could not be modeled. [Figure 11] Figure 11 shows the superposition of the crystal structures of the ABAM004 Fab fragment alone in complex with AMP, and the ternary complex with AMP and CTLA4, with the structure of the antibody and AMP extracted from the crystal structures, as described in Reference Example 2-15. In the figure, the heavy chain of the antibody is shown in black, the light chain in gray, and AMP as a ball-and-stick model. The structure of the ABAM004 Fab fragment alone is shown in thin lines, the structure of the binary complex with AMP in medium-thick lines, and the structure of the ternary complex in thick lines. [Figure 12] 12 is a graph showing the CTLA-4 binding activity of the anti-CTLA-4 antibody ABAM004 and its variants 04H0150 / 04L0072 depending on the ATP, ADP, or AMP concentration, as described in Reference Example 3-2. In the figure, WT represents ABAM004, and H150L072 represents 04H0150 / 04L0072, respectively. [Figure 13] FIG. 13 shows the ATP concentration-dependent neutralizing activity of anti-CTLA-4 antibody SW1077 against CTLA-4, as described in Reference Example 3-6. [Figure 14]Figure 14 shows the antitumor 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 0.01 mg / kg, 0.1 mg / kg, 0.25 mg / kg, 1 mg / kg, 10 mg / kg, 30 mg / kg, or 100 mg / kg. Each point represents the mean tumor volume for n=4 mice per group. [Figure 15] Figure 15 shows the antitumor effect of 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 0.1 mg / kg, 1 mg / kg, 10 mg / kg, 100 mg / kg, or 500 mg / kg. Each point represents the mean tumor volume for n=4 groups. [Figure 16] Figure 16 shows the change in the proportion of effector Treg cells in tumors following administration of the anti-CTLA-4 antibodies mNS-mFa55 (control antibody) and SW1208-mFa55 (switch antibody) 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, or 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, or 500 mg / kg. Tumors were harvested 6 days after administration, and the increase or decrease in effector Tregs was assessed by FACS analysis. The vertical axis represents the proportion of effector Tregs (CD4+ FoxP3+ KLRG1+) relative to CD45+ cells. The average value for n = 3 is shown. [Figure 17]Figure 17 shows the change in the percentage of activated helper T cells in the spleen following administration of the anti-CTLA-4 antibodies mNS-mFa55 (control antibody) and SW1208-mFa55 (switch antibody) in 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, or 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, or 500 mg / kg. Six days after administration, spleens were collected and the increase or decrease in activated helper T cells was assessed by FACS analysis. The vertical axis represents the percentage of activated helper T cells (CD4+ Foxp3- ICOS+) relative to CD45+ cells. The average value for n = 3 is shown. [Figure 18] Figure 18 shows the antitumor effect of 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, or 100 mg / kg. Each point represents the mean tumor volume for n=4 groups. [Figure 19] Figure 19 shows the antitumor effect of the anti-CTLA-4 antibody hNS-mFa55 (control 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, or 30 mg / kg. Each point represents the mean tumor volume for n=4 groups. [Figure 20]Figure 20 shows the change in the proportion of effector Treg cells in tumors following administration of the anti-CTLA-4 antibodies hNS-mFa55 (control antibody) and SW1389-mFa55 (switch antibody) in a mouse model transplanted with the Hepa1-6 / hGPC3 cell line, as described in Reference Example 4-3-8. hNS-mFa55 was administered via the tail vein at 0.1 mg / kg, 1 mg / kg, 10 mg / kg, or 30 mg / kg, and SW1389-mFa55 was administered at 0.1 mg / kg, 1 mg / kg, 10 mg / kg, 100 mg / kg, or 500 mg / kg. Tumors were harvested 6 days after administration, and the increase or decrease in effector Tregs was assessed by FACS analysis. The vertical axis represents the proportion of effector Tregs (CD4+ FoxP3+ CCR7lowKLRG1+) relative to CD45+ cells. The average value is shown for n = 3. [Figure 21] Figure 21 shows the change in the percentage of activated helper T cells in the spleen following administration of the anti-CTLA-4 antibodies hNS-mFa55 (control antibody) and SW1389-mFa55 (switch antibody) in a mouse model transplanted with the Hepa1-6 / hGPC3 cell line, as described in Reference Example 4-3-9. hNS-mFa55 was administered at 0.1 mg / kg, 1 mg / kg, 10 mg / kg, or 30 mg / kg, and SW1389-mFa55 was administered at 0.1 mg / kg, 1 mg / kg, 10 mg / kg, 100 mg / kg, or 500 mg / kg via the tail vein. Six days after administration, spleens were collected and the increase or decrease in activated helper T cells was assessed by FACS analysis. The vertical axis represents the percentage of activated helper T cells (CD4+ Foxp3- ICOS+) relative to CD45+ cells. The average value for n = 3 is shown. [Figure 22] Figure 22 shows the antitumor effect of anti-CTLA-4 antibody SW1610-mFa55 (switch antibody) in a mouse model implanted with the Hepa1-6 / hGPC3 cell line, as described in Reference Example 5-4-5. The antibody was administered via the tail vein at 0.3 mg / kg, 1 mg / kg, or 3 mg / kg. Each point represents the mean tumor volume for a group of 5 (n = 5). [Figure 23] Figure 23 shows the antitumor effect of anti-CTLA-4 antibody SW1612-mFa55 (switch antibody) in a mouse model implanted with the Hepa1-6 / hGPC3 cell line, as described in Reference Example 5-4-5. The antibody was administered via the tail vein at 0.3 mg / kg, 1 mg / kg, or 3 mg / kg. Each point represents the mean tumor volume for a group of 5 (n = 5). [Figure 24] Figure 24 shows the antitumor effect of 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 0.3 mg / kg, 1 mg / kg, or 3 mg / kg. Each point represents the mean tumor volume for a group of 5 (n = 5). [Figure 25] 25 shows the change in the proportion of effector Treg cells in tumors following administration of the anti-CTLA-4 antibodies SW1610-mFa55, SW1612-mFa55, and SW1615-mFa55 (all switch antibodies) in a mouse model implanted with the Hepa1-6 / hGPC3 cell line, as described in Reference Example 5-4-8. SW1610-mFa55 was administered at 50 mg / kg, 100 mg / kg, or 200 mg / kg; SW1612-mFa55 was administered at 50 mg / kg, 100 mg / kg, or 200 mg / kg; SW1615-mFa55 was administered at 50 mg / kg, 100 mg / kg, 200 mg / kg, or 400 mg / kg; and the negative control antibody KLH-mFa55 was administered at 400 mg / kg via the tail vein. Tumors were harvested 6 days after administration and the increase or decrease in effector Tregs was assessed by FACS analysis. The vertical axis represents the ratio of effector Tregs (CD4+ FoxP3+ CCR7lowKLRG1+) to CD45+ cells. The mean value of n=3 is shown. [Figure 26]26 shows the change in the percentage of activated helper T cells in the spleen following administration of the anti-CTLA-4 antibodies SW1610-mFa55, SW1612-mFa55, and 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 at 50 mg / kg, 100 mg / kg, or 200 mg / kg; SW1612-mFa55 was administered at 50 mg / kg, 100 mg / kg, or 200 mg / kg; SW1615-mFa55 was administered at 50 mg / kg, 100 mg / kg, 200 mg / kg, or 400 mg / kg; and the negative control antibody KLH-mFa55 was administered at 400 mg / kg via the tail vein. Six days after administration, spleens were collected and the increase or decrease in activated helper T cells was evaluated by FACS analysis. The vertical axis represents the ratio of activated helper T cells (CD4+ Foxp3- ICOS+) to CD45+ cells. The mean value of n=3 is shown. [Figure 27] Figure 27 shows a comparison of the in vitro ADCC activity of antibodies with various altered constant regions that exhibit enhanced FcγR binding, as described in Reference Example 6-2. 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 represents an antibody with a control constant region, GASDALIE represents an antibody with a constant region described in a prior publication, and ART6 and ART8 represent antibodies with altered constant regions prepared in Reference Example 6-1. [Figure 28]Figure 28 shows a comparison of the in vitro ADCP activity of antibodies with various altered constant regions that exhibit enhanced FcγR binding, as described in Reference Example 6-3. 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 represents an antibody with a control constant region, GASDIE represents an antibody with a constant region described in a prior publication, and ART6 and ART8 represent antibodies with altered constant regions prepared in Reference Example 6-1. [Figure 29] Figure 29 shows the in vitro ADCC activity of anti-CTLA4 switch antibody SW1389-ART6, which has a modified constant region with enhanced binding to FcγR, as described in Reference Example 6-4. [Figure 30] Figure 30 shows the in vitro ADCC activity of anti-CTLA4 switch antibody SW1610-ART6, which has a modified constant region with enhanced binding to FcγR, as described in Reference Example 6-4. [Figure 31] Figure 31 shows the in vitro ADCC activity of anti-CTLA4 switch antibody SW1612-ART6, which has a modified constant region with enhanced binding to FcγR, as described in Reference Example 6-4. [Figure 32] Figure 32 shows the neutralizing activity of anti-CTLA4 switch antibody SW1389 against CTLA4 (the activity of canceling the CTLA4 signal that acts suppressively on the activation of effector cells), as described in Reference Example 6-5. [Figure 33] Figure 33 shows the neutralizing activity of anti-CTLA4 switch antibody SW1610 against CTLA4 (the activity of canceling the CTLA4 signal that acts suppressively on the activation of effector cells), as described in Reference Example 6-5. [Figure 34] Figure 34 shows the neutralizing activity of anti-CTLA4 switch antibody SW1612 against CTLA4 (the activity of canceling the CTLA4 signal that acts suppressively on the activation of effector cells), as described in Reference Example 6-5. [Figure 35] Figure 35 shows the neutralizing activity of anti-CTLA4 switch antibody SW1615 against CTLA4 (the activity of canceling the CTLA4 signal that acts suppressively on the activation of effector cells), as described in Reference Example 6-5. [Figure 36] Figure 36 shows the in vitro cytotoxic activity of anti-CTLA4 switch antibody SW1389-ART5+ACT1 against CTLA4-positive regulatory T cells, as described in Reference Example 6-6. [Figure 37] Figure 37 shows the in vitro cytotoxic activity of anti-CTLA4 switch antibody SW1389-ART6+ACT1 against CTLA4-positive regulatory T cells, as described in Reference Example 6-6. [Figure 38] Figure 38 shows the in vitro cytotoxic activity of anti-CTLA4 switch antibody SW1610-ART5+ACT1 against CTLA4-positive regulatory T cells, as described in Reference Example 6-6. [Figure 39] Figure 39 shows the in vitro cytotoxic activity of anti-CTLA4 switch antibody SW1610-ART6+ACT1 against CTLA4-positive regulatory T cells, as described in Reference Example 6-6.

[0016] [Figure 40] Figure 40 shows the results of an ADCC reporter gene assay using Hepa1-6 / hEREG cells as target cells and hFcγRIIIaV-expressing Jurkat cells as effector cells, as described in Reference Example 9-2. Each point represents the average fold induction value for n=2. [Figure 41]Figure 41 shows the results of an ADCP reporter gene assay using Hepa1-6 / hEREG cells as target cells and Jurkat cells expressing hFcγRIIaH as effector cells, as described in Reference Example 10. Each point represents the average fold induction value (n=3). [Figure 42] Figure 42 shows the antitumor effects of EGL-G1d, EGL-afucosyl, 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. The antibodies were administered via the tail vein at 10 mg / kg. Each point represents the mean tumor volume for a group of n=5. [Figure 43] Figure 43 shows the binding activity of each antibody with a modified Fc to hC1q, as described in Reference Example 12. Each point represents the average ELISA color development value (n=2). [Figure 44] Figure 44 is a continuation of the graph showing the binding activity to hC1q of each antibody having a modified Fc, as described in Reference Example 12. Each point represents the average ELISA color development value for n=2. DETAILED DESCRIPTION OF THE INVENTION

[0017] The techniques and procedures described or cited herein are generally well understood and can be found, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd edition (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; Animal Cell Culture (RI Freshney), ed., 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 C.C.Blackwell, eds.);Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987);PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994);Current Protocols in Immunology (JE Coligan et al., eds., 1991);Short Protocols in Molecular Biology (Wiley and Sons, 1999);Immunobiology (CA 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, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (VT DeVita et al., eds., JB Lippincott Company, 1993), using conventional techniques commonly used by those skilled in the art.

[0018] I. Definition Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994), and March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, NY 1992) provide those skilled in the art with general guidance for 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.

[0019] For purposes of interpreting this specification, the following definitions will apply, and wherever applicable, terms used in the singular will also include the plural and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. In the event that any of the definitions below conflict with any document incorporated herein by reference, the definition below shall control.

[0020] As used herein, the term "and / or" refers to each of the objects listed before and after "and / or" or any combination thereof. For example, "A, B and / or C" includes each of the objects "A," "B," and "C," as well as any combination selected from among "A and B," "A and C," "B and C," and "A and B and C."

[0021] An "acceptor human framework," for purposes of this specification, is a framework that comprises 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 may comprise those same amino acid sequences or may contain 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.

[0022] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" (antibody-dependent cell-mediated cytotoxicity) refers to a form of cytotoxicity in which secreted immunoglobulins bind to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., NK cells, neutrophils, and macrophages), thereby enabling these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill them with cytotoxins. NK cells, the primary cells for mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. The expression of FcRs on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991). To assess the ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that 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, the ADCC activity of a molecule of interest can be assessed in vivo in an animal model, such as that disclosed in Clynes et al. PNAS (USA) 95: 652-656 (1998).

[0023] Examples of "cytotoxic activity" include the above-mentioned antibody-dependent cell-mediated cytotoxicity (ADCC) activity, the below-mentioned complement-dependent cytotoxicity (CDC) activity, and T-cell cytotoxic activity. CDC activity refers to cytotoxic activity mediated by the complement system. On the other hand, ADCC activity refers to the activity of an antibody binding to an antigen present on the surface of a target cell, and then effector cells binding to the antibody, causing the effector cells to injure the target cell. Whether an antibody of interest has ADCC activity or 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)).

[0024] "Neutralizing activity" refers to the activity of an antibody that inhibits a biological activity by binding to a molecule involved in that biological activity. In some embodiments, the biological activity is brought about by the binding of a ligand to a receptor. In certain embodiments, the antibody inhibits the binding of the ligand to the receptor by binding to the ligand or receptor. An antibody having such neutralizing activity is called a neutralizing antibody. The neutralizing activity of a test substance can be measured by comparing the biological activity in the presence of a ligand with that in the presence or absence of the test substance.

[0025] The term "antibody-dependent cellular phagocytosis" or "ADCP" refers to the process by which antibody-coated cells, either in whole or in part, are internalized by phagocytic immune cells (e.g., macrophages, neutrophils, and dendritic cells) that bind to the Fc region of immunoglobulin.

[0026] The terms "binding activity" and "binding capacity" are used interchangeably herein and refer to the strength of the sum of noncovalent interactions between one or more binding sites (e.g., variable region or Fc region) of a molecule (e.g., an antibody or other polypeptide) and the molecule's binding partner (e.g., an antigen or Fcγ receptor). Here, "binding activity" is not strictly limited to 1:1 interactions between members of a binding pair (e.g., an antibody and an antigen, or an Fc region and an Fcγ receptor). For example, when members of a binding pair reflect a monovalent 1:1 interaction, binding activity refers to the intrinsic binding affinity ("affinity"). When members of a binding pair are capable of both monovalent and multivalent binding, binding activity is the sum of these avidities. The binding activity of a molecule X for its partner Y can generally be expressed as a dissociation constant (KD) or "amount of analyte bound per unit amount of ligand." Avidity can be measured by conventional methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring avidity are described below.

[0027] An "affinity matured" antibody refers to an antibody with one or more modifications in one or more hypervariable regions (HVRs) that result in improved affinity of the antibody for antigen, compared to a parent antibody that does not possess such modifications.

[0028] The term "anti-CTLA-4 antibody" or "antibody that binds to CTLA-4" refers to an antibody that can bind to CTLA-4 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent when targeted to CTLA-4. In one embodiment, the extent of binding of the anti-CTLA-4 antibody to an unrelated, non-CTLA-4 protein is less than about 10% of the binding of the antibody to CTLA-4, as measured (e.g., by radioimmunoassay (RIA)). In certain embodiments, an antibody that binds to CTLA-4 has a binding affinity of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., ≦10 -8 M or less, e.g. 10 -8 M~10 -13 M, e.g., 10 -9 M~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.

[0029] The term "antibody" is used herein in the broadest sense and encompasses 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.

[0030] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of the 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.

[0031] An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks the binding of the reference antibody to its own antigen in a competition assay, e.g., by 50% or more, and / or the reference antibody blocks the binding of the antibody to its own antigen in a competition assay, e.g., by 50% or more. Exemplary competition assays are provided herein.

[0032] "Autoimmune disease" refers to a non-malignant disease or disorder arising from and directed against an individual's own tissues. As used herein, autoimmune disease specifically excludes malignant or cancerous diseases or conditions, and specifically excludes B-cell lymphoma, acute lymphoblastic 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 sclerosis 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 conditions, such as eczema and asthma and other conditions involving T-cell infiltration and chronic inflammatory responses; atherosclerosis; leukocyte adhesion deficiency; rheumatoid arthritis; and systemic lupus erythematosus (SLE). (including, but not limited to, lupus nephritis, cutaneous lupus); diabetes (e.g., type 1 diabetes or insulin-dependent diabetes); multiple sclerosis; Raynaud's syndrome; autoimmune thyroiditis; Hashimoto's thyroiditis; allergic encephalomyelitis; Sjogren's syndrome; juvenile-onset diabetes; and immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T lymphocytes, typically seen in tuberculosis, sarcoidosis, polymyositis, granulomatosis, and vasculitis; pernicious anemia (Addison's disease); diseases associated with leukocyte leakage; and the central nervous system (CNS). Inflammatory disorders; multiple organ injury syndrome; hemolytic anemia (including but not limited to cryoglobulinemia or Coombs' positive anemia); myasthenia gravis; antigen-antibody complex-mediated disease; antiglomerular basement membrane disease; antiphospholipid syndrome; allergic neuritis; Graves' disease; Lambert-Eaton myasthenic syndrome; bullous pemphigoid; pemphigus; autoimmune polyendocrinopathy; Reiter's disease; Stiffman syndrome; Behçet's disease; giant cell arteritis; immune complex nephritis; IgA nephropathy; IgM polyneuropathy; immune thrombocytopenic purpura (ITP) or autoimmune thrombocytopenia.

