Anti-CTLA-4 antibody

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 an anti-CTLA-4 antibody and a method for using the same. The present invention also relates to a polypeptide comprising a mutant Fc region including an amino acid modification in the parent Fc region, and a method for producing the polypeptide. [Background technology]

[0002] Cells that have mutated due to genetic mutations or other causes in the body are monitored and eliminated by the immune surveillance system. On the other hand, a persistent excessive immune response can be harmful to the body itself, such as damaging normal tissues through autoimmunity. Therefore, the immune system is equipped with a negative feedback mechanism (immune checkpoint) to suppress an activated immune response (see, for example, Non-Patent Document 1). Immune checkpoints are thought to play an important role in maintaining homeostasis in the immune system. Meanwhile, it has become clear that some tumors use immune checkpoints to evade the immune response. Currently, research into 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) is being widely conducted. CTLA-4 is a glycoprotein belonging to the immunoglobulin superfamily, whose gene was cloned in 1987 from a cDNA library of killer T cell clones derived from mice (see, for example, Non-Patent Document 2). It is known that the immune response of T cells is suppressed via CTLA-4. Based on the idea that suppressing the function of CTLA-4 and promoting T cell activation leads to cancer regression, it was reported in 1996 that the administration of anti-CTLA-4 antibodies to tumor-bearing mice resulted in tumor regression (see, for example, Non-Patent Document 3). Since 2000, the effectiveness of anti-CTLA-4 antibodies in humans has been evaluated, and in 2011, the anti-human CTLA-4 monoclonal antibody (ipilimumab) was approved by the U.S. Food and Drug Administration (FDA) as the world's first immunoactivating 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 their development as pharmaceuticals is being attempted. Drugs that inhibit immune checkpoints, thereby disabling the immunosuppressive mechanism and ultimately enhancing immune activity, are called immune checkpoint inhibitors. On the other hand, it had long been known that some T cells possess immunosuppressive functions, but in 1995 these were identified as CD25-positive and CD4-positive T cells and named regulatory T cells (see, for example, Non-Patent Literature 4). In 2003, the Foxp3 gene, a master gene that is specifically expressed in regulatory T cells and controls their development and function, was identified. Foxp3 is a transcription factor that regulates the expression of various immune response-related genes. In particular, Foxp3 is involved in the constitutive expression of CTLA-4 in regulatory T cells, and it is thought that this plays an important role in the immunosuppressive function of regulatory T cells (see, for example, Non-Patent Literature 5). It is believed that the infiltration of regulatory T cells into tumor tissue leads to a weakening or inhibition of the immune surveillance mechanism against the tumor. In fact, it has been shown that regulatory T cells are increased in many human cancers (see, for example, Non-Patent Literature 6), and it has been reported that local infiltration of regulatory T cells into tumors may be a poor prognostic factor in cancer patients. Conversely, it is expected that removing or reducing regulatory T cells from tumor tissue will lead to an enhancement of anti-tumor immunity. Currently, the development of cancer immunotherapies targeting regulatory T cells is being actively pursued. While ipilimumab, an anti-CTLA-4 antibody, enhances antitumor immunity, it has also been reported to cause autoimmune diseases due to systemic enhancement of immune activity. In one clinical trial, 60% of patients treated with ipilimumab experienced adverse events, many of which were autoimmune diseases related to the skin or gastrointestinal tract. Other clinical trials have also reported that approximately half of patients treated with ipilimumab developed similar autoimmune diseases. To mitigate these side effects, immunosuppressants are sometimes administered to patients treated with ipilimumab. There is a need for the development of new drugs that can maintain the antitumor immune response while suppressing the side effects of these immune checkpoint inhibitors. When therapeutic antibodies are administered into the body, 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 normal tissue, which is not a lesion site, and this can cause undesirable side effects from a therapeutic standpoint. For example, antibodies against tumor antigens can exert toxic activity against tumor cells through ADCC, etc., but if the same antigen is also expressed in normal tissue, it may also damage normal cells. To solve the above problems, a technology has been developed to create antigen-binding molecules whose binding activity to the antigen changes depending on the concentration of the target tissue (e.g., tumor tissue), focusing on the phenomenon of certain compounds being present in large quantities in the target tissue (e.g., tumor tissue) (see, for example, Patent Document 11).

[0003] Antibodies are attracting attention as pharmaceuticals due to their high stability in the blood and low incidence of 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, namely antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cell-mediated cytotoxicity (CDC). It has been reported that antibodies of the human IgG class, specifically the IgG1 subclass, have the highest ADCC and CDC activity (Non-Patent Document 14). Furthermore, antibody-dependent cell-mediated phagocytosis (ADCP), which is 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 IgG antibodies ADCC, CDC, and ADCP 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 allotypes for each have also been reported (Non-Patent Literature 17).

[0005] The enhancement of cytotoxic effector functions such as ADCC, ADCP, and CDC is attracting attention as a promising means of enhancing the antitumor effects of antibodies. The importance of FcγR-mediated effector function for the antitumor effects of antibodies has been reported using mouse models (Non-Patent Literature 7, Non-Patent Literature 8). Furthermore, a correlation has been observed between clinical efficacy in humans and the high-affinity polymorphic allotype (V158) and low-affinity polymorphic allotype (F158) of FcγRIIIa (Non-Patent Literature 18). Similarly, it has been shown that clinical efficacy differs depending on the allotype (H131 and R131) of FcγRIIa (Non-Patent Literature 19). These reports suggest that antibodies with Fc regions optimized for binding to specific FcγRs mediate more potent effector function, thereby exerting effective antitumor effects.

[0006] The balance of antibody binding activity to the activating receptor (consisting of FcγRIa, FcγRIIa, FcγRIIIa, and FcγRIIIb) and the inhibitory receptor (consisting of FcγRIIb) is a crucial factor in optimizing the effector function of an antibody. By using an Fc region that enhances binding activity to the activating receptor and reduces binding activity to the inhibitory receptor, it may be possible to confer optimal effector function to the antibody (Non-Patent Document 20). Regarding the binding of the Fc region to FcγR, it has been shown that the hinge region of the antibody, several amino acid residues within the CH2 domain, and the glycan attached to the EU numbering 297th Asn bound to the CH2 domain are important (Non-Patent Documents 14, 21, and 22). Focusing on this binding site, various Fc region variants with different FcγR binding characteristics have been studied, and Fc region variants with higher activating FcγR binding activity have been obtained (Patent Documents 5, 6, 9, and 10). For example, Lazar et al. succeeded in increasing the binding to human FcγRIIIa (V158) by approximately 370 times by substituting Ser at EU numbering position 239, Ala at EU numbering position 330, and Ile at EU numbering position 332 of human IgG1 with Asp, Leu, and Glu, respectively (Non-Patent Document 9, Patent Document 6). Shinkawa et al. succeeded in increasing the binding to FcγRIIIa by approximately 100 times by deleting the fucose in the sugar chain attached to Asn at EU numbering position 297 (Non-Patent Document 23). These methods introduce the same modification or the same sugar chain modification to the Fc region of both H chains of the antibody. On the other hand, it has been reported that even though the Fc of the antibody is a homodimer, it binds to FcγR in a 1:1 ratio and recognizes FcγR asymmetrically in the lower hinge and CH2 region (Non-Patent Document 11). Considering that the Fc region interacts asymmetrically with FcγR, it is thought that introducing different modifications to each H chain would allow for more precise optimization of the interaction between IgG and FcγR. Based on this idea, a method has been reported in which different modifications are made to the Fc region of each H chain of an antibody, thereby modifying Fc asymmetrically and optimizing the interaction with FcγR (Patent Documents 7, 8, 9, and 10).In fact, by asymmetrically modifying the Fc region, modified antibodies exhibiting higher ADCC activity compared to existing ADCC-enhancing antibodies such as afucosylated antibodies have been obtained (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 Literature 24). ADCP activity can be enhanced by inhibiting the "Don't eat me" signal represented by CD47 (Non-Patent Literature 24), as well as by enhancing the ability to bind to FcγRIIa (Non-Patent Literature 25). However, the active form of FcγR, FcγRIIa, and the repressive form, FcγRIIb, have very high homology in the amino acid sequences of their extracellular regions, making it difficult to selectively enhance the ability to bind to FcγRIIa (Non-Patent Literature 26). Therefore, enhancing the ability to bind to FcγRIIa may also enhance the ability to bind to the repressive receptor, FcγRIIb, potentially weakening the effector function. In fact, in modified antibodies with significantly improved ability to bind to FcγRIIa, the ability to bind to FcγRIIb is also enhanced compared to natural IgG1 (Patent Literature 9, Patent Literature 10). Therefore, in order to exhibit high ADCC / ADCP activity, it is preferable to enhance binding to FcγRIIIa and FcγRIIa as much as possible without enhancing binding ability to FcγRIIb, but no such modified compounds have 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]

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[0010] This invention provides an anti-CTLA-4 antibody and a method for using the same. The invention also provides polypeptides containing mutated Fc regions and methods for 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) Mutant Fc region containing multiple amino acid modifications in the parent Fc region An anti-CTLA-4 antibody comprising the following, wherein the parent Fc region is composed of two polypeptide chains, and the mutant Fc region contains an amino acid modification at the following position: (i) Positions 234, 235, 236, 239, 268, 270, 298, and 330 in the first polypeptide of the parent Fc region, as represented by EU numbering, and (ii) Positions 270, 298, 326, 330, and 334 in the second polypeptide of the parent Fc region, as represented by EU numbering. [2] The anti-CTLA-4 antibody described in [1], wherein the variable region has at least one feature selected from (a) to (i) below: (a) The binding activity in the presence of a 100 μM adenosine-containing compound is more than twice as high as the binding activity in the absence of the adenosine-containing compound. (b) KD value of 5 × 10 in the presence of a 100 μM adenosine-containing compound -7 It is less than or equal to M. (c) KD value of 1 × 10 in the absence of adenosine-containing compounds -6 It is M or higher. (d) Forms a ternary complex with adenosine-containing compounds 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) Regarding binding to CTLA-4, it competes with ABAM004 (VH, SEQ ID NO: 10; and VL, SEQ ID NO: 11), (g) binds to the same epitope as 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 human and mouse-derived CTLA-4. [3] A monoclonal anti-CTLA-4 antibody as described in [1] or [2]. [4] An anti-CTLA-4 antibody as described in any one of items [1] to [3], which is a human antibody, a humanized antibody, or a chimeric antibody. [5] An anti-CTLA-4 antibody according to any one of the items [1] to [4], comprising (a) HVR-H1 (SEQ ID NO: 223) comprising the amino acid sequence SX1TMN, where X1 is H, A, R, or K; (b) 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) HVR-H3 (SEQ ID NO: 225) comprising the amino acid sequence YGX1REDMLWVFDY, where X1 is K or A. [6] The anti-CTLA-4 antibody described in [5], further comprising: (a) 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) 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) 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 according to [5], further comprising a heavy chain variable domain FR1 containing any one amino acid sequence of SEQ ID NO: 229 to 232, FR2 containing the amino acid sequence of SEQ ID NO: 233, FR3 containing the amino acid sequence of SEQ ID NO: 234, and FR4 containing the amino acid sequence of SEQ ID NO: 235. [8] The anti-CTLA-4 antibody according to [6], further comprising a light chain variable domain FR1 containing any one amino acid sequence of SEQ ID NOs: 236-238, FR2 containing any one amino acid sequence of SEQ ID NOs: 240-241, FR3 containing any one amino acid sequence of SEQ ID NOs: 242-244, and FR4 containing any one amino acid sequence of SEQ ID NOs: 245-246. [9] (a) A VH sequence having at least 95% sequence identity with 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 one of the amino acid sequences of SEQ ID NOs: 88-95, 97, 99, 134, 144-149; or (c) An anti-CTLA-4 antibody according to any one of items [1] to [4], comprising a VH sequence having one of the amino acid sequences of SEQ ID NOs: 83-86, 98, 135-141, and a VL sequence having one of the amino acid sequences of SEQ ID NOs: 88-95, 97, 99, 134, 144-149.

[10] (1) VH sequence of sequence number 98 and VL sequence of sequence number 99, (2) VH sequence of sequence number 83 and VL sequence of sequence number 88, (3) VH sequence of sequence number 83 and VL sequence of sequence number 89, (4) VH sequence of sequence number 83 and VL sequence of sequence number 90, (5) VH sequence of sequence number 83 and VL sequence of sequence number 91, (6) VH sequence of sequence number 83 and VL sequence of sequence number 92, (7) VH sequence of sequence number 83 and VL sequence of sequence number 93, (8) VH sequence of sequence number 83 and VL sequence of sequence number 94, (9) VH sequence of sequence number 83 and VL sequence of sequence number 97, (10) VH sequence of sequence number 83 and VL sequence of sequence number 95, (11) VH sequence of sequence number 84 and VL sequence of sequence number 97, (12) VH sequence of sequence number 85 and VL sequence of sequence number 97, (13) VH sequence of sequence number 86 and VL sequence of sequence number 97, (14) VH sequence of sequence number 86 and VL sequence of sequence number 134, (15) VH sequence of sequence number 136 and VL sequence of sequence number 97, (16) VH sequence of sequence number 135 and VL sequence of sequence number 97, (17) VH sequence of sequence number 136 and VL sequence of sequence number 95, (18) VH sequence of sequence number 137 and VL sequence of sequence number 97, (19) VH sequence of sequence number 138 and VL sequence of sequence number 97, (20) VH sequence of sequence number 138 and VL sequence of sequence number 144, (21) VH sequence of sequence number 138 and VL sequence of sequence number 145, (22) VH sequence of sequence number 138 and VL sequence of sequence number 146, (23) VH sequence of sequence number 139 and VL sequence of sequence number 146, (24) VH sequence of sequence number 140 and VL sequence of sequence number 146, (25) VH sequence of sequence number 141 and VL sequence of sequence number 146, (26) VH sequence of sequence number 140 and VL sequence of sequence number 147, (27) VH sequence of sequence number 141 and VL sequence of sequence number 147, (28) VH sequence of sequence number 140 and VL sequence of sequence number 148, (29) VH sequence of sequence number 141 and VL sequence of sequence number 148, (30) VH sequence of sequence number 136 and VL sequence of sequence number 149, (31) A first variable region containing the VH sequence of sequence number 140 and the VL sequence of sequence number 146, and a second variable region containing the VH sequence of sequence number 141 and the VL sequence of sequence number 146, or (32) A first variable region containing the VH sequence of sequence number 140 and the VL sequence of sequence number 147, and a second variable region containing the VH sequence of sequence number 141 and the VL sequence of sequence number 147, The anti-CTLA-4 antibody described in [9], including the following:

[11] A full-length IgG1 antibody, which is an anti-CTLA-4 antibody as described in any one of items [1] to

[10] .

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

[11] , wherein the mutated Fc region further comprises an amino acid modification at position 332, as represented by EU numbering, in the first polypeptide of the parent Fc region.

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

[12] , wherein the mutated Fc region further comprises an amino acid modification at position 332, as represented by EU numbering, in the second polypeptide of the parent Fc region.

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

[13] , wherein the mutated Fc region further comprises an amino acid modification at position 236, as represented by EU numbering, in the second polypeptide of the parent Fc region.

[15] An anti-CTLA-4 antibody according to 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 primary polypeptide of the parent Fc region.

[16] An anti-CTLA-4 antibody according to 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] An anti-CTLA-4 antibody according to any one of items [1] to

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

[18] An anti-CTLA-4 antibody according to any one of items [1] to

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

[19] An anti-CTLA-4 antibody according to any one of items [1] to

[18] , wherein the mutant Fc region further comprises one of the following amino acid modifications in the primary polypeptide and / or secondary polypeptide of the parental Fc region: (a) Ala at position 434 as represented by EU numbering, (b) Ala at position 434, Thr at position 436, Arg at position 438, and Glu at position 440, as represented by EU numbering. (c) Leu at position 428, Ala at position 434, Thr at position 436, Arg at position 438, and Glu at position 440, represented by EU numbering. (d) Leu at position 428, Ala at position 434, Arg at position 438, and Glu at position 440, as represented by EU numbering.

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

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

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

[20] , including the following: 〔twenty two〕 (1) The first H chain polypeptide of SEQ ID NO: 335, the second H chain polypeptide of SEQ ID NO: 336, and the L chain polypeptide of SEQ ID NO: 161, or (2) The first H chain polypeptide of SEQ ID NO: 337, the second H chain polypeptide of SEQ ID NO: 338, and the L chain polypeptide of SEQ ID NO: 161, An anti-CTLA-4 antibody, including [this component].

[23] An isolated nucleic acid encoding an anti-CTLA-4 antibody as described in any one of items [1] to

[22] .

[24] A host cell containing the nucleic acid described in

[23] .

[25] A method for producing an anti-CTLA-4 antibody, comprising culturing the host cells described in

[24] so as to produce an anti-CTLA-4 antibody.

[26] A pharmaceutical preparation comprising an anti-CTLA-4 antibody as described in any one of paragraphs [1] to

[22] and a pharmaceutically acceptable carrier.

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

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

[102] The polypeptide according to

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

[103] The polypeptide according to

[101] or

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

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

[105] The polypeptide according to any one of

[101] to

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

[106] The polypeptide according to any one of

[101] to

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

[107] The polypeptide according to any one of

[101] to

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

[108] The polypeptide according to any one of

[101] to

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

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

[110] The polypeptide according to any one 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 primary polypeptide of the parent Fc region.

[111] The polypeptide according to any one 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 the second polypeptide of the parent Fc region.

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

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

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

[115] Polypeptides according to any one of

[101] to

[114] , comprising at least one amino acid modification selected from the amino acid modifications listed below: (i) In the first polypeptide of the 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, Glu at position 332, 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, or Glu at position 334 in the second polypeptide of the parent Fc region, as represented by EU numbering.

[116] The polypeptide described in any of

[101] to

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

[117] Polypeptides according to any one of

[101] to

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

[118] A polypeptide according to any one of

[101] to

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

[119] The polypeptide described in

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

[120] A polypeptide according to any one of

[101] to

[119] , wherein the mutant Fc region exhibits improved selectivity between the active Fcγ receptor and the repressive Fcγ receptor compared to the parent Fc region. [120-2] A polypeptide according to any one of

[101] to

[119] , wherein, compared to the parent Fc region, the binding activity to the active Fcγ receptor is selectively enhanced in the mutant Fc region compared to the binding activity to the repressive Fcγ receptor. [120-3] A polypeptide according to any one of

[101] to

[119] , wherein the ratio of binding activity to the active Fcγ receptor to the binding activity to the inhibitory Fcγ receptor (A / I ratio) is larger in the mutant Fc region compared to the parent Fc region. [120-4] The ratio (A / I ratio) in the polypeptide containing the mutant Fc region is 1.1 times or more, 1.2 times or more, 1.3 times or more, 1.4 times or more, 1.5 times or more, 1.6 times or more, 1.7 times or more, 1.8 times or more, 1.9 times or more, 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times or more, 7 times or more, 8 times or more, 9 times or more, 10 times or more, 20 times or more, 30 times or more, 40 times or more, 50 times or more, 60 times or more compared to the polypeptide containing the parent Fc region. The polypeptide described in [120-3] is larger 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. [120-5] The polypeptide according to [120-3], wherein the ratio (A / I ratio) in the polypeptide containing the mutated 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] A polypeptide according to any one of

[120] to [120-5], wherein the active 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 according to any one of

[101] to

[121] , wherein the polypeptide containing the mutated Fc region is an antibody.

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

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

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

[0013]

[126] An isolated nucleic acid encoding a polypeptide as described in any of

[101] to

[122] .

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

[126] .

[128] A method for producing a polypeptide, comprising culturing the host cells described in

[127] so as to produce a polypeptide.