[0033] 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.

[0034] The term "complement-dependent cytotoxicity" or "CDC" refers to a mechanism for inducing cell death in which the Fc effector domain of a target-bound antibody activates a series of enzymatic reactions that result in the formation of holes in the target cell's membrane. Typically, antigen-antibody complexes formed on the target cell bind to and activate complement component C1q, which then activates the complement cascade, leading to target cell death. Complement activation can also result in the deposition of complement components on the surface of target cells, which promote ADCC by binding to complement receptors (e.g., CR3) on leukocytes.

[0035] "Chemotherapeutic agent" refers to a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include: alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN®); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylolmelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicine; betulinic acid; camptothecin (including the synthetic analogs topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®)), acetylcamptothecin, and scopolectin. (including scopolectin, and 9-aminocamptothecin); bryostatin; kallistatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); podophyllotoxin; podophyllic acid; teniposide; cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictiin; spongistatin; chlorambucil, chlornaphazine, chlorophosphamide (chlorophosphamide), estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobuenbiquine, fenesterine, prednimustine, trofosfamide, nitrogen mustards, such as uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine;Enediyne antibiotics {e.g., calicheamicin, particularly calicheamicin gamma 11 and calicheamicin omega 11 (e.g., Nicolaou et al., Angew. Chem Intl. Ed. Engl., 33: 183-186 (1994)) See also); CDP323, an oral alpha-4 integrin inhibitor; dynemicins, including dynemicin A; esperamicin; and neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores, aclacinomycin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, carubicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (ADRIAMYCIN®), morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HCl liposomal for injection (DOXIL®), liposomal doxorubicin TCL D-99 (including MYOCET®), pegylated liposomal doxorubicin (including CAELYX®, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, quelamycin, and lodorubicin antibiotics, such as rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites, such as methotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), epothilones, and 5-fluorouracil (5-FU); folic acid analogs, such as denopterin, methotrexate, pteropterin, and trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine;Pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calucelone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; antiadrenal agents such as aminoglutethimide, mitotane, and trilostane. (anti-adrenals); folic acid supplements, such as folinic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatrexate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydrochloride; lentinan; lonidamine; maytansinoids, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR; razoxane; rhizoxin; schizophyllan; spirogermanium; tenuazonic acid; triaziquone; 2,2',2'-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verraculin A) A), Roridin A, and Anguidine; urethane; vindesine (ELDISINE®, FILDESIN®); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); thiotepa; taxoids, such as paclitaxel (TAXOL®), albumin-modified nanoparticle formulations of paclitaxel (ABRAXANE™), and docetaxel (TAXOTERE®); chlorambucil; 6-thioguanine; mercaptopurine; methotrexate; platinum agents, such as cisplatin, oxaliplatin (e.g., ELOXATIN®), and carboplatin;Vinces, which interfere with the formation of microtubules by tubulin polymerization, include vinblastine (VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®, FILDESIN®), and vinorelbine (NAVELBINE®); etoposide (VP-16); ifosfamide; mitoxantrone; leucovorin; novantrone; edatrexate; daunomycin; aminopterin; ibandronate; the topoisomerase inhibitor RFS 2000; difluoromethylornithine; DMFO; retinoids, such as retinoic acids, including bexarotene (TARGRETIN®); bisphosphonates, such as clodronate (e.g., BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid / zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®); troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit the expression of genes in signaling pathways associated with abnormal cell proliferation, such as PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGFR). EGF-R; vaccines such as THERATOPE® vaccine and gene therapy vaccines (e.g., ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine); topoisomerase 1 inhibitors (e.g., LURTOTECAN®); rmRH (e.g., ABARELIX®); BAY439006 (sorafenib; Bayer); SU-11248 (sunitinib, SUTENT®, Pfizer); perifosine, COX-2 inhibitors (e.g., celecoxib or etoricoxib), proteosome inhibitors (e.g., PS341);Bcl-2 inhibitors such as bortezomib (VELCADE®); CCI-779; tipifarnib (R11577); sorafenib, ABT510; oblimersen sodium (GENASENSE®); pixantrone; EGFR inhibitors (see definition below); tyrosine kinase inhibitors (see definition below); serine-threonine kinase inhibitors such as rapamycin (sirolimus, RAPAMUNE®); lonafarnib (SCH 6636, SARASAR™); and pharmaceutically acceptable salts, acids, or derivatives of any of the above; and combinations of two or more of the above, such as CHOP, which is an abbreviation for combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone; and FOLFOX, which is an abbreviation for a treatment regimen with oxaliplatin (ELOXANTIN™) in combination with 5-FU and leucovorin;

[0036] 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.

[0037] The "class" of an antibody refers to the type of constant domain or constant region present in the antibody's heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM. Some of these may be 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.

[0038] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents the function of cells and / or causes the death or destruction of cells. Cytotoxic agents include, but are not limited to, radioisotopes (e.g., 211 At, 131 I, 125I, 90 Y, 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P, 212 radioactive isotopes of Pb and Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents); growth inhibitors; enzymes and fragments thereof, such as nucleases; antibiotics; toxins, such as, for example, small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin (including fragments and / or variants thereof); and the various chemotherapeutic agents disclosed above.

[0039] "Effector cell" refers to a leukocyte that expresses one or more FcRs and exerts effector function. In certain embodiments, the cell expresses at least FcγRIII and exerts ADCC effector function. 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, e.g., from blood. In certain embodiments, effector cells can be human effector cells.

[0040] "Effector function" refers to a biological activity attributable to the Fc region of an antibody, which varies depending on 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), down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0041] The term "epitope" includes any determinant that can be bound by an antibody. An epitope is a region of an antigen that is bound by an antibody that targets that antigen and includes specific amino acids that directly contact the antibody. Epitopic determinants can include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and can have specific three-dimensional structural characteristics and / or specific charge characteristics. Generally, antibodies specific for a particular target antigen will preferentially recognize an epitope on that target antigen in a complex mixture of proteins and / or macromolecules.

[0042] "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR is a native human FcR. In some embodiments, the FcR 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 (an "activating receptor") and FcγRIIB (an "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. The 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 reviewed, 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). Other FcRs, including those identified in the future, are also encompassed by the term "FcR" herein.

[0043] The term "Fc receptor" or "FcR" also includes the neonatal receptor FcRn, which is responsible for regulating maternal IgG transfer to the fetus (Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J. Immunol. 24: 249 (1994)) and immunoglobulin homeostasis. Methods for measuring binding to FcRn are known (see, for example, 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); WO2004 / 92219 (Hinton et al.)).

[0044] The in vivo binding to human FcRn and serum half-life of human FcRn high-affinity binding polypeptides can be measured, for example, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates to which polypeptides with mutated Fc regions are administered. WO2000 / 42072 (Presta) describes antibody mutants with improved or reduced binding to FcR. See, for example, Shields et al. J. Biol. Chem. 9(2): 6591-6604 (2001).

[0045] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the constant region. This term includes native-sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain, except that the C-terminal lysine (Lys447) or glycine-lysine (Gly446-Lys447) residues of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is 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 Ed. Public Health Service, National Institutes of Health, Bethesda, MD 1991.

[0046] The term "Fc region-containing antibody" refers to an antibody that contains 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 a nucleic acid encoding the antibody. Thus, a composition containing an antibody with an Fc region according to the present invention can contain an antibody with G446-K447, an antibody with G446 but without K447, an antibody with G446-K447 completely removed, or a mixture of the above three types of antibodies.

[0047] 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 heavy and light chain variable domains (VH and VL, respectively) of natural antibodies typically have similar structures, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR) (see, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen may be isolated by screening a complementary library of VL or VH domains, respectively, using a VH or VL domain from an antibody that binds to the antigen. See, e.g., Portolano et al., J. Immunol. 150: 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991).

[0048] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain typically consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences typically appear in VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0049] The terms "full length antibody," "complete antibody," and "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain that includes an Fc region as defined herein.

[0050] A "functional Fc region" comprises an "effector function" 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 receptors (BCRs)); and the like. Such effector functions generally require that the Fc region be combined with a binding domain (e.g., an antibody variable domain) and can be assessed using various assays, for example, as disclosed within the definitions herein.

[0051] A "human antibody" is an antibody with an amino acid sequence that corresponds to that of an antibody produced by a human or human cell, or an antibody derived from a human antibody repertoire or other non-human source that uses human antibody coding sequences. This definition of a human antibody specifically excludes humanized antibodies, which contain non-human antigen-binding residues.

[0052] 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 subgroup of sequences is a subgroup in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one embodiment, for VL, the subgroup is subgroup κI according to Kabat et al., supra. In one embodiment, for VH, the subgroup is subgroup III according to Kabat et al., supra.

[0053] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and human FRs. In certain embodiments, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, in which all or substantially all HVRs (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all FRs correspond to those of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization.

[0054] As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain that is hypervariable in sequence (the "complementarity determining region" or "CDR") and / or forms structurally defined loops (the "hypervariable loops") and / or contains antigen-contacting residues (the "antigen contacts"). Typically, antibodies contain six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Exemplary HVRs herein include the following: (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)); (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)); (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 (d) A combination of (a), (b), and / or (c), comprising 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). Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

[0055] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, including but not limited to, cytotoxic agents.

[0056] An "isolated" antibody is one that has been separated from a component of its original environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, for example, as measured by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848: 79-87 (2007).

[0057] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its original environment. Isolated nucleic acid includes a nucleic acid molecule contained in cells that normally contain the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or in a chromosomal location that is different from its natural chromosomal location.

[0058] An "isolated nucleic acid encoding an antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of an antibody, including nucleic acid molecules carried on a single vector or separate vectors, and including nucleic acid molecules present in one or more locations in a host cell.

[0059] As used herein, "first polypeptide" and "second polypeptide" refer to polypeptides that constitute the Fc region of an antibody. "First polypeptide" and "second polypeptide" mean that they have different sequences, and preferably differ in at least the sequence of their CH2 regions. They may also differ in the sequence of their CH3 regions. Such polypeptides may be, for example, polypeptides that constitute the Fc region of native IgG, or may be polypeptides in which modifications have been made to the polypeptides that constitute the Fc region of native IgG.

[0060] Native IgG refers to a polypeptide that includes the same amino acid sequence as IgG found in nature and belongs to the class of antibodies substantially encoded by immunoglobulin gamma genes. For example, native human IgG refers to native human IgG1, native human IgG2, native human IgG3, native human IgG4, etc. Native IgG also includes naturally occurring mutants thereof.

[0061] In the present invention, the term "polypeptide" generally refers to a peptide or protein having a length of about 10 amino acids or more. It is generally a polypeptide derived from a living organism, but is not particularly limited thereto, and may be, for example, a polypeptide consisting of an artificially designed sequence. It may also be a natural polypeptide, a synthetic polypeptide, a recombinant polypeptide, or the like. In addition, the term "protein molecule" in the present invention refers to a molecule containing such a polypeptide.

[0062] A preferred example of the polypeptide of the present invention is an antibody. Further preferred examples include natural IgG and antibodies obtained by modifying natural IgG. An example of natural IgG is, in particular, natural human IgG. Natural IgG refers to a polypeptide that includes the same amino acid sequence as an IgG found in nature and belongs to the class of antibodies substantially encoded by immunoglobulin gamma genes. For example, natural human IgG refers to natural human IgG1, natural human IgG2, natural human IgG3, natural human IgG4, etc. Natural IgG also includes naturally occurring mutants thereof.

[0063] The term "polypeptide comprising an Fc region" is not particularly limited as long as it refers to a polypeptide containing an Fc region, including, 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 a nucleic acid encoding the polypeptide. Thus, a composition comprising a polypeptide having an Fc region of the present invention can include a polypeptide comprising an Fc region with G446-K447, a polypeptide comprising an Fc region with G446 but without K447, a polypeptide with an Fc region from which G446-K447 has been completely removed, or a mixture of the above three types of polypeptides.

[0064] An "isolated" polypeptide is one that has been separated from a component of its original environment. In some embodiments, the polypeptide is purified to greater than 95% or 99% purity, for example, as measured by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). For a review of methods for assessing polypeptide purity, see, e.g., Flatman et al., J. Chromatogr. B 848: 79-87 (2007).

[0065] An "isolated nucleic acid encoding a polypeptide" refers to one or more nucleic acid molecules that encode the polypeptide (e.g., an Fc region of an antibody, or the heavy and light chains or fragments thereof of an antibody), and includes nucleic acid molecules carried on a single vector or separate vectors, and nucleic acid molecules present in one or more locations in a host cell.

[0066] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. This term includes vectors as self-replicating nucleic acid structures and vectors that are integrated into the genome of a host cell into which they are introduced. Some vectors are capable of conferring expression of a nucleic acid to which they are operatively linked. Such vectors are also referred to herein as "expression vectors." Vectors can be introduced into host cells by viral or electroporation methods, but vector introduction is not limited to ex vivo introduction; vectors can also be introduced directly into a living body.

[0067] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and 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 originally transformed cell and progeny derived from that cell regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as that for which the original transformed cell was screened or selected are also included herein.

[0068] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies. That is, the individual antibodies comprising the population are identical and / or bind to the same epitope, except for possible variants (e.g., variants containing naturally occurring mutations or variants that arise during the production of a monoclonal antibody preparation; such variants are usually present in small amounts). In contrast 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 an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be produced by 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 making monoclonal antibodies are described herein.

[0069] A "naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or a radiolabel. Naked antibodies may be present in a pharmaceutical formulation.

[0070] "Native antibodies" refer to immunoglobulin molecules with various naturally occurring structures. For example, native IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light domain or light chain variable domain, followed by a constant light (CL) domain. The light chain of an antibody can be assigned to one of two types, called kappa (κ) or lambda (λ), based on the amino acid sequence of its constant domain.

[0071] A "native-sequence Fc region" comprises an amino acid sequence identical to that 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, as well as naturally occurring variants thereof.

[0072] A "mutant Fc region" comprises an amino acid sequence that differs from that of a native-sequence Fc region by at least one amino acid modification (alteration), preferably one or more amino acid substitutions. Preferably, a mutant Fc region has at least one amino acid substitution, 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, in the native-sequence Fc region or parent Fc region compared to the native-sequence Fc region or parent Fc region. The mutant Fc region herein preferably has at least about 80% homology with the native-sequence Fc region or 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.

[0073] "Percent (%) amino acid sequence identity" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after aligning the sequences to achieve the maximum percent sequence identity and introducing gaps, if necessary, and excluding any conservative substitutions from the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved by a variety of methods within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software, or GENETYX® (Genetyx Corporation). Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared.

[0074] The ALIGN-2 sequence comparison computer program is the copyright of Genentech, Inc., and its source code, together with user documentation, has been filed with the U.S. Copyright Office, Washington, DC 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 may be compiled from the source code. The ALIGN-2 program is compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary. In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (alternatively, one can say that a given amino acid sequence A has or contains a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in its alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that if 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 equal the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph.

[0075] The terms "pharmaceutical formulation" and "pharmaceutical composition" are used interchangeably and refer to a preparation in a form that allows the biological activity of the active ingredient contained therein to be effective, and that does not contain additional components that are unacceptably toxic to the subject to which it is administered.

[0076] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, 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 human.

[0077] A "pharmaceutically acceptable carrier" refers to an ingredient, other than an active ingredient, in a pharmaceutical formulation or composition that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0078] An "effective amount" of an agent (e.g., a pharmaceutical formulation) refers to an amount, at dosages and for periods of time necessary, effective to achieve a desired therapeutic or prophylactic result.

[0079] The term "package insert" is used to refer to instructions typically included in commercial packaging of therapeutic products that contain information about the indications, usage, dosage, method of administration, concomitant therapy, contraindications, and / or warnings regarding the use of such therapeutic product.

[0080] As used herein, the term "CTLA-4," unless otherwise indicated, refers to any native form of CTLA-4 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full-length," unprocessed CTLA-4 as well as any form of CTLA-4 resulting from processing in cells. The term also encompasses naturally occurring variants of CTLA-4, such as splice variants and allelic variants. An exemplary amino acid sequence of human CTLA-4 is set forth in SEQ ID NO:214, mouse CTLA-4 in SEQ ID NO:247, monkey CTLA-4 in SEQ ID NO:248, and the amino acid sequence of the extracellular domain of human CTLA-4 in SEQ ID NO:28. CTLA-4 may also be referred to herein as CTLA4.

[0081] The term "regulatory T (Treg) cells" refers to a subpopulation of T cells that regulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune diseases. These cells generally suppress 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 + These Tregs are distinct from helper T cells. Several different methods are used to identify and monitor Treg cells. These are defined by CD4 and CD25 expression (CD4 + CD25 +cells), Treg cells are mature CD4 + They comprise approximately 5-10% of the T cell subpopulation, while approximately 1-2% of Tregs can be measured in whole blood. Expression of Foxp3 may also be measured (CD4 + CD25 + Foxp3 + Alternatively, the absence or low expression of CD127, in combination with the presence of CD4 and CD25, may be used as another marker. Treg cells also express high levels of CTLA-4 and GITR. Tregs can also be identified by the methods described in the Examples below.

[0082] As used herein, the terms "substantially similar," "substantially equal," or "substantially the same" refer to a similarity between two numerical values ​​(e.g., between one relating to an antibody of the invention and one relating to a reference / comparator antibody) that is sufficiently high that one of skill in the art would consider the difference between the two numerical values ​​to have little or no biological and / or statistical significance in terms of the biological characteristic measured by the numerical values ​​(e.g., KD value).

[0083] As used herein, "treatment" (and its grammatical derivatives, such as "treat," "treating," etc.) refers to a clinical intervention intended to alter the natural course of the individual being treated and can be performed prophylactically or during the course of a clinical condition. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, attenuation of any direct or indirect pathological effects of the disease, prevention of metastasis, reduction in the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, antibodies of the present invention are used to delay the onset of disease or slow the progression of disease. In some embodiments, polypeptides comprising a variant Fc region of the present invention are used to delay the onset of disease or slow the progression of disease.

[0084] The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive as used herein.