[129] A polypeptide according to any one of

[101] to

[122] for use in the treatment of tumors.

[130] A polypeptide according to any one of

[101] to

[122] for use in cell injury.

[131] The polypeptide described in

[130] , wherein cell damage is due to ADCC activity, CDC activity, or ADCP activity.

[132] A pharmaceutical composition comprising a polypeptide described in any of

[101] to

[122] and a pharmaceutically acceptable carrier.

[133] The pharmaceutical composition described in

[132] , which is a pharmaceutical composition for the treatment of tumors.

[134] The pharmaceutical composition described in

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

[135] The pharmaceutical composition according to

[134] wherein cell damage is due to ADCC activity, CDC activity, or ADCP activity.

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

[101] to

[122] or a pharmaceutical composition according to

[132] .

[137] A method of damaging cells, comprising administering a polypeptide according to any one of

[101] to

[122] or a pharmaceutical composition according to

[132] .

[138] The method according to

[137] wherein the cell damage is due to ADCC activity, CDC activity, or ADCP activity.

[139] Use of a polypeptide described in any of

[101] to

[122] in the manufacture of an anti-tumor agent.

[140] Use of a polypeptide described in any of

[101] to

[122] in the manufacture of a cytotoxic agent.

[141] Use as described in

[140] , where cell damage is due to ADCC activity, CDC activity, or ADCP activity.

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

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

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

[145] The method according to any one of

[142] to

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

[146] The method according to any one of

[142] to

[144] , wherein the modification of the function is an improvement in selectivity between the active Fcγ receptor and the inhibitory Fcγ receptor.

[147] The method according to any one of

[142] to

[144] , wherein the modification of the function is a selective enhancement of binding activity to the active Fcγ receptor compared to binding activity to the inhibitory Fcγ receptor.

[148] The method according to any one of

[142] to

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

[149] The ratio (A / I ratio) 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, 70 times or more compared to the polypeptide containing the parent Fc region.

[148] The increase of 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.

[150] The method according to any one of

[142] to

[149] , wherein the active 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 modification of the function is an enhancement of ADCC activity, CDC activity, or ADCP activity. [Brief explanation of the drawing]

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

[0015] [Figure 5] Figure 5 shows the ATP, ADP, or AMP concentration-dependent binding activity of the anti-CTLA-4 antibody ABAM004 to CTLA-4, as described in Reference Examples 1-9. [Figure 6] Figure 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] Figure 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 Examples 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 is shown as a ball-and-stick model. Amino acid residues that form interactions with AMP are shown as stick models. The dashed lines and their values ​​indicate the distance (Å) between each amino acid residue and AMP. [Figure 9] Figure 9 shows the binding modes of the ABAM004 Fab fragment, 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 as a ball-and-stick model. An amino acid residue of hCTLA4 containing one or more non-hydrogen atoms located within 4.2 Å of either the antibody or AMP is used as an epitope and is shown as a stick model. [Figure 10] Figure 10 shows the mapping of the epitopes of the ABAM004 Fab fragment into the amino acid sequence of hCTLA4, as described in Reference Example 2-14. In the figure, amino acid residues shown in black represent amino acid residues of hCTLA4 that contain one or more non-hydrogen atoms located within 4.2 Å of either the ABAM004 or AMP portion in the crystal structure. Amino acid residues shown in gray represent residues for which a model could not be constructed because they were disordered in the crystal structure. [Figure 11] Figure 11 is a diagram showing the superimposed structures of the antibody and AMP extracted from the crystal structures of the ABAM004 Fab fragment alone and its complex with AMP, and the ternary complex with AMP and CTLA4, 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. Thin lines show the structure of the ABAM004 Fab fragment alone, medium-thick lines show the structure of the binary complex with AMP, and thick lines show the structure of the ternary complex. [Figure 12] Figure 12 shows the ATP, ADP, or AMP concentration-dependent binding activity of the anti-CTLA-4 antibody ABAM004 and its variant 04H0150 / 04L0072 to CTLA-4, as described in Reference Example 3-2. In the figure, WT represents ABAM004 and H150L072 represents 04H0150 / 04L0072. [Figure 13] Figure 13 shows the ATP concentration-dependent neutralizing activity of the 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 tail vein at doses of 0.01 mg / kg, 0.1 mg / kg, 0.25 mg / kg, 1 mg / kg, 10 mg / kg, 30 mg / kg, and 100 mg / kg. Each point represents the average tumor volume for a group of n=4. [Figure 15] Figure 15 shows the antitumor effect of the anti-CTLA-4 antibody SW1208-mFa55 (switch antibody) in a mouse model transplanted with the FM3A cell line, as described in Reference Example 3-7-4. The antibody was administered via tail vein at doses of 0.1 mg / kg, 1 mg / kg, 10 mg / kg, 100 mg / kg, and 500 mg / kg. Each point represents the average tumor volume for a group of n=4. [Figure 16] Figure 16 shows the change in the percentage of effector Treg cells in a mouse model transplanted with the FM3A cell line, as described in Reference Example 3-7-7, after administration of the anti-CTLA-4 antibody mNS-mFa55 (control antibody) and SW1208-mFa55 (switch antibody). mNS-mFa55 was administered via tail vein at doses of 0.1 mg / kg, 1 mg / kg, 10 mg / kg, and 100 mg / kg, and SW1208-mFa55 was administered via tail vein at doses of 0.1 mg / kg, 1 mg / kg, 10 mg / kg, 100 mg / kg, and 500 mg / kg. Tumors were collected 6 days after administration, and the increase or decrease in effector Tregs was evaluated by FACS analysis. The vertical axis represents the percentage of effector Tregs (CD4+ FoxP3+ KLRG1+) relative to CD45+ cells. The mean value for n=3 is shown. [Figure 17]Figure 17 shows the change in the percentage of activated helper T cells in the spleen of a mouse model transplanted with the FM3A cell line, as described in Reference Example 3-7-8, after administration of the anti-CTLA-4 antibody mNS-mFa55 (control antibody) and SW1208-mFa55 (switch antibody). mNS-mFa55 was administered via tail vein at doses of 0.1 mg / kg, 1 mg / kg, 10 mg / kg, and 100 mg / kg, and SW1208-mFa55 was administered via tail vein at doses of 0.1 mg / kg, 1 mg / kg, 10 mg / kg, 100 mg / kg, and 500 mg / kg. The spleen was collected 6 days after administration, and the increase or decrease in activated helper T cells was evaluated by FACS analysis. The vertical axis represents the percentage of activated helper T cells (CD4+ Foxp3- ICOS+) relative to CD45+ cells. The mean value for n=3 is shown. [Figure 18] Figure 18 shows the antitumor effect of the anti-CTLA-4 antibody SW1389-mFa55 (switch antibody) in a mouse model transplanted with the Hepa1-6 / hGPC3 cell line, as described in Reference Example 4-3-5. The antibody was administered via tail vein at doses of 0.1 mg / kg, 1 mg / kg, 10 mg / kg, and 100 mg / kg. Each point represents the average tumor volume for a group of n=4. [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 tail vein at doses of 0.1 mg / kg, 1 mg / kg, 10 mg / kg, and 30 mg / kg. Each point represents the average tumor volume for a group of n=4. [Figure 20]Figure 20 shows the change in the percentage of effector Treg cells in a mouse model transplanted with the Hepa1-6 / hGPC3 cell line, as described in Reference Example 4-3-8, after administration of the anti-CTLA-4 antibody hNS-mFa55 (control antibody) and SW1389-mFa55 (switch antibody). hNS-mFa55 was administered via tail vein at doses of 0.1 mg / kg, 1 mg / kg, 10 mg / kg, and 30 mg / kg, and SW1389-mFa55 was administered at doses of 0.1 mg / kg, 1 mg / kg, 10 mg / kg, 100 mg / kg, and 500 mg / kg. Tumors were collected 6 days after administration, and the increase or decrease in effector Tregs was evaluated by FACS analysis. The vertical axis represents the percentage of effector Tregs (CD4+ FoxP3+ CCR7lowKLRG1+) relative to CD45+ cells. The mean value for n=3 is shown. [Figure 21] Figure 21 shows the change in the percentage of activated helper T cells in the spleen of a mouse model transplanted with the Hepa1-6 / hGPC3 cell line, as described in Reference Example 4-3-9, after administration of the anti-CTLA-4 antibody hNS-mFa55 (control antibody) and SW1389-mFa55 (switch antibody). hNS-mFa55 was administered via tail vein at doses of 0.1 mg / kg, 1 mg / kg, 10 mg / kg, and 30 mg / kg, and SW1389-mFa55 was administered at doses of 0.1 mg / kg, 1 mg / kg, 10 mg / kg, 100 mg / kg, and 500 mg / kg. The spleen was collected 6 days after administration, and the increase or decrease in activated helper T cells was evaluated by FACS analysis. The vertical axis represents the percentage of activated helper T cells (CD4+ Foxp3- ICOS+) relative to CD45+ cells. The mean value for n=3 is shown. [Figure 22] Figure 22 shows the antitumor effect of the anti-CTLA-4 antibody SW1610-mFa55 (switch antibody) in a mouse model transplanted with the Hepa1-6 / hGPC3 cell line, as described in Reference Example 5-4-5. The antibody was administered via tail vein at doses of 0.3 mg / kg, 1 mg / kg, and 3 mg / kg. Each point represents the average tumor volume for a group of n=5. [Figure 23] Figure 23 shows the antitumor effect of the anti-CTLA-4 antibody SW1612-mFa55 (switch antibody) in a mouse model transplanted with the Hepa1-6 / hGPC3 cell line, as described in Reference Example 5-4-5. The antibody was administered via tail vein at doses of 0.3 mg / kg, 1 mg / kg, and 3 mg / kg. Each point represents the average tumor volume for a group of n=5. [Figure 24] Figure 24 shows the antitumor effect of the anti-CTLA-4 antibody SW1615-mFa55 (switch antibody) in a mouse model transplanted with the Hepa1-6 / hGPC3 cell line, as described in Reference Example 5-4-5. The antibody was administered via tail vein at doses of 0.3 mg / kg, 1 mg / kg, and 3 mg / kg. Each point represents the average tumor volume for a group of n=5. [Figure 25] Figure 25 shows the changes in the percentage of effector Treg cells in a mouse model transplanted with the Hepa1-6 / hGPC3 cell line, as described in Reference Example 5-4-8, after administration of the anti-CTLA-4 antibodies SW1610-mFa55, SW1612-mFa55, and SW1615-mFa55 (all switch antibodies). SW1610-mFa55 was administered via tail vein at doses of 50 mg / kg, 100 mg / kg, and 200 mg / kg; SW1612-mFa55 at 50 mg / kg, 100 mg / kg, and 200 mg / kg; SW1615-mFa55 at 50 mg / kg, 100 mg / kg, 200 mg / kg, and 400 mg / kg; and the negative control antibody KLH-mFa55 was administered at 400 mg / kg. Tumor samples were collected 6 days after administration, and the increase or decrease in effector Treg cells was evaluated by FACS analysis. The vertical axis represents the percentage of effector Treg cells (CD4+ FoxP3+ CCR7lowKLRG1+) relative to CD45+ cells. The mean value for n=3 is shown. [Figure 26]Figure 26 shows the changes in the percentage of activated helper T cells in the spleen of a mouse model transplanted with the Hepa1-6 / hGPC3 cell line, as described in Reference Example 5-4-9, after administration of the anti-CTLA-4 antibodies SW1610-mFa55, SW1612-mFa55, and SW1615-mFa55 (all switch antibodies). SW1610-mFa55 was administered via tail vein at doses of 50 mg / kg, 100 mg / kg, and 200 mg / kg; SW1612-mFa55 at 50 mg / kg, 100 mg / kg, and 200 mg / kg; SW1615-mFa55 at 50 mg / kg, 100 mg / kg, 200 mg / kg, and 400 mg / kg; and the negative control antibody KLH-mFa55 was administered at 400 mg / kg. Spleen samples were collected six days after administration, and the increase or decrease in activated helper T cells was evaluated by FACS analysis. The vertical axis represents the percentage of activated helper T cells (CD4+ Foxp3- ICOS+) relative to CD45+ cells. The mean value for n=3 is shown. [Figure 27] Figure 27 shows a comparison of in vitro ADCC activity of various antibodies having modified constant regions with enhanced binding to FcγR, 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-Hl445 / MDX10D1L-k0MT, and ART8 represents MDX10D1H-Kn461 / MDX10D1H-Hl443 / MDX10D1L-k0MT. Here, IgG1 is the control antibody with a constant region, GASDALIE is the antibody with a constant region described in the prior art, and ART6 and ART8 are antibodies with modified constant regions prepared in Reference Example 6-1. [Figure 28]Figure 28 shows a comparison of in vitro ADCP activity of various antibodies having modified constant regions with enhanced binding to FcγR, 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-Hl445 / MDX10D1L-k0MT, and ART8 represents MDX10D1H-Kn461 / MDX10D1H-Hl443 / MDX10D1L-k0MT. Here, IgG1 is the control antibody with a constant region, GASDIE is the antibody with a constant region described in the prior art, and ART6 and ART8 are antibodies with modified constant regions prepared in Reference Example 6-1. [Figure 29] Figure 29 shows the in vitro ADCC activity of the 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 the 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 the 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 the anti-CTLA4 switch antibody SW1389 against CTLA4 (activity that cancels the CTLA4 signal that has an inhibitory effect on the activation of effector cells), as described in Reference Example 6-5. [Figure 33] Figure 33 shows the neutralizing activity of the anti-CTLA4 switch antibody SW1610 against CTLA4 (activity that cancels the CTLA4 signal that has an inhibitory effect on the activation of effector cells), as described in Reference Example 6-5. [Figure 34] Figure 34 shows the neutralizing activity of the anti-CTLA4 switch antibody SW1612 against CTLA4 (activity that cancels the CTLA4 signal that has an inhibitory effect on the activation of effector cells), as described in Reference Example 6-5. [Figure 35] Figure 35 shows the neutralizing activity of the anti-CTLA4 switch antibody SW1615 against CTLA4 (activity that cancels the CTLA4 signal that has an inhibitory effect on the activation of effector cells), as described in Reference Example 6-5. [Figure 36] Figure 36 shows the in vitro cytotoxic activity of the 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 the 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 the 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 the 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 Jurkat cells expressing hFcγRIIIaV 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 for 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. Antibodies were administered via tail vein at a dose of 10 mg / kg. Each point represents the average tumor volume for a group of n=5. [Figure 43] Figure 43 shows the binding activity of each antibody with modified Fc to hC1q, as described in Reference Example 12. Each point represents the average value of the ELISA colorimetric values ​​for n=2. [Figure 44] Figure 44 is a continuation of the diagram showing the binding activity of each antibody with modified Fc to hC1q, as described in Reference Example 12. Each point represents the average value of the ELISA colorimetric values ​​for n=2. [Modes for carrying out the invention]

[0017] The methods and procedures described or cited herein are generally well understood, for example, 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 (FM Ausubel, et al. eds., (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (RI Freshney, ed. (1987)); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JE 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. Conventional techniques, such as those widely used by those skilled in the art, are commonly employed by those skilled in the art, as described in Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (VT DeVita et al., eds., JB Lippincott Company, 1993).

[0018] I. Definition Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art in which this invention pertains. 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 general guidance for many of the terms used herein. All references cited herein, including patent applications and publications, are incorporated herein by reference in their entirety.

[0019] For the purpose of interpreting this Spec., the following definitions apply, and wherever applicable, a term used in the singular also includes the plural, and vice versa. It should be understood that the terms used herein are intended solely to describe a particular aspect and not to limit it. In the event of any conflict between the following definitions and any document incorporated herein by reference, the following definitions shall prevail.

[0020] In this specification, the term "and / or" refers to each subject or any combination thereof that is listed before or after "and / or". For example, "A, B and / or C" includes not only the subjects "A", "B", and "C", but also "A and B", "A and C", "B and C", and any combination selected from "A, B, and C".

[0021] In the spirit of this specification, “acceptor human framework” is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from the human immunoglobulin framework or human consensus framework as defined below. An acceptor human framework “derived” from the human immunoglobulin framework or human consensus framework may contain the same amino acid sequence or may contain a modification of the amino acid sequence. In some embodiments, the number of amino acid modifications 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 sequence-identical to the VL human immunoglobulin framework sequence or human consensus framework sequence.

[0022] Antibody-dependent cell-mediated cytotoxicity (ADCC) is a form of cytotoxicity in which secreted immunoglobulins bind to Fc receptors (FcRs) present on specific cytotoxic cells (e.g., NK cells, neutrophils, and macrophages), thereby enabling these cytotoxic effector cells to specifically bind to target cells containing antigens and subsequently kill those target cells with cytotoxicity. NK cells, the primary cells mediating ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991). To evaluate the ADCC activity of the molecule of interest, in vitro ADCC assays, such as those described in U.S. Patent No. 5,500,362, No. 5,821,337, or No. 6,737,056 (Presta), may be performed. Effector cells useful for such assays include PBMCs and NK cells. Alternatively, the ADCC activity of the molecule of interest may be evaluated in vivo in animal models, such as the animal models disclosed in Clynes et al. PNAS (USA) 95: 652-656 (1998).

[0023] Examples of "cytotoxic activity" include antibody-dependent cell-mediated cytotoxicity (ADCC) activity, complement-dependent cytotoxicity (CDC) activity, and T cell-mediated cytotoxicity. CDC activity refers to cytotoxic activity mediated by the complement system. ADCC activity, on the other hand, refers to the activity in which an antibody binds to an antigen present on the surface of a target cell, and then effector cells bind to that antibody, causing damage to the target cell by the effector cells. Whether or not a target antibody has ADCC activity or CDC activity can be measured by known methods (e.g., Current Protocols in Immunology, Chapter 7. Immunologic studies in humans, Coligan et al. (1993), etc.).

[0024] "Neutralizing activity" refers to the activity of an antibody that inhibits a molecule involved in some biological activity by binding to that molecule. In some embodiments, 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. Antibodies possessing such neutralizing activity are called neutralizing antibodies. The neutralizing activity of a test substance can be measured by comparing its biological activity in the presence of a ligand with that substance in the presence or absence of the substance.

[0025] The term "antibody-dependent cell phagocytosis" or "ADCP" refers to the process by which either all or part of an antibody-coated cell is taken up into phagocytic immune cells (e.g., macrophages, neutrophils, and dendritic cells) that bind to the immunoglobulin Fc region.

[0026] The terms “binding activity” and “binding ability” are used interchangeably herein and refer to the total strength of non-covalent interactions between one or more binding sites (e.g., variable regions or Fc regions) of a molecule (e.g., an antibody or other polypeptide) and its binding partner (e.g., an antigen or Fcγ receptor). Here, “binding activity” is not strictly limited to a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen, or an Fc region and an Fcγ receptor). For example, if the members of a binding pair reflect a monovalent 1:1 interaction, binding activity refers to the intrinsic binding affinity ("affinity"). If the members of a binding pair are capable of both monovalent and polyvalent binding, binding activity is the sum of these binding forces. The binding activity of molecule X to its partner Y can generally be expressed by its dissociation constant (KD) or “analyte binding per unit amount of ligand.” Binding activity can be measured by conventional methods known in the art, including those described herein. Specific examples and exemplary embodiments for measuring binding activity are described below.

[0027] An "affinity-matured" antibody is an antibody that, compared to a parent antibody without modifications, has one or more modifications in one or more hypervariable regions (HVRs) that result in improved affinity of the antibody to the antigen.

[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, and as a result, is useful as a diagnostic and / or therapeutic agent when it targets CTLA-4. In one embodiment, the degree of binding of an anti-CTLA-4 antibody to unrelated non-CTLA-4 proteins is less than approximately 10% of the antibody’s binding to CTLA-4, as measured (e.g., by radioimmunoassay (RIA)). In a particular embodiment, an antibody that binds to CTLA-4 has a concentration 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, for example, 10 -8 M~10 -13 M, for example, 10 -9 M~10 -13 It has a dissociation constant (KD) of M). In certain embodiments, the anti-CTLA-4 antibody binds to CTLA-4 epitopes that are conserved among CTLA-4 from different species.

[0029] In this specification, the term “antibody” is used in its broadest sense and encompasses a variety of antibody structures, including monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0030] An "antibody fragment" refers to a molecule other than the complete antibody, containing a portion of the complete antibody that binds to the antigen to which the complete antibody binds. Examples of antibody fragments, but not limited to these, include 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 the reference antibody" is an antibody that, in a competitive assay, blocks, for example, 50% or more of the binding of the reference antibody to its own antigen, and / or, the reference antibody blocks, for example, 50% or more of the binding of the aforementioned antibody to its own antigen in a competitive assay. An exemplary competitive assay is provided herein.

[0032] An "autoimmune disease" is a non-malignant disease or disorder that originates from and is directed toward the tissues of an individual. In this specification, an autoimmune disease explicitly excludes malignant or cancerous diseases or conditions, and in particular 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, the following: inflammatory reactions such as inflammatory skin diseases including psoriasis and dermatitis (e.g., atopic dermatitis); systemic scleroderma and sclerosis; reactions associated with inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis); respiratory distress syndromes (including adult respiratory distress syndrome: ARDS); dermatitis; meningitis; encephalitis; uveitis; colitis; glomerulonephritis; allergic conditions such as eczema and asthma and other conditions with T-cell infiltration and chronic inflammatory reactions; atherosclerosis; leukocyte adhesion deficiency; rheumatoid arthritis; and systemic lupus erythematosus (SLE). (Including but not limited to lupus nephritis and cutaneous lupus); diabetes mellitus (e.g., type 1 diabetes or insulin-dependent diabetes mellitus); multiple sclerosis; Raynaud's syndrome; autoimmune thyroiditis; Hashimoto's thyroiditis; allergic encephalomyelitis; Sjögren's syndrome; juvenile-onset diabetes mellitus; 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 involving leukocyte leakage; central nervous system (CNS) Inflammatory disorders; multiple organ injury syndromes; hemolytic anemia (including but not limited to cryoglobulinemia or Coombs-positive anemia); myasthenia gravis; antigen-antibody complex-mediated disorders; anti-glomerular basement membrane disorders; antiphospholipid syndromes; allergic neuritis; Graves' disease; Lambert-Eaton myasthenic syndrome; bullous pemphigoid; pemphigus; autoimmune polyglandular endocrine disorders; 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 a physiological condition in mammals typically characterized by unregulated cell growth / proliferation. Examples of cancer include breast cancer and liver cancer.

[0034] The term "complement-dependent cell injury" or "CDC" refers to a mechanism for inducing cell death in which the Fc effector domain of an antibody bound to a target activates a series of enzymatic reactions, resulting in the formation of holes in the membrane of the target cell. Typically, an antigen-antibody complex formed on a target cell binds to and activates complement component C1q, which then activates the complement cascade, leading to the death of the target cell. Complement activation may also result in the deposition of complement components on the surface of the target cell, which in turn promotes ADCC by binding to complement receptors (e.g., CR3) on leukocytes.

[0035] "Chemotherapy agents" refer to chemical compounds that are useful in treating 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, carbocone, meturedopa, and uredopa; ethyleneimines and methylolmelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylomellamine; acetogenins (especially bratacin and bratacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapacone; lapachol; colchicine; betulinic acid; camptothecin (synthetic analogs include topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®)), acetylcamptothecin, and scopolectin. (scopolectin), and 9-aminocamptothecin; bryostatin; calistatin; CC-1065 (including its adzeresin, karzeresin, and bizeresin synthetic analogs); podophyllotoxin; podophyllic acid; teniposide; cryptophycin (especially cryptophycin 1 and cryptophycin 8); dorastatin; duocalmycin (including synthetic analogs KW-2189 and CB1-TM1); eloiterobin; pancratistatin; sarcodictiin; spongistatin; chlorambucil, chlornafadin, chlorophosphamide (chlorophosphamide), estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, nobuenvicin, fenesterine, prednimustine, trophosphamide, uracil mustard, and other nitrogen mustards; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine;Endiye antibiotics {e.g., calicheamicin, especially calicheamicin gamma 1I and calicheamicin omega I1 (e.g., Nicolaou et al., Angew. Chem Intl. Ed. Engl., 33: 183-186 (1994)} See also); oral alpha-4 integrin inhibitors such as CDP323; dinemycin containing dinemycin A; esperamycin; and neocardinostatin chromophore and related pigment protein enediin antibiotics: chromophore, acrasinomycin, actinomycin, anthramycin, azaserin, bleomycin, kactinomycin, carbicin, carminomycin, cardinophilin, chromomycin, dactinomycin, daunorubicin, detrubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (ADRIAMYCIN®), morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HCl for liposomal injection (DOXIL®), liposomal doxorubicin TCL D-99 (MYOCET®), pegylated liposomal doxorubicin (CAELYX®), and deoxydoxorubicin, epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin such as mitomycin C, mycophenolic acid, nogaramycin, olibomycin, peplomycin, porphyromycin, puromycin, quelamycin, rhodorubicin Antibiotics such as rodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, and zolubicin; antimetabolites such as methotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), epotilon, and 5-fluorouracil (5-FU); folate analogs such as denopterin, methotrexate, pteropterin, and trimethrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine;Pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and phloxuridine; androgens such as carsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; and anti-adrenal substances such as aminoglutethimide, mitotane, and trilostane. (anti-adrenals); folic acid supplements such as folic acid; acegraton; aldofamide glycoside; aminolevulinic acid; enyluracil; amsacrin; bestrabusil; bisanthren; edatrexate; defofamine; demecoltin; diaziquan; elfornithine; eriptinium acetate; epotilon; etogluside; gallium nitrate; hydrociurea; lentinan; ronidamin; mayansinoids such as mayansin and anthamitocin; mitogluazone; mitoxantrone; mopidamol; nitraerine; pentostatin; fenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK (registered trademark) polysaccharide complex (JHS Natural Products, Eugene, OR); Lazoxane; Rhizoxin; Schizophyllan; Spirogermanium; Tenuazonic acid; Triadiquan; 2,2',2'-Trichlorotriethylamine; Trichothecene (especially T-2 toxin, Veracurin A) A) Loridine A and Anguidine); Urethanes; Vindesine (ELDISINE®, FILDESIN®); Dacarbazine; Mannomustine; Mitobronitol; Mitractol; Pipobroman; Gacytosine; Arabinoside ("Ara-C"); Thiotepa; Taxoids, e.g., Paclitaxel (TAXOL®), Albumin-Modified Nanoparticle Formulations of Paclitaxel (ABRAXANE®), and Docetaxel (TAXOTERE®); Chlorambucil; 6-Thiogunine; Mercaptopurine; Methotrexate; Platinum preparations such as Cisplatin, Oxaliplatin (e.g., ELOXATIN®), and Carboplatin;Vinca derivatives that inhibit microtubule formation by tubulin polymerization, including vinblastine (VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®, FILDESIN®), and vinorelbine (NAVELBINE®); etoposide (VP-16); ifosfamide; mitoxantrone; leucovorin; novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine: DMFO); Retinoids such as retinoic acid containing bexarotene (TARGRETIN®); Bisphosphonates such as clodronate (e.g., BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid / zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDIA®), tildronate (SKELID®), or risedronate (ACTONEL®); Troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); Antisense oligonucleotides, in particular those that inhibit gene expression in signaling pathways associated with abnormal cell proliferation, e.g., PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EPDRO) Vaccines such as EGF-R; 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);Bortezomib (VELCADE®); CCI-779; Tipifarnib (R11577); Sorafenib, ABT510; Oblimersen sodium (GENASENSE®), and other Bcl-2 inhibitors; Pixantrone; EGFR inhibitors (see definition below); tyrosine kinase inhibitors (see definition below); rapamycin (sirolimus, RAPAMUNE®), and other serine-threonine kinase inhibitors; lonafarnib (SCH 6636, SARASAR™), and other farnesyl transferase inhibitors; and pharmaceutically acceptable salts, acids, or derivatives of any of the above; and combinations of two or more of the above, for example CHOP, an abbreviation for the combination therapy of cyclophosphamide, doxorubicin, vincristine and prednisolone; and FOLFOX, an abbreviation for the 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 remaining portion 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 heavy chain of the antibody. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM. And some of these may be further divided into subclasses (isotypes). For example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to 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 interferes with the function of cells and / or causes cell death or destruction. 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 Radioisotopes of Pb and Lu); chemotherapeutic agents or chemotherapeutic drugs (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents); growth inhibitors; enzymes such as nucleases and their fragments; antibiotics; toxins such as low molecular weight toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin (including their fragments and / or variants); and various chemotherapeutic agents disclosed above.

[0039] "Effector cells" refer to leukocytes that express one or more FcRs and exert effector function. In certain embodiments, these cells express at least FcγRIII and exert 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, for example, from blood. In certain embodiments, effector cells may be human effector cells.

[0040] "Effector function" refers to the biological activity that varies depending on the antibody isotype, stemming from the Fc region of the antibody. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); antibody-dependent cell-mediated phagocytosis (ADCP); downregulation of cell surface receptors (e.g., B cell 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 targeting that antigen and contains specific amino acids that are in direct contact with the antibody. Epitope determinants can include a group of chemically active surface molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and can possess specific three-dimensional structural properties and / or specific charge properties. Generally, antibodies specific to a particular target antigen preferentially recognize epitopes on that target antigen in a complex mixture of proteins and / or macromolecules.

[0042] An "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR is the native human FcR. In some embodiments, the FcR is one that binds to an IgG antibody (gamma receptor) and includes the FcγRI, FcγRII, and FcγRIII subclass receptors, including allelic variants and alternative splicing forms of these receptors. The FcγRII receptor includes FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immune receptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (see, e.g., Daeron, Annu. Rev. Immunol. 15: 203-234 (1997)). FcRs have been 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 to be identified in the future, are also included in the term "FcR" as used herein.

[0043] The term “Fc receptor” or “FcR” also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J. Immunol. 24: 249 (1994)) and the regulation of immunoglobulin homeostasis. Methods for measuring binding to FcRn are known (see, e.g., Ghetie and Ward., Immunol. Today 18(12): 592-598 (1997); Ghetie et al., Nature Biotechnology, 15(7): 637-640 (1997); Hinton et al., J. Biol. Chem. 279(8): 6213-6216 (2004); WO2004 / 92219 (Hinton et al.)).

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

[0045] In this specification, the term “Fc region” is used to define the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the constant region. This term includes both the native sequence Fc region and mutant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain, provided that the C-terminal lysine (Lys447) or glycine-lysine (Gly446-Lys447) of the Fc region is present or absent. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system (also known as the EU index) 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 of the Fc region (residue 447 according to the EU numbering system) or the C-terminal glycine-lysine of the Fc region (residues 446-447) can be removed, for example, during antibody purification or by recombination operations of the nucleic acid encoding the antibody. Therefore, a composition containing an antibody having an Fc region according to the present invention may include an antibody with G446-K447, an antibody with G446 but without K447, an antibody from which G446-K447 has been completely removed, or a mixture of the above three types of antibodies.

[0047] The term "variable region" or "variable domain" refers to a domain in the heavy or light chain of an antibody that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of native antibodies (VH and VL, respectively) typically have similar structures, with each domain containing four conserved framework regions (FRs) and three hypervariable regions (HVRs) (see, for example, Kindt et al. Kuby Immunology, 6th ed., WH 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 particular antigen may be isolated by screening complementary libraries of VL or VH domains, respectively, using the VH or VL domains from antibodies that bind to that antigen. See, for example, Portolano et al., J. Immunol. 150: 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991).

[0048] The "framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The variable domain FR typically consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the sequences of HVR and FR usually 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 antibodies having a structure substantially similar to that of a native antibody, or having a heavy chain containing an Fc region as defined herein.

[0050] A "functional Fc region" possesses "effector functions" of a native sequence Fc region. Exemplary "effector functions" include C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; and downregulation of cell surface receptors (e.g., B cell receptors: BCRs). Such effector functions generally require the Fc region to be combined with a binding domain (e.g., an antibody-variable domain) and can be evaluated using various measurement methods disclosed, for example, within the definitions herein.

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

[0052] The "Human Consensus Framework" is a framework that shows the most commonly occurring amino acid residues in selected human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from subgroups of variable domain sequences. Typically, the sequence subgroups are those described 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 by Kabat et al. As described above. In another embodiment, for VH, the subgroup is subgroup III by Kabat et al. As described above.

[0053] A “humanized” antibody is a chimeric antibody that contains amino acid residues from a non-human HVR and amino acid residues from a human FR. In some embodiments, a humanized antibody contains substantially all of at least one, 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 contain at least a portion of the antibody constant region derived from a human antibody. The “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 the variable domain of an antibody that is hypervariable in sequence (a “complementarity determining region” or “CDR”), and / or forms a structurally defined loop (a “hypervariable loop”), and / or contains an antigen contact residue (a “antigen contact”). Typically, an antibody contains six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Illustrative HVRs as used herein include: (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) Antigen contact 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), including HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3). Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein in accordance with Kabat et al.

[0055] An "immunoconjugate" is an antibody that has been conjugated to one or more heterologous molecules (the heterologous molecules may include, but are not limited to, cytotoxic agents).

[0056] "Isolated" antibodies are those separated from the components of their original environment. In some embodiments, antibodies are purified to a purity of over 95% or 99% by, for example, 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 evaluating antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848: 79-87 (2007).

[0057] "Isolated" nucleic acids are nucleic acid molecules that have been separated from the components of their original environment. Isolated nucleic acids include nucleic acid molecules that would normally be found in the cell containing them, but these nucleic acid molecules are located outside the chromosome or in a chromosomal location different from their original 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, and includes nucleic acid molecules mounted on one or more vectors, and nucleic acid molecules present at one or more locations within a host cell.

[0059] In this specification, "first polypeptide" and "second polypeptide" mean polypeptides that constitute the Fc region of an antibody. "First polypeptide" and "second polypeptide" mean that their sequences are different from each other, preferably that at least the CH2 region sequence is different. The CH3 region sequence may also be different. The polypeptide may be, for example, a polypeptide that constitutes the Fc region of natural IgG, or a polypeptide that has been modified from a polypeptide that constitutes the Fc region of natural IgG.

[0060] Natural IgG refers to polypeptides belonging to a class of antibodies that contain the same amino acid sequence as naturally occurring IgG and are substantially encoded by the immunoglobulin gamma gene. 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 variants.

[0061] In this invention, "polypeptide" usually refers to peptides and proteins having a length of about 10 amino acids or more. While it is usually a polypeptide of biological origin, it is not particularly limited; for example, it may be a polypeptide consisting of an artificially designed sequence. It may also be a natural polypeptide, synthetic polypeptide, recombinant polypeptide, etc. In this invention, a protein molecule refers to a molecule containing the polypeptide.

[0062] A preferred example of the polypeptide of the present invention is an antibody. A more preferred example is natural IgG or an antibody modified from natural IgG. A particularly good example of natural IgG is natural human IgG. Natural IgG refers to a polypeptide that contains the same amino acid sequence as naturally occurring IgG and belongs to a class of antibodies substantially encoded by the immunoglobulin gamma gene. 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 variants.

[0063] The term "polypeptide containing an Fc region" is not particularly limited as long as it contains an Fc region, but an example is an antibody containing an Fc region. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) or the C-terminal glycine-lysine of the Fc region (residues 446-447) can be removed, for example, during the purification of the polypeptide (e.g., antibody) or by a recombination operation of the nucleic acid encoding the polypeptide. Therefore, a composition containing a polypeptide having an Fc region according to the present invention may include a polypeptide containing an Fc region with G446-K447, a polypeptide containing an Fc region with G446 but without K447, a polypeptide containing an Fc region from which G446-K447 has been completely removed, or a mixture of the above three types of polypeptides.

[0064] "Isolated" polypeptides are those separated from the components of their original environment. In some embodiments, polypeptides are purified to a purity of over 95% or 99% by, for example, 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, for example, Flatman et al., J. Chromatogr. B 848: 79-87 (2007).

[0065] "Isolated nucleic acid encoding a polypeptide" means one or more nucleic acid molecules encoding the polypeptide (e.g., the Fc region of an antibody, or the heavy and light chains or fragments thereof of an antibody), and includes nucleic acid molecules mounted on one vector or separate vectors, and nucleic acid molecules present at one or more locations in a host cell.

[0066] As used herein, the term "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked. This term includes vectors as self-replicating nucleic acid structures, and vectors that are incorporated into the genome of a host cell into which they are introduced. Some vectors can result in the expression of the nucleic acid to which they are operationally linked. Such vectors are also referred to herein as "expression vectors." Vectors can be introduced into host cells by methods such as viruses or electroporation, but vector introduction is not limited to in vitro; it is also possible to introduce vectors directly into living organisms.

[0067] The terms “host cell,” “host cell line,” and “host cell culture” refer to cells (including their offspring) that are interchangeably used and into which foreign nucleic acids have been introduced. Host cells include “transformed organisms” and “transformed cells,” which include primary transformed cells and their offspring, regardless of passage number. Offspring do not have to be completely identical to the parent cells in terms of nucleic acid content and may contain mutations. Mutant offspring that have the same function or biological activity as those used when the original transformed cells were screened or selected are also included herein.

[0068] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies. That is, the individual antibodies constituting that population are identical and / or bind to the same epitope, except for any mutant antibodies that may occur (e.g., mutant antibodies containing naturally occurring mutations, or mutant antibodies that occur during the manufacture of a monoclonal antibody preparation; such variants are usually present in small amounts). In contrast to polyclonal antibody preparations, which typically contain different antibodies against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is against a single determinant on an antigen. Therefore, the modifier “monoclonal” indicates a characteristic of the antibody that it is obtained from a substantially homogeneous population of antibodies, and should not be interpreted as requiring the production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be prepared by a variety of methods, including, but are not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of a human immunoglobulin locus, and such methods and other exemplary methods for producing monoclonal antibodies are described herein.

[0069] A "naked antibody" is an antibody that is not conjugated with a different part (e.g., a cytotoxic part) or a radioactive label. Naked antibodies may be present in pharmaceutical preparations.

[0070] "Natural antibodies" refer to immunoglobulin molecules with various structures that occur naturally. For example, a natural IgG antibody is a heterotetrameric glycoprotein with approximately 150,000 daltons, composed of two identical light chains and two identical heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy chain 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 chain domain or light chain variable domain, followed by a constant light chain (CL) domain. Based on the amino acid sequence of its constant domain, the light chains of an antibody may be assigned to one of two types, called kappa (κ) and lambda (λ).

[0071] The "natural-type Fc region" contains amino acid sequences identical to those of Fc regions found in nature. The natural-type human Fc region includes the natural-type human IgG1 Fc region (non-A and A allotypes); the natural-type human IgG2 Fc region; the natural-type human IgG3 Fc region; and the natural-type human IgG4 Fc region, as well as naturally occurring variants thereof.

[0072] A "mutant Fc region" includes an amino acid sequence that differs from that of the native sequence Fc region by at least one amino acid modification (alteration), preferably one or more amino acid substitutions. Preferably, the mutant Fc region has at least one amino acid substitution in the native sequence Fc region or the parent Fc region compared to the native sequence Fc region or the parent Fc region, for example, about 1 to about 30 amino acid substitutions, preferably about 1 to about 20 amino acid substitutions, more preferably about 1 to about 10 amino acid substitutions, and most preferably about 1 to about 5 amino acid substitutions. The mutant Fc regions described herein preferably have at least about 80% homology to the native sequence Fc region or the parent Fc region, preferably at least about 85% homology to them, more preferably at least about 90% homology to them, and most preferably at least about 95% homology to them.