[0085] The term "tumor tissue" refers to tissue containing at least one tumor cell. Tumor tissue usually consists of a group of tumor cells (parenchyma), which form the main body of the tumor, and the connective tissue and blood vessels (stroma) that exist between them and support the tumor. In some cases, the distinction between the two is clear, while in other cases, the two are mixed. Immune cells and other substances may infiltrate tumor tissue. On the other hand, "non-tumor tissue" refers to tissue other than tumor tissue in the body. Healthy tissue / normal tissue that is not in a diseased state is a typical example of non-tumor tissue.

[0086] II. Compositions and Methods 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 are useful, for example, for the diagnosis or treatment of cancer.

[0087] A. Exemplary anti-CTLA-4 antibodies In one aspect, the present invention provides isolated antibodies that bind to CTLA-4. In certain embodiments, the anti-CTLA-4 antibodies of the present 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 the adenosine-containing compound. Alternatively, in other embodiments, 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 the adenosine-containing compound. In further embodiments, the difference in binding activity to CTLA-4 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 More than twice, 2×10 3More than double, 3×10 3 More than twice, 5×10 3 More than twice, 1×10 4 More than twice, 2×10 4 More than double, 3×10 4 More than twice, 5×10 4 More than double, or 1×10 5 It is more than double.

[0088] In some embodiments, the binding activity of an anti-CTLA-4 antibody can be expressed as a KD (Dissociation constant) value. In a further embodiment, the KD value of the anti-CTLA-4 antibody is smaller in the presence of an adenosine-containing compound than in the absence of the adenosine-containing compound. Alternatively, in another embodiment, the KD value of the anti-CTLA-4 antibody is smaller in the presence of a high concentration of an adenosine-containing compound than in the presence of a low concentration of the adenosine-containing compound. In a further embodiment, the difference in the KD values ​​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 More than twice, 2×10 3 More than double, 3×10 3 More than twice, 5×10 3 More than twice, 1×10 4 More than twice, 2×10 4 More than double, 3×10 4 More than twice, 5×10 4 More than double, or 1×10 5 The KD value of an anti-CTLA-4 antibody in the presence of an adenosine-containing compound or in the presence of a high concentration of an adenosine-containing compound is, 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 x 10 -10 The KD value of an 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 can 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 above, 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 larger, or 9 x 10 -6 It can be M or more.

[0089] In another embodiment, the binding activity of an anti-CTLA-4 antibody may be expressed as a kd (Dissociation rate constant) value instead of a KD value.

[0090] In another embodiment, the binding activity of an anti-CTLA-4 antibody may be expressed as 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 a response unit (RU). The value obtained by dividing the amount of antigen bound thereto by the amount of antibody bound thereto can be defined as the amount of antigen bound per unit amount of antibody. Specific methods for measuring 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 that in the absence of an 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 that in the presence of a low concentration of an adenosine-containing compound. In further embodiments, the difference in the amount of CTLA-4 binding 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 More than twice, 2×10 3 More than double, 3×10 3 More than twice, 5×10 3 More than twice, 1×10 4 More than twice, 2×10 4 More than double, 3×10 4 More than twice, 5×10 4 More than double, or 1×10 5The value of the amount of CTLA-4 binding 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 binding 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.

[0091] In some embodiments, the KD values, kd values, binding amounts, etc., described herein are measured or calculated by performing a surface plasmon resonance assay at 25°C or 37°C (see, e.g., Reference Example 3 herein).

[0092] The concentration of the adenosine-containing compound can be selected at any concentration as long as it detects a difference in the binding activity of anti-CTLA-4 antibodies. In certain embodiments, high concentrations 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, 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 a sufficient amount such that each anti-CTLA-4 antibody exhibits maximum binding activity. In one embodiment, the high concentration herein can be selected to be 1 μM, 10 μM, 100 μM, 1 mM, or a sufficient amount such that each anti-CTLA-4 antibody exhibits maximum binding activity. In a specific embodiment, the low concentration can include, for example, 1 mM or lower, 300 μM or lower, 100 μM or lower, 30 μM or lower, 10 μM or lower, 3 μM or lower, 1 μM or lower, 300 nM or lower, 100 nM or lower, 30 nM or lower, 10 nM or lower, 3 nM or lower, 1 nM or lower, 300 pM or lower, 100 pM or lower, 30 pM or lower, 10 pM or lower, 3 pM or lower, 1 pM or lower, etc. Alternatively, the low concentration here can be the concentration at which each anti-CTLA-4 antibody exhibits minimal binding activity.A substantially zero concentration (in the absence of an adenosine-containing compound) can also be selected as an embodiment of a low concentration. In one embodiment, 1 mM, 100 μM, 10 μM, or 1 μM, the concentration at which each anti-CTLA-4 antibody exhibits minimal binding activity, or the absence of an adenosine compound, can be selected as a low concentration. In another embodiment, the ratio of the high concentration to the low concentration can be, for example, 3-fold or more, 10-fold or more, 30-fold or more, 100-fold or more, 300-fold or more, or 1×10. 3 Double or more, 3 x 10 3 Double or more, 1 x 10 4 Double or more, 3 x 10 4 Double or more, 1 x 10 5 Double or more, 3 x 10 5 Double or more, 1 x 10 6 Double or more, 3 x 10 6 Double or more, 1 x 10 7 Double or more, 3 x 10 7 Double or more, 1 x 10 8 Double or more, 3 x 10 8 Double or more, 1 x 10 9 Double or more, 3 x 10 9 Double or more, 1 x 10 10 Double or more, 3 x 10 10 Double or more, 1 x 10 11 Double or more, 3 x 10 11 Double or more, 1 x 10 12 A value of double or more can be selected.

[0093] In another embodiment, the anti-CTLA-4 antibody of the present invention also has binding activity to adenosine-containing compounds. Using the above-mentioned method, the amount of adenosine-containing compounds bound per unit antibody amount can be calculated, and this can be used as the binding activity of the anti-CTLA-4 antibody to adenosine-containing compounds. Specific methods for measuring and calculating such binding amounts are described in the Examples below. The amount of adenosine-containing compound bound per unit antibody amount of an 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.

[0094] In another embodiment, the anti-CTLA-4 antibody of the present invention forms a ternary complex with an adenosine-containing compound and CTLA-4. In one embodiment, the anti-CTLA-4 antibody binds to an adenosine-containing compound via 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 may consist of at least one amino acid at positions 33, 52, 52a, 53, 56, 58, 95, 96, 100a, 100b, and 100c, for example, according to the Kabat numbering system. In a further embodiment, the anti-CTLA-4 antibody binds to an adenosine-containing compound via at least one amino acid selected from the group consisting of positions 33, 52, 52a, 53, 56, 58, 95, 96, 100a, 100b, and 100c, for example, according to the Kabat numbering system. 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, as determined by the Kabat numbering system. CTLA-4 may also bind to the complex formed by binding of the anti-CTLA-4 antibody and the adenosine-containing compound. Alternatively, the adenosine-containing compound may be present at the interface where the anti-CTLA-4 antibody and CTLA-4 interact, binding to both of them. The formation of a ternary complex between the anti-CTLA-4 antibody, the adenosine-containing compound, and CTLA-4 can be confirmed, for example, by techniques such as crystal structure analysis described below (see Examples).

[0095] In another embodiment, the anti-CTLA-4 antibodies of the present invention bind to at least one amino acid selected from the group consisting of amino acid 3 (Met), amino acid 33 (Glu), amino acid 35 (Arg), amino acid 53 (Thr), amino acid 97 (Glu), amino acid 99 (Met), amino acid 100 (Tyr), amino acid 101 (Pro), amino acid 102 (Pro), amino acid 103 (Pro), amino acid 104 (Tyr), amino acid 105 (Tyr), and amino acid 106 (Leu) of human CTLA-4 (extracellular domain, SEQ ID NO: 28). These amino acids may constitute the epitope of the anti-CTLA-4 antibodies of the present invention. In another embodiment, the anti-CTLA-4 antibodies of the present invention bind to the region from amino acid 97 (Glu) to amino acid 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).

[0096] In another embodiment, an anti-CTLA-4 antibody of the invention competes 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) for binding to CTLA-4. In another embodiment, an anti-CTLA-4 antibody of the invention binds to the same epitope as ABAM004. When an anti-CTLA-4 antibody is present in excess, the binding of ABAM004 to CTLA-4 can be reduced by, 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. Exemplary competition assays are provided herein.

[0097] In another embodiment, the anti-CTLA-4 antibodies of the present invention exhibit cytotoxic activity against CTLA-4-expressing cells. When CTLA-4 is expressed on the surface of a target cell and an anti-CTLA-4 antibody binds to the cell, the cell can be cytotoxic. Cell cytotoxicity may be induced by effector cells bound to the antibody, such as through antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP), or by complement bound to the antibody, such as through complement-dependent cytotoxicity (CDC). Alternatively, the cytotoxicity may be induced by a cytotoxic agent (e.g., a radioisotope or a chemotherapeutic agent) bound to the antibody, such as in an immunoconjugate. Here, cytotoxicity may include effects such as inducing cell death, inhibiting cell proliferation, or impairing cell function. When present in sufficient amounts, an anti-CTLA-4 antibody can cause cytotoxicity in, 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 CTLA-4-expressing cells. Such cytotoxicity measurements can be compared to measurements in the absence of the antibody or in the presence of a negative control antibody. Exemplary cytotoxicity assays are provided herein.

[0098] In another embodiment, the anti-CTLA-4 antibody of the present invention exhibits neutralizing activity against CTLA-4. CTLA-4 is known to function by interacting with its ligands, CD80 (B7-1) or CD86 (B7-2). In certain embodiments, the anti-CTLA-4 antibody inhibits the interaction of CTLA-4 with CD80 (B7-1) or CD86 (B7-2). When present in a sufficient amount, the anti-CTLA-4 antibody 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. Such inhibitory activity measurements can be made relative to measurements in the absence of the antibody or in the presence of a negative control antibody. Specific methods for measuring neutralizing activity are provided herein.

[0099] In another embodiment, the anti-CTLA-4 antibodies of the present invention bind to CTLA-4 derived from multiple animal species. Exemplary animal species include mammals, such as humans, monkeys, mice, rats, hamsters, guinea pigs, rabbits, pigs, cows, goats, horses, sheep, camels, dogs, and cats. In a specific embodiment, the anti-CTLA-4 antibodies bind to CTLA-4 derived from humans and non-humans (e.g., monkeys, mice, rats, etc.). The amino acid sequence of human CTLA-4 is set forth in SEQ ID NO: 214, the amino acid sequence of monkey CTLA-4 is set forth in SEQ ID NO: 247, and the amino acid sequence of mouse CTLA-4 is set forth in SEQ ID NO: 248. The amino acid sequences of CTLA-4 derived from other animal species can also be determined appropriately by methods known to those skilled in the art.

[0100] In certain embodiments, examples of adenosine-containing compounds of the present invention include 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), adenosine gamma thiotriphosphate (ATPγS), and the like.

[0101] In one aspect, the invention provides antibodies comprising at least one, at least two, or all three VH HVR sequences selected from: (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.

[0102] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from: (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.

[0103] In another aspect, an antibody of the present invention comprises: (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (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 (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.

[0104] 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.

[0105] In one aspect, the invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences 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. 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.

[0106] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences 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. 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.

[0107] In another aspect, an antibody of the present invention comprises: (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (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 (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.

[0108] 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.

[0109] In one aspect, the invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences 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: 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.

[0110] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from: (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.

[0111] In another aspect, an antibody of the present invention comprises: (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (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 (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.

[0112] 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.

[0113] In one aspect, the invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from: (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.

[0114] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from: (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.

[0115] In another aspect, an antibody of the present invention comprises: (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (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 (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.

[0116] 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.

[0117] In one aspect, the invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from: (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.

[0118] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from: (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.

[0119] In another aspect, an antibody of the present invention comprises: (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (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 (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.

[0120] 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.

[0121] In one aspect, the invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from: (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.

[0122] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from: (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.

[0123] In another aspect, an antibody of the present invention comprises: (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (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 (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.

[0124] 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.

[0125] In one aspect, the invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from: (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.

[0126] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from: (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.

[0127] In another aspect, an antibody of the present invention comprises: (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (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 (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.

[0128] 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.

[0129] In one aspect, the invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from: (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.

[0130] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from: (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.

[0131] In another aspect, an antibody of the present invention comprises: (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (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 (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.

[0132] 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.

[0133] In one aspect, the invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from: (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.

[0134] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from: (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.

[0135] In another aspect, an antibody of the present invention comprises: (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (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 (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.

[0136] 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.

[0137] In one aspect, the invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from: (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.

[0138] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from: (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.

[0139] In another aspect, an antibody of the present invention comprises: (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (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 (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.

[0140] 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.

[0141] In one aspect, the invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from: (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.

[0142] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences 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. 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.

[0143] In another aspect, an antibody of the present invention comprises: (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from (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 (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.

[0144] 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.

[0145] In certain embodiments, any one or more amino acids of the above-described anti-CTLA-4 antibodies are substituted at the following HVR positions: - in HVR-H1 (SEQ ID NO: 223): position 2 - in HVR-H2 (SEQ ID NO: 224): positions 4, 5, 7, 13, and 16 - in HVR-H3 (SEQ ID NO: 225): position 3 - in HVR-L1 (SEQ ID NO: 226): positions 1, 3, 6, 11, 12, and 14 - in HVR-L2 (SEQ ID NO: 227): positions 1, 3, 4, and 7 - in HVR-L3 (SEQ ID NO: 228): positions 1 and 10

[0146] In certain embodiments, the substitutions provided herein are conservative substitutions. In certain embodiments, any one or more of the following substitutions may be made in any combination: - in HVR-H1 (SEQ ID NO: 100): H2A, R or K - in HVR-H2 (SEQ ID NO: 101): S4T; R5Q; G7H; D13E or R; K16R - in HVR-H3 (SEQ ID NO: 102): K3A - in HVR-L1 (SEQ ID NO: 113): T1D, Q or E; T3P; D6G; N11T; Y12W; S14H - in HVR-L2 (SEQ ID NO: 114): E1F or Y; S3I; K4S; S7E or K - in HVR-L3 (SEQ ID NO: 115): S1Q;M10T

[0147] All possible combinations of the above substitutions are encompassed in the consensus sequences of SEQ ID NOs: 223, 224, 225, 226, 227, and 228 for HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-L3, respectively.

[0148] In any of the above-described embodiments, the anti-CTLA-4 antibody is humanized. In one embodiment, the anti-CTLA-4 antibody comprises the HVR of any of the above-described embodiments and further comprises an acceptor human framework (e.g., a human immunoglobulin framework or a human consensus framework). In another embodiment, the anti-CTLA-4 antibody comprises the HVR of any of the above-described embodiments and further comprises a VH or VL comprising FR sequences. 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:229-232, 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 any one of the amino acid sequences of SEQ ID NOs: 236 to 238, FR2 comprises any one of the amino acid sequences of SEQ ID NOs: 240 to 241, FR3 comprises any one of the amino acid sequences of SEQ ID NOs: 242 to 244, and FR4 comprises any one of the amino acid sequences of SEQ ID NOs: 245 to 246.

[0149] In another aspect, an anti-CTLA-4 antibody comprises a heavy chain variable domain (VH) sequence 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: 10. In certain embodiments, a 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 such a 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., within the FRs). Optionally, the anti-CTLA-4 antibody comprises the VH sequence in SEQ ID NO: 10, including post-translational modifications of that sequence. In certain 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 glutamine or glutamic acid at the N-terminus of the heavy or light chain to pyroglutamic acid by pyroglutamylation.

[0150] In another aspect, an anti-CTLA-4 antibody is provided that 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, a 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 such a 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., within the FRs). Optionally, the anti-CTLA-4 antibody comprises the VL sequence in SEQ ID NO: 11, including post-translational modifications of that sequence. In certain 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 glutamine or glutamic acid at the N-terminus of the heavy or light chain to pyroglutamic acid by pyroglutamylation.

[0151] In another aspect, an anti-CTLA-4 antibody is provided that 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, a 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 such a 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., within the FRs). Optionally, the anti-CTLA-4 antibody comprises the VL sequence in SEQ ID NO: 149, including post-translational modifications of that sequence. In certain 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 glutamine or glutamic acid at the N-terminus of the heavy or light chain to pyroglutamic acid by pyroglutamylation.

[0152] In another aspect, an anti-CTLA-4 antibody is provided, comprising the VH of any of the above embodiments and the VL of any of the above embodiments. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:10 and SEQ ID NO:11, respectively, including post-translational modifications of said sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:98 and SEQ ID NO:99, respectively, including post-translational modifications of said sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:83 and SEQ ID NO:88, respectively, including post-translational modifications of said sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:83 and SEQ ID NO:89, respectively, including post-translational modifications of said sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:83 and SEQ ID NO:90, respectively, including post-translational modifications of said sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:83 and SEQ ID NO:91, respectively, including post-translational modifications of said sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:83 and SEQ ID NO:92, respectively, including post-translational modifications of said sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:83 and SEQ ID NO:93, respectively, including post-translational modifications of said sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:83 and SEQ ID NO:94, respectively, including post-translational modifications of said sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:83 and SEQ ID NO:97, respectively, including post-translational modifications of said sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:83 and SEQ ID NO:95, respectively, including post-translational modifications of said sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:84 and SEQ ID NO:97, respectively, including post-translational modifications of said sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:85 and SEQ ID NO:97, respectively, including post-translational modifications of said sequences.In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:86 and SEQ ID NO:97, respectively, including post-translational modifications of said sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:86 and SEQ ID NO:134, respectively, including post-translational modifications of said sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:136 and SEQ ID NO:97, respectively, including post-translational modifications of said sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:135 and SEQ ID NO:97, respectively, including post-translational modifications of said sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:136 and SEQ ID NO:95, respectively, including post-translational modifications of said sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:137 and SEQ ID NO:97, respectively, including post-translational modifications of said sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 138 and SEQ ID NO: 97, respectively, including post-translational modifications of said sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 138 and SEQ ID NO: 144, respectively, including post-translational modifications of said sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 138 and SEQ ID NO: 145, respectively, including post-translational modifications of said sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 138 and SEQ ID NO: 146, respectively, including post-translational modifications of said sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 139 and SEQ ID NO: 146, respectively, including post-translational modifications of said sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 140 and SEQ ID NO: 146, respectively, including post-translational modifications of said sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 141 and SEQ ID NO: 146, respectively, including post-translational modifications of said sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 140 and SEQ ID NO: 147, respectively, including post-translational modifications of said sequences.In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 141 and SEQ ID NO: 147, respectively, including post-translational modifications of said sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 140 and SEQ ID NO: 148, respectively, including post-translational modifications of said 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 said 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 said sequences. In a further aspect, heterologous anti-CTLA-4 antibodies are provided that comprise at least two different variable regions selected from among the variable regions comprising the VH and VL sequences described above. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 140 and SEQ ID NO: 146, and the VH and VL sequences in SEQ ID NO: 141 and SEQ ID NO: 146, respectively, including post-translational modifications of said sequences. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 140 and SEQ ID NO: 147, and the VH and VL sequences in SEQ ID NO: 141 and SEQ ID NO: 147, respectively, including post-translational modifications of said sequences. Post-translational modifications include, but are not limited to, modification of glutamine or glutamic acid at the N-terminus of the heavy or light chain to pyroglutamic acid by pyroglutamylation.