[0073] "Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage ratio of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference polypeptide sequence, after the sequences have been aligned to obtain the greatest possible percentage sequence identity and gaps have been introduced where necessary, and no conservative substitutions are considered part of the sequence identity. Alignment for the purpose of determining percentage amino acid sequence identity can be achieved by using various methods within the scope of the art, such as publicly available computer software, including BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software, or GENETYX® (Genetics Co., Ltd.). A person skilled in the art can determine appropriate parameters for sequence alignment, including any algorithm necessary to achieve the greatest possible alignment over the entire length of the sequences being compared.

[0074] The ALIGN-2 sequence comparison computer program is copyrighted by Genentech, Inc., and its source code, along with user documentation, is filed with the U.S. Copyright Office (Washington DC, 20559) and registered under U.S. Copyright Registration Number TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, and may also 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 change. 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, or with, or relative to, a given amino acid sequence B (or, a given amino acid sequence A having or containing a certain % amino acid sequence identity to, or with, or relative to, 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 in the alignment of A and B by the sequence alignment program ALIGN-2, 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 is not equal to 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 preceding paragraph.

[0075] The terms "pharmaceutical preparation" and "pharmaceutical composition" refer to preparations that are interchangeable, in a form in which the biological activity of the active ingredients contained herein can exert its effect, and that do not contain additional elements that are toxic to an unacceptable degree to the subject to which they are administered.

[0076] The “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, 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 a human.

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

[0078] The “effective dose” of a drug (for example, a pharmaceutical formulation) refers to the amount in the required dosage and over the required period of time that is effective in achieving the desired therapeutic or prophylactic outcome.

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

[0080] As used herein, the term "CTLA-4" refers to any naturally occurring CTLA-4 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. This term encompasses both "full-length" CTLA-4 that has not undergone processing and any form of CTLA-4 resulting from intracellular processing. The term also encompasses naturally occurring CTLA-4 variants, such as splice variants and allele variants. The amino acid sequence of an exemplary human CTLA-4 is shown in SEQ ID NO: 214, the amino acid sequence of mouse CTLA-4 in SEQ ID NO: 247, the amino acid sequence of 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. In this specification, CTLA-4 may also be denoted as CTLA4.

[0081] The term "regulatory T (Treg) cells" refers to a subpopulation of T cells that modulate the immune system, maintain tolerance to autoantigens, and suppress autoimmune diseases. These cells generally suppress or downregulate the induction and proliferation of effector T cells. The most well understood Treg cells are those that express CD4, CD25, and Foxp3 (CD4 + CD25 + These are Treg cells. These Tregs are different from helper T cells. Several different methods are used to identify and monitor Treg cells. When defined by CD4 and CD25 expression (CD4 + CD25 +Treg cells are mature CD4 cells in mice and humans. + T cells constitute approximately 5-10% of the T cell subpopulation, while about 1-2% of Tregs can be measured in whole blood. Foxp3 expression may also be added to the measurement (CD4). + CD25 + Foxp3 + (Cells). Additionally, the absence or low level of CD127 expression may be used as another marker, in combination with the presence of CD4 and CD25. Furthermore, 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” mean that the similarity between two numerical values ​​(e.g., between the antibody of the present invention and the reference / comparative antibody) is sufficiently high that a person skilled in the art would consider the difference between the two numerical values ​​to be little or no biological and / or statistically significant in terms of the biological characteristics measured by the numerical value (e.g., the KD value).

[0083] As used herein, “treatment” (and its grammatical derivatives, e.g., “to treat,” “to treat,” etc.) means a clinical intervention intended to modify the natural course of the individual being treated, and may be carried out for preventive purposes or during the course of a clinical condition. Desired effects of treatment include, but are not limited to, prevention of disease onset or recurrence, reduction of symptoms, attenuation of any direct or indirect pathological effects of the disease, prevention of metastasis, reduction of the rate of disease progression, recovery or mitigation of the disease state, and remission or improved prognosis. In some embodiments, the antibodies of the present invention are used to delay the onset of disease or slow the progression of disease. In some embodiments, polypeptides comprising the mutated 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 precancerous and cancerous cells and tissues, regardless of malignancy or benignity. The terms "cancer", "cancerous", "cell proliferative disorder", "proliferative disorder", and "tumor" are not mutually exclusive as used herein.

[0085] The term "tumor tissue" means a tissue containing at least one tumor cell. Tumor tissue usually consists of a population of tumor cells (parenchyma) that forms the main body of the tumor and connective tissue and blood vessels (stroma) that exist between them and support the tumor. There are cases where the distinction between the two is clear, and there are also cases where the two are mixed. Immune cells and the like may infiltrate into the tumor tissue. On the other hand, "non-tumor tissue" means tissue other than tumor tissue in the living body. Healthy tissue / normal tissue without a disease state is a typical example of non-tumor tissue.

[0086] II. Compositions and Methods In one aspect, the present invention is partly based 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 an isolated antibody that binds to CTLA-4. In certain embodiments, the anti-CTLA-4 antibody of the present invention has CTLA-4 binding activity that depends on the concentration of the adenosine-containing compound. In some embodiments, the binding activity to CTLA-4 in the presence of the adenosine-containing compound is higher than that in the absence of the adenosine-containing compound. Alternatively, in another embodiment, the binding activity to CTLA-4 in the presence of a high concentration of the adenosine-containing compound is higher than that in the presence of a low concentration of the adenosine-containing compound. In a further embodiment, the difference in the 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 -fold or more, 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 x 10 5 It's more than double.

[0088] In some embodiments, the binding activity of an anti-CTLA-4 antibody can be expressed by its KD (Dissociation Constant) value. In a further embodiment, the KD value of an anti-CTLA-4 antibody is lower in the presence of an adenosine-containing compound compared to the absence of the adenosine-containing compound. Or, in another embodiment, the KD value of an anti-CTLA-4 antibody is lower in the presence of a high concentration of an adenosine-containing compound compared to the presence of a low concentration of the adenosine-containing compound. In a further embodiment, the difference in the KD value of an anti-CTLA-4 antibody can be, for example, 2 times or more, 3 times or more, 5 times or more, 10 times or more, 20 times or more, 30 times or more, 50 times or more, 100 times or more, 200 times or more, 300 times or more, 500 times or more, or 1 × 10⁻¹⁶ 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 x 10 5 It is more than double. The KD value of anti-CTLA-4 antibodies in the presence of adenosine-containing compounds, or in the presence of high concentrations of adenosine-containing compounds, is, for example, 9 × 10⁻⁶. -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 Less than M, 6×10 -8 Less than M, 5×10 -8 Less than M, 4×10 -8 Less than M, 3×10 -8 Less than M, 2×10 -8 Less than M, 1×10 -8 Less than M, 9×10 -9 Less than M, 8×10 -9 Less than M, 7×10 -9 Less than M, 6×10 -9 Less than M, 5×10 -9 Less than M, 4×10 -9 Less than M, 3×10 -9 Less than M, 2×10 -9 Less than M, 1×10 -9 Less than M, 9×10 -10 Less than M, 8×10 -10 Less than M, 7×10 -10 Less than M, 6×10 -10 Less than M, 5×10 -10 Less than M, 4×10 -10 Less than M, 3×10 -10 Less than M, 2×10 -10 Less than M, or 1×10 -10 Less than M can be. The KD value of the anti-CTLA-4 antibody in the absence of an adenosine-containing compound or in the presence of a low concentration of an adenosine-containing compound is, for example, 1×10 -8 Greater than or equal to M, 2×10 -8 Greater than or equal to M, 3×10 -8 Greater than or equal to M, 4×10 -8 Greater than or equal to M, 5×10 -8 Greater than or equal to M, 6×10 -8 Greater than or equal to M, 7×10 -8 Greater than or equal to M, 8×10 -8 Greater than or equal to M, 9×10 -8 Greater than or equal to M, 1×10 -7 Greater than or equal to M, 2×10 -7 Greater than or equal to M, 3×10 -7 Greater than or equal to M, 4×10 -7 Greater than or equal to M, 5×10 -7 Greater than or equal to M, 6×10 -7 Greater than or equal to M, 7×10 -7 Greater than or equal to M, 8×10 -7 Greater than or equal to M, 9×10 -7 Greater than or equal to M, 1×10 -6 Greater than or equal to M, 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 9×10 -6 It can be M or higher.

[0089] In another embodiment, the binding activity of an anti-CTLA-4 antibody may be expressed in terms of the kd (Dissociation rate constant) value instead of the 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 immobilized on a sensor chip and the amount of antigen bound to it are each measured as a resonance unit (RU). The amount of antigen bound and the amount of antibody bound can be divided to define 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 is greater in the presence of an adenosine-containing compound than in the absence of the adenosine-containing compound. Or, in another embodiment, the amount of CTLA-4 bound is greater 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 aspect, the difference in the amount of CTLA-4 bound can be, for example, 2 times or more, 3 times or more, 5 times or more, 10 times or more, 20 times or more, 30 times or more, 50 times or more, 100 times or more, 200 times or more, 300 times or more, 500 times or more, or 1 × 10⁻¹⁶ 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 x 10 5It is more than double. The value of the amount of CTLA-4 bound in the presence of an adenosine-containing compound, or in the presence of a high concentration of an adenosine-containing compound, can be, for example, 0.01 or higher, 0.02 or higher, 0.03 or higher, 0.04 or higher, 0.05 or higher, 0.06 or higher, 0.07 or higher, 0.08 or higher, 0.09 or higher, 0.1 or higher, 0.2 or higher, 0.3 or higher, 0.4 or higher, 0.5 or higher, 0.6 or higher, 0.7 or higher, 0.8 or higher, 0.9 or higher, or 1 or higher. The amount of CTLA-4 bound in the absence of an adenosine-containing compound, or in the presence of a low concentration of an adenosine-containing compound, can be, for example, 0.5 or less, 0.4 or less, 0.3 or less, 0.2 or less, 0.1 or less, 0.09 or less, 0.08 or less, 0.07 or less, 0.06 or less, 0.05 or less, 0.04 or less, 0.03 or less, 0.02 or less, 0.01 or less, 0.009 or less, 0.008 or less, 0.007 or less, 0.006 or less, 0.005 or less, 0.004 or less, 0.003 or less, 0.002 or less, or 0.001 or less.

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

[0092] The concentration of the adenosine-containing compound can be any concentration as long as a difference in the binding activity of the anti-CTLA-4 antibody is detected. In a particular embodiment, 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, 300 mM or higher, 100 mM or higher, 300 mM or higher, and 1 M or higher. Alternatively, a sufficient amount that shows the maximum binding activity of each anti-CTLA-4 antibody can be considered a high concentration. In one embodiment, high concentrations can be selected as 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 particular embodiment, low concentrations can include, for example, 1 mM or less, 300 μM or less, 100 μM or less, 30 μM or less, 10 μM or less, 3 μM or less, 1 μM or less, 300 nM or less, 100 nM or less, 30 nM or less, 10 nM or less, 3 nM or less, 1 nM or less, 300 pM or less, 100 pM or less, 30 pM or less, 10 pM or less, 3 pM or less, 1 pM or less, and so on. Alternatively, the low concentration used here could be defined as the concentration at which each anti-CTLA-4 antibody exhibits minimum binding activity.A case where the effective concentration is zero (in the absence of an adenosine-containing compound) can also be selected as one embodiment of a low concentration. In one embodiment, the low concentrations selected here can be the concentrations at which 1 mM, 100 μM, 10 μM, and 1 μM anti-CTLA-4 antibodies exhibit minimal binding activity, or the absence of an adenosine compound. In another embodiment, the ratio of high to low concentration can be, for example, 3 times or more, 10 times or more, 30 times or more, 100 times or more, 300 times or more, or 1 × 10⁻⁶. 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 You can choose a value that is double or greater than that.

[0093] In another embodiment, the anti-CTLA-4 antibody of the present invention also has binding activity to adenosine-containing compounds. Using the method described above, the amount of adenosine-containing compound bound per unit amount of antibody 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 value of the amount of adenosine-containing compound bound per unit antibody amount of the anti-CTLA-4 antibody of the present invention can be, for example, 0.0001 or more, 0.0002 or more, 0.0003 or more, 0.0004 or more, 0.0005 or more, 0.0006 or more, 0.0007 or more, 0.0008 or more, 0.0009 or more, 0.001 or more, 0.002 or more, 0.003 or more, 0.004 or more, 0.005 or more, 0.006 or more, 0.007 or more, 0.008 or more, 0.009 or more, or 0.01 or more.

[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 the adenosine-containing compound via heavy chains CDR1, CDR2, and CDR3. In one embodiment, the anti-CTLA-4 antibody has a binding motif to the adenosine-containing compound. The binding motif to the adenosine-containing compound may consist of at least one amino acid located at positions 33, 52, 52a, 53, 56, 58, 95, 96, 100a, 100b, and 100c, for example, as represented by Kabat numbering. In a further embodiment, the anti-CTLA-4 antibody binds to the 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, as represented by Kabat numbering. In certain embodiments, the anti-CTLA-4 antibody has at least one amino acid selected from the group consisting of Thr at position 33, Ser at position 52, Ser at position 52a, Arg at position 53, Tyr at position 56, Tyr at position 58, Tyr at position 95, Gly at position 96, Met at position 100a, Leu at position 100b, and Trp at position 100c, as represented by Kabat numbering. CTLA-4 may further bind to the complex formed by the 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, and may bind to both. The formation of a ternary complex with the anti-CTLA-4 antibody, the adenosine-containing compound, and CTLA-4 can be confirmed, for example, by methods such as crystal structure analysis described later (see Examples).

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

[0096] In another embodiment, the anti-CTLA-4 antibody of the present 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, the anti-CTLA-4 antibody of the present invention binds to the same epitope as ABAM004. In the presence of an excess of anti-CTLA-4 antibody, 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 competitive assays are provided herein.

[0097] In another embodiment, the anti-CTLA-4 antibody of the present invention exhibits cytotoxic activity against CTLA-4-expressing cells. When CTLA-4 is expressed on the surface of target cells and the anti-CTLA-4 antibody binds to them, those cells may be damaged. The damage to the cells may be caused by antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP), which are effector cells bound to the antibody, or by complement bound to the antibody, which are complement-dependent cytotoxicity (CDC), which are complement-bound cells. Alternatively, it may be caused by an antibody-bound cytotoxic agent (e.g., a radioisotope or chemotherapeutic agent), such as an immunoconjugate. The cytotoxicity here may include effects that induce cell death, inhibit cell proliferation, or impair cellular function. When sufficient amounts of anti-CTLA-4 antibody are present, it can cause damage to, for example, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of CTLA-4 expressing cells. Such cytotoxic activity can be measured by comparison with measurements in the absence of the antibody or in the presence of a negative control antibody. Exemplary cytotoxic 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 ligand, CD80(B7-1) or CD86(B7-2). In a particular embodiment, the anti-CTLA-4 antibody inhibits the interaction between CTLA-4 and CD80(B7-1) or CD86(B7-2). When the anti-CTLA-4 antibody is present in sufficient quantities, it can inhibit the interaction between CTLA-4 and CD80(B7-1) or CD86(B7-2) 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. Such inhibitory activity can be measured by comparing it with 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 antibody of the present invention binds to CTLA-4 derived from multiple animal species. Exemplary animal species include mammals such as humans, monkeys, mice, rats, hamsters, guinea pigs, rabbits, pigs, cattle, goats, horses, sheep, camels, dogs, and cats. In a particular embodiment, the anti-CTLA-4 antibody binds to CTLA-4 derived from humans and non-human species (e.g., monkeys, mice, rats, etc.). The amino acid sequence of human CTLA-4 is shown in SEQ ID NO: 214, the amino acid sequence of monkey CTLA-4 is shown in SEQ ID NO: 247, and the amino acid sequence of mouse CTLA-4 is shown in SEQ ID NO: 248. The amino acid sequences of CTLA-4 derived from other animal species can also be appropriately determined by methods known to those skilled in the art.

[0100] In certain embodiments, examples of adenosine-containing compounds in 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), and adenosine γ-thiotriphosphate (ATPγS).

[0101] In one aspect, the present 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: 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, the 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 present 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, the 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 present 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, the 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 present 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, the 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 present 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, the 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 present 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, the 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 antibodies 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 present 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, the 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 present 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, the 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 antibodies 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 present 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, the 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 present 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, the 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 present 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, the 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 a specific embodiment, one or more amino acids of the above-mentioned anti-CTLA-4 antibody are substituted at the following HVR positions: -HVR-H1 (Sequence ID: 223): Position 2 -In HVR-H2 (Sequence ID: 224): Positions 4, 5, 7, 13, and 16 -HVR-H3 (Sequence ID: 225): Position 3 -In HVR-L1 (Sequence ID: 226): Positions 1, 3, 6, 11, 12, and 14 -In HVR-L2 (Sequence ID: 227): Positions 1, 3, 4, and 7 -In HVR-L3 (Sequence ID: 228): Position 1, and 10

[0146] In certain embodiments, the substitutions provided herein are conservative substitutions. In certain embodiments, one or more of the following substitutions may be performed in any combination: -In HVR-H1 (SEQ ID NO: 100): H2A, R, or K -In HVR-H2 (Sequence ID: 101): S4T;R5Q;G7H;D13E or R;K16R -In HVR-H3 (Sequence ID: 102): K3A -In HVR-L1 (Sequence ID: 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 (Sequence ID: 115): S1Q;M10T

[0147] All possible substitution combinations described above are included in the consensus sequences of sequence numbers 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 embodiments described above, the anti-CTLA-4 antibody is humanized. In one embodiment, the anti-CTLA-4 antibody comprises HVR in any of the embodiments described above 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 HVR in any of the embodiments described above and further comprises VH or VL containing an FR sequence. In a further embodiment, the anti-CTLA-4 antibody comprises the following heavy chain and / or light chain variable domain FR sequences: for the heavy chain variable domain, FR1 comprises one amino acid sequence 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. Regarding the light chain variable domains, FR1 contains one amino acid sequence from sequence numbers 236 to 238, FR2 contains one amino acid sequence from sequence numbers 240 to 241, FR3 contains one amino acid sequence from sequence numbers 242 to 244, and FR4 contains one amino acid sequence from sequence numbers 245 to 246.

[0149] In another aspect, the anti-CTLA-4 antibody contains a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 10. In a particular embodiment, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect to the reference sequence includes substitutions (e.g., conservative substitutions), insertions, or deletions, but the anti-CTLA-4 antibody containing such sequence retains the ability to bind to CTLA-4. In a particular embodiment, 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, substitutions, insertions, or deletions occur in the region outside the HVR (i.e., within the FR). Optionally, the anti-CTLA-4 antibody includes the VH sequence in SEQ ID NO: 10, including those with post-translational modifications. In certain embodiments, the VH includes one, two, or three HVRs selected from (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 100, (b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 101, and (c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 102. Post-translational modifications include, but are not limited to, modifications to pyroglutamate by pyroglutamylation of glutamine or glutamic acid at the N-terminus of the heavy or light chain.

[0150] In another aspect, an anti-CTLA-4 antibody is provided comprising a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 11. In a particular embodiment, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect to the reference sequence includes substitutions (e.g., conservative substitutions), insertions, or deletions, but the anti-CTLA-4 antibody comprising such sequence retains the ability to bind to CTLA-4. In a particular embodiment, 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, substitutions, insertions, or deletions occur in the outer region of the HVR (i.e., within the FR). Optionally, the anti-CTLA-4 antibody includes the VL sequence in SEQ ID NO: 11, including those with post-translational modifications of said sequence. In certain embodiments, the VL includes one, two, or three HVRs selected from (a) HVR-L1 containing the amino acid sequence of SEQ ID NO: 113, (b) HVR-L2 containing the amino acid sequence of SEQ ID NO: 114, and (c) HVR-L3 containing the amino acid sequence of SEQ ID NO: 115. Post-translational modifications include, but are not limited to, modifications to pyroglutamate by pyroglutamylation of glutamine or glutamic acid at the N-terminus of the heavy or light chain.

[0151] In another aspect, an anti-CTLA-4 antibody is provided comprising a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 149. In a particular embodiment, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect to the reference sequence includes substitutions (e.g., conservative substitutions), insertions, or deletions, but the anti-CTLA-4 antibody comprising such sequence retains the ability to bind to CTLA-4. In a particular embodiment, 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, substitutions, insertions, or deletions occur in the outer region of the HVR (i.e., within the FR). Optionally, the anti-CTLA-4 antibody includes the VL sequence in SEQ ID NO: 149, including those with post-translational modifications of said sequence. In certain embodiments, the VL includes one, two, or three HVRs selected from (a) HVR-L1 containing the amino acid sequence of SEQ ID NO: 130, (b) HVR-L2 containing the amino acid sequence of SEQ ID NO: 117, and (c) HVR-L3 containing the amino acid sequence of SEQ ID NO: 133. Post-translational modifications include, but are not limited to, modifications to pyroglutamate by pyroglutamylation of glutamine or glutamic acid at the N-terminus of the heavy or light chain.

[0152] In another aspect, an anti-CTLA-4 antibody is provided, comprising VH in any of the above embodiments and VL in 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 those with post-translational modifications. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 98 and SEQ ID NO: 99, respectively, including those with post-translational modifications. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 83 and SEQ ID NO: 88, respectively, including those with post-translational modifications. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 83 and SEQ ID NO: 89, respectively, including those with post-translational modifications. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 83 and SEQ ID NO: 90, respectively, including those with post-translational modifications. In one embodiment, the antibody contains the VH and VL sequences in SEQ ID NO: 83 and SEQ ID NO: 91, respectively, including those with post-translational modifications. In one embodiment, the antibody contains the VH and VL sequences in SEQ ID NO: 83 and SEQ ID NO: 92, respectively, including those with post-translational modifications. In one embodiment, the antibody contains the VH and VL sequences in SEQ ID NO: 83 and SEQ ID NO: 93, respectively, including those with post-translational modifications. In one embodiment, the antibody contains the VH and VL sequences in SEQ ID NO: 83 and SEQ ID NO: 94, respectively, including those with post-translational modifications. In one embodiment, the antibody contains the VH and VL sequences in SEQ ID NO: 83 and SEQ ID NO: 97, respectively, including those with post-translational modifications. In one embodiment, the antibody contains the VH and VL sequences in SEQ ID NO: 83 and SEQ ID NO: 95, respectively, including those with post-translational modifications. In one embodiment, the antibody contains the VH and VL sequences in SEQ ID NO: 84 and SEQ ID NO: 97, respectively, including those with post-translational modifications. In another embodiment, the antibody contains the VH and VL sequences in SEQ ID NO: 85 and SEQ ID NO: 97, respectively, including those with post-translational modifications.In one embodiment, the antibody contains the VH and VL sequences in SEQ ID NO: 86 and SEQ ID NO: 97, respectively, including those with post-translational modifications. In one embodiment, the antibody contains the VH and VL sequences in SEQ ID NO: 86 and SEQ ID NO: 134, respectively, including those with post-translational modifications. In one embodiment, the antibody contains the VH and VL sequences in SEQ ID NO: 136 and SEQ ID NO: 97, respectively, including those with post-translational modifications. In one embodiment, the antibody contains the VH and VL sequences in SEQ ID NO: 135 and SEQ ID NO: 97, respectively, including those with post-translational modifications. In one embodiment, the antibody contains the VH and VL sequences in SEQ ID NO: 136 and SEQ ID NO: 95, respectively, including those with post-translational modifications. In one embodiment, the antibody contains the VH and VL sequences in SEQ ID NO: 137 and SEQ ID NO: 97, respectively, including those with post-translational modifications. In one embodiment, the antibody contains the VH and VL sequences in SEQ ID NO: 138 and SEQ ID NO: 97, respectively, including those with post-translational modifications. In one embodiment, the antibody contains the VH and VL sequences in SEQ ID NO: 138 and SEQ ID NO: 144, respectively, including those with post-translational modifications. In one embodiment, the antibody contains the VH and VL sequences in SEQ ID NO: 138 and SEQ ID NO: 145, respectively, including those with post-translational modifications. In one embodiment, the antibody contains the VH and VL sequences in SEQ ID NO: 138 and SEQ ID NO: 146, respectively, including those with post-translational modifications. In one embodiment, the antibody contains the VH and VL sequences in SEQ ID NO: 139 and SEQ ID NO: 146, respectively, including those with post-translational modifications. In one embodiment, the antibody contains the VH and VL sequences in SEQ ID NO: 140 and SEQ ID NO: 146, respectively, including those with post-translational modifications. In one embodiment, the antibody contains the VH and VL sequences in SEQ ID NO: 141 and SEQ ID NO: 146, respectively, including those with post-translational modifications. In one embodiment, the antibody contains the VH and VL sequences in SEQ ID NO: 140 and SEQ ID NO: 147, respectively, including those with post-translational modifications.In one embodiment, the antibody contains the VH and VL sequences in SEQ ID NO: 141 and SEQ ID NO: 147, respectively, including those with post-translational modifications. In one embodiment, the antibody contains the VH and VL sequences in SEQ ID NO: 140 and SEQ ID NO: 148, respectively, including those with post-translational modifications. In one embodiment, the antibody contains the VH and VL sequences in SEQ ID NO: 141 and SEQ ID NO: 148, respectively, including those with post-translational modifications. In one embodiment, the antibody contains the VH and VL sequences in SEQ ID NO: 136 and SEQ ID NO: 149, respectively, including those with post-translational modifications. In a further aspect, a heterogeneous anti-CTLA-4 antibody is provided that contains at least two different variable regions selected from the variable regions containing 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, respectively, and the VH and VL sequences in SEQ ID NO: 141 and SEQ ID NO: 146, including those with post-translational modifications. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 140 and SEQ ID NO: 147, respectively, and the VH and VL sequences in SEQ ID NO: 141 and SEQ ID NO: 147, respectively, including those with post-translational modifications. Post-translational modifications include, but are not limited to, modification to pyroglutamic acid by pyroglutamylation of glutamine or glutamic acid at the N-terminus of the heavy chain or light chain.

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

[0154] In a further aspect, the present invention provides antibodies that bind to the same epitopes as the anti-CTLA-4 antibodies provided herein. For example, in certain embodiments, antibodies that bind to the same epitopes as the antibodies described in Tables 7, 12, 17, and 22 are provided. In certain embodiments, antibodies that bind to epitopes in a fragment of CTLA-4 containing at least one amino acid selected from the group consisting of the 3rd amino acid (Met), 33rd amino acid (Glu), 35th amino acid (Arg), 53rd amino acid (Thr), 97th amino acid (Glu), 99th amino acid (Met), 100th amino acid (Tyr), 101st amino acid (Pro), 102nd amino acid (Pro), 103rd amino acid (Pro), 104th amino acid (Tyr), 105th amino acid (Tyr), and 106th amino acid (Leu) of SEQ ID NO: 28 are provided. In a particular embodiment, an antibody is provided that binds to an epitope in a CTLA-4 fragment consisting of amino acids 97 (Glu) to 106 (Leu) of SEQ ID NO: 28. In a particular embodiment, an antibody is provided that binds to an epitope in a CTLA-4 fragment consisting of amino acids 99 (Met) to 106 (Leu) of SEQ ID NO: 28.

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

[0156] In a further aspect, the anti-CTLA-4 antibody of the present invention includes an Fc region. In a further aspect, the anti-CTLA-4 antibody of the present invention includes a constant region. The constant region may be a heavy chain constant region (including the Fc region), a light chain constant region, or both. In some embodiments, the Fc region is the Fc region of the native sequence. Examples of heavy chain constant regions derived from native antibodies include, for example, human IgG1 (SEQ ID NO: 249), human IgG2 (SEQ ID NO: 250), human IgG3 (SEQ ID NO: 251), and human IgG4 (SEQ ID NO: 252). Other examples of heavy chain constant regions include, for example, SEQ ID NO: 82, SEQ ID NO: 158, and SEQ ID NO: 334. Examples of light chain constant regions derived from native antibodies include, for example, human κ chain (SEQ ID NO: 33, SEQ ID NO: 63, SEQ ID NO: 159) and human λ chain (SEQ ID NO: 53, SEQ ID NO: 87).

[0157] In another embodiment, the Fc region is a mutant Fc region created by modifying the amino acid sequence of the Fc region of the native sequence. In a particular embodiment, the mutant Fc region exhibits enhanced binding activity to at least one Fcγ receptor selected from the group consisting of FcγRIa, FcγRIIa, FcγRIIb, and FcγRIIIa compared to the Fc region of the native sequence. In a further embodiment, the mutant Fc region exhibits enhanced binding activity to FcγRIIa and FcγRIIIa compared to the Fc region of the native sequence. Examples of heavy chain constant regions containing such mutant Fc regions include, for example, 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] The Fc region of the native sequence is typically composed of a homodimer consisting of two identical polypeptide chains. In certain embodiments, the mutant Fc region may be a homodimer composed of polypeptide chains with the same sequence, or a heterodimer composed of polypeptide chains with different sequences. Similarly, the heavy chain constant region containing the Fc region may be a homodimer composed of polypeptide chains with the same sequence, or a heterodimer composed of polypeptide chains with different sequences. Examples of heterogeneous heavy chain constant regions include, for example, a heavy chain constant region containing the polypeptide chain of SEQ ID NO: 31 and the polypeptide chain of SEQ ID NO: 32, a heavy chain constant region containing the polypeptide chain of SEQ ID NO: 43 and the polypeptide chain of SEQ ID NO: 44, a heavy chain constant region containing the polypeptide chain of SEQ ID NO: 45 and the polypeptide chain of SEQ ID NO: 46, a heavy chain constant region containing the polypeptide chain of SEQ ID NO: 254 and the polypeptide chain of SEQ ID NO: 256, a heavy chain constant region containing the polypeptide chain of SEQ ID NO: 257 and the polypeptide chain of SEQ ID NO: 258, a heavy chain constant region containing the polypeptide chain of SEQ ID NO: 259 and the polypeptide chain of SEQ ID NO: 260, a heavy chain constant region containing the polypeptide chain of SEQ ID NO: 261 and the polypeptide chain of SEQ ID NO: 263, a heavy chain constant region containing the polypeptide chain of SEQ ID NO: 262 and the polypeptide chain of SEQ ID NO: 264, and the polypeptide chain of SEQ ID NO: 265 Heavy chain constant region including the heavy chain and the polypeptide chain of SEQ ID NO: 267, heavy chain constant region including the polypeptide chain of SEQ ID NO: 266 and the polypeptide chain of SEQ ID NO: 268, heavy chain constant region including the polypeptide chain of SEQ ID NO: 269 and the polypeptide chain of SEQ ID NO: 270, heavy chain constant region including the polypeptide chain of SEQ ID NO: 271 and the polypeptide chain of SEQ ID NO: 81, heavy chain constant region including the polypeptide chain of SEQ ID NO: 65 and the polypeptide chain of SEQ ID NO: 66, heavy chain constant region including the polypeptide chain of SEQ ID NO: 239 and the polypeptide chain of SEQ ID NO: 207, heavy chain constant region including the polypeptide chain of SEQ ID NO: 259 and the polypeptide chain of SEQ ID NO: 276, heavy chain constant region including the polypeptide chain of SEQ ID NO: 65 and the polypeptide chain of SEQ ID NO: 278, heavy chain constant region including the polypeptide chain of SEQ ID NO: 308 and the polypeptide chain of SEQ ID NO: 309,Heavy chain constant region containing polypeptide chains of SEQ ID NO: 311 and SEQ ID NO: 312, heavy chain constant region containing polypeptide chains of SEQ ID NO: 313 and SEQ ID NO: 314, heavy chain constant region containing polypeptide chains of SEQ ID NO: 315 and SEQ ID NO: 316, heavy chain constant region containing polypeptide chains of SEQ ID NO: 317 and SEQ ID NO: 318, heavy chain constant region containing polypeptide chains of SEQ ID NO: 319 and SEQ ID NO: 320, heavy chain constant region containing polypeptide chains of SEQ ID NO: 321 and SEQ ID NO: 322, heavy chain constant region containing polypeptide chains of SEQ ID NO: 323 and SEQ ID NO: 324, heavy chain constant region containing polypeptide chains of SEQ ID NO: 325 and SEQ ID NO: 326, SEQ ID NO: 3 Examples include heavy chain constant regions containing polypeptide chains 27 and 328; heavy chain constant regions containing polypeptide chains 330 and 331; heavy chain constant regions containing polypeptide chains 332 and 333; heavy chain constant regions containing polypeptide chains 358 and 359; heavy chain constant regions containing polypeptide chains 360 and 361; heavy chain constant regions containing polypeptide chains 362 and 363; heavy chain constant regions containing polypeptide chains 364 and 366; and heavy chain constant regions containing polypeptide chains 365 and 367.

[0159] In further contexts, anti-CTLA-4 antibodies in any of the above embodiments may, alone or in combination, incorporate any of the features described in items 1 to 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, for example, 10 -8 M~10 -13 M, for example 10 -9 M~10 -13 This is the dissociation constant (KD) of M).

[0161] In one embodiment, the binding activity of an antibody is measured by a radiolabeled antigen binding assay (RIA). In one embodiment, the RIA is performed using the Fab version of the antibody of interest and its antigen. For example, the solution binding affinity of Fab to an antigen is measured in the presence of a gradual increase in the concentration of the unlabeled antigen. 125 I) Fab is equilibrated with a labeled antigen, and then the bound antigen is captured by a plate coated with anti-Fab antibody (see, for example, Chen et al., J. Mol. Biol. 293: 865-881 (1999)). To establish the measurement conditions, a MICROTITER® multiwell plate (Thermo Scientific) is coated overnight with 5 μg / ml of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and then blocked with 2% (w / v) bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23°C). In a non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [ 125Mix the [I]-antigen with serial dilutions of the Fab of interest (e.g., as in the evaluation of anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57: 4593-4599 (1997)). Then incubate the Fab of interest overnight, although this incubation may be extended for a longer period (e.g., about 65 hours) to ensure equilibrium is achieved. Subsequently, transfer the mixture to a capture plate for incubation at room temperature (e.g., 1 hour). Then remove the solution and wash the plate eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. Once the plate is dry, add 150 μl / well of scintillant (MICROSCINT-20®, Packard) and count the plate for 10 minutes on a TOPCOUNT® gamma counter (Packard). Select concentrations of each Fab that give less than 20% of maximum binding for use in competitive binding assays.

[0162] In one embodiment, antibody binding activity is measured using a ligand capture method employing surface plasmon resonance spectroscopy, such as BIACORE® T200 or BIACORE® 4000 (GE Healthcare, Uppsala, Sweden). BIACORE® Control Software is used for instrument operation. In one embodiment, an amine coupling kit (GE Healthcare, Uppsala, Sweden) is used according to the supplier's instructions, and ligand capture molecules, such as anti-tag antibodies, anti-IgG antibodies, or protein A, are immobilized on a carboxymethyl dextran-coated sensor chip (GE Healthcare, Uppsala, Sweden). The ligand capture molecules are diluted with a 10 mM sodium acetate solution at an appropriate pH and injected at an appropriate flow rate and injection time. Binding activity is measured using a buffer containing 0.05% polysorbate 20 (also known as Tween(trademark)-20) as the measurement buffer, with a flow rate of 10-30 μL / min and a measurement temperature preferably of 25°C or 37°C. When measuring by capturing an antibody as a ligand with a ligand-capturing molecule, the antibody is injected to capture the desired amount, and then serial dilutions (analytes) of the antigen or Fc receptor prepared using the measurement buffer are injected. When measuring by capturing an antigen or Fc receptor as a ligand with a ligand-capturing molecule, the antigen or Fc receptor is injected to capture the desired amount, and then serial dilutions (analytes) of the antibody prepared using the measurement buffer are injected.

[0163] In one embodiment, the measurement results are analyzed using BIACORE® Evaluation Software. The kinetic parameters are calculated by simultaneously fitting binding and dissociation sensorgrams using a 1:1 Binding model, and the binding rate (kon or ka), dissociation rate (koff or kd), and equilibrium dissociation constant (KD) can be calculated. If the binding activity is weak, especially if dissociation is rapid and it is difficult to calculate the kinetic parameters, the equilibrium dissociation constant (KD) may be calculated using a Steady-state model. As another parameter of binding activity, the "analyte binding amount per unit amount of ligand" may be calculated by dividing the amount of analyte bound (RU) at a particular concentration by the amount of ligand captured (RU).

[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, for example, Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); in addition, see WO93 / 16185; and U.S. Patents 5,571,894 and 5,587,458. For a discussion on the Fab and F(ab')2 fragments containing salvage receptor-binding epitope residues and exhibiting extended in vivo half-lives, see U.S. Patent No. 5,869,046.

[0165] A diabody is an antibody fragment containing two antigen-binding sites, which may be bivalent or bispecific. See, for example, EP404,097; WO1993 / 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 containing all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (see, for example, Domantis, Inc., Waltham, MA; U.S. Patent No. 6,248,516B1).

[0167] Antibody fragments can be produced by various methods, including, but are not limited to, the proteolytic digestion of complete antibodies and production by recombinant host cells (e.g., Escherichia coli or phages) as described herein.

[0168] 3. Chimeric and humanized antibodies In certain embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and in Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984). In one example, a chimeric antibody includes 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 further examples, a chimeric antibody is a “class-switched” antibody in which the class or subclass of the parent antibody has been changed. A chimeric antibody also includes its antigen-binding fragment.

[0169] In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce its immunogenicity to 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 HVR (e.g., CDR (or a portion thereof)) is derived from the non-human antibody and the FR (or a portion thereof) is derived from the human antibody sequence. The humanized antibody optionally includes at least a portion of the human constant region. In some embodiments, several FR residues in the humanized antibody are replaced with corresponding residues from the non-human antibody (e.g., the antibody from which the HVR residues originated) to restore or improve the specificity or affinity of the antibody, for example.

[0170] Humanized antibodies and their production methods have been reviewed in Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008), and also in, for example, Riechmann et al., Nature 332: 323-329 (1988); Queen et al., Proc. Natl. Acad. Sci. USA 86: 10029-10033 (1989); U.S. Patents No. 5,821,337, No. 7,527,791, No. 6,982,321, and No. 7,087,409; Kashmiri et al., Methods 36: 25-34 (2005) (describes specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28: 489-498 (1991). (Resurfacing is described); Dall'Acqua et al., Methods 36: 43-60 (2005) (FR shuffling is described); and further described in Osbourn et al., Methods 36: 61-68 (2005) and Klimka et al., Br. J. Cancer, 83: 252-260 (2000) ("Guided Selection" approach for FR shuffling is described).

[0171] The human framework regions that can be used for humanization are not limited to these, but include: 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 chain 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 maturation (somatic mutation) 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 (Baca et al., J. Biol. Chem. 272: 10678-10684). (See also 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 methods known in the art. Human antibodies are outlined 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 immunogens to transgenic animals modified to produce fully human antibodies or fully human antibodies with human variable regions in response to antigen challenge (loading). Such animals typically contain all or part of the human immunoglobulin locus, which either replaces endogenous immunoglobulin loci or is randomly incorporated extrachromosomally or within the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin locus is 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 describing XENOMOUSE® technology; U.S. Patent No. 5,770,429 describing HUMAB® technology; U.S. Patent No. 7,041,870 describing KM MOUSE® technology; and U.S. Patent Application Publication 2007 / 0061900 describing VELOCIMOUSE® technology. Human variable regions from complete antibodies produced by such animals may be further modified, for example, by combining them 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 already been described (see, for example, 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, for example, those described in U.S. Patent No. 7,189,826 (describes the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4): 265-268 (2006) (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 selected Fv clone variable domain sequences from a human-derived phage display library. Such variable domain sequences can then be combined with a desired human constant domain. A method for selecting human antibodies from an antibody library is described below.