[0153] When the N-terminal amino acid of the heavy or light chain of an anti-CTLA-4 antibody provided herein is glutamine, the amino acid may be substituted with glutamic acid. When the N-terminal amino acid of the heavy or light chain of an anti-CTLA-4 antibody provided herein is glutamic acid, the amino acid may be substituted with glutamine.

[0154] In a further 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 are provided that bind to the same epitope as the antibodies set forth in Tables 7, 12, 17, and 22. In certain embodiments, antibodies are provided that bind to an epitope in a fragment of CTLA-4 that contains at least one amino acid selected from the group consisting of amino acid 3 (Met), amino acid 33 (Glu), amino acid 35 (Arg), amino acid 53 (Thr), amino acid 97 (Glu), amino acid 99 (Met), amino acid 100 (Tyr), amino acid 101 (Pro), amino acid 102 (Pro), amino acid 103 (Pro), amino acid 104 (Tyr), amino acid 105 (Tyr), and amino acid 106 (Leu) of SEQ ID NO:28. In certain embodiments, antibodies are provided that bind to an epitope in a fragment of CTLA-4 consisting of amino acid 97 (Glu) to amino acid 106 (Leu) of SEQ ID NO: 28. In certain embodiments, antibodies are provided that bind to an epitope in a fragment of CTLA-4 consisting of amino acid 99 (Met) to amino acid 106 (Leu) of SEQ ID NO: 28.

[0155] In further aspects of the invention, the anti-CTLA-4 antibody according to any of the above embodiments is a monoclonal antibody, including a chimeric, humanized, or human antibody. In one embodiment, the anti-CTLA-4 antibody is an antibody fragment, such as, for example, an Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In another embodiment, the antibody is a full-length antibody, such as, for example, a full IgG1 antibody or a full IgG4 antibody, or other antibody classes or isotypes defined herein.

[0156] In a further aspect, the anti-CTLA-4 antibody of the present invention comprises an Fc region. In a further aspect, the anti-CTLA-4 antibody of the present invention comprises a constant region. The constant region may be a heavy chain constant region (including an 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 native antibodies include heavy chain constant regions of human IgG1 (SEQ ID NO: 249), human IgG2 (SEQ ID NO: 250), human IgG3 (SEQ ID NO: 251), human IgG4 (SEQ ID NO: 252), etc. Other exemplary heavy chain constant regions include heavy chain constant regions of SEQ ID NO: 82, SEQ ID NO: 158, SEQ ID NO: 334, etc. Exemplary light chain constant regions derived from native antibodies include light chain constant regions of human κ chain (SEQ ID NO: 33, SEQ ID NO: 63, SEQ ID NO: 159), human λ chain (SEQ ID NO: 53, SEQ ID NO: 87), etc.

[0157] In another embodiment, the Fc region is a mutant Fc region created by adding amino acid modifications to a native-sequence Fc region. In a specific embodiment, the mutant Fc region has enhanced binding activity to at least one Fcγ receptor selected from the group consisting of FcγRIa, FcγRIIa, FcγRIIb, and FcγRIIIa, compared to a native-sequence Fc region. In a further embodiment, the mutant Fc region has enhanced binding activity to FcγRIIa and FcγRIIIa, compared to a native-sequence Fc region. Examples of heavy chain constant regions comprising such mutant Fc regions include the heavy chain constant regions listed in Tables 29 to 33, and the heavy chain constant regions listed in SEQ ID NOs: 31, 32, 41 to 46, 65, 66, 81, 207, 239, 253 to 271, 276, 277, 278, 308, 309, 311 to 333, and 358 to 367.

[0158] Native sequence Fc regions are usually structured as homodimers consisting of two identical polypeptide chains. In certain embodiments, mutant Fc regions may be homodimers consisting of polypeptide chains with the same sequence, or may be heterodimers consisting of polypeptide chains with different sequences. Similarly, heavy chain constant regions comprising Fc regions may be homodimers consisting of polypeptide chains with the same sequence, or may be heterodimers consisting of polypeptide chains with different sequences. Examples of heterologous heavy chain constant regions include a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 31 and a polypeptide chain of SEQ ID NO: 32, a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 43 and a polypeptide chain of SEQ ID NO: 44, a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 45 and a polypeptide chain of SEQ ID NO: 46, a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 254 and a polypeptide chain of SEQ ID NO: 256, a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 257 and a polypeptide chain of SEQ ID NO: 258, a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 259 and a polypeptide chain of SEQ ID NO: 260, a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 261 and a polypeptide chain of SEQ ID NO: 263, a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 262 and a polypeptide chain of SEQ ID NO: 264, a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 265, a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 269 and a polypeptide chain of SEQ ID NO: 270; a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 271 and a polypeptide chain of SEQ ID NO: 81; a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 65 and a polypeptide chain of SEQ ID NO: 66; a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 239 and a polypeptide chain of SEQ ID NO: 207; a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 259 and a polypeptide chain of SEQ ID NO: 276; a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 65 and a polypeptide chain of SEQ ID NO: 278; a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 308 and a polypeptide chain of SEQ ID NO: 309;a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 311 and a polypeptide chain of SEQ ID NO: 312; a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 313 and a polypeptide chain of SEQ ID NO: 314; a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 315 and a polypeptide chain of SEQ ID NO: 316; a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 317 and a polypeptide chain of SEQ ID NO: 318; a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 319 and a polypeptide chain of SEQ ID NO: 320; a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 321 and a polypeptide chain of SEQ ID NO: 322; a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 323 and a polypeptide chain of SEQ ID NO: 324; a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 325 and a polypeptide chain of SEQ ID NO: 326; Examples of such heavy chain constant regions include a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 27 and a polypeptide chain of SEQ ID NO: 328, a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 330 and a polypeptide chain of SEQ ID NO: 331, a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 332 and a polypeptide chain of SEQ ID NO: 333, a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 358 and a polypeptide chain of SEQ ID NO: 359, a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 360 and a polypeptide chain of SEQ ID NO: 361, a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 362 and a polypeptide chain of SEQ ID NO: 363, a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 364 and a polypeptide chain of SEQ ID NO: 366, and a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 365 and a polypeptide chain of SEQ ID NO: 367.

[0159] In further aspects, anti-CTLA-4 antibodies according to any of the above embodiments may incorporate, alone or in combination, any of the features described in items 1-7 below.

[0160] 1. Antibody binding activity In certain embodiments, the binding activity of the antibodies provided herein is ≦10 μM, ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g. 10 -8 M~10 -13 M, e.g. 10 -9 M~10 -13 is the dissociation constant (KD) of the ATPase (M).

[0161] In one embodiment, antibody binding activity is measured by 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 measured by measuring the binding affinity of the Fab to the antigen at a minimum concentration ( 125 I) Measurement is performed by equilibrating Fab with labeled antigen and then capturing the bound antigen using a plate coated with anti-Fab antibody (see, e.g., Chen et al., J. Mol. Biol. 293: 865-881 (1999)). To establish the measurement conditions, MICROTITER® multiwell plates (Thermo Scientific) are coated overnight with 5 μg / ml of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), followed by blocking with 2% (w / v) bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23°C). In non-adsorbent plates (Nunc #269620), 100 pM or 26 pM [ 125[I]-antigen is mixed with serial dilutions of the Fab of interest (e.g., as in 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, although this incubation can be continued for longer periods (e.g., approximately 65 hours) to ensure equilibrium is reached. The mixture is then transferred to a capture plate for incubation at room temperature (e.g., 1 hour). The solution is then removed, and the plate is washed eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. Once the plate has dried, 150 μl / well of scintillant (MICROSCINT-20™, Packard) is added, and the plate is counted for 10 minutes in a TOPCOUNT™ gamma counter (Packard). The concentration of each Fab that gives 20% or less of maximum binding is selected for use in the competitive binding assay.

[0162] In one embodiment, antibody binding activity is measured using a ligand capture method based on surface plasmon resonance analysis, for example, using a BIACORE™ T200 or BIACORE™ 4000 (GE Healthcare, Uppsala, Sweden). BIACORE™ Control Software is used to operate the instrument. In one embodiment, an amine coupling kit (GE Healthcare, Uppsala, Sweden) is used according to the supplier's instructions to immobilize a ligand capture molecule, such as an anti-tag antibody, anti-IgG antibody, or protein A, on a carboxymethyldextran-coated sensor chip (GE Healthcare, Uppsala, Sweden). 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. Binding activity measurements are performed using a buffer containing 0.05% polysorbate 20 (also known as Tween®-20) as the measurement buffer, at a flow rate of 10-30 μL / min, and at a temperature of preferably 25°C or 37°C. When measurements are performed by capturing an antibody as a ligand on a ligand capture molecule, the antibody is injected and captured in a desired amount, and then a serial dilution (analyte) of an antigen or Fc receptor prepared using the measurement buffer is injected. When measurements are performed by capturing an antigen or Fc receptor as a ligand on a ligand capture molecule, the antigen or Fc receptor is injected and captured in a desired amount, and then a serial dilution (analyte) of an antibody prepared using the measurement buffer is injected.

[0163] In one embodiment, the measurement results are analyzed using BIACORE® Evaluation Software. Kinetic parameters are calculated by simultaneously fitting the binding and dissociation sensorgrams using a 1:1 binding model, and the binding rate (k or ka), dissociation rate (k or k), and equilibrium dissociation constant (KD) can be calculated. When the binding activity is weak, particularly when dissociation is rapid and calculation of kinetic parameters is difficult, the equilibrium dissociation constant (KD) may be calculated using a steady state model. Another parameter of binding activity may be calculated by dividing the amount of analyte bound (RU) at a specific concentration by the amount of ligand captured (RU), i.e., the "amount of analyte bound per unit amount of ligand."

[0164] 2. Antibody fragment 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 specific antibody fragments, see Hudson et al. Nat. Med. 9: 129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 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. See US Pat. No. 5,869,046 for a discussion of Fab and F(ab')2 fragments containing salvage receptor binding epitope residues and having increased half-lives in vivo.

[0165] Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, e.g., EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med. 9: 129-134 (2003); 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).

[0166] A single-domain antibody is an antibody fragment that contains all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 B1).

[0167] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of whole antibodies and production by recombinant host cells (e.g., E. coli or phage), as described herein.

[0168] 3. Chimeric and humanized antibodies In certain embodiments, the antibody provided herein is a chimeric antibody. 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 non-human primate such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In another example, a chimeric antibody is a "class-switched" antibody whose class or subclass is changed from that of the parent antibody. Chimeric antibodies also include antigen-binding fragments thereof.

[0169] In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity in humans while maintaining the specificity and affinity of the parent non-human antibody. A humanized antibody usually comprises one or more variable domains, in which the HVRs (e.g., CDRs (or portions thereof)) are derived from a non-human antibody and the FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally comprises at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues were derived), e.g., to restore or improve the specificity or affinity of the antibody.

[0170] Humanized antibodies and methods for their production are reviewed in Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008), and also 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 resurfacing); 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 a "guide selection" approach for FR shuffling).

[0171] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best-fit" method (see Sims et al. J. Immunol. 151: 2296 (1993)); framework regions derived from consensus sequences of human antibodies of specific subgroups of light or heavy chain variable regions (see 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 of FR libraries (see Baca et al., J. Biol. Chem. 272: 10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271: 22611-22618 (1996)).

[0172] 4. Human antibodies In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be produced by various techniques known in the art. Human antibodies are reviewed in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-374 (2001) and Lonberg, Curr. Opin. Immunol. 20: 450-459 (2008).

[0173] Human antibodies may be prepared by administering an immunogen to transgenic animals that have been engineered to produce fully human antibodies or complete antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or a portion of human immunoglobulin loci, which either replace endogenous immunoglobulin loci or are present extrachromosomally or randomly integrated into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin loci are usually 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, U.S. Patent Nos. 6,075,181 and 6,150,584, which describe XENOMOUSE™ technology; U.S. Patent No. 5,770,429, which describes HUMAB® technology; U.S. Patent No. 7,041,870, which describes KM MOUSE® technology; and U.S. Patent Application Publication No. 2007 / 0061900, which describes VELOCIMOUSE® technology. The human variable regions from whole antibodies produced by such animals may be further modified, for example, by combining with different human constant regions.

[0174] Human antibodies can also be produced using hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of 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 via human B cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103: 3557-3562 (2006). Additional methods include those described, for example, in U.S. Patent No. 7,189,826 (which describes the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4): 265-268 (2006) (which describes 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).

[0175] Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. These variable domain sequences can then be combined with desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0176] 5. Library-derived Antibodies Antibodies of the invention may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies with the desired binding characteristics. Such methods are reviewed in Hoogenboom et al. in Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and further see, for example, 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, in 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).

[0177] In a specific phage display method, VH and VL gene repertoires are separately cloned by polymerase chain reaction (PCR) and randomly recombined into phage libraries, which can be screened for antigen-binding phage as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the need for hybridoma construction. Alternatively, naive repertoires can be cloned (e.g., from humans) to provide a single source of antibodies to a wide range of non-self and self antigens without immunization, as described in Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, naive libraries can be generated synthetically by cloning unrearranged V-gene segments from stem cells and using PCR primers encoding the hypervariable CDR3 regions and containing random sequences to achieve rearrangement in vitro, as described in Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patent literature describing human antibody phage libraries includes, for example: U.S. Pat. No. 5,750,373, and U.S. Patent Application Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0178] Antibodies or antibody fragments isolated from a human antibody library are considered herein to be human antibodies or human antibody fragments.

[0179] 6. Multispecific antibodies In certain embodiments, the antibodies provided herein are multispecific antibodies (e.g., bispecific antibodies). Multispecific antibodies are monoclonal antibodies that have binding specificities at at least two different sites. In certain embodiments, one of the binding specificities is for CTLA-4 and the other is for any other antigen. In certain embodiments, bispecific antibodies may bind to two different epitopes of CTLA-4. Bispecific antibodies may be used to localize cytotoxic agents to cells expressing CTLA-4. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0180] Techniques for producing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature 305: 537 (1983), WO93 / 08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), and knob-in-hole technology (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can be produced by manipulating electrostatic steering effects to create Fc heterodimeric molecules (WO2009 / 089004A1); cross-linking two or more antibodies or fragments (see U.S. Pat. No. 4,676,980 and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to generate antibodies with two specificities (see Kostelny et al., J. Immunol., 148(5): 1547-1553 (1992)); using "diabody" technology to create bispecific antibody fragments (see Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993)); and using single-chain Fv (scFv) dimers (Gruber et al., J. Immunol., 152: 6444-6448 (1993)). 5368 (1994)); and by preparing trispecific antibodies as described, for example, in Tutt et al. J. Immunol. 147: 60 (1991).

[0181] Engineered antibodies with three or more functional antigen binding sites, including "octopus antibodies," are also included herein (see, eg, US Patent Application Publication No. 2006 / 0025576 A1).

[0182] As used herein, antibody or fragment also includes a "dual-acting Fab" or "DAF" that contains one antigen-binding site that binds to CTLA-4 and another distinct antigen (see, e.g., U.S. Patent Application Publication No. 2008 / 0069820).

[0183] 7. Antibody Variants In certain embodiments, amino acid sequence variants of the antibodies provided herein are also contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics (e.g., antigen binding).

[0184] a) Substitution, insertion, and deletion mutants In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Target sites for substitutional mutagenesis include HVRs and FRs. Conservative substitutions are shown in Table 1 under the heading of "Preferred Substitutions." More substantial changes are provided in Table 1 under the heading of "Exemplary Substitutions" and are detailed below with reference to classes of amino acid side chains. Amino acid substitutions may be introduced into the antibody of interest, and the products may be screened for a desired activity, such as, for example, retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0185] [Table 1]

[0186] Amino acids can be divided into groups according to common side chain properties: (1) Hydrophobic: norleucine, methionine (Met), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile); (2) neutral hydrophilic: cysteine ​​(Cys), serine (Ser), threonine (Thr), asparagine (Asn), glutamine (Gln); (3) Acidic: aspartic acid (Asp), glutamic acid (Glu); (4) Basic: histidine (His), lysine (Lys), arginine (Arg); (5) residues that affect chain orientation: glycine (Gly), proline (Pro); (6) Aromatic: tryptophan (Trp), tyrosine (Tyr), phenylalanine (Phe). Non-conservative substitutions refer to the exchange of a member of one of these classes for one from another class.

[0187] 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 a modified (e.g., improved) specific biological property compared to the parent antibody (e.g., increased affinity, decreased immunogenicity) and / or will substantially retain the specific biological property of the parent antibody. An exemplary substitutional variant is an affinity-matured antibody, which can be conveniently generated using, for example, phage-display-based affinity maturation techniques (e.g., those described herein). Briefly, one or more HVR residues are mutated, and the mutated antibodies are displayed on phage and screened for a specific biological activity (e.g., binding affinity).

[0188] Modifications (e.g., substitutions) can be made in HVRs, for example, to improve antibody affinity. Such modifications can be made in HVR "hot spots," i.e., residues encoded by codons that frequently mutate during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)), and / or residues that contact antigen, and the resulting mutant VH or VL can be tested for binding affinity. Affinity maturation by construction and reselection from secondary libraries is described, for example, in Hoogenboom et al. in Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., 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 generated. This library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves an HVR-directed approach, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

[0189] In certain embodiments, substitutions, insertions, or deletions may be made within one or more HVRs, as long as such modifications do not substantially reduce the antibody's ability to bind to antigen. For example, conservative modifications (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs. Such modifications may, for example, be outside the antigen-contacting residues of the HVRs. In certain embodiments of the above-described mutant VH and VL sequences, each HVR is unaltered or contains only one, two, or three amino acid substitutions.