[0176] 5. Library-derived antibodies The antibodies of the present invention may be isolated by screening a combinatorial library for antibodies exhibiting one or more desired activities. For example, various methods are known in the art for generating phage display libraries and for screening such libraries for antibodies possessing desired binding properties. Such methods have been 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, 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. This is described in 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 certain phage display methods, the VH and VL gene repertoires are cloned separately by polymerase chain reaction (PCR), randomly recombined in a phage library, and screened for antigen-binding phages as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). The phages typically present antibody fragments either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies against the immunosource without requiring the construction of hybridomas. Alternatively, as described in Griffiths et al., EMBO J, 12: 725-734 (1993), naive repertoires (e.g., from humans) can be cloned to provide a single source of antibodies against a wide range of non-self and self-antigens without immunization. Finally, naive libraries can also be synthesized synthetically, as described in Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992), by cloning the pre-reorganization V-gene segment from stem cells and using PCR primers containing random sequences that encode the hypervariable CDR3 region and achieve in vitro rearrangement. Patent documents describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373, as well as U.S. Patent Application Publications 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 human antibodies or human antibody fragments in this specification.

[0179] 6. Multispecific antibodies In certain embodiments, the antibodies provided herein are multispecific antibodies (e.g., bispecific antibodies). A multispecific antibody is a monoclonal antibody having binding specificity to at least two different sites. In certain embodiments, one of the binding specificities is to CTLA-4 and the other is to any other antigen. In certain embodiments, the bispecific antibody may bind to two different epitopes of CTLA-4. The bispecific antibody may be used to localize a cytotoxic agent to cells expressing CTLA-4. The bispecific antibody may be prepared as a full-length antibody or as an antibody fragment.

[0180] Methods for producing multispecific antibodies are not limited to these, but include, the recombinant co-expression of two immunoglobulin heavy-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 techniques (see, for example, U.S. Patent No. 5,731,168). Multispecific antibodies can be produced by manipulating electrostatic steering effects to create Fc heterodimer molecules (WO2009 / 089004A1); crosslinking two or more antibodies or fragments (see U.S. Patent No. 4,676,980 and Brennan et al., Science, 229: 81 (1985)); creating antibodies with two specificities using a leucine zipper (see Kostelny et al., J. Immunol., 148(5): 1547-1553 (1992)); producing bispecific antibody fragments using "diabody" technology (see Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993)); and using single-chain Fv (scFv) dimers (see Gruber et al., J. Immunol., 152: See 5368 (1994); and may also be prepared by preparing a trispecific antibody as described, for example, in Tutt et al. J. Immunol. 147: 60 (1991).

[0181] Modified antibodies containing three or more functional antigen-binding sites, including "octopus antibodies," are also included herein (see, for example, U.S. Patent Application Publication 2006 / 0025576A1).

[0182] In this specification, an antibody or fragment also includes a “dual-acting Fab” or “DAF” comprising one antigen-binding site that binds to CTLA-4 and another different antigen (see, for example, U.S. Patent Application Publication No. 2008 / 0069820).

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

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

[0185] [Table 1]

[0186] Amino acids can be grouped according to their common side-chain characteristics: (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 substitution refers to replacing one member of one class with one of another.

[0187] One type of substitution mutant involves the substitution of one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Typically, the resulting mutants, and those selected for further study, will have modifications (e.g., improvements) in specific biological properties (e.g., increased affinity, decreased immunogenicity) compared to the parent antibody, and / or will substantially retain certain biological properties of the parent antibody. An exemplary substitution mutant is an affinity-matured antibody, which can be appropriately produced using, for example, a phage display-based affinity-mature technique (e.g., one described herein). Briefly, one or more HVR residues are mutated, and the mutant antibody is displayed on a phage and screened for specific biological activity (e.g., binding affinity).

[0188] Modifications (e.g., substitutions) may be made in HVRs, for example, to improve antibody affinity. Such modifications may be made in HVR "hot spots," i.e., residues encoded by codons that frequently mutate during the somatic cell maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)) and / or residues that come into contact with the antigen, and the resulting mutant VH or VL may 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 variable genes selected for maturation by any variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). Next, a secondary library is prepared. This library is then screened to identify any antibody variant with the desired affinity. Another method for introducing diversity involves an HVR-directed approach that randomizes several HVR residues (e.g., 4-6 residues at a time). 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, provided that such modifications do not substantially reduce the antibody's ability to bind to the antigen. For example, conservative modifications that do not substantially reduce binding affinity (e.g., conservative substitutions as provided herein) may be made within an HVR. Such modifications may be, for example, outside the antigen-contact residue of the HVR. In certain embodiments of the mutant VH and VL sequences described above, each HVR is either unmodified 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 mutation introduction is called "alanine scanning mutagenesis," described by Cunningham and Wells (1989) Science, 244: 1081-1085. In this method, one or a group of target residues (e.g., charged residues, e.g., arginine, aspartic acid, histidine, lysine, and glutamic acid) are identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the antibody-antigen interaction is affected. Further substitutions may be introduced at amino acid positions that show functional sensitivity to this initial substitution. Alternatively, the crystal structure of the antigen-antibody complex may be analyzed to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted as substitution candidates or excluded from the list of substitution candidates. Mutants may be screened to determine whether they possess the desired properties.

[0191] Amino acid sequence insertions include not only the insertion of single or multiple amino acid residues within a sequence, but also the fusion of polypeptides ranging in length from one to over 100 residues at the amino and / or carboxyl terminals. An example of terminal insertion is an antibody with a methionyl residue at the N-terminus. Other insertion variants of antibody molecules include those in which an enzyme (e.g., for ADEPT) or a polypeptide that increases the plasma half-life of the antibody is fused to the N- or C-terminus of the antibody.

[0192] b) Glycosylated mutants In certain embodiments, the antibodies provided herein are modified to increase or decrease the degree to which the antibody is glycosylated. Adding or removing glycosylation sites to an antibody can be easily achieved by modifying the amino acid sequence to create or remove one or more glycosylation sites.

[0193] If the antibody contains an Fc region, the carbohydrate to which it is attached may be modified. Native antibodies produced by mammalian cells typically contain branched oligosaccharides, which are usually attached to Asn297 of the CH2 domain of the Fc region by N-linkage. See, for example, 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 branched oligosaccharide structure. In some embodiments, the modification of the oligosaccharide in the antibody of the present invention may be carried out to produce antibody variants with specific improved properties.

[0194] In one embodiment, antibody variants are provided having a carbohydrate structure lacking fucose (directly or indirectly) attached to the Fc region. For example, the amount of fucose in such an antibody may be 1%–80%, 1%–65%, 5%–65%, or 20%–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 sugar structures (e.g., complex, hybrid, and high-mannose structures) attached to Asn297, measured by MALDI-TOF mass spectrometry, as described, for example, in WO2008 / 077546. Asn297 represents an asparagine residue located around position 297 of the Fc region (EU numbering of Fc region residues). However, due to slight sequence variability among multiple antibodies, Asn297 may also be located ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylated variants may have improved ADCC function. See, for example, U.S. Patent Application Publication No. 2003 / 0157108 (Presta, L.) and No. 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications concerning "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 lacking protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249: 533-545 (1986); U.S. Patent Application Publication US2003 / 0157108A1, Presta, L; and WO2004 / 056312A1, Adams et al., particularly Example 11) and knockout cell lines, such as alpha-1,6-fucosyltransferase gene FUT8 knockout CHO cells (see, for example, 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 antibody variants are provided having a bifid oligosaccharide, for example, in which a bifid branched oligosaccharide attached to the Fc region of the antibody is bifid 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 U.S.2005 / 0123546 (Umana et al.). Antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO1997 / 30087 (Patel et al.); WO1998 / 58964 (Raju, S.); and WO1999 / 22764 (Raju, S.).

[0196] c) Fc region variant In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of the antibodies provided herein to generate Fc region variants. The Fc region variants may include a human Fc region sequence (e.g., the Fc region of human IgG1, IgG2, IgG3, or IgG4) that includes amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0197] In certain embodiments, antibody variants possessing some, but not all, effector functions are also within consideration of the present invention, such effector functions making the antibody a desirable candidate for application when its in vivo half-life is important, but certain effector functions (such as complement and ADCC) are unnecessary or harmful. In vitro and / or in vivo cytotoxicity measurements can be performed to confirm reduced / deficient CDC and / or ADCC activity. For example, Fc receptor (FcR) binding measurements may be performed to confirm that the antibody lacks FcγR binding (and therefore is likely to lack ADCC activity) while maintaining FcRn binding ability. NK cells, the primary cells that mediate ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-492 (1991). Non-limiting examples of in vitro assays for evaluating the ADCC activity of the target molecule are described in U.S. Patent 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); and U.S. Patent No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166: 1351-1361 (1987)). Alternatively, non-radioactive measurement methods may be used (see, for example, ACT1® non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc., Mountain View, CA); and CytoTox 96® non-radioactive cytotoxicity assays (Promega, Madison, WI)).Effector cells useful for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, the ADCC activity of the molecule of interest may be evaluated in vivo in animal models, such as those 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 cannot bind to C1q and therefore lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402. Furthermore, CDC measurements may be performed to evaluate complement activation (see, for example, 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)). In addition, FcRn binding and in vivo clearance / half-life can be determined using methods known in the art (see, for example, 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 the so-called "DANA" Fc variant with alanine substitutions at residues 265 and 297 (U.S. Patent No. 7,332,581), and Fc variants with two or more substitutions at amino acid positions 265, 269, 270, 297, and 327.

[0199] Certain antibody variants with increased or decreased binding affinity to FcRs have been described (see U.S. Patent 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 includes an Fc region with one or more amino acid substitutions that improve ADCC (e.g., substitutions at positions 298, 333, and / or 334 (residue in 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 cell injury (CDC), as described, for example, in U.S. Patent No. 6,194,551, WO99 / 51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

[0202] Antibodies with increased half-life and increased binding affinity to the neonatal Fc receptor (FcRn: which plays a role in transferring 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 / 0014934A1 (Hinton et al.). These antibodies contain an Fc region with one or more substitutions therein that increase the binding affinity of the Fc region to FcRn. Such Fc variants include those involving substitutions at one or more of the Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434 (for example, substitution of Fc region residue 434 (U.S. 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 WO94 / 29351.

[0204] d) Cysteine-modified antibody variants In certain embodiments, it would be desirable to produce cysteine-modified antibodies (e.g., "thioMAbs") in which one or more residues of the antibody are substituted with cysteine ​​residues. In certain embodiments, the residues to be substituted occur in accessible sites of the antibody. By substituting these residues with cysteine, a reactive thiol group is located in an accessible site of the antibody, and this reactive thiol group may be used to conjugate the antibody to other parts (such as a drug part or a linker-drug part) to create an immunoconjugate as further detailed herein. In certain embodiments, any one or more of the following residues may be substituted with 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-modified antibodies may be produced, for example, as described in U.S. Patent No. 7,521,541.

[0205] e) Antibody derivative In certain embodiments, the antibodies provided herein may be further modified to include additional non-protein moieties known and readily available in the art. Suitable moieties for antibody derivatization 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 homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde would be advantageous in production due to its stability in water. The polymers may have any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and if one or more polymers are attached, they may be the same molecule or different molecules. Generally, the number and / or type of polymers used in derivatization can be determined based on considerations such as the specific properties or functions of the antibody to be improved, and whether the antibody derivative will be used for therapy under specified conditions, although these are not limited to these.

[0206] In another embodiment, a conjugate is provided of an antibody and a non-protein moiety that can be selectively heated by exposure to radiation. In one embodiment, the non-protein moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation may be of any wavelength, but is not limited thereto, and includes wavelengths that heat the non-protein moiety to a temperature that does not harm normal cells but kills cells adjacent to the antibody-non-protein moiety.

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

[0208] As an example of a non-limiting technique that can be combined with the anti-CTLA-4 antibody described herein, the creation of a T-cell redirecting antibody using the anti-CTLA-4 antibody is exemplified (Nature (1985) 314 (6012), 628-31, Int J Cancer (1988) 41 (4), 609-15, Proc Natl Acad Sci USA (1986) 83 (5), 1453-7). One non-limiting embodiment of the T-cell redirecting antibody is a bispecific antibody containing a binding domain to any of the constituent subunits of the T-cell receptor (TCR) complex on T cells, particularly a binding domain to the CD3 epsilon chain within CD3, and the antigen-binding domain of the anti-CTLA-4 antibody.

[0209] B. Method and configuration of rearrangement For example, as described in U.S. Patent No. 4,816,567, antibodies can be manufactured using recombinant methods or compositions. In one embodiment, an isolated nucleic acid encoding the anti-CTLA-4 antibody described herein is provided. Such nucleic acid may encode an amino acid sequence containing VL and / or VH of the antibody (e.g., the light chain and / or heavy chain of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) containing such nucleic acid are provided. In a further embodiment, a host cell containing such nucleic acid is provided. In one such embodiment, the host cell comprises (1) a vector containing nucleic acid encoding an amino acid sequence containing VL of the antibody and an amino acid sequence containing VH of the antibody, or (2) a first vector containing nucleic acid encoding an amino acid sequence containing VL of the antibody and a second vector containing nucleic acid encoding an amino acid sequence containing VH of the antibody (e.g., transformed). In one embodiment, the host cell is eukaryotic (e.g., Chinese hamster ovary (CHO) cells) or lymphoid cells (e.g., Y0, NS0, Sp2 / 0 cells). In one embodiment, a method for producing an anti-CTLA-4 antibody is provided, comprising culturing host cells containing the nucleic acid encoding the antibody as described above under conditions suitable for the expression of the anti-CTLA-4 antibody, and optionally recovering the antibody from the host cells (or host cell culture medium).

[0210] For the recombinant production of anti-CTLA-4 antibodies, nucleic acids encoding the antibody (e.g., those described above) are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids will be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the antibody).

[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, especially when glycosylation and Fc effector function are not required. For the expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patents 5,648,237, 5,789,199, and 5,840,523 (and also see Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, for the expression of antibody fragments in Escherichia coli). After expression, antibodies may be isolated from bacterial cell paste into soluble fractions and further purified.

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

[0213] Cells derived from multicellular organisms (invertebrates and vertebrates) are also suitable host cells for the expression of 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 the transformation of Spodoptera frugiperda cells.

[0214] Plant cell cultures can also be used as hosts. See, for example, U.S. Patents 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLANTIBODIES® technology for antibody production in transgenic plants).

[0215] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in a suspension state would be useful. Other examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 cell line (COS-7); human embryonic kidney cell line (293 or 293 cells as described in Graham et al., J. Gen Virol. 36: 59 (1977), etc.); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells as described in Mather, Biol. Reprod. 23: 243-251 (1980), etc.); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); Buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary cancer cells (MMT 060562); and TRI cells (e.g., Mather et al., Annals NY Acad. Sci. 383: 44-68 (1982)). These include MRC5 cells and FS4 cells, as described in [reference]. 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, for example, 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 produced in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and adjuvant. The relevant antigen is converted to an immunogenic protein in the immunized species, for example, keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soy trypsin inhibitor, and a bifunctional substance or derivatizer, for example, maleimide benzoyl sulfosuccinimide (conjugation via cysteine ​​residue), N-hydroxysuccinimide (conjugation via lysine residue), glutaraldehyde, succinic anhydride, SOCl2, or R 1 N=C=NR(where R and R 1 It may be useful to conjugate them using different alkyl groups.

[0217] Animals (usually non-human mammals) are immunized to an antigen, immunogenic conjugate, or derivative by intradermal injection at multiple sites of a solution of 100 μg or 5 μg of protein or conjugate (for rabbits or mice, respectively) combined with three times the volume of Freund's complete adjuvant. After one month, the animals are boost-immunized with 1 / 5 to 1 / 10 of the initial amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. After 7 to 14 days, blood is collected from the animals and the serum is assayed for antibody titer. The animals are boost-immunized until the titer reaches a plateau. Preferably, the animals are boost-immunized with conjugates that are the same antigen but conjugated to a different protein and / or conjugated via a different crosslinking reagent. The conjugates can also be prepared as protein fusions in recombinant cell cultures. Coagulants such as alum are also preferably used to enhance the immune response.

[0218] Monoclonal antibodies are obtained from a substantially homogeneous population of antibodies; that is, the individual antibodies constituting the population are identical except for some naturally occurring potential mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in small amounts. Thus, the modifier "monoclonal" indicates a characteristic of the antibody that it is not a mixture of distinct 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, mice or other suitable host animals, such as hamsters, are immunized as described herein to induce lymphocytes capable of producing antibodies that specifically bind to the proteins used for immunization. Alternatively, lymphocytes can be immunized in vitro.

[0220] Immunotherapeutic agents typically include antigen proteins or their fusion variants. Generally, peripheral blood lymphocytes (PBLs) are used when human-derived cells are desired, and spleen cells or lymph node cells are used when non-human mammalian sources are desired. The lymphocytes are then fused with immortalized cell lines using appropriate fusion agents such as polyethylene glycol to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986), pp. 59-103).

[0221] Immortalized cell lines are typically transformed mammalian cells, particularly myeloma cells of rodent, bovine, and human origin. Rat or mouse myeloma cell lines are usually used. The hybridoma cells thus produced are seeded and grown in a suitable culture medium preferably containing one or more substances that inhibit the proliferation or survival of unfused parent myeloma cells. For example, if the parent myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomas would typically contain hypoxanthine, aminopterin, and thymidine (HAT medium), which are substances that inhibit the proliferation of HGPRT-deficient cells.

[0222] Preferred immortalized myeloma cells are those that efficiently fuse, facilitate stable and high-level antibody production by selected antibody-producing cells, and are sensitive to culture media such as HAT medium. Among these, mouse myeloma strains, 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, e.g., X63-Ag8-653) available from the American Type Culture Collection in Manassas, Virginia, USA, are preferred. For the production of human monoclonal antibodies, human myeloma cell lines and mouse-human heteromyeloma cell lines have also been described (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 hybridoma cells are proliferating is assayed for the production of monoclonal antibodies against the antigen. Preferably, the binding specificity of the monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art. For example, binding affinity can be determined by Scatchard analysis as described in Munson, Anal Biochem. 107(1): 220-239 (1980).

[0224] After hybridoma cells producing antibodies with the desired specificity, affinity, and / or activity are identified, these clones can be subcloned by limiting dilution and grown using standard methods (Goding, cited above). 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 within mammals.

[0225] Monoclonal antibodies secreted by subclones can be appropriately isolated from culture medium, ascites fluid, or serum by conventional immunoglobulin purification methods such as protein A-Sepharose chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0226] Antibodies may be produced by immunizing a suitable host animal with an antigen. In one embodiment, the antigen is a polypeptide containing full-length CTLA-4. In one embodiment, the antigen is a polypeptide containing soluble CTLA-4. In one embodiment, the antigen is a polypeptide containing a region corresponding to amino acids 97 (Glu) to 106 (Leu) of human CTLA-4 (extracellular domain, SEQ ID NO: 28). In one embodiment, the antigen is a polypeptide containing a region corresponding to amino acids 99 (Met) to 106 (Leu) of human CTLA-4 (extracellular domain, SEQ ID NO: 28). The present invention also encompasses antibodies produced by immunizing an animal with an antigen. Antibodies may incorporate any of the features described above for exemplary anti-CTLA-4 antibodies, either alone or in combination.

[0227] C. Measurement Method (Assay) The anti-CTLA-4 antibodies provided herein may be identified, screened, or have their physical / chemical properties and / or biological activity elucidated by various assay methods known in the art.

[0228] 1. Combined measurement methods and other measurement methods In one aspect, the antibodies of the present invention are tested for their antigen-binding activity by known methods such as ELISA, Western blotting, and surface plasmon resonance assays.

[0229] In another context, a competitive assay may 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, if such competing antibodies are present in excess, the binding of the reference antibody to CTLA-4 is blocked (e.g., reduced) 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 epitopes (e.g., linear or structural epitopes) that are bound by the anti-CTLA-4 antibodies described herein (e.g., the anti-CTLA-4 antibodies described in Tables 7, 12, 17, and 22). Detailed exemplary methods for mapping the epitopes to which antibodies bind are provided in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ).

[0230] In an exemplary competition assay, immobilized CTLA-4 is incubated in a solution containing a first labeled antibody that binds to CTLA-4, and a second unlabeled antibody to be tested for its ability to compete with the first antibody for binding to CTLA-4. The second antibody may be present in the hybridoma supernatant. As a control, immobilized CTLA-4 is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow the first antibody to bind to CTLA-4, any excess unbound antibody is removed, and the amount of label bound to the immobilized CTLA-4 is measured. If the amount of label bound to the immobilized CTLA-4 is substantially reduced in the test sample compared to the control sample, it indicates that the second antibody is competing with the first antibody for binding to CTLA-4. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

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

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

[0233] In certain embodiments, the antibodies of the present invention are tested for their ability to inhibit cell growth or proliferation in vitro. Methods for measuring inhibition of cell growth or proliferation are well known in the art. Certain cell proliferation assays, exemplified by the “cytotoxic” assays 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 a culture based on the abundance 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. The assay may be performed in a 96 or 384-well format to accommodate automated high-throughput screening (HTS). See Cree et al (1995) AntiCancer Drugs 6: 398-404. The assay procedure involves directly adding a single reagent (CellTiter-Glo® reagent) to cultured cells. This lyses the cells, and a luciferase reaction generates a luminescence signal. The luminescence signal is proportional to the amount of ATP present, which is directly proportional to the number of viable cells in the culture. Data can be recorded using a luminometer or a CCD camera imaging device. The luminescence value is 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-dimethylthiazole-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 the cell culture. See, for example, Mosmann (1983) J. Immunol. Meth. 65: 55-63 and Zhang et al. (2005) Cancer Res. 65: 3877-3882.

[0235] The cells used in any of the in vitro assays described above include cells or cell lines that naturally express CTLA-4 or have been engineered to express CTLA-4. Such cells also include cell lines that express CTLA-4 and cell lines that do not normally express CTLA-4 but have been transfected with nucleic acids that encode CTLA-4.

[0236] In one aspect, anti-CTLA-4 antibodies are tested for their ability to inhibit cell growth or proliferation in vivo. In a specific embodiment, anti-CTLA-4 antibodies are tested for their ability to inhibit tumor growth in vivo. In vivo model systems, such as xenograft models, can be used for such tests. In an exemplary xenograft system, human tumor cells are introduced into a appropriately immunodeficient non-human animal, e.g., an athymoid "nude" mouse. The antibody of the present invention is administered to this animal. The ability of the antibody to inhibit or reduce tumor growth is measured. In a specific embodiment 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 a specific embodiment, human tumor cells are introduced into an appropriately immunodeficient non-human animal by subcutaneous injection or by transplantation into an appropriate site, such as a mammary gland fat pad.

[0237] It is understood that any of the above measurement methods may be performed using the immunoconjugate of the present invention in place of or in addition to the anti-CTLA-4 antibody.

[0238] A typical assay for measuring the ADCC activity of therapeutic antibodies is: 51 This is based on a Cr release assay and includes the following steps: target cells [ 51 The steps are: labeling with Cr]Na2CrO4; opsonizing target cells expressing the antigen on their cell surface with an antibody; combining opsonized radiolabeled target cells and effector cells in a suitable ratio in a microtiter plate, with or without 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. Subsequently, the cytotoxicity of the test antibody is determined, for example, by the following formula: relative cytotoxicity (%) = (radioactivity in the presence of antibody - radioactivity in the absence of antibody) / (maximum radioactivity - radioactivity in the absence of antibody) × 100. Graphs can be created by changing the target cell:effector cell ratio or antibody concentration.

[0239] To evaluate 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). In short, various concentrations of polypeptide variants and human complement are diluted in buffer. Cells expressing the antigen to which the polypeptide variant binds are then subjected to a assay of approximately 1 × 10⁻¹⁶ cells. 6Dilute to a cell / ml density. Add the polypeptide variant, diluted human complement, and antigen-expressing cells mixture to a 96-well flat-bottom tissue culture plate and incubate at 37°C and 5% CO2 for 2 hours to promote complement-mediated cell lysis. Then, add 50 μl of Alamer Blue (Accumed International) to each well and incubate overnight at 37°C. Measure absorbance 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 standard curves, and the percentage of activity compared to the non-mutant polypeptide is reported for the polypeptide variant of interest.

[0240] An exemplary assay method for ADCP activity may include: coating target biological particles such as E. coli-labeled FITC (Molecular Probes) or Staphylococcus aureus-FITC with a test antibody; forming opsonized particles; adding the opsonized particles to THP-1 effector cells (monocyte cell line, available from ATCC) in a ratio of 1:1, 10:1, 30:1, 60:1, 75:1, or 100:1 to induce FcγR-mediated phagocytosis; preferably incubating the cells and E. coli-FITC / antibody at 37°C for 1.5 hours; after incubation, adding trypan blue to the cells (preferably at room temperature for 2-3 minutes) to eliminate the fluorescence of bacteria that have adhered to the outside of the cell surface without being taken up; transferring the cells to FACS buffer (e.g., 0.1% BSA and 0.1% sodium azide in PBS) and assaying the fluorescence of THP-1 cells using FACS (e.g., BD FACS Calibur). To assay the degree of ADCP, the 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 (control), E. coli-FITC and THP-1 cells (used as FcγR-independent ADCP activity), E. coli-FITC, THP-1 cells and test antibody (used as FcγR-dependent ADCP activity) in culture medium.

[0241] Antibody-mediated cytotoxic activity typically involves the binding of antibodies to the cell surface. Whether or not an antigen is expressed on the surface of target cells can be appropriately confirmed by methods 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 levels of cytokines (e.g., IL-6, G-CSF, IL-12, TNFα, and IFNγ) or their receptors, enhanced proliferation of immune cells (e.g., B cells, T cells, NK cells, macrophages, monocytes, etc.), increased activation, enhanced function, or increased cytotoxic activity. In particular, T cell activation can be detected by measuring increased expression of activation markers such as CD25, CD69, and ICOS. For example, in patients administered the anti-CTLA-4 antibody ipilimumab, ICOS levels in peripheral blood increased after administration. + CD4 + An increase in T cells is known, which is thought to be due to the activation of the systemic immune state by administration of anti-CTLA-4 antibodies (Cancer Immunol. Res. (2013) 1(4): 229-234).

[0243] T cell activation requires not only stimulation via antigen receptors (TCRs) but also co-stimulation via CD28. When CD28 on the surface of T cells binds to B7-1 (CD80) or B7-2 (CD86) present on the surface of antigen-presenting cells, a co-signal is transmitted into the T cell, activating it. On the other hand, CTLA-4 is expressed on the surface of activated T cells. Because CTLA-4 binds to CD80 and CD86 with stronger affinity than CD28, it preferentially interacts with CD80 and CD86 over CD28, and consequently suppresses T cell activation.

[0244] Based on this mechanism of action, inhibitory activity against CTLA-4 can be measured as activity that inhibits the binding of CTLA-4 to CD80 or CD86. In one embodiment, an assay for measuring inhibitory activity against CTLA-4 includes 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 or not the 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 that detects T cell activation as described above. For example, in a system that measures T cell activation by stimulating a T cell population with cells expressing CD80 or CD86, adding a test antibody having CTLA-4 inhibitory activity will result in a further enhancement of T cell activation.

[0245] D. immunoconjugate The present invention also provides an immunoconjugate comprising one or more cytotoxic agents (e.g., chemotherapeutic agents or chemotherapeutic drugs, growth inhibitors, toxins (e.g., protein toxins of bacterial, fungal, plant or animal origin, enzymatically active toxins, or fragments thereof) or radioisotopes) conjugated with the anti-CTLA-4 antibodies of this specification.

[0246] In one embodiment, an immunoconjugate is an antibody-drug conjugate (ADC) in which an antibody is conjugated to one or more drugs, including, but not limited to, the following: These include: meitansinoids (see U.S. Patent Nos. 5,208,020, 5,416,064, and European Patent No. 0,425,235B1); auristatins such as monomethyl auristatin drug parts DE and DF (MMAE and MMAF) (see U.S. Patents Nos. 5,635,483, 5,780,588, and 7,498,298); drastatin; calicheamycin or its derivatives (see U.S. Patents 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 includes antibodies described herein, conjugated to enzymatically active toxins or fragments thereof, including, but not limited to, diphtheria A chain, unbound active fragments of diphtheria toxin, exotoxin A chain (derived from Pseudomonas aeruginosa), lysine A chain, abrin A chain, modesine A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolacca americana protein (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crocin, saponaria officinalis inhibitor, geronin, mitogellin, restrictosin, phenomycin, enomycin, and trichothecene.

[0248] In another embodiment, the immunoconjugate comprises an antibody described herein that has been conjugated to a radioactive atom to form a radioactive conjugate. Various radioisotopes are available for the production of radioactive conjugates. For example, 211 At, 131 I, 125 I, 90 Y, 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P, 212 Contains radioactive isotopes of Pb and Lu. When using a radioactive conjugate for detection, the radioactive conjugate contains radioactive atoms (e.g., Tc-99m or) for scintigraphy examination. 123 I) or may include spin labels for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging or MRI) (e.g., iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron).

[0249] Antibody and cytotoxic agent conjugates can be prepared using a variety of bifunctional protein conjugates. Examples include N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imide esters (e.g., dimethyl HCl adipiimidoate), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azide 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, lysine 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 the conjugation of radionuclides to antibodies. See WO94 / 11026. Linkers may be “cleavable linkers” that facilitate the release of cytotoxic drugs within cells. For example, acid-unstable linkers, peptidase-sensitive linkers, photo-unstable linkers, dimethyl linkers, or disulfide-containing linkers (Chari et al., Cancer Res. 52: 127-131 (1992); U.S. Patent No. 5,208,020) may be used.

[0250] The immunoconjugates or ADCs described herein expressly consider conjugates prepared using crosslinking 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, as well as SVSB (succinimidyl-(4-vinylsulfone)benzoate), which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL., USA).

[0251] E. Methods and compositions for diagnosis 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” includes quantitative or qualitative detection. In certain embodiments, the biological sample includes cells or tissues, such as serum, whole blood, blood, biopsy specimens, tissue specimens, cell suspensions, saliva, sputum, oral fluid, cerebrospinal fluid, amniotic fluid, ascites, milk, colostrum, mammary secretions, lymph, urine, sweat, tears, gastric juice, synovial fluid, ascites, 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 a particular embodiment, the method comprises contacting a biological sample with the anti-CTLA-4 antibody described herein under conditions in which binding of the anti-CTLA-4 antibody to CTLA-4 is permissible, and detecting whether a complex has been formed between the anti-CTLA-4 antibody and CTLA-4. Such a method may be an in vitro or in vivo method. In one embodiment, the anti-CTLA-4 antibody is used to select subjects suitable for treatment using the 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 state of the immune response or to diagnose dysfunction of the immune system.

[0254] In certain embodiments, labeled anti-CTLA-4 antibodies are provided. Labeling includes, but is not limited to, directly detectable labels or moieties (e.g., fluorescent labels, chromogenic labels, high-electron-density labels, chemiluminescent labels, and radioactive labels) as well as moieties indirectly detectable through, for example, enzymatic reactions or intermolecular interactions (e.g., enzymes or ligands). Exemplary labels, but are not limited to, include: radioisotopes. 32 P, 14 C, 125 I, 3 H and 131 I, fluorescent phosphopoides such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, luciferases such as dansyl, umbelliferone, firefly luciferase and bacterial luciferase (U.S. Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, 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 linked to oxidizing pigment precursors with hydrogen peroxide (e.g., HRP, lactoperoxidase, or microperoxidase), biotin / avidin, spin-labeled, bacteriophage-labeled, stable free radicals, and similar substances.

[0255] F. Pharmaceutical preparations The pharmaceutical formulations of anti-CTLA-4 antibodies described herein are prepared in the form of lyophilized formulations or aqueous solutions by mixing the antibody of 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 the recipient at the doses and concentrations used, and include, but are not limited to, the following: buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and Examples of pharmaceutically acceptable carriers herein include: m-cresol, etc.; low molecular weight (less than approximately 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 nonionic surfactants such as polyethylene glycol (PEG). Specific exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersants such as soluble neutral active hyaluronidase glycoprotein (sHASEGP). Specific exemplary sHASEGPs and their uses are described in U.S. Patent Application Publications 2005 / 0260186 and 2006 / 0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycans, such as chondroitinase.

[0256] An exemplary lyophilized antibody preparation is described in U.S. Patent No. 6,267,958. Aqueous aqueous antibody preparations include those described in U.S. Patent No. 6,171,586 and WO2006 / 044908, the latter of which contains a histidine-acetate buffer.

[0257] The formulations described herein may contain one or more active ingredients if necessary for the specific indication being treated. Preferably, these active ingredients have complementary activities that do not adversely affect each other. Such active ingredients are preferably present in combination in amounts effective for the intended purpose.

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

[0259] A sustained-release formulation may be prepared. A preferred example of a sustained-release formulation is one comprising a semipermeable matrix of a solid hydrophobic polymer containing an antibody, the matrix being in the form of a fabricated product such as a film or microcapsule.

[0260] Preparations used for in vivo administration are typically sterile. Sterility can be easily achieved, for example, by filtering through a sterile filtration membrane.

[0261] G. Therapeutic methods and therapeutic 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 the treatment of 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, comprising the step of administering an effective amount of an anti-CTLA-4 antibody to the individual. In a further embodiment, the present invention provides an anti-CTLA-4 antibody for use in cell injury. In a particular embodiment, the present invention provides an anti-CTLA-4 antibody for use in a method of injuring cells in an individual, comprising the step of administering an effective amount of an anti-CTLA-4 antibody to the individual in order to injure cells. In any of the above embodiments, “individual” is preferably a human.

[0262] In many cases, immune cells such as lymphocytes infiltrate tumor tissue in living organisms, and these also constitute part of the tumor tissue. In several embodiments, immune cells, particularly regulatory T (Treg) cells, infiltrate the 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 the tumor tissue are damaged.

[0263] In a further aspect, the degree of the pharmaceutical effect induced by the anti-CTLA-4 antibody of the present invention varies depending on the tissue within the individual. In certain embodiments, the degree changes depending on the concentration of the adenosine-containing compound in the tissue. In a further embodiment, the effect is increased in tissues with a higher concentration of the adenosine-containing compound compared to tissues with a lower concentration. Examples of tissues with a high concentration of the adenosine-containing compound include tumor tissue. Examples of tissues with a low concentration of the adenosine-containing compound include non-tumor tissues such as normal tissue. In some embodiments, the immune system is activated more strongly in tumor tissue than in non-tumor tissue. Such differences in response do not need to be observed at all doses of the anti-CTLA-4 antibody, but only within a specific range of doses. In another embodiment, the immune system is activated at lower doses in tumor tissue compared to non-tumor tissue. In yet another embodiment, the therapeutic effect is observed at a dose lower than at which side effects are observed. In certain embodiments, therapeutic effect refers to the manifestation of an antitumor effect (e.g., tumor regression, induction of cell death or inhibition of proliferation of tumor cells), and side effect refers to the development of an autoimmune disease (including damage to normal tissue due to an excessive immune response).

[0264] In a particular manner, tumors are selected from a group consisting of breast cancer and liver cancer.

[0265] In a further aspect, the present invention provides the use of an anti-CTLA-4 antibody in the manufacture or preparation of a pharmaceutical product. In one embodiment, the pharmaceutical product is for the treatment of a tumor. In a further embodiment, the pharmaceutical product is for use in a method of treating a tumor, comprising the step of administering an effective amount of the pharmaceutical product to an individual having a tumor. In a further embodiment, the pharmaceutical product is for cell injury. In a further embodiment, the pharmaceutical product is for use in a method of injuring cells in an individual, comprising the step of administering an effective amount of the pharmaceutical product to the individual in order to injure cells. In any of the above embodiments, “individual” may be a human.

[0266] In a further aspect, the present invention provides a method for treating a tumor. In one embodiment, the method comprises the step of administering an effective amount of an anti-CTLA-4 antibody to an individual having such a tumor. The “individual” in 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 organism. In one embodiment, the method comprises the step of administering an effective amount of an anti-CTLA-4 antibody to an organism to damage cells. In one embodiment, “organism” is a human.

[0268] In a further aspect, the present invention provides a pharmaceutical formulation (pharmaceutical composition) comprising any of the anti-CTLA-4 antibodies provided herein (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 a pharmaceutical formulation (pharmaceutical composition) for use in the treatment of tumors. In one embodiment, the present invention provides a pharmaceutical formulation (pharmaceutical composition) for use in cell injury.

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

[0270] The antibodies of the present invention may be administered by any preferred means, including parenteral administration, intrapulmonary administration, and nasal administration, and, if desired for local treatment, intrafocal administration. Parenteral administration includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Dosage may be made by any preferred route, such as injection, including intravenous or subcutaneous injection, depending in part on whether the administration is short-term or long-term. Various dosing schedules, including single doses, repeated doses over various time points, bolus administration, and pulse infusion, are within consideration herein, but are not limited to these.

[0271] The antibodies of the present invention are formulated, administered, and given in a manner consistent with good medical practice. Factors to be considered from this perspective include the specific disorder being treated, the specific mammal being treated, the clinical symptoms of the individual patient, the cause of the disorder, the site of drug delivery, the method of administration, the schedule of administration, and other factors known to healthcare professionals.

[0272] For the prevention or treatment of a disease, the appropriate dose of the antibody of the present 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 for prophylactic or therapeutic purposes, the patient's medical history, clinical history and response to the antibody, and the discretion of the attending physician. The antibody is preferably administered to the patient as a single dose or over a series of treatments. Depending on the type and severity of the disease, for example, whether by single or multiple separate doses or by continuous infusion, an antibody dose of about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg) may be the initial candidate dose for administration to the patient. A typical daily dose may range from about 1 μg / kg to 100 mg / kg or more, depending on the factors described above. In the case of repeated administration over several days or longer, treatment is usually maintained, depending on the situation, until the desired suppression of disease symptoms occurs. One exemplary dose of the antibody is in the range of about 0.05 mg / kg to about 10 mg / kg. Therefore, one or more doses (or any combination thereof) of approximately 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, or 10 mg / kg may be administered to the patient. Such doses may be administered intermittently, for example, every week or every three weeks (for example, so that the patient receives approximately 2 to approximately 20, or for example, approximately 6, doses of antibody). One or more low doses may be administered after a high initial loading dose. The course of this therapy can be easily monitored by conventional methods and measurements.