[0190] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis," described by Cunningham and Wells (1989) Science, 244: 1081-1085. In this method, a residue or group of target residues (e.g., charged residues, such as arginine, aspartic acid, histidine, lysine, and glutamic acid) is identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine), and it is determined whether the antibody-antigen interaction is affected. Further substitutions can be introduced at amino acid positions that show functional sensitivity to this initial substitution. Alternatively or additionally, a crystal structure of the antigen-antibody complex can be analyzed to identify contact points between the antibody and antigen. Such contact residues and neighboring residues can be targeted as substitution candidates or can be excluded from the list. Mutants can be screened to determine whether they contain desired properties.

[0191] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as internal insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of antibody molecules include the fusion to the N- or C-terminus of the antibody of an enzyme (e.g., for ADEPT) or a polypeptide which increases the plasma half-life of the antibody.

[0192] b) Glycosylation variants In certain embodiments, the antibodies provided herein have been modified to increase or decrease the extent to which the antibody is 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.

[0193] If the antibody contains an Fc region, the carbohydrate attached thereto may be modified. Natural antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides, which are usually attached to Asn297 in the CH2 domain of the Fc region via an N-linkage. See, e.g., Wright et al. TIBTECH 15: 26-32 (1997). Oligosaccharides include various 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, modifications of the oligosaccharides in the antibodies of the present invention may be performed to create antibody variants with specific improved properties.

[0194] In one embodiment, antibody variants are provided that have carbohydrate structures lacking fucose added (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can 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 in the glycan at Asn297 relative to the sum of all glycostructures (e.g., complex, hybrid, and high-mannose structures) added to Asn297, as measured by MALDI-TOF mass spectrometry, for example, as described in WO2008 / 077546. Asn297 represents an asparagine residue located approximately at position 297 in the Fc region (EU numbering of Fc region residues). However, due to slight sequence variability between multiple antibodies, Asn297 may also be located ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylation variants may have improved ADCC function. See, for example, U.S. Patent Application Publication Nos. 2003 / 0157108 (Presta, L.); 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include US2003 / 0157108; WO2000 / 61739; WO2001 / 29246; US2003 / 0115614; US2002 / 0164328; US2004 / 0093621; US2004 / 0132140; US2004 / 0110704; US2004 / 0110282; US2004 / 0109865; WO2003 / 085119; WO2003 / 084570; WO2005 / 035586; WO2005 / 035778; WO2005 / 053742; WO2002 / 031140; Okazaki et al. 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, which lack protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249: 533-545 (1986); U.S. Patent Application Publication No. US2003 / 0157108 A1, Presta, L; and WO2004 / 056312A1, Adams et al., especially Example 11), and knockout cell lines, such as alpha-1,6-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 WO2003 / 085107).

[0195] Further provided are antibody variants having bisected oligosaccharides, for example, bisected oligosaccharides attached to the Fc region of the antibody by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US2005 / 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 WO1997 / 30087 (Patel et al.); WO1998 / 58964 (Raju, S.); and WO1999 / 22764 (Raju, S.).

[0196] c) Fc region mutants In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0197] In certain embodiments, antibody variants that retain some, but not all, effector functions are also contemplated by the present invention, making them desirable candidates for applications where in vivo half-life is important but certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / lack of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to confirm that an antibody lacks FcγR binding (and thus likely lacks ADCC activity) while retaining FcRn binding ability. NK cells, the primary cells for mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. Expression of FcRs 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 to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 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'l Acad. Sci. USA 82: 1499-1502 (1985); U.S. Pat. No. 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166: 1351-1361 (1987)). Alternatively, non-radioactive assays may be used (see, e.g., ACT1™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA); and CytoTox 96® non-radioactive cytotoxicity assays (Promega, Madison, WI)).Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of a molecule of interest may be assessed in vivo in an animal model, e.g., as described in Clynes et al. Proc. Nat'l Acad. Sci. USA 95: 652-656 (1998). C1q binding assays may also be performed to confirm that the antibody is unable to bind C1q and thus lacks CDC activity. See, e.g., the C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402. CDC measurements may also be performed to assess complement activation (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)). Furthermore, determination of FcRn binding and in vivo clearance / half-life may also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12): 1759-1769 (2006)).

[0198] Antibodies with reduced effector function include those with one or more substitutions at Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants with two or more substitutions at amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant with substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581).

[0199] Certain antibody variants with increased or decreased binding to FcRs have been described (see U.S. Pat. No. 6,737,056; WO2004 / 056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001)).

[0200] In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC (e.g., substitutions at positions 298, 333, and / or 334 (EU numbering) of the Fc region).

[0201] In some embodiments, modifications are made in the Fc region that result in altered (i.e., either increased or decreased) C1q 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).

[0202] Antibodies with increased half-lives and increased binding to the neonatal Fc receptor (FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117: 587 (1976); Kim et al., J. Immunol. 24: 249 (1994)) are described in U.S. Patent Application Publication No. 2005 / 0014934 A1 (Hinton et al.). These antibodies comprise an Fc region with one or more substitutions therein that increase binding of the Fc region to FcRn. Such Fc variants include those with 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 (e.g., substitution of Fc region residue 434 (U.S. Patent No. 7,371,826)).

[0203] For other examples of Fc region variants, see also 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.

[0204] d) Cysteine ​​Engineered Antibody Variants In certain embodiments, it may be desirable to generate cysteine-engineered antibodies (e.g., "thioMAbs") in which one or more residues of an antibody have been substituted with a cysteine ​​residue. In certain embodiments, the substituted residues occur at accessible sites of the antibody. By substituting these residues with cysteine, reactive thiol groups are placed at accessible sites of the antibody, which may be used to conjugate the antibody to other moieties (such as drug moieties or linker-drug moieties) to generate immunoconjugates, as further detailed herein. In certain embodiments, any one or more of the following residues may be substituted with a cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region. Cysteine-engineered antibodies may be generated, for example, as described in U.S. Pat. No. 7,521,541.

[0205] e) Antibody derivative In certain embodiments, the antibodies provided herein may be further modified to contain additional nonproteinaceous moieties known in the art and readily available. Suitable moieties for derivatization of 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), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, 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-vinylpyrrolidone), polyethylene glycol, polypropylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in manufacturing due to its stability in water. Polymers may be of any molecular weight and may be branched or unbranched. The number of polymers attached to an antibody can vary, and if more than one polymer is attached, they can be the same molecule or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined based on considerations such as, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used in therapy under defined conditions, etc.

[0206] In another embodiment, a conjugate of an antibody and a non-protein moiety that can be selectively heated by exposure to radiation is provided. 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, including but not limited to, wavelengths that heat the non-protein moiety to temperatures that are not harmful to normal cells but that kill cells in close proximity to the antibody-non-protein moiety.

[0207] The anti-CTLA-4 antibodies described herein can be combined with various existing technologies. A non-limiting example of such a combination of technologies is the creation of cells expressing chimeric antigen receptors (CARs) using anti-CTLA-4 antibodies. Examples of cells include T cells, γδT 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). One non-limiting method for creating T cells expressing the CAR (CAR-T) is to use genetic engineering to introduce a CAR containing the antigen-binding domain (e.g., scFv) of an anti-CTLA-4 antibody, the transmembrane domain of a TCR, and the signaling domain of a costimulatory molecule such as CD28 to enhance T cell activation into effector cells such as T cells.

[0208] A non-limiting example of a technique that can be combined with the anti-CTLA-4 antibody of the present specification is the production of a T cell redirecting antibody using an anti-CTLA-4 antibody (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 of the constituent subunits of the T cell receptor (TCR) complex on T cells, particularly a binding domain for the CD3 epsilon chain of CD3, and the antigen-binding domain of an anti-CTLA-4 antibody.

[0209] B. Recombinant Methods and Constructs Antibodies can be produced using recombinant methods or constructs, for example, as described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an anti-CTLA-4 antibody described herein is provided. Such a nucleic acid may encode an amino acid sequence comprising the VL and / or an amino acid sequence comprising the VH of the antibody (e.g., the light and / or heavy chains 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, a host cell comprising such nucleic acids is provided. In one such embodiment, the host cell comprises (e.g., is transformed with) (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 eukaryotic (e.g., a Chinese hamster ovary (CHO) cell) or a lymphoid cell (e.g., a Y0, NS0, or Sp2 / 0 cell)). In one aspect, a method for producing an anti-CTLA-4 antibody is provided, comprising culturing a host cell containing nucleic acid encoding the antibody as described 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).

[0210] For recombinant production of an anti-CTLA-4 antibody, nucleic acid encoding the antibody (e.g., such as those described above) is isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acid may be readily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes capable of binding specifically to genes encoding the antibody heavy and light chains).

[0211] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies may be produced in bacteria, particularly if glycosylation and Fc effector functions are not required. For the expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523 (also see Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli). After expression, the antibody may be isolated in a soluble fraction from the bacterial cell paste and further purified.

[0212] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been "humanized," resulting in the production of antibodies with partial or fully human glycosylation patterns. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004) and Li et al., Nat. Biotech. 24: 210-215 (2006).

[0213] Host cells derived from multicellular organisms (invertebrates and vertebrates) are also suitable for expressing glycosylated antibodies. Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified for use in conjugation with insect cells, particularly for transformation of Spodoptera frugiperda cells.

[0214] Plant cell cultures can also be used as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants).

[0215] Vertebrate cells can also be used as hosts. For example, mammalian cell lines that have been adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 (COS-7); human embryonic kidney (293 or 293 cells, e.g., as described in Graham et al., J. Gen Virol. 36: 59 (1977)); baby hamster kidney (BHK) cells; mouse Sertoli cells (TM4 cells, e.g., as described in Mather, Biol. Reprod. 23: 243-251 (1980)); monkey kidney (CV1); African green monkey kidney (VERO-76); human cervical carcinoma (HELA); canine kidney (MDCK); Buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary carcinoma (MMT 060562); TRI cells (e.g., Mather et al., Annals NY Acad. Sci. 383: 44-68 (1982)). (described in

[1999] ); MRC5 cells; and FS4 cells. 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 specific 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).

[0216] Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. The relevant antigen is conjugated to a protein that is immunogenic in the species being immunized, such as keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor, with a bifunctional or derivatizing agent, such as maleimidobenzoyl sulfosuccinimide ester (conjugation via cysteine ​​residues), N-hydroxysuccinimide (via lysine residues), glutaraldehyde, succinic anhydride, SOCl, or R. 1 N=C=NR (where R and R 1 are different alkyl groups).

[0217] Animals (usually non-human mammals) are immunized against an antigen, immunogenic conjugate, or derivative by combining 100 μg or 5 μg of 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 of the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. Seven to 14 days later, the animal is bled and the serum is assayed for antibody titer. The animal is boosted until the titer plateaus. Preferably, the animal is boosted with a conjugate of the same antigen but conjugated to a different protein and / or via a different cross-linking reagent. Conjugates can also be prepared as protein fusions in recombinant cell culture. Aggregating agents, such as alum, are also suitably used to enhance the immune response.

[0218] Monoclonal antibodies are 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 small amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies.

[0219] For example, monoclonal antibodies can be produced using the hybridoma method first described in 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 hereinabove to induce 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.

[0220] The immunizing agent typically includes an antigen protein or a fusion variant thereof. Generally, peripheral blood lymphocytes (PBLs) are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986), pp. 59-103).

[0221] Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine, or human origin. Rat or mouse myeloma cell lines are commonly used. The hybridoma cells thus generated are seeded and grown in an appropriate culture medium, preferably containing 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 for the hybridoma will typically contain hypoxanthine, aminopterin, and thymidine (HAT medium), substances that prevent the growth of HGPRT-deficient cells.

[0222] Preferred immortalized myeloma cells are those that fuse efficiently, support stable high-level antibody production by selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these, mouse myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center in San Diego, California, USA, and SP-2 cells (and their derivatives, such as X63-Ag8-653) available from the American Type Culture Collection in Manassas, Virginia, USA, are preferred. Human myeloma cell lines and mouse-human heteromyeloma cell lines have also been described for the production of 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, pp. 51-63 (1987)).

[0223] The culture medium in which the hybridoma cells are growing is assayed for the production of monoclonal antibodies against the antigen. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, 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 the Scatchard analysis of Munson, Anal Biochem. 107(1): 220-239 (1980).

[0224] After hybridoma cells producing antibodies of the desired specificity, affinity, and / or activity are identified, the clones can be subcloned by limiting dilution and grown by standard methods (Goding, supra). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. Hybridoma cells can also be grown in vivo as tumors in mammals.

[0225] 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.

[0226] Antibodies may be produced by immunizing a suitable host animal against 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 acid 97 (Glu) to amino acid 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 acid 99 (Met) to amino acid 106 (Leu) of human CTLA-4 (extracellular domain, SEQ ID NO:28). The present invention also encompasses antibodies produced by immunizing an animal against an antigen. The antibody may incorporate any of the features described above for the exemplary anti-CTLA-4 antibodies, alone or in combination.

[0227] C. Assay The anti-CTLA-4 antibodies provided herein may be identified, screened, or characterized for their physical / chemical properties and / or biological activity by a variety of assays known in the art.

[0228] 1. Binding and other assays In one aspect, the antibodies of the invention are tested for their antigen binding activity by known methods, such as ELISA, Western blot, surface plasmon resonance assay, and the like.

[0229] In another aspect, a competition assay 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 Tables 7, 12, 17, and 22) for binding to CTLA-4. In certain embodiments, when such a competing antibody is present in excess, it prevents (e.g., reduces) binding of the reference antibody to CTLA-4 by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more. In some examples, binding is inhibited 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 that bound by an anti-CTLA-4 antibody described herein (e.g., an anti-CTLA-4 antibody described in Table 7, Table 12, Table 17, and Table 22). Detailed exemplary methods for mapping antibody-binding epitopes are provided in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology, vol. 66 (Humana Press, Totowa, NJ).

[0230] In an exemplary competitive assay, immobilized CTLA-4 is incubated in a solution containing a first, labeled antibody that binds to CTLA-4 and a second, unlabeled antibody to be tested for its ability to compete with the first antibody for binding to CTLA-4. The second antibody may be present in hybridoma supernatant. As a control, immobilized CTLA-4 is incubated in a solution containing the first, labeled antibody but not the second, unlabeled antibody. After incubation under conditions that allow binding of the first antibody to CTLA-4, excess unbound antibody is removed and the amount of label bound to immobilized CTLA-4 is measured. A substantial decrease in the amount of label bound to immobilized CTLA-4 in the test sample compared to the control sample indicates that the second antibody competes 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).

[0231] 2.Activity measurement method In one aspect, a method for identifying an anti-CTLA-4 antibody having biological activity is provided. Biological activity may include, for example, cell growth inhibitory activity, cytotoxic activity (e.g., ADCC / CDC activity, ADCP activity), immunostimulatory activity, and CTLA-4 inhibitory activity. Also provided are antibodies having such biological activity in vivo and / or in vitro.

[0232] In certain embodiments, antibodies of the invention are tested for such biological activities.

[0233] In certain embodiments, antibodies of the present invention are tested for their ability to inhibit cell growth or proliferation in vitro. Assays for cell growth or proliferation inhibition are well known in the art. Certain cell proliferation assays, exemplified by the "cell killing" assay described herein, measure cell viability. One such assay is the CellTiter-Glo™ Luminescent Cell Viability Assay, commercially available from Promega (Madison, WI). This assay determines the number of viable cells in culture based on the presence of ATP, an indicator of metabolically active cells. See Crouch et al. (1993) J. Immunol. Meth. 160: 81-88; U.S. Patent No. 6,602,677. Assays may also be performed in 96- or 384-well formats, making them amenable to 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® Reagent) directly to cultured cells, which lyses the cells and generates a luminescent signal via the luciferase reaction. The luminescent signal is proportional to the amount of ATP present, which is directly proportional to the number of viable cells present in the culture. Data can be recorded by a luminometer or a CCD camera imager. Luminescence values ​​are expressed in relative light units (RLU).

[0234] Another cell proliferation assay is the "MTT" assay, a colorimetric assay that measures the oxidation of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to formazan by mitochondrial reductase. Similar to the CellTiter-Glo™ assay, this assay indicates the number of metabolically active cells present in cell culture. See, for example, Mosmann (1983) J. Immunol. Meth. 65: 55-63 and Zhang et al. (2005) Cancer Res. 65: 3877-3882.

[0235] Cells for use in any of the above in vitro assays include cells or cell lines that naturally express CTLA-4 or that 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 that have been transfected with a nucleic acid encoding CTLA-4.

[0236] In one aspect, anti-CTLA-4 antibodies are tested for their ability to inhibit cell growth or proliferation in vivo. In certain embodiments, anti-CTLA-4 antibodies are tested for their ability to inhibit tumor growth in vivo. An in vivo model system, such as a xenograft model, can be used for such testing. In an exemplary xenograft system, human tumor cells are introduced into an appropriately immunocompromised non-human animal, such as an athymic "nude" mouse. An antibody of the present invention is administered to the animal. The ability of the antibody to inhibit or reduce tumor growth is measured. In certain embodiments of the above xenograft system, the human tumor cells are tumor cells derived from a human patient. Such xenograft models are commercially available from Oncotest GmbH (Frieberg, Germany). In certain embodiments, human tumor cells are introduced into an appropriately immunocompromised non-human animal by subcutaneous injection or by implantation into a suitable site, such as the mammary fat pad.

[0237] It will be understood that any of the above assays can be performed using an immunoconjugate of the invention in place of or in addition to an anti-CTLA-4 antibody.

[0238] A representative assay for measuring the ADCC activity of a therapeutic antibody is 51 It is based on a Cr release assay and includes the following steps: 51 The method involves labeling the cells with [Cr]Na2CrO4; opsonizing target cells expressing the antigen on their surface with the antibody; combining the opsonized radiolabeled target cells with effector cells at a suitable ratio in a microtiter plate in the presence or absence of the test antibody; incubating the cell mixture for 16-18 hours, preferably at 37°C; collecting the supernatant; and analyzing the radioactivity in the supernatant sample. The cytotoxicity of the test antibody is then determined, for example, using the following formula: specific cytotoxicity (%) = (radioactivity in the presence of antibody - radioactivity in the absence of antibody) / (maximum radioactivity - radioactivity in the absence of antibody) x 100. Graphs can be generated by varying the target cell:effector cell ratio or antibody concentration.

[0239] To assess complement activation, a complement-dependent cytotoxicity (CDC) assay can be performed, for example, as described in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996). Briefly, various concentrations of the polypeptide variant and human complement are diluted in buffer. Approximately 1 x 10 cells expressing the antigen to which the polypeptide variant binds are cultured. 6The mixture is diluted to a density of 1000 cells / ml. The mixture of polypeptide variant, diluted human complement, and antigen-expressing cells is added to a flat-bottom 96-well tissue culture plate and incubated at 37°C and 5% CO2 for 2 hours to promote complement-mediated cell lysis. 50 μl of Alamar Blue (Accumed International) is then added to each well and incubated overnight at 37°C. Absorbance is measured using a 96-well fluorometer with excitation at 530 nm and emission at 590 nm. Results are expressed in relative fluorescence units (RFU). Sample concentrations can be calculated from the standard curve, and percent activity compared to the non-mutant polypeptide is reported for the polypeptide variant of interest.

[0240] An exemplary assay for ADCP activity may include coating a target bioparticle, such as E. coli-labeled FITC (Molecular Probes) or Staphylococcus aureus-FITC, with a test antibody to form an opsonized particle; adding the opsonized particle to THP-1 effector cells (a monocytic cell line available from the American College of Cancer Research) at a ratio of 1:1, 10:1, 30:1, 60:1, 75:1, or 100:1 to allow FcγR-mediated phagocytosis to occur; preferably incubating the cells with E. coli-FITC / antibody at 37°C for 1.5 hours; after incubation, adding trypan blue to the cells (preferably for 2-3 minutes at room temperature) to quench the fluorescence of bacteria that are not internalized but adhere to the outer cell surface; transferring the cells to FACS buffer (e.g., 0.1% BSA, 0.1% sodium azide in PBS) and assaying the fluorescence of the THP-1 cells using FACS (e.g., BD FACS Calibur). To assay the degree of ADCP, a gate is preferably set on THP-1 cells and the median fluorescence intensity is measured. In the most preferred embodiment, the ADCP assay is performed using E. coli-FITC in culture medium (control), E. coli-FITC and THP-1 cells (used as FcγR-independent ADCP activity), or E. coli-FITC, THP-1 cells, and the test antibody (used as FcγR-dependent ADCP activity).

[0241] The cytotoxic activity of an antibody is usually accompanied by binding of the antibody to the cell surface. Whether or not an antigen is expressed on the surface of a target cell can be appropriately confirmed by techniques known to those skilled in the art, such as FACS.

[0242] Immune activation can be detected using cellular or humoral immune responses as indicators. Specifically, this includes increased expression of cytokines (e.g., IL-6, G-CSF, IL-12, TNFα, and IFNγ) or their receptors, and enhanced proliferation, activation, function, or cytotoxic activity of immune cells (e.g., B cells, T cells, NK cells, macrophages, monocytes). In particular, T cell activation can be detected by measuring increased expression of activation markers such as CD25, CD69, and ICOS. For example, patients administered the anti-CTLA-4 antibody ipilimumab showed significantly increased ICOS levels in peripheral blood after administration. + CD4 + It is known that T cells increase, which is thought to be the result of activating the systemic immune state by administering anti-CTLA-4 antibodies (Cancer Immunol. Res. (2013) 1(4): 229-234).

[0243] T cell activation requires not only stimulation via the antigen receptor (TCR) but also costimulation via CD28. When CD28 on the T cell surface binds to B7-1 (CD80) or B7-2 (CD86) present on the surface of an antigen-presenting cell, a costimulatory signal is transmitted within the T cell, resulting in T cell activation. CTLA-4 is also expressed on the surface of activated T cells. CTLA-4 binds to CD80 and CD86 with stronger affinity than CD28, so it interacts with CD80 and CD86 preferentially over CD28, ultimately suppressing T cell activation.

[0244] Based on this mechanism of action, inhibitory activity against CTLA-4 can be measured as the activity of inhibiting the binding of CTLA-4 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 the bound labeled CD80 or CD86. Whether a test antibody cross-reacts with CD28 can be confirmed by performing a similar assay in which CTLA-4 is replaced with CD28. In another embodiment, inhibitory activity against CTLA-4 can also be measured using a functional assay for detecting T cell activation, as described above. For example, in a system in which a T cell population is stimulated with cells expressing CD80 or CD86 to measure T cell activation, the addition of a test antibody with CTLA-4 inhibitory activity results in further enhancement of T cell activation.

[0245] D. Immunoconjugates The present invention also provides immunoconjugates comprising the anti-CTLA-4 antibodies herein conjugated to one or more cytotoxic agents (e.g., a chemotherapeutic agent or drug, a growth inhibitory agent, a toxin (e.g., a protein toxin of bacterial, fungal, plant, or animal origin, an enzymatically active toxin, or fragment thereof), or a radioactive isotope).

[0246] In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC) in which an antibody is conjugated to one or more drugs, including but not limited to: maytansinoids (see U.S. Pat. Nos. 5,208,020, 5,416,064, and European Patent No. 0,425,235 B1); auristatins, such as the monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483, 5,780,588, and 7,498,298); dolastatins; 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 see Lode et al., Cancer Res. 58: 2925-2928 (1998)); anthracyclines such as daunomycin or doxorubicin (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, tesetaxel, and ortataxel; trichothecenes; and CC1065.

[0247] In another embodiment, the immunoconjugate comprises an antibody described herein conjugated to an enzymatically active toxin or fragment thereof, including, but not limited to, diphtheria A chain, nonbinding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolacca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, saponaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and a trichothecene.

[0248] In another embodiment, the immunoconjugate comprises an antibody described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes are available for the production of radioconjugates. Examples include: 211 At, 131 I, 125 I, 90 Y, 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P, 212 Radioactive isotopes include Pb and Lu. When radioactive conjugates are used for detection, they are used in combination with radioactive atoms for scintigraphic examinations (e.g., Tc-99m or 123 I), or spin labels (again, e.g., iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron) for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI).

[0249] Conjugates of antibodies and cytotoxic agents can be prepared using a variety of bifunctional protein linking agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins 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 facilitates release of the cytotoxic drug inside the cell. For example, an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker, or disulfide-containing linker (Chari et al., Cancer Res. 52: 127-131 (1992); U.S. Patent No. 5,208,020) can be used.

[0250] The immunoconjugates or ADCs herein expressly contemplate, but are not limited to, conjugates prepared using cross-linking reagents including, but not limited to, 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), which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL, USA).

[0251] E. Methods and Compositions for Diagnostics and Detection In certain embodiments, any of the anti-CTLA-4 antibodies provided herein are useful for detecting the presence of CTLA-4 in a biological sample. As used herein, the term "detection" encompasses quantitative or qualitative detection. In certain embodiments, the biological sample comprises cells or tissues, such as serum, whole blood, plasma, biopsy sample, tissue sample, cell suspension, saliva, sputum, oral fluid, cerebrospinal fluid, amniotic fluid, ascites, milk, colostrum, mammary gland secretions, lymph, urine, sweat, tears, gastric juice, synovial fluid, peritoneal fluid, ocular fluid, or mucus.

[0252] In one embodiment, an anti-CTLA-4 antibody is provided for use in a diagnostic or detection method. In a further aspect, a method for detecting the presence of CTLA-4 in a biological sample is provided. In certain embodiments, the method comprises contacting a biological sample with an anti-CTLA-4 antibody described herein under conditions that allow binding of the anti-CTLA-4 antibody to CTLA-4, and detecting whether a complex is formed between the anti-CTLA-4 antibody and CTLA-4. Such a method can be an in vitro method or an in vivo method. In one embodiment, the anti-CTLA-4 antibody is used to select subjects suitable for treatment with an anti-CTLA-4 antibody, for example, when CTLA-4 is a biomarker for patient selection.

[0253] The antibodies of the present invention can be used, for example, to check the status of the immune response or to diagnose immune system dysfunction.

[0254] In certain embodiments, labeled anti-CTLA-4 antibodies are provided. Labels include, but are not limited to, labels or moieties that are directly detected (e.g., fluorescent labels, chromogenic labels, electron-dense labels, chemiluminescent labels, and radioactive labels) and moieties that are indirectly detected, for example, through enzymatic reactions or molecular interactions (e.g., enzymes or ligands). Exemplary labels include, but are not limited to, radioisotopes 32 P, 14 C. 125 I, 3 H and 131 Fluorophores such as I, rare earth chelates or those linked to 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-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, monosaccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases such as uricase and xanthine oxidase, enzymes that oxidize dye precursors using hydrogen peroxide (e.g., HRP, lactoperoxidase, or microperoxidase), biotin / avidin, spin labels, bacteriophage labels, stable free radicals, and the like.

[0255] F. Pharmaceutical Formulations Pharmaceutical formulations of the anti-CTLA-4 antibodies described herein are prepared in the form of lyophilized formulations or aqueous solutions by mixing the antibody having the desired purity with one or more pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers are generally non-toxic 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 (octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol, etc.); small (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 dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, and sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersing agents, such as soluble neutral activated hyaluronidase glycoproteins (sHASEGPs). Certain exemplary sHASEGPs and methods of use thereof are described in U.S. Patent Application Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one aspect, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0256] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO2006 / 044908, the latter formulations including a histidine-acetate buffer.

[0257] The formulations herein may contain more than one active ingredient as necessary for the particular indication being treated, preferably with complementary activities that do not adversely affect each other. Such active ingredients are present in suitable combinations and in amounts that are effective for the purpose intended.

[0258] The active ingredient may be incorporated into microcapsules (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively) prepared, for example, by droplet formation (coacervation) techniques or by interfacial polymerization, into colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or into macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0259] Sustained-release preparations may also 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.

[0260] Preparations to be used for in vivo administration are generally sterile, and sterility is readily accomplished, for example, by filtration through sterile filtration membranes.

[0261] G. Therapeutic Methods and Compositions Any of the anti-CTLA-4 antibodies provided herein may be used in therapeutic methods. In one aspect, an anti-CTLA-4 antibody is provided for use as a pharmaceutical. In a further aspect, an anti-CTLA-4 antibody is provided for use in treating tumors. In a particular embodiment, an anti-CTLA-4 antibody is provided for use in a therapeutic method. In a particular embodiment, the present invention provides an anti-CTLA-4 antibody for use in a method of treating an individual having a tumor, the method comprising administering to the individual an effective amount of an anti-CTLA-4 antibody. In a further embodiment, the present invention provides an anti-CTLA-4 antibody for use in cell damage. In a particular embodiment, the present invention provides an anti-CTLA-4 antibody for use in a method of cell damage in an individual, the method comprising administering to the individual an effective amount of an anti-CTLA-4 antibody to damage the cells. An "individual" according to any of the above embodiments is preferably a human.

[0262] Tumor tissue in vivo is often infiltrated with immune cells such as lymphocytes, which also constitute a part of the tumor tissue. In some embodiments, immune cells, particularly regulatory T (Treg) cells, are infiltrated into tumor tissue. In one embodiment, cell damage is induced by ADCC activity, CDC activity, or ADCP activity. In one embodiment, cells expressing CTLA-4 on their cell surface are damaged. In a further embodiment, the damaged cells are Treg cells. In a specific embodiment, Treg cells infiltrating into tumor tissue are damaged.

[0263] In a further aspect, the extent of the pharmaceutical effect provided by the anti-CTLA-4 antibody of the present invention varies depending on the tissue within an individual. In certain embodiments, the extent varies depending on the concentration of adenosine-containing compounds in the tissue. In further embodiments, the effect is enhanced 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, stronger immune activation is observed in tumor tissue compared to non-tumor tissue. Such a difference in response need not be observed at all doses of the anti-CTLA-4 antibody, but may be observed within a certain range of doses. In another embodiment, immune activation is observed in tumor tissue compared to non-tumor tissue at a lower dose. In yet another embodiment, a therapeutic effect is observed at a dose lower than that at which side effects are observed. In a specific embodiment, the therapeutic effect is the manifestation of an anti-tumor effect (e.g., tumor regression, induction of tumor cell death or inhibition of tumor cell proliferation, etc.), and the side effect is the onset of an autoimmune disease (including damage to normal tissues due to an excessive immune response).

[0264] In certain embodiments, the tumor is selected from the group consisting of breast cancer and liver cancer.

[0265] In a further aspect, the present invention provides use of an anti-CTLA-4 antibody in the manufacture or preparation of a medicament. In one embodiment, the medicament is for the treatment of a tumor. In a further embodiment, the medicament is for use in a method of treating a tumor, the method comprising administering an effective amount of the medicament to an individual having a tumor. In a further embodiment, the medicament is for cell damage. In a further embodiment, the medicament is for use in a method of cell damage in an individual, the method comprising administering an effective amount of the medicament to the individual to damage the cells. An "individual" according to any of the above embodiments may be a human.

[0266] In a further aspect, the present invention provides a method of treating a tumor. In one embodiment, the method comprises administering to an individual having such a tumor an effective amount of an anti-CTLA-4 antibody. The "individual" according to any of the above embodiments may be a human.

[0267] In a further aspect, the present invention provides a method for 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 one embodiment, the "individual" is a human.

[0268] In a further aspect, 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 therapeutic methods described above). 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 use in treating tumors. In one embodiment, the present invention provides pharmaceutical formulations (pharmaceutical compositions) for use in damaging cells.

[0269] In a further aspect, the present invention provides a method for preparing a medicament or pharmaceutical formulation (e.g., for use in any of the therapeutic methods described above) comprising mixing any of the anti-CTLA-4 antibodies provided herein with a pharmaceutically acceptable carrier.

[0270] The antibodies of the present invention can be administered by any suitable means, including parenteral, pulmonary, and nasal administration, and, if desired for localized treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether administration is brief or chronic. Various dosing schedules are contemplated herein, including, but not limited to, single administration or repeated administration over various time periods, bolus administration, and pulse infusion.

[0271] The antibodies of the present invention will be formulated, dosed, and administered in a manner consistent with good medical practice. Factors to be considered in this regard include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the schedule of administration, and other factors known to medical practitioners.

[0272] For disease prevention or treatment, the appropriate dose of an antibody of the invention will depend on the type of disease being treated, the type of antibody, the severity and course of the disease, whether the antibody is administered prophylactically or therapeutically, previous medical history, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, for example, about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg) of antibody may be the initial candidate dose for administration to a patient, whether by one or more separate administrations or by continuous infusion. A typical daily dose may range from about 1 μg / kg to 100 mg / kg or more, depending on the factors discussed above. For repeated administrations over several days or longer, depending on the situation, treatment is usually maintained until a desired suppression of disease symptoms occurs. An exemplary dose of antibody is 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) may be administered to the patient. Such doses may be administered intermittently, for example, every week or every three weeks (e.g., so that the patient receives about 2 to about 20, or, for example, about 6 doses of antibody). A high initial loading dose may be followed by one or more lower doses. The progress of this therapy is easily monitored by conventional techniques and measurements.

[0273] It will be understood that any of the above-described formulations or therapeutic methods may be practiced using an immunoconjugate of the invention in place of, or in addition to, an anti-CTLA-4 antibody.

[0274] The anti-CTLA-4 antibody of the present specification can be administered by administering or incorporating a nucleic acid encoding the anti-CTLA-4 antibody into a living body using a vector or the like, thereby directly expressing the anti-CTLA-4 antibody in the living body. However, the antibody can also be administered without a vector. Examples of vectors include viral vectors, plasmid vectors, and even adenoviral vectors. The nucleic acid encoding the anti-CTLA-4 antibody can be administered directly to a living body, or cells transfected with a nucleic acid encoding the anti-CTLA-4 antibody can be administered to a living body. For example, an anti-CTLA-4 antibody can be administered by chemically modifying mRNA encoding the anti-CTLA-4 antibody to increase the stability of the mRNA in the living body, directly administering the mRNA to a human, and expressing the anti-CTLA-4 antibody in the living body (see EP2101823B, WO2013 / 120629). Alternatively, B cells transfected with a nucleic acid encoding the anti-CTLA-4 antibody can be administered (Sci Immunol. (2019) 4(35), eaax0644). Alternatively, bacteria containing nucleic acids encoding anti-CTLA-4 antibodies may be administered (Nature Reviews Cancer (2018) 18, 727-743).

[0275] A non-limiting example of a technique that can be combined with the anti-CTLA-4 antibody of the present specification is the creation of T cells that secrete a T cell redirecting antibody using an anti-CTLA-4 antibody (Trends Immunol. (2019) 40(3) 243-257). One non-limiting method for creating such a bispecific antibody is to use gene modification technology to introduce nucleic acid encoding a bispecific antibody that contains a binding domain for one of the constituent subunits of the T cell receptor (TCR) complex on T cells, particularly a binding domain for the CD3 epsilon chain of CD3, and the antigen-binding domain of an anti-CTLA-4 antibody into effector cells such as T cells.

[0276] H.Product In another aspect of the present invention, an article of manufacture containing materials useful for the treatment, prevention, and / or diagnosis of the aforementioned disorders is provided. The article of manufacture includes a container and a label on the container or a package insert associated with the container. Preferred containers include, for example, bottles, vials, syringes, and intravenous (IV) solution bags. The containers may be formed from a variety of materials, such as glass or plastic. The container may hold the composition alone or in combination with another composition effective for the treatment, prevention, and / or diagnosis of a condition, and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial having 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 the condition of choice. The article of manufacture in this aspect of the invention may further include a package insert indicating that the composition can be used to treat the particular condition. Alternatively, or additionally, the article of manufacture may further comprise a second container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. The article of manufacture may further include other materials desirable from a commercial or user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0277] It will be understood that any of the above-described products may include an immunoconjugate of the present invention in place of or in addition to an anti-CTLA-4 antibody. <Polypeptides containing mutant Fc regions>

[0278] In one aspect, the present invention provides an isolated polypeptide comprising a mutant Fc region. In some aspects, the polypeptide is an antibody. In some aspects, the polypeptide is an Fc fusion protein. In certain embodiments, the mutant Fc region comprises at least one amino acid residue modification (e.g., substitution) compared to the corresponding sequence in the Fc region of a native sequence or a reference mutant sequence (sometimes collectively referred to herein as a "parent" Fc region). Native sequence Fc regions are typically organized as homodimers consisting of two identical polypeptide chains. The amino acid modification in the mutant Fc region of the present invention may be introduced into either one of the two polypeptide chains of the parent Fc region, or into both of the two polypeptide chains.