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

[0274] The anti-CTLA-4 antibody described herein can be administered by administering or incorporating nucleic acids encoding the anti-CTLA-4 antibody into a living body using a vector, etc., and directly expressing the anti-CTLA-4 antibody in the living body; however, it may also be administered without using a vector. Examples of vectors include viral vectors, plasmid vectors, and adenovirus vectors. The nucleic acids encoding the anti-CTLA-4 antibody may be administered directly to the living body, or cells into which nucleic acids encoding the anti-CTLA-4 antibody have been introduced may be administered to the living body. For example, the anti-CTLA-4 antibody can be administered by chemically modifying mRNA encoding the anti-CTLA-4 antibody to enhance its stability in vivo, and then directly administering the mRNA to a human to express the anti-CTLA-4 antibody in vivo (see EP2101823B, WO2013 / 120629). Alternatively, B cells into which nucleic acids encoding the anti-CTLA-4 antibody have been introduced may be administered (Sci Immunol. (2019) 4(35), eaax0644). Alternatively, bacteria into which nucleic acids encoding anti-CTLA-4 antibodies have been introduced may be administered (Nature Reviews Cancer (2018) 18, 727-743).

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

[0276] H.Product In another aspect of the present invention, a product is provided comprising equipment useful for the treatment, prevention, and / or diagnosis of the above-mentioned disorders. The product comprises a container and a label on the container or accompanying documentation attached to the container. Preferred containers include, for example, bottles, vials, syringes, and intravenous (IV) solution bags. Containers may be formed from a variety of materials, such as glass or plastic. A container may hold a composition alone or in combination with another composition effective for the treatment, prevention, and / or diagnosis of a symptom, and may have a sterile access port (for example, the container may be an intravenous solution bag or vial with a stopper that can be punctured by a subcutaneous needle). At least one active ingredient in the composition is the antibody of the present invention. The label or accompanying documentation indicates that the composition is used to treat a selected symptom. The product in this aspect of the present invention may further include accompanying documentation indicating that the composition may be used to treat a particular symptom. Alternatively, the product may further include a second container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other commercially or user-desirable equipment, such as other buffers, diluents, filters, needles, and syringes.

[0277] It will be understood that any of the above-mentioned products may contain the immunoconjugate of the present invention in place of or in addition to the anti-CTLA-4 antibody. <Polypeptide containing a mutated Fc region>

[0278] In one aspect, the present invention provides an isolated polypeptide comprising a mutant Fc region. In several aspects, the polypeptide is an antibody. In several 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 the native sequence or a reference mutant sequence (which may be collectively referred to herein as the “parent” Fc region). The Fc region of the native sequence is typically composed of a homodimer 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 polypeptide chains.

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

[0280] In some embodiments, the variant Fc region of the present invention includes 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, as represented by EU numbering. Alternatively, amino acid modifications described in International Publications WO2013 / 002362 and WO2014 / 104165 may also be used in the present invention.

[0281] In certain embodiments, the binding activity of the parental Fc region and the mutant Fc region can be expressed by a KD (Dissociation constant) value. In one embodiment, the ratio of [KD value of the parental Fc region to FcγRIIa] / [KD value of the mutant Fc region to FcγRIIa] is, for example, 1.5 or greater, 2 or greater, 3 or greater, 4 or greater, 5 or greater, 6 or greater, 7 or greater, 8 or greater, 9 or greater, 10 or greater, 15 or greater, 20 or greater, 25 or greater, 30 or greater, 40 or greater, or 50 or greater. In further embodiments, FcγRIIa may be FcγRIIa R, FcγRIIa H, or both. Therefore, the KD value of the Fc region to FcγRIIa may be the KD value of the Fc region to FcγRIIa R, or the KD value of the Fc region to FcγRIIa H, or the sum or average of both. In one aspect, the ratio of [binding activity of the parental Fc region to FcγRIIIa] / [binding activity of the 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 The above is 2 x 10 3 The above is 3 x 10 3 Above, or 5 x 10 3 That concludes the explanation. 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, or the sum or average of both.

[0282] In one embodiment, the KD value of the mutated Fc region for FcγRIIa is, for example, 1.0 × 10⁻⁶. -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 × 10 -9 It is less than or equal to M. In a further embodiment, FcγRIIa may be FcγRIIa R, FcγRIIa H, or both. In one embodiment, the KD value of the mutant Fc region for FcγRIIIa is, for example, 1.0 × 10⁻⁶. -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 × 10 -10 It is less than or equal to M. In a further embodiment, FcγRIIIa may be FcγRIIIa F, or FcγRIIIa V, or both.

[0283] In another embodiment, the binding activity of the parental Fc region and the mutant Fc region may be expressed in terms of the kd (Dissociation rate constant) value instead of the KD value.

[0284] In another embodiment, the binding activity of the parental Fc region and the mutant Fc region may be expressed as the amount of Fc region binding to the Fcγ receptor per unit amount. For example, in a surface plasmon resonance assay, the amount of Fc region immobilized on a sensor chip, and the amount of Fcγ receptor bound to it, are each measured as a resonance unit (RU). The amount of Fcγ receptor binding there, divided by the amount of Fc region binding, can be defined as the amount of Fc region binding to the 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 parental Fc region binding to FcγRIIa] is, for example, 1.5 or greater, 2 or greater, 3 or greater, 4 or greater, 5 or greater, 6 or greater, 7 or greater, 8 or greater, 9 or greater, 10 or greater, 15 or greater, 20 or greater, 25 or greater, 30 or greater, 40 or greater, or 50 or greater. In some aspects, the ratio of [binding amount to FcγRIIIa in the mutant Fc region] / [binding amount to FcγRIIIa in the parent Fc region] 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 The above is 2 x 10 3 The above is 3 x 10 3 Above, or 5 x 10 3 That's all.

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

[0286] In certain aspects, the mutant Fc region of the present invention exhibits improved selectivity between active and inhibitory Fcγ receptors compared to the parental Fc region. In other words, the mutant Fc region of the present invention exhibits significantly enhanced binding activity to active Fcγ receptors compared to the parental Fc region. In certain embodiments, the active 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 region of the present invention exhibits improved selectivity between FcγRIIa and FcγRIIb. In some embodiments, the mutant Fc region of the present invention exhibits improved selectivity between FcγRIIIa and FcγRIIb. In a further embodiment, the mutant Fc region of the present invention exhibits improved selectivity between FcγRIIa and FcγRIIb, and between FcγRIIIa and FcγRIIb.

[0287] In some embodiments, the variant 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 236, 239, 268, 270, and 326, as represented by EU numbering. Alternatively, amino acid modifications described in International Publications WO2013 / 002362 and WO2014 / 104165 may also be used in the present invention.

[0288] In certain embodiments, the binding activity of the parental Fc region and the mutant Fc region can be expressed by a KD (Dissociation constant) value. The embodiments of the binding activity to FcγRIIa and FcγRIIIa are as described above. In one embodiment, the ratio of [KD value of the parental Fc region to FcγRIIb] / [KD value of the mutant Fc region to FcγRIIb] is, for example, 10 or less, 5 or less, 3 or less, 2 or less, 1 or less, 0.5 or less, 0.3 or less, 0.2 or less, or 0.1 or less. In another embodiment, the binding activity of the parental Fc region and the mutant Fc region may be expressed by a kd (Dissociation rate constant) value instead of a KD value.

[0289] In another embodiment, the binding activity of the parental Fc region and the mutant Fc region may be expressed as the amount of the Fc region binding to the Fcγ receptor per unit amount as described above. In some embodiments, the ratio of [binding amount of mutant Fc region to FcγRIIb] / [binding amount of parental 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 binding amount 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 certain embodiments, improved selectivity between active and repressive Fcγ receptors is a selective enhancement of binding activity to active Fcγ receptors compared to binding activity to repressive Fcγ receptors; in other words, an increase in the ratio of binding activity to active Fcγ receptors to binding activity to repressive Fcγ receptors (A / I ratio). Such a ratio (A / I ratio) is an indicator of superior effector function, and polypeptides with a large A / I ratio can be evaluated as having superior effector function. The binding activity of parental and mutant Fc regions to Fcγ receptors can be expressed as KD value, kd value, or the amount of Fc region bound to Fcγ receptors 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 active Fcγ receptor], [kd value for inhibitory Fcγ receptor] / [kd value for active Fcγ receptor], or [binding amount for active Fcγ receptor] / [binding amount for inhibitory Fcγ receptor].

[0291] In one embodiment, the A / I ratio of the mutated 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. The above figures represent increases of 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. In one aspect, the A / I ratio value of the mutated 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 binding activity to FcγRIa to binding activity to FcγRIIb, the ratio of binding activity to FcγRIIa to binding activity to FcγRIIb, the ratio of binding activity to FcγRIIIa to binding activity to FcγRIIb, or the ratio of the sum or average of two or three of the binding activities to FcγRIa, FcγRIIa, and FcγRIIIa to binding activity to FcγRIIb. In a particular embodiment, FcγRIIa is FcγRIIa R, FcγRIIa H, or both, and therefore the binding activity to FcγRIIa is the sum or average of the binding activity to FcγRIIa R, the binding activity to FcγRIIa H, or both.In certain embodiments, FcγRIIIa is FcγRIIIa F, FcγRIIIa V, or both, and therefore the binding activity to FcγRIIIa is the sum or mean of the binding activity to FcγRIIIa F, the binding activity to FcγRIIIa V, or both.

[0292] In some embodiments, the mutant Fc region of the present invention includes amino acid modifications at the following positions: (i) Positions 234, 235, 236, 239, 268, 270, and 298 in the first polypeptide of the parent Fc region, as represented by EU numbering, and (ii) Positions 270, 298, 326, and 334 in the second polypeptide of the parent Fc region, as represented by EU numbering. In certain embodiments, the mutant Fc region of the present invention further includes an amino acid modification at position 326, represented by EU numbering, in the first polypeptide of the parent Fc region. In certain embodiments, the mutant Fc region of the present invention further includes an amino acid modification at position 236, represented by EU numbering, in the second polypeptide of the parent Fc region. In certain embodiments, the mutant Fc region of the present invention further includes an amino acid modification at position 332, represented by EU numbering, in the first polypeptide of the parent Fc region. In certain embodiments, the mutant Fc region of the present invention further includes an amino acid modification at position 330, represented by EU numbering, in the first polypeptide of the parent Fc region. In certain embodiments, the mutant Fc region of the present invention further includes an amino acid modification at position 332, represented by EU numbering, in the second polypeptide of the parent Fc region. In certain embodiments, the mutant Fc region of the present invention further includes an amino acid modification at position 330, represented by EU numbering, in the second polypeptide of the parent Fc region. Alternatively, amino acid modifications described in International Publications WO2013 / 002362 and WO2014 / 104165 can also be used in the present invention.

[0293] In some embodiments, the mutant Fc region of the present invention includes amino acid modifications at the following positions: (i) Positions 234, 235, 236, 239, 268, 270, 298, and 330 in the first polypeptide of the parent Fc region, as represented by EU numbering, and (ii) Positions 270, 298, 326, 330, and 334 in the second polypeptide of the parent Fc region, as represented by EU numbering. In certain embodiments, the mutant Fc region of the present invention further includes an amino acid modification at position 326, represented by EU numbering, in the first polypeptide of the parent Fc region. In certain embodiments, the mutant Fc region of the present invention further includes an amino acid modification at position 236, represented by EU numbering, in the second polypeptide of the parent Fc region. In certain embodiments, the mutant Fc region of the present invention further includes an amino acid modification at position 332, represented by EU numbering, in the first polypeptide of the parent Fc region. In certain embodiments, the mutant Fc region of the present invention further includes an amino acid modification at position 332, represented by EU numbering, in the second polypeptide of the parent Fc region. In certain embodiments, or the amino acid modifications described in International Publications WO2013 / 002362 and WO2014 / 104165, may also be used in the present invention.

[0294] In certain aspects, the mutant Fc region of the present invention exhibits 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 using indicators such as the Tm value. The Tm value can be measured using methods known to those skilled in the art, such as CD (circular dichroism), DSC (scanning scan calorimeter), and DSF (scanning scan fluorescence quantitative analysis). In one embodiment, the mutant Fc region of the present invention exhibits a Tm value of 0.1 degrees or more, 0.2 degrees or more, 0.3 degrees or more, 0.4 degrees or more, 0.5 degrees or more, 1 degree or more, 2 degrees or more, 3 degrees or more, 4 degrees or more, 5 degrees or more, and 10 degrees or more higher in the CH2 region compared to the parent Fc region.

[0295] In some embodiments, 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 250 and 307, as represented by EU numbering, in the first 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 certain aspects, the mutant Fc region of the present invention is composed of two polypeptide chains with different sequences. In further aspects, heterodimerization between the first polypeptide and the second polypeptide is promoted in the mutant Fc region of the present invention. When producing a heterodimer protein using a recombinant method, it is preferable that different peptide chains preferentially associate to form a heterodimer rather than identical polypeptide chains associating to form a homodimer. Whether heterodimerization of the mutant Fc region is promoted can be determined, for example, by separating the homodimer and heterodimer from the produced mutant Fc region using a method such as chromatography and determining the ratio of each component.

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

[0298] In certain aspects, the mutant Fc region of the present invention exhibits enhanced binding activity to FcRn under acidic pH conditions. In some embodiments, acidic pH refers to a pH of 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, 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 at at least one position selected from the group consisting of positions 428, 434, 436, 438, and 440, as represented by EU numbering, in the first 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 includes 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 represented by EU numbering.

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

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

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

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

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

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

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

[0308] In a further embodiment, the mutant Fc region of the present invention comprises (i) at least one amino acid modification selected from the group consisting of Tyr or Phe at position 234, represented by EU numbering, 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 in the first polypeptide of the parent Fc region, and (ii) at least one amino acid modification selected from the group consisting of 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, or Lys, Asp at position 332, or Glu at position 334 in the 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) Lys at position 356 in EU numbering in the first polypeptide of the parent Fc region, and Glu at position 439 in EU numbering in the second polypeptide of the parent Fc region, (b) Glu at position 439 in EU numbering in the first polypeptide of the parent Fc region, and Lys at position 356 in EU numbering in the second polypeptide of the parent Fc region. (c) Trp at position 366 in EU numbering in the first polypeptide of the parent Fc region, and Ser at position 366, Ala at position 368, and Val at position 407 in EU numbering in the second polypeptide of the parent Fc region, (d) Ser at position 366, Ala at position 368, and Val at position 407 in the first polypeptide of the parent Fc region, as represented by EU numbering, and Trp at position 366 in the second polypeptide of the parent Fc region, (e) Cys at position 349 and Trp at position 366 in the first polypeptide of the parent Fc region, as represented by EU numbering, and Cys at position 356, Ser at position 366, Ala at position 368, and Val at position 407 in the second polypeptide of the parent Fc region, as represented by EU numbering, (f) Cys at position 356, Ser at position 366, Ala at position 368, and Val at position 407 in the first polypeptide of the parent Fc region, as represented by EU numbering, and Cys at position 349 and Trp at position 366 in the second polypeptide of the parent Fc region, as represented by EU numbering.

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

[0311] In a particular embodiment, the polypeptide comprising the mutated Fc region of the present invention is the constant heavy chain region of an antibody.

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

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

[0314] "Fcγ receptor" (hereinafter referred to as Fcγ receptor, FcγR, or FcgR) refers to a receptor capable of binding to the Fc region of IgG1, IgG2, IgG3, and IgG4 monoclonal antibodies, and effectively means any member of the protein family encoded by the Fcγ receptor gene. In humans, this family includes, but is not limited to, Fcγ...

Claims

1. Anti-CTLA-4 antibody, (A) (1) (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 112; (c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 102; (d) HVR-L1 containing the amino acid sequence of SEQ ID NO: 128; (e) HVR-L2 containing the amino acid sequence of SEQ ID NO: 117; (f) HVR-L3 containing the amino acid sequence of SEQ ID NO: 133 (2) (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 111; (c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 152; (d) HVR-L1 containing the amino acid sequence of SEQ ID NO: 128; (e) HVR-L2 containing the amino acid sequence of SEQ ID NO: 117; (f) HVR-L3 containing the amino acid sequence of SEQ ID NO: 133 (3) (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 112; (c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 102; (d) HVR-L1 containing the amino acid sequence of SEQ ID NO: 129; (e) HVR-L2 containing the amino acid sequence of SEQ ID NO: 117; (f) HVR-L3 containing the amino acid sequence of SEQ ID NO: 133, or (4) (a) HVR-H1 containing the amino acid sequence of SEQ ID NO: 107; (b) HVR-H2 containing the amino acid sequence of SEQ ID NO: 111; (c) HVR-H3 containing the amino acid sequence of SEQ ID NO: 152; (d) HVR-L1 containing the amino acid sequence of SEQ ID NO: 129; (e) HVR-L2 containing the amino acid sequence of SEQ ID NO: 117; (f) HVR-L3 containing the amino acid sequence of SEQ ID NO: 133 A variable region including, (B) (i) Amino acid modifications in the first polypeptide of the parent Fc region, which is composed of two polypeptide chains, to Phe at position 234, Gln at position 235, Trp at position 236, Met at position 239, Asp at position 268, Glu at position 270, Ala at position 298, and Met at position 330, as represented by EU numbering, and (ii) Amino acid modifications in the second polypeptide of the parent Fc region to Glu at position 270, Ala at position 298, Asp at position 326, Met at position 330, and Glu at position 334, as represented by EU numbering. A mutated Fc region including, Isolated nucleic acids encoding an anti-CTLA-4 antibody, including [specific component].

2. The nucleic acid according to claim 1, wherein the anti-CTLA-4 antibody comprises (a) a VH sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 140 or 141; (b) a VL sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 146 or 147; or (c) a VH sequence having the amino acid sequence of SEQ ID NO: 140 or 141, and a VL sequence having the amino acid sequence of SEQ ID NO: 146 or 147.

3. Anti-CTLA-4 antibody, (1) VH sequence with sequence number 140 and VL sequence with sequence number 146, (2) VH sequence of sequence number 141 and VL sequence of sequence number 146, (3) VH sequence of sequence number 140 and VL sequence of sequence number 147, (4) VH sequence of sequence number 141 and VL sequence of sequence number 147, (5) A first variable region containing the VH sequence of sequence number 140 and the VL sequence of sequence number 146, and a second variable region containing the VH sequence of sequence number 141 and the VL sequence of sequence number 146, or (6) A first variable region containing the VH sequence of sequence number 140 and the VL sequence of sequence number 147, and a second variable region containing the VH sequence of sequence number 141 and the VL sequence of sequence number 147, The nucleic acid according to claim 1, comprising:

4. The mutated Fc region further (i) Amino acid modifications in the first polypeptide of the parent Fc region to Val at position 250 and Pro at position 307, as represented by EU numbering, and (ii) Amino acid modifications in the second polypeptide of the parent Fc region to Val at position 250 and Pro at position 307, as represented by EU numbering. The nucleic acid according to claim 1, comprising:

5. The nucleic acid according to claim 1, wherein the anti-CTLA-4 antibody comprises a heavy chain constant region including a mutated Fc region.

6. The heavy chain constant region is (1) The first polypeptide of SEQ ID NO: 358, and the second polypeptide of SEQ ID NO: 359, or (2) The first polypeptide of Sequence ID: 360, and the second polypeptide of Sequence ID: 361, The nucleic acid according to claim 5, comprising:

7. (1) The first H chain polypeptide of SEQ ID NO: 335, the second H chain polypeptide of SEQ ID NO: 336, and the L chain polypeptide of SEQ ID NO: 161, or (2) The first H chain polypeptide of SEQ ID NO: 337, the second H chain polypeptide of SEQ ID NO: 338, and the L chain polypeptide of SEQ ID NO: 161, Isolated nucleic acids encoding an anti-CTLA-4 antibody, including [specific component].

8. A host cell comprising the nucleic acid according to any one of claims 1 to 7.

9. A method for producing an anti-CTLA-4 antibody, comprising culturing the host cells described in claim 8 so as to produce the anti-CTLA-4 antibody.

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