[0279] In some aspects, the present invention provides mutant Fc regions with altered function compared to their parent Fc regions. In certain aspects, the mutant Fc regions of the present invention have enhanced binding activity to Fcγ receptors compared to their parent Fc regions. In certain embodiments, the mutant Fc regions of the present invention have enhanced binding activity to at least one Fcγ receptor selected from the group consisting of FcγRIa, FcγRIIa, FcγRIIb, and FcγRIIIa compared to their parent Fc regions. In some embodiments, the mutant Fc regions of the present invention have enhanced binding activity to FcγRIIa. In some embodiments, the mutant Fc regions of the present invention have enhanced binding activity to FcγRIIIa. In a further embodiment, the mutant Fc regions of the present invention have enhanced binding activity to FcγRIIa and FcγRIIIa. In another aspect, the mutant Fc regions of the present invention have enhanced ADCC activity, CDC activity, or ADCP activity compared to their parent Fc regions.

[0280] In some embodiments, the mutant Fc regions of the present invention comprise at least one amino acid modification at at least one position selected from the group consisting of positions 234, 235, 236, 298, 330, 332, and 334 (EU numbering). Alternatively, amino acid modifications such as those described in International Publication WO2013 / 002362 and WO2014 / 104165 may also be used in the present invention.

[0281] In a specific embodiment, the binding activity of the parent Fc region and the mutant Fc region can be expressed as KD (Dissociation constant) values. In one embodiment, the ratio of [KD value of the parent Fc region for FcγRIIa] / [KD value of the mutant 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 a further embodiment, FcγRIIa may be FcγRIIa R or FcγRIIa H, or both. Therefore, the KD value of the Fc region for FcγRIIa may be the KD value of the Fc region for FcγRIIa R or the KD value of the Fc region for FcγRIIa H. Alternatively, it may be the sum or average of both. In one embodiment, the ratio of [binding activity of parent Fc region to FcγRIIIa] / [binding activity of mutant Fc region to FcγRIIIa] is, 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 × 10 3 That's it, 2 x 10 3 That's it, 3 x 10 3 or more, or 5 x 10 3 That is all. In a further embodiment, FcγRIIIa may be FcγRIIIa F or FcγRIIIa V, or both. Therefore, the KD value of the Fc region for FcγRIIIa may be the KD value of the Fc region for FcγRIIIa F or the KD value of the Fc region for FcγRIIIa V. Alternatively, it may be the sum or average of both.

[0282] In one embodiment, the KD value of the mutant Fc region for FcγRIIa is, for example, 1.0×10 -6 M or less, 5.0×10 -7 M or less, 3.0×10 -7 M or less, 2.0×10 -7 M or less, 1.0×10-7 M or less, 5.0×10 -8 M or less, 3.0×10 -8 M or less, 2.0×10 -8 M or less, 1.0×10 -8 M or less, 5.0×10 -9 M or less, 3.0×10 -9 M or less, 2.0×10 -9 M or less, or 1.0 x 10 -9 In a further embodiment, FcγRIIa may be FcγRIIa R or FcγRIIa H, or both. In one embodiment, the KD value of the mutant Fc region for FcγRIIIa is, for example, 1.0 × 10 -6 M or less, 5.0×10 -7 M or less, 3.0×10 -7 M or less, 2.0×10 -7 M or less, 1.0×10 -7 M or less, 5.0×10 -8 M or less, 3.0×10 -8 M or less, 2.0×10 -8 M or less, 1.0×10 -8 M or less, 5.0×10 -9 M or less, 3.0×10 -9 M or less, 2.0×10 -9 M, 1.0 × 10 -9 M or less, 5.0×10 -10 M or less, 3.0×10 -10 M or less, 2.0×10 -10 M, or 1.0 x 10 -10 In a further embodiment, FcγRIIIa may be FcγRIIIa F or FcγRIIIa V, or both.

[0283] In another embodiment, the binding activity of the parent Fc region and the mutant Fc region may be expressed as kd (Dissociation rate constant) values ​​instead of KD values.

[0284] In another embodiment, the binding activity of the parent Fc region and the mutant Fc region may be expressed as the amount of Fcγ receptor binding per unit amount. For example, in a surface plasmon resonance assay, the amount of Fc region bound to a sensor chip and the amount of Fcγ receptor binding thereto are each measured in resonance units (RU). The amount of Fcγ receptor binding thereto divided by the amount of Fc region binding can be defined as the amount of Fc region binding to Fcγ receptor per unit amount. Specific methods for measuring and calculating such binding amounts are described in the Examples below. In some embodiments, the ratio of [amount of mutant Fc region binding to FcγRIIa] / [amount of parent Fc region binding 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, 40 or more, or 50 or more. In some embodiments, the ratio of [amount of binding of mutant Fc region to FcγRIIIa] / [amount of binding of parent Fc region to FcγRIIIa] is, 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 × 10 3 That's it, 2 x 10 3 That's it, 3 x 10 3 or more, or 5 x 10 3 That's all.

[0285] In certain embodiments, the KD values, kd values, binding amounts, etc., presented herein are measured or calculated by performing a surface plasmon resonance assay at 25°C or 37°C (see, e.g., Reference Example 8 herein).

[0286] In certain aspects, the mutant Fc regions of the present invention have improved selectivity between activating Fcγ receptors and inhibitory Fcγ receptors compared to the parent Fc region. In other words, the mutant Fc regions of the present invention have greater enhanced binding activity to activating Fcγ receptors than 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 the group consisting of FcγRIa, FcγRIIa R, FcγRIIa H, FcγRIIIa F, and FcγRIIIa V, and the inhibitory Fcγ receptor is FcγRIIb. In some embodiments, the mutant Fc regions of the present invention have improved selectivity between FcγRIIa and FcγRIIb. In some embodiments, the mutant Fc regions of the present invention have improved selectivity between FcγRIIIa and FcγRIIb. In a further aspect, the mutant Fc regions of the present invention have improved selectivity between FcγRIIa and FcγRIIb and between FcγRIIIa and FcγRIIb.

[0287] In some embodiments, the mutant Fc regions of the present invention comprise at least one amino acid modification at at least one position selected from the group consisting of positions 236, 239, 268, 270, and 326 (EU numbering). Alternatively, the amino acid modifications described in International Publication Nos. WO2013 / 002362 and WO2014 / 104165 can also be used in the present invention.

[0288] In certain embodiments, the binding activity of the parent Fc region and the mutant Fc region can be expressed as KD (Dissociation constant) values. The binding activity for FcγRIIa and FcγRIIIa is as described above. In one embodiment, the ratio of [KD value of the parent Fc region for FcγRIIb] / [KD value of the mutant Fc region for 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 Fc region and the mutant Fc region may be expressed as kd (Dissociation rate constant) values ​​instead of KD values.

[0289] In another embodiment, the binding activity of the parent Fc region and the mutant Fc region may be expressed as the amount of binding of the Fc region to an Fcγ receptor per unit amount as described above. In some embodiments, the ratio of [amount of binding of the mutant Fc region to FcγRIIb] / [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 mutant 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.

[0290] In a specific embodiment, improved selectivity between activating Fcγ receptors and inhibitory Fcγ receptors refers to a selective enhancement of binding activity to activating Fcγ receptors compared to binding activity to inhibitory Fcγ receptors; in other words, an increase in the ratio of binding activity to activating Fcγ receptors to binding activity to inhibitory Fcγ receptors (A / I ratio). Such a ratio (A / I ratio) is an indicator of the exertion of superior effector function, and polypeptides with a large A / I ratio can be evaluated as having superior effector function. The binding activity of a parent Fc region and a mutant Fc region to an Fcγ receptor can be expressed as a KD value, kd value, or the amount of Fc region binding to an Fcγ receptor per unit amount. The A / I ratio can be expressed using the KD value, kd value, or binding amount as follows: [KD value for inhibitory Fcγ receptor] / [KD value for activating Fcγ receptor], [kd value for inhibitory Fcγ receptor] / [kd value for activating Fcγ receptor], or [amount of binding to activating Fcγ receptor] / [amount of binding to inhibitory Fcγ receptor].

[0291] In one aspect, the A / I ratio of the mutant Fc region of the present invention 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, 50 times or more, 60 times or more compared to the parent Fc region. or more than 70 times, 80 times, 90 times, 100 times, 200 times, 300 times, 400 times, 500 times, 600 times, 700 times, 800 times, 900 times, 1000 times, 2000 times, 3000 times, 4000 times, 5000 times, 6000 times, 7000 times, 8000 times, 9000 times, or 10000 times. In one aspect, the A / I ratio of the mutant Fc region of the present invention 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. In one embodiment, the A / I ratio is the ratio of FcγRIa-binding activity to FcγRIIb-binding activity, the ratio of FcγRIIa-binding activity to FcγRIIb-binding activity, the ratio of FcγRIIIa-binding activity to FcγRIIb-binding activity, or the ratio of [the sum or average of two or three of FcγRIa-binding activity, FcγRIIa-binding activity, and FcγRIIIa-binding activity] to FcγRIIb-binding activity. In a specific embodiment, FcγRIIa is FcγRIIa R, FcγRIIa H, or both, and therefore, the binding activity to FcγRIIa is the sum or average of FcγRIIa R-binding activity, FcγRIIa H-binding activity, or both.In a specific embodiment, FcγRIIIa is FcγRIIIa F, FcγRIIIa V, or both, and thus, the binding activity to FcγRIIIa is the sum or average of the binding activity to FcγRIIIa F, the binding activity to FcγRIIIa V, or both.

[0292] In some embodiments, the variant Fc regions of the present invention comprise amino acid modifications at the following positions: (i) positions 234, 235, 236, 239, 268, 270, and 298, as represented by EU numbering, in the first polypeptide of the parent Fc region; and (ii) positions 270, 298, 326, and 334, as expressed in EU numbering, in the second polypeptide of the parent Fc region. In a specific embodiment, a variant Fc region of the present invention further comprises an amino acid modification at position 326 (EU numbering) in a first polypeptide of the parent Fc region. In a specific embodiment, a variant Fc region of the present invention further comprises an amino acid modification at position 236 (EU numbering) in a second polypeptide of the parent Fc region. In a specific embodiment, a variant Fc region of the present invention further comprises an amino acid modification at position 332 (EU numbering) in a first polypeptide of the parent Fc region. In a specific embodiment, a variant Fc region of the present invention further comprises an amino acid modification at position 330 (EU numbering) in a first polypeptide of the parent Fc region. In a specific embodiment, a variant Fc region of the present invention further comprises an amino acid modification at position 332 (EU numbering) in a second polypeptide of the parent Fc region. In a specific embodiment, a variant Fc region of the present invention further comprises an amino acid modification at position 330 (EU numbering) in a second polypeptide of the parent Fc region. Alternatively, the amino acid modifications described in International Publication Nos. WO2013 / 002362 and WO2014 / 104165 can also be used in the present invention.

[0293] In some embodiments, the variant Fc regions of the present invention comprise amino acid modifications at the following positions: (i) positions 234, 235, 236, 239, 268, 270, 298, and 330, as indicated by EU numbering, in the first polypeptide of the parent Fc region; and (ii) positions 270, 298, 326, 330, and 334, as expressed in EU numbering, in the second polypeptide of the parent Fc region. In a specific embodiment, a mutant Fc region of the present invention further comprises an amino acid modification at position 326 (EU numbering) in a first polypeptide of the parent Fc region. In a specific embodiment, a mutant Fc region of the present invention further comprises an amino acid modification at position 236 (EU numbering) in a second polypeptide of the parent Fc region. In a specific embodiment, a mutant Fc region of the present invention further comprises an amino acid modification at position 332 (EU numbering) in a first polypeptide of the parent Fc region. In a specific embodiment, a mutant Fc region of the present invention further comprises an amino acid modification at position 332 (EU numbering) in a second polypeptide of the parent Fc region. Alternatively, the amino acid modifications described in International Publication Nos. WO2013 / 002362 and WO2014 / 104165 can also be used in the present invention.

[0294] In certain aspects, the mutant Fc regions of the present invention have improved stability compared to the parent Fc region. In certain embodiments, the stability is thermodynamic stability. The thermodynamic stability of a polypeptide can be determined, for example, using the Tm value as an indicator. The Tm value can be measured using techniques known to those skilled in the art, such as CD (circular dichroism), DSC (differential scanning calorimetry), and DSF (differential scanning fluorometry). In one embodiment, the Tm value of the CH2 region of the mutant Fc regions of the present invention is increased by 0.1° or more, 0.2° or more, 0.3° or more, 0.4° or more, 0.5° or more, 1° or more, 2° or more, 3° or more, 4° or more, 5° or more, or 10° or more compared to the parent Fc region.

[0295] In some embodiments, the mutant Fc regions of the present invention comprise at least one amino acid modification in at least one position selected from the group consisting of positions 250 and 307 (EU numbering) in the first polypeptide and / or second polypeptide of the parent Fc region. Alternatively, the amino acid modifications described in International Publication WO2013 / 118858 may also be used in the present invention.

[0296] In a specific aspect, the mutant Fc region of the present invention is composed of two polypeptide chains with different sequences. In a further aspect, the mutant Fc region of the present invention exhibits enhanced heterodimerization between a first polypeptide and a second polypeptide. When producing heterodimeric proteins using recombinant techniques, it is preferable that different peptide chains preferentially associate to form heterodimers, rather than identical polypeptide chains associating to form homodimers. Whether or not heterodimerization of the mutant Fc region has been enhanced can be determined, for example, by separating homodimers and heterodimers from the produced mutant Fc region using techniques such as chromatography and determining the ratio of each component.

[0297] In some embodiments, the mutant Fc regions of the present invention comprise at least one amino acid modification in at least one position selected from the group consisting of positions 349, 356, 366, 368, 407, and 439 (EU numbering) in the first polypeptide and / or second polypeptide of the parent Fc region. Alternatively, the amino acid modifications described in International Publication WO2006 / 106905 and WO1996 / 027011 can also be used in the present invention.

[0298] In certain aspects, the mutant Fc regions of the present invention have enhanced FcRn-binding activity at acidic pH. In some embodiments, acidic pH refers to pH 4.0 to 6.5. In further embodiments, acidic pH is at least one selected from the group consisting of pH 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, and 6.5. In certain embodiments, the acidic pH is pH 5.8.

[0299] In some embodiments, the mutant Fc region of the present invention comprises at least one amino acid modification in at least one position selected from the group consisting of positions 428, 434, 436, 438, and 440 (EU numbering) in the first polypeptide and / or second polypeptide of the parent Fc region. Alternatively, the amino acid modifications described in International Publication WO2016 / 125495 may also be used in the present invention.

[0300] In one aspect, the mutant Fc region of the present invention comprises at least one amino acid modification at at least one position selected from the group consisting of positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 330, 332, 334, 349, 356, 366, 368, 407, 428, 434, 436, 438, 439, and 440, as expressed in EU numbering.

[0301] In one embodiment, a variant Fc region of the invention comprises amino acid modifications at positions 234, 235, 236, 239, 268, 270, 298, 326, and 334, as indicated by EU numbering. In a particular embodiment, a variant Fc region of the invention comprises amino acid modifications at (i) positions 234, 235, 236, 239, 268, 270, and 298, as indicated by EU numbering, in a first polypeptide of a parent Fc region, and (ii) positions 270, 298, 326, and 334, as indicated by EU numbering, in a second polypeptide of a parent Fc region. In another specific embodiment, the variant Fc region of the invention comprises amino acid modifications at (i) positions 234, 235, 236, 239, 268, 270, 298, and 326, as indicated by EU numbering, in a first polypeptide of the parent Fc region, and (ii) positions 236, 270, 298, 326, and 334, as indicated by EU numbering, in a second polypeptide of the parent Fc region.

[0302] In one embodiment, a variant Fc region of the invention comprises amino acid modifications at positions 234, 235, 236, 239, 268, 270, 298, 326, 330, and 334, as indicated by EU numbering. In a particular embodiment, a variant Fc region of the invention comprises amino acid modifications at (i) positions 234, 235, 236, 239, 268, 270, 298, and 330, as indicated by EU numbering, in a first polypeptide of a parent Fc region, and (ii) positions 270, 298, 326, 330, and 334, as indicated by EU numbering, in a second polypeptide of a parent Fc region.

[0303] In one embodiment, a variant Fc region of the invention comprises amino acid modifications at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, and 334, as indicated by EU numbering. In a particular embodiment, a variant Fc region of the invention comprises amino acid modifications at (i) positions 234, 235, 236, 239, 250, 268, 270, 298, and 307, as indicated by EU numbering, in a first polypeptide of a parent Fc region, and (ii) positions 250, 270, 298, 307, 326, and 334, as indicated by EU numbering, in a second polypeptide of a parent Fc region. In another specific embodiment, the variant Fc region of the invention comprises amino acid modifications at (i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, and 326, as indicated by EU numbering, in a first polypeptide of the parent Fc region, and (ii) positions 236, 250, 270, 298, 307, 326, and 334, as indicated by EU numbering, in a second polypeptide of the parent Fc region.

[0304] In one embodiment, a variant Fc region of the invention comprises amino acid modifications at positions 234, 235, 236, 239, 268, 270, 298, 326, 330, 332, and 334, as indicated by EU numbering. In a particular embodiment, a variant Fc region of the invention comprises amino acid modifications at (i) positions 234, 235, 236, 239, 268, 270, 298, 330, and 332, as indicated by EU numbering, in a first polypeptide of a parent Fc region, and (ii) positions 236, 270, 298, 326, 330, 332, and 334, as indicated by EU numbering, in a second polypeptide of a parent Fc region. In one embodiment, a variant Fc region of the invention comprises amino acid modifications at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 330, 332, and 334, as indicated by EU numbering. In a particular embodiment, a variant Fc region of the invention comprises amino acid modifications at (i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 330, and 332, as indicated by EU numbering, in a first polypeptide of a parent Fc region, and (ii) positions 236, 250, 270, 298, 307, 326, 330, 332, and 334, as indicated by EU numbering, in a second polypeptide of a parent Fc region. In another specific embodiment, the variant Fc region of the invention comprises amino acid modifications at (i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, and 326, as indicated by EU numbering, in a first polypeptide of the parent Fc region, and (ii) positions 236, 250, 270, 298, 307, 326, 330, 332, and 334, as indicated by EU numbering, in a second polypeptide of the parent Fc region.

[0305] In one embodiment, a variant Fc region of the invention comprises amino acid modifications at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 332, and 334, as indicated by EU numbering. In a particular embodiment, a variant Fc region of the invention comprises amino acid modifications at (i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, and 332, as indicated by EU numbering, in a first polypeptide of a parent Fc region, and (ii) positions 236, 250, 270, 298, 307, 326, 332, and 334, as indicated by EU numbering, in a second polypeptide of a parent Fc region.

[0306] In one embodiment, a variant Fc region of the invention comprises amino acid modifications at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 332, and 334, as indicated by EU numbering. In a particular embodiment, a variant Fc region of the invention comprises amino acid modifications at (i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, and 332, as indicated by EU numbering, in a first polypeptide of a parent Fc region, and (ii) positions 250, 270, 298, 307, 326, 332, and 334, as indicated by EU numbering, in a second polypeptide of a parent Fc region.

[0307] In one embodiment, a variant Fc region of the invention comprises amino acid modifications at positions 234, 235, 236, 239, 250, 268, 270, 298, 307, 326, 330, and 334, as indicated by EU numbering. In a particular embodiment, a variant Fc region of the invention comprises amino acid modifications at (i) positions 234, 235, 236, 239, 250, 268, 270, 298, 307, and 330, as indicated by EU numbering, in a first polypeptide of a parent Fc region, and (ii) positions 250, 270, 298, 307, 326, 330, and 334, as indicated by EU numbering, in a second polypeptide of a parent Fc region.

[0308] In a further embodiment, the variant Fc region of the present invention comprises at least one amino acid modification selected from the group consisting of: (i) Tyr or Phe at position 234, Gln or Tyr 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, and Glu at position 332, as indicated by EU numbering, in a first polypeptide of the parent Fc region; and (ii) Ala at position 236, Val at position 250, Val at position 270, Glu at position 298, Ala at position 307, Pro at position 326, Asp at position 326, Met or Lys at position 330, Asp or Glu at position 332, and Glu at position 334, as indicated by EU numbering, in a second polypeptide of the parent Fc region.

[0309] In a further embodiment, the mutant Fc region of the present invention further comprises any of the following amino acid modifications (a) to (f): (a) a Lys at position 356 (EU numbering) in a first polypeptide of a parent Fc region and a Glu at position 439 (EU numbering) in a second polypeptide of a parent Fc region; (b) Glu at position 439 (EU numbering) in the first polypeptide of the parent Fc region, and Lys at position 356 (EU numbering) in the second polypeptide of the parent Fc region. (c) a Trp at position 366, as indicated by EU numbering, in a first polypeptide of a parent Fc region, and a Ser at position 366, an Ala at position 368, and a Val at position 407, as indicated by EU numbering, in a second polypeptide of a parent Fc region; (d) Ser at position 366, Ala at position 368, and Val at position 407, as indicated by EU numbering, in a first polypeptide of the parent Fc region, and Trp at position 366, as indicated by EU numbering, in a second polypeptide of the parent Fc region; (e) a Cys at position 349 and a Trp at position 366, as indicated by EU numbering, in a first polypeptide of a parent Fc region, and a Cys at position 356, a Ser at position 366, an Ala at position 368, and a Val at position 407, as indicated by EU numbering, in a second polypeptide of a parent Fc region; (f) Cys at position 356, Ser at position 366, Ala at position 368, and Val at position 407, as indicated by EU numbering, in a first polypeptide of the parent Fc region, and Cys at position 349 and Trp at position 366, as indicated by EU numbering, in a second polypeptide of the parent Fc region.

[0310] In a further aspect, the variant Fc region of the present invention further comprises any of the following amino acid modifications (a) to (d) in the first polypeptide and / or second polypeptide of the parent Fc region: (a) Ala at position 434, as expressed in EU numbering; (b) Ala at position 434, Thr at position 436, Arg at position 438, and Glu at position 440, as indicated by EU numbering; (c) Leu at position 428, Ala at position 434, Thr at position 436, Arg at position 438, Glu at position 440, as indicated by EU numbering; (d) Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440, as indicated by EU numbering.

[0311] In certain embodiments, the polypeptide comprising a variant Fc region of the present invention is an antibody heavy chain constant region.

[0312] In a further aspect, the present invention provides a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 280, 281, 283-305, 308, 309, and 311-333.

[0313] In a further aspect, the present invention provides a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 308 and a polypeptide chain of SEQ ID NO: 309, a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 311 and a polypeptide chain of SEQ ID NO: 312, a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 313 and a polypeptide chain of SEQ ID NO: 314, a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 315 and a polypeptide chain of SEQ ID NO: 316, a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 317 and a polypeptide chain of SEQ ID NO: 318, a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 319 and a polypeptide chain of SEQ ID NO: 320, a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 321 and a polypeptide chain of SEQ ID NO: 322, a heavy chain constant region comprising a polypeptide chain of SEQ ID NO: 323 and a polypeptide chain of SEQ ID NO: 324, a polypeptide chain of SEQ ID NO: 325, and and a heavy chain constant region comprising the polypeptide chain of SEQ ID NO: 326, a heavy chain constant region comprising the polypeptide chain of SEQ ID NO: 327 and the polypeptide chain of SEQ ID NO: 328, a heavy chain constant region comprising the polypeptide chain of SEQ ID NO: 330 and the polypeptide chain of SEQ ID NO: 331, a heavy chain constant region comprising the polypeptide chain of SEQ ID NO: 332 and the polypeptide chain of SEQ ID NO: 333, a heavy chain constant region comprising the polypeptide chain of SEQ ID NO: 358 and the polypeptide chain of SEQ ID NO: 359, a heavy chain constant region comprising the polypeptide chain of SEQ ID NO: 360 and the polypeptide chain of SEQ ID NO: 361, a heavy chain constant region comprising the polypeptide chain of SEQ ID NO: 362 and the polypeptide chain of SEQ ID NO: 363, a heavy chain constant region comprising the polypeptide chain of SEQ ID NO: 364 and the polypeptide chain of SEQ ID NO: 366, and a heavy chain constant region comprising the polypeptide chain of SEQ ID NO: 365 and the polypeptide chain of SEQ ID NO: 367.

[0314] The term "Fcγ receptor" (herein referred to as Fcγ receptor, FcγR, or FcgR) refers to a receptor that can bind to the Fc region of IgG1, IgG2, IgG3, and IgG4 monoclonal antibodies, and effectively refers to any member of a family of proteins encoded by Fcγ receptor genes. In humans, this family includes FcγRI (CD64), which includes the isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), which includes the isoforms FcγRIIa (including allotypes H131 (H type) and R131 (R type)), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16), which includes the isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), as well as any unidentified human FcγR, FcγR isoform, or allotype. FcγRIIb1 and FcγRIIb2 have been reported as splice variants of human FcγRIIb. Furthermore, a splice variant designated FcγRIIb3 has been reported (J Exp Med, 1989, 170: 1369-1385). In addition to these splice variants, human FcγRIIb includes all splice variants registered with NCBI: NP_001002273.1, NP_001002274.1, NP_001002275.1, NP_001177757.1, and NP_003992.3. Furthermore, human FcγRIIb includes, in addition to FcγRIIb, all genetic polymorphisms that have been reported in the past (Arthritis Rheum. 48: 3242-3252 (2003); Kono et al., Hum. Mol. Genet. 14: 2881-2892 (2005); and Kyogoju et al., Arthritis Rheum. 46: 1242-1254 (2002)), as well as all genetic polymorphisms that will be reported in the future.

[0315] There are two allotypes of FcγRIIa: one in which the amino acid at position 131 of FcγRIIa is histidine (H type), and the other in which the amino acid at position 131 is substituted with arginine (R type) (Warrmerdam, J. Exp. Med. 172: 19-25(1990)).

[0316] FcγRs may be derived from any organism, including, but not limited to, FcγRs from humans, mice, rats, rabbits, and monkeys. Mouse FcγRs include, but are not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2), as well as any mouse FcγR or FcγR isoform.

[0317] The amino acid sequence of human FcγRI is set forth in SEQ ID NO: 131 (NP_000557.1); the amino acid sequence of human FcγRIIa is set forth in SEQ ID NO: 132 (AAH20823.1), SEQ ID NO: 142, SEQ ID NO: 143, or SEQ ID NO: 150; the amino acid sequence of human FcγRIIb is set forth in SEQ ID NO: 151 (AAI46679.1), SEQ ID NO: 169, or SEQ ID NO: 172; the amino acid sequence of human FcγRIIIa is set forth in SEQ ID NO: 174 (AAH33678.1), SEQ ID NO: 175, SEQ ID NO: 176, or SEQ ID NO: 177; and the amino acid sequence of human FcγRIIIb is set forth in SEQ ID NO: 178 (AAI28563.1).

[0318] Unlike Fcγ receptors, which belong to the immunoglobulin superfamily, human FcRn is structurally similar to major histocompatibility complex (MHC) class I polypeptides, sharing 22–29% sequence identity with class I MHC molecules (Ghetie et al., Immunol. Today (1997) 18(12), 592–598). FcRn is expressed as a heterodimer consisting of a transmembrane α or heavy chain complexed with a soluble β or light chain (β2-microglobulin). Like MHC, the α chain of FcRn consists of three extracellular domains (α1, α2, and α3), and a short cytoplasmic domain anc...

Claims

**Claim 1** (A) a variable region having CTLA-4 binding activity that depends on the concentration of an adenosine-containing compound, and (B) a mutant Fc region containing a plurality of amino acid modifications in the parental Fc region An isolated nucleic acid encoding an anti-CTLA-4 antibody comprising, wherein the parental Fc region is composed of two polypeptide chains, and the mutant Fc region contains amino acid modifications at the following positions: (i) positions 234, 235, 236, 239, 268, 270, 298, and 330 represented by EU numbering in the first polypeptide of the parental Fc region, and (ii) positions 270, 298, 326, 330, and 334 represented by EU numbering in the second polypeptide of the parental Fc region. **Claim 2** The nucleic acid according to claim 1, wherein the variable region has at least one characteristic selected from the following (a) to (i): (a) The binding activity in the presence of 100 μM of an adenosine-containing compound is at least 2-fold higher than the binding activity in the absence of the adenosine-containing compound, (b) The KD value is 5×10 -7 M or less in the presence of a 100 μM adenosine-containing compound, (c) The KD value in the absence of the adenosine-containing compound is 1 × 10 -6 M or more, (d) Forms a ternary complex with an adenosine-containing compound and CTLA-4, (e) Binds to the region of amino acids 97 to 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 that bound by ABAM004 (VH, SEQ ID NO: 10; and VL, SEQ ID NO: 11), (h) Exhibits cytotoxic activity against CTLA-4-expressing cells, and (i) Binds to CTLA-4 derived from human and mouse.

3. The anti-CTLA-4 antibody comprises (a) the amino acid sequence SX 1 TMN, where X 1 is H, A, R, or K, HVR-H1 (SEQ ID NO: 223), (b) the amino acid sequence SISX 1 X 2 SX 3 YIYYAX 4 SVX 5 G, where X 1 is S or T, X 2 is R or Q, X 3 is G or H, X 4 is D, E, or R, X 5 is K or R, HVR-H2 (SEQ ID NO: 224), and (c) the amino acid sequence YGX 1 REDMLWVFDY, where X 1 is K or A, HVR-H3 (SEQ ID NO: 225), the nucleic acid according to claim 1.

4. The anti-CTLA-4 antibody comprises (a) the amino acid sequence X 1 GX 2 STX 3 VGDYX 4 X 5 VX 6 wherein X 1 is T, D, Q, or E, X 2 is T or P, X 3 is D or G, X 4 is N or T, X 5 is Y or W, X 6 is S or H, and is HVR-L1 (SEQ ID NO: 226), (b) the amino acid sequence X 1 TX 2 X 3 KPX 4 wherein X 1 is E, F, or Y, X 2 is S or I, X 3 is K or S, X 4 is S, E, or K, and is HVR-L2 (SEQ ID NO: 227), and (c) the amino acid sequence X 1 TYAAPLGPX 2 wherein X 1 is S or Q, X 2 is M or T, and further comprises HVR-L3 (SEQ ID NO: 228), the nucleic acid according to claim 3. **Claim 5** The anti-CTLA-4 antibody is (1) (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; (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 115, (2) (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; (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 115, (3) (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; (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133, (4) (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; (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 153, (5) (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; (f) HVR-L3 comprising the amino acid sequence of 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: 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; (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133, (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: 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; (f) HVR-L3 comprising the amino acid sequence of 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: 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; (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133, or (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; (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 133, The nucleic acid according to claim 4, comprising **Claim 6** The anti-CTLA-4 antibody has (a) a VH sequence having at least 95% sequence identity with any one of the amino acid sequences of SEQ ID NOs: 83-86, 98, 135-141; (b) a VL sequence having at least 95% sequence identity with any one of the amino acid sequences of SEQ ID NOs: 88-95, 97, 99, 134, 144-149; or (c) a VH sequence having any one of the amino acid sequences of SEQ ID NOs: 83-86, 98, 135-141 and a VL sequence having any one of the amino acid sequences of SEQ ID NOs: 88-95, 97, 99, 134, 144-149, the nucleic acid according to claim 1. **Claim 7** The anti-CTLA-4 antibody is (1) the VH sequence of SEQ ID NO: 98 and the VL sequence of SEQ ID NO: 99, (2) the VH sequence of SEQ ID NO: 83 and the VL sequence of SEQ ID NO: 88, (3) the VH sequence of SEQ ID NO: 83 and the VL sequence of SEQ ID NO: 89, (4) the VH sequence of SEQ ID NO: 83 and the VL sequence of SEQ ID NO: 90, (5) VH sequence of SEQ ID NO: 83 and VL sequence of SEQ ID NO: 91, (6) VH sequence of SEQ ID NO: 83 and VL sequence of SEQ ID NO: 92, (7) VH sequence of SEQ ID NO: 83 and VL sequence of SEQ ID NO: 93, (8) VH sequence of SEQ ID NO: 83 and VL sequence of SEQ ID NO: 94, (9) VH sequence of SEQ ID NO: 83 and VL sequence of SEQ ID NO: 97, (10) VH sequence of SEQ ID NO: 83 and VL sequence of SEQ ID NO: 95, (11) VH sequence of SEQ ID NO: 84 and VL sequence of SEQ ID NO: 97, (12) VH sequence of SEQ ID NO: 85 and VL sequence of SEQ ID NO: 97, (13) VH sequence of SEQ ID NO: 86 and VL sequence of SEQ ID NO: 97, (14) VH sequence of SEQ ID NO: 86 and VL sequence of SEQ ID NO: 134, (15) VH sequence of SEQ ID NO: 136 and VL sequence of SEQ ID NO: 97, (16) VH sequence of SEQ ID NO: 135 and VL sequence of SEQ ID NO: 97, (17) VH sequence of SEQ ID NO: 136 and VL sequence of SEQ ID NO: 95, (18) VH sequence of SEQ ID NO: 137 and VL sequence of SEQ ID NO: 97, (19) VH sequence of SEQ ID NO: 138 and VL sequence of SEQ ID NO: 97, (20) VH sequence of SEQ ID NO: 138 and VL sequence of SEQ ID NO: 144, (21) VH sequence of SEQ ID NO: 138 and VL sequence of SEQ ID NO: 145, (22) VH sequence of SEQ ID NO: 138 and VL sequence of SEQ ID NO: 146, (23) VH sequence of SEQ ID NO: 139 and VL sequence of SEQ ID NO: 146, (24) VH sequence of SEQ ID NO: 140 and VL sequence of SEQ ID NO: 146, (25) VH sequence of SEQ ID NO: 141 and VL sequence of SEQ ID NO: 146, (26) VH sequence of SEQ ID NO: 140 and VL sequence of SEQ ID NO: 147, (27) VH sequence of SEQ ID NO: 141 and VL sequence of SEQ ID NO: 147, (28) VH sequence of SEQ ID NO: 140 and VL sequence of SEQ ID NO: 148, (29) VH sequence of SEQ ID NO: 141 and VL sequence of SEQ ID NO: 148, (30) VH sequence of SEQ ID NO: 136 and VL sequence of SEQ ID NO: 149, (31) a first variable region comprising the VH sequence of SEQ ID NO: 140 and the VL sequence of SEQ ID NO: 146, and a second variable region comprising the VH sequence of SEQ ID NO: 141 and the VL sequence of SEQ ID NO: 146, or (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, The nucleic acid according to claim 6, comprising the same. **Claim 8** The nucleic acid according to claim 1, wherein the mutated Fc region further comprises amino acid modifications at positions 250 and 307 represented by EU numbering in the first polypeptide of the parental Fc region. **Claim 9** The nucleic acid according to claim 1, wherein the mutated Fc region further comprises amino acid modifications at positions 250 and 307 represented by EU numbering in the second polypeptide of the parental Fc region. **Claim 10** The nucleic acid according to claim 1, wherein the anti-CTLA-4 antibody comprises a heavy chain constant region comprising a mutated Fc region. **Claim 11** The heavy chain constant region is (1) a first polypeptide of SEQ ID NO: 358 and a second polypeptide of SEQ ID NO: 359, or (2) a first polypeptide of SEQ ID NO: 360 and a second polypeptide of SEQ ID NO: 361, The nucleic acid according to claim 10, comprising the same. **Claim 12** (1) a first H-chain polypeptide of SEQ ID NO: 335, a second H-chain polypeptide of SEQ ID NO: 336, and an L-chain polypeptide of SEQ ID NO: 161, or (2) a first H-chain polypeptide of SEQ ID NO: 337, a second H-chain polypeptide of SEQ ID NO: 338, and an L-chain polypeptide of SEQ ID NO: 161, An isolated nucleic acid encoding an anti-CTLA-4 antibody, comprising the same. **Claim 13** A host cell comprising the nucleic acid according to any one of claims 1 to 12. **Claim 14** A method for producing an anti-CTLA-4 antibody, the method comprising culturing the host cell according to claim 13 such that the anti-CTLA-4 antibody is produced.

